**Greenwashing and Cleaning**

Develter Dirk1 and Malaise Peter2

*1Ecover Coordination Center NV, 2Meta Fellowship npo Belgium* 

## **1. Introduction**

VIII Preface

source of energy.

silver nanoparticles.

an introduction to the overall aspects of Knoevenagel Reaction; Chapter 3 is concerned with the use of nanometals fabricated in cancer diagnosis; Chapter 4 addresses the formation of NaHSO4 by the green conversion of SO2 through electrochemical forces; and chapter 5 involves the synthesis of azoles with the replacement of traditional, environmentally unattractive methodologies by the utilization of ultrasonic process as

Another key topic - greener solvent-free reactions on ZnO – is addressed in Chapter 6. The use of nano-ZnO as a catalyst under solvent-free conditions for organic reactions is referred as green reactions. Chapter 7 deals with the new green oil-field agents. Finally, this book covers a growing field of green chemistry in Chapter 8 which describes a number of greener techniques to synthesize and characterize the gold and

It is clear that many industries and the research of many academics recognize the

It was impossible to meet all our goals or cover all areas of green chemistry in this monograph. However, we believe that this book will provide both researchers and

> **Professor M. Kidwai, Ph.D, FEnA** and **Dr. Neeraj Kumar Mishra, M.Sc., Ph.D**

> > Department of Chemistry, University of Delhi, Delhi,

> > > India

significance of green chemistry. However, more work remains to be done.

scientists with ideas for future developments in the field of green chemistry.

The world population is rapidly growing: estimates tell us that from nearly seven billion actually, we're on our way to some eight to ten billion in 2050 (US Census Bureau, 2011). This ongoing growth generates a forever growing demand in products and services: raw materials, energy, transport and transformation capacity, waste disposal. Unfortunately, the backbone of all of these activities is mainly dependent of a single fossil source, crude oil and its derivatives. However, these have a double disadvantage:


Parallel to this huge growth and rapid depletion there is a growing consciousness on hygiene and personal deployment in all countries worldwide, but mainly in developing ones. The need for products and services is still growing exponentially.

Basically, this complex growth is already transcending the capacity of the planet, making the largest part of the average economic activities *unsustainable*. Some market segments already suffer from it, metals e.g.: prices for ores and metals have rocketed the last couple of years and the market for recycled materials is on an all time high. For some of them we are quite close to a shortage. It won't stop there: with decreasing mineral and fossil sourcing capabilities, producers will be forced to turn to non-fossil, non-mineral, renewable sources, that means in the first place plant sources and to a lesser extent, animal sources.

These raw material sources are actually and nearly exclusively the providers of food, but they will inevitably suffer a strong competition from non-food production demand. We already saw the consequences of unplanned and unregulated behavior on the matter when, in 2007, there was a sudden huge demand in renewable raw materials for the production of biofuel, the "Food vs. Fuel" crisis, also called the "Tortilla Flap". Tortilla prices doubled for the already poor Mexican population, causing riots. The real causes were not even a raw material shortage, but mainly speculations in the globalised markets (Kaufman, 2010; Nelson, 2008; Western Organisation of Resource Councils, 2007).

When one day, next to food, a large part of clothing, housing and utensils will forcedly have to be derived from plant and animal sources, there will not only be substantial shortages,

Greenwashing and Cleaning 3

mortality amongst honey bees. Hardly noticed by most people but for their honey we consume, they are primarily responsible for pollination in nature. A declining pollination will, amongst others, have an immediate negative influence on agriculture and everything depending on it, but also on pollination in the wild. This will at the end turn the planet into

The production processes for most goods and services are mainly using unsustainable energy, involve unsustainable processing techniques, unsustainable transport methods and generate an unacceptable amount of problematic waste. Only very few countries have managed to switch a substantial part of their energy production to sustainable energy sources. Some of the actions that have been taken on the matter are questionable, because they only touch a small part of the problem. Saving bulbs are such an example: they leave about 81 % of the household consumption of electricity with fossil sources and make the consumer believe that he is solving a problem. Politically spoken the venue of saving bulbs was a quick and dirty decision, but in the meantime new health concerns have risen in relation to saving bulbs. Unfortunately, they are now becoming compulsory in many countries. We don't hear anything though on saving fridges, saving deep freezers, saving washing machines, saving dry tumblers, saving fryers, saving stoves; all of these devices are in their actual form the real culprits, counting for 81% of

Saving cars exist to some extent: the hybrids are on sale since some years and some prototypes of electric cars are presented increasingly. However, their promotion is not taken serious enough, neither by their producers, nor by politicians. Most cars still have fuel consumption and CO2 emission rates which are unacceptably high, although the improvement technology is available. Some car companies are known to actively lobby against stricter emission laws (Greenpeace, 2011). Public transport offers an interesting solution to a large part of the private traffic, but it is actually legging far behind as to comfort, frequency and efficiency. It will nevertheless be one of the main choices to realize a

When we look at production processes the situation is even worse. Up to recently there was hardly any attention for sustainable industrial processing techniques. More or less as a rule such processes involve high temperature and high pressure, often accompanied by other energy demanding techniques such as vacuum generation, freezing or desiccation. The production and transformation of aluminum or the cracking of crude oil and the processing of many of their derivatives e.g., are such energy devouring activities. Some other sectors such as the production of chemicals for household and industrial uses can cause huge environmental and health problems. Hopefully we will not forget Bhopal (India), still an unsolved problem for the local victims, 20 years after the catastrophy occurred. Nor more recently the *Deepwater Horizon* (Mexico) and *Ganeth Alpha* (Aberdeen) ridge oil spills and the flood of poisonous aluminum sludge in the *Ajkai Timföldgyár* factory (Ajka, Hungary). These

are just a few examples of the consequences of unsustainable production methods.

The production of commodities for the mass market is equally tainted. Detergents and their raw materials are for the essential part made from fossil sources, although they could easily be made from renewables – as a matter of fact they have been, up to about 1930. A large part

an infertile, uneconomic desert.

**3. Unsustainable processing** 

the energy consumption in households.

sustainable transport system for future society.

high prices, fights and even wars to be expected in relation to those materials, but it will simply be impossible to generate such an amount of raw materials on this planet. At the actual consumption rate, available agricultural space is simply not big enough to provide all the necessary. The only reasonable outcome is a double action. We must at the same time substantially reduce our needs in raw materials and energy, and hugely increase research in new ways of raw material sourcing, higher efficiency and better transformation processes.

Such a type of development has already been proposed and documented by the Wuppertal Institute (http://www.wupperinst.org), Germany, under the names of "Factor Four" and "Factor Ten", targeting a fourfold, respectively tenfold reduction in the bulk of our needs. Their theme is still: *"We use resources as though we had four earths at our disposal",* which describes the actual problem quite well. The Wuppertal Institute also developed instruments to quantify such developments, amongst them the *Material Input Per Service* tool (MIPS). This approach measures how much earthly substance is needed to generate all the necessary for one service of a given product.

As always with such developments we can discuss until Doomsday if this is the right thing to do here and now and if there are no better solutions (read: less compulsory, less drastic, financially cheaper etc.). Perhaps – but can we afford to wait? Do we really have the time to abide such ideal solutions? We, the authors, are convinced we don't and we prefer in this to marry the approach of the Swedish 'Natural Step' organization (http://www.naturalstep.org): *let's not be stuck on endless discussions about the twigs of problems, but let's agree about the trunk and the branches*.

## **2. Global pollution consequences**

One of the main consequences of unsustainable economical activities is the continuous exposure of man to man-made chemicals, and to high levels of mixed, persistent pollution. Especially children, young people and the elderly are vulnerable. It's not that much the spectacular catastrophes, such as oil spills and sinking tankers, which are quite visible; but the silent, insidious spreading of chemicals that should not reside in nature at all. Heavy metals are notorious, but there are even more risky compounds, such as:


All of the consequences become only indirectly visible, through degrading biodiversity and fertility, making species disappear at an abnormal rate and speed, through high incidences of uncommon pathologies or even through the transmission of risky genetic properties. Unfortunately, economists and investors don't see these phenomena as relevant for economic life. It's true that when economic life is just looking at the next fiscal quarter, hardly anything is relevant. When our ancestors would have thought and acted like that, we wouldn't be here.

But there *is* a relationship with those phenomena - and a tight one too. One out of many immediate consequences of man-made pollution for example is the actual worldwide

high prices, fights and even wars to be expected in relation to those materials, but it will simply be impossible to generate such an amount of raw materials on this planet. At the actual consumption rate, available agricultural space is simply not big enough to provide all the necessary. The only reasonable outcome is a double action. We must at the same time substantially reduce our needs in raw materials and energy, and hugely increase research in new ways of raw material sourcing, higher efficiency and better transformation processes. Such a type of development has already been proposed and documented by the Wuppertal Institute (http://www.wupperinst.org), Germany, under the names of "Factor Four" and "Factor Ten", targeting a fourfold, respectively tenfold reduction in the bulk of our needs. Their theme is still: *"We use resources as though we had four earths at our disposal",* which describes the actual problem quite well. The Wuppertal Institute also developed instruments to quantify such developments, amongst them the *Material Input Per Service* tool (MIPS). This approach measures how much earthly substance is needed to generate all the

As always with such developments we can discuss until Doomsday if this is the right thing to do here and now and if there are no better solutions (read: less compulsory, less drastic, financially cheaper etc.). Perhaps – but can we afford to wait? Do we really have the time to abide such ideal solutions? We, the authors, are convinced we don't and we prefer in this to marry the approach of the Swedish 'Natural Step' organization (http://www.naturalstep.org): *let's not be stuck on endless discussions about the twigs of* 

One of the main consequences of unsustainable economical activities is the continuous exposure of man to man-made chemicals, and to high levels of mixed, persistent pollution. Especially children, young people and the elderly are vulnerable. It's not that much the spectacular catastrophes, such as oil spills and sinking tankers, which are quite visible; but the silent, insidious spreading of chemicals that should not reside in nature at all. Heavy


All of the consequences become only indirectly visible, through degrading biodiversity and fertility, making species disappear at an abnormal rate and speed, through high incidences of uncommon pathologies or even through the transmission of risky genetic properties. Unfortunately, economists and investors don't see these phenomena as relevant for economic life. It's true that when economic life is just looking at the next fiscal quarter, hardly anything is relevant. When our ancestors would have thought and acted like that, we

But there *is* a relationship with those phenomena - and a tight one too. One out of many immediate consequences of man-made pollution for example is the actual worldwide


metals are notorious, but there are even more risky compounds, such as:


necessary for one service of a given product.

**2. Global pollution consequences** 


book *Silent Spring* 

wouldn't be here.

*problems, but let's agree about the trunk and the branches*.

mortality amongst honey bees. Hardly noticed by most people but for their honey we consume, they are primarily responsible for pollination in nature. A declining pollination will, amongst others, have an immediate negative influence on agriculture and everything depending on it, but also on pollination in the wild. This will at the end turn the planet into an infertile, uneconomic desert.

### **3. Unsustainable processing**

The production processes for most goods and services are mainly using unsustainable energy, involve unsustainable processing techniques, unsustainable transport methods and generate an unacceptable amount of problematic waste. Only very few countries have managed to switch a substantial part of their energy production to sustainable energy sources. Some of the actions that have been taken on the matter are questionable, because they only touch a small part of the problem. Saving bulbs are such an example: they leave about 81 % of the household consumption of electricity with fossil sources and make the consumer believe that he is solving a problem. Politically spoken the venue of saving bulbs was a quick and dirty decision, but in the meantime new health concerns have risen in relation to saving bulbs. Unfortunately, they are now becoming compulsory in many countries. We don't hear anything though on saving fridges, saving deep freezers, saving washing machines, saving dry tumblers, saving fryers, saving stoves; all of these devices are in their actual form the real culprits, counting for 81% of the energy consumption in households.

Saving cars exist to some extent: the hybrids are on sale since some years and some prototypes of electric cars are presented increasingly. However, their promotion is not taken serious enough, neither by their producers, nor by politicians. Most cars still have fuel consumption and CO2 emission rates which are unacceptably high, although the improvement technology is available. Some car companies are known to actively lobby against stricter emission laws (Greenpeace, 2011). Public transport offers an interesting solution to a large part of the private traffic, but it is actually legging far behind as to comfort, frequency and efficiency. It will nevertheless be one of the main choices to realize a sustainable transport system for future society.

When we look at production processes the situation is even worse. Up to recently there was hardly any attention for sustainable industrial processing techniques. More or less as a rule such processes involve high temperature and high pressure, often accompanied by other energy demanding techniques such as vacuum generation, freezing or desiccation. The production and transformation of aluminum or the cracking of crude oil and the processing of many of their derivatives e.g., are such energy devouring activities. Some other sectors such as the production of chemicals for household and industrial uses can cause huge environmental and health problems. Hopefully we will not forget Bhopal (India), still an unsolved problem for the local victims, 20 years after the catastrophy occurred. Nor more recently the *Deepwater Horizon* (Mexico) and *Ganeth Alpha* (Aberdeen) ridge oil spills and the flood of poisonous aluminum sludge in the *Ajkai Timföldgyár* factory (Ajka, Hungary). These are just a few examples of the consequences of unsustainable production methods.

The production of commodities for the mass market is equally tainted. Detergents and their raw materials are for the essential part made from fossil sources, although they could easily be made from renewables – as a matter of fact they have been, up to about 1930. A large part

Greenwashing and Cleaning 5

Starting in 1970, several high-level reports continued warning for the consequences of such an unsustainable development: *The Predicament of Mankind (*Christakis et al., 1970); *Limits to Growth* from the Club of Rome (Meadows et al., 1972); *Our Common Future* from the Brundtland Commission (World Commission on Environment and Development, 1987). But in spite of all these serious efforts and a series of follow-up initiatives such as the *Rio Conference*, *Agenda 21, Rio+10* and many more, very little systematic action has been taken. The global principle to tackle what became a *global problem,* is *Sustainable Development*. The Brundtland report, in which the term 'sustainable development' was first used, describes this in a much cited quote as *"development that meets the needs of the present without compromising the ability of future generations to meet their own needs."* It has three focus points,

It will be obvious that each of these three members has its own specific rules and laws, which might be influenced by the others, but not overruled or replaced by them. It is not possible that economic principles will become more important than social or environmental ones; but they cannot become less important either. An essential fact - often misunderstood even by fervent followers – is that Sustainable Development is a life style, not a status that one can reach some time. You can't be, or can't become 'sustainable', there will always be a

However, to cut short any misunderstanding: when we will in the following write about 'sustainable raw materials' or 'sustainable energy' we are not pointing at a status those items are supposed to be in, but at the whole *process* that leads to their existence. The raw material is a crystallization point of a generative process which fits – or doesn't fit, or only partially

It's also obvious that, on the short term, it will not be possible to realize all elements of Sustainable Development to an equal degree of fulfillment, immediately and at the same time. Many things will only be partially realized through compromises between societal

Sustainable Development encompasses sustainable design, sustainable raw materials, sustainable production processes, sustainable energy and services, green taxes, as well as sustainable consumption. In short, it's a *cradle-to-grave* approach at all times and a *cradle-tocradle* approach whenever possible (more on this theme is to be found in Braungart and McDonough (2002). *Cradle-to-grave* means that all partners are part of the whole process, from the design of the product or service until the disposal of possible leftovers, and anything in between. Each step has to be optimized: it should involve the lowest amount of earthly substance and energy possible, have the highest efficiency and user friendliness possible and generate as little leftovers as feasible (that what we still call 'waste'). This should be featured without compromising elements such as the availability, the efficacy or

*Cradle-to-cradle* goes even a step further: whatever substance that is not fully destroyed at use (such as food), has to be made reusable for a similar, or even for a completely different

which are inextricably intertwined and should not be separated at any time:

 a social focus an economical focus an ecological focus

further stage of development to attend.

the price of the product or service.

fits – into principles of Sustainable Development.

partners, in a slow process of involvement and comprehending.

of paper derived disposables still use freshly cut trees instead of recycled fibers. In many countries industries get an explicit advantage on their electricity bill because of their high consumption – whereas the opposite should be the case when we would apply the rule 'the pollutor pays'. There is hardly an incentive for companies to take serious action on the matter.

Production processes in the agricultural realm have, on top of high energy demands in some sectors, such as greenhouses, a huge impact on health and environment through the chemicals they introduce in the food web: synthetic fertilizer, insecticides and pesticides, as well as aftertreatments for preservation and pest control. Potato culture knows an average of about 11 chemical treatments before harvesting. The production and use of banana pesticides and insecticides causes heavy health and environmental impacts (Chua, 2007). Soy production for animal feedstock, and palm oil production for food and non-food applications, are still devastating huge areas of virgin forest, destroying important natural CO2 dumps.

The policy of subsidizing agricultural and other produce for export are still in place, even for such goods that can easily be produced in the destination countries. There is no sensible reason why Austrians should eat Belgian green peas instead of the ones from their own agriculture, and Belgians the Austrian ones, unless in case of a shortage on either side. Consuming as much as possible produce and products from where one lives could substantially reduce primary fuel consumption, traffic jam, pollution and health impacts. Farmers should be subsidized for maintaining the natural fertility of the soil and the preservation of biodiversity – not for overproduction within monocultures, as it is the case now. That would lead to a broad support and promotion of certified organic farming, rather than fighting it with prejudice. Organic farming has maintaining the natural fertility and preserving biodiversity in its basic principles – you can't have organic farming without them. The secondary effects of such measures would in the middle and long term be very important as well: no synthetic fertilizer, little to no chemical insecticides and pesticides, a healthy soil and a healthier water system.

The professional transport of food and non-food commodities knows comparable problems. But there is in the transport sector even less interest for sustainable transport solutions than with individuals: maximum load, high speed and low financial cost are the sole drivers. Some isolated projects, such as the one set up by the Belgian distributor Colruyt with an innovative lorry, goes further and tries to reduce fuel consumption and emissions by proactively financing, testing and adopting hybrid equipment that will respond to the newest EU requirements (Colruyt Group, 2010).

For any man made activity we should since long have adopted the *Precautionary Principle*  (European Commission, 2000): when we don't know the consequences, or have difficulties in estimating the extent of health and environmental impacts – including the depletion of raw material sources – we just shouldn't do it.

## **4. Old stuff**

These facts are not new. Rachel Carson wrote her book *Silent Spring* in 1960 (Carson,1960). She warned for a thoughtless, large scale use of man made, highly effective chemicals and documented the then already visible consequences for health and environment.

of paper derived disposables still use freshly cut trees instead of recycled fibers. In many countries industries get an explicit advantage on their electricity bill because of their high consumption – whereas the opposite should be the case when we would apply the rule 'the pollutor pays'. There is hardly an incentive for companies to take serious action

Production processes in the agricultural realm have, on top of high energy demands in some sectors, such as greenhouses, a huge impact on health and environment through the chemicals they introduce in the food web: synthetic fertilizer, insecticides and pesticides, as well as aftertreatments for preservation and pest control. Potato culture knows an average of about 11 chemical treatments before harvesting. The production and use of banana pesticides and insecticides causes heavy health and environmental impacts (Chua, 2007). Soy production for animal feedstock, and palm oil production for food and non-food applications, are still

The policy of subsidizing agricultural and other produce for export are still in place, even for such goods that can easily be produced in the destination countries. There is no sensible reason why Austrians should eat Belgian green peas instead of the ones from their own agriculture, and Belgians the Austrian ones, unless in case of a shortage on either side. Consuming as much as possible produce and products from where one lives could substantially reduce primary fuel consumption, traffic jam, pollution and health impacts. Farmers should be subsidized for maintaining the natural fertility of the soil and the preservation of biodiversity – not for overproduction within monocultures, as it is the case now. That would lead to a broad support and promotion of certified organic farming, rather than fighting it with prejudice. Organic farming has maintaining the natural fertility and preserving biodiversity in its basic principles – you can't have organic farming without them. The secondary effects of such measures would in the middle and long term be very important as well: no synthetic fertilizer, little to no chemical insecticides and pesticides, a

The professional transport of food and non-food commodities knows comparable problems. But there is in the transport sector even less interest for sustainable transport solutions than with individuals: maximum load, high speed and low financial cost are the sole drivers. Some isolated projects, such as the one set up by the Belgian distributor Colruyt with an innovative lorry, goes further and tries to reduce fuel consumption and emissions by proactively financing, testing and adopting hybrid equipment that will respond to the newest

For any man made activity we should since long have adopted the *Precautionary Principle*  (European Commission, 2000): when we don't know the consequences, or have difficulties in estimating the extent of health and environmental impacts – including the depletion of

These facts are not new. Rachel Carson wrote her book *Silent Spring* in 1960 (Carson,1960). She warned for a thoughtless, large scale use of man made, highly effective chemicals and

documented the then already visible consequences for health and environment.

devastating huge areas of virgin forest, destroying important natural CO2 dumps.

on the matter.

healthy soil and a healthier water system.

EU requirements (Colruyt Group, 2010).

**4. Old stuff** 

raw material sources – we just shouldn't do it.

Starting in 1970, several high-level reports continued warning for the consequences of such an unsustainable development: *The Predicament of Mankind (*Christakis et al., 1970); *Limits to Growth* from the Club of Rome (Meadows et al., 1972); *Our Common Future* from the Brundtland Commission (World Commission on Environment and Development, 1987). But in spite of all these serious efforts and a series of follow-up initiatives such as the *Rio Conference*, *Agenda 21, Rio+10* and many more, very little systematic action has been taken.

The global principle to tackle what became a *global problem,* is *Sustainable Development*. The Brundtland report, in which the term 'sustainable development' was first used, describes this in a much cited quote as *"development that meets the needs of the present without compromising the ability of future generations to meet their own needs."* It has three focus points, which are inextricably intertwined and should not be separated at any time:


It will be obvious that each of these three members has its own specific rules and laws, which might be influenced by the others, but not overruled or replaced by them. It is not possible that economic principles will become more important than social or environmental ones; but they cannot become less important either. An essential fact - often misunderstood even by fervent followers – is that Sustainable Development is a life style, not a status that one can reach some time. You can't be, or can't become 'sustainable', there will always be a further stage of development to attend.

However, to cut short any misunderstanding: when we will in the following write about 'sustainable raw materials' or 'sustainable energy' we are not pointing at a status those items are supposed to be in, but at the whole *process* that leads to their existence. The raw material is a crystallization point of a generative process which fits – or doesn't fit, or only partially fits – into principles of Sustainable Development.

It's also obvious that, on the short term, it will not be possible to realize all elements of Sustainable Development to an equal degree of fulfillment, immediately and at the same time. Many things will only be partially realized through compromises between societal partners, in a slow process of involvement and comprehending.

Sustainable Development encompasses sustainable design, sustainable raw materials, sustainable production processes, sustainable energy and services, green taxes, as well as sustainable consumption. In short, it's a *cradle-to-grave* approach at all times and a *cradle-tocradle* approach whenever possible (more on this theme is to be found in Braungart and McDonough (2002). *Cradle-to-grave* means that all partners are part of the whole process, from the design of the product or service until the disposal of possible leftovers, and anything in between. Each step has to be optimized: it should involve the lowest amount of earthly substance and energy possible, have the highest efficiency and user friendliness possible and generate as little leftovers as feasible (that what we still call 'waste'). This should be featured without compromising elements such as the availability, the efficacy or the price of the product or service.

*Cradle-to-cradle* goes even a step further: whatever substance that is not fully destroyed at use (such as food), has to be made reusable for a similar, or even for a completely different

Greenwashing and Cleaning 7

sustainable washing and cleaning products, as staff member and retired staff member with

A traditional situation for commodities is that there is long trail of experience, starting in the past and ending today. But – as banks use to state lately – gains from the past are no guarantee for future gains. On the contrary; each and every conventional commodity is anchored in an unsustainable past and can by no means give reliable clues for a future that is headed by Sustainable Development. The trail for sustainable products and services has no past, it starts today and leads far into the future. Only, we don't know anything about that future. Keeping in mind what the Brundtland Report says: *"Sustainable Development is development that meets the needs of the present without compromising the ability of future generations to meet their own needs"* (World Commission on Environment and Development, 1987), we have the responsibility from now on to design products and services *in the wake of issues which lie in the future, in front of us!* It's a complete, revolutionary turning around of the

Products and services which have been conceived this way can be called *Future Capable*. Although not yet part of that future, they have the potential to fit in a future context. It will be clear that *unsustainable product design, unsustainable raw materials, unsustainable processing and energy, next to substantial waste generation, will altogether lead to a product that is not Future Capable*. That is a huge risk for the operations and investments of any company that would act in such a way. A producing company or developing lab will, on the contrary, try to the

But that is not easy and not immediately rewarding in terms of profit. Therefore, we will more often than not see some form of greenwashing popping up. There's all kinds of flavors, from smoothing some edges to sheer fraud, but always with the purpose to be perceived as "green". You have green petrol (it's toxic, carcinogenic and highly flammable), green apple perfume (green apples really don't have any perfume stuff) or natural soap (there is no soap

Unfortunately, many publications targeting a "green" consumer try to pick their grain as well. They start making product evaluations without having the technical knowledge to do so and without any real knowledge about environmental issues or Sustainable Development – except for the pure legal things, but those are always legging far behind

This mustn't be, however. It is perfectly possible to select - or even develop - sustainable gauges. The starting point has to be to select or develop gauges for raw material sourcing, process technique selection and energy, product design, and finally the health and environmental impacts at use and after disposal. None of these can claim to be the ultimate complete tool, a "green meter", that gives you once and for all the mathematically exact ranking of whatever. Nature doesn't function like that, it's not a machine, and neither are

The Eco-costs/Value Ratio (EVR) developed by the Technical University Delft, The

reality. Thus is the consumer more or less left to himself in a no mans land.

we. But these tools can give us fairly reliable estimations.

Netherlands (http://www.ecocostsvalue.com).

best of their knowledge and capabilities to become *Future Capable* as an organization.

the worldwide market leader in the trade.

way we used to think – and very challenging.

tree on which that grows).

Just two examples:

application by similar or different producers. In doing so, 'waste' becomes non-existent, as it will be a raw material for a new process. This is the way nature acts, and nature is never short of raw materials, unless humans degrade its ways.

Can we secure such a development, such type of products and services, can we guarantee that this will work and that everything will be true and honest? No! One of the important elements to be redeveloped in parallel, is *trust*. Trust mustn't be blind, though; there are several mechanisms that can be put to work to coach Sustainable Development. *Green Taxes* are one of those, but they are sort of an end-of-pipe solution and they should preferably only be used as temporary, corrective measures. It makes no sense to implement such a huge beast as Sustainable Development by means of force. Another useful mechanism are *Green Labels*. We know a whole bunch of them all over the planet, they have since a couple of years grown like mushrooms, and not always for the good. Unfortunately these Green Labels have mostly been developed by an amalgamate of politics and industry, and we should not forget that this is the tandem that pushed us into *Unsustainability*. It's comparable to farmers and butchers deciding about the criteria for veggie burgers; that makes no sense, really.

When Green Labels have to play a proficient role - and we think they can and should - the consumers have to get far more grip on the process of green labeling, from designing over controlling to improving them. Politics can check their correct and equitable implementation, but should not decide on the content or the format. Industry has to listen to what the consumer wants, not enforce what they themselves want to produce and sell. When it goes like that, we end up with a mishap, such as the actual EU *Ecolabel* on detergents. It lacks all kinds of arms and legs: the raw material sourcing is evaluated on its ecological merits in a crippled way, there is no social element present in the model, product efficiency is stubbornly compared against conventional, thus unsustainable products (in other words: race car vs. bike). Very weird influencing from conventional industry circles crept in through doors and windows, and one of the consequences is that fragrances based on plants are in practice *prohibited* in ecolabeled products!

*Public procurement* is another extremely powerful mechanism to implement Sustainable Development. When all layers of public power should systematically include ecological and social criteria in their evaluations and tenders for products and services, and not just take the lowest price as a gauge, the usual product and service ranking might be turned upside down.

Companies and offices of all kinds can use the same strategy. New knowledge and understanding would be instilled slowly into society as a whole, because consumers would comprehend and follow the example.

Hovering back over what we wrote up to now, we can see that Sustainable Development is not just about some kind of environmental conservation, but clearly encompasses the economical, social and environmental issues we tried to describe.

## **5. Washing and cleaning**

Why washing and cleaning? In the next two chapters we will mainly deal with ways to deploy, expand and improve the characteristics of Sustainable Development within commodity products and services, and the models used to do so. Both the authors have a longstanding experience in designing and modeling concepts, formulas and strategies for

application by similar or different producers. In doing so, 'waste' becomes non-existent, as it will be a raw material for a new process. This is the way nature acts, and nature is never

Can we secure such a development, such type of products and services, can we guarantee that this will work and that everything will be true and honest? No! One of the important elements to be redeveloped in parallel, is *trust*. Trust mustn't be blind, though; there are several mechanisms that can be put to work to coach Sustainable Development. *Green Taxes* are one of those, but they are sort of an end-of-pipe solution and they should preferably only be used as temporary, corrective measures. It makes no sense to implement such a huge beast as Sustainable Development by means of force. Another useful mechanism are *Green Labels*. We know a whole bunch of them all over the planet, they have since a couple of years grown like mushrooms, and not always for the good. Unfortunately these Green Labels have mostly been developed by an amalgamate of politics and industry, and we should not forget that this is the tandem that pushed us into *Unsustainability*. It's comparable to farmers and butchers deciding

When Green Labels have to play a proficient role - and we think they can and should - the consumers have to get far more grip on the process of green labeling, from designing over controlling to improving them. Politics can check their correct and equitable implementation, but should not decide on the content or the format. Industry has to listen to what the consumer wants, not enforce what they themselves want to produce and sell. When it goes like that, we end up with a mishap, such as the actual EU *Ecolabel* on detergents. It lacks all kinds of arms and legs: the raw material sourcing is evaluated on its ecological merits in a crippled way, there is no social element present in the model, product efficiency is stubbornly compared against conventional, thus unsustainable products (in other words: race car vs. bike). Very weird influencing from conventional industry circles crept in through doors and windows, and one of the consequences is that fragrances based

*Public procurement* is another extremely powerful mechanism to implement Sustainable Development. When all layers of public power should systematically include ecological and social criteria in their evaluations and tenders for products and services, and not just take the lowest price as a gauge, the usual product and service ranking might be turned upside down. Companies and offices of all kinds can use the same strategy. New knowledge and understanding would be instilled slowly into society as a whole, because consumers would

Hovering back over what we wrote up to now, we can see that Sustainable Development is not just about some kind of environmental conservation, but clearly encompasses the

Why washing and cleaning? In the next two chapters we will mainly deal with ways to deploy, expand and improve the characteristics of Sustainable Development within commodity products and services, and the models used to do so. Both the authors have a longstanding experience in designing and modeling concepts, formulas and strategies for

short of raw materials, unless humans degrade its ways.

about the criteria for veggie burgers; that makes no sense, really.

on plants are in practice *prohibited* in ecolabeled products!

economical, social and environmental issues we tried to describe.

comprehend and follow the example.

**5. Washing and cleaning** 

sustainable washing and cleaning products, as staff member and retired staff member with the worldwide market leader in the trade.

A traditional situation for commodities is that there is long trail of experience, starting in the past and ending today. But – as banks use to state lately – gains from the past are no guarantee for future gains. On the contrary; each and every conventional commodity is anchored in an unsustainable past and can by no means give reliable clues for a future that is headed by Sustainable Development. The trail for sustainable products and services has no past, it starts today and leads far into the future. Only, we don't know anything about that future. Keeping in mind what the Brundtland Report says: *"Sustainable Development is development that meets the needs of the present without compromising the ability of future generations to meet their own needs"* (World Commission on Environment and Development, 1987), we have the responsibility from now on to design products and services *in the wake of issues which lie in the future, in front of us!* It's a complete, revolutionary turning around of the way we used to think – and very challenging.

Products and services which have been conceived this way can be called *Future Capable*. Although not yet part of that future, they have the potential to fit in a future context. It will be clear that *unsustainable product design, unsustainable raw materials, unsustainable processing and energy, next to substantial waste generation, will altogether lead to a product that is not Future Capable*. That is a huge risk for the operations and investments of any company that would act in such a way. A producing company or developing lab will, on the contrary, try to the best of their knowledge and capabilities to become *Future Capable* as an organization.

But that is not easy and not immediately rewarding in terms of profit. Therefore, we will more often than not see some form of greenwashing popping up. There's all kinds of flavors, from smoothing some edges to sheer fraud, but always with the purpose to be perceived as "green". You have green petrol (it's toxic, carcinogenic and highly flammable), green apple perfume (green apples really don't have any perfume stuff) or natural soap (there is no soap tree on which that grows).

Unfortunately, many publications targeting a "green" consumer try to pick their grain as well. They start making product evaluations without having the technical knowledge to do so and without any real knowledge about environmental issues or Sustainable Development – except for the pure legal things, but those are always legging far behind reality. Thus is the consumer more or less left to himself in a no mans land.

This mustn't be, however. It is perfectly possible to select - or even develop - sustainable gauges. The starting point has to be to select or develop gauges for raw material sourcing, process technique selection and energy, product design, and finally the health and environmental impacts at use and after disposal. None of these can claim to be the ultimate complete tool, a "green meter", that gives you once and for all the mathematically exact ranking of whatever. Nature doesn't function like that, it's not a machine, and neither are we. But these tools can give us fairly reliable estimations.

Just two examples:

 The Eco-costs/Value Ratio (EVR) developed by the Technical University Delft, The Netherlands (http://www.ecocostsvalue.com).

Greenwashing and Cleaning 9

by the consumer, did by no means automatically guarantee a satisfactory product performance. But such was the understanding of the post-hippie generation: a product was considered "environmental", or even worse, "natural" when it did not contain certain ingredients which were on a relatively vague blacklist. Over the years and in the wake of the appearance of a forever growing number of renewable raw materials, it was replaced by a more pragmatic approach, the in the meantime quite well known and respected "Ecover

Today, Ecover's environmental product profile is maximized to achieve market standard performance. If the balance of a basic set of criteria (price, performance, convenience, human safety, environmental profile) of a functional ingredient, is considered prone to improvement, an ingredient development project is set up, usually in cooperation with academic and/or industrial partners. This approach necessitates a quantitative tool to measure ingredient and product strengths and weaknesses and to allow a company wide evaluation of innovation progress. Ironically enough, a large part of the market where Ecover acquired sales strength over the last decade is reluctant to even try to understand the full story; yet they desire to make the best environmental purchase. They are looking for a simple approach or even some authority who can tell them what is right and what is wrong; as we learned however, there is no such situation in the real world. But some ecolabel schemes deliver exactly this pass/fail endorsement, without necessarily featuring a coherent

The challenge for Ecover therefore was to develop a model based on externally verifiable data, encompassing the largest part of the Ecover concept, yet easy to understand for the non-chemist and allowing almost instant appreciation of a product's profile, both within the Ecover company and among its consumers. By furthermore incorporating European ecolabel criteria into the model and having this model validated and controlled on a yearly basis by an independent third party (in this case the Belgian NPO *Vinçotte Environnement*) the Ecover "diamond" model (fig.1) has become a strong and difficult to dispute communication tool. It takes Ecolabel criteria to a higher level by incorporating criteria on ingredient sourcing and adhering to stricter standards with regard to environmental impact and leftover fate. The "Ecover diamond" model (thus named because of the diamond-like structure of its visualisation) can be considered as a self-declared environmental claim according to ISO 14021. Self-declarations are more often than not unreliable and untrustworthy, but here we have one that responds to strict external regulations and controls. An important requirement for environmental claims and their evaluation is their

scientific basis, with only clearly referenced methods, calculations and standards.

become the written compilation of the Ecover concept.

The diamond model has proven to be a useful tool in product development, product benchmarking (comparing Ecover products with market references) and in communication. The Ecover diamond is the methodological translation of most of the Ecover environmental concept as it has been around for more than a decade. The procedure describing the diamond compilation also includes "focal drivers" mainly pertaining to qualitative criteria and to criteria which are hard if not impossible to assess for competing products. These focal drivers thus embody additional Ecover commitments not visualised in the diamond and often not communicated in any way. In this respect the diamond procedure has in fact

Concept".

product picture.

 The Eco-Footprint, originally developed in Canada, in the meantime in use in different forms (http://www.myfootprint.org)

When held against such sustainable gauges, actual solutions can be evaluated and be put in a priority ranking for further improvement. Compromises will have to be made and a time frame accepted: not every critical element can be instantly replaced, for very different reasons, such as unavailability of materials or processing, technical incompatibilities or financial constraints – or all of them together. There are many examples from the recent past.


Because of the complexity of the issues, each market segment will have to develop its own models and time frames. These models will need frequent revisions to fit in a forever changing context. All of us will have to learn to operate in a very different context. Where we are now in a closed circuit, with proprietary knowledge and confidentiality issues, we will have to adhere to Open Access, validation and control by external parties and sharing of know-how. It seems that the idea of competition in the old sense is getting quite rusty in this changing world and asks rather for models based on communication and collaboration.

In the next chapter, we describe the elements and the backbone of such a model for washing and cleaning commodities as developed and used by Ecover, based on the above ideas.

## **6. Ecover's "diamond" model**

Ecover is a medium sized, Belgium based company and is one of the foremost pioneers in developing and manufacturing washing and cleaning products with respect for the environment. It started off three decades ago by deleting environmentally troublesome ingredients (such as phosphates, alkyl phenol ethoxylates and the like) from standard frame formulas. This resulted in the so-called "No-code" (product doesn't contain such and doesn't contain so), which was communicated on the packaging.

Oleochemical based alternatives to petrochemicals were used wherever possible – there were not very many, 30 years ago. This black-or-white approach, though easy to understand

The Eco-Footprint, originally developed in Canada, in the meantime in use in different

When held against such sustainable gauges, actual solutions can be evaluated and be put in a priority ranking for further improvement. Compromises will have to be made and a time frame accepted: not every critical element can be instantly replaced, for very different reasons, such as unavailability of materials or processing, technical incompatibilities or financial constraints – or all of them together. There are many

 Until recently, fridges used to function on chlorinated compounds (CFC's) which are ozone depleting, persistent, toxic and carcinogenic. They have been exchanged for one or two less risky compounds - which could have been done since long. Nevertheless,

 Hybrid cars mainly use two different engines, a combustion one and an electric one. They have low consumption and low emissions. But hybrids are neither the solution for the mobility problem, nor are they the ultimate green cars; they just feature the Best

 Ecosurfactants are a class of washing agents from renewable raw materials, made via fermentation, at low temperature, low pressure and zero waste. They outperform both petrochemical and plant based surfactants on efficiency. But not all needs of detergent

 In almost each country there are organizations which defend consumer interests. But they are more often than not axed on quite superficial, practical and price issues and hardly on sustainable ones. They mostly take a Calimero standpoint and don't really try

Because of the complexity of the issues, each market segment will have to develop its own models and time frames. These models will need frequent revisions to fit in a forever changing context. All of us will have to learn to operate in a very different context. Where we are now in a closed circuit, with proprietary knowledge and confidentiality issues, we will have to adhere to Open Access, validation and control by external parties and sharing of know-how. It seems that the idea of competition in the old sense is getting quite rusty in this changing world and asks rather for models based on communication and collaboration. In the next chapter, we describe the elements and the backbone of such a model for washing and cleaning commodities as developed and used by Ecover, based on the above ideas.

Ecover is a medium sized, Belgium based company and is one of the foremost pioneers in developing and manufacturing washing and cleaning products with respect for the environment. It started off three decades ago by deleting environmentally troublesome ingredients (such as phosphates, alkyl phenol ethoxylates and the like) from standard frame formulas. This resulted in the so-called "No-code" (product doesn't contain such and doesn't

Oleochemical based alternatives to petrochemicals were used wherever possible – there were not very many, 30 years ago. This black-or-white approach, though easy to understand

to mediate between consumers and industry to develop a common ground.

'less risky' is not 'good' and other solutions have to be developed.

forms (http://www.myfootprint.org)

Available Technology (BAT) of the moment.

examples from the recent past.

concepts can yet be covered.

**6. Ecover's "diamond" model** 

contain so), which was communicated on the packaging.

by the consumer, did by no means automatically guarantee a satisfactory product performance. But such was the understanding of the post-hippie generation: a product was considered "environmental", or even worse, "natural" when it did not contain certain ingredients which were on a relatively vague blacklist. Over the years and in the wake of the appearance of a forever growing number of renewable raw materials, it was replaced by a more pragmatic approach, the in the meantime quite well known and respected "Ecover Concept".

Today, Ecover's environmental product profile is maximized to achieve market standard performance. If the balance of a basic set of criteria (price, performance, convenience, human safety, environmental profile) of a functional ingredient, is considered prone to improvement, an ingredient development project is set up, usually in cooperation with academic and/or industrial partners. This approach necessitates a quantitative tool to measure ingredient and product strengths and weaknesses and to allow a company wide evaluation of innovation progress. Ironically enough, a large part of the market where Ecover acquired sales strength over the last decade is reluctant to even try to understand the full story; yet they desire to make the best environmental purchase. They are looking for a simple approach or even some authority who can tell them what is right and what is wrong; as we learned however, there is no such situation in the real world. But some ecolabel schemes deliver exactly this pass/fail endorsement, without necessarily featuring a coherent product picture.

The challenge for Ecover therefore was to develop a model based on externally verifiable data, encompassing the largest part of the Ecover concept, yet easy to understand for the non-chemist and allowing almost instant appreciation of a product's profile, both within the Ecover company and among its consumers. By furthermore incorporating European ecolabel criteria into the model and having this model validated and controlled on a yearly basis by an independent third party (in this case the Belgian NPO *Vinçotte Environnement*) the Ecover "diamond" model (fig.1) has become a strong and difficult to dispute communication tool. It takes Ecolabel criteria to a higher level by incorporating criteria on ingredient sourcing and adhering to stricter standards with regard to environmental impact and leftover fate. The "Ecover diamond" model (thus named because of the diamond-like structure of its visualisation) can be considered as a self-declared environmental claim according to ISO 14021. Self-declarations are more often than not unreliable and untrustworthy, but here we have one that responds to strict external regulations and controls. An important requirement for environmental claims and their evaluation is their scientific basis, with only clearly referenced methods, calculations and standards.

The diamond model has proven to be a useful tool in product development, product benchmarking (comparing Ecover products with market references) and in communication. The Ecover diamond is the methodological translation of most of the Ecover environmental concept as it has been around for more than a decade. The procedure describing the diamond compilation also includes "focal drivers" mainly pertaining to qualitative criteria and to criteria which are hard if not impossible to assess for competing products. These focal drivers thus embody additional Ecover commitments not visualised in the diamond and often not communicated in any way. In this respect the diamond procedure has in fact become the written compilation of the Ecover concept.

Greenwashing and Cleaning 11

The *Primary Efficiency* is the immediately perceivable performance of a product. This axis represents the percentage of "performance score", relative to a reference formula and

*Secondary Efficiency* is the performance of the product at lower temperature (in automated appliance products) or a second performance attribute (such as speed of drying, gloss

*Consumer Safety* covers the use of surfactants that are safe for the user. Several attributing points towards consumer safety are defined. The absence of certain danger classes (e.g.

The **Absorption Phase** involves Aquatic Safety, Limited Aquatic Impact, Aerobically Degradable Ingredients, Anaerobically Degradable Surfactants, Phosphorus Absence, VOC

*Aquatic Safety* covers the use of ingredients that are safe for the aquatic environment and is determined experimentally at Ecover as aquatic toxicity tests and expressed as a dose

*Limited Aquatic Impact* is calculated as the Critical Dilution Volume (CDV), a concept developed within the EU ecolabel and expressing the theoretical amount of liters required to dilute a single product dose down to environmentally harmless concentrations, provided

*Aerobic Biodegradability* is an important and desirable property of any ingredient in washing and cleaning products. This diamond axis visualizes the amount of persistent chemicals in the product, i.e. the chemicals that are not inherently degradable by microorganisms when oxygen is present. An ingredient can be readily biodegradable, inherently biodegradable or persistent in aerobic conditions. This clearly differentiates the diamond model from ecolabel criteria.

*Anaerobically Degradable Surfactants* excludes surfactants which are not biodegradable in anaerobic conditions, i.e. in oxygen deprived environments should be avoided to the extent possible since aerobic conditions are not always the case, such as in many rivers, marine

*Phosphorus Absence* documents possible amounts of phosphorus, which in the aquatic environment causes eutrophication. Hence, the use of phosphorus-based ingredients should

The environmental relevance of *Volatile Organic Carbons* (VOC ) is the contribution to indoor

The *Primary Packaging* axis aims at reducing this waste according to several references and

For more detailed information on the Diamond Model, see at www.ecover.com (or specific

Braungart M.& McDonough, B. (2002). Cradle to Cradle: Remaking the Way We Make

determined according to EU Ecolabel standards.

retention, …), again relative to a reference formula.

Absence and Primary Packaging Optimisation.

sewage treatment systems are in place.

sediments or sewage sludge.

air pollution and smog formation.

Things, North Point Press, New York.

be minimized.

assumptions.

**7. References** 

URL).

related LC50 quotient.

corrosive, toxic, …) attributes a percentage to the total score.

Fig. 1. Ecover diamond model with 13 axes distributed over 3 life cycle phases.

The model involves the total life cycle of a product, the **Extraction Phase**, the **Usage Phase** and the **Absorption Phase**. The latter phase is termed "Absorption" rather than the standard LCA "Disposal phase" terminology, to refer to a cradle-to-cradle, closed carbon loop, without persistent chemicals and without lasting ecosystem perturbation. It is visualized as a spidergram with 13 quantitative axes distributed over the three said phases.

The **Extraction Phase** involves *Renewable Resources*, *Green Chemistry* and *Material Proximity*. *Renewable Resources* are defined as animal, vegetable or microbial derived feedstocks, as opposed to water, mineral and petrochemical resources. This axis represents the percentage of renewable matter over the total organic dry matter of the end product and correlates very good with experimental C14 carbon dating results.

The *Green Chemistry* axis reflects Ecover's striving for efficient resource transformation at low temperature and pressure, without potential run away reactions or risk of explosion, while making use of chemicals currently considered as safe, with limited risk of undesirable by-product formation. The axis is calculated by a weighted sum of "green chemistry scores" across all ingredients over the total organic dry matter.

*Resource Proximity* covers the CO2 contribution of the complete product formula, from the source of the ingredient constituents, to the ingredient manufacturer, to the Ecover factory in Malle, taking into account the distance traveled by all individual ingredients and their transport mode.

The **Usage Phase** involves Primary Efficiency, Secondary Efficiency and Consumer Safety.

Fig. 1. Ecover diamond model with 13 axes distributed over 3 life cycle phases.

good with experimental C14 carbon dating results.

across all ingredients over the total organic dry matter.

transport mode.

The model involves the total life cycle of a product, the **Extraction Phase**, the **Usage Phase** and the **Absorption Phase**. The latter phase is termed "Absorption" rather than the standard LCA "Disposal phase" terminology, to refer to a cradle-to-cradle, closed carbon loop, without persistent chemicals and without lasting ecosystem perturbation. It is visualized as a spidergram with 13 quantitative axes distributed over the three said phases. The **Extraction Phase** involves *Renewable Resources*, *Green Chemistry* and *Material Proximity*. *Renewable Resources* are defined as animal, vegetable or microbial derived feedstocks, as opposed to water, mineral and petrochemical resources. This axis represents the percentage of renewable matter over the total organic dry matter of the end product and correlates very

The *Green Chemistry* axis reflects Ecover's striving for efficient resource transformation at low temperature and pressure, without potential run away reactions or risk of explosion, while making use of chemicals currently considered as safe, with limited risk of undesirable by-product formation. The axis is calculated by a weighted sum of "green chemistry scores"

*Resource Proximity* covers the CO2 contribution of the complete product formula, from the source of the ingredient constituents, to the ingredient manufacturer, to the Ecover factory in Malle, taking into account the distance traveled by all individual ingredients and their

The **Usage Phase** involves Primary Efficiency, Secondary Efficiency and Consumer Safety.

The *Primary Efficiency* is the immediately perceivable performance of a product. This axis represents the percentage of "performance score", relative to a reference formula and determined according to EU Ecolabel standards.

*Secondary Efficiency* is the performance of the product at lower temperature (in automated appliance products) or a second performance attribute (such as speed of drying, gloss retention, …), again relative to a reference formula.

*Consumer Safety* covers the use of surfactants that are safe for the user. Several attributing points towards consumer safety are defined. The absence of certain danger classes (e.g. corrosive, toxic, …) attributes a percentage to the total score.

The **Absorption Phase** involves Aquatic Safety, Limited Aquatic Impact, Aerobically Degradable Ingredients, Anaerobically Degradable Surfactants, Phosphorus Absence, VOC Absence and Primary Packaging Optimisation.

*Aquatic Safety* covers the use of ingredients that are safe for the aquatic environment and is determined experimentally at Ecover as aquatic toxicity tests and expressed as a dose related LC50 quotient.

*Limited Aquatic Impact* is calculated as the Critical Dilution Volume (CDV), a concept developed within the EU ecolabel and expressing the theoretical amount of liters required to dilute a single product dose down to environmentally harmless concentrations, provided sewage treatment systems are in place.

*Aerobic Biodegradability* is an important and desirable property of any ingredient in washing and cleaning products. This diamond axis visualizes the amount of persistent chemicals in the product, i.e. the chemicals that are not inherently degradable by microorganisms when oxygen is present. An ingredient can be readily biodegradable, inherently biodegradable or persistent in aerobic conditions. This clearly differentiates the diamond model from ecolabel criteria.

*Anaerobically Degradable Surfactants* excludes surfactants which are not biodegradable in anaerobic conditions, i.e. in oxygen deprived environments should be avoided to the extent possible since aerobic conditions are not always the case, such as in many rivers, marine sediments or sewage sludge.

*Phosphorus Absence* documents possible amounts of phosphorus, which in the aquatic environment causes eutrophication. Hence, the use of phosphorus-based ingredients should be minimized.

The environmental relevance of *Volatile Organic Carbons* (VOC ) is the contribution to indoor air pollution and smog formation.

The *Primary Packaging* axis aims at reducing this waste according to several references and assumptions.

For more detailed information on the Diamond Model, see at www.ecover.com (or specific URL).

## **7. References**

Braungart M.& McDonough, B. (2002). Cradle to Cradle: Remaking the Way We Make Things, North Point Press, New York.

**2** 

*Brazil* 

**Green Chemistry –** 

Ricardo Menegatti

O

O R

*Universidade Federal de Goiás* 

**Aspects for the Knoevenagel Reaction** 

O

hydrolysis

O

O R

O R

Knoevenagel condensation is a classic C-C bond formation reaction in organic chemistry (Laue & Plagens, 2005). These condensations occur between aldehydes or ketones and active methylene compounds with ammonia or another amine as a catalyst in organic solvents (Knoevenagel, 1894). The Knoevenagel reaction is considered to be a modification of the aldol reaction; the main difference between these approaches is the higher acidity of the active methylene hydrogen when compared to an -carbonyl hydrogen (Smith & March, 2001).

Figure 1 illustrates the condensation of a ketone (1) with a malonate compound (2) to form the Knoevenagel condensation product (3), which is then used to form the ,-unsaturated

Subsequent to the first description of the Knoevenagel reaction, changes were introduced using pyridine as the solvent and piperidine as the catalyst, which was named the Doebner Modification (Doebner, 1900). The Henry reaction is another variation of the Knoevenagel condensation that utilises compounds with an -nitro active methylene (Henry, 1895). The general mechanism for the Knoevenagel reaction, which involves deprotonation of the malonate derivative (6) by piperidine (5) and attack by the formed carbanion (8) on the carbonyl subunit (9) as an aldol reaction that forms the product (10) of the addition step is illustrated in Fig. 2. After the proton transfer step between the protonated base (7) and compound (10), intermediate (11) forms and is then deprotonated to (12), which forms the

(1) (2) (3) (4)

carboxylic compounds (3) and (4) (Laue & Plagens, 2005).

R

O

H H

O

R

O

O

base

Fig. 1. An example of the Knoevenagel reaction.

<sup>O</sup> +

elimination product (13) in the last step.

**1. Introduction** 

Carson, R. (1960). Silent Spring, First Mariner Books, ISBN 0-618-24906-0, edition 2002


http://www.treehugger.com/files/2007/08/pesticide\_lawsuit.php


http://worldinbalance.net/intagreements/1987-brundtland.php

## **Green Chemistry – Aspects for the Knoevenagel Reaction**

Ricardo Menegatti *Universidade Federal de Goiás Brazil* 

## **1. Introduction**

12 Green Chemistry – Environmentally Benign Approaches

Chua, J. (2007). Latin American Banana farmers sue over pesticides. In: TreeHugger,

Colruyt Group, 2010. Groep Colruyt ontwikkelt hybride trekker, press release 21/06/2010,

European Commission, 2000. Communication from the Commission of 2 February 2000 on

http://europa.eu/legislation\_summaries/consumers/consumer\_safety/l32042\_en.

Greenpeace, 2011. Turn VW away from the Dark Side. Press Campaign, Available from:

Kaufman F. (2010). The Food Bubble: How Wall Street starved millions and got away with

Meadows, D.; Meadows, D.; Randers, J.& Behrens III, W. (1972). The Limits to Growth.

Nelson, S. (2008). Ethanol no longer seen as big driver of food price, Reuters Press Release

World Commission on Environment and Development, 1987. Our Common Future, Report

23/10/08, Available from: http://uk.reuters.com/article/2008/10/23/food-corn-

of the World Commission on Environment and Development, Published as Annex to General Assembly document A/42/427, Development and International Co-

Available from: http://www.colruytgroup.be/colruytgroup/static/energiebeleid-

Carson, R. (1960). Silent Spring, First Mariner Books, ISBN 0-618-24906-0, edition 2002 Christakis, H.; Jantsch, E.& Özbekhan, H. (1970). The Predicament of Mankind, Date of

http://sunsite.utk.edu/FINS/loversofdemocracy/Predicament.PTI.pdf

http://www.treehugger.com/files/2007/08/pesticide\_lawsuit.php

the precautionary principle, Available from:

Universe Books , ISBN 0-87663-165-0, New York:

US Census Bureau, 2011. World POPClock Projection, Available from : http://www.census.gov/population/popclockworld.html

http://worldinbalance.net/intagreements/1987-brundtland.php

Western Organisation of Resource Councils (WORC), 2007. Fact sheet October 2007

http://www.vwdarkside.com/en

it, Harper's Magazine, July 23, 2010.

ethanol-idUKN2338007820081023

operation: Environment, Available from:

access 27/10/11, Available from:

27/10/11, Available from:

hybride\_be-nl.shtml

htm

Knoevenagel condensation is a classic C-C bond formation reaction in organic chemistry (Laue & Plagens, 2005). These condensations occur between aldehydes or ketones and active methylene compounds with ammonia or another amine as a catalyst in organic solvents (Knoevenagel, 1894). The Knoevenagel reaction is considered to be a modification of the aldol reaction; the main difference between these approaches is the higher acidity of the active methylene hydrogen when compared to an -carbonyl hydrogen (Smith & March, 2001).

Figure 1 illustrates the condensation of a ketone (1) with a malonate compound (2) to form the Knoevenagel condensation product (3), which is then used to form the ,-unsaturated carboxylic compounds (3) and (4) (Laue & Plagens, 2005).

Fig. 1. An example of the Knoevenagel reaction.

Subsequent to the first description of the Knoevenagel reaction, changes were introduced using pyridine as the solvent and piperidine as the catalyst, which was named the Doebner Modification (Doebner, 1900). The Henry reaction is another variation of the Knoevenagel condensation that utilises compounds with an -nitro active methylene (Henry, 1895). The general mechanism for the Knoevenagel reaction, which involves deprotonation of the malonate derivative (6) by piperidine (5) and attack by the formed carbanion (8) on the carbonyl subunit (9) as an aldol reaction that forms the product (10) of the addition step is illustrated in Fig. 2. After the proton transfer step between the protonated base (7) and compound (10), intermediate (11) forms and is then deprotonated to (12), which forms the elimination product (13) in the last step.

Green Chemistry – Aspects for the Knoevenagel Reaction 15

based on the ratio of the molecular weight of the desired product to the sum of the molecular weights of all stoichiometric reagents. This indicator enables the evaluation of atom utilisation in a reaction (Trost, 1991). As illustrated in Table 1, the pharmaceutical industry produces 25->100 kg of waste per kg of drug produced, which is the worst E factor observed among the surveyed industrial sectors (Sheldon, 2007). This result is problematic as the pharmaceutical market is among the major sectors of the global economy, accounting

tonnage

Among the 20 top-selling drugs of 2010, atorvastatin (14) is at the top of the list, corresponding to US \$ 12.6 billion in sales (Gatyas, 2011b). One step in the synthesis of atorvastatin (14) (Fig. 3) uses a Knoevenagel condensation between methylene compound (15) and benzaldehyde (9) to produce an intermediate (16) in yields of 85.0% (Li et al., 2004;

H

(15) (9) (16)


O

N H

O

Fig. 3. A Knoevenagel condensation used during the synthesis of atorvastatin (14).

In addition to atorvastatin (14), many others drugs and pharmacological tools use the Knoevenagel reaction during their syntheses. Figure 4 illustrates the synthesis of pioglitazone (17), a benzylthiazolidinedione derivative approved as a drug for the

Oil refining 106-108 ca. 0.1 Bulk chemicals 104-106 <1-5 Fine chemicals 102-104 5->50 Pharmaceuticals 10-103 25->100

kg waste/ kg product

O

N

H

F

N

(14)

O

OH OH

OH

O

for US \$ 856 billion in 2010 (Gatyas, 2011a).

O

Roth, 1993).

N

H

O

Industrial sector Annual product

Table 1. The E Factor for selected industrial sectors, left justified.

+

Fig. 2. General mechanism for the Knoevenagel reaction.

## **2. Green chemistry and new synthetic approaches**

In the past two decades, classic organic chemistry had been rewritten around new approaches that search for products and processes in the chemical industry that are environmentally acceptable (Okkerse & Bekkum, 1999; Sheldon et al., 2007). With the emergence of Green Chemistry, a term coined in 1993 by Anastas at the US Environmental Protection Agency (EPA), a set of principles was proposed for the development of environmentally safer products and processes: waste prevention instead of remediation; atom efficiency; less hazardous/toxic chemicals; safer products by design; innocuous solvents and auxiliaries; energy efficiency by design; preference for renewable raw materials; shorter syntheses; catalytic rather than stoichiometric reagents; products designed for degradation; analytical methodologies for pollution prevention; and inherently safer processes (Anastas & Warner, 2000).

Consequently, many classic reactions, such as the Knoevenagel reaction, have been studied based upon the green chemistry perspective, which is very important in the context of the pharmaceutical industry. Currently, two indicators are used to evaluate environmental acceptability of products and chemical processes. The first is the Environmental factor (E factor), which measures the mass ratio of kg of waste to kg of desired product, as described by Sheldon in 1992 (Sheldon, 2007). The second indicator is a measure of atom economy

+

O

H

O

R H

R

O

O O

N H

+ + H2O

O

O

(9)

O


O

R

O

H

<sup>N</sup><sup>+</sup> <sup>H</sup> <sup>H</sup>

R

+

O

(7) (10)

R

R

O O

N <sup>+</sup> H H

Fig. 2. General mechanism for the Knoevenagel reaction.

OH

processes (Anastas & Warner, 2000).

N H

N H

N <sup>+</sup> H H +

+

(11) (5)

O

O

OH

O O

O

R H

O

R

R

<sup>+</sup> - <sup>O</sup>

(7) (12) (13) (5)

O

R

O

(5) (6) (7) (8)

O

H H R

O

R

**2. Green chemistry and new synthetic approaches** 

O O

In the past two decades, classic organic chemistry had been rewritten around new approaches that search for products and processes in the chemical industry that are environmentally acceptable (Okkerse & Bekkum, 1999; Sheldon et al., 2007). With the emergence of Green Chemistry, a term coined in 1993 by Anastas at the US Environmental Protection Agency (EPA), a set of principles was proposed for the development of environmentally safer products and processes: waste prevention instead of remediation; atom efficiency; less hazardous/toxic chemicals; safer products by design; innocuous solvents and auxiliaries; energy efficiency by design; preference for renewable raw materials; shorter syntheses; catalytic rather than stoichiometric reagents; products designed for degradation; analytical methodologies for pollution prevention; and inherently safer

Consequently, many classic reactions, such as the Knoevenagel reaction, have been studied based upon the green chemistry perspective, which is very important in the context of the pharmaceutical industry. Currently, two indicators are used to evaluate environmental acceptability of products and chemical processes. The first is the Environmental factor (E factor), which measures the mass ratio of kg of waste to kg of desired product, as described by Sheldon in 1992 (Sheldon, 2007). The second indicator is a measure of atom economy based on the ratio of the molecular weight of the desired product to the sum of the molecular weights of all stoichiometric reagents. This indicator enables the evaluation of atom utilisation in a reaction (Trost, 1991). As illustrated in Table 1, the pharmaceutical industry produces 25->100 kg of waste per kg of drug produced, which is the worst E factor observed among the surveyed industrial sectors (Sheldon, 2007). This result is problematic as the pharmaceutical market is among the major sectors of the global economy, accounting for US \$ 856 billion in 2010 (Gatyas, 2011a).


Table 1. The E Factor for selected industrial sectors, left justified.

Among the 20 top-selling drugs of 2010, atorvastatin (14) is at the top of the list, corresponding to US \$ 12.6 billion in sales (Gatyas, 2011b). One step in the synthesis of atorvastatin (14) (Fig. 3) uses a Knoevenagel condensation between methylene compound (15) and benzaldehyde (9) to produce an intermediate (16) in yields of 85.0% (Li et al., 2004; Roth, 1993).

Fig. 3. A Knoevenagel condensation used during the synthesis of atorvastatin (14).

In addition to atorvastatin (14), many others drugs and pharmacological tools use the Knoevenagel reaction during their syntheses. Figure 4 illustrates the synthesis of pioglitazone (17), a benzylthiazolidinedione derivative approved as a drug for the

Green Chemistry – Aspects for the Knoevenagel Reaction 17

MDL 103371 (25) is an *N*-methyl-*D*-aspartate-type glycine receptor antagonist for the treatment of stroke (Watson et al., 2000). As illustrated in Fig. 6, synthesis of MDL 103371 (25) involves production of key intermediate (28) in yields of 91.0% via the condensation of 4,6-dichloro-3-formyl-1*H*-indole-2-carboxylate (26) with 3-nitrophenylacetonitrile (27); this piperidine-catalysed step is carried out using ethanol under reflux conditions for 70 hours

(28) (26) (27)

EtOH 91.0%

piperidine

Cl

Cl

N

NO2

CO2Et

H

CN

H

NH2

(25)

OH

H

O

O

OH

N

H

Cl

Cl

(*S*)-(+)-3-Aminomethyl-5-methylhexanoic acid, or pregabalin (29), is a lipophilic GABA (*γ*aminobutyric acid) analogue used for the treatment of several central nervous system (CNS) disorders, such as epilepsy, neuropathic pain, anxiety and social phobia (Martinez et al., 2008). As shown in Fig. 7, intermediate (32), which is produced during the synthesis of pregabalin (29), is formed in yields of 95.0% from the reaction between isovaleraldehyde (30) and diethyl malonate (31) using acetic acid as the solvent and di-*n*-propylamine as the

(Walker et al., 2011).

Cl

H

Cl

+

CHO

CO2Et

N CN

NO2

Fig. 6. MDL 103371 (25) synthesis.

catalyst.

management of diabetes (Madivada et al., 2009). In this synthesis, the key intermediate (20) was formed in yields of 94.5% through the piperidine-catalysed reaction of aldehyde intermediate (18) and 2,4-thiozolidinedione (19) (Madivada et al., 2009).

Fig. 4. Selected steps of the pioglitazone (17) synthesis.

AMG 837 (21) is a novel agonist of GPR40; this compound is being investigated as apotentially new therapeutic agent for the treatment of type 2 diabetes (Walker et al., 2011). As shown in Fig. 5, the synthetic route for AMG 837 (21) involves the production of intermediate (24), which is formed in yields of 97.0% via the reaction between aldehyde (22) with Meldrum's acid (23), using water/toluene (10/1) as a catalytic solvent (Walker et al., 2011).

Fig. 5. AMG 837 (21) synthesis.

management of diabetes (Madivada et al., 2009). In this synthesis, the key intermediate (20) was formed in yields of 94.5% through the piperidine-catalysed reaction of aldehyde

> piperidine EtOH

> > N

N

O

O

HO O

O

(21)

CF3 CH3

O

S

S

O

OH

O

O

(17)

Pd/C/H2 dioxane N

O

N

O

H

O

H

O

AMG 837 (21) is a novel agonist of GPR40; this compound is being investigated as apotentially new therapeutic agent for the treatment of type 2 diabetes (Walker et al., 2011). As shown in Fig. 5, the synthetic route for AMG 837 (21) involves the production of intermediate (24), which is formed in yields of 97.0% via the reaction between aldehyde (22) with Meldrum's

(22) (23) (24)

H2O/toluene

97.0%

acid (23), using water/toluene (10/1) as a catalytic solvent (Walker et al., 2011).

O (10/1)

O

O

O

intermediate (18) and 2,4-thiozolidinedione (19) (Madivada et al., 2009).

O

H

(18) (19) (20)

+ <sup>S</sup> <sup>N</sup>

O

Fig. 4. Selected steps of the pioglitazone (17) synthesis.

CHO

N

HO

O

Fig. 5. AMG 837 (21) synthesis.

CHO

+

MDL 103371 (25) is an *N*-methyl-*D*-aspartate-type glycine receptor antagonist for the treatment of stroke (Watson et al., 2000). As illustrated in Fig. 6, synthesis of MDL 103371 (25) involves production of key intermediate (28) in yields of 91.0% via the condensation of 4,6-dichloro-3-formyl-1*H*-indole-2-carboxylate (26) with 3-nitrophenylacetonitrile (27); this piperidine-catalysed step is carried out using ethanol under reflux conditions for 70 hours (Walker et al., 2011).

Fig. 6. MDL 103371 (25) synthesis.

(*S*)-(+)-3-Aminomethyl-5-methylhexanoic acid, or pregabalin (29), is a lipophilic GABA (*γ*aminobutyric acid) analogue used for the treatment of several central nervous system (CNS) disorders, such as epilepsy, neuropathic pain, anxiety and social phobia (Martinez et al., 2008). As shown in Fig. 7, intermediate (32), which is produced during the synthesis of pregabalin (29), is formed in yields of 95.0% from the reaction between isovaleraldehyde (30) and diethyl malonate (31) using acetic acid as the solvent and di-*n*-propylamine as the catalyst.

Green Chemistry – Aspects for the Knoevenagel Reaction 19

Coartem is an antimalarial drug that is a combination of artemether and lumefantrine (37) (Beulter et al., 2007). This combination greatly benefits patients because it facilitates treatment compliance and supports optimal clinical effectiveness. As shown in Fig. 9, crude lumefantrine (37) was produced in yields of 88.0% via the reaction of 4-chlorobenzaldehyde (39) with methylene compound (38) in ethanol with sodium hydroxide as a catalyst. After crystallisation in heptane, pure lumefantrine (37) was generated in yields of 93.0% (Beulter

Entacapone (40) is a catechol-O-methyltransferase (COMT) inhibitor used in combination with *L*-DOPA for the treatment of Parkinson's disease (Mukarram et al., 2007). This combination prevents *L*-DOPA degradation through COMT inhibition. As illustrated in Fig. 10, this drug (40) is synthesised in yields of 73.0% from aldehyde (41) and methylene

Cl 88.0%

EtOH

NaOH

Cl Cl

H

HO Bu2

Cl

N

O

CN

(38) (37)

(39)

CHO

The examples illustrated above for selected drug syntheses emphasise the on-going necessity of finding new approaches to carry out classic reactions that are essential to developing environmentally responsible products and chemical processes. Some new, green

piperidine

73.0%

(42) (40)

HO

HO

NO2

Microwave irradiation is a method used to speed up reactions with potential uses under the guidelines of Green Chemistry principles. Microwave radiation utilises wavelengths of 0.001 – 1 m and frequencies of 0.3 – 300 GHz. When a polar organic reaction is irradiated in a microwave, energy is transferred to the sample, and the result is an increase in the rate of

compound (42) in ethanol with a piperidine catalyst (Mukarram et al., 2007).

O

CN

N

+

et al., 2007).

Fig. 9. Lumefantrine (37) synthesis.

Cl Cl

HO Bu2

Fig. 10. Entacapone (40) synthesis.

(41)

NO2

HO CHO

+

EtOH HO

approaches are presented below.

**2.1 Microwave-promoted Knoevenagel reactions** 

Fig. 7. Pregabalin (29) synthesis.

(*E*)-4-Cyclobutyl-2-[2-(3-nitrophenyl)ethenyl] thiazole, or Ro 24-5913 (33), is a leukotriene antagonist that has been utilised as a pharmacological tool to study asthma as well as other inflammatory diseases (Kuzemko et al., 2007). One method used to prepare Ro 24-5913 (33), illustrated in Fig. 8, is under Doebner conditions in which 3-nitrobenzaldehyde (34) reacts in the first step with malonic acid to produce intermediate (36) in yields of 65.6% (Kuzemko et al., 2007).

Fig. 8. Ro 24-5913 (33) synthesis.

AcOH 95.0%

EtO (32)

CO2Et

(29) NH2

OH

S

N

O

(33)

NO2

CO2Et

CO2H

(*E*)-4-Cyclobutyl-2-[2-(3-nitrophenyl)ethenyl] thiazole, or Ro 24-5913 (33), is a leukotriene antagonist that has been utilised as a pharmacological tool to study asthma as well as other inflammatory diseases (Kuzemko et al., 2007). One method used to prepare Ro 24-5913 (33), illustrated in Fig. 8, is under Doebner conditions in which 3-nitrobenzaldehyde (34) reacts in the first step with malonic acid to produce intermediate (36) in yields of 65.6% (Kuzemko et

> Pyridine 65.6%

HO (36)

NO2

Fig. 7. Pregabalin (29) synthesis.

CHO

+

HO

O

O

(34) (35)

+

*<sup>n</sup>*-Pr2NH CHO <sup>O</sup>

EtO

O

(30) (31)

Fig. 8. Ro 24-5913 (33) synthesis.

al., 2007).

NO2

Coartem is an antimalarial drug that is a combination of artemether and lumefantrine (37) (Beulter et al., 2007). This combination greatly benefits patients because it facilitates treatment compliance and supports optimal clinical effectiveness. As shown in Fig. 9, crude lumefantrine (37) was produced in yields of 88.0% via the reaction of 4-chlorobenzaldehyde (39) with methylene compound (38) in ethanol with sodium hydroxide as a catalyst. After crystallisation in heptane, pure lumefantrine (37) was generated in yields of 93.0% (Beulter et al., 2007).

Fig. 9. Lumefantrine (37) synthesis.

Entacapone (40) is a catechol-O-methyltransferase (COMT) inhibitor used in combination with *L*-DOPA for the treatment of Parkinson's disease (Mukarram et al., 2007). This combination prevents *L*-DOPA degradation through COMT inhibition. As illustrated in Fig. 10, this drug (40) is synthesised in yields of 73.0% from aldehyde (41) and methylene compound (42) in ethanol with a piperidine catalyst (Mukarram et al., 2007).

Fig. 10. Entacapone (40) synthesis.

The examples illustrated above for selected drug syntheses emphasise the on-going necessity of finding new approaches to carry out classic reactions that are essential to developing environmentally responsible products and chemical processes. Some new, green approaches are presented below.

#### **2.1 Microwave-promoted Knoevenagel reactions**

Microwave irradiation is a method used to speed up reactions with potential uses under the guidelines of Green Chemistry principles. Microwave radiation utilises wavelengths of 0.001 – 1 m and frequencies of 0.3 – 300 GHz. When a polar organic reaction is irradiated in a microwave, energy is transferred to the sample, and the result is an increase in the rate of

Green Chemistry – Aspects for the Knoevenagel Reaction 21

In recent years, ionic liquids (ILs) have attracted increasing interest as environmentally benign solvents and catalysts due to their relatively low viscosities, low vapour pressures and high thermal and chemical stabilities (Hajipour & Rafiee, 2010; Wasserscheid & Welton,

As illustrated in Fig 13, the pyrazolonic compound (48) was produced in yields of 71.0% from the reaction between benzaldehyde (9) and 3-methyl-1-phenylpyrazolin-5-(4*H*)-one (49) after 30 minutes using ethylammonium nitrate as an ionic liquid at room temperature

Other reactions between aromatic aldehydes and methylene compounds that were catalysed by 1,3-dimethylimidazolium methyl sulphate [MMIm][MSO4] and 2.16% water have been

71.0%

ethylammonium nitrate

<sup>N</sup> <sup>O</sup> <sup>N</sup>

(48)

O O

(50)

O

<sup>N</sup> <sup>O</sup>

Historically, microorganisms have been of enormous social and economic importance (Liese et al., 2006). In the pharmaceutical industry, companies are using biotechnology to develop 901 medicines and vaccines targeting more than 100 diseases (Castellani, 2001a). In 2010, 26 new treatments were approved, and five of these treatments were based on biotechnology

Using a biotechnology-based approach, coumarin (50) was produced in yields of 58.0% when the reaction was catalysed by alkaline protease from *Bacillus licheniformis* (BLAP) in a

DMSO/H2O

BLAP

(9:1), 550C

58.0%

DMSO:H2O (9:1) solvent at a temperature of 550C (Fig. 14) (Wang et al., 2011).

O

O

**2.3 The use of ionic liquids in Knoevenagel reactions** 

(Hangarge et al., 2002).

Fig. 13. Compound (48) synthesis.

(Castellani, 2001b).

carried out in good yields (Verdía et al., 2011),

+

CHO

(9) (49)

Fig. 14. Coumarin (50) synthesis using BLAP.

OH

CHO

+

EtO

(51) (44)

**2.4 Catalysis of Knoevenagel reactions using biotechnology** 

2002). ILs have been successfully used in a variety of reactions.

N

reaction. The transference of energy from microwave radiation to the sample is accomplished through dipolar polarisation and conduction mechanisms (Lidström et al. 2001; Loupy, 2002). As illustrated in Fig. 11, the coumarinic derivative (42) is produced in yields of 75.0% after eight minutes of irradiation. This reaction was carried out using aldehyde (43) and methylene compound (44) and was catalysed by piperidine without solvent present (Bogdal, 1998). Following the Knoevenagel condensation, the transesterification reaction to form the ring quickly occurs.

Fig. 11. Coumarinic derivative (42) synthesis.

There are many examples in the literature involving the use of microwave radiation to promote Knoevenagel reactions. In these examples, several different aldehydes, methylene compounds and catalysts were used for the syntheses involving cinnamic acids on silica gel (Kumar et al., 2000), ammonium acetate (Kumar et al., 1998; Mitra et al., 1999) and lithium chloride as catalysts (Mogilaiah & Reddy, 2004).

## **2.2 Clays as catalysts for Knoevenagel reactions**

Clays are abundant in nature, and their high surface area, utility as supports and ionexchange properties have been exploited for catalytic applications (Dasgupta & Török, 2008; Varma, 2002). As shown in Fig. 12, the product of Knoevenagel reaction (45) from the reaction between ninhydrin (46) and malononitrile (47) can be formed in yields of 85.0% after five minutes. This reaction was carried out at room temperature without solvent using K10 as a catalyst (Chakrabarty et al., 2009).

Fig. 12. Knoevenagel product (45) synthesis.

Other Knoevenagel reactions between aromatic aldehydes and malononitrile (47) have also performed successfully without solvent using calcite or fluorite catalysts prepared using a ball mill (Wada & Suzuki, 2003).

reaction. The transference of energy from microwave radiation to the sample is accomplished through dipolar polarisation and conduction mechanisms (Lidström et al. 2001; Loupy, 2002). As illustrated in Fig. 11, the coumarinic derivative (42) is produced in yields of 75.0% after eight minutes of irradiation. This reaction was carried out using aldehyde (43) and methylene compound (44) and was catalysed by piperidine without solvent present (Bogdal, 1998). Following the Knoevenagel condensation, the

There are many examples in the literature involving the use of microwave radiation to promote Knoevenagel reactions. In these examples, several different aldehydes, methylene compounds and catalysts were used for the syntheses involving cinnamic acids on silica gel (Kumar et al., 2000), ammonium acetate (Kumar et al., 1998; Mitra et al., 1999) and lithium

piperidine

o, 10%, 8'

O O

(45)

O

O

CN

CN

(42)

O

75.0%

Clays are abundant in nature, and their high surface area, utility as supports and ionexchange properties have been exploited for catalytic applications (Dasgupta & Török, 2008; Varma, 2002). As shown in Fig. 12, the product of Knoevenagel reaction (45) from the reaction between ninhydrin (46) and malononitrile (47) can be formed in yields of 85.0% after five minutes. This reaction was carried out at room temperature without solvent using

Other Knoevenagel reactions between aromatic aldehydes and malononitrile (47) have also performed successfully without solvent using calcite or fluorite catalysts prepared using a

CN

CN

85.0%

r.t., 5'

k10

transesterification reaction to form the ring quickly occurs.

EtO

O

O

Fig. 11. Coumarinic derivative (42) synthesis.

+

OH

CHO

(43) (44)

chloride as catalysts (Mogilaiah & Reddy, 2004).

K10 as a catalyst (Chakrabarty et al., 2009).

OH

OH

O

O

Fig. 12. Knoevenagel product (45) synthesis.

(46) (47)

ball mill (Wada & Suzuki, 2003).

**2.2 Clays as catalysts for Knoevenagel reactions** 

+

## **2.3 The use of ionic liquids in Knoevenagel reactions**

In recent years, ionic liquids (ILs) have attracted increasing interest as environmentally benign solvents and catalysts due to their relatively low viscosities, low vapour pressures and high thermal and chemical stabilities (Hajipour & Rafiee, 2010; Wasserscheid & Welton, 2002). ILs have been successfully used in a variety of reactions.

As illustrated in Fig 13, the pyrazolonic compound (48) was produced in yields of 71.0% from the reaction between benzaldehyde (9) and 3-methyl-1-phenylpyrazolin-5-(4*H*)-one (49) after 30 minutes using ethylammonium nitrate as an ionic liquid at room temperature (Hangarge et al., 2002).

Fig. 13. Compound (48) synthesis.

Other reactions between aromatic aldehydes and methylene compounds that were catalysed by 1,3-dimethylimidazolium methyl sulphate [MMIm][MSO4] and 2.16% water have been carried out in good yields (Verdía et al., 2011),

### **2.4 Catalysis of Knoevenagel reactions using biotechnology**

Historically, microorganisms have been of enormous social and economic importance (Liese et al., 2006). In the pharmaceutical industry, companies are using biotechnology to develop 901 medicines and vaccines targeting more than 100 diseases (Castellani, 2001a). In 2010, 26 new treatments were approved, and five of these treatments were based on biotechnology (Castellani, 2001b).

Using a biotechnology-based approach, coumarin (50) was produced in yields of 58.0% when the reaction was catalysed by alkaline protease from *Bacillus licheniformis* (BLAP) in a DMSO:H2O (9:1) solvent at a temperature of 550C (Fig. 14) (Wang et al., 2011).

Fig. 14. Coumarin (50) synthesis using BLAP.

Green Chemistry – Aspects for the Knoevenagel Reaction 23

approximately 90 minutes, producing yields of 95.0% (Ratan, 2007). Thus, it's clear that there are green approaches for carrying out organic reactions in water to prepare

morpholine

84.5%

(54) (57)

HO

75.0%

H2O, 15'

grinding

O

HO

O

O

OEt

CN

N

(61)

O

CN

N

NC

H

O

CN H

O

CN

EtO

Entacapone (40), a COMT inhibitor drug whose synthesis is illustrated above in Fig. 10, is another example of the synthesis of important industrial compounds using green conditions. As shown in Fig. 18, Knoevenagel reaction product (59) is formed in yields of 88.0% after two hours under reflux in water with piperidine as a catalyst (McCluskey, 2002).

There are others examples of Knoeveganel reactions carried out in water that are catalysed by *L*-histidine and *L*-arginine (Rhamati & Vakili, 2010). Isatin compounds (61) can also be produced in water at room temperature after fifteen minutes in yields of 75.0% (Fig. 19)

88.0%

H2O, 2h, reflux

piperidine

<sup>O</sup> (59)

O

CN

+

(62) (47)

O

Knoevenagel reactions can also be used to assemble a benzo[*b*]pyrane [4,3-*d*][1,2]oxazine-2 oxide skeleton (63) and (64) via a domino-effect Knoevenagel–Diels–Alder process (Fig. 20)

CN

CN

compounds of industrial interest.

(58)

O

Fig. 18. Compound (59) synthesis.

Fig. 19. Isatin compound (61) synthesis.

N

H

O

(Demchuk, 2011).

HO

Fig. 17. Morphonile-catalysed synthesis of sunscreen in water.

+

+

H N

CHO

(58) (60)

CHO

H2O HO

Because cells are chemical systems that must conform to all chemical and physical laws, whole microorganisms may be used (Alberts et al., 2002). Figure 15 illustrates examples of other Knoevenagel products (52) and (53) resulting from reactions between benzaldehyde (9) and methylene compounds (44) and (19) that were catalysed by baker's yeast. These reactions were carried out under mild conditions, e.g., room temperature and in ethanol as the solvent, with moderate to good yields (Pratap et al., 2011).

Fig. 15. Knoevenagel reactions using a biotechnology-based approach.

#### **2.5 Knoevenagel reactions in water**

Water as a solvent is not only inexpensive and environmentally benign but also provides completely different reactivity (Li & Chen, 2006). It has been suggested that the effect of water on organic reactions may be due to the high internal pressure exerted by a water solution, which results from the high cohesive energy of water (Breslow, 1991).

As illustrated in Fig. 16, the Knoevenagel reaction product (55) is formed in yields of 97.0% when condensation between aldehyde (56) and malononitrile (47) was carried out in water at a temperature of 650C in the absence of catalyst (Bigi et al., 2000).

Fig. 16. A Knoevenagel reaction carried out in water.

The product of the reaction between vanillin (58) and ethyl cyanoacetate (54) was formed in yields of 84.5% in water at room temperature (Fig. 17) (Gomes et al., 2011). This compound was patented in 2007 by Merck & Co. for use in sunscreen compositions containing a UVA sunscreen, photostabliser and antioxidant. The reaction was carried out using piperidine as the catalyst and acetic acid/benzene as the solvent under reflux conditions for

Because cells are chemical systems that must conform to all chemical and physical laws, whole microorganisms may be used (Alberts et al., 2002). Figure 15 illustrates examples of other Knoevenagel products (52) and (53) resulting from reactions between benzaldehyde (9) and methylene compounds (44) and (19) that were catalysed by baker's yeast. These reactions were carried out under mild conditions, e.g., room temperature and in ethanol as

O 70.0%

CN CHO CN

EtOH, r.t.

EtOH, r.t. 45.0%

N Baker's yeast

Baker's yeast

O OEt

S

N

H

O

CN

CN

O

(52)

(53)

the solvent, with moderate to good yields (Pratap et al., 2011).

EtO

+

(9) (54)

+

(9) (19)

Fig. 15. Knoevenagel reactions using a biotechnology-based approach.

S

O

at a temperature of 650C in the absence of catalyst (Bigi et al., 2000).

H2O, 650C, 2h Cl

CN

CN

Fig. 16. A Knoevenagel reaction carried out in water.

CHO

+

solution, which results from the high cohesive energy of water (Breslow, 1991).

O

H

Water as a solvent is not only inexpensive and environmentally benign but also provides completely different reactivity (Li & Chen, 2006). It has been suggested that the effect of water on organic reactions may be due to the high internal pressure exerted by a water

As illustrated in Fig. 16, the Knoevenagel reaction product (55) is formed in yields of 97.0% when condensation between aldehyde (56) and malononitrile (47) was carried out in water

The product of the reaction between vanillin (58) and ethyl cyanoacetate (54) was formed in yields of 84.5% in water at room temperature (Fig. 17) (Gomes et al., 2011). This compound was patented in 2007 by Merck & Co. for use in sunscreen compositions containing a UVA sunscreen, photostabliser and antioxidant. The reaction was carried out using piperidine as the catalyst and acetic acid/benzene as the solvent under reflux conditions for

(47) (55)

97.0%

Cl

**2.5 Knoevenagel reactions in water** 

(56)

CHO

approximately 90 minutes, producing yields of 95.0% (Ratan, 2007). Thus, it's clear that there are green approaches for carrying out organic reactions in water to prepare compounds of industrial interest.

Fig. 17. Morphonile-catalysed synthesis of sunscreen in water.

Entacapone (40), a COMT inhibitor drug whose synthesis is illustrated above in Fig. 10, is another example of the synthesis of important industrial compounds using green conditions. As shown in Fig. 18, Knoevenagel reaction product (59) is formed in yields of 88.0% after two hours under reflux in water with piperidine as a catalyst (McCluskey, 2002).

Fig. 18. Compound (59) synthesis.

There are others examples of Knoeveganel reactions carried out in water that are catalysed by *L*-histidine and *L*-arginine (Rhamati & Vakili, 2010). Isatin compounds (61) can also be produced in water at room temperature after fifteen minutes in yields of 75.0% (Fig. 19) (Demchuk, 2011).

Fig. 19. Isatin compound (61) synthesis.

Knoevenagel reactions can also be used to assemble a benzo[*b*]pyrane [4,3-*d*][1,2]oxazine-2 oxide skeleton (63) and (64) via a domino-effect Knoevenagel–Diels–Alder process (Fig. 20)

Green Chemistry – Aspects for the Knoevenagel Reaction 25

piperidine

pyridine, r.t., US

91.0%

Method A

Method B

O O

(35) (68)

O

OH <sup>O</sup>

H3O+

O (70)

(72)

OH

O

O

O

O

Figure 22 illustrates reactions between benzaldehyde (9) and coumarin (71) that can also be conducted in water under ultrasound irradiation at a temperature of 400C for 90 minutes, forming product (70) in yields of 88.0% (Method B) (Palmisano et al., 2011). In the absence of ultrasound irradiation, formation of product (70) occurs in yields of 62.0% under anhydric

Fig. 22. Synthesis of coumarinic compound (70) with and without ultrasound irradiation.

As mentioned previously, the reduction or elimination of volatile organic solvents in organic syntheses is one of the main goals in green chemistry. Solvent-free organic reactions result in syntheses that are simpler and less energy-intensive, and these conditions also

One method of solvent-free organic synthesis uses high pressure, as shown in Fig. 22 (Jenner, 2001). The piperidine-catalysed reaction between 2-butanone (75) and ethyl cyanoacetate (54) was carried out using two methods in which formation of E(73)/Z(74) Knoevenagel reaction products was observed. When the pressure was increased from 0.10

reduce or eliminate solvent waste, hazards, and toxicity (Tanaka, 2003).

Fig. 21. Ultrasound-catalysed synthesis of Knoevenagel reaction product (68).

O

O

HO

HO

O

Method A =Hantzsch's ester 1.5 eq, *L*-proline 0.2 eq, N2, EtOH,

Method B =Hantzsch's ester 1.05 eq, DBSA 0.1 eq, H2O, US

OH

conditions (Method A) (Palmisano et al., 2011).

+

CHO

reflux 6 h, 62.0%

(9) (71)

+

(69)

O

O CHO

**2.7 Solvent-free Knoevenagel reactions** 

(19.6 kHz, 60W), 400C, 1.5 h, 88.0%

(Amantini, 2001). When the prenylated phenolic aldehyde (65) reacts with methylene compound (66) in water at room temperature for three hours, Knoevenagel intermediate (67) forms, which then reacts to form Diels-Alder product (63) and (64) in yields of 75.0% at a 16:1 ratio (Amantini, 2001).

Fig. 20. Synthesis of benzo[*b*]pyrane [4,3-*d*][1,2]oxazine-2-oxide skeletons (63) and (64).

### **2.6 Ultrasound-catalysed Knoevenagel reactions**

The application of ultrasound waves triggers high-energy chemistry, which is thought to occur through the process of acoustic cavitation, i.e., the formation, growth and implosive collapse of bubbles in a liquid. During cavitational collapse, intense heating of the bubbles occurs (Suslick, 1990).

The piperidine-catalysed reaction between piperonal (69) and malonic acid (35) at room temperature with pyridine as the solvent was carried out under ultrasound irradiation, and Knoevenagel reaction product (68) formed in yields of 91.0% after three hours (Fig. 21) (McNulty et al., 1998). When carried out under reflux conditions, the same reaction forms the Knoevenagel reaction product in yields of 52.0% after three hours (McNulty et al., 1998).

(Amantini, 2001). When the prenylated phenolic aldehyde (65) reacts with methylene compound (66) in water at room temperature for three hours, Knoevenagel intermediate (67) forms, which then reacts to form Diels-Alder product (63) and (64) in yields of 75.0% at

O

CN

NO2

O

H

(63) (64)

O N +

+ CN

H

O

(67)

Fig. 20. Synthesis of benzo[*b*]pyrane [4,3-*d*][1,2]oxazine-2-oxide skeletons (63) and (64).

75.0%

H2O, 3h, r.t.

The application of ultrasound waves triggers high-energy chemistry, which is thought to occur through the process of acoustic cavitation, i.e., the formation, growth and implosive collapse of bubbles in a liquid. During cavitational collapse, intense heating of the bubbles

O

H

O N +

H

O

CN

The piperidine-catalysed reaction between piperonal (69) and malonic acid (35) at room temperature with pyridine as the solvent was carried out under ultrasound irradiation, and Knoevenagel reaction product (68) formed in yields of 91.0% after three hours (Fig. 21) (McNulty et al., 1998). When carried out under reflux conditions, the same reaction forms the Knoevenagel reaction product in yields of 52.0% after three hours (McNulty et

**2.6 Ultrasound-catalysed Knoevenagel reactions** 

occurs (Suslick, 1990).

al., 1998).

a 16:1 ratio (Amantini, 2001).

O

+

CN

NO2

(65) (66)

CHO

Fig. 21. Ultrasound-catalysed synthesis of Knoevenagel reaction product (68).

Figure 22 illustrates reactions between benzaldehyde (9) and coumarin (71) that can also be conducted in water under ultrasound irradiation at a temperature of 400C for 90 minutes, forming product (70) in yields of 88.0% (Method B) (Palmisano et al., 2011). In the absence of ultrasound irradiation, formation of product (70) occurs in yields of 62.0% under anhydric conditions (Method A) (Palmisano et al., 2011).

Fig. 22. Synthesis of coumarinic compound (70) with and without ultrasound irradiation.

### **2.7 Solvent-free Knoevenagel reactions**

As mentioned previously, the reduction or elimination of volatile organic solvents in organic syntheses is one of the main goals in green chemistry. Solvent-free organic reactions result in syntheses that are simpler and less energy-intensive, and these conditions also reduce or eliminate solvent waste, hazards, and toxicity (Tanaka, 2003).

One method of solvent-free organic synthesis uses high pressure, as shown in Fig. 22 (Jenner, 2001). The piperidine-catalysed reaction between 2-butanone (75) and ethyl cyanoacetate (54) was carried out using two methods in which formation of E(73)/Z(74) Knoevenagel reaction products was observed. When the pressure was increased from 0.10

Green Chemistry – Aspects for the Knoevenagel Reaction 27

pyridine, 16h,

piperidine

DMF:MeOH (10:1), r.t. 12h

piperidine

(79)

(82)

N

O

CN

OH

O O

O

O

O

r.t.

Solid phase Knoevenagel reactions were also utilised to produce triphostin protein tyrosine kinases inhibitors. As illustrated in Fig. 26, the piperidine-catalysed reaction between 4 hydroxybenzaldehyde (22) and a resin-bonded methylene compound (83) was carried out using DMF:MeOH (10:1) as the solvent over a period of twelve hours (Guo et al., 1999).

As illustrated by the examples presented herein, classic reactions such as the Knoevenagel condensation can be modernised through new approaches related to Green Chemistry. Particularly in the area of drug synthesis, these new approaches have been being very useful in the development of more environmentally supportable products and chemical processes in the pharmaceutical industry, which works with compounds with high added

OH

Alberts, B., Johonson, A., Lewis, J., Raff, M., Roberts, K. & Walter, P. (2002). *Molecular Biology* 

Amantini, D., Fringuelli, F., Piermatti, O., Pizzo, F. & Vaccaro, L. (2001). Water, a clean,

inexpensive, and re-usable reaction medium. One-pot synthesis of (*E*)-2-aryl-1 cyano-1-nitroethenes. *Green Chemistry,* Vol.3, No.5, (September 2001), pp. 229-232,

The author is grateful to FAPEG, INCT-INOFAR and UFG for financial support.

*of The Cell,* Garland Science, ISBN 0-8153-3218-1, New York, USA

Fig. 25. Synthesis of coumarin (79) via solid phase organic synthesis.

OHC

HO O

+

(80) (81)

+

OHC

CN

(83) (22)

O

O

Fig. 26. Synthesis of triphostin (82) via solid phase organic synthesis.

**3. Conclusions** 

N

O

O

O

**4. Acknowledgments** 

ISSN 1463-9262

**5. References** 

values.

MPa to 300 MPa, the yield increased from 28.1% to 99.0%. However, significant changes were not observed in the ratio of E(73)/Z(74) Knoevenagel reaction products (Jenner, 2001).

Fig. 23. High-pressure Knoevenagel reactions.

Solvent-free Knoevenagel reactions have also been carried out using a mortar and pestle. Under these conditions, the reaction between benzaldehyde (9) diethyl malonate (31), which was catalysed by triethylbenzylammonium chloride (TEBA), resulted in product yields of 87.5% after ten minutes (Rong et al., 2006). Similarly, the domino-effect Friedlander condensation reaction, which can also be conducted using a mortar and pestle, was observed between aldehyde (77) and methylene compound (78). This sodium fluoridecatalysed reaction formed the aromatic Knoevenagel reaction product (76) in yields of 92.0% after eight minutes (Fig. 24) (Mogilaiah & Reddy, 2003).

Fig. 24. Synthesis of the Friedlander condensation product (76).

## **2.8 Knoevenagel reactions using solid phase organic synthesis**

An innovative and important field of organic synthesis involves the use of solid phase organic synthesis (Czarnik, 2001). This new methodology was introduced by Merrifield in 1963 when he used it to synthesise amino acids (Merrifield, 1963). Solid phase organic synthesis uses insoluble polymers that covalently bond organic substrates to the solid surface until the synthesis is complete, at which point the compound of interest is separated from the solid matrix (Czarnik, 2001).

This approach has been used to synthesise coumaric compound (79) from the reaction of aldehyde (81) and methylene compound (80) bonded to Wang resin. The reaction was complete after sixteen hours under Doebner conditions, as illustrated in Fig. 25 (Xia et al., 1999).

Fig. 25. Synthesis of coumarin (79) via solid phase organic synthesis.

Solid phase Knoevenagel reactions were also utilised to produce triphostin protein tyrosine kinases inhibitors. As illustrated in Fig. 26, the piperidine-catalysed reaction between 4 hydroxybenzaldehyde (22) and a resin-bonded methylene compound (83) was carried out using DMF:MeOH (10:1) as the solvent over a period of twelve hours (Guo et al., 1999).

Fig. 26. Synthesis of triphostin (82) via solid phase organic synthesis.

## **3. Conclusions**

26 Green Chemistry – Environmentally Benign Approaches

MPa to 300 MPa, the yield increased from 28.1% to 99.0%. However, significant changes were not observed in the ratio of E(73)/Z(74) Knoevenagel reaction products (Jenner, 2001).

O

+

N N

(76)

O

O

CN

OEt

CN

EtO

Solvent-free Knoevenagel reactions have also been carried out using a mortar and pestle. Under these conditions, the reaction between benzaldehyde (9) diethyl malonate (31), which was catalysed by triethylbenzylammonium chloride (TEBA), resulted in product yields of 87.5% after ten minutes (Rong et al., 2006). Similarly, the domino-effect Friedlander condensation reaction, which can also be conducted using a mortar and pestle, was observed between aldehyde (77) and methylene compound (78). This sodium fluoridecatalysed reaction formed the aromatic Knoevenagel reaction product (76) in yields of 92.0%

Method A =piperidine 0.10 MPa 28.1% (73):(74) 58:42 Method B =piperidine 300 MPa 99.0% (73):(74) 57:43

(75) (74)

Method B

(54) (73)

An innovative and important field of organic synthesis involves the use of solid phase organic synthesis (Czarnik, 2001). This new methodology was introduced by Merrifield in 1963 when he used it to synthesise amino acids (Merrifield, 1963). Solid phase organic synthesis uses insoluble polymers that covalently bond organic substrates to the solid surface until the synthesis is complete, at which point the compound of interest is separated

NaF

92.0%

pestle and mortar, 8'

This approach has been used to synthesise coumaric compound (79) from the reaction of aldehyde (81) and methylene compound (80) bonded to Wang resin. The reaction was complete after sixteen hours under Doebner conditions, as illustrated in Fig. 25 (Xia et al.,

Fig. 23. High-pressure Knoevenagel reactions.

EtO

O

CN Method A

+

O

after eight minutes (Fig. 24) (Mogilaiah & Reddy, 2003).

+

N NH2

CHO

(77) (78)

Fig. 24. Synthesis of the Friedlander condensation product (76).

from the solid matrix (Czarnik, 2001).

1999).

**2.8 Knoevenagel reactions using solid phase organic synthesis** 

O

O

As illustrated by the examples presented herein, classic reactions such as the Knoevenagel condensation can be modernised through new approaches related to Green Chemistry. Particularly in the area of drug synthesis, these new approaches have been being very useful in the development of more environmentally supportable products and chemical processes in the pharmaceutical industry, which works with compounds with high added values.

## **4. Acknowledgments**

The author is grateful to FAPEG, INCT-INOFAR and UFG for financial support.

## **5. References**


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**3** 

**Application of Nanometals** 

Srinath Palakurthi3 and Jingbo Liu4,5

*3Department of Pharmaceutical Sciences,* 

*Texas A&M University-Kingsville, TX,* 

*Texas A&M University, College Station, TX,* 

*4Nanotech and Cleantech Group,* 

*1Department of Chemistry,* 

*2Department of Chemistry,* 

*5Department of Chemistry,* 

*1Mexico 2,3,4,5USA* 

**Cancer Diagnosis and Therapy** 

Iliana Medina-Ramirez1, Maribel Gonzalez-Garcia2,

*Universidad Autonoma de Aguascalientes, Aguascalientes,* 

*Texas A&M University-Kingsville, Kingsville, TX,* 

*Texas A&M Health Science Center, Kingssville, TX,* 

**Fabricated Using Green Synthesis in** 

The interest for the development of new materials for biomedical applications has steadly increased over the past ten years, due to the numerous advances made in the field of cancer diagnosis and therapy using nanoparticles (NPs). Nowadays, these NPs, such as noble metal gold (Au) and silver (Ag), are considered as valuable starting materials for the construction of innovative nanodevices and nanosystems that are built based on the rational design and precise integration of the tailored-functional properties of NPs. The two main goals of this investigation are to: (1) conduct multidisciplinary project and emerging research in the biological and physical sciences to develop new diagnostic methods or cancer therapy tools (health aspect); and (2) optimize the fabrication variables of nano-metals using green colloidal chemistry method (nanotechnological aspect). To accomplish the above goals, we have extensively investigated the fabrication of nano-structured metal(s) using green colloidal (sol-gel) approaches to formulate particles of specific size with defined homogeneity at molecular level; characterized the fabricated nanostructured materials using state-of-the-art instrumentation; and evaluated their *in vitro* cytotoxicity using model cell lines (such as ovarian adenocarcinoma and normal ovarian cell line), and related hemocompatibility of Au and Ag NPs with human red blood cells. The scope of this article will focus on introductory nanoscience, green synthesis strategies, and structural analysis

techniques, followed by specific examples related to diagnostics and cancer therapy.

**1. Introduction** 

Treatment of Stroke. *Organic Process Research & Development,* Vol.4, No.6, (December 2000), pp. 477-487, ISSN 1083-6160

Xia, Y., Yang, Z., Brossi, A. & Lee, K. (1999). Asymmetric Solid-Phase Synthesis of (3'*R*,4'*R*)- Di-*O*-*cis*-acyl 3-Carboxyl Khellactones. *Organic Letters,* Vol.1, No.13, (December 1999), pp. 2113-2115, ISSN 1523-7060

## **Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy**

Iliana Medina-Ramirez1, Maribel Gonzalez-Garcia2, Srinath Palakurthi3 and Jingbo Liu4,5 *1Department of Chemistry, Universidad Autonoma de Aguascalientes, Aguascalientes, 2Department of Chemistry, Texas A&M University-Kingsville, Kingsville, TX, 3Department of Pharmaceutical Sciences, Texas A&M Health Science Center, Kingssville, TX, 4Nanotech and Cleantech Group, Texas A&M University-Kingsville, TX, 5Department of Chemistry, Texas A&M University, College Station, TX, 1Mexico 2,3,4,5USA* 

## **1. Introduction**

32 Green Chemistry – Environmentally Benign Approaches

Xia, Y., Yang, Z., Brossi, A. & Lee, K. (1999). Asymmetric Solid-Phase Synthesis of (3'*R*,4'*R*)-

(December 2000), pp. 477-487, ISSN 1083-6160

1999), pp. 2113-2115, ISSN 1523-7060

Treatment of Stroke. *Organic Process Research & Development,* Vol.4, No.6,

Di-*O*-*cis*-acyl 3-Carboxyl Khellactones. *Organic Letters,* Vol.1, No.13, (December

The interest for the development of new materials for biomedical applications has steadly increased over the past ten years, due to the numerous advances made in the field of cancer diagnosis and therapy using nanoparticles (NPs). Nowadays, these NPs, such as noble metal gold (Au) and silver (Ag), are considered as valuable starting materials for the construction of innovative nanodevices and nanosystems that are built based on the rational design and precise integration of the tailored-functional properties of NPs. The two main goals of this investigation are to: (1) conduct multidisciplinary project and emerging research in the biological and physical sciences to develop new diagnostic methods or cancer therapy tools (health aspect); and (2) optimize the fabrication variables of nano-metals using green colloidal chemistry method (nanotechnological aspect). To accomplish the above goals, we have extensively investigated the fabrication of nano-structured metal(s) using green colloidal (sol-gel) approaches to formulate particles of specific size with defined homogeneity at molecular level; characterized the fabricated nanostructured materials using state-of-the-art instrumentation; and evaluated their *in vitro* cytotoxicity using model cell lines (such as ovarian adenocarcinoma and normal ovarian cell line), and related hemocompatibility of Au and Ag NPs with human red blood cells. The scope of this article will focus on introductory nanoscience, green synthesis strategies, and structural analysis techniques, followed by specific examples related to diagnostics and cancer therapy.

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 35

Fig. 1. The band gap between HOMO and LUMO (note: the band gap of nanosystem was

The development of cost-effective environmental friendly methods for large-scale synthesis of benign, highly efficient nanomaterials represents a critical challenge to their practical applications in biomedical research. [14] Some nanoporous materials with regular shapes such as porous nanowires, nanotubes, spheres, and nanoparticles have been successfully prepared by chemical or physical methods which can be carried out in a variety of ways, such as in the gas phase, or in solution, or supported on a substrate, or in a matrix. [15] Although a comprehensive comparison of these approaches does not exist, significant differences in the physicochemical properties, and therefore performance of the resulting materials does exist, allowing for some quantative assessment. In general, physical methods (also known as "top-down" techniques, *Fig. 2*) are highly energy demanding, besides, it is difficult to control the size and composition of the fabricated materials. [16] In the top-down method, the bulk is "broken" down to the nanometer length scale by lithographic or laser ablation-condensation techniques. [17] Chemical methods (also known as "Bottom-up" techniques, *Fig. 2*) are the most popular methods of manufacturing nanomaterials. [18] They are characterized by narrow nanoparticles size distribution, relative simplicity of control over synthesis, and reliable stabilization of nanoparticles in the systems; besides, kinetically controlled mixing of elements using low temperature approaches might yield nanocrystalline phases that are not otherwise accessible. [19] These methods are based on various reduction procedures involving surfactants or templating molecules, as well as thermal decomposition of metal or metal-organic precursors. [20] The sol-gel process has proved to be very effective in the preparation of diverse metal oxide nanomaterials, such as films, particles or monoliths. [21] The sol-gel process consists of the hydrolysis of metal alkoxides and subsequent polycondensation to form the metal oxide gel. [22] One means of achieving shape control is by using a static template to enhance the growth rate of one crystallographic phase over another. [23] The organic surfactants may be undesired for many

increased compared with macrosystem).

**1.1.3 Synthesis of nanomaterials** 

## **1.1 Source and properties of engineered nanomaterials**

## **1.1.1 General view of nanotechnology**

The development of engineered nanomaterials (ENMs) is considered as one of the major achievements of the twentieth century. [1] The novel and outstanding physicochemical properties (which are distinctively different from that of bulk materials) of these ENMs have led to their use in numerous current and emerging technologies. [2] Nowadays, it is practically impossible to find any field of knowledge that is not in some way or another related with nanomaterials; for instance, development of diagnostic sensors for biomedical and environmental applications. [3] In biology and medicine, sensors are being used as DNA/protein markers for disease identification, or as novel drug carriers with little or no immunogenicity and high cell specificity. [4] In materials science, ENMs are currently being used for the development of solar cells, light-emitting diodes (LEDs), information recording systems and non-linear optical devices. [5] Although ENMs represent numerous advantages in their applications, a number of significant challenges still remain in order to ensure the implementation of synthetic pathways that allow for controlled production of all nanomaterials with desired size, uniform size distribution, morphology, crystallinity, chemical composition, and microstructure, which altogether result in desired physical properties. [6] Another important consideration for the practical application of these materials is the high cost associated to their large scale production, coupled with the tremendous difficulties in separation, recovery, and recycling in industrial applications. [7]

## **1.1.2 Properties of nanomaterials**

Nanostructured materials display three major unique properties not observed in their bulk counterparts. [8] They possess: 1) "ultra high surface effect" allowing for dramatic increase in the number of atoms in the surface. [9] When the nanosize is reduced to about 10 nm, the surface atoms account for 20 % of the total atoms composed of the perfect particles. If the particle size is further decreased to 1 nm, the surface atoms account up to 99 % of the total number of atoms. [10] Due to the lack of adjacent atoms, there exist large amount of dangling bonds, which are not saturated. Those atoms will bind with others to be stabilized. This process results in lower than the maximum coordination number and increased surface energy, collectively resulting in high chemical reactivities of the generated nanomaterials; 2) "ultrahigh volume effect" allowing for light weight of small particles. [11] Due to the reduction in the diameter of the nanomaterials, the energy gap was increased. Herein, the electrons are mobile relative to the bulk. This causes unique physical, chemical, electronic and biological properties of nanomaterials compared with macroscopic systems; and 3) "quantum size effect" allowing for nanosize decrease and quasi-discrete energy of electron orbital around the Femi energy level. [12] This will increase the band gap between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), shown in *Fig. 1*. Therefore, the electromagnetic quantum properties of solids are altered. When the nanometer size range is reached, the quantum size effect will become pronounced, resulting in abnormal optical, acoustic, electronic, magnetic, thermal and dynamic properties. The above energy gap () of conduction and valence band of metals was determined by Kubo using an electronic model, =4Ef/3N, where the Ef stands for Fermi enegy, N the total electrons in the particles. [13]

Fig. 1. The band gap between HOMO and LUMO (note: the band gap of nanosystem was increased compared with macrosystem).

#### **1.1.3 Synthesis of nanomaterials**

34 Green Chemistry – Environmentally Benign Approaches

The development of engineered nanomaterials (ENMs) is considered as one of the major achievements of the twentieth century. [1] The novel and outstanding physicochemical properties (which are distinctively different from that of bulk materials) of these ENMs have led to their use in numerous current and emerging technologies. [2] Nowadays, it is practically impossible to find any field of knowledge that is not in some way or another related with nanomaterials; for instance, development of diagnostic sensors for biomedical and environmental applications. [3] In biology and medicine, sensors are being used as DNA/protein markers for disease identification, or as novel drug carriers with little or no immunogenicity and high cell specificity. [4] In materials science, ENMs are currently being used for the development of solar cells, light-emitting diodes (LEDs), information recording systems and non-linear optical devices. [5] Although ENMs represent numerous advantages in their applications, a number of significant challenges still remain in order to ensure the implementation of synthetic pathways that allow for controlled production of all nanomaterials with desired size, uniform size distribution, morphology, crystallinity, chemical composition, and microstructure, which altogether result in desired physical properties. [6] Another important consideration for the practical application of these materials is the high cost associated to their large scale production, coupled with the tremendous difficulties in separation, recovery, and recycling in industrial applications. [7]

Nanostructured materials display three major unique properties not observed in their bulk counterparts. [8] They possess: 1) "ultra high surface effect" allowing for dramatic increase in the number of atoms in the surface. [9] When the nanosize is reduced to about 10 nm, the surface atoms account for 20 % of the total atoms composed of the perfect particles. If the particle size is further decreased to 1 nm, the surface atoms account up to 99 % of the total number of atoms. [10] Due to the lack of adjacent atoms, there exist large amount of dangling bonds, which are not saturated. Those atoms will bind with others to be stabilized. This process results in lower than the maximum coordination number and increased surface energy, collectively resulting in high chemical reactivities of the generated nanomaterials; 2) "ultrahigh volume effect" allowing for light weight of small particles. [11] Due to the reduction in the diameter of the nanomaterials, the energy gap was increased. Herein, the electrons are mobile relative to the bulk. This causes unique physical, chemical, electronic and biological properties of nanomaterials compared with macroscopic systems; and 3) "quantum size effect" allowing for nanosize decrease and quasi-discrete energy of electron orbital around the Femi energy level. [12] This will increase the band gap between highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO), shown in *Fig. 1*. Therefore, the electromagnetic quantum properties of solids are altered. When the nanometer size range is reached, the quantum size effect will become pronounced, resulting in abnormal optical, acoustic, electronic, magnetic, thermal and dynamic properties. The above energy gap () of conduction and valence band of metals was determined by Kubo using an electronic model, =4Ef/3N, where the Ef stands for

**1.1 Source and properties of engineered nanomaterials** 

**1.1.1 General view of nanotechnology** 

**1.1.2 Properties of nanomaterials** 

Fermi enegy, N the total electrons in the particles. [13]

The development of cost-effective environmental friendly methods for large-scale synthesis of benign, highly efficient nanomaterials represents a critical challenge to their practical applications in biomedical research. [14] Some nanoporous materials with regular shapes such as porous nanowires, nanotubes, spheres, and nanoparticles have been successfully prepared by chemical or physical methods which can be carried out in a variety of ways, such as in the gas phase, or in solution, or supported on a substrate, or in a matrix. [15] Although a comprehensive comparison of these approaches does not exist, significant differences in the physicochemical properties, and therefore performance of the resulting materials does exist, allowing for some quantative assessment. In general, physical methods (also known as "top-down" techniques, *Fig. 2*) are highly energy demanding, besides, it is difficult to control the size and composition of the fabricated materials. [16] In the top-down method, the bulk is "broken" down to the nanometer length scale by lithographic or laser ablation-condensation techniques. [17] Chemical methods (also known as "Bottom-up" techniques, *Fig. 2*) are the most popular methods of manufacturing nanomaterials. [18] They are characterized by narrow nanoparticles size distribution, relative simplicity of control over synthesis, and reliable stabilization of nanoparticles in the systems; besides, kinetically controlled mixing of elements using low temperature approaches might yield nanocrystalline phases that are not otherwise accessible. [19] These methods are based on various reduction procedures involving surfactants or templating molecules, as well as thermal decomposition of metal or metal-organic precursors. [20] The sol-gel process has proved to be very effective in the preparation of diverse metal oxide nanomaterials, such as films, particles or monoliths. [21] The sol-gel process consists of the hydrolysis of metal alkoxides and subsequent polycondensation to form the metal oxide gel. [22] One means of achieving shape control is by using a static template to enhance the growth rate of one crystallographic phase over another. [23] The organic surfactants may be undesired for many

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 37

studies by Wagner *et al* showed that Al NPs exhibit higher toxicity in rat alveolar macrophages and also their phagocytic ability is diminished after 24 h of exposure. [29] Al NPs have produced significant increase in lactate dehydrogensae (LDH) leakage and were shown to induce apoptosis after exposing them to mammalian germline stem cells. [30] *In vivo* toxicity experiments of aluminum oxide nanoparticles in imprinting control region (ICR) strained mice indicated that nano-alumina impaired neurobehavioral functions. Furthermore, these defects in neurobehavioral functions were shown to be mediated by

Gold (Au) NPs are recently widely used in cellular imaging and photodynamic therapy. Au NPs exhibited size-dependent toxicity with smaller-sized particles showing more toxicity than larger-sized particles in various cell lines *in vitro*, and *in vivo* similar pattern of size dependent toxicity was observed. [32] The effect of shape of the Au NPs on toxicity was also assessed and it was reported that Au nanorods were more toxic than spherical Au NPs. [33] The effect of surface chemistry of Au NPs was investigated in monkey kidney cells (CV-1) in and cells carrying SV40 genetic material (Cos-1 cells), and *Escherichia coli* (*E. coli*) bacteria. The results indicated that cationic Au NPs were more toxic compared to their anionic counterparts. [34]. Lately, biofuncionalization (Lysine capped) of Au NPs has been explored in an attempt to reduce their toxicity. These lysines capped Au NPs were not toxic to macrophages at concentrations up to 100 µM after 72 h exposure. Moreover, they did not elicit the secretion of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) or

Silicon microparticles were investigated for their biomedical application; the results have shown that vascular endothelial cells which internalized silicon microparticles maintained their cellular integrity as demonstrated by cellular morphology, viability, and intact mitotic trafficking of vesicles bearing silicon microparticles. [36] Silica (SiO2) NPs were also investigated for their toxicity as they promise effective biomedical applications. When SiO2 NPs were treated on normal human mesothelial cells at concentration of 26.7 µg/mL, there was only 3% LDH leakage after 3 hr exposure. When mice were treated with SiO2-nanoparticle coated magnetic nanoparticles for 4 weeks, the NPs were shown to be taken up by the liver and then redistributed to other organs such as spleen, lungs, heart, and kidney. It was also reported that NPs (<50 nm) bypassed various biological barriers (blood-brain and blood-testis) without inducing any toxicity. [37] The Ag NPs have also shown cellular toxicity; *in vitro* they exhibited size and dose dependent toxicity in neuroendocrine cells, liver cells, lung cells and germline stem cells, and the toxicity was reported to be mediated mainly through oxidative stress. *In vivo,* these NPs of 60 nm size were investigated at various doses (30, 300 and 1000 mg/kg) and these NPs showed a dose dependent liver toxicity. [38] Copper (Cu) nanoparticles are widely investigated for their antimicrobial properties. Cu NPs, though very effective antimicrobials, exhibited severe toxicological effects including heavy injuries in kidney, liver, and spleen of

Titanium dioxide (TiO2) nanoparticles are very widely manufactured nanomaterials for various applications including cosmetics, paints and as additives to surface coatings. TiO2 NPs have shown to induce inflammatory responses and reactive oxygen species (ROS) in various cell types and tissues. [41] However, Renwick *et al* reported that TiO2 NPs were not directly toxic to macrophages but significantly reduced the ability of macrophages to phagocytose other particles. [42] Cerium oxide (CeO2) nanoparticles were recently used in computer chip manufacturing, polishing and as an additive to decrease diesel emissions.

mitochondrial impairment, oxidative damage and neural cell loss. [31]

interleukin-1 beta (IL-1β). [35]

mice after administration. [39,40]

applications, and a relatively high temperature is needed to decompose the material. [24] Unfortunately, such thermal treatment generally induces dramatic growth of nanoparticles such that ultrafine nanoparticles free of templating and stabilizing agents could not be obtained [25] Lately, novel and simple methods to prepare metal oxides with controllable morphologies by simply varying the hydrolytic conditions have been reported. [26]

Fig. 2. Schematic of Bottom-up and Top-down Fabrication.

### **1.2 Toxicological effects of engineered nanomaterials**

Metal based nanoparticles (NPs) have been widely used in various applications including biological diagnostics, cell labeling, targeted drug delivery, cancer therapy, and biological sensors and also as antiviral, antibacterial and antifungal agents. [27] An understanding of the potential toxicity induced by these NPs to human health and environment is of prime importance in development of these NPs for biomedical applications. The metal NPs can enter the body via routes such as the gastrointestinal tract, lungs, intravenous injection and exposure in skin. When NPs come to contact with biological membranes, they pose a threat by affecting physiology of the body. For example, silver (Ag), copper (Cu) and aluminum (Al) NPs may induce oxidative stress and generate free radicals that could disrupt the endothelial cell membrane. It was reported recently that in utero exposure to NPs present in exhaust of diesel affects testicular function of the male fetus by inhibiting testosterone production. [28] In this section toxicity issues of each metal nanoparticle will be discussed, starting with aluminum followed by gold, silicon, copper, titania and ceria. Al NPs are widely used in military applications such as fuels, propellants, and coatings. Thus, exposure of aluminum to soldiers and other defense personnel is on the rise. Recent

applications, and a relatively high temperature is needed to decompose the material. [24] Unfortunately, such thermal treatment generally induces dramatic growth of nanoparticles such that ultrafine nanoparticles free of templating and stabilizing agents could not be obtained [25] Lately, novel and simple methods to prepare metal oxides with controllable

morphologies by simply varying the hydrolytic conditions have been reported. [26]

Fig. 2. Schematic of Bottom-up and Top-down Fabrication.

**1.2 Toxicological effects of engineered nanomaterials** 

Metal based nanoparticles (NPs) have been widely used in various applications including biological diagnostics, cell labeling, targeted drug delivery, cancer therapy, and biological sensors and also as antiviral, antibacterial and antifungal agents. [27] An understanding of the potential toxicity induced by these NPs to human health and environment is of prime importance in development of these NPs for biomedical applications. The metal NPs can enter the body via routes such as the gastrointestinal tract, lungs, intravenous injection and exposure in skin. When NPs come to contact with biological membranes, they pose a threat by affecting physiology of the body. For example, silver (Ag), copper (Cu) and aluminum (Al) NPs may induce oxidative stress and generate free radicals that could disrupt the endothelial cell membrane. It was reported recently that in utero exposure to NPs present in exhaust of diesel affects testicular function of the male fetus by inhibiting testosterone production. [28] In this section toxicity issues of each metal nanoparticle will be discussed, starting with aluminum followed by gold, silicon, copper, titania and ceria. Al NPs are widely used in military applications such as fuels, propellants, and coatings. Thus, exposure of aluminum to soldiers and other defense personnel is on the rise. Recent studies by Wagner *et al* showed that Al NPs exhibit higher toxicity in rat alveolar macrophages and also their phagocytic ability is diminished after 24 h of exposure. [29] Al NPs have produced significant increase in lactate dehydrogensae (LDH) leakage and were shown to induce apoptosis after exposing them to mammalian germline stem cells. [30] *In vivo* toxicity experiments of aluminum oxide nanoparticles in imprinting control region (ICR) strained mice indicated that nano-alumina impaired neurobehavioral functions. Furthermore, these defects in neurobehavioral functions were shown to be mediated by mitochondrial impairment, oxidative damage and neural cell loss. [31]

Gold (Au) NPs are recently widely used in cellular imaging and photodynamic therapy. Au NPs exhibited size-dependent toxicity with smaller-sized particles showing more toxicity than larger-sized particles in various cell lines *in vitro*, and *in vivo* similar pattern of size dependent toxicity was observed. [32] The effect of shape of the Au NPs on toxicity was also assessed and it was reported that Au nanorods were more toxic than spherical Au NPs. [33] The effect of surface chemistry of Au NPs was investigated in monkey kidney cells (CV-1) in and cells carrying SV40 genetic material (Cos-1 cells), and *Escherichia coli* (*E. coli*) bacteria. The results indicated that cationic Au NPs were more toxic compared to their anionic counterparts. [34]. Lately, biofuncionalization (Lysine capped) of Au NPs has been explored in an attempt to reduce their toxicity. These lysines capped Au NPs were not toxic to macrophages at concentrations up to 100 µM after 72 h exposure. Moreover, they did not elicit the secretion of pro-inflammatory cytokines tumor necrosis factor-alpha (TNF-α) or interleukin-1 beta (IL-1β). [35]

Silicon microparticles were investigated for their biomedical application; the results have shown that vascular endothelial cells which internalized silicon microparticles maintained their cellular integrity as demonstrated by cellular morphology, viability, and intact mitotic trafficking of vesicles bearing silicon microparticles. [36] Silica (SiO2) NPs were also investigated for their toxicity as they promise effective biomedical applications. When SiO2 NPs were treated on normal human mesothelial cells at concentration of 26.7 µg/mL, there was only 3% LDH leakage after 3 hr exposure. When mice were treated with SiO2-nanoparticle coated magnetic nanoparticles for 4 weeks, the NPs were shown to be taken up by the liver and then redistributed to other organs such as spleen, lungs, heart, and kidney. It was also reported that NPs (<50 nm) bypassed various biological barriers (blood-brain and blood-testis) without inducing any toxicity. [37] The Ag NPs have also shown cellular toxicity; *in vitro* they exhibited size and dose dependent toxicity in neuroendocrine cells, liver cells, lung cells and germline stem cells, and the toxicity was reported to be mediated mainly through oxidative stress. *In vivo,* these NPs of 60 nm size were investigated at various doses (30, 300 and 1000 mg/kg) and these NPs showed a dose dependent liver toxicity. [38] Copper (Cu) nanoparticles are widely investigated for their antimicrobial properties. Cu NPs, though very effective antimicrobials, exhibited severe toxicological effects including heavy injuries in kidney, liver, and spleen of mice after administration. [39,40]

Titanium dioxide (TiO2) nanoparticles are very widely manufactured nanomaterials for various applications including cosmetics, paints and as additives to surface coatings. TiO2 NPs have shown to induce inflammatory responses and reactive oxygen species (ROS) in various cell types and tissues. [41] However, Renwick *et al* reported that TiO2 NPs were not directly toxic to macrophages but significantly reduced the ability of macrophages to phagocytose other particles. [42] Cerium oxide (CeO2) nanoparticles were recently used in computer chip manufacturing, polishing and as an additive to decrease diesel emissions.

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 39

n is an integer determined by the order given, λ is the wavelength of x-rays (nm), and moving electrons, protons and neutrons, d is the spacing between the planes in the atomic lattice (nm), θ is the angle between the incident ray, and the scattering planes (rad) , L is the crystallite size (nm), is the full width

In this study, the morphology and crystalline structure of the engineered nanomaterials were characterized using scanning and transmission electron microscopy (SEM and TEM). [51] Both techniqes are based on the use of a microscope that uses high energy electrons to form an image. Due to their advantages of higher magnification, larger depth of focus, greater resolution, and ease of sample observation, both facilities have been employed widely to determine the crystalline phase, defects, and texture of materials. [52] High resolution field emission TEM was employed to achieve high spatial resolution, high contrast, and unsurpassed versatility. Particularly, the Tecnai F20 G2 TEM used in this study, which includes a Schottky field emission source, provides ultra-high brightness, low energy spread and very small probe sizes. The Tecnai F20 G2 used in this study has been designed and preconfigured specifically to meet the strict requirements of nanomaterials. This TEM is also equipped with a robust high-brightness field emission gun, allowing for a

wide range of applications, from morphological analysis to defect characterisation. [53]

As complementary techniques, energy dispersive spectroscopy (EDS, equiped with TEM) and X-ray photoelectron spectroscopy were used to accurately determine the elemental composition. [54] The EDS is normally used as a semi-quantitative analysis that allows for determination of the amount and identity of the different elements. The EDS can stimulate

**1.3.4 Elemental composition study of engineered nanomaterials** 

at half maximum (rad), and K is a constant, that varies with the method of taking the breadth

(0.89<K<1).

Fig. 3. XRD characterization of nano-materials.

**1.3.3 Fine-structure study of engineered nanomaterials** 

The toxicity studies of CeO2 nanoparticles involving human lung adenocarcinoma epithelial cell line (A549 lung cells) have shown that CeO2 NPs did not result in any significant change in LDH leakage or cell morphology, but exhibited a NP induced oxidative stress resulting in altered gene expression. [43]

Hemocompatibility of metal nanoparticles is a very important property for biomedical applications. Metal nanoparticles with good hemocompatibility are often desirable since red blood cells are the primary cells that come in contact with metal nanoparticle when administered intravenously. Various attempts were made to improve the hemocompatibility of metal nanoparticles. It was reported that when Zein, a natural polymer, was incorporated into silver NPs, the hemocompatibility was easily achieved. The results indicated that zeinsilver nanocomposites have shown better hemocompatibility when compared with Ag NPs alone. [44,48-49] Ren *et al* have investigated the hemocompatibility of cisplatin loaded Au-Au2S nanoaparticles. The results indicated that bare Au-Au2S NPs have shown hemolysis ratio below 2 % at 100 µg/mL concentration. The cisplatin loaded Au-Au2S nanoparticles have shown hemolysis ratio < 5% at 80 µg/mL, indicating their hemocompatibility and potential use for cancer therapy. [45]

## **1.3 Nanostructural characterization of engineered nanomaterials**

We have been able to synthesize several metallic and semiconducting NPs, which have been evaluated using several state-of-the-art instrumentation techniques. Spectroscopic and microscopic techniques were employed in order to determine the stability, crystalline phase, morphology and size distribution of the synthesized NPs.

## **1.3.1 Surface energy study of engineered nanomaterials**

The electrokinetics (expressed by zetapotential, ) of the colloidal suspension was measured using a ZetPALS approach to evaluate particle size and its distribution, from which the stability of engineered nanomaterials can be further determined. [46] Based on the sign of particle's, , the charge can be also determined. [47] The time dependence of the zetapotential on the course of measurement is another technique to understand the formation mechanism of nanoparticles. It was encountered that the large zetapotential of the like (negative) charges enabled to minimize the particles agglomeration due to electrostatic repulsion.

## **1.3.2 Crystalline phase study of engineered nanomaterials**

X-ray powder diffraction (XRD) analysis was used to identify the crystalline phase of the NPs due to its capability to provide rapid, non-destructive analysis of multi-component mixtures, allowing quick and accurate analysis of phase, crystallinity, lattice parameters, expandition tensors and bulk modules, and aperiodical arranged clusters. Based on the peak broadening, the cyrstallite size can also be calculated using Scherrer equation. [50] XRD characterization is widely used in various fields as metallurgy, mineralogy, forensic science, archeology, condensed matter physics, and the biological and pharmaceutical sciences. *Fig. 3* display the working principle and major information obtained from XRD.

n is an integer determined by the order given, λ is the wavelength of x-rays (nm), and moving electrons, protons and neutrons, d is the spacing between the planes in the atomic lattice (nm), θ is the angle between the incident ray, and the scattering planes (rad) , L is the crystallite size (nm), is the full width at half maximum (rad), and K is a constant, that varies with the method of taking the breadth (0.89<K<1).

Fig. 3. XRD characterization of nano-materials.

38 Green Chemistry – Environmentally Benign Approaches

The toxicity studies of CeO2 nanoparticles involving human lung adenocarcinoma epithelial cell line (A549 lung cells) have shown that CeO2 NPs did not result in any significant change in LDH leakage or cell morphology, but exhibited a NP induced oxidative stress resulting in

Hemocompatibility of metal nanoparticles is a very important property for biomedical applications. Metal nanoparticles with good hemocompatibility are often desirable since red blood cells are the primary cells that come in contact with metal nanoparticle when administered intravenously. Various attempts were made to improve the hemocompatibility of metal nanoparticles. It was reported that when Zein, a natural polymer, was incorporated into silver NPs, the hemocompatibility was easily achieved. The results indicated that zeinsilver nanocomposites have shown better hemocompatibility when compared with Ag NPs alone. [44,48-49] Ren *et al* have investigated the hemocompatibility of cisplatin loaded Au-Au2S nanoaparticles. The results indicated that bare Au-Au2S NPs have shown hemolysis ratio below 2 % at 100 µg/mL concentration. The cisplatin loaded Au-Au2S nanoparticles have shown hemolysis ratio < 5% at 80 µg/mL, indicating their hemocompatibility and potential

We have been able to synthesize several metallic and semiconducting NPs, which have been evaluated using several state-of-the-art instrumentation techniques. Spectroscopic and microscopic techniques were employed in order to determine the stability, crystalline phase,

The electrokinetics (expressed by zetapotential, ) of the colloidal suspension was measured using a ZetPALS approach to evaluate particle size and its distribution, from which the stability of engineered nanomaterials can be further determined. [46] Based on the sign of particle's, , the charge can be also determined. [47] The time dependence of the zetapotential on the course of measurement is another technique to understand the formation mechanism of nanoparticles. It was encountered that the large zetapotential of the like (negative) charges enabled to minimize the particles agglomeration due to

X-ray powder diffraction (XRD) analysis was used to identify the crystalline phase of the NPs due to its capability to provide rapid, non-destructive analysis of multi-component mixtures, allowing quick and accurate analysis of phase, crystallinity, lattice parameters, expandition tensors and bulk modules, and aperiodical arranged clusters. Based on the peak broadening, the cyrstallite size can also be calculated using Scherrer equation. [50] XRD characterization is widely used in various fields as metallurgy, mineralogy, forensic science, archeology, condensed matter physics, and the biological and pharmaceutical sciences. *Fig.* 

**1.3 Nanostructural characterization of engineered nanomaterials** 

morphology and size distribution of the synthesized NPs.

**1.3.1 Surface energy study of engineered nanomaterials** 

**1.3.2 Crystalline phase study of engineered nanomaterials** 

*3* display the working principle and major information obtained from XRD.

altered gene expression. [43]

use for cancer therapy. [45]

electrostatic repulsion.

## **1.3.3 Fine-structure study of engineered nanomaterials**

In this study, the morphology and crystalline structure of the engineered nanomaterials were characterized using scanning and transmission electron microscopy (SEM and TEM). [51] Both techniqes are based on the use of a microscope that uses high energy electrons to form an image. Due to their advantages of higher magnification, larger depth of focus, greater resolution, and ease of sample observation, both facilities have been employed widely to determine the crystalline phase, defects, and texture of materials. [52] High resolution field emission TEM was employed to achieve high spatial resolution, high contrast, and unsurpassed versatility. Particularly, the Tecnai F20 G2 TEM used in this study, which includes a Schottky field emission source, provides ultra-high brightness, low energy spread and very small probe sizes. The Tecnai F20 G2 used in this study has been designed and preconfigured specifically to meet the strict requirements of nanomaterials. This TEM is also equipped with a robust high-brightness field emission gun, allowing for a wide range of applications, from morphological analysis to defect characterisation. [53]

## **1.3.4 Elemental composition study of engineered nanomaterials**

As complementary techniques, energy dispersive spectroscopy (EDS, equiped with TEM) and X-ray photoelectron spectroscopy were used to accurately determine the elemental composition. [54] The EDS is normally used as a semi-quantitative analysis that allows for determination of the amount and identity of the different elements. The EDS can stimulate

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 41

that Ti was well-indexed with the standard Ti 2p binding energies and their difference (2p3/2

The most important driving force behind many changes in the field of medical research is the advent of nanotechnology. Progress in nanotechnology is not only aiming at miniaturization but also at systems with increased complexity. This is not just a matter of geometrical structurization but also a matter of specific functionalities that are positioned at discrete locations and in defined distances. Whereas many NMs exhibit localization to diseased tissues via intrinsic targeting, the addition of targeting ligands, such as antibodies, peptides, aptamer, and small molecules, facilitates far more sensitive cancer detection. Nanoparticles with certain specific surface characteristics such as charge and hydrophobicity along with enhanced permeability and retention effect (EPR) have shown higher bioavailability at the target site. EPR is a result of disorganized angiogenesis leading to production of "leaky" blood vessels. EPR effect and lack of effective lymphatic drainage in the tumor tissue have improved the chances of nanoparticle imaging agents with sizes 10-

Use of nanoparticles in imaging for cancer broadly encompasses two wide areas: (1) detection of certain protein or cancer cells using nanoparticles and, (2) formulation of nanoimaging agents to improve the specificity and to provide high-contrast imaging. Capturing of circulating tumor cells has great potential in cancer diagnosis and therapy. It is very challenging because of the fact that on an average there may be only 1-2 cancer cells per milliliter of blood. Nanotechnology devices based on molecular biomarkers have shown great promise in improving the yield of cancer cell captured. [60] For example, to improve the detection sensitivity and specificity, folate conjugated gold-plated carbon nanotubes were

= 454.1 eV, 2p1/2 = 460.3 eV, = 6.2 eV). [58]

Fig. 5. Demonstration of how XPs works and information obtained.

**1.4 Biomedical application of engineered nanomaterials** 

100 nm to be retained in tumor. [59]

used as contrast agent for photoacoustic imaging. [61]

the emission of characteristic X-rays from a specimen, herein, a high energy beam of charged particles (electrons, protons) is then produced and focused into the sample. An atom within the sample contains ground state electrons in discrete energy levels or electron shells bound to the nucleus. [55] The incident beam form EDS may excite an electron in an inner shell, ejecting it from the shell and creating an electron hole. An electron from an outer, higher-energy shell then fills the hole. The difference in energy may be released in the form of an X-ray. The number and energy of the X-rays emitted from a specimen can be measured by an energy dispersive spectrometer. As the energy of the X-rays is characteristic of the difference in energy between the two shells, and of the atomic structure of the element from which they were emitted, this allows the elemental composition of the specimen to be measured. The principle of EDS is also shown in *Fig. 4* (see § 1.3.3 *Fine-structure study of engineered nanomaterials*).

Fig. 4. Schematic demonstrates the working principle of TEM (SEM included).

XPS is a surface chemical analysis technique (*Fig. 5*), which can irradiate a material with a beam of aluminum (Al) or magnesium (Mg) X-rays. [56] Measurement of kinetic energy (KE) and binding energy (BE) can be completed by determining the number of electrons that escape from the top within 1 to 10 nm. Both EDS and XPS require ultra-high vacuum (UHV) conditions to provide uniformity of elemental composition across the top of the surface. [57] Additionally, XPS can also provide uniformity of elemental composition as a function of depth by ion beam ablation and by tilting the sample, empirical formula of pure materials, elements that contaminate a surface, and chemical or electronic state of each element in the surface. An analysis of titanium (Ti) is selected for demonstration. Based on the binding energy, the element can be determined; such as Ti can be identified via measurement of its binding energies of Ti 2p3/2 and Ti 2p1/2 electron configurations located at 456.3 electronvolt (eV, ~1.602×10−19 C) and 462.2 eV, respectively. In addition, the difference in binding energy can be used for indexing, since the difference in binding energy for the peak splitting of Ti 2p3/2 and Ti 2p1/2 was calculated to be 5.9 eV. From this analysis, it can be concluded that Ti was well-indexed with the standard Ti 2p binding energies and their difference (2p3/2 = 454.1 eV, 2p1/2 = 460.3 eV, = 6.2 eV). [58]

Fig. 5. Demonstration of how XPs works and information obtained.

## **1.4 Biomedical application of engineered nanomaterials**

40 Green Chemistry – Environmentally Benign Approaches

the emission of characteristic X-rays from a specimen, herein, a high energy beam of charged particles (electrons, protons) is then produced and focused into the sample. An atom within the sample contains ground state electrons in discrete energy levels or electron shells bound to the nucleus. [55] The incident beam form EDS may excite an electron in an inner shell, ejecting it from the shell and creating an electron hole. An electron from an outer, higher-energy shell then fills the hole. The difference in energy may be released in the form of an X-ray. The number and energy of the X-rays emitted from a specimen can be measured by an energy dispersive spectrometer. As the energy of the X-rays is characteristic of the difference in energy between the two shells, and of the atomic structure of the element from which they were emitted, this allows the elemental composition of the specimen to be measured. The principle of EDS is also shown in *Fig. 4* (see § 1.3.3 *Fine-structure study of* 

Fig. 4. Schematic demonstrates the working principle of TEM (SEM included).

XPS is a surface chemical analysis technique (*Fig. 5*), which can irradiate a material with a beam of aluminum (Al) or magnesium (Mg) X-rays. [56] Measurement of kinetic energy (KE) and binding energy (BE) can be completed by determining the number of electrons that escape from the top within 1 to 10 nm. Both EDS and XPS require ultra-high vacuum (UHV) conditions to provide uniformity of elemental composition across the top of the surface. [57] Additionally, XPS can also provide uniformity of elemental composition as a function of depth by ion beam ablation and by tilting the sample, empirical formula of pure materials, elements that contaminate a surface, and chemical or electronic state of each element in the surface. An analysis of titanium (Ti) is selected for demonstration. Based on the binding energy, the element can be determined; such as Ti can be identified via measurement of its binding energies of Ti 2p3/2 and Ti 2p1/2 electron configurations located at 456.3 electronvolt (eV, ~1.602×10−19 C) and 462.2 eV, respectively. In addition, the difference in binding energy can be used for indexing, since the difference in binding energy for the peak splitting of Ti 2p3/2 and Ti 2p1/2 was calculated to be 5.9 eV. From this analysis, it can be concluded

*engineered nanomaterials*).

The most important driving force behind many changes in the field of medical research is the advent of nanotechnology. Progress in nanotechnology is not only aiming at miniaturization but also at systems with increased complexity. This is not just a matter of geometrical structurization but also a matter of specific functionalities that are positioned at discrete locations and in defined distances. Whereas many NMs exhibit localization to diseased tissues via intrinsic targeting, the addition of targeting ligands, such as antibodies, peptides, aptamer, and small molecules, facilitates far more sensitive cancer detection. Nanoparticles with certain specific surface characteristics such as charge and hydrophobicity along with enhanced permeability and retention effect (EPR) have shown higher bioavailability at the target site. EPR is a result of disorganized angiogenesis leading to production of "leaky" blood vessels. EPR effect and lack of effective lymphatic drainage in the tumor tissue have improved the chances of nanoparticle imaging agents with sizes 10- 100 nm to be retained in tumor. [59]

Use of nanoparticles in imaging for cancer broadly encompasses two wide areas: (1) detection of certain protein or cancer cells using nanoparticles and, (2) formulation of nanoimaging agents to improve the specificity and to provide high-contrast imaging. Capturing of circulating tumor cells has great potential in cancer diagnosis and therapy. It is very challenging because of the fact that on an average there may be only 1-2 cancer cells per milliliter of blood. Nanotechnology devices based on molecular biomarkers have shown great promise in improving the yield of cancer cell captured. [60] For example, to improve the detection sensitivity and specificity, folate conjugated gold-plated carbon nanotubes were used as contrast agent for photoacoustic imaging. [61]

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 43

One major difficulty in the treatment of cancer is that the tumor cannot often be detected early enough in patients. Early stage tumors usually have favorable prognosis, while many larger, more developed tumors are frequently refractory to current anti-cancer therapies. Another major problem of many current therapies is the lack of adequate selectivity for cancer cells. This inability to adequately distinguish between cancer and normal, healthy cells, leads to toxic effects on the normal cell population and, therefore, detrimental side effects on the cancer patient. The use of NPs in anti-cancer therapies may improve tumor

Different types of metal nanoparticles can be modified to expose molecules that confer them selective binding to specific molecules on the surface of cancer cells, providing probes for the detection and monitoring of cancer cells in tissues (*Fig. 6*). Au NPs in particular have long been utilized in the specific detection of molecules at the surface or inside cells. [74] Other nanomaterials with different chemical reactivity characteristics, including reactivity towards specific functional groups or surface oxidation properties, could provide certain advantages for performing modifications that confer targeting properties to the NPs. The changes in surface plasmon resonance (SPR) properties of Au and Ag NPs caused by interactions with molecules in the surrounding environment can be applied in sensing

Detectors based on such sensors can be very useful for the early detection of cancer cells (*Fig. 7*). The use of Au NPs is also being explored for the early detection of tumors by other techniques such as X-ray scatter imaging . [75] In this research, hepatocellular carcinoma cells containing Au NPs coated with two layers of charged polymers showed enhanced X-ray scattering over cells containing no gold. Hepatocellular carcinoma is the most common cancer that affects the liver and has an estimated 5-year survival rate of only 10%, since current detection methods can only spot the tumors when they have grown to about 5 centimeters in diameter. The imaging technique utilized by this research group (spatial harmonic imaging, SHI) in combination with targeting of the Au NPs to hepatocellular carcinoma cells via attachment to a specific antibody (FB50) has the potential to detect tumors of only a few millimeters in diameter, which would very significantly improve the

therapies in these two fronts.

Fig. 6. Detection of cancer cells using NPs.

biomolecules on the surface of tumor cells.

prognosis of this cancer type.

Many nanoparticle based devices were investigated for their potential applications for imaging in tumor therapy. Most of these were investigated at preclinical stage with few reaching clinical trials. The nanoparticles investigated are either monofunctional or multifunctional. Quantum dots (QDs), which are colloidal fluorescent semiconductor nanocrystals, have sizes ranging from 2-10 nm were investigated in visualization of colon cancer using fiber optics. [62] Iron oxide Nanoparticles conjugated with herceptin as targeting ligand were investigated for detection of small tumors of breast cancer using magnetic resonance imaging (MRI). [63] Dendrimers which are highly branched synthetic polymers were conjugated with prostate specific antigen and were used for imaging in prostate cancer. [64]

Multifunctional nanoparticles are the nanoparticles which combine various functionalities to improve specificity to cancer cells at a single component. Silica based nanoparticles are considered to be promising candidates for development of multifunctional nanoparticles because of their ability to host various materials such as fluorescent dyes, metal ions and drugs. [65] Supramagnetic iron oxide nanoparticles coated with silica were conjugated with fluorescein isothiocyanate to label human bone marrow mesenchymal stem cells. [66]

The most recent nanoparticles also termed as third-generation nanoparticles involve a disease-inspired approach of the "nanocell" which are nanoparticle constructs that comprise a polymeric nanoparticle core enclosed in a lipid-based nanoparticle. For example, an antiangiogenic agent (combretastatin) will be trapped in the lipid envelope and polymeric nanoparticle core will be loaded with a conventional chemotherapeutic agent like doxorubicin. This nanocell when accumulated in tumor by EPR effect will effectively disrupt the tumor vascular growth by releasing the anti-angiogenic agent followed by release of cytotoxic agent for effective tumor inhibition. [67]

## **1.4.1 Mechanistic study of cancer theragnostics**

Relevant biomedical applications of new nanomaterials are cancer diagnosis and treatment. Nanotechnology offers opportunities to enhance our understanding of the mechanisms of cancer, such as by detecting the generation and distribution of cancer cells in tissues, which can lead to improved diagnosis of this dreadful disease. Furthermore, by harnessing and targeting the toxic properties of nanoparticles, therapeutic agents that are more effective for treating cancer will be developed through molecular targeting.

Cancer encompassess a set of complex and diverse diseases that arise generally when normal cells are transformed into tumorogenic cells, which grow in an uncontrolled fashion to the detriment of the surrounding tissues and eventually the organism. Several factors can play a role in the generation and aggressiveness of the tumor cells, such as accelerated growth, lack of need for growth factors or absence of response to growth inhibitors, unresponsiveness to factors that trigger cell death by apoptosis, enhanced migration properties and ability to invade blood circulation, evasion of the immune response, etc. [68] Mechanistically, the transformed phenotype is derived from the progressive accumulation of genetic mutations, including base changes, nucleotide additions and deletions, insertions, duplications, and chromosomal translocations. [69] Many types of genes have been associated with the development of cancer, such as oncogenes, tumor suppressor genes, apoptosis regulatory genes, cell cycle genes, invasiveness and metastasis related genes, etc. [68-73]

One major difficulty in the treatment of cancer is that the tumor cannot often be detected early enough in patients. Early stage tumors usually have favorable prognosis, while many larger, more developed tumors are frequently refractory to current anti-cancer therapies. Another major problem of many current therapies is the lack of adequate selectivity for cancer cells. This inability to adequately distinguish between cancer and normal, healthy cells, leads to toxic effects on the normal cell population and, therefore, detrimental side effects on the cancer patient. The use of NPs in anti-cancer therapies may improve tumor therapies in these two fronts.

Fig. 6. Detection of cancer cells using NPs.

42 Green Chemistry – Environmentally Benign Approaches

Many nanoparticle based devices were investigated for their potential applications for imaging in tumor therapy. Most of these were investigated at preclinical stage with few reaching clinical trials. The nanoparticles investigated are either monofunctional or multifunctional. Quantum dots (QDs), which are colloidal fluorescent semiconductor nanocrystals, have sizes ranging from 2-10 nm were investigated in visualization of colon cancer using fiber optics. [62] Iron oxide Nanoparticles conjugated with herceptin as targeting ligand were investigated for detection of small tumors of breast cancer using magnetic resonance imaging (MRI). [63] Dendrimers which are highly branched synthetic polymers were conjugated with prostate specific antigen and were used for imaging in prostate

Multifunctional nanoparticles are the nanoparticles which combine various functionalities to improve specificity to cancer cells at a single component. Silica based nanoparticles are considered to be promising candidates for development of multifunctional nanoparticles because of their ability to host various materials such as fluorescent dyes, metal ions and drugs. [65] Supramagnetic iron oxide nanoparticles coated with silica were conjugated with

The most recent nanoparticles also termed as third-generation nanoparticles involve a disease-inspired approach of the "nanocell" which are nanoparticle constructs that comprise a polymeric nanoparticle core enclosed in a lipid-based nanoparticle. For example, an antiangiogenic agent (combretastatin) will be trapped in the lipid envelope and polymeric nanoparticle core will be loaded with a conventional chemotherapeutic agent like doxorubicin. This nanocell when accumulated in tumor by EPR effect will effectively disrupt the tumor vascular growth by releasing the anti-angiogenic agent followed by

Relevant biomedical applications of new nanomaterials are cancer diagnosis and treatment. Nanotechnology offers opportunities to enhance our understanding of the mechanisms of cancer, such as by detecting the generation and distribution of cancer cells in tissues, which can lead to improved diagnosis of this dreadful disease. Furthermore, by harnessing and targeting the toxic properties of nanoparticles, therapeutic agents that are more effective for

Cancer encompassess a set of complex and diverse diseases that arise generally when normal cells are transformed into tumorogenic cells, which grow in an uncontrolled fashion to the detriment of the surrounding tissues and eventually the organism. Several factors can play a role in the generation and aggressiveness of the tumor cells, such as accelerated growth, lack of need for growth factors or absence of response to growth inhibitors, unresponsiveness to factors that trigger cell death by apoptosis, enhanced migration properties and ability to invade blood circulation, evasion of the immune response, etc. [68] Mechanistically, the transformed phenotype is derived from the progressive accumulation of genetic mutations, including base changes, nucleotide additions and deletions, insertions, duplications, and chromosomal translocations. [69] Many types of genes have been associated with the development of cancer, such as oncogenes, tumor suppressor genes, apoptosis

regulatory genes, cell cycle genes, invasiveness and metastasis related genes, etc. [68-73]

fluorescein isothiocyanate to label human bone marrow mesenchymal stem cells. [66]

release of cytotoxic agent for effective tumor inhibition. [67]

treating cancer will be developed through molecular targeting.

**1.4.1 Mechanistic study of cancer theragnostics** 

cancer. [64]

Different types of metal nanoparticles can be modified to expose molecules that confer them selective binding to specific molecules on the surface of cancer cells, providing probes for the detection and monitoring of cancer cells in tissues (*Fig. 6*). Au NPs in particular have long been utilized in the specific detection of molecules at the surface or inside cells. [74] Other nanomaterials with different chemical reactivity characteristics, including reactivity towards specific functional groups or surface oxidation properties, could provide certain advantages for performing modifications that confer targeting properties to the NPs. The changes in surface plasmon resonance (SPR) properties of Au and Ag NPs caused by interactions with molecules in the surrounding environment can be applied in sensing biomolecules on the surface of tumor cells.

Detectors based on such sensors can be very useful for the early detection of cancer cells (*Fig. 7*). The use of Au NPs is also being explored for the early detection of tumors by other techniques such as X-ray scatter imaging . [75] In this research, hepatocellular carcinoma cells containing Au NPs coated with two layers of charged polymers showed enhanced X-ray scattering over cells containing no gold. Hepatocellular carcinoma is the most common cancer that affects the liver and has an estimated 5-year survival rate of only 10%, since current detection methods can only spot the tumors when they have grown to about 5 centimeters in diameter. The imaging technique utilized by this research group (spatial harmonic imaging, SHI) in combination with targeting of the Au NPs to hepatocellular carcinoma cells via attachment to a specific antibody (FB50) has the potential to detect tumors of only a few millimeters in diameter, which would very significantly improve the prognosis of this cancer type.

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 45

vendors. Silver nitrate (AgNO3) and gold (III) chloride trihydrate were purchased from Sigma–Aldrich (St Louis, MO), L-ascorbic acid and other reducing agens from Fisher (Thermo scientific, Pittsburgh, PA), Arabic gum from M.P. Biomedicals, Morgan Irvine, CA), and used without modification. The experiments are carried out for the construction of NPs derived from a bottom-up colloidal method. Non-toxic chemicals and two strong reducants are used in various formulations of NPs, allowing for mitigation of pollution. The structural characterization is by electron microscopy and X-ray diffraction. We also investigated the toxicity of some of the synthesized NPs. Importantly, as previously indicated in the introduction, this approach can also be applied to the toxicity and cancer diagnosis, along

The experimental workflow to engineer nanoparticles is shown in *Fig. 2* (see §1.1.3 *Synthesis of nanomaterials*). Briefly, the nanometal was created by dissolving different concentrations of silver nitrate (AgNO3) and/or gold (III) chloride trihydrate (HAuCl4·3H2O) in distilled water. Concentration was controlled at 0.005-1.0 M according to application. This aqueous solution was mixed continuously with a magnetic stirrer/mechanical agitation for 30 min. *Arabica* gum (AG, as a surfactant) was added (2 % by mass) to the above solution to improve the dispersion of NPs and to control the particle size. Reducing agents (four selected ones) were then incrementally added into the solution. The molar ratio of metallic cations to reducing agent was controlled at 1:2 to 1:8 and to produce 16 formulations. The colloids were directly used for zetapotential, cytotoxicity and hemocompatibility analyses. The precursor sols were heated between 60 and 80 C for 2 hrs and filtered and rinsed with ethanol/deionized (DI) water to produce powders used for XRD and and TEM analysis.

In tandem to §1.3 (*Nanostructural Characterization of Engineered Nanomaterials*), this section briefly discusses the technical variables or the characterization methods. A ZetaPALS™ (Brookhaven Instruments Corporation, NY) was used to measure the particle size distribution and zeta-potential to evaluate the stability of NPs. The ZetaPALS operating conditions are as follows: An electric field (2 V•cm) was applied to obtain good results. This electric field was used and an alternating current (AC) waveform with the frequency control of 2-20 Hz was applied; measurement duration was kept at 30 to 60 s; laser input was 5 V and laser output power was 35 mW; and an automatic selection of field strength was used. The Ultima III X-ray powder Diffraction (XRD) was employed to determine the crystalline phase and average crystallite size of the NPs. The XRD patterns were collected using a Rigaku multiflex diffractometer, equipped with a copper (Cu) target. The scanning range varied between 30 and 80 degrees at a scanning rate of 2 /min. The phase structure was then identified through the Jade 7.0 database. A Tecnai F20-G2 TEM (FEI Company, Tecnai F20-G2 Hillsboro, OR), equipped with ED and EDS techniques were also employed to obtain nanostructural information, crystalline phase and elemental composition of nanometals. In the EDS analysis, the peak intensities were converted to percent weight and then to final molar ratio via the DAX ZAF Quantification tool. The spectra were acquired to determine elemental composition. The HRTEM images were taken at a direct magnification of 600 thousand magnitudes with the point resolution of 0.2 nm. An Axis ULTRA XPS (Kratos

the "theragnostics mechanism" described in the following sections.

**2.1 Fabrication of nanometals** 

**2.2 Structural characterization** 

Fig. 7. Schematic design for cancer imaging via optically/electrochemically active gold-NPs.

As indicated, the use of nanomaterials is being investigated not only for the early detection of tumors, but also for the specific destruction of cancerous cells. Ag NPs disrupt the function of proteins and other macromolecules on the surface of bacterial and eukaryotic cells, which can lead to permanent damage and death of the cells. [76-77] Other metallic NPs have different toxicity effects on cells. TiO2 NPs for example do generate reactive oxygen species (ROS) upon absorption of ultraviolet (UV) light. Additionally, specific modifications of the metallic NPs can attach other toxic molecules to the nanomaterial. Combining the toxicity effects of novel nanomaterials with specific targeting to tumor cells can lead to the development of highly specialized therapeutic NPs that can be introduced into the body of cancer patients, where they could detect and destroy cancer cells at early stages, preventing the growth of tumors.

Molecular targeting of NPs was utilized in the generation of TiO2 NPs coupled with an antibody, which targeted the nanomaterial to glioblastoma multiforme cancer cells (glioblastoma multiforme is a type of brain cancer). [78] The antibody used recognizes the interleukin-13 receptor 2 protein chain (IL13R2), which has been shown to be almost exclusively overexpressed on the plasma membrane of certain brain tumors, including glioblastoma multiforme. [78-80] The nanomaterial accumulated inside tumor cells and upon exposure to UV light, the NPs led to the formation of ROS and killed the cancerous cells, while they did not show citotoxicity toward normal brain cells.

An example of NPs application for drug delivery is the formation of "Trojan horse" like hybrid NPs. Glycan NPs were synthesized using cyclodextrins as stabilizing agent (polymeric backbone). These glycan NPs were used as envelope for the encapsulation of camptothecin. The formulation of the NPs allowed its internalization on tumor cells; inside the tumor cells, the coating is desintegrated, releasing the toxic molecule that destoys specifically the tumor cell without harming healthy cells. [81] This glycan NPs containing camptothecin, designated as IT-101, have been shown to eliminate or significantly reduce tumors in several mice models of human cancer, and are currently under investigation in clinical trials for the treatment of cancer patients. Delivery of these glycan NPs to tumors is based on the fact that newly formed tumor blood vessels are leaky and let particles as large as 400 to 700 nm into the cancerous mass.

## **2. Experimental approach**

Here, we describe design, synthesis and characterization of nanoscaled metals and their nanostructures. All chemicals used are of analytical grade and were obtained from various vendors. Silver nitrate (AgNO3) and gold (III) chloride trihydrate were purchased from Sigma–Aldrich (St Louis, MO), L-ascorbic acid and other reducing agens from Fisher (Thermo scientific, Pittsburgh, PA), Arabic gum from M.P. Biomedicals, Morgan Irvine, CA), and used without modification. The experiments are carried out for the construction of NPs derived from a bottom-up colloidal method. Non-toxic chemicals and two strong reducants are used in various formulations of NPs, allowing for mitigation of pollution. The structural characterization is by electron microscopy and X-ray diffraction. We also investigated the toxicity of some of the synthesized NPs. Importantly, as previously indicated in the introduction, this approach can also be applied to the toxicity and cancer diagnosis, along the "theragnostics mechanism" described in the following sections.

#### **2.1 Fabrication of nanometals**

44 Green Chemistry – Environmentally Benign Approaches

Fig. 7. Schematic design for cancer imaging via optically/electrochemically active gold-NPs.

As indicated, the use of nanomaterials is being investigated not only for the early detection of tumors, but also for the specific destruction of cancerous cells. Ag NPs disrupt the function of proteins and other macromolecules on the surface of bacterial and eukaryotic cells, which can lead to permanent damage and death of the cells. [76-77] Other metallic NPs have different toxicity effects on cells. TiO2 NPs for example do generate reactive oxygen species (ROS) upon absorption of ultraviolet (UV) light. Additionally, specific modifications of the metallic NPs can attach other toxic molecules to the nanomaterial. Combining the toxicity effects of novel nanomaterials with specific targeting to tumor cells can lead to the development of highly specialized therapeutic NPs that can be introduced into the body of cancer patients, where they could detect and destroy cancer cells at early stages, preventing the growth of tumors.

Molecular targeting of NPs was utilized in the generation of TiO2 NPs coupled with an antibody, which targeted the nanomaterial to glioblastoma multiforme cancer cells (glioblastoma multiforme is a type of brain cancer). [78] The antibody used recognizes the interleukin-13 receptor 2 protein chain (IL13R2), which has been shown to be almost exclusively overexpressed on the plasma membrane of certain brain tumors, including glioblastoma multiforme. [78-80] The nanomaterial accumulated inside tumor cells and upon exposure to UV light, the NPs led to the formation of ROS and killed the cancerous cells,

An example of NPs application for drug delivery is the formation of "Trojan horse" like hybrid NPs. Glycan NPs were synthesized using cyclodextrins as stabilizing agent (polymeric backbone). These glycan NPs were used as envelope for the encapsulation of camptothecin. The formulation of the NPs allowed its internalization on tumor cells; inside the tumor cells, the coating is desintegrated, releasing the toxic molecule that destoys specifically the tumor cell without harming healthy cells. [81] This glycan NPs containing camptothecin, designated as IT-101, have been shown to eliminate or significantly reduce tumors in several mice models of human cancer, and are currently under investigation in clinical trials for the treatment of cancer patients. Delivery of these glycan NPs to tumors is based on the fact that newly formed tumor blood vessels are leaky and let particles as large

Here, we describe design, synthesis and characterization of nanoscaled metals and their nanostructures. All chemicals used are of analytical grade and were obtained from various

while they did not show citotoxicity toward normal brain cells.

as 400 to 700 nm into the cancerous mass.

**2. Experimental approach** 

The experimental workflow to engineer nanoparticles is shown in *Fig. 2* (see §1.1.3 *Synthesis of nanomaterials*). Briefly, the nanometal was created by dissolving different concentrations of silver nitrate (AgNO3) and/or gold (III) chloride trihydrate (HAuCl4·3H2O) in distilled water. Concentration was controlled at 0.005-1.0 M according to application. This aqueous solution was mixed continuously with a magnetic stirrer/mechanical agitation for 30 min. *Arabica* gum (AG, as a surfactant) was added (2 % by mass) to the above solution to improve the dispersion of NPs and to control the particle size. Reducing agents (four selected ones) were then incrementally added into the solution. The molar ratio of metallic cations to reducing agent was controlled at 1:2 to 1:8 and to produce 16 formulations. The colloids were directly used for zetapotential, cytotoxicity and hemocompatibility analyses. The precursor sols were heated between 60 and 80 C for 2 hrs and filtered and rinsed with ethanol/deionized (DI) water to produce powders used for XRD and and TEM analysis.

#### **2.2 Structural characterization**

In tandem to §1.3 (*Nanostructural Characterization of Engineered Nanomaterials*), this section briefly discusses the technical variables or the characterization methods. A ZetaPALS™ (Brookhaven Instruments Corporation, NY) was used to measure the particle size distribution and zeta-potential to evaluate the stability of NPs. The ZetaPALS operating conditions are as follows: An electric field (2 V•cm) was applied to obtain good results. This electric field was used and an alternating current (AC) waveform with the frequency control of 2-20 Hz was applied; measurement duration was kept at 30 to 60 s; laser input was 5 V and laser output power was 35 mW; and an automatic selection of field strength was used. The Ultima III X-ray powder Diffraction (XRD) was employed to determine the crystalline phase and average crystallite size of the NPs. The XRD patterns were collected using a Rigaku multiflex diffractometer, equipped with a copper (Cu) target. The scanning range varied between 30 and 80 degrees at a scanning rate of 2 /min. The phase structure was then identified through the Jade 7.0 database. A Tecnai F20-G2 TEM (FEI Company, Tecnai F20-G2 Hillsboro, OR), equipped with ED and EDS techniques were also employed to obtain nanostructural information, crystalline phase and elemental composition of nanometals. In the EDS analysis, the peak intensities were converted to percent weight and then to final molar ratio via the DAX ZAF Quantification tool. The spectra were acquired to determine elemental composition. The HRTEM images were taken at a direct magnification of 600 thousand magnitudes with the point resolution of 0.2 nm. An Axis ULTRA XPS (Kratos

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 47

as the control samples. The concentration of the reducing agent was varied from 1:1, 1:2, 1:4 and to 1:8 molar ratios with respect to metallic ion. Table 1 tabulated the 44 formulations of the nanopartciles via bottom-up "green" colloidal chemistry method. In this study, the reduction of noble metal cation occurs spontaneously. The reactions are shown as follows:

(aq) + 3 e- Au (s) + H+ (aq) + 4 Cl-

 C6H8O6 (aq) + 2 e- C6H6O6 (aq) + 2H+(aq) E = +0.06V (3) According to these three half reaction standard reduction potentials, the overall reaction is determined to have a potential of 0.74 and 0.93 V, respectively. This indicates that the redox reaction between Ag+ and C6H8O6, and HAuCl4 and C6H8O6 occurs spontaneously since they are favored, thermodynamically. The released proton H+ (from both half reactions, the

In this research, the dispersing agent, Arabic gum (AG), was used as a size directing agent in the synthesis of the nanometals and to prevent the agglomeration of the fine particles (< 10 nm). Because the complex between surfactant and metal ions can be formed, the growth of the central particle is prevented and terminates at a size in the nanoscale regime (1-100 nm). In short, AG regulates stability of nanoparticles. The synthesis of noble metal nanocomposite via reduction of metal ions in aqueous solutions of AG is based on the

20 mL

20 mL

20 mL

20 mL

20 mL

20 mL

Table 1. Potential set of values of volume of Mn+,and reducing agents at designed molar

· · ·

AuCl4

Cross Product of conc. and molar ratio

Concentration of reducing agents and Ag+ or

Au3+

(mol/L)


redox process) was responsible for acidity and resulting decrease in pH.

formation of a stable metal particle-macromolecular complex.

0.005 10 mL \* 10 mL\*\*

0.01 10 mL 10 mL

0.02 10 mL 10 mL

0.03 10 mL 10 mL

0.5 10 mL 10 mL

1.0 10 mL 10 mL

· · ·

Note: \* represent the volume of metallic cation solution and \*\* the volume of reducants.

ratio when temperature is maintained at 60 C and agitation at 1000 rpm.

· · · Ag+ (aq) + e- Ag (s) E = +0.80V (1)

Molar ratio of Mn+ : reducing agent

40 mL

40 mL

40 mL

40 mL

40 mL

40 mL

· ·

10 mL

10 mL

10 mL

10 mL

10 mL

10 mL

10 mL

10 mL

10 mL

10 mL

10 mL

10 mL

80 mL

80 mL

80 mL

80 mL

80 mL

80 mL

· ·

1:1 1:2 1:4 1:8

10 mL

10 mL

10 mL

10 mL

10 mL

10 mL

(aq) E = +0.99V (2)

Analytical Inc, NY) was employed to identify the elemental composition of the products. The operating specifications were: high vacuum was controlled under 10-8 Torr; anode mode was aluminum (Al, Kα) monochromatic energy source with the power of 10 mA by 12 kV; the lens was used in hybrid mode; resolution of the individual element analysis was of pass energy of 40 eV and 160 eV for survey. [88]

## **2.3 Evaluation of** *in vitro* **cytotoxic activity**

The cytotoxicity of Au and Ag NPs was performed in two cell lines: ovarian adenocarcinoma cell line (NCI/ADR-RES), and normal ovarian cell line (NCI/CHO). A total of 2×104 cells in 200 µL of medium per well were placed in a 96-well plate. After incubation overnight, the medium was replaced with media containing nanometals at different concentrations (0.1, 0.5, 1, 10, and 100 µM) in separate wells. After 24 hrs of incubation, the medium was removed and the cells were washed with ice-cold phosphate-buffered saline (PBS) three times to remove NPs. A volume of 50 µL of 3-[4, 5-dimethylthiazol-2-yl]-2,5, diphenyl tetrazolium bromide (MTT) at a concentration of 5 mg/mL was then added to each well. Following incubation for 4 hrs, formazan crystals formed were dissolved in 150 µL of dimethylsulfoxide and absorbance was measured at 530 nm using a NOVOstar plate reader (BMG lab technologies, Cary, NC, USA). The percentage viability was calculated by comparing absorbance of treated cells versus untreated control cells which were assigned 100 % viability.

## **2.4 Evaluation of hemocompatibility**

To evaluate hemocompatibility, human red blood cells (HBCs), which composed of reduced leukocytes with adenine-saline were used. HBCs were obtained from coastal bend blood center, Corpus Christi, Texas as a kind gift from. The HBCs were separated from plasma by centrifugation at 4,000 rpm for 5 min at 4 °C. HBCs were washed three times with physiological saline solution and re-suspended in saline to obtain HBCs suspension at 2% (v/v) hematocrit. The so-prepared HBC-suspension was used within 24 hrs upon preparation.

## **3. Results and discussion**

The optimal fabrication variables of NPs will be discussed first (described in § 3.1), followed by electrokinetic properties analysis and crystalline phase strucure of NPs (described in § 3.2-3.3). The *in-vitro* toxicicity and hemocompatibility will be discussed next (described in § 3.5) and conclusion on bioapplication of nanomaterials to close the chapter (§ 4.0).

## **3.1 Fabrication optimization of engineered nanomaterials**

The generation process of nanoparticles through facile chemical approach is as follows: (1) A complex between a metal ion and a ligand, that is the functional group of a protecting polymer, is formed in an aqueous solution; (2) The metal ion is reduced to a metal atom with a reducing agent; and (3) The metal atoms aggregate and grow into nanoparticles. The effect of the nature and concentration of the reducing agent was evaluated. As "green" reducing agents, ascorbic acid and sodium citrate were employed. As strong reducing agents, sodium borohydride (NaBH4) and dimethylaminoborane (DMAB) were also tested

Analytical Inc, NY) was employed to identify the elemental composition of the products. The operating specifications were: high vacuum was controlled under 10-8 Torr; anode mode was aluminum (Al, Kα) monochromatic energy source with the power of 10 mA by 12 kV; the lens was used in hybrid mode; resolution of the individual element analysis was of pass

The cytotoxicity of Au and Ag NPs was performed in two cell lines: ovarian adenocarcinoma cell line (NCI/ADR-RES), and normal ovarian cell line (NCI/CHO). A total of 2×104 cells in 200 µL of medium per well were placed in a 96-well plate. After incubation overnight, the medium was replaced with media containing nanometals at different concentrations (0.1, 0.5, 1, 10, and 100 µM) in separate wells. After 24 hrs of incubation, the medium was removed and the cells were washed with ice-cold phosphate-buffered saline (PBS) three times to remove NPs. A volume of 50 µL of 3-[4, 5-dimethylthiazol-2-yl]-2,5, diphenyl tetrazolium bromide (MTT) at a concentration of 5 mg/mL was then added to each well. Following incubation for 4 hrs, formazan crystals formed were dissolved in 150 µL of dimethylsulfoxide and absorbance was measured at 530 nm using a NOVOstar plate reader (BMG lab technologies, Cary, NC, USA). The percentage viability was calculated by comparing absorbance of treated cells versus untreated control cells which were assigned

To evaluate hemocompatibility, human red blood cells (HBCs), which composed of reduced leukocytes with adenine-saline were used. HBCs were obtained from coastal bend blood center, Corpus Christi, Texas as a kind gift from. The HBCs were separated from plasma by centrifugation at 4,000 rpm for 5 min at 4 °C. HBCs were washed three times with physiological saline solution and re-suspended in saline to obtain HBCs suspension at 2% (v/v) hematocrit. The so-prepared HBC-suspension was used within 24 hrs upon

The optimal fabrication variables of NPs will be discussed first (described in § 3.1), followed by electrokinetic properties analysis and crystalline phase strucure of NPs (described in § 3.2-3.3). The *in-vitro* toxicicity and hemocompatibility will be discussed next (described in §

The generation process of nanoparticles through facile chemical approach is as follows: (1) A complex between a metal ion and a ligand, that is the functional group of a protecting polymer, is formed in an aqueous solution; (2) The metal ion is reduced to a metal atom with a reducing agent; and (3) The metal atoms aggregate and grow into nanoparticles. The effect of the nature and concentration of the reducing agent was evaluated. As "green" reducing agents, ascorbic acid and sodium citrate were employed. As strong reducing agents, sodium borohydride (NaBH4) and dimethylaminoborane (DMAB) were also tested

3.5) and conclusion on bioapplication of nanomaterials to close the chapter (§ 4.0).

**3.1 Fabrication optimization of engineered nanomaterials** 

energy of 40 eV and 160 eV for survey. [88]

**2.4 Evaluation of hemocompatibility** 

**3. Results and discussion** 

100 % viability.

preparation.

**2.3 Evaluation of** *in vitro* **cytotoxic activity** 

as the control samples. The concentration of the reducing agent was varied from 1:1, 1:2, 1:4 and to 1:8 molar ratios with respect to metallic ion. Table 1 tabulated the 44 formulations of the nanopartciles via bottom-up "green" colloidal chemistry method. In this study, the reduction of noble metal cation occurs spontaneously. The reactions are shown as follows:

$$\text{Ag} + \text{(aq)} + \text{e}^{\cdot} \rightarrow \text{Ag}^{\circ} \text{ (s)} \qquad\qquad \qquad \text{E}^{\circ} = +0.80 \text{V} \quad \text{(1)}$$

$$\text{AuCl} \cdot \text{(aq)} + \text{3 e} \cdot \text{e} \rightarrow \text{Au}^{\circ} \text{(s)} + \text{H}^{+} \text{(aq)} + 4 \text{ Cl} \cdot \text{(aq)} \qquad \text{E}^{\circ} = +0.99 \text{V} \tag{2}$$

$$\rm C\_6H\_8O\_6 \text{ (aq)} + 2\text{ e} \rightarrow \rm C\_6H\_6O\_6 \text{ (aq)} + 2H^+ \text{(aq)} \qquad E^\circ = +0.06V \tag{3}$$

According to these three half reaction standard reduction potentials, the overall reaction is determined to have a potential of 0.74 and 0.93 V, respectively. This indicates that the redox reaction between Ag+ and C6H8O6, and HAuCl4 and C6H8O6 occurs spontaneously since they are favored, thermodynamically. The released proton H+ (from both half reactions, the redox process) was responsible for acidity and resulting decrease in pH.

In this research, the dispersing agent, Arabic gum (AG), was used as a size directing agent in the synthesis of the nanometals and to prevent the agglomeration of the fine particles (< 10 nm). Because the complex between surfactant and metal ions can be formed, the growth of the central particle is prevented and terminates at a size in the nanoscale regime (1-100 nm). In short, AG regulates stability of nanoparticles. The synthesis of noble metal nanocomposite via reduction of metal ions in aqueous solutions of AG is based on the formation of a stable metal particle-macromolecular complex.


Note: \* represent the volume of metallic cation solution and \*\* the volume of reducants.

Table 1. Potential set of values of volume of Mn+,and reducing agents at designed molar ratio when temperature is maintained at 60 C and agitation at 1000 rpm.

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 49

controlled through incorporation of GA as the dispersing agent, which also aids wetting of the metal salt. The large negative zetapotential would promote repulsion between the nanoparticles, which in turn would minimize particles agglomeration, which is schematically summarised in *Fig. 8e* (see above figure). The contribution of this green synthesis lies in the following: 1) Finding the best fabrication parameters to achieve complete de-aggregation of the nanoscaled catalyst; 2) Identifying non-toxic dispersing agents to prevent aggregation of the nanocatalyst; and 3) Identifying the suitable reducing agent that ensures complete reduction of metal ions and improves the mono-dispersion of

XRD results can provide figure-print characterization of the crystalline phase and the lattice constants. *Fig. 9a* indicates that Ag-NPs display highly crystalline face centered cubic (fcc) space group. The Ag NPs derived from green colloidal chemistry is well indexed with PDF 01-089-3722 (lattice constants, a = 4.0855 Å and = 90°). In addition, the crystallite size was calculated to be averaged at 30.2 nm. According to the full width at half maximum, the crystallite size was calculated using Scherrer equation (also see Fig 3). Similarly, the Au NPs correspond to the standard PDF 04-0784 (*Fig. 9b*: lattice constants, a = 4.078 Å and = 90°). The crystallite size of Au nanoparticles was averaged at 10.4 nm; suggesting both Au and

Fig. 9. XRD patterns for nanometals, including XRD patterns for two selected samples, cyrstallite size distribution and cubic centered phase structure, a: XRD pattern of silver and

gold nanoparticles and b: particle size distribution of silver and gold nanoparticles.

**3.2.2 Crystalline phase analyses of engineered nanomaterials** 

nanocatalyst.

## **3.2 Nanocharacterization and bio-application of engineered nanomaterials**

The surface energies of nanomaterials are described first in §3.2.1. The crystalline structural, morphological and elemental composition characterization are described next from §3.2.2 to -3.2.4. Lastly, the cytotoxic activity and hemocompatibility of nanometals is described in §3.3 and 3.4, respectively.

## **3.2.1 Electrokinetic behavior of metallic colloidal suspension**

The measured zetapotentials (, with ZetaPALS™) of nanosized metal colloidal suspension were averaged at -44.00 mV. The negative sign indicates large repulsive forces between nanoparticles of either silver or gold preventing flocculation and aggregation of particles and the numerical value indicates samples had colloidal stability (*Fig. 8*). results indicate that the colloid of Ag and Au are stable and agglomeration is successfully prevented using GA as the surfactant. The electrokinetic study also confirms that nano-dispersion of Au with size of 10-15 nm (correspondingly, the particle size of NP powders were found by TEM, see *Fig. 10 a & b*) has been achieved. Fluctuations in the measured value of during the experiment were not observed; therefore the measurements were time independent in the present study, which confirms the stability of nanometal colloid.

Fig. 8. Relationship between particle size and zetapotential, a: the zetapotential measurement to determine the surface energy of silver nanoparticles, b: the zetapotential measurement to determine the surface energy of gold nanoparticles, c: redox reaction to obtain nanometal particles, d: repulsive and attractive forces in colloidal suspension, and e: schematic of repulsive or attractive forces on nanoparticles, the magnitude which defines aggregation, or separation.

The main goal during the synthesis phase was to control the particle size and its morphology followed by nanocharacterization of the nanoparticles. These particles were synthesized using a bottom-up colloidal chemistry approach. Ag and Au-NP size was controlled through incorporation of GA as the dispersing agent, which also aids wetting of the metal salt. The large negative zetapotential would promote repulsion between the nanoparticles, which in turn would minimize particles agglomeration, which is schematically summarised in *Fig. 8e* (see above figure). The contribution of this green synthesis lies in the following: 1) Finding the best fabrication parameters to achieve complete de-aggregation of the nanoscaled catalyst; 2) Identifying non-toxic dispersing agents to prevent aggregation of the nanocatalyst; and 3) Identifying the suitable reducing agent that ensures complete reduction of metal ions and improves the mono-dispersion of nanocatalyst.

### **3.2.2 Crystalline phase analyses of engineered nanomaterials**

48 Green Chemistry – Environmentally Benign Approaches

The surface energies of nanomaterials are described first in §3.2.1. The crystalline structural, morphological and elemental composition characterization are described next from §3.2.2 to -3.2.4. Lastly, the cytotoxic activity and hemocompatibility of nanometals is described in §3.3

The measured zetapotentials (, with ZetaPALS™) of nanosized metal colloidal suspension were averaged at -44.00 mV. The negative sign indicates large repulsive forces between nanoparticles of either silver or gold preventing flocculation and aggregation of particles and the numerical value indicates samples had colloidal stability (*Fig. 8*). results indicate that the colloid of Ag and Au are stable and agglomeration is successfully prevented using GA as the surfactant. The electrokinetic study also confirms that nano-dispersion of Au with size of 10-15 nm (correspondingly, the particle size of NP powders were found by TEM, see *Fig. 10 a & b*) has been achieved. Fluctuations in the measured value of during the experiment were not observed; therefore the measurements were time independent in the

**3.2 Nanocharacterization and bio-application of engineered nanomaterials** 

**3.2.1 Electrokinetic behavior of metallic colloidal suspension** 

present study, which confirms the stability of nanometal colloid.

Fig. 8. Relationship between particle size and zetapotential, a: the zetapotential

measurement to determine the surface energy of silver nanoparticles, b: the zetapotential measurement to determine the surface energy of gold nanoparticles, c: redox reaction to obtain nanometal particles, d: repulsive and attractive forces in colloidal suspension, and e: schematic of repulsive or attractive forces on nanoparticles, the magnitude which defines

The main goal during the synthesis phase was to control the particle size and its morphology followed by nanocharacterization of the nanoparticles. These particles were synthesized using a bottom-up colloidal chemistry approach. Ag and Au-NP size was

and 3.4, respectively.

aggregation, or separation.

XRD results can provide figure-print characterization of the crystalline phase and the lattice constants. *Fig. 9a* indicates that Ag-NPs display highly crystalline face centered cubic (fcc) space group. The Ag NPs derived from green colloidal chemistry is well indexed with PDF 01-089-3722 (lattice constants, a = 4.0855 Å and = 90°). In addition, the crystallite size was calculated to be averaged at 30.2 nm. According to the full width at half maximum, the crystallite size was calculated using Scherrer equation (also see Fig 3). Similarly, the Au NPs correspond to the standard PDF 04-0784 (*Fig. 9b*: lattice constants, a = 4.078 Å and = 90°). The crystallite size of Au nanoparticles was averaged at 10.4 nm; suggesting both Au and

Fig. 9. XRD patterns for nanometals, including XRD patterns for two selected samples, cyrstallite size distribution and cubic centered phase structure, a: XRD pattern of silver and gold nanoparticles and b: particle size distribution of silver and gold nanoparticles.

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 51

which occurred at 2.10 KeV and the K peak which occurred at 2.18 KeV, respectively, which is in agreement with Au characteristic peaks. The Au NP appearance is near-spherical with the average particle size from 15 to 20 nm in diameter. The high resolution image also shows the lattice fringe which was distinguished, which also confirms the formation of Au crystals.

Fig. 10. TEM images and elemental analysis of nanoparticles, a: morphology, ring pattern and composition of nanosilver; b: morphology, ring pattern and composition of nanogold.

The XPS results (*Fig. 11*) show that Ag and Au are characterized by an asymmetric line shape with the peak tailing to the higher BE. The BE may be varied due to its extra columbic interaction between the photoemitted electron and ion core. The binding energies for Ag electron configurations of 3d5/2 and 3d3/2 were found to be 366.0 eV and 372.0 eV with the difference of 6.0 eV (Fig. 11a). This measurement was corresponding to the standard Ag 3d binding energies (3d5/2 = 368.3 eV, 3d3/2 = 374.3 eV, = 6.0 eV). Apparently, the 3d

The element Cu peak detected is resulting from Cu grid sample holder.

Ag particle agglomeration was successfully prevented. In both circumstances, the reducing agent, ascorboic acid was selected for demonstration. Other reducing agents provided the same crystalline phase; however, the crystallite size of the engineering particles varies accordingly. It is found that the NaBH4 and DMAB provided essentially the same size, averaged at 12.7 nm for Ag and 4.8 nm for Au nanoparticles, which is also confirmed by TEM measurement. It is important to point out that the lattice occupancy is near ideal (99.0 %), suggesting the Ag and Au nanoparticles possessed highly crystalline structure with less lattice distortion. Both nanoparticles also display high frenquecy of merit (0.2) when using Jade 7.1 database to index with the standard poser diffraction files. The crystalinity index was also determined via taking the ratio of particle size from TEM vs the crystallite size calculated from XRD data. It can be seen that Ag and Au nanoparticles are highly crystalline. Ag showed monoparticles and Au on the other hand polyparticles (see table 2) according to different fabrication methods.


Table 2. Summary of the crystalinity index of Ag and Au nanoparticles (six samples are selected for demonstration)

## **3.2.3 Fine-structure study of engineered nanomaterials**

The morphological and elemental studies through HRTEM technique are shown *(Fig.10a*  and *10b*). HRTEM was used as a complementary technique to SEM, which revealed that mono-dispersed and highly crystalline Ag and Au nanoparticles were synthesized. The appearances of both Ag and Au NPs are found to be near-spherical. The particle sizes are varied according to different reducing agents. Generally, the sizes for Ag and Au via reduction of ascorbic acid and citrate are larger than those produced from strong reducing agents, DMAB and NaBH4, with the average particle size from 4.8, to 32 nm in diameter. For both Ag and Ag, ring pattern of the particles indicated that the Ag and Au were well indexed with the standard metallic pattern, which correlated with the XRD analysis.

### **3.2.4 Elemental composition analyses of engineered nanomaterials**

EDS and XPS were used as supplemental techniques to determine the composition of the engineered nanomaterials. EDS spectra of the metal nanoparticles (*Fig. 10a* & *b*, lower righthand-side panel for elemental analysis) indicated the presence of the Ag and Au elements, noting that metallic Ag displays two major emission lines at K: 22.162 keV and L: 2.984 keV, respectively. While Au displays two major peaks of L at 9.711 keV and M 2.123 keV, respectively. The characteristic peaks of Au were obtained, and were correlated to K peak

Ag particle agglomeration was successfully prevented. In both circumstances, the reducing agent, ascorboic acid was selected for demonstration. Other reducing agents provided the same crystalline phase; however, the crystallite size of the engineering particles varies accordingly. It is found that the NaBH4 and DMAB provided essentially the same size, averaged at 12.7 nm for Ag and 4.8 nm for Au nanoparticles, which is also confirmed by TEM measurement. It is important to point out that the lattice occupancy is near ideal (99.0 %), suggesting the Ag and Au nanoparticles possessed highly crystalline structure with less lattice distortion. Both nanoparticles also display high frenquecy of merit (0.2) when using Jade 7.1 database to index with the standard poser diffraction files. The crystalinity index was also determined via taking the ratio of particle size from TEM vs the crystallite size calculated from XRD data. It can be seen that Ag and Au nanoparticles are highly crystalline. Ag showed monoparticles and Au on the other hand polyparticles (see table 2)

> Particles size (nm)

Ag-Ascobic acid 30.2 33.1 1.1 monocrystalline Ag- DMAB 12.7 30.4 2.4 twincrystalline Ag-DMAB 11.2 34.1 3.1 polycrystalline Au-Ascobic acid 10.4 15.6 1.5 monocrystalline Au-DMAB 5.8 14.4 2.2 twincrystalline Au-DMAB 4.8 15.4 3.2 polycrystalline

Table 2. Summary of the crystalinity index of Ag and Au nanoparticles (six samples are

indexed with the standard metallic pattern, which correlated with the XRD analysis.

EDS and XPS were used as supplemental techniques to determine the composition of the engineered nanomaterials. EDS spectra of the metal nanoparticles (*Fig. 10a* & *b*, lower righthand-side panel for elemental analysis) indicated the presence of the Ag and Au elements, noting that metallic Ag displays two major emission lines at K: 22.162 keV and L: 2.984 keV, respectively. While Au displays two major peaks of L at 9.711 keV and M 2.123 keV, respectively. The characteristic peaks of Au were obtained, and were correlated to K peak

**3.2.4 Elemental composition analyses of engineered nanomaterials** 

The morphological and elemental studies through HRTEM technique are shown *(Fig.10a*  and *10b*). HRTEM was used as a complementary technique to SEM, which revealed that mono-dispersed and highly crystalline Ag and Au nanoparticles were synthesized. The appearances of both Ag and Au NPs are found to be near-spherical. The particle sizes are varied according to different reducing agents. Generally, the sizes for Ag and Au via reduction of ascorbic acid and citrate are larger than those produced from strong reducing agents, DMAB and NaBH4, with the average particle size from 4.8, to 32 nm in diameter. For both Ag and Ag, ring pattern of the particles indicated that the Ag and Au were well

Crystallinity Particle Type

according to different fabrication methods.

Selected samples Crystallite size

selected for demonstration)

(nm)

**3.2.3 Fine-structure study of engineered nanomaterials** 

which occurred at 2.10 KeV and the K peak which occurred at 2.18 KeV, respectively, which is in agreement with Au characteristic peaks. The Au NP appearance is near-spherical with the average particle size from 15 to 20 nm in diameter. The high resolution image also shows the lattice fringe which was distinguished, which also confirms the formation of Au crystals. The element Cu peak detected is resulting from Cu grid sample holder.

Fig. 10. TEM images and elemental analysis of nanoparticles, a: morphology, ring pattern and composition of nanosilver; b: morphology, ring pattern and composition of nanogold.

The XPS results (*Fig. 11*) show that Ag and Au are characterized by an asymmetric line shape with the peak tailing to the higher BE. The BE may be varied due to its extra columbic interaction between the photoemitted electron and ion core. The binding energies for Ag electron configurations of 3d5/2 and 3d3/2 were found to be 366.0 eV and 372.0 eV with the difference of 6.0 eV (Fig. 11a). This measurement was corresponding to the standard Ag 3d binding energies (3d5/2 = 368.3 eV, 3d3/2 = 374.3 eV, = 6.0 eV). Apparently, the 3d

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 53

of hemolysis increased with increase in dose. The Au NPs have shown 9.5 % and 12.5 % hemolysis at 0.1 and 1 µM concentrations, respectively. However, at 10 µM concentrations the % hemolysis was found to be 22.4 %. Similarly, Ag NPs have shown hemolytic profile of 9.47 % and 13.07 % at 0.1 and 1 µM. Interestingly, Ag NPs have also shown higher hemolytic activity 29.15 % at 10 µM. These data suggest that at lower concentrations (0.1, 1 µM), Au and Ag NPs can be used for *in vivo* applications. The *in vitro* hemolysis data are presented in

> *In vitro* **cytotoxicity of Au and Ag NPs in OVCAR-8 Cells**

> > 0.1 0.5 1 10 100

Concentration (µM)

*In vitro* **cytotoxicity of Ag and Au NPs in CHO cells**

0.1 0.5 1 10 100

Concentration (µM)

Fig. 12. *In vitro* cytotoxic activity of nanometals, percentage viability was calculated by comparing absorbance of treated cells with untreated cells (100 % Viability), **top panel**: The

OVCAR-8 cells were used; **bottom panel**: CHO cells were used.

Ag Au

Ag Au

*Fig. 13*.

% Cell Viability

% Cell Viability

photoemission of Ag is split between two peaks with an intensity ratio at about 6:5. It was also found that Au (Fig. 11b) displayed two principal emissions at 4f7/2 = 82.2 eV, 4f5/2 = 85.8 eV, respectively. The spectrum was also well-indexed with the standard metallic Au spectra (4f7/2 = 82.2 eV, 4f5/2 = 85.8 eV, = 3.6 eV).

Fig. 11. XPS analyses of NPs, a: determination of nanosilver; b: determination of nanogold.

#### **3.3** *In vitro* **cytotoxic activity of nanometals**

In addition to nanostructural characterization, the cytotoxicity in normal ovary and ovarian cancer cells using Au and Ag NPs in human red blood cells has been systematically investigated. The *in vitro* cytotoxicity of Ag and Au NPs was assessed in NCI/ADR - RES (OVCAR -8) cells, which are ovarian adenocarcinoma cells, and Chinese hamster ovary (CHO) cells. Samples with 2×104 OVCAR-8 and CHO cells were seeded in a 96-well plate. Percentage viability was calculated by comparing absorbance of treated cells with untreated cells (100 % Viability). These results have indicated that at all concentrations (0.1, 0.5, 1, 10 and 100 µM), Au nanoparticles have not shown any toxicity in both normal and ovarian cancer cell lines indicating their biocompatibility. Ag NPs have shown no toxicity at lower concentrations (0.1, 0.5 and 1 µM) in both cell lines. However, at higher concentrations (10 and 100 µM), Ag NPs have shown only 55.9 % and 32.59 % viability in OVCAR-8 cells for 10 and 100 µM doses, respectively. In CHO cells, the Ag NPs have shown 37.88 % and 15.37 % viability for 10 and 100 µM doses, respectively. Based on the data, it may be concluded that the Ag NPs can potentially be used Carriers for cancer therapy. The *in vitro* cytotoxicity data is presented in *Fig. 12a* and *12b*.

#### **3.4 Hemocompatibility of nanometals**

Ag and Au NPs were incubated with human red blood cells (RBCs) suspension for 6 hrs in a 96 well plate. The plate was then centrifuged and supernatant was collected and measured for release of hemoglobin by measuring absorbance at 540 nm. RBCs treated with Triton X-100 was used as the reference standard. RBCs are the first point of contact of NPs when administered systemically. In the present study, the Au NPs have shown that the percentage

photoemission of Ag is split between two peaks with an intensity ratio at about 6:5. It was also found that Au (Fig. 11b) displayed two principal emissions at 4f7/2 = 82.2 eV, 4f5/2 = 85.8 eV, respectively. The spectrum was also well-indexed with the standard metallic Au spectra

Fig. 11. XPS analyses of NPs, a: determination of nanosilver; b: determination of nanogold.

In addition to nanostructural characterization, the cytotoxicity in normal ovary and ovarian cancer cells using Au and Ag NPs in human red blood cells has been systematically investigated. The *in vitro* cytotoxicity of Ag and Au NPs was assessed in NCI/ADR - RES (OVCAR -8) cells, which are ovarian adenocarcinoma cells, and Chinese hamster ovary (CHO) cells. Samples with 2×104 OVCAR-8 and CHO cells were seeded in a 96-well plate. Percentage viability was calculated by comparing absorbance of treated cells with untreated cells (100 % Viability). These results have indicated that at all concentrations (0.1, 0.5, 1, 10 and 100 µM), Au nanoparticles have not shown any toxicity in both normal and ovarian cancer cell lines indicating their biocompatibility. Ag NPs have shown no toxicity at lower concentrations (0.1, 0.5 and 1 µM) in both cell lines. However, at higher concentrations (10 and 100 µM), Ag NPs have shown only 55.9 % and 32.59 % viability in OVCAR-8 cells for 10 and 100 µM doses, respectively. In CHO cells, the Ag NPs have shown 37.88 % and 15.37 % viability for 10 and 100 µM doses, respectively. Based on the data, it may be concluded that the Ag NPs can potentially be used Carriers for cancer therapy. The *in vitro* cytotoxicity data

Ag and Au NPs were incubated with human red blood cells (RBCs) suspension for 6 hrs in a 96 well plate. The plate was then centrifuged and supernatant was collected and measured for release of hemoglobin by measuring absorbance at 540 nm. RBCs treated with Triton X-100 was used as the reference standard. RBCs are the first point of contact of NPs when administered systemically. In the present study, the Au NPs have shown that the percentage

(4f7/2 = 82.2 eV, 4f5/2 = 85.8 eV, = 3.6 eV).

**3.3** *In vitro* **cytotoxic activity of nanometals** 

is presented in *Fig. 12a* and *12b*.

**3.4 Hemocompatibility of nanometals** 

of hemolysis increased with increase in dose. The Au NPs have shown 9.5 % and 12.5 % hemolysis at 0.1 and 1 µM concentrations, respectively. However, at 10 µM concentrations the % hemolysis was found to be 22.4 %. Similarly, Ag NPs have shown hemolytic profile of 9.47 % and 13.07 % at 0.1 and 1 µM. Interestingly, Ag NPs have also shown higher hemolytic activity 29.15 % at 10 µM. These data suggest that at lower concentrations (0.1, 1 µM), Au and Ag NPs can be used for *in vivo* applications. The *in vitro* hemolysis data are presented in *Fig. 13*.

Fig. 12. *In vitro* cytotoxic activity of nanometals, percentage viability was calculated by comparing absorbance of treated cells with untreated cells (100 % Viability), **top panel**: The OVCAR-8 cells were used; **bottom panel**: CHO cells were used.

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 55

most important component of a theragnositc is its targeting ligand. The targeting ligand includes small molecule ligands like short peptides, aptamers, and large molecule ligands

Metallic nanoparticles because of their unique optical properties and their capability to transit therapeutics to tumor have gained wide importance as theragnostic agents. Au nanoparticles possess unique optical properties such as photothermal and surface-plasmon effects, and these properties can be utilized for various clinical applications in cancer diagnostics. Rod shaped Au NPs (Au nanorods) when irradiated in near-infrared region show a surface Plasmon band. This property can be used for bioimaging, or as heating devices for photothermal therapy. Thus, Au nanorods can be used as potential theragnostic devices for bioimaging, thermal therapy and a drug delivery system. When these Au Nanorods coated with as thermosensitive gel were administered to tumor bearing animals and the tumor was irradiated externally, a significantly larger amount of gold was detected in the irradiated tumor. [91] Ag nanosystems have also shown promise as theragnostic agents because of their optical absorption properties. Ag nanosystems were reported to be potential contrast agents for photoacoustic imaging and image-guided therapy. Ag nanosystems consisting of PEGylated Ag layer deposited on silica Cores were investigated and was shown to be non-toxic *in vitro* at concentrations up to 2 mg/mL. Ag nanosystems also have shown strong concentration-dependent photoacoustic signal when embedded in an *ex vivo* tissue sample. [92] Thus, Ag nanosystems which have capability to carry therapeutic payload and exhibit very strong signal when used for image-guided therapy can be widely used for

Theragnostics advanced with advent of nanotechnology. Nanotechnology, by virtue of their unique physicochemical properties like quantum confinement in quantum dots, superparamagnetism in certain oxide nanoparticles and surface-enhanced Raman scattering (SERS) in metallic nanoparticles, resulted in emergence of sensitive and cost effective imaging agents. Similarly, their properties like large surface area to volume ratio, capability to control size, hydrophobicity and surface charge according to intended application made them valuable carriers for therapeutic drugs and genes. [93] Many nanocarriers with anticancer drugs are currently investigated clinically for their targeting capability. Immunoliposomes of doxorubicin are in Phase I clinical trials for targeting human metastatic stomach cancer. [94] Polymeric micelles with Paclitaxel are currently in Phase II clinical trials for targeting stomach cancer. [95] Thus, nanoparticles have the required attributes to house therapeutic payload along with diagnostic imaging agent for real-time

The Au/Ag-NPs exhibit potential for use for molecular-targeted cancer diagnosis and therapeutics. The Au-NPs were biocompatible with the cell lines utilized in our studies. They exhibit strong absorption at 530 nm in the visible light spectrum, with the absorption maximum wavelength displaying shifts upon interaction with other molecules. They are therefore well suited for the development of diagnostic tools aimed at molecular-targeted detection of cancer cells in samples or *in vivo* through imaging technologies. To achieve molecular targeting, modification of the Au-NPs by conjugation with diverse macromolecules (polypeptides, polysaccharides, and even polynucleotides), is being

like antibodies.

theragnostic applications.

monitoring of treatment response.

Fig. 13. The hemocompatibility of Ag and Au NPs with human red blood cells, the hollow collum represents the hemocopatibility of Ag, and the filled black colum represents hemocompatibility of Au.

## **4. Mechanistic studies of nanometals for cancer diagnosis and therapy**

A new concept in cancer therapy called "Theragnostics" is emerging recently, which summarizes information based on various biotechnologies involving genomics, transcriptomics, proteomics and metabolomics. [89] The term "Theragnostics" encompasses wide array of topics which includes predictive medicine, personalized medicine, integrated medicine, and pharmacodiagnostics. Major applications of theragnostics in biomedical research include profiling of subgroups of patients based on the likelihood of occurrence of positive outcome to a given treatment so as to entail them to targeted therapies (efficacy), identification of subgroups of patients who are at risk of side effects during a treatment using pharmacogenomics (safety), and monitoring therapeutic response after treatment (efficacy and safety). [90]

The various components of nanotheragnostics each having their own advantage are: signal emitter which emits signal upon excitation by external source, therapeutic moiety can be a drug, or a nucleic acid like small interfering ribonucleic acid (siRNA), nanocarrier is a polymeric carrier capable of carrying high drug payload, and targeting ligand is the entity which can bind to a disease marker with high specificity so as to deliver the entire system to target cell. Non-invasive imaging techniques like magnetic resonance imaging (MRI), X-ray computed tomography (CT), positron emission tomography (PET), single photon emission computed tomography (SPECT), and ultrasound can be used to capture response of signal emitter in real-time. The therapeutic moiety as well as the signal emitter can be encapsulated or covalently attached to the polymeric carrier. Many synthetic and natural polymers were proved to be effective carriers but polymers that are approved for clinical application or currently under clinical trials are poly (ethylene glycol) (PEG), dextran, carboxydextran, βcyclodextrin, poly (Dextro Levo-lactide-co-glycolide) (PLGA), and poly (L-lysine) (PLL). The

Ag Au

**Hemocompatibility of Ag and Au NPs**

Fig. 13. The hemocompatibility of Ag and Au NPs with human red blood cells, the hollow collum represents the hemocopatibility of Ag, and the filled black colum represents

0.1 1 10

Concentration (µM)

A new concept in cancer therapy called "Theragnostics" is emerging recently, which summarizes information based on various biotechnologies involving genomics, transcriptomics, proteomics and metabolomics. [89] The term "Theragnostics" encompasses wide array of topics which includes predictive medicine, personalized medicine, integrated medicine, and pharmacodiagnostics. Major applications of theragnostics in biomedical research include profiling of subgroups of patients based on the likelihood of occurrence of positive outcome to a given treatment so as to entail them to targeted therapies (efficacy), identification of subgroups of patients who are at risk of side effects during a treatment using pharmacogenomics (safety), and monitoring therapeutic response after treatment

The various components of nanotheragnostics each having their own advantage are: signal emitter which emits signal upon excitation by external source, therapeutic moiety can be a drug, or a nucleic acid like small interfering ribonucleic acid (siRNA), nanocarrier is a polymeric carrier capable of carrying high drug payload, and targeting ligand is the entity which can bind to a disease marker with high specificity so as to deliver the entire system to target cell. Non-invasive imaging techniques like magnetic resonance imaging (MRI), X-ray computed tomography (CT), positron emission tomography (PET), single photon emission computed tomography (SPECT), and ultrasound can be used to capture response of signal emitter in real-time. The therapeutic moiety as well as the signal emitter can be encapsulated or covalently attached to the polymeric carrier. Many synthetic and natural polymers were proved to be effective carriers but polymers that are approved for clinical application or currently under clinical trials are poly (ethylene glycol) (PEG), dextran, carboxydextran, βcyclodextrin, poly (Dextro Levo-lactide-co-glycolide) (PLGA), and poly (L-lysine) (PLL). The

**4. Mechanistic studies of nanometals for cancer diagnosis and therapy** 

hemocompatibility of Au.

% Hemolysis

(efficacy and safety). [90]

most important component of a theragnositc is its targeting ligand. The targeting ligand includes small molecule ligands like short peptides, aptamers, and large molecule ligands like antibodies.

Metallic nanoparticles because of their unique optical properties and their capability to transit therapeutics to tumor have gained wide importance as theragnostic agents. Au nanoparticles possess unique optical properties such as photothermal and surface-plasmon effects, and these properties can be utilized for various clinical applications in cancer diagnostics. Rod shaped Au NPs (Au nanorods) when irradiated in near-infrared region show a surface Plasmon band. This property can be used for bioimaging, or as heating devices for photothermal therapy. Thus, Au nanorods can be used as potential theragnostic devices for bioimaging, thermal therapy and a drug delivery system. When these Au Nanorods coated with as thermosensitive gel were administered to tumor bearing animals and the tumor was irradiated externally, a significantly larger amount of gold was detected in the irradiated tumor. [91] Ag nanosystems have also shown promise as theragnostic agents because of their optical absorption properties. Ag nanosystems were reported to be potential contrast agents for photoacoustic imaging and image-guided therapy. Ag nanosystems consisting of PEGylated Ag layer deposited on silica Cores were investigated and was shown to be non-toxic *in vitro* at concentrations up to 2 mg/mL. Ag nanosystems also have shown strong concentration-dependent photoacoustic signal when embedded in an *ex vivo* tissue sample. [92] Thus, Ag nanosystems which have capability to carry therapeutic payload and exhibit very strong signal when used for image-guided therapy can be widely used for theragnostic applications.

Theragnostics advanced with advent of nanotechnology. Nanotechnology, by virtue of their unique physicochemical properties like quantum confinement in quantum dots, superparamagnetism in certain oxide nanoparticles and surface-enhanced Raman scattering (SERS) in metallic nanoparticles, resulted in emergence of sensitive and cost effective imaging agents. Similarly, their properties like large surface area to volume ratio, capability to control size, hydrophobicity and surface charge according to intended application made them valuable carriers for therapeutic drugs and genes. [93] Many nanocarriers with anticancer drugs are currently investigated clinically for their targeting capability. Immunoliposomes of doxorubicin are in Phase I clinical trials for targeting human metastatic stomach cancer. [94] Polymeric micelles with Paclitaxel are currently in Phase II clinical trials for targeting stomach cancer. [95] Thus, nanoparticles have the required attributes to house therapeutic payload along with diagnostic imaging agent for real-time monitoring of treatment response.

The Au/Ag-NPs exhibit potential for use for molecular-targeted cancer diagnosis and therapeutics. The Au-NPs were biocompatible with the cell lines utilized in our studies. They exhibit strong absorption at 530 nm in the visible light spectrum, with the absorption maximum wavelength displaying shifts upon interaction with other molecules. They are therefore well suited for the development of diagnostic tools aimed at molecular-targeted detection of cancer cells in samples or *in vivo* through imaging technologies. To achieve molecular targeting, modification of the Au-NPs by conjugation with diverse macromolecules (polypeptides, polysaccharides, and even polynucleotides), is being

Application of Nanometals Fabricated Using Green Synthesis in Cancer Diagnosis and Therapy 57

Phase 2 includes phase I study but on a larger sample size, inculding formulation and

The Trojan Horse is a stratagem that causes a the nanomaterial to be delivered without

The authors are grateful to Texas A&M University-Kingsville (TAMUK), the College of Arts and Sciences Research and Development Fund for funding this research activity. The technical support from the National Science Foundation, Major Research Instrumentation program is duly acknowledged to allow the use of JEOL field emission scanning electron microscopy and (TAMUK) Rigaku Ultima III X-ray powder diffraction (TAMU-Corpus Christi). The Academia Mexicana de Ciencias (AMC), Fundación México Estados Unidos para la Ciencia (FUMEC) is also duly acknowledged for their financial support for Dr. Medina-Ramirez to prepare Ag and Au nanoparticles. The Microscope and Imaging Center and the Center of Materials Characterization Facility at TAMU-College Station are also duly acknowledged for technical support and access to the advanced instrumentation, respectively. Last but not least, the authors wish to thank Dr. Sajid Bashir for copy-editing

I. Medina-Ramirez conducted the synthesis, and electrokinetic study of nanomaterials. M. Gonzalez-Garcia jointly conceived the conceptual framework with J. Liu and contributed specifically to the application of nanomaterials for cancer diagnosis and treatment. S. Palakurthi completed the toxicicity and hemocompatibility studies. J. Liu prepared Ag and Au nanoparticles based on Medina-Ramirez's discoveries. She completed the spectroscopic and data collection and analysis. She also collected microscopic data with the other investigators (e.g. Dr. Luo at the Materials Characterization Facility and Microscopy

[1] J. Zhang, Z. Liu, B. Han, D. Liu, J. Chen, J. He, T. Jiang, (2004), Chemistry - A European

[2] Eric Drexler, Engines of Creation: The Coming Era of Nanotechnology, Bantam Dell Publishing Group Inc (Random House), New York, USA, 1990, pp57-141. [3] Eric Drexler, Nanotechnology, Molecular Manufacturing, and Productive Nanosystems,

[4] Y. Yao, Y. Ohko, Y. Sekiguchi, A. Fujishima, Y. Kubota, Journal of Biomedical Materials

[5] A. Borras, Á. Barranco, J. P. Espinós, J. Cotrino, J. P. Holgado, A. R. González-Elipe,

John Wiley and Sons Inc, New Jersey, USA, 1991, pp.71-89.

Research Part B: Applied Biomaterials, 2008, 85B, 453.460.

Plasma Processes and Polymers, 2007, 4, 515-527.

Journal, 10 (14), 3531-3536; Drexler E., Peterson C., Pergamit G. (1991) Unbounding the Future: the Nanotechnology Revolution. William Morrow and Company, New

dosage.

detection of the host immune system.

**7. Acknowledgements** 

this book chapter.

**9. References** 

York.

**8. Author contributions** 

Imaging Center) at Texas A&M University-College Station.

explored in our laboratory. The conjugated Au-NPs can be used in the development of contrast agents for optical imaging to identify the cancerous tissues. On the other hand, Ag-NPs showed cytotoxic effects on both the ovarian adenocarcinoma cell line and the nontumorogenic ovary cell line in the low micromolar range, as well as stronger hemolytic effects with red blood cells. The toxicity properties of Ag-NPs could be utilized for the development of therapeutic agents to eliminate cancer cells. Once again, conjugation with macromolecules could result in novel nanomaterials with enhanced specificity of action. In this case, the macromolecules should either block the toxicity on normal cells, or target the Ag-NPs to the surface of cancer cells specifically. In the latter case, a lower overall concentration of the targeted Ag-NPs could be utilized, which would reach only toxic levels on the surface or within the cancer cells. Oligosaccharide coatings are currently being investigated in our laboratory. The hypothesis is that cancer cells need to grow fast and would have an enhanced requirement for carbohydrates, driving them to accelerated endocytosis of the nanomaterial, reaching therefore toxic levels of Ag-NPs within the cell upon degradation of the oligosaccharide coat. Furthermore, in advanced cancers the leaky blood vessels would release these NPs right near the cancerous cells, providing for another mechanism for concentration of the therapeutic agent at the target sites.

Our laboratory has focused on the coating of NPs with macromolecules that provide a first level of specificity to distinguish cancer cells from healthy cells. Further specificity for diagnosis or treatment of specific cancer types could be achieved by forming hybrid materials that include specific ligands or antibodies that bind to specific cancer cell types. As described earlier, an antibody against the IL13R2 receptor has been utilized to direct TiO2- NPs to certain brain tumors. [78] Our knowledge of molecular markers of specific tumors is increasing steadily, providing tools for the development of a larger collection of highspecificity therapeutic agents that can be utilized in tailored personalized treatment regimens.

## **5. Conclusion**

The green colloidal approach provides stable metallic nanoparticles (Au and Ag). The asprepared nanometals are determined to be ultrafine, pseudo-spherical and monodispersed. The technical advantages of the colloidal method lie in cost and time effectiveness, simplicity, ability to control homogeneity from molecular level, and high precision of aggregation under use-friendly fabrication variables. The nano-characterization by state-ofthe-art instrumentation techniques allow for in-depth understanding of control in morphological, structural and elemental composition via varying the fabrication parameters. Additionally, the Au and Ag nanometals displayed 100 % percentage viability when *in vitro* cytotoxicity was tested. Both metal NPs were also found to be applicable for *in vivo* applications at lower concentrations (0.1, 1 µM).

## **6. Useful definitions**

Phase 0 trials are designed to speed up the development of promising drugs by establishing early on whether they have a similar profile as in the early preclinical studies.

Phase 1 includes trials designed to assess the safety, kinetics, and pharma of a drug.

Phase 2 includes phase I study but on a larger sample size, inculding formulation and dosage.

The Trojan Horse is a stratagem that causes a the nanomaterial to be delivered without detection of the host immune system.

## **7. Acknowledgements**

56 Green Chemistry – Environmentally Benign Approaches

explored in our laboratory. The conjugated Au-NPs can be used in the development of contrast agents for optical imaging to identify the cancerous tissues. On the other hand, Ag-NPs showed cytotoxic effects on both the ovarian adenocarcinoma cell line and the nontumorogenic ovary cell line in the low micromolar range, as well as stronger hemolytic effects with red blood cells. The toxicity properties of Ag-NPs could be utilized for the development of therapeutic agents to eliminate cancer cells. Once again, conjugation with macromolecules could result in novel nanomaterials with enhanced specificity of action. In this case, the macromolecules should either block the toxicity on normal cells, or target the Ag-NPs to the surface of cancer cells specifically. In the latter case, a lower overall concentration of the targeted Ag-NPs could be utilized, which would reach only toxic levels on the surface or within the cancer cells. Oligosaccharide coatings are currently being investigated in our laboratory. The hypothesis is that cancer cells need to grow fast and would have an enhanced requirement for carbohydrates, driving them to accelerated endocytosis of the nanomaterial, reaching therefore toxic levels of Ag-NPs within the cell upon degradation of the oligosaccharide coat. Furthermore, in advanced cancers the leaky blood vessels would release these NPs right near the cancerous cells, providing for another

Our laboratory has focused on the coating of NPs with macromolecules that provide a first level of specificity to distinguish cancer cells from healthy cells. Further specificity for diagnosis or treatment of specific cancer types could be achieved by forming hybrid materials that include specific ligands or antibodies that bind to specific cancer cell types. As described earlier, an antibody against the IL13R2 receptor has been utilized to direct TiO2- NPs to certain brain tumors. [78] Our knowledge of molecular markers of specific tumors is increasing steadily, providing tools for the development of a larger collection of highspecificity therapeutic agents that can be utilized in tailored personalized treatment

The green colloidal approach provides stable metallic nanoparticles (Au and Ag). The asprepared nanometals are determined to be ultrafine, pseudo-spherical and monodispersed. The technical advantages of the colloidal method lie in cost and time effectiveness, simplicity, ability to control homogeneity from molecular level, and high precision of aggregation under use-friendly fabrication variables. The nano-characterization by state-ofthe-art instrumentation techniques allow for in-depth understanding of control in morphological, structural and elemental composition via varying the fabrication parameters. Additionally, the Au and Ag nanometals displayed 100 % percentage viability when *in vitro* cytotoxicity was tested. Both metal NPs were also found to be applicable for *in vivo*

Phase 0 trials are designed to speed up the development of promising drugs by establishing

early on whether they have a similar profile as in the early preclinical studies.

Phase 1 includes trials designed to assess the safety, kinetics, and pharma of a drug.

mechanism for concentration of the therapeutic agent at the target sites.

regimens.

**5. Conclusion** 

**6. Useful definitions** 

applications at lower concentrations (0.1, 1 µM).

The authors are grateful to Texas A&M University-Kingsville (TAMUK), the College of Arts and Sciences Research and Development Fund for funding this research activity. The technical support from the National Science Foundation, Major Research Instrumentation program is duly acknowledged to allow the use of JEOL field emission scanning electron microscopy and (TAMUK) Rigaku Ultima III X-ray powder diffraction (TAMU-Corpus Christi). The Academia Mexicana de Ciencias (AMC), Fundación México Estados Unidos para la Ciencia (FUMEC) is also duly acknowledged for their financial support for Dr. Medina-Ramirez to prepare Ag and Au nanoparticles. The Microscope and Imaging Center and the Center of Materials Characterization Facility at TAMU-College Station are also duly acknowledged for technical support and access to the advanced instrumentation, respectively. Last but not least, the authors wish to thank Dr. Sajid Bashir for copy-editing this book chapter.

## **8. Author contributions**

I. Medina-Ramirez conducted the synthesis, and electrokinetic study of nanomaterials. M. Gonzalez-Garcia jointly conceived the conceptual framework with J. Liu and contributed specifically to the application of nanomaterials for cancer diagnosis and treatment. S. Palakurthi completed the toxicicity and hemocompatibility studies. J. Liu prepared Ag and Au nanoparticles based on Medina-Ramirez's discoveries. She completed the spectroscopic and data collection and analysis. She also collected microscopic data with the other investigators (e.g. Dr. Luo at the Materials Characterization Facility and Microscopy Imaging Center) at Texas A&M University-College Station.

## **9. References**


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[66] O. Akhavan, Journal of Colloid and Interface Science, 2009, 336, 117-124. [67] M. S. Chun, H. I. Cho, I. K. Song, Desalination, 2002, 148, 363-367. [68] Hanahan D, Weinberg RA, 2000. The hallmarks of cancer. Cell 100, 57-70

[71] Green DR, Evan GI, 2002. A matter of life and death. Cancer Cell 1, 19-30

Imaging of Hepatocellular Carcinoma. Nano Lett 11, 2678-2683

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Chapter 1.

2014–2021.

13955–13961.

077106-1 to 077106-4.

2009, 27, 461 -468.

118-133

Photobiology A: Chemistry, 2005, 169, 131-137.


**4** 

*China* 

**Electrochemically-Driven** 

**and Green Conversion of SO2** 

Hong Liu1,2,\*, Chuan Wang1,2 and Yuan Liu1 *1Chongqing Institute of Green and Intelligent Technology,* 

> *Chinese Academy of Sciences, Chongqing, 2School of Chemistry and Chemical engineering,*

> > *Sun Yat-sen University, Guangzhou,*

**to NaHSO4 in Aqueous Solution** 

The world has widely resorted to fossil fuels to power the industry and everyday life. In China, above 70% of the energy is extracted from coal. Emission of SO2 due to burning of fossil fuels, in particularly of coal, causes harmful impacts on the environment, human health, livestock, and plants *(1,2)*. Many measures have been taken to cut off the emission of SO2 during the last generations. It can be seen that the reduction of SO2 emission in developed countries such as the United States has been witnessed *(3)*. However, due to the aspiration of energy to drive the economic increase and sustain the expanded population, the SO2 emission is estimated to augment sharply in the rapidly developing Asian areas,

Basically, the SO2 emission is reduced after the burning processes through various flue gas desulfurization (FGD) processes *(5-8)*, which serves to transform the S(IV) to S(VI) and frequently to immobilize the SO2 waste in the form of a solid. Of them, a wet limestone FGD process *(6,9,10)* using CaCO3 mineral, represents over 90% of the installed desulfurization

> 2 3 2 2 42 2 <sup>1</sup> 2 2 2

Eq 1 illustrates that the SO2 is transformed and immobilized in the form of CaSO4·2H2O, which may be commercialized as gypsum, but the incentive is little in areas including the United Sates *(11)* and China because of its rich natural sources. To our knowledge, only 3% of the FGD byproduct gypsum can be reused in China*.* In fact, once treated improperly, the solid waste becomes a secondary pollutant, and thereby is of a great environmental concern. Meanwhile, eq 1 shows that 1 mole of SO2 leads to 1 mole of CO2, whose discharge and

*SO CaCO H O O CaSO H O CO* (1)

and will still pose as a worldwide environmental problem in the next 30 years *(4)*.

capacities in the world *(9)*, and can be chemically expressed below :

**1. Introduction** 

 \*

Corresponding Author

[96] Chen CS (2008) Biotechnology: Remote control of living cells. Nature Nanotechnology 3:13 – 14; Li Z, Jin R, Mirkin CA, Letsinger RL (2002) Multiple thiol-anchor capped DNA–gold nanoparticle conjugates. Nucleic Acids Research 30:1558-1562.

## **Electrochemically-Driven and Green Conversion of SO2 to NaHSO4 in Aqueous Solution**

Hong Liu1,2,\*, Chuan Wang1,2 and Yuan Liu1

*1Chongqing Institute of Green and Intelligent Technology, Chinese Academy of Sciences, Chongqing, 2School of Chemistry and Chemical engineering, Sun Yat-sen University, Guangzhou, China* 

### **1. Introduction**

62 Green Chemistry – Environmentally Benign Approaches

[96] Chen CS (2008) Biotechnology: Remote control of living cells. Nature Nanotechnology

DNA–gold nanoparticle conjugates. Nucleic Acids Research 30:1558-1562.

3:13 – 14; Li Z, Jin R, Mirkin CA, Letsinger RL (2002) Multiple thiol-anchor capped

The world has widely resorted to fossil fuels to power the industry and everyday life. In China, above 70% of the energy is extracted from coal. Emission of SO2 due to burning of fossil fuels, in particularly of coal, causes harmful impacts on the environment, human health, livestock, and plants *(1,2)*. Many measures have been taken to cut off the emission of SO2 during the last generations. It can be seen that the reduction of SO2 emission in developed countries such as the United States has been witnessed *(3)*. However, due to the aspiration of energy to drive the economic increase and sustain the expanded population, the SO2 emission is estimated to augment sharply in the rapidly developing Asian areas, and will still pose as a worldwide environmental problem in the next 30 years *(4)*.

Basically, the SO2 emission is reduced after the burning processes through various flue gas desulfurization (FGD) processes *(5-8)*, which serves to transform the S(IV) to S(VI) and frequently to immobilize the SO2 waste in the form of a solid. Of them, a wet limestone FGD process *(6,9,10)* using CaCO3 mineral, represents over 90% of the installed desulfurization capacities in the world *(9)*, and can be chemically expressed below :

$$\rm{CaSO}\_2 + \rm{CaCO}\_3 + 2H\_2O + \frac{1}{2}O\_2 \rightarrow \rm{CaSO}\_4 \cdot 2H\_2O + CO\_2 \tag{1}$$

Eq 1 illustrates that the SO2 is transformed and immobilized in the form of CaSO4·2H2O, which may be commercialized as gypsum, but the incentive is little in areas including the United Sates *(11)* and China because of its rich natural sources. To our knowledge, only 3% of the FGD byproduct gypsum can be reused in China*.* In fact, once treated improperly, the solid waste becomes a secondary pollutant, and thereby is of a great environmental concern. Meanwhile, eq 1 shows that 1 mole of SO2 leads to 1 mole of CO2, whose discharge and

<sup>\*</sup> Corresponding Author

Electrochemically-Driven and Green Conversion of SO2 to NaHSO4 in Aqueous Solution 65

**Oxidation of SO2 to SO42-.** After the absorption, the absorbed SO2 is oxidized by air in

and SO32- to SO4

2

*HSO O SO H* (4)

*SO O SO* (5)

2 2 <sup>2</sup> *O H e HO* 2 2 (6)

<sup>2</sup> 2 2 *H eH* (7)

*HO O H e* (8)

<sup>2</sup> *SO H bisul* <sup>4</sup> *fate ion* (9)

ions. All these

2 2 2 2 *O H O e H O OH* 22 2 (6')

2- ions. This oxidation process


32 4 1 2

2 2 3 24 1 2

**ESH.** The H+ ions released through eqs 2~4 need to be scavenged due to their hindrance of the SO2 absorption. Otherwise, the continuous absorption of SO2 will be terminated. It can be noted that while O2 in air is utilized for the SO2 oxidation through eqs 4 and 5, the cathodic reduction of O2 can be employed to scavenge the process-released H+ ions. The reactions of O2 reduction through a 2-electron process at acidic and neutral/alkaline

is expressed as follows, of which eq 4 releases H+ ions, but eq 5 does not*(19)*:

conditions are expressed in eqs 6 and 6', respectively *(20-22)*:

At the same time, a side reaction co-exists with eq 6 below:

reaction is shown below:

**3. Experimental section** 

all experiments.

formed in eqs 4 and 5 to form a bisulfate:

formation of NaHSO4 as a product of desulfurization.

It can be seen that eqs 6 and 7 consume H+ ions and eq 6' supplies OH-

2 2

2

**Transformation of SO42- to Bisulfate.** As coupled to the cathodic reactions, an anodic

Under an extremely acidic condition, the H+ ions in eq 8 are combined with the SO42- ions

As a result, a model experiment becomes necessary to chemically substantiate this design by disclosing the ESH effect on the SO2 absorption and oxidation, and by confirming the

**Chemicals and Reagents.** SO2 gas (99.9%) was obtained from KEDI, Foshan, China. Other chemicals as analytical reagents were used as obtained. Double distilled water was used in

<sup>1</sup> 2 2

reactions can be utilized to scavenge the H+ ions released through eqs 2~4.

aqueous solution at moderate pH from HSO3

accumulation in the atmosphere is recognized to aggravate a greenhouse effect *(12,13)*. Actually, it is considered that most wet FGD processes have an inherent shortcoming of secondary pollution, or of high running cost if the secondary pollutant is avoided. A challenge to overcome such shortcomings still remains *(14)*.

To meet the challenge, novel green technologies with no/less secondary pollution and with a value-added product become essential. Fan et al. have developed a process of converting the SO2 to polymeric ferric sulfate, which can be employed as a common coagulant for water and wastewater treatment *(12)*.

Electrochemical techniques, utilizing electrons as a clean reagent, exactly enjoy the sustainability. Since most wet FGD processes embrace a sub-process of electron transfer for the oxidation of S(IV) to S(VI), the electrochemical techniques appear to fitfully work there. The electrochemical cleanup of flue gas has already been tested. For example, SO2 can be anodically oxidized to H2SO4 in aqueous solution *(14-16)*, and regeneration of FGD agents is developed by using electrodialysis with a bipolar membrane *(17,18)*. It should be noted that as air coexists with the SO2 in flue gas, electrochemical utilization of the molecular oxygen from air to further oxide the SO2 is indispensable and should be encouraged. Such a new concept, however, has not been implemented so far.

To convert the SO2 to be a value-added product without secondary pollution, this study aims at developing such a novel and green process by designing a series of electrochemical reactions through a SO2 absorption-and-conversion process. In the process, a few considerations in the process can benefit the attempt. (i) The cathodic reaction utilizes O2 from air to scavenge the process-released H+ ions, while the anodic reaction uses H2O to supply H+ ions. (ii) The H+ scavenging benefits the SO2 absorption and its further oxidation. (iii) The H+ supply benefits the formation of a bisulfate. Consequently, the SO2 conversion is driven electrochemically to form NaHSO4 as a sulfur-containing product.

NaHSO4 is a valuable chemical and widely used as an additive in manufacture of dye stuff, a soil amender in agriculture, and replacement of H2SO4 in industry for pH adjustment and catalytic reactions. This study focused on the chemical and sustainable fundamentals as well as the pH optimization for the SO2 oxidation. The findings are expected to lay a basis of understanding this new design with potential to convert the SO2 from flue gas to NaHSO4 as a value-added product in a green way.

## **2. Chemical fundamentals of the process**

In this process, the SO2 is designed to be absorbed into aqueous solution with alkaline, then oxidized to sulfate, and then transformed into bisulfate. The chemical fundamentals should be clarified to understand how the process works.

**Absorption of SO2.** In the wet FGD processes, H+ ions are released upon the absorption of SO2 into the aqueous solution *(5)*:

$$4SO\_2 + H\_2O \Leftrightarrow H^+ + HSO\_3^- \tag{2}$$

$$+\text{HSO}\_3^- \Leftrightarrow H^+ + \text{SO}\_3^{2-} \tag{3}$$

accumulation in the atmosphere is recognized to aggravate a greenhouse effect *(12,13)*. Actually, it is considered that most wet FGD processes have an inherent shortcoming of secondary pollution, or of high running cost if the secondary pollutant is avoided. A

To meet the challenge, novel green technologies with no/less secondary pollution and with a value-added product become essential. Fan et al. have developed a process of converting the SO2 to polymeric ferric sulfate, which can be employed as a common coagulant for water

Electrochemical techniques, utilizing electrons as a clean reagent, exactly enjoy the sustainability. Since most wet FGD processes embrace a sub-process of electron transfer for the oxidation of S(IV) to S(VI), the electrochemical techniques appear to fitfully work there. The electrochemical cleanup of flue gas has already been tested. For example, SO2 can be anodically oxidized to H2SO4 in aqueous solution *(14-16)*, and regeneration of FGD agents is developed by using electrodialysis with a bipolar membrane *(17,18)*. It should be noted that as air coexists with the SO2 in flue gas, electrochemical utilization of the molecular oxygen from air to further oxide the SO2 is indispensable and should be encouraged. Such a new

To convert the SO2 to be a value-added product without secondary pollution, this study aims at developing such a novel and green process by designing a series of electrochemical reactions through a SO2 absorption-and-conversion process. In the process, a few considerations in the process can benefit the attempt. (i) The cathodic reaction utilizes O2 from air to scavenge the process-released H+ ions, while the anodic reaction uses H2O to supply H+ ions. (ii) The H+ scavenging benefits the SO2 absorption and its further oxidation. (iii) The H+ supply benefits the formation of a bisulfate. Consequently, the SO2 conversion is

NaHSO4 is a valuable chemical and widely used as an additive in manufacture of dye stuff, a soil amender in agriculture, and replacement of H2SO4 in industry for pH adjustment and catalytic reactions. This study focused on the chemical and sustainable fundamentals as well as the pH optimization for the SO2 oxidation. The findings are expected to lay a basis of understanding this new design with potential to convert the SO2 from flue gas to NaHSO4 as

In this process, the SO2 is designed to be absorbed into aqueous solution with alkaline, then oxidized to sulfate, and then transformed into bisulfate. The chemical fundamentals should

**Absorption of SO2.** In the wet FGD processes, H+ ions are released upon the absorption of

*SO H O H HSO* 2 2 <sup>3</sup>

<sup>2</sup> *HSO H SO* 3 3

(2)

(3)

driven electrochemically to form NaHSO4 as a sulfur-containing product.

challenge to overcome such shortcomings still remains *(14)*.

concept, however, has not been implemented so far.

a value-added product in a green way.

SO2 into the aqueous solution *(5)*:

**2. Chemical fundamentals of the process** 

be clarified to understand how the process works.

and wastewater treatment *(12)*.

**Oxidation of SO2 to SO42-.** After the absorption, the absorbed SO2 is oxidized by air in aqueous solution at moderate pH from HSO3 and SO3 2- to SO4 2- ions. This oxidation process is expressed as follows, of which eq 4 releases H+ ions, but eq 5 does not*(19)*:

$$HSO\_3^- + \frac{1}{2}O\_2 \rightarrow SO\_4^{2-} + H^+ \tag{4}$$

$$\mathrm{SO}\_3^{2-} + \frac{1}{2}\mathrm{O}\_2 \rightarrow \mathrm{SO}\_4^{2-} \tag{5}$$

**ESH.** The H+ ions released through eqs 2~4 need to be scavenged due to their hindrance of the SO2 absorption. Otherwise, the continuous absorption of SO2 will be terminated. It can be noted that while O2 in air is utilized for the SO2 oxidation through eqs 4 and 5, the cathodic reduction of O2 can be employed to scavenge the process-released H+ ions. The reactions of O2 reduction through a 2-electron process at acidic and neutral/alkaline conditions are expressed in eqs 6 and 6', respectively *(20-22)*:

$$2\text{ O}\_2 + 2\text{H}^+ + 2\text{e} \rightarrow \text{H}\_2\text{O}\_2\tag{6}$$

$$2H\_2 + 2H\_2O + 2e \to H\_2O\_2 + 2OH^- \tag{6'}$$

At the same time, a side reaction co-exists with eq 6 below:

$$2H^{+} + 2e \to H\_{2} \uparrow \tag{7}$$

It can be seen that eqs 6 and 7 consume H+ ions and eq 6' supplies OH ions. All these reactions can be utilized to scavenge the H+ ions released through eqs 2~4.

**Transformation of SO42- to Bisulfate.** As coupled to the cathodic reactions, an anodic reaction is shown below:

$$H\_2O \to \frac{1}{2}O\_2\uparrow + 2H^+ + 2e\tag{8}$$

Under an extremely acidic condition, the H+ ions in eq 8 are combined with the SO42- ions formed in eqs 4 and 5 to form a bisulfate:

$$\text{lSO}\_4^{2-} + \text{H}^+ \Leftrightarrow \text{bisulfate ion} \tag{9}$$

As a result, a model experiment becomes necessary to chemically substantiate this design by disclosing the ESH effect on the SO2 absorption and oxidation, and by confirming the formation of NaHSO4 as a product of desulfurization.

#### **3. Experimental section**

**Chemicals and Reagents.** SO2 gas (99.9%) was obtained from KEDI, Foshan, China. Other chemicals as analytical reagents were used as obtained. Double distilled water was used in all experiments.

Electrochemically-Driven and Green Conversion of SO2 to NaHSO4 in Aqueous Solution 67

simultaneously in the SR chamber. This start-up procedure continued till the pH increase to ≥ 9.0 in the DS chamber. Then, the solution in the SR chamber was discarded, and the

Thereafter, the SO2 absorption of Step I was performed without current application. Initially, nitrogen gas was bubbled into the solution to remove any oxygen, then gaseous SO2 was

In the A-SO2 oxidation of Step I, an air flow at 100 mL min-1 was purged onto the cathode placed in the A-SO2 solution. And a cathodic current was applied to maintain the electrochemical reactions. The A-SO2 oxidation proceeded till the solution pH recovered to

To optimize the pH for the A-SO2 oxidation, a set of experiments was performed at 1.0 mM A-SO2 concentration, and different pHs in the range of 4.0~8.0 were maintained by chemical

In the transformation of SO42- to bisulfate of Step II, the reacted solution of Step I was relocated from the DS chamber to the SR chamber. Step II proceeded under a cathodic current with solution pH decrease in the SR chamber, while it stopped upon that the pH in

In Step I, the A-SO2 concentrations were monitored by taking 1.0 mL of solution samples at pre-set time intervals, and 5 µL of methanol was injected into the samples taken during the A-SO2 oxidation to quench any possible radical reaction (*23)*. After Step I, SO42 concentrations were measured. After Step II, H+, Na+, and SO42- concentrations were

Notably, in actual wet FGD processes, the SO2 absorption and oxidation in Step I occur concurrently. However, to understand the ESH effect on the SO2 absorption and its oxidation independently, the two experiments were conducted separately. At the same time, the air content in actual FGD processes should be minimized, and thus a small rate of 100 mL min-1 was fixed without further optimization. It was believed that an alkaline condition at pH > 7.0 was beneficial to the SO2 absorption, and an acidic condition at pH < 3.0 was beneficial to the NaHSO4 formation, while the A-SO2 oxidation relied on pH, so the pH

**Chemical Analysis.** Measurement of the concentrations of electrochemically-generated OHand H+ ions is accomplished by counting the dose of added H+ solution with a known concentration to determine the amount of OH-, and by counting that of OH- to determine that of H+. The A-SO2 concentration was determined in terms of S(IV) concentration using a UV-VIS spectrosphotometer (TU1810, Universal Analysis, Beijing, China) according to a reported procedure *(24)*. The H2O2 concentration was determined by spectrophotometry according to a potassium titanium (IV) oxalate method *(20)*. The SO42- and Na+ concentrations were measured by ion chromatography (Dionex DX-600, U.S.). The cyclic voltammetry of A-SO2 solution was performed in a N2-saturated solution with 0.05 mM Na2SO4 as electrolyte on a CHI work station (Chenhua, Shanghai, China) with 50 mV s-1 scanning rate. The pH was monitored by a pH meter (PB-10, Sartorius, Shanghai, China).

introduced into the alkaline solution to form A-SO2 till pH decreased below 7.0.

ions was utilized to absorb the

solution in the DS chamber containing the pre-stored OH-

SO2 gas in Step I of the model experiment.

dosing of 0.01 M NaOH solution except at pH0 8.0.

the DS chamber reached the pH0 value at the start-up step.

optimization for the A-SO2 oxidation was performed.

neutral pH (7.0).

measured.

**Experimental Procedure.** The model experiment to substantiate this design was performed in an experimental setup as schemed in Figure 1, which consists of two 200 mL chambers: a DS chamber with a graphite rod (ø = 6.4 mm, and L = 200 mm, Chenhua, Shanghai, China) as the cathode and a saturated calomel electrode as the reference electrode, and an SR chamber with a Pt flake (2 × 1.5 cm2, Chenhua, Shanghai, China) as the anode. Both chambers were connected by a salt bridge containing saturated Na2SO4 solution with agar. A PS-1 potentiostat/galvanostat (Zhongfu, Beijing, China) was employed to apply a cathodic current. The solution temperature was kept by a water bath at 25.0 ± 0.5 oC and monitored by a thermometer. Air was purged onto the cathode surface by an air pump through a glass frit diffuser, and a needle valve was used to control its flow rate.

Fig. 1. Scheme of the experimental setup: DS chamber is desulfurization chamber, and SR chamber is sulfur-recovery chamber.

To quantify the OH- ions electrochemically generated in the DS chamber and the H+ ions in the SR chamber, electrochemical reactions of eqs 6, 6', 7 and 8 were performed in 0.01 M Na2SO4 solution without SO2 at pH0 6.0, in which an air flow of 100 mL min-1 and different current densities of 0.10, 0.15, 0.20, 0.25, and 0.30 mA cm-2 were applied. During the reactions, 0.01 M HCl and 0.01 M NaOH solutions were fed by a pump (Longer BTOQ-50M, Baoding, China) into the DS chamber and SR chamber, respectively, to maintain the pH at 6.0 ± 0.2. At the same time, 1.0 mL of solution sample was taken from the DS chamber for the quantification of H2O2 generation.

After that, two steps of the model experiment were performed in a batch mode. Step I was performed in the DS chamber, and Step II, in the SR chamber. Each experiment was performed three times and the values of experimental data in average are presented.

Prior to Step I, a start-up procedure was carried out to pre-store OH- ions in the DS chamber through eqs 6', and thus a solution pH0 ≥ 9.0 in this chamber was obtained. The start-up procedure was described below, which was performed in the setup as schemed in Figure 1. Upon application of a cathodic current density as large as 0.60 mA cm-1 to preclude the generation of cathodic byproduct H2O2 *(20)*, air with 100 mL min-1 flow rate was bubbled into water in the DS chamber to allow eq 6' to occur. The coupled reaction of eq 8 occurred

**Experimental Procedure.** The model experiment to substantiate this design was performed in an experimental setup as schemed in Figure 1, which consists of two 200 mL chambers: a DS chamber with a graphite rod (ø = 6.4 mm, and L = 200 mm, Chenhua, Shanghai, China) as the cathode and a saturated calomel electrode as the reference electrode, and an SR chamber with a Pt flake (2 × 1.5 cm2, Chenhua, Shanghai, China) as the anode. Both chambers were connected by a salt bridge containing saturated Na2SO4 solution with agar. A PS-1 potentiostat/galvanostat (Zhongfu, Beijing, China) was employed to apply a cathodic current. The solution temperature was kept by a water bath at 25.0 ± 0.5 oC and monitored by a thermometer. Air was purged onto the cathode surface by an air pump

through a glass frit diffuser, and a needle valve was used to control its flow rate.

Fig. 1. Scheme of the experimental setup: DS chamber is desulfurization chamber, and SR

the SR chamber, electrochemical reactions of eqs 6, 6', 7 and 8 were performed in 0.01 M Na2SO4 solution without SO2 at pH0 6.0, in which an air flow of 100 mL min-1 and different current densities of 0.10, 0.15, 0.20, 0.25, and 0.30 mA cm-2 were applied. During the reactions, 0.01 M HCl and 0.01 M NaOH solutions were fed by a pump (Longer BTOQ-50M, Baoding, China) into the DS chamber and SR chamber, respectively, to maintain the pH at 6.0 ± 0.2. At the same time, 1.0 mL of solution sample was taken from the DS chamber for

After that, two steps of the model experiment were performed in a batch mode. Step I was performed in the DS chamber, and Step II, in the SR chamber. Each experiment was

Prior to Step I, a start-up procedure was carried out to pre-store OH- ions in the DS chamber through eqs 6', and thus a solution pH0 ≥ 9.0 in this chamber was obtained. The start-up procedure was described below, which was performed in the setup as schemed in Figure 1. Upon application of a cathodic current density as large as 0.60 mA cm-1 to preclude the generation of cathodic byproduct H2O2 *(20)*, air with 100 mL min-1 flow rate was bubbled into water in the DS chamber to allow eq 6' to occur. The coupled reaction of eq 8 occurred

performed three times and the values of experimental data in average are presented.

ions electrochemically generated in the DS chamber and the H+ ions in

chamber is sulfur-recovery chamber.

the quantification of H2O2 generation.

To quantify the OH-

simultaneously in the SR chamber. This start-up procedure continued till the pH increase to ≥ 9.0 in the DS chamber. Then, the solution in the SR chamber was discarded, and the solution in the DS chamber containing the pre-stored OH ions was utilized to absorb the SO2 gas in Step I of the model experiment.

Thereafter, the SO2 absorption of Step I was performed without current application. Initially, nitrogen gas was bubbled into the solution to remove any oxygen, then gaseous SO2 was introduced into the alkaline solution to form A-SO2 till pH decreased below 7.0.

In the A-SO2 oxidation of Step I, an air flow at 100 mL min-1 was purged onto the cathode placed in the A-SO2 solution. And a cathodic current was applied to maintain the electrochemical reactions. The A-SO2 oxidation proceeded till the solution pH recovered to neutral pH (7.0).

To optimize the pH for the A-SO2 oxidation, a set of experiments was performed at 1.0 mM A-SO2 concentration, and different pHs in the range of 4.0~8.0 were maintained by chemical dosing of 0.01 M NaOH solution except at pH0 8.0.

In the transformation of SO42- to bisulfate of Step II, the reacted solution of Step I was relocated from the DS chamber to the SR chamber. Step II proceeded under a cathodic current with solution pH decrease in the SR chamber, while it stopped upon that the pH in the DS chamber reached the pH0 value at the start-up step.

In Step I, the A-SO2 concentrations were monitored by taking 1.0 mL of solution samples at pre-set time intervals, and 5 µL of methanol was injected into the samples taken during the A-SO2 oxidation to quench any possible radical reaction (*23)*. After Step I, SO4 2 concentrations were measured. After Step II, H+, Na+, and SO42- concentrations were measured.

Notably, in actual wet FGD processes, the SO2 absorption and oxidation in Step I occur concurrently. However, to understand the ESH effect on the SO2 absorption and its oxidation independently, the two experiments were conducted separately. At the same time, the air content in actual FGD processes should be minimized, and thus a small rate of 100 mL min-1 was fixed without further optimization. It was believed that an alkaline condition at pH > 7.0 was beneficial to the SO2 absorption, and an acidic condition at pH < 3.0 was beneficial to the NaHSO4 formation, while the A-SO2 oxidation relied on pH, so the pH optimization for the A-SO2 oxidation was performed.

**Chemical Analysis.** Measurement of the concentrations of electrochemically-generated OHand H+ ions is accomplished by counting the dose of added H+ solution with a known concentration to determine the amount of OH-, and by counting that of OH- to determine that of H+. The A-SO2 concentration was determined in terms of S(IV) concentration using a UV-VIS spectrosphotometer (TU1810, Universal Analysis, Beijing, China) according to a reported procedure *(24)*. The H2O2 concentration was determined by spectrophotometry according to a potassium titanium (IV) oxalate method *(20)*. The SO4 2- and Na+ concentrations were measured by ion chromatography (Dionex DX-600, U.S.). The cyclic voltammetry of A-SO2 solution was performed in a N2-saturated solution with 0.05 mM Na2SO4 as electrolyte on a CHI work station (Chenhua, Shanghai, China) with 50 mV s-1 scanning rate. The pH was monitored by a pH meter (PB-10, Sartorius, Shanghai, China).

Electrochemically-Driven and Green Conversion of SO2 to NaHSO4 in Aqueous Solution 69

increased first quickly, then slowly to a plateau. Comparatively, at pH0 9.0, the A-SO2 concentration increased most rapidly and ended at the highest level. The acceleration of SO2 absorption at pH0 9.0 was caused by the lifted pH, which was realized by pre-storing the OH- ions electrochemically. Obviously, the pre-stored OH- ions served to scavenge the SO2 absorption-released H+ ions as a means of ESH. Thus, the SO2 absorption was accelerated by

**0 10 20 30 40 50 60**

**Time (min)**

Fig. 3. Buildup of A-SO2 concentration in the SO2 absorption at solution pH0 5.0, 7.0, and 9.0.

**ESH Effect on A-SO2 Oxidation.** The oxidation-released H+ ions were in situ scavenged through eqs 6, 6' or 7. To disclose the ESH effect on the A-SO2 oxidation, two sets of SO2 oxidation experiments in Step I were carried out. In the first set with ESH, a current density at 0.20 mA cm-2 was applied to maintain the electrochemical reactions, while in the second

Figure 4 showed that with ESH, 100% and 95.8% of A-SO2 disappearances were achieved at 30 min for the 1.0 mM A-SO2 and 1.5 mM A-SO2 solutions, respectively. Following the oxidation reaction, SO42- concentrations were detected, and the results as listed in Table 1 indicated that 95.0% and 88.0% of the A-SO2 were converted to SO42- ions. By contrast without SH, only 70.7% of the 1.0 mM A-SO2 and 60.9% of the 1.5 mM A-SO2 disappeared after 30 min, and the SO42- concentrations in the reacted solution were significantly lower than those with ESH (Table 1). From these it could be understood that the A-SO2 oxidation with ESH proceeded more rapidly than that without SH, since ESH was beneficial to the

**pH0 9.0 pH0 7.0 pH0 5.0**

the ESH.

**0.0**

set without SH, no current density was applied.

conversion of A-SO2 oxidation to SO42-.

**0.2**

**A-SO**

**2**

**Concentration (mM)**

**0.4**

**0.6**

**0.8**

**1.0**

## **4. Results and discussion**

**Electrochemical Generation of OH and H+ Ions.** This designed process underlined (i) that the electrochemical scavenging of process-released H+ ions due to eqs 2~4 would benefit the SO2 absorption and oxidation, and (ii) that the electrochemical supply of H+ ions through eq 8 would realize the NaHSO4 formation. Three cathodic reactions of eqs 6, 6', and 7 served to increase the solution pH and electrochemical generation of OH ions should be considered. Figure 2A illustrated that the OH concentration in the DS chamber increased proportionally to the reaction time and the accumulative rate of OH ions depended on the applied current density. In the meantime, H2O2 was generated through eq 6 and 6'. Figure 1B revealed that the H2O2 concentration increased against the reaction time. As paired to the cathodic reactions, anodic reaction of eq 8 occurred to supply H+ ions in the SR chamber. Measurements of the H+ ions revealed that the H+ concentration increased at the same rate as that of OH ions in the DS chamber (not shown here).

The electrochemically-generated OH ions in the DS chamber and H+ ions in the SR chamber carried electrons, which must be balanced electrically to keep the electrical neutralization of solution in each chamber. An analysis of electron balance will be described later.

Fig. 2. Buildup of electrochemically-generated OH- ions (A), and H2O2 (B) in the DS chamber.

**ESH Effect on SO2 Absorption.** The above electrochemically-generated OH- ions could be utilized to scavenge the absorption-released H+ ions through eqs 2 and 3. ESH has two functions. One was the pre-storage of OH- ions through eq 6', which later served to scavenge the SO2-absorption-released H+ ions. The other was the *in situ* scavenging of the process-released H+ ions through eqs 6 and 7 or *in situ* supplying OH- ions through eq 6', which also served to scavenge the A-SO2-oxidation-released H+ ions.

To disclose the ESH effect on the SO2 absorption, one SO2 absorption experiment in Step I of the model experiment was performed at pH0 9.0 with pre-stored OH- ions, and then two additional SO2 absorption experiments without pre-stored OH- ions were performed at pH0 5.0 and 7.0, respectively. Figure 3 revealed that under the three pH conditions, the A-SO2

the electrochemical scavenging of process-released H+ ions due to eqs 2~4 would benefit the SO2 absorption and oxidation, and (ii) that the electrochemical supply of H+ ions through eq 8 would realize the NaHSO4 formation. Three cathodic reactions of eqs 6, 6', and 7 served to

density. In the meantime, H2O2 was generated through eq 6 and 6'. Figure 1B revealed that the H2O2 concentration increased against the reaction time. As paired to the cathodic reactions, anodic reaction of eq 8 occurred to supply H+ ions in the SR chamber. Measurements of the H+ ions revealed that the H+ concentration increased at the same rate

carried electrons, which must be balanced electrically to keep the electrical neutralization of

**0.0**

**0.1**

**H2O2**

Fig. 2. Buildup of electrochemically-generated OH- ions (A), and H2O2 (B) in the DS

which also served to scavenge the A-SO2-oxidation-released H+ ions.

**ESH Effect on SO2 Absorption.** The above electrochemically-generated OH- ions could be utilized to scavenge the absorption-released H+ ions through eqs 2 and 3. ESH has two functions. One was the pre-storage of OH- ions through eq 6', which later served to scavenge the SO2-absorption-released H+ ions. The other was the *in situ* scavenging of the process-released H+ ions through eqs 6 and 7 or *in situ* supplying OH- ions through eq 6',

To disclose the ESH effect on the SO2 absorption, one SO2 absorption experiment in Step I of the model experiment was performed at pH0 9.0 with pre-stored OH- ions, and then two additional SO2 absorption experiments without pre-stored OH- ions were performed at pH0 5.0 and 7.0, respectively. Figure 3 revealed that under the three pH conditions, the A-SO2

**concentration (mM)**

**0.2**

**0.3**

**0.4**

**0.5**

solution in each chamber. An analysis of electron balance will be described later.

increase the solution pH and electrochemical generation of OH-

ions in the DS chamber (not shown here).

**A**

to the reaction time and the accumulative rate of OH-

**0 10 20 30 40 50 60**

**Time (min)**

 **and H+ Ions.** This designed process underlined (i) that

concentration in the DS chamber increased proportionally

ions in the DS chamber and H+ ions in the SR chamber

 **I = 0.10 mA cm-2 I = 0.15 mA cm-2 I = 0.20 mA cm-2 I = 0.25 mA cm-2 I = 0.30 mA cm-2**

ions should be considered.

**B**

ions depended on the applied current

**0 10 20 30 40 50 60**

**Time (min)**

**4. Results and discussion** 

**Electrochemical Generation of OH-**

Figure 2A illustrated that the OH-

The electrochemically-generated OH-

 **I = 0.10 mA cm-2 I = 0.15 mA cm-2 I = 0.20 mA cm-2 I = 0.25 mA cm-2 I = 0.30 mA cm-2**

as that of OH-

**0.0**

chamber.

**0.5**

**1.0**

 **OH- concentration (mM)**

**1.5**

**2.0**

**2.5**

increased first quickly, then slowly to a plateau. Comparatively, at pH0 9.0, the A-SO2 concentration increased most rapidly and ended at the highest level. The acceleration of SO2 absorption at pH0 9.0 was caused by the lifted pH, which was realized by pre-storing the OH- ions electrochemically. Obviously, the pre-stored OH- ions served to scavenge the SO2 absorption-released H+ ions as a means of ESH. Thus, the SO2 absorption was accelerated by the ESH.

Fig. 3. Buildup of A-SO2 concentration in the SO2 absorption at solution pH0 5.0, 7.0, and 9.0.

**ESH Effect on A-SO2 Oxidation.** The oxidation-released H+ ions were in situ scavenged through eqs 6, 6' or 7. To disclose the ESH effect on the A-SO2 oxidation, two sets of SO2 oxidation experiments in Step I were carried out. In the first set with ESH, a current density at 0.20 mA cm-2 was applied to maintain the electrochemical reactions, while in the second set without SH, no current density was applied.

Figure 4 showed that with ESH, 100% and 95.8% of A-SO2 disappearances were achieved at 30 min for the 1.0 mM A-SO2 and 1.5 mM A-SO2 solutions, respectively. Following the oxidation reaction, SO42- concentrations were detected, and the results as listed in Table 1 indicated that 95.0% and 88.0% of the A-SO2 were converted to SO4 2- ions. By contrast without SH, only 70.7% of the 1.0 mM A-SO2 and 60.9% of the 1.5 mM A-SO2 disappeared after 30 min, and the SO42- concentrations in the reacted solution were significantly lower than those with ESH (Table 1). From these it could be understood that the A-SO2 oxidation with ESH proceeded more rapidly than that without SH, since ESH was beneficial to the conversion of A-SO2 oxidation to SO42-.

Electrochemically-Driven and Green Conversion of SO2 to NaHSO4 in Aqueous Solution 71

**Transformation of Na2SO4 to NaHSO4.** Beyond the utilization of cathodic reaction to scavenge the H+ ions released in eqs 2~4, this new process utilized an anodic reaction (eq 8) to supply H+ ions which combined the SO42- to form bisulfate. In both Steps I and II, the H+ ions were produced in the SR chamber. Step II was performed by relocating the reacted solution of Step I from the DS chamber to the SR chamber where eq 8 occurred. Thus after this step, three ions of Na+, H+, and SO42- presented in the SR chamber, and the analysis of mass balance of electrons as would be shown later suggested that a mixture of the three ions

To confirm the NaHSO4 formation, after the two-step model experiments with 1.0 mM and 1.5 mM A-SO2 concentrations, the concentrations of the three ions in the SR chamber were measured. The results as shown in Figure 5 demonstrated that a mass balance of Na+, H+

**Na H SO**

**Optimization of pH for A-SO2 Oxidation with ESH.** Figure 6A demonstrated that pH 5.0~6.0 was optimal for the A-SO2 oxidation, which was in good agreement with others'

seawater FGD process *(24)*. Figure 6B showed the records of cyclic voltammetry in A-SO2

Figure 6B illustrates the cyclic voltammetry recorded in the 1.0 mM A-SO2 solution, and the peaks at 0.12~0.15 V and 0.81~0.83 V were associated with the HSO3- oxidation and the SO3

oxidation, respectively. On the other hand, the A-SO2 solution consists of two major species

occupies 100%, 92%, 38% and 10% of A-SO2 at pH0 5.0, 6.0, 7.0 and 8.0, respectively *(27)*.

Fig. 5. Ion concentrations measured in the SR chamber after the model experiment.

solution, which confirmed that the optimal pH for the SO2 oxidation was 5.0~6.0.

of HSO3- and SO32- in the pH range of 4.0~9.5. It has been documented that the HSO3-

**+**

 **1.0 mM A-SO2 1.5 mM A-SO2**

> **4 2-**

ions in the SO2 oxidation by air during a

2-

species

and SO42- ions was approximately 1:1:1, which ensured the NaHSO4 formation.

might result in the formation of NaHSO4 through eq 9.

**0**

**+**

results of optimal pH 6.0 kept by addition of OH-

**1**

**2**

**Ion concentration (mM)**

**3**

**4**

Fig. 4. Temporal disappearance of A-SO2 concentration in the A-SO2 oxidation at pH0 6.0, with ESH at 0.20 mA cm-2 current density for 1.0 mM A-SO2 and 0.25 mA cm-2 current density for 1.5 mM A-SO2, with CSH, and without SH.


Table 1. SO4 2- concentrations after the A-SO2 oxidation at pH0 6.0, with ESH at 0.20 mA cm-2 current density for 1.0 mM A-SO2 and 0.25 mA cm-2 current density for 1.5 mM A-SO2, with CSH, and without SH.

To further disclose the ESH effect, a set of experiments in 1.0 mM and 1.5 mM A-SO2 solutions was carried out with CSH, and the results are added in Figure 4. Clearly, the A-SO2 oxidations with CSH proceeded more rapidly than those without SH, but more slowly than those with ESH. Therefore, it was further confirmed that the SH benefited the A-SO2 oxidation, while the ESH was more effective than the CSH.

Moreover, the results in Table 1 showed that more SO4 2- ions were obtained in the reacted solution with ESH than those with CSH. It was believed that the H2O2 produced through eq 6 or 6' could enhance the SO2 oxidation as an oxidizing reagent *(27,28)*. After the reaction, no H2O2 residue left as impurity in the final sulfur-containing product. Therefore, the ESH benefited the A-SO2 oxidation with two advantages of (i) scavenging the absorption- and oxidation-released H+ ions, and (ii) simultaneously generating H2O2 to facilitate the conversion of A-SO2 to SO4 2-.

**1.5 mM without SH 1.0 mM without SH 1.5 mM with CSH 1.0 mM with CSH 1.5 mM with ESH 1.0 mM with ESH**

**0 5 10 15 20 25 30**

Fig. 4. Temporal disappearance of A-SO2 concentration in the A-SO2 oxidation at pH0 6.0, with ESH at 0.20 mA cm-2 current density for 1.0 mM A-SO2 and 0.25 mA cm-2 current

> 1.0 0.98 0.78 0.66 1.5 1.32 1.15 0.97

current density for 1.0 mM A-SO2 and 0.25 mA cm-2 current density for 1.5 mM A-SO2, with

To further disclose the ESH effect, a set of experiments in 1.0 mM and 1.5 mM A-SO2 solutions was carried out with CSH, and the results are added in Figure 4. Clearly, the A-SO2 oxidations with CSH proceeded more rapidly than those without SH, but more slowly than those with ESH. Therefore, it was further confirmed that the SH benefited the A-SO2

Moreover, the results in Table 1 showed that more SO42- ions were obtained in the reacted solution with ESH than those with CSH. It was believed that the H2O2 produced through eq 6 or 6' could enhance the SO2 oxidation as an oxidizing reagent *(27,28)*. After the reaction, no H2O2 residue left as impurity in the final sulfur-containing product. Therefore, the ESH benefited the A-SO2 oxidation with two advantages of (i) scavenging the absorption- and oxidation-released H+ ions, and (ii) simultaneously generating H2O2 to facilitate the

2- concentrations after the A-SO2 oxidation at pH0 6.0, with ESH at 0.20 mA cm-2

**Time (min)**

**SO42- with CSH (mM)** 

**SO42- without SH (mM)** 

**0**

density for 1.5 mM A-SO2, with CSH, and without SH.

oxidation, while the ESH was more effective than the CSH.

**SO42- with ESH (mM)** 

**20**

**A-SO**

**A-SO2 (mM)** 

CSH, and without SH.

conversion of A-SO2 to SO42-.

Table 1. SO4

**2 residual (%)**

**40**

**60**

**80**

**100**

**Transformation of Na2SO4 to NaHSO4.** Beyond the utilization of cathodic reaction to scavenge the H+ ions released in eqs 2~4, this new process utilized an anodic reaction (eq 8) to supply H+ ions which combined the SO42- to form bisulfate. In both Steps I and II, the H+ ions were produced in the SR chamber. Step II was performed by relocating the reacted solution of Step I from the DS chamber to the SR chamber where eq 8 occurred. Thus after this step, three ions of Na+, H+, and SO42- presented in the SR chamber, and the analysis of mass balance of electrons as would be shown later suggested that a mixture of the three ions might result in the formation of NaHSO4 through eq 9.

To confirm the NaHSO4 formation, after the two-step model experiments with 1.0 mM and 1.5 mM A-SO2 concentrations, the concentrations of the three ions in the SR chamber were measured. The results as shown in Figure 5 demonstrated that a mass balance of Na+, H+ and SO4 2- ions was approximately 1:1:1, which ensured the NaHSO4 formation.

Fig. 5. Ion concentrations measured in the SR chamber after the model experiment.

**Optimization of pH for A-SO2 Oxidation with ESH.** Figure 6A demonstrated that pH 5.0~6.0 was optimal for the A-SO2 oxidation, which was in good agreement with others' results of optimal pH 6.0 kept by addition of OH ions in the SO2 oxidation by air during a seawater FGD process *(24)*. Figure 6B showed the records of cyclic voltammetry in A-SO2 solution, which confirmed that the optimal pH for the SO2 oxidation was 5.0~6.0.

Figure 6B illustrates the cyclic voltammetry recorded in the 1.0 mM A-SO2 solution, and the peaks at 0.12~0.15 V and 0.81~0.83 V were associated with the HSO3 - oxidation and the SO3 2 oxidation, respectively. On the other hand, the A-SO2 solution consists of two major species of HSO3- and SO32- in the pH range of 4.0~9.5. It has been documented that the HSO3- species occupies 100%, 92%, 38% and 10% of A-SO2 at pH0 5.0, 6.0, 7.0 and 8.0, respectively *(27)*.

Electrochemically-Driven and Green Conversion of SO2 to NaHSO4 in Aqueous Solution 73

In the DS chamber, Step I, the absorption-released H+ ions through eqs 2 and 3 were

solution at pH 5.0~6.0 *(27)*. Clearly, the HSO3- ions were electrically balanced by the pre-

In the DS chamber, Step I, the oxidation-released H+ ions through eq 4 were scavenged by

eq 4 generated SO42- ions as product of Step I. Concurrently, Na+ ions were released from the salt bridge to balance the SO42- ions electrically. In the SR chamber, Step I, accompanied generation of H+ ions occurred through eq 8. The H+ ions were balanced electrically by the

In the SR chamber, Step II, the H+ and SO42- ions from Step I that were electrically balanced mutually from Step I stayed there. And the mutually balanced Na+ and SO42 ions from Step I were relocated to this chamber. At the same time, H+ ions continued to be generated through eq 8. Accordingly, SO42- ions continued to release from the salt bridge to balance the H+ ions. Therefore, the electron balance resulted in a mixture of H2SO4 and Na2SO4 in the SR chamber after Step II. Since a reaction of Na2SO4 and H2SO4 is often adopted to manufacture NaHSO4 in industry, NaHSO4 might be obtained as a product in

through eq 6'. The OH- ions were balanced electrically by the Na+ released from the salt

**Process of Electrochemically-driven Conversion of SO2 to NaHSO4.** Up to now, this process design has been chemically substantiated, and the oxidation reaction of A-SO2 can be optimized. Accordingly, a process of the SO2 conversion to NaHSO4 was schemed in

are presented in Figure 8. Figures 7 and 8 illustrate that the SO2 gas is converted to NaHSO4 through two stages: (i) SO2 absorption plus oxidation to Na2SO4 in the DS chamber, and (ii)

Additionally in the DS chamber, Step II, accompanied generation of OH-

ions that were in situ generated through eq 6, 6', or 7. The A-SO2 oxidation through

H+ from eq 8

Na+ and SO4

of Step I, H+ from eq 8

SO42- from salt bridge

SO42- from salt bridge

ions. After Step I, HSO3- ions predominated in the A-SO2

H+ and SO42- coming from Step I

2- relocated from DS chamber

ions occurred

and H+ ions, while those of Na+ and SO42- ions

**DS chamber SR chamber** 

Table 2. Ion species after each step in the DS chamber and SR chamber.

Step I HSO3-

SO42- from eq 4

Step II OH- from eq 6'


scavenged by the pre-stored OH-

the SR chamber after Step II.

Figure 5 to show the mass flows of SO2, OH-

transformation of Na2SO4 to NaHSO4 in the SR chamber.

bridge (see Figure 1).

stored Na+ ions.

the OH-

Na+ (pre-stored)

Na+ from salt bridge OH- from eq 6'

Na+ from salt bridge

SO42- ions released from the salt bridge (Figure 1).

Clearly, the peaks associated with the HSO3- oxidation were not pronounced at pH 7.0 and 8.0, while the peaks were mature at pH 5.0 and 6.0. On the contrary, the peaks associated with the SO32- oxidation were mature at pH 7.0 and 8.0, while no peak was observed at 5.0 and 6.0 since the SO32- species were only 8% and 0, respectively. These results implied that at optimal pH 5.0~6.0, the HSO3 - predominated in the A-SO2. Moreover, the HSO3 oxidation proceeded more rapidly at this pH range than at pHs beyond this range. Thus, the optimal pH for the A-SO2 oxidation was pH 5.0~6.0.

Fig. 6. Dependence of 1.0 mM A-SO2 disappearance after 30 min oxidation on the solution pH (A), and cyclic voltammetry recorded in 1.0 mM A-SO2 solution (B).

In real application, the current density should be adjusted to maintain the optimal range of pH 5.0~6.0 for the A-SO2 oxidation. Consequently, a set of 1.0 mM A-SO2 oxidation experiments was performed at pH0 5.0, 6.0, 7.0, and 8.0. It was found that when the current densities were adjusted to 0.20, 0.25, and 0.30 mA cm-2, the optimal pH 5.0~6.0 could be maintained for pH0 7.0, 6.0, and 5.0, respectively. Evidently, the current density to maintain the optimal pH increased with the pH0 decrease of A-SO2 solution. The lower pH0 meant more H+ ions in the solution, so the higher current density was required to scavenge the H+ ions. At pH0 8.0, the SO32- species predominated in the A-SO2 solution *(27)*, and no pH decrease was observed in the A-SO2 oxidation by air as indicated by eq 5. Thus no application of cathodic current to generate OH- ions was required.

**Analysis of Electron Balance in DS Chamber and SR Chamber.** In this study, OH ions were generated through eq 6' in the DS chamber, and H+ ions were generated through eq 8 in the SR chamber. The electrons carried by the OH- , H+, and other emerged ions must be balanced electrically to keep the electrical neutralization of solution in either chamber. Table 2 lists the ion species after each step in the DS chamber and SR chamber. From the ion species, the analysis of electron balance could be made.

8.0, while the peaks were mature at pH 5.0 and 6.0. On the contrary, the peaks associated with the SO32- oxidation were mature at pH 7.0 and 8.0, while no peak was observed at 5.0 and 6.0 since the SO32- species were only 8% and 0, respectively. These results implied that at

proceeded more rapidly at this pH range than at pHs beyond this range. Thus, the optimal

**-10**

Fig. 6. Dependence of 1.0 mM A-SO2 disappearance after 30 min oxidation on the solution

In real application, the current density should be adjusted to maintain the optimal range of pH 5.0~6.0 for the A-SO2 oxidation. Consequently, a set of 1.0 mM A-SO2 oxidation experiments was performed at pH0 5.0, 6.0, 7.0, and 8.0. It was found that when the current densities were adjusted to 0.20, 0.25, and 0.30 mA cm-2, the optimal pH 5.0~6.0 could be maintained for pH0 7.0, 6.0, and 5.0, respectively. Evidently, the current density to maintain the optimal pH increased with the pH0 decrease of A-SO2 solution. The lower pH0 meant more H+ ions in the solution, so the higher current density was required to scavenge the H+ ions. At pH0 8.0, the SO32- species predominated in the A-SO2 solution *(27)*, and no pH decrease was observed in the A-SO2 oxidation by air as indicated by eq 5. Thus no

**Analysis of Electron Balance in DS Chamber and SR Chamber.** In this study, OH-

were generated through eq 6' in the DS chamber, and H+ ions were generated through eq 8

balanced electrically to keep the electrical neutralization of solution in either chamber. Table 2 lists the ion species after each step in the DS chamber and SR chamber. From the ion

pH (A), and cyclic voltammetry recorded in 1.0 mM A-SO2 solution (B).

application of cathodic current to generate OH- ions was required.

in the SR chamber. The electrons carried by the OH-

species, the analysis of electron balance could be made.

**-5**

**0**

**Current** (**10-5 mA**)

**5**

**B**

**10**


**-1.0 -0.5 0.0 0.5 1.0 1.5 2.0**

, H+, and other emerged ions must be

**Voltage (vs SHE)**

**pH 8.0 pH 7.0** **Blank**

ions

predominated in the A-SO2. Moreover, the HSO3- oxidation

**pH 6.0 pH 5.0**

**pH 8.0 pH 6.0**

**pH 5.0 pH 7.0**

Clearly, the peaks associated with the HSO3

pH for the A-SO2 oxidation was pH 5.0~6.0.

**3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0**

**pH**

**0**

**20**

**40**

**60**

**A-SO2 disappearance** (**%**)

**80**

**A**

**100**


optimal pH 5.0~6.0, the HSO3


Table 2. Ion species after each step in the DS chamber and SR chamber.

In the DS chamber, Step I, the absorption-released H+ ions through eqs 2 and 3 were scavenged by the pre-stored OH ions. After Step I, HSO3- ions predominated in the A-SO2 solution at pH 5.0~6.0 *(27)*. Clearly, the HSO3- ions were electrically balanced by the prestored Na+ ions.

In the DS chamber, Step I, the oxidation-released H+ ions through eq 4 were scavenged by the OH ions that were in situ generated through eq 6, 6', or 7. The A-SO2 oxidation through eq 4 generated SO42- ions as product of Step I. Concurrently, Na+ ions were released from the salt bridge to balance the SO42- ions electrically. In the SR chamber, Step I, accompanied generation of H+ ions occurred through eq 8. The H+ ions were balanced electrically by the SO42- ions released from the salt bridge (Figure 1).

In the SR chamber, Step II, the H+ and SO42- ions from Step I that were electrically balanced mutually from Step I stayed there. And the mutually balanced Na+ and SO42 ions from Step I were relocated to this chamber. At the same time, H+ ions continued to be generated through eq 8. Accordingly, SO42- ions continued to release from the salt bridge to balance the H+ ions. Therefore, the electron balance resulted in a mixture of H2SO4 and Na2SO4 in the SR chamber after Step II. Since a reaction of Na2SO4 and H2SO4 is often adopted to manufacture NaHSO4 in industry, NaHSO4 might be obtained as a product in the SR chamber after Step II.

Additionally in the DS chamber, Step II, accompanied generation of OH ions occurred through eq 6'. The OH- ions were balanced electrically by the Na+ released from the salt bridge (see Figure 1).

**Process of Electrochemically-driven Conversion of SO2 to NaHSO4.** Up to now, this process design has been chemically substantiated, and the oxidation reaction of A-SO2 can be optimized. Accordingly, a process of the SO2 conversion to NaHSO4 was schemed in Figure 5 to show the mass flows of SO2, OH- and H+ ions, while those of Na+ and SO42- ions are presented in Figure 8. Figures 7 and 8 illustrate that the SO2 gas is converted to NaHSO4 through two stages: (i) SO2 absorption plus oxidation to Na2SO4 in the DS chamber, and (ii) transformation of Na2SO4 to NaHSO4 in the SR chamber.

Electrochemically-Driven and Green Conversion of SO2 to NaHSO4 in Aqueous Solution 75

Figure 7 illustrates that in the first stage, the SO2 gas with the co-existing air is introduced into the aqueous solution where the reactions through eqs 2~7 occur to form Na2SO4. The SO2 absorption occurs effectively at high pH, and the A-SO2 oxidation occurs rapidly at pH 5.0~6.0 but quite slowly at pH 8.0. Thus, although occurring concurrently, the SO2 absorption predominates first at pH above 6.0, and then the A-SO2 oxidation becomes the main reaction at pH 5.0~6.0. In the second stage, the solution containing Na2SO4 is relocated from the DS chamber to the SR chamber, and then the Na2SO4 is transformed into NaHSO4

Accompanied with the A-SO2 oxidation in the DS chamber, H+ ions are generated (eq 8) in the SR chamber for the subsequent formation of NaHSO4. Accompanied with the NaHSO4 transformation in the SR chamber, OH- ions are generated (eq 6') in the DS chamber and

To practice this new process, some findings from this study may be summarized as tips: (i) alkaline condition at pH > 7.0 in the DS chamber is beneficial to the SO2 absorption, which can be achieved by the electrochemical pre-storage of OH- ions through eq 6', (ii) electrochemical generation of H2O2 (eqs 6 and 6' ) in the DS chamber can be designed as a concurrent reaction with the A-SO2 oxidation reaction by air, since the H2O2 can accelerate the A-SO2 oxidation reaction significantly, and (iii) a suitable current density shall be

Thus by combination of the mass flow in Figure 8, an overall reaction can be expressed below to illustrate the SO2 conversion into NaHSO4 under the electrical driving force:

> 2 2 2 24 4 <sup>1</sup> <sup>2</sup>

Eq 10 indicates that from a thermodynamic point of view, the electrochemical reactions of eqs 6, 6', 7 and 8 may not be essential, but from an engineering point of view, they are critical. The cathodic reactions scavenge the absorption- and oxidation-released H+ ions to drive the SO2 absorption and oxidation, and the anodic reaction provides H+ ions to drive the NaHSO4 formation. Therefore, the electrochemical reactions ensure the process of SO2

**Sustainability Evaluation of The Process.** Green chemistry has a set of principles to minimize pollution as far as possible in chemical processes, including achieving high value of atom economy in the synthetic process, eliminating toxicity to human health and the environment, minimizing energy consumption, utilizing clean materials, and so on *(28,29)*. Although the aspiration of green chemistry is preferably realized in the manufacturing processes rather than the subsequent cleanup of effluent, the principles are still applicable in the wet FGD process. In this study, sustainability of this new process was evaluated from

The atom economy is expressed by *AU*, which is defined by a ratio of mole mass between

*mole mass in desired product AU*

tan ( )

*electricity SO O H O Na SO NaHSO* (10)

100%

*mole mass in reac t s* (11)

adjusted to maintain the optimal pH 5.0~6.0 of the A-SO2 oxidation reaction.

2

the desired product and the reactant(s) as shown below:

(eq 9) under an acidic condition through eq 8.

pre-stored for the operation of next run.

conversion to NaHSO4 through eq 10.

the perspective of green chemistry.

Fig. 7. Mass flow of SO2, OH-, and H+ ions in the process of SO2 conversion to NaHSO4 in aqueous solution: the number before the species designates their mole mass.

Fig. 8. Mass flow of Na+ (A) and SO42- (B) ions in the proposed process of SO2 conversion to NaHSO4 in aqueous solution: the number before the species designates their mole mass; chambers in left hand are DS chamber, and in right hand, SR chamber.

Fig. 7. Mass flow of SO2, OH-, and H+ ions in the process of SO2 conversion to NaHSO4 in

Fig. 8. Mass flow of Na+ (A) and SO42- (B) ions in the proposed process of SO2 conversion to NaHSO4 in aqueous solution: the number before the species designates their mole mass;

chambers in left hand are DS chamber, and in right hand, SR chamber.

aqueous solution: the number before the species designates their mole mass.

Figure 7 illustrates that in the first stage, the SO2 gas with the co-existing air is introduced into the aqueous solution where the reactions through eqs 2~7 occur to form Na2SO4. The SO2 absorption occurs effectively at high pH, and the A-SO2 oxidation occurs rapidly at pH 5.0~6.0 but quite slowly at pH 8.0. Thus, although occurring concurrently, the SO2 absorption predominates first at pH above 6.0, and then the A-SO2 oxidation becomes the main reaction at pH 5.0~6.0. In the second stage, the solution containing Na2SO4 is relocated from the DS chamber to the SR chamber, and then the Na2SO4 is transformed into NaHSO4 (eq 9) under an acidic condition through eq 8.

Accompanied with the A-SO2 oxidation in the DS chamber, H+ ions are generated (eq 8) in the SR chamber for the subsequent formation of NaHSO4. Accompanied with the NaHSO4 transformation in the SR chamber, OH- ions are generated (eq 6') in the DS chamber and pre-stored for the operation of next run.

To practice this new process, some findings from this study may be summarized as tips: (i) alkaline condition at pH > 7.0 in the DS chamber is beneficial to the SO2 absorption, which can be achieved by the electrochemical pre-storage of OH ions through eq 6', (ii) electrochemical generation of H2O2 (eqs 6 and 6' ) in the DS chamber can be designed as a concurrent reaction with the A-SO2 oxidation reaction by air, since the H2O2 can accelerate the A-SO2 oxidation reaction significantly, and (iii) a suitable current density shall be adjusted to maintain the optimal pH 5.0~6.0 of the A-SO2 oxidation reaction.

Thus by combination of the mass flow in Figure 8, an overall reaction can be expressed below to illustrate the SO2 conversion into NaHSO4 under the electrical driving force:

$$\text{SO}\_2 + \frac{1}{2}\text{O}\_2 + \text{H}\_2\text{O} + \text{Na}\_2\text{SO}\_4 \xrightarrow{\text{electroity}} 2\text{Na}\text{HSO}\_4 \tag{10}$$

Eq 10 indicates that from a thermodynamic point of view, the electrochemical reactions of eqs 6, 6', 7 and 8 may not be essential, but from an engineering point of view, they are critical. The cathodic reactions scavenge the absorption- and oxidation-released H+ ions to drive the SO2 absorption and oxidation, and the anodic reaction provides H+ ions to drive the NaHSO4 formation. Therefore, the electrochemical reactions ensure the process of SO2 conversion to NaHSO4 through eq 10.

**Sustainability Evaluation of The Process.** Green chemistry has a set of principles to minimize pollution as far as possible in chemical processes, including achieving high value of atom economy in the synthetic process, eliminating toxicity to human health and the environment, minimizing energy consumption, utilizing clean materials, and so on *(28,29)*. Although the aspiration of green chemistry is preferably realized in the manufacturing processes rather than the subsequent cleanup of effluent, the principles are still applicable in the wet FGD process. In this study, sustainability of this new process was evaluated from the perspective of green chemistry.

The atom economy is expressed by *AU*, which is defined by a ratio of mole mass between the desired product and the reactant(s) as shown below:

$$ALI = \frac{mole \text{ mass in desired product}}{mole \text{ mass in react} \,\text{at(s)}} \times 100\% \tag{11}$$

Electrochemically-Driven and Green Conversion of SO2 to NaHSO4 in Aqueous Solution 77

L. Thus, in both cases, 1.28 kWh of electricity was consumed in the oxidative conversion of

Experimental results showed that the H+ ions scavenged by eqs 6, 6', and 7 in the A-SO2 oxidation of Step I were equivalent to the H+ ions electrochemically generated through eq 8 in Step II. Thus, the *E* values of the A-SO2 oxidation and Step II were equal, and doubled electricity of A-SO2 oxidation, i.e. 2.56 kWh was consumed to convert every of kg A-SO2 to

Therefore, a total electricity of 2.56 kWh was needed for the overall conversion of every kg of gaseous SO2 to NaHSO4 in aqueous solution, which was only slightly higher than the electricity consumption between 1.8 and 2.4 kWh for the anodic oxidation conversion of one kg of SO2 into H2SO4 in aqueous solution *(14)*. In comparison, the electricity consumption by

Also important, the final product of NaHSO4 has an added value. Eq 10 indicates that 2.06 kg of Na2SO4 needs to be consumed to convert one kg of SO2 to attain 3.75 kg of NaHSO4 with 82% increase in mass. It is learned from the current market that NaHSO4 has an approximately quadruple commercial value of Na2SO4. In addition, the alkaline and acid demanded by this new process are provided on site electrochemically instead of addition of chemicals. Considering that the alkaline and acid are manufactured at the price of

Therefore, this new process can fully comply with the principles of green chemistry and shows promising feasibility. If it is flexibly applied in a wet FGD process for SO2 removal, it could be an environmentally-sustainable technique. In fact, few of environmental processes, which serve to decompose the pollutants, have high value of atom economy. Fortunately, all the atoms from the raw materials in this process end up in the product of NaHSO4. During the process, some intermediates are involved, while they are reusable and environmentally

**Further Investigations.** Before this process is practiced for reduction of SO2 in flue gas, more investigations remain. First, effect of CO2 which abounds in the flue gas should be precluded. We will demonstrate elsewhere that the CO2 can be separated advisably from the SO2 in this process, and then the CO2 can be further captured and recovered electrochemically in the manner of synchronous supply of alkaline and acid. Second, a side reaction that accompanies the cathodic reaction of O2 reduction is the H2 evolution (eq 7). A coupling of this reaction with the anodic oxidation of H2O (eq 8) shows the well-know

consumes extra energy. Thus, the H2O electrolysis must be avoided by a careful operation of the electrochemical reactor and selection of cathode on which the H2 evolution can be inhibited. Third, eq 6 has a byproduct of H2O2, which serves to accelerate the S(IV) oxidation. Under some circumstances such as on Pt modified carbon electrode, the O2

the electrochemical reactions of this new process seems quite competitive.

electricity, we can presume that this approach appears clean and cheap.

reaction of H2O electrolysis. While eq 7 also outputs OH-

reduction proceeded in a 4-electron pathway to solely output OH-

ions were pre-stored through eq 6' in Step II. Obviously,

ions for Step I was included in Step II.

, the reaction of H2O electrolysis

free of H2O2. In this case,

= 4.0 V, *t* = 0.5 h, and *S* =

= 4.3 V, *t* = 0.5 h, and *S* = 0.20

= 1.0 10-3 M, *Ct* = 0.02 10-3 M, *I* = 8.0 10-3 A, *U*

0.20 L; *C0* = 1.5 10-3 M, *Ct* = 0.18 10-3 M, *I* = 10.0 10-3 A, *U*

every kg of A-SO2 to Na2SO4 in Step I.

Figure 7 demonstrates that the OH-

the electricity consumption to pre-store the OH-

In this study, *C0*

NaHSO4.

benign.


The *AU* values of eqs 1~10 are listed in Table 3. Clearly, eqs 5, 6, 9, and 10 received 100% of *AU*. Eqs 2, 3, and 4 received *AU* as high as 98.8%, 98.8%, and 90.0%, respectively. Only eqs 7 and 8 received 50.0% and 11.1%, respectively.

Table 3. Values of Atom Utilization (*AU*), desired products, and byproducts in eqs 1~10.

In addition to the individual *AU* values of single reaction, the byproducts of eqs 1~10 are listed in Table 3, and their fates and environmental impacts are discussed as follows. The H+ ions as byproduct in eqs 2~4 are scavenged to form H2O, and the H2O can be reused through eq 8 to generate H+ ions that entirely end up in the NaHSO4 through eq 9. The H2O2 in eq 6 or 6' is reused to enhance the A-SO2 oxidation and also enters into the NaHSO4. The gaseous byproducts of H2 in eq 6' and O2 in eq 8 escape into the environment, whereas it is not hazardous to human health and the environment, while some safety measure should be taken to deal with the H2. However, all other atoms are kept in the final product through reusing the byproducts in the same setup.

Therefore, it can be seen that except the portion of oxygen and hydrogen atoms that released as gases through eqs 7 and 8, respectively, all other atoms are remained in the final product. This process in essence utilizes the raw materials of Na2SO4, O2 in ambient air, and water which are all environmentally clean. As a result, secondary pollution can be avoided.

The electricity consumption (*E*) in terms of kWh through eqs 6, 6', 7 and 8 is calculated in light of the two-step model experiment.

In the A-SO2 oxidation of Step I, electricity was consumed through the cathodic reactions of eqs 6, 6', and 7 to scavenge the H+ irons released in eqs 2~4, and its consumption was calculated by:

$$E = \frac{\bar{\underline{U}} \cdot \underline{I} \cdot \underline{t} \cdot 10^{-3}}{64 \cdot \{\mathcal{C}\_0 - \mathcal{C}\_t\} \cdot 10^{-3} \cdot \mathcal{S}} \tag{S1}$$

where *U* and *I* were the voltage in average and current that kept constant, respectively, the *C0* and *Ct* were the A-SO2 concentrations before and after the oxidation reaction, respectively, and *S* was the volume.

The *AU* values of eqs 1~10 are listed in Table 3. Clearly, eqs 5, 6, 9, and 10 received 100% of *AU*. Eqs 2, 3, and 4 received *AU* as high as 98.8%, 98.8%, and 90.0%, respectively. Only eqs 7

> **eq** *AU* **(%) desired product byproduct**  1 75.5 CaSO4·2H2O CO2 2 98.8 HSO3- H+

4 90.0 SO42- H+ 5 100 SO42- no 6 100 H2O2 no 7 50.0 OH- H2O2 6' ---- no H2 8 11.1 H+ O2 9 100 bisulfate no 10 100 NaHSO4 no

Table 3. Values of Atom Utilization (*AU*), desired products, and byproducts in eqs 1~10.

In addition to the individual *AU* values of single reaction, the byproducts of eqs 1~10 are listed in Table 3, and their fates and environmental impacts are discussed as follows. The H+ ions as byproduct in eqs 2~4 are scavenged to form H2O, and the H2O can be reused through eq 8 to generate H+ ions that entirely end up in the NaHSO4 through eq 9. The H2O2 in eq 6 or 6' is reused to enhance the A-SO2 oxidation and also enters into the NaHSO4. The gaseous byproducts of H2 in eq 6' and O2 in eq 8 escape into the environment, whereas it is not hazardous to human health and the environment, while some safety measure should be taken to deal with the H2. However, all other atoms are kept in the final product through

Therefore, it can be seen that except the portion of oxygen and hydrogen atoms that released as gases through eqs 7 and 8, respectively, all other atoms are remained in the final product. This process in essence utilizes the raw materials of Na2SO4, O2 in ambient air, and water which are all environmentally clean. As a result, secondary pollution can be avoided.

The electricity consumption (*E*) in terms of kWh through eqs 6, 6', 7 and 8 is calculated in

In the A-SO2 oxidation of Step I, electricity was consumed through the cathodic reactions of eqs 6, 6', and 7 to scavenge the H+ irons released in eqs 2~4, and its consumption was

0

*UI t <sup>E</sup>*

64 ( ) 10 *<sup>t</sup>*

*C0* and *Ct* were the A-SO2 concentrations before and after the oxidation reaction,

3 3

(S1)

10

*CC S*

and *I* were the voltage in average and current that kept constant, respectively, the


and 8 received 50.0% and 11.1%, respectively.

reusing the byproducts in the same setup.

light of the two-step model experiment.

respectively, and *S* was the volume.

calculated by:

where *U* 

3 98.8 SO3

In this study, *C0* = 1.0 10-3 M, *Ct* = 0.02 10-3 M, *I* = 8.0 10-3 A, *U* = 4.0 V, *t* = 0.5 h, and *S* = 0.20 L; *C0* = 1.5 10-3 M, *Ct* = 0.18 10-3 M, *I* = 10.0 10-3 A, *U* = 4.3 V, *t* = 0.5 h, and *S* = 0.20 L. Thus, in both cases, 1.28 kWh of electricity was consumed in the oxidative conversion of every kg of A-SO2 to Na2SO4 in Step I.

Experimental results showed that the H+ ions scavenged by eqs 6, 6', and 7 in the A-SO2 oxidation of Step I were equivalent to the H+ ions electrochemically generated through eq 8 in Step II. Thus, the *E* values of the A-SO2 oxidation and Step II were equal, and doubled electricity of A-SO2 oxidation, i.e. 2.56 kWh was consumed to convert every of kg A-SO2 to NaHSO4.

Figure 7 demonstrates that the OH- ions were pre-stored through eq 6' in Step II. Obviously, the electricity consumption to pre-store the OH- ions for Step I was included in Step II. Therefore, a total electricity of 2.56 kWh was needed for the overall conversion of every kg of gaseous SO2 to NaHSO4 in aqueous solution, which was only slightly higher than the electricity consumption between 1.8 and 2.4 kWh for the anodic oxidation conversion of one kg of SO2 into H2SO4 in aqueous solution *(14)*. In comparison, the electricity consumption by the electrochemical reactions of this new process seems quite competitive.

Also important, the final product of NaHSO4 has an added value. Eq 10 indicates that 2.06 kg of Na2SO4 needs to be consumed to convert one kg of SO2 to attain 3.75 kg of NaHSO4 with 82% increase in mass. It is learned from the current market that NaHSO4 has an approximately quadruple commercial value of Na2SO4. In addition, the alkaline and acid demanded by this new process are provided on site electrochemically instead of addition of chemicals. Considering that the alkaline and acid are manufactured at the price of electricity, we can presume that this approach appears clean and cheap.

Therefore, this new process can fully comply with the principles of green chemistry and shows promising feasibility. If it is flexibly applied in a wet FGD process for SO2 removal, it could be an environmentally-sustainable technique. In fact, few of environmental processes, which serve to decompose the pollutants, have high value of atom economy. Fortunately, all the atoms from the raw materials in this process end up in the product of NaHSO4. During the process, some intermediates are involved, while they are reusable and environmentally benign.

**Further Investigations.** Before this process is practiced for reduction of SO2 in flue gas, more investigations remain. First, effect of CO2 which abounds in the flue gas should be precluded. We will demonstrate elsewhere that the CO2 can be separated advisably from the SO2 in this process, and then the CO2 can be further captured and recovered electrochemically in the manner of synchronous supply of alkaline and acid. Second, a side reaction that accompanies the cathodic reaction of O2 reduction is the H2 evolution (eq 7). A coupling of this reaction with the anodic oxidation of H2O (eq 8) shows the well-know reaction of H2O electrolysis. While eq 7 also outputs OH- , the reaction of H2O electrolysis consumes extra energy. Thus, the H2O electrolysis must be avoided by a careful operation of the electrochemical reactor and selection of cathode on which the H2 evolution can be inhibited. Third, eq 6 has a byproduct of H2O2, which serves to accelerate the S(IV) oxidation. Under some circumstances such as on Pt modified carbon electrode, the O2 reduction proceeded in a 4-electron pathway to solely output OH free of H2O2. In this case,

Electrochemically-Driven and Green Conversion of SO2 to NaHSO4 in Aqueous Solution 79

[9] Hrastel, I.; Gerbec, M; Stergaršek, A. Technology optimization of wet flue gas

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the S(IV) oxidation is accomplished by the air oxidation while the overall reaction of NaHSO4 production remains. Since the air oxidation of S(IV) is fast to some extent, and thus in practice, whether the H2O2 is essential needs further scrutiny. Forth, a salt bridge is employed in this study to spatially separate the cathodic and anodic chambers. In real application, a membrane *(17,18)* that is commercially available can be employed. Anyway, this new process is promising as an alternative FGD process that immobilizes the SO2 waste in the form of non-calcium product by means of a cheap and non-toxic material, and thereby avoids the concern over any secondary pollution *(30)*.

## **5. Nomenclature**

A-SO2 = absorbed SO2 in aqueous solution

AU = atom utilization in %

CSH = chemical scavenging of H+ ions through addition of NaOH solution

DS = desulfurization chamber, as cathodic chamber

ESH = electrochemical scavenging of H+ ions through eqs 6, 6' or 7

FGD = flue gas desulfurization

SH = scavenging of H+ ions

SR = sulfur-recovery chamber, as anodic chamber

## **6. Acknowledgements**

This work was supported by Natural Science Foundation of China (Project No: 50978260, 21077136).

## **7. References**


the S(IV) oxidation is accomplished by the air oxidation while the overall reaction of NaHSO4 production remains. Since the air oxidation of S(IV) is fast to some extent, and thus in practice, whether the H2O2 is essential needs further scrutiny. Forth, a salt bridge is employed in this study to spatially separate the cathodic and anodic chambers. In real application, a membrane *(17,18)* that is commercially available can be employed. Anyway, this new process is promising as an alternative FGD process that immobilizes the SO2 waste in the form of non-calcium product by means of a cheap and non-toxic material, and thereby

This work was supported by Natural Science Foundation of China (Project No: 50978260,

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[3] Lynch, J. A.; Bowersox, V. C.; Grimm, J. W. Acid rain reduced in eastern United States.

[4] Cofala, J.; Amann, M.; Gyarfas, F.; Schoepp, W.; Boudri, J. C.; Hordijk, L.; Kroeze, C.; Li,

[5] Kikkawa, H.; Nakamoto, T.; Morishita, M.; Yamada, K. New wet FGD process using

[6] Gutiérrez Ortiz, F. J.; Vidal, F.; Ollero, P.; Salvador, L.; Cortés, V.; Giménez, A. Pilot-

[7] Karatza, D.; Prisciandaro, M.; Lancia, A.; Musmarra, D. Calcium bisulfite oxidation in

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J.; Lin, D.; Panwar, T. S.; Gupta, S. Cost-effective control of SO2 emissions in Asia. *J.* 

plant technical assessment of wet flue gas desulfurization using limestone. *Ind. Eng.* 

the flue gas desulfurization process catalyzed by iron and manganese ions. *Ind.* 

filter-bioreactor system. *Environ. Sci. Technol.* 2003, *37*, 1978-1982.

granular limestone. *Ind. Eng. Chem. Res.* 2002, *41*, 3028-3036.

avoids the concern over any secondary pollution *(30)*.

DS = desulfurization chamber, as cathodic chamber

SR = sulfur-recovery chamber, as anodic chamber

*Manage. Assoc.* 2001, *51*, 1676-1688.

*Environ. Manage.* 2004, *72*, 149-161.

*Chem. Res*. 2006, *45*, 1466-1477.

*Eng. Chem. Res*. 2004, *43*, 4876-4882.

*Environ. Sci. Technol.* 2000, *34*, 940-949.

CSH = chemical scavenging of H+ ions through addition of NaOH solution

ESH = electrochemical scavenging of H+ ions through eqs 6, 6' or 7

A-SO2 = absorbed SO2 in aqueous solution

**5. Nomenclature** 

AU = atom utilization in %

FGD = flue gas desulfurization SH = scavenging of H+ ions

**6. Acknowledgements** 

21077136).

**7. References** 


**5** 

*Brazil* 

**Recent Advances in** 

**the Ultrasound-Assisted Synthesis of Azoles** 

The ever increasing awareness of the need to protect natural resources through the development of environmentally sustainable processes and the optimization of energy consumption has guided the actions of both the private and governmental sectors of society. Economic planning has been strongly impacted by this new paradigm, which has led to increasing demands by society for products produced in a sustainable way and to more stringent governmental regulatory policies. Thus, while in the past profit was often the major concern, in the current economic context more sustainable production processes are preferred. This has triggered a demand in both industry and academy for the development

In the field of chemistry and chemical technology, the 12 principles of Green Chemistry provide a set of clear guidelines for the development of new synthetic methodologies and chemical processes and for the evaluation of their potential for environmental impact. As a consequence, in organic chemistry, numerous investigations now routinely use nontradicional synthetic metodologies such as solvent-free reactions, the application of alternative activation techniques like microwaves or ultrasound, the replacement of volatile

In medicinal chemistry, experience has shown that compounds with biological activity are often based on heterocyclic structures. In particular, azoles and their derivatives have attracted increasing interest as versatile intermediates for the synthesis of biologically active compounds such as potent antitumour, antibacterial, antifungal, antiviral and antioxidizing agents. Azoles are a large class of 5-membered ring heterocyclic compounds containing at least one nitrogen atom and one heteroatom in their structure. The construction of this type of molecule has received great attention due to the wide spectrum of biological activities that have been attributed to structurally distinct azoles. Fluconazole, itraconazole, voriconazole and posaconazole are antifungal agents commercially available that contain a triazole nucleus. Celecoxib is a non-steroidal anti-inflammatory and analgesic agent of the

organic solvents by water, ionic liquids, or supercritical CO2, etc.

**1. Introduction** 

of new, cleaner technologies.

Lucas Pizzuti1, Márcia S.F. Franco1, Alex F.C. Flores2,

*1Universidade Federal da Grande Dourados, Mato Grosso do Sul 2Universidade Federal de Santa Maria, Rio Grande do Sul 3Instituto de Química, Universidade de São Paulo, São Paulo* 

Frank H. Quina3 and Claudio M.P. Pereira4

*4Universidade Federal de Pelotas, Rio Grande do Sul* 


## **Recent Advances in the Ultrasound-Assisted Synthesis of Azoles**

Lucas Pizzuti1, Márcia S.F. Franco1, Alex F.C. Flores2, Frank H. Quina3 and Claudio M.P. Pereira4 *1Universidade Federal da Grande Dourados, Mato Grosso do Sul 2Universidade Federal de Santa Maria, Rio Grande do Sul 3Instituto de Química, Universidade de São Paulo, São Paulo 4Universidade Federal de Pelotas, Rio Grande do Sul Brazil* 

## **1. Introduction**

80 Green Chemistry – Environmentally Benign Approaches

[29] Lankey, R. L.; Anastas, P. T. Life-cycle approaches for assessing green chemistry

[30] Wang C., Liu H., Li X.Z., Shi J.Y., Ouyang G.F., Peng M., Jiang C.C., Cui H.N., A new

concept of desulfurization: the electrochemically driven and green conversion of SO2 to NaHSO4 in aqueous solution. Environ. Sci. Technol., 2008, 42, 8585-8590.

technologies. *Ind. Eng. Chem. Res.* 2002; *41*, 4498-4502.

The ever increasing awareness of the need to protect natural resources through the development of environmentally sustainable processes and the optimization of energy consumption has guided the actions of both the private and governmental sectors of society. Economic planning has been strongly impacted by this new paradigm, which has led to increasing demands by society for products produced in a sustainable way and to more stringent governmental regulatory policies. Thus, while in the past profit was often the major concern, in the current economic context more sustainable production processes are preferred. This has triggered a demand in both industry and academy for the development of new, cleaner technologies.

In the field of chemistry and chemical technology, the 12 principles of Green Chemistry provide a set of clear guidelines for the development of new synthetic methodologies and chemical processes and for the evaluation of their potential for environmental impact. As a consequence, in organic chemistry, numerous investigations now routinely use nontradicional synthetic metodologies such as solvent-free reactions, the application of alternative activation techniques like microwaves or ultrasound, the replacement of volatile organic solvents by water, ionic liquids, or supercritical CO2, etc.

In medicinal chemistry, experience has shown that compounds with biological activity are often based on heterocyclic structures. In particular, azoles and their derivatives have attracted increasing interest as versatile intermediates for the synthesis of biologically active compounds such as potent antitumour, antibacterial, antifungal, antiviral and antioxidizing agents. Azoles are a large class of 5-membered ring heterocyclic compounds containing at least one nitrogen atom and one heteroatom in their structure. The construction of this type of molecule has received great attention due to the wide spectrum of biological activities that have been attributed to structurally distinct azoles. Fluconazole, itraconazole, voriconazole and posaconazole are antifungal agents commercially available that contain a triazole nucleus. Celecoxib is a non-steroidal anti-inflammatory and analgesic agent of the

Recent Advances in the Ultrasound-Assisted Synthesis of Azoles 83

sonochemical preparation of organolithium and Grignard reagents and their coupling with carbonyls. In the ensuing four decades, numerous efficient and innovative applications of ultrasound in organic synthesis have appeared, which have established sonochemistry as an

The two main sources of ultrasound in organic synthesis are ultrasonic cleaning baths and ultrasonic immersion probes, which typically operate at frequencies of 40 and 20 kHz, respectively (Mason, 1997). The former are more commonly employed in organic synthesis simply because they are less expensive and hence more widely available to chemists, even though the amount of energy transferred to the reaction medium is lower than that of ultrasonic probe systems, which deposit the acoustic energy directly into the reaction medium.

1,5-Diarylpyrazoles (**3**) can be prepared by the reaction between Baylis-Hillman adducts (**1**) and phenylhydrazine hydrochloride (**2**) in 1,2-dichloroetane under sonication with reaction times of 60-180 minutes (Scheme 1) (Mamaghani & Dastmard, 2009). The reactions proceeded regioselectively to afford the desired products in 80-90% yields. The same reaction carried out by simply heating the reaction mixture (80 °C) produced the products in

In 2009, Pathak and co-workers (Pathak et al., 2009) conducted a comparative study between four activating methods for obtaining *N*-acetyl-pyrazolines (**7**), including reflux, solvent-free conditions, microwave irradiation and ultrasonic irradiation. Microwave irradiation was found to be the most efficient activating method, followed by ultrasound. Employing ultrasound, the reactions of 1,4-pentadien-3-ones (**4**) with hydrazine (**5**) and acetic acid (**6**) in ethanol went to

<sup>R</sup> <sup>R</sup>

completion in 10-25 minutes and afforded the products (**7**) in good yields (Scheme 2).

NH2NH2H2O

**5**

CH3COOH

**6**

ClCH2CH2Cl

EtOH )))), 30 C, 10-25 min

Ar NHNH2HCl

7 examples 80-90%

**3**

10 examples 76-91%

O CH3

R

**7**

N N

N N

Et Me

lower yields (60-75%) and required longer reaction times (6-9 h).

)))), 60 C, 60-180 min Ar

**2**

important tool in the arsenal of Green Chemistry.

**3. Azoles with two heteroatoms** 

OH O

**1**

O

**4**

Scheme 1.

R

Scheme 2.

Et

**3.1 Pyrazole derivatives** 

pyrazole class. Isoxazole compounds such as valdecoxib are selective COX-2 inhibitors used in the treatment of pain. In this context, much attention has been given by researchers in universities and pharmaceutical industries to the development of new, energy saving, costeffective, environmentally safe technologies for the synthesis of azoles.

In this context, the use of ultrasound to accelerate reactions has proven to be a particularly important tool for meeting the Green Chemistry goals of minimization of waste and reduction of energy requirements (Cintas & Luche, 1999). Applications of ultrasonic irradiation are playing an increasing role in chemical processes, especially in cases where classical methods require drastic conditions or prolonged reaction times. The excellent review of Cella and Stefani (Cella & Stefani, 2009), which covered the available literature up to about three years ago, clearly showed the importance of taking advantage of the unique features of ultrasound-assisted reactions to synthesize heterocyclic ring systems. The present chapter will therefore limit its coverage of the literature to the period of the last three years and focus on the use of ultrasound to promote the cyclization reactions employed to obtain azoles. Modifications of side chains are not covered in this work. After a brief consideration of ultrasound and the origin of its effects on chemical reactions, a total of 42 reports of the preparation of azoles under conditions of ultrasonic irradiation are reviewed, including several of our own contributions to this field of research. These reports were grouped together according to the number of heteroatoms present in the ring (2, 3 or 4) and each group subdivided by the azole class. Reports in which more than one class of azoles were prepared were collected in the last section, labelled "Miscellaneous".

## **2. Ultrasound and its chemical effects**

The discovery of the piezoelectric effect in the 1880s provided the basis for the construction of modern ultrasonic devices. Piezoelectric materials generate mechanical vibrations in response to an applied alternating electrical potential. If the potential is applied at sufficiently high frequency, ultrasonic waves are generated. The phenomenon responsible for the beneficial effects of ultrasound on chemical reactions is cavitation. Ultrasonic waves are propagated *via* alternating compressions and rarefactions induced in the transmitting medium through which they pass. During the rarefaction cycle of the sound wave, the molecules of the liquid are separated, generating bubbles that subsequently collapse in the compression cycle. These rapid and violent implosions generate short-lived regions with local temperatures of roughly 5000 °C, pressures of about 1000 atm and heating and cooling rates that can exceed 10 billion °C per second. Such localized hot spots can be thought of as micro reactors in which the mechanical energy of sound is transformed into a useful chemical form. In addition to the generation of such hot spots, there can also be mechanical effects produced as a result of the violent collapse (Mason & Lorimer, 2002).

More than 80 years have passed since the effect of ultrasound on reaction rates was first reported by Richards and Loomis (Richards & Loomis, 1927). However, this work received little attention at the time because it used a high-frequency apparatus that was not commonly available to chemists. According to the review of Cravotto and Cintas (Cravotto & Cintas, 2006), two classical papers, published in 1978 and 1980, provided a major stimulus for the development of modern sonochemistry: (1) the report by Fry and Herr (Fry & Herr, 1978) of the reductive dehalogenation of dibromoketones with mercury dispersed by ultrasound; and (2) the work of Luche and Damiano (Luche & Damiano, 1980) on the sonochemical preparation of organolithium and Grignard reagents and their coupling with carbonyls. In the ensuing four decades, numerous efficient and innovative applications of ultrasound in organic synthesis have appeared, which have established sonochemistry as an important tool in the arsenal of Green Chemistry.

The two main sources of ultrasound in organic synthesis are ultrasonic cleaning baths and ultrasonic immersion probes, which typically operate at frequencies of 40 and 20 kHz, respectively (Mason, 1997). The former are more commonly employed in organic synthesis simply because they are less expensive and hence more widely available to chemists, even though the amount of energy transferred to the reaction medium is lower than that of ultrasonic probe systems, which deposit the acoustic energy directly into the reaction medium.

## **3. Azoles with two heteroatoms**

#### **3.1 Pyrazole derivatives**

82 Green Chemistry – Environmentally Benign Approaches

pyrazole class. Isoxazole compounds such as valdecoxib are selective COX-2 inhibitors used in the treatment of pain. In this context, much attention has been given by researchers in universities and pharmaceutical industries to the development of new, energy saving, cost-

In this context, the use of ultrasound to accelerate reactions has proven to be a particularly important tool for meeting the Green Chemistry goals of minimization of waste and reduction of energy requirements (Cintas & Luche, 1999). Applications of ultrasonic irradiation are playing an increasing role in chemical processes, especially in cases where classical methods require drastic conditions or prolonged reaction times. The excellent review of Cella and Stefani (Cella & Stefani, 2009), which covered the available literature up to about three years ago, clearly showed the importance of taking advantage of the unique features of ultrasound-assisted reactions to synthesize heterocyclic ring systems. The present chapter will therefore limit its coverage of the literature to the period of the last three years and focus on the use of ultrasound to promote the cyclization reactions employed to obtain azoles. Modifications of side chains are not covered in this work. After a brief consideration of ultrasound and the origin of its effects on chemical reactions, a total of 42 reports of the preparation of azoles under conditions of ultrasonic irradiation are reviewed, including several of our own contributions to this field of research. These reports were grouped together according to the number of heteroatoms present in the ring (2, 3 or 4) and each group subdivided by the azole class. Reports in which more than one class of azoles were

The discovery of the piezoelectric effect in the 1880s provided the basis for the construction of modern ultrasonic devices. Piezoelectric materials generate mechanical vibrations in response to an applied alternating electrical potential. If the potential is applied at sufficiently high frequency, ultrasonic waves are generated. The phenomenon responsible for the beneficial effects of ultrasound on chemical reactions is cavitation. Ultrasonic waves are propagated *via* alternating compressions and rarefactions induced in the transmitting medium through which they pass. During the rarefaction cycle of the sound wave, the molecules of the liquid are separated, generating bubbles that subsequently collapse in the compression cycle. These rapid and violent implosions generate short-lived regions with local temperatures of roughly 5000 °C, pressures of about 1000 atm and heating and cooling rates that can exceed 10 billion °C per second. Such localized hot spots can be thought of as micro reactors in which the mechanical energy of sound is transformed into a useful chemical form. In addition to the generation of such hot spots, there can also be mechanical

More than 80 years have passed since the effect of ultrasound on reaction rates was first reported by Richards and Loomis (Richards & Loomis, 1927). However, this work received little attention at the time because it used a high-frequency apparatus that was not commonly available to chemists. According to the review of Cravotto and Cintas (Cravotto & Cintas, 2006), two classical papers, published in 1978 and 1980, provided a major stimulus for the development of modern sonochemistry: (1) the report by Fry and Herr (Fry & Herr, 1978) of the reductive dehalogenation of dibromoketones with mercury dispersed by ultrasound; and (2) the work of Luche and Damiano (Luche & Damiano, 1980) on the

effective, environmentally safe technologies for the synthesis of azoles.

prepared were collected in the last section, labelled "Miscellaneous".

effects produced as a result of the violent collapse (Mason & Lorimer, 2002).

**2. Ultrasound and its chemical effects** 

1,5-Diarylpyrazoles (**3**) can be prepared by the reaction between Baylis-Hillman adducts (**1**) and phenylhydrazine hydrochloride (**2**) in 1,2-dichloroetane under sonication with reaction times of 60-180 minutes (Scheme 1) (Mamaghani & Dastmard, 2009). The reactions proceeded regioselectively to afford the desired products in 80-90% yields. The same reaction carried out by simply heating the reaction mixture (80 °C) produced the products in lower yields (60-75%) and required longer reaction times (6-9 h).

Scheme 1.

In 2009, Pathak and co-workers (Pathak et al., 2009) conducted a comparative study between four activating methods for obtaining *N*-acetyl-pyrazolines (**7**), including reflux, solvent-free conditions, microwave irradiation and ultrasonic irradiation. Microwave irradiation was found to be the most efficient activating method, followed by ultrasound. Employing ultrasound, the reactions of 1,4-pentadien-3-ones (**4**) with hydrazine (**5**) and acetic acid (**6**) in ethanol went to completion in 10-25 minutes and afforded the products (**7**) in good yields (Scheme 2).

Scheme 2.

Recent Advances in the Ultrasound-Assisted Synthesis of Azoles 85

A multicomponent ultrasound-assisted protocol for the synthesis of bridgehead pyrazole derivatives (**20**) was developed by Nabid and co-workers (Nabid et al., 2010). Under ultrasonic irradiation, the reaction between phthalhydrazide (**17**), malononitrile or ethyl cyanoacetate (**18**), and aromatic aldehydes (**19**) in the presence of triethylamine furnished

TEA, EtOH

HCl, EtOH

)))), rt, 45-165 min <sup>N</sup>

)))) 65 C, 10 min

Ar **18**

)))), 50 C, 60 min <sup>N</sup>

21 examples 85-97%

> 10 examples 69-99%

R

**23**

N

1 example

<sup>S</sup> <sup>N</sup> O O

**26**

N NH

O

N O

NH2

R

1*H*-pyrazolo[1,2-*b*]phthalazine-5,10-diones (**20**) in very good yields (Scheme 6).

**17 19 20**

Ar H O

3-Aryl-2,3-epoxy-1-phenyl-1-propanone (**21**) reacted under ultrasonic irradiation with phenylhydrazine (**22**) catalyzed by HCl at room temperature to produce 1,3,5 triarylpyrazoles (**23**) in 69-99% yields (Scheme 7) (Li et al., 2010). The same reactions

NHNH2

3,4-Dimethyl-2,4-dihydropyrazolo[4,3-*c*][1,2]benzothiazine 5,5-dioxide (**26**) was prepared in only 10 minutes by the cyclization of 1-(4-hydroxy-2-methyl-1,1-dioxido-2*H*-1,2 benzothiazin-3-yl)ethanone (**24**) with hydrazine (**25**) under ultrasonic irradiation (Scheme 8) (Ahmad et al., 2010). The product was used for the preparation of acetohydrazide

In 2010, ultrasound was employed to promote the synthesis of pyrazolones (**29**) *via* the reaction of *β*-keto esters (**27**) with hydrazine derivatives (**28**) in ethanol. The reactions went to completion in short times (2-25 min) and afforded the products in 4-93% yields (Scheme

**22**

**25**

NH2NH2H2O

performed in the absence of sonication gave substantially poorer yields.

derivatives with potential antioxidant and antibacterial activities.

R CN

NH NH

O

O

S N

O O

**24**

OH O

**21**

O

O

Scheme 6.

R

Scheme 7.

Scheme 8.

9) (Al-Mutairi et al., 2010).

Previously, we described a greener, ultrasound-assisted synthesis of 1-thiocarbamoyl-3,5 diaryl-4,5-dihydropyrazoles (**10**) from chalcones (**8**) and thiosemicarbazide (**9**) catalyzed by KOH (Scheme 3) (Pizzuti et al., 2009). The products were obtained in high purity and in good yields in only 20 minutes *via* a simple filtration of the reaction mixture.

Scheme 3.

Similarly, in 2010, we reported the cyclization of chalcones (**11**) with aminoguanidine hydrochloride (**12**) under essentially the same conditions (Scheme 4) (Pizzuti et al., 2010). The 4,5-dihydropyrazole derivatives (**13**) were obtained in high yields in 30 minutes employing sonication. The same reactions carried out under reflux without ultrasonic irradiation afforded the products in lower yields (57-69%) and required substantially longer reaction times (3-6 h).

Gupta and co-workers utilized an ultrasonic cleaning-bath to promote the cyclization reaction between chalcones (**14**) and phenylhydrazine (**15**) under acid conditions, giving the desired cyclization products (**16**) in good yields (Scheme 5) (Gupta et al., 2010).

Scheme 5.

A multicomponent ultrasound-assisted protocol for the synthesis of bridgehead pyrazole derivatives (**20**) was developed by Nabid and co-workers (Nabid et al., 2010). Under ultrasonic irradiation, the reaction between phthalhydrazide (**17**), malononitrile or ethyl cyanoacetate (**18**), and aromatic aldehydes (**19**) in the presence of triethylamine furnished 1*H*-pyrazolo[1,2-*b*]phthalazine-5,10-diones (**20**) in very good yields (Scheme 6).

Scheme 6.

84 Green Chemistry – Environmentally Benign Approaches

Previously, we described a greener, ultrasound-assisted synthesis of 1-thiocarbamoyl-3,5 diaryl-4,5-dihydropyrazoles (**10**) from chalcones (**8**) and thiosemicarbazide (**9**) catalyzed by KOH (Scheme 3) (Pizzuti et al., 2009). The products were obtained in high purity and in

)))), rt, 20 min

Similarly, in 2010, we reported the cyclization of chalcones (**11**) with aminoguanidine hydrochloride (**12**) under essentially the same conditions (Scheme 4) (Pizzuti et al., 2010). The 4,5-dihydropyrazole derivatives (**13**) were obtained in high yields in 30 minutes employing sonication. The same reactions carried out under reflux without ultrasonic irradiation afforded the products in lower yields (57-69%) and required substantially longer

)))), rt, 30 min

Gupta and co-workers utilized an ultrasonic cleaning-bath to promote the cyclization reaction between chalcones (**14**) and phenylhydrazine (**15**) under acid conditions, giving the

)))), 25-40 C, 30-100 min

NH2HCl

**<sup>11</sup> <sup>12</sup> <sup>13</sup>**

N H

desired cyclization products (**16**) in good yields (Scheme 5) (Gupta et al., 2010).

NHNH2

**15**

NH

R R

H2N

NH2

N H

**<sup>8</sup> <sup>9</sup> <sup>10</sup>**

S

KOH, EtOH

KOH, EtOH

AcOH

12 examples 60-78%

10 examples 75-99%

5 examples 80-90%

Cl

**16**

N N R

N N

HN NH2

N N

S NH2

good yields in only 20 minutes *via* a simple filtration of the reaction mixture.

H2N

R R

O

Scheme 3.

Scheme 4.

Scheme 5.

reaction times (3-6 h).

O

O

Cl <sup>R</sup>

**14**

3-Aryl-2,3-epoxy-1-phenyl-1-propanone (**21**) reacted under ultrasonic irradiation with phenylhydrazine (**22**) catalyzed by HCl at room temperature to produce 1,3,5 triarylpyrazoles (**23**) in 69-99% yields (Scheme 7) (Li et al., 2010). The same reactions performed in the absence of sonication gave substantially poorer yields.

Scheme 7.

3,4-Dimethyl-2,4-dihydropyrazolo[4,3-*c*][1,2]benzothiazine 5,5-dioxide (**26**) was prepared in only 10 minutes by the cyclization of 1-(4-hydroxy-2-methyl-1,1-dioxido-2*H*-1,2 benzothiazin-3-yl)ethanone (**24**) with hydrazine (**25**) under ultrasonic irradiation (Scheme 8) (Ahmad et al., 2010). The product was used for the preparation of acetohydrazide derivatives with potential antioxidant and antibacterial activities.

Scheme 8.

In 2010, ultrasound was employed to promote the synthesis of pyrazolones (**29**) *via* the reaction of *β*-keto esters (**27**) with hydrazine derivatives (**28**) in ethanol. The reactions went to completion in short times (2-25 min) and afforded the products in 4-93% yields (Scheme 9) (Al-Mutairi et al., 2010).

Recent Advances in the Ultrasound-Assisted Synthesis of Azoles 87

Both microwave and ultrasonic irradiation promoted de reaction of nitrile derivatives (**42**) with hydrazines (**43**) (Rodrigues-Santos & Echevarria, 2011). The reaction was highly regioselective and produced only one isomer (**44**) in 70-95% yields after 3 hours under sonication (Scheme 13). Although microwave irradiation required a much shorter reaction time (15 min), the yields were much lower (40-65%) than with ultrasound. However,

Very recently, Shekouhy and Hasaninejad (Shekouhy & Hasaninejad, 2012) reported the rapid and efficient preparation of 2*H*-indazolo[2,1-*b*]phthalazine-triones (**49**) (Scheme 14). Their four-component one-pot methodology consisted of the reaction of phthalic anhydride (**45**), dimedone (**46**), and hydrazine hydrate (**47**) with several aromatic aldehydes (**48**) in an ionic liquid under sonication. The products were obtained in good to excellent yields in very

[bmim]Br

**45** O

In connection with reactions in aqueous media, low potency (50 Watts) sonochemistry has been used to prepare 2-imidazolines (**52**) from the reaction of aldehydes (**50**) with

)))), rt, 3-15 min <sup>N</sup>

EtOH

)))), 50 C, 15-40 min

ultrasound was not effective for the preparation of phenylhydrazine derivatives.

)))), 35 C, 3 h <sup>N</sup> NC

O O

<sup>O</sup>

H

**48**

**46**

NH2NHR2

**43**

NC CN

O H

Ar

NH2NH2H2O

O

OEt

**37 38**

**39 40**

OCH3 H

**42**

O

O

NH2NH2H2O

**47**

**3.2 Imidazole derivatives** 

O

O

O

Scheme 12.

R1

short reaction times.

Scheme 13.

Scheme 14.

H2O

13 examples 79-95%

O

Ar

CN

NH2

6 examples 70-95%

18 examples 89-95%

O

**49**

N O

N N R2

R1

H2N

<sup>N</sup> <sup>H</sup> O

**44**

**41**

N N H

Scheme 9.

Recently, Machado and co-workers (Machado et al., 2011) reported the preparation of several ethyl 1-(2,4-dichlorophenyl)-1*H*-pyrazole-3-carboxylates (**32**) that are structurally analogous to the CB1 receptor antagonists used in the treatment of obesity. Cyclization of 4 alkoxy-2-oxo-3-butenoic ester (**30**) and 2,4-dicholorophenyl hydrazine hydrochloride (**31**) under sonication (10-12 min) or conventional thermal conditions (2.5-3 h) regioselectivelly afforded the desired products (Scheme 10). The use of ultrasound proved to be fundamental for reducing the reaction time.

Scheme 10.

A rapid procedure for obtaining acetylated *bis*-pyrazole derivatives (**36**) was based on the sonication of *bis*-chalcones (**33**) and hydrazine (**34**) in the presence of acetic anhydride (**35**) during 10-20 minutes (Scheme 11) (Kanagarajan et al., 2011). The same reactions required 5-8 h to go to completion when carried out under heating in the absence of ultrasound and afforded lower yields (55-70%) than those of sonochemical-assisted reaction.

Scheme 11.

A four-component one-pot reaction of ethyl acetoacetate (**37**), aromatic aldehydes (**38**), hydrazine (**39**), and malononitrile (**40**) in water afforded dihydropyrano[2,3-*c*]pyrazoles (**41**) in good yields under ultrasonic irradiation (79-95%) (Scheme 12) (Zou et al., 2011). Again, a comparative study in the absence of ultrasound showed that the products were obtained in lower yields (70-86%) and demanded longer reaction times (1-5 h).

#### Scheme 12.

86 Green Chemistry – Environmentally Benign Approaches

)))), 2-25 min

Recently, Machado and co-workers (Machado et al., 2011) reported the preparation of several ethyl 1-(2,4-dichlorophenyl)-1*H*-pyrazole-3-carboxylates (**32**) that are structurally analogous to the CB1 receptor antagonists used in the treatment of obesity. Cyclization of 4 alkoxy-2-oxo-3-butenoic ester (**30**) and 2,4-dicholorophenyl hydrazine hydrochloride (**31**) under sonication (10-12 min) or conventional thermal conditions (2.5-3 h) regioselectivelly afforded the desired products (Scheme 10). The use of ultrasound proved to be fundamental

**27 28 29**

NH2NHR<sup>2</sup>

)))), 68-72 C, 10-12 min <sup>O</sup>

afforded lower yields (55-70%) than those of sonochemical-assisted reaction.

NH2NH2H2O

**34**

(CH3CO)2O

**35**

Cl

Cl

O

R

lower yields (70-86%) and demanded longer reaction times (1-5 h).

NHNH2HCl

**31**

A rapid procedure for obtaining acetylated *bis*-pyrazole derivatives (**36**) was based on the sonication of *bis*-chalcones (**33**) and hydrazine (**34**) in the presence of acetic anhydride (**35**) during 10-20 minutes (Scheme 11) (Kanagarajan et al., 2011). The same reactions required 5-8 h to go to completion when carried out under heating in the absence of ultrasound and

)))), 45 C, 10-20 min

A four-component one-pot reaction of ethyl acetoacetate (**37**), aromatic aldehydes (**38**), hydrazine (**39**), and malononitrile (**40**) in water afforded dihydropyrano[2,3-*c*]pyrazoles (**41**) in good yields under ultrasonic irradiation (79-95%) (Scheme 12) (Zou et al., 2011). Again, a comparative study in the absence of ultrasound showed that the products were obtained in

CH3COONa

R OEt <sup>1</sup> O

O

for reducing the reaction time.

CO2Et

**30**

Scheme 10.

O

R

Scheme 11.

R1

**33**

R2 R<sup>3</sup>

Scheme 9.

EtOH

EtOH

11 examples 4-93%

N N R2

R1

O

14 examples 71-92%

6 examples 88-98%

H3C O O CH3

**36**

N N

R R

N

Cl

N

R1

N R CO2Et <sup>2</sup>

Cl

**32**

N

Both microwave and ultrasonic irradiation promoted de reaction of nitrile derivatives (**42**) with hydrazines (**43**) (Rodrigues-Santos & Echevarria, 2011). The reaction was highly regioselective and produced only one isomer (**44**) in 70-95% yields after 3 hours under sonication (Scheme 13). Although microwave irradiation required a much shorter reaction time (15 min), the yields were much lower (40-65%) than with ultrasound. However, ultrasound was not effective for the preparation of phenylhydrazine derivatives.

Scheme 13.

Very recently, Shekouhy and Hasaninejad (Shekouhy & Hasaninejad, 2012) reported the rapid and efficient preparation of 2*H*-indazolo[2,1-*b*]phthalazine-triones (**49**) (Scheme 14). Their four-component one-pot methodology consisted of the reaction of phthalic anhydride (**45**), dimedone (**46**), and hydrazine hydrate (**47**) with several aromatic aldehydes (**48**) in an ionic liquid under sonication. The products were obtained in good to excellent yields in very short reaction times.

Scheme 14.

#### **3.2 Imidazole derivatives**

In connection with reactions in aqueous media, low potency (50 Watts) sonochemistry has been used to prepare 2-imidazolines (**52**) from the reaction of aldehydes (**50**) with

Recent Advances in the Ultrasound-Assisted Synthesis of Azoles 89

and the desired products obtained in satisfactory yields. Ionic liquids have shown great potential as catalysts and are a particularly attractive alternative to conventional catalysts. Their ability to dissolve a wide variety of substance and their potential for recyclability are

Joshi and co-workers (Joshi et al., 2010) reported the synthesis of 1,3-imidazoles (**68**) by the reaction of substituted aldehydes (**67**) with *o*-phenylenediamine (**66**) catalysed by 5 mol% of tetrabutylammonium fluoride (TBAF) in water under ultrasonic irradiation at room temperature (Scheme 19). Quaternary ammonium fluoride salts are inexpensive and relatively non-toxic reagents and water is generally recognized to be a green solvent in organic synthesis. The reported synthesis thus represents a mild, chemoselective method for

TBAF, H2O

KOH, DMSO/H2O

)))), rt, 30-65 min <sup>R</sup> <sup>H</sup>

Very recently, Arani and Safari (Arani & Safari, 2011) reported a very efficient high yield (96-98%) synthesis of 5,5-diphenylhydantoin (**72**) and 5,5-diphenyl-2-thiohydantoin (**73**) derivatives under mild conditions (Scheme 20). The reactions were performed in

)))), rt, 90-260 s

**71**

R2

R1 **<sup>69</sup>**,X=O

Recently, Li and co-workers described a facile and economical procedure for the synthesis of spiro azole compounds (**77**) (Li et al., 2010). The one-pot synthesis of 3-aza-6,10-diaryl-2-oxa-

O

O

NH4OAc

[emim]OAc, EtOH )))), rt, 45-90 min

**64 65**

10 examples 70-96%

Ph <sup>R</sup>

N N H

Ph

13 examples 82-94%

N H

**68**

R2

R2

12 examples 96-99%

**72**,X=O **73**,X=S

O

N

H N X

N R

among the attributes responsible for their recent popularity.

H O

<sup>O</sup>

NH2

NH2

R2

**66 67**

DMSO/H2O with ultrasonic irradiation and catalyzed by KOH.

preparing these heterocycles.

H2N NHR<sup>1</sup> X

**70**,X=S

**3.3 Isoxazole derivatives** 

Scheme 20.

R

**62 63**

Ph O

Ph O

Scheme 18.

Scheme 19.

ethylenediamine (**51**) and NBS (*N*-bromosuccinimide) as catalyst (Scheme 15) (Sant'Anna et al., 2009). The compounds were isolated in high yields (80-99%) and required only short reaction times (12-18 minutes). The isolated compounds showed bioactivity as monoamine oxidase (MAO) inhibitors.

Scheme 15.

Shelke and co-workers (Shelke et al., 2009) reported the synthesis of 2,4,5-triaryl-1*H*imidazoles (**57**) from the three-component one-pot condensation of benzil (**53**)/benzoin (**54**), aldehydes (**55**) and ammonium acetate (**56**) in aqueous media under ultrasound at room temperature (Scheme 16). BO3H3 (5 mol%) was used as catalyst. The reaction, performed under conventional stirring without ultrasound, required a reaction time (180 minutes) clearly longer than those require when ultrasound was used.

Scheme 16.

Li and co-workers (Li et al., 2010) reported the synthesis of glycoluril derivatives catalyzed by potassium hydroxide in EtOH under ultrasonic irradiation (Scheme 17). Although the reaction was relatively efficient, it was not selective. Two products were isolated, the desired glycoluril (**60**) (17-75%) together with a hydantoin co-product (**61**) (1-37%).

Scheme 17.

Zang and co-workers (Zang et al., 2010) reported a three-component one-pot synthesis of 2 aryl-4,5-diphenyl imidazoles (**65**) at room temperature under ultrasonic irradiation (Scheme 18). The ionic liquid 1-ethyl-3-methylimidazole acetate ([emim]OAc) was used as catalyst and the desired products obtained in satisfactory yields. Ionic liquids have shown great potential as catalysts and are a particularly attractive alternative to conventional catalysts. Their ability to dissolve a wide variety of substance and their potential for recyclability are among the attributes responsible for their recent popularity.

Scheme 18.

88 Green Chemistry – Environmentally Benign Approaches

ethylenediamine (**51**) and NBS (*N*-bromosuccinimide) as catalyst (Scheme 15) (Sant'Anna et al., 2009). The compounds were isolated in high yields (80-99%) and required only short reaction times (12-18 minutes). The isolated compounds showed bioactivity as monoamine

**<sup>50</sup> <sup>51</sup> <sup>52</sup>**

Shelke and co-workers (Shelke et al., 2009) reported the synthesis of 2,4,5-triaryl-1*H*imidazoles (**57**) from the three-component one-pot condensation of benzil (**53**)/benzoin (**54**), aldehydes (**55**) and ammonium acetate (**56**) in aqueous media under ultrasound at room temperature (Scheme 16). BO3H3 (5 mol%) was used as catalyst. The reaction, performed under conventional stirring without ultrasound, required a reaction time (180 minutes)

)))), rt, 30-70 min Ar <sup>H</sup>

NH4OAc

**56**

NH4OAc

**56**

glycoluril (**60**) (17-75%) together with a hydantoin co-product (**61**) (1-37%).

H2O, NBS )))), 65-70 C, 12-18 min

BO3H3, H2O/EtOH

BO3H3, H2O/EtOH )))), rt, 50-95 min

Li and co-workers (Li et al., 2010) reported the synthesis of glycoluril derivatives catalyzed by potassium hydroxide in EtOH under ultrasonic irradiation (Scheme 17). Although the reaction was relatively efficient, it was not selective. Two products were isolated, the desired

)))), 38-42 C, 2-8 h

Zang and co-workers (Zang et al., 2010) reported a three-component one-pot synthesis of 2 aryl-4,5-diphenyl imidazoles (**65**) at room temperature under ultrasonic irradiation (Scheme 18). The ionic liquid 1-ethyl-3-methylimidazole acetate ([emim]OAc) was used as catalyst

16 examples 80-99%

12 examples 92-98% from benzil 85-94% from benzoin

N H Ar

**57**

HN NH

Ar1 Ar<sup>2</sup>

O

9 examples 1-37%

**61**

O

N

Ph

Ph

9 examples 17-75%

O

HN NH

HN NH

Ar<sup>2</sup> Ar1

O

KOH, EtOH

**60**

HN N R

oxidase (MAO) inhibitors.

Ph O

Ph O

Ph O

Ph OH

O

**58 59**

Ar<sup>2</sup> Ar<sup>1</sup>

O

Scheme 17.

Scheme 16.

Scheme 15.

clearly longer than those require when ultrasound was used.

<sup>O</sup>

**53 55**

**54 55**

Ar H <sup>O</sup>

> H2N NH2 O

H2N NH2

R H O

> Joshi and co-workers (Joshi et al., 2010) reported the synthesis of 1,3-imidazoles (**68**) by the reaction of substituted aldehydes (**67**) with *o*-phenylenediamine (**66**) catalysed by 5 mol% of tetrabutylammonium fluoride (TBAF) in water under ultrasonic irradiation at room temperature (Scheme 19). Quaternary ammonium fluoride salts are inexpensive and relatively non-toxic reagents and water is generally recognized to be a green solvent in organic synthesis. The reported synthesis thus represents a mild, chemoselective method for preparing these heterocycles.

Scheme 19.

Very recently, Arani and Safari (Arani & Safari, 2011) reported a very efficient high yield (96-98%) synthesis of 5,5-diphenylhydantoin (**72**) and 5,5-diphenyl-2-thiohydantoin (**73**) derivatives under mild conditions (Scheme 20). The reactions were performed in DMSO/H2O with ultrasonic irradiation and catalyzed by KOH.

Scheme 20.

### **3.3 Isoxazole derivatives**

Recently, Li and co-workers described a facile and economical procedure for the synthesis of spiro azole compounds (**77**) (Li et al., 2010). The one-pot synthesis of 3-aza-6,10-diaryl-2-oxa-

Recent Advances in the Ultrasound-Assisted Synthesis of Azoles 91

In 2009, Noei and Khosropour (Noei & Khosropour, 2009) reported a high yield, green protocol for the synthesis of 2,4-diarylthiazole derivatives (**89** and **91**) *via* the reaction of arylthioamides (**88** and **90**) with *α*-bromoacetophenones (**87**) under ultrasonic irradiation in

> Ar NH2 S

> > NH2

EtOH, )))) 30-35 C, 60-150 min

S

**90**

Among the natural products containing a 1,3-thiazole ring, thiamine (aneurine, vitamin B1) is of great importance (Eicher & Hauptmann, 2003). Several 2-(*N*-arylamino)-4-arylthiazoles (**94**) were prepared by the reaction of *α*-bromoacetophenones (**92**) with *N*-aryl substituted thioureas (**93**), as in the classical Hantzsch synthesis, but using ultrasonic irradiation (Scheme 26) (Gupta et al., 2010). This further confirmed that thiazole heterocycles can be conveniently synthesized in good yields (88-97%) by the application of sonochemistry. The

**88**

catalyst

R )))), 30-45 min

Catalyst = highly sulfonated carbon solid acid

H2N

S

R2

Recently, we reported an ultrasound-based procedure for the synthesis of pyrazolylsubstituted thiazoles (**97**) by the cyclization reaction between thiocarbamoyl-pyrazoles (**95**) and *α*-bromoacetophenone (**96**) (Venzke et al., 2011). The reactions occurred in only 15

**<sup>84</sup> <sup>85</sup>**

HO

H2N

CN

Scheme 24.

**3.5 Thiazole derivatives** 

Ar

Scheme 25.

Scheme 26.

O

**87**

O

Br

**92 93**

<sup>H</sup> <sup>R</sup><sup>1</sup>

Br

[bmim]BF4 )))), rt, 2-8.5 min

insecticidal activity of these 1,3-thiazoles was evaluated.

S N

H2N

the ionic liquid [bmim]BF4 (Scheme 25).

11 examples 76-95%

**86**

N

R O

S N

Ar Ar

12 examples 91-98%

S

R1

N N

8 examples 84-95%

Ar Ar

**89**

S

**91**

16 examples 88-97%

S N HN

R2

**94**

spiro[4.5]decane-1,4,8-trione (**77**) from 1,5-diaryl-1,4-pentadien-3-one (**74**), dimethyl malonate (**75**), and hydroxylamine hydrochloride (**76**) in the presence of sodium hydroxide gave good yields at 50 °C under ultrasound irradiation (Scheme 21).

Scheme 21.

A series of dihydroisoxazole derivatives (**80**) were prepared by the ultrasound-promoted cyclization reaction between chalcones (**78**) bearing a quinolinyl substituent and hydroxylamine hydrochloride (**79**) in the presence of sodium acetate in aqueous acetic acid solution (Scheme 22) (Tiwari et al., 2011). The sonochemical method gave better yields (87- 90%) of the products in shorter times (90-120 min) than the corresponding thermal reactions (72-78% in 6-7 h).

Scheme 22.

#### **3.4 Oxazole derivatives**

Ultrasound proved to be efficient for accelerating the cyclization reaction of aryl and methyl nitriles (**81**) with ethanolamine (**82**) catalyzed by InCl3 to give oxazole derivatives (**83**) (Scheme 23) (Moghadam et al., 2009). Products were obtained in 81-97% yields after 5-45 minutes under sonication at room temperature.

Scheme 23.

The same research group developed a new highly sulfonated carbon-based solid acid and employed it to catalyze reactions similar to those presented above under ultrasonic irradiation (Scheme 24) (Mirkhani et al., 2009). Reactions performed with a combination of the new catalyst and sonication were more efficient than those run without ultrasonic irradiation.

Scheme 24.

90 Green Chemistry – Environmentally Benign Approaches

spiro[4.5]decane-1,4,8-trione (**77**) from 1,5-diaryl-1,4-pentadien-3-one (**74**), dimethyl malonate (**75**), and hydroxylamine hydrochloride (**76**) in the presence of sodium hydroxide

<sup>O</sup> <sup>O</sup> **<sup>74</sup> <sup>75</sup>**

A series of dihydroisoxazole derivatives (**80**) were prepared by the ultrasound-promoted cyclization reaction between chalcones (**78**) bearing a quinolinyl substituent and hydroxylamine hydrochloride (**79**) in the presence of sodium acetate in aqueous acetic acid solution (Scheme 22) (Tiwari et al., 2011). The sonochemical method gave better yields (87- 90%) of the products in shorter times (90-120 min) than the corresponding thermal reactions

)))), 90-120 min

NH2OHHCl

**79**

N O

Ultrasound proved to be efficient for accelerating the cyclization reaction of aryl and methyl nitriles (**81**) with ethanolamine (**82**) catalyzed by InCl3 to give oxazole derivatives (**83**) (Scheme 23) (Moghadam et al., 2009). Products were obtained in 81-97% yields after 5-45

The same research group developed a new highly sulfonated carbon-based solid acid and employed it to catalyze reactions similar to those presented above under ultrasonic irradiation (Scheme 24) (Mirkhani et al., 2009). Reactions performed with a combination of the new catalyst and sonication were more efficient than those run without ultrasonic irradiation.

 InCl3 )))), 5-45 min <sup>R</sup><sup>1</sup> CN HO

R3

**82**

H2N

AcONa, AcOH, H2O

<sup>R</sup> <sup>N</sup> <sup>2</sup>

2. NH2OHHCl (**76**), )))), 50 C, 5-8 h <sup>O</sup>

OMe

O

1. NaOH, MeOH, )))), 50 C

9 examples 49-91%

HN O

**77**

8 examples 87-90%

Cl

N

17 examples 81-97%

O

R2 R3

**83**

R1

N

**80**

R

Ar Ar

O

gave good yields at 50 °C under ultrasound irradiation (Scheme 21).

O

Ar Ar MeO

O

R

minutes under sonication at room temperature.

**81**

Cl

**78**

**3.4 Oxazole derivatives** 

Scheme 21.

(72-78% in 6-7 h).

Scheme 22.

Scheme 23.

#### **3.5 Thiazole derivatives**

In 2009, Noei and Khosropour (Noei & Khosropour, 2009) reported a high yield, green protocol for the synthesis of 2,4-diarylthiazole derivatives (**89** and **91**) *via* the reaction of arylthioamides (**88** and **90**) with *α*-bromoacetophenones (**87**) under ultrasonic irradiation in the ionic liquid [bmim]BF4 (Scheme 25).

#### Scheme 25.

Among the natural products containing a 1,3-thiazole ring, thiamine (aneurine, vitamin B1) is of great importance (Eicher & Hauptmann, 2003). Several 2-(*N*-arylamino)-4-arylthiazoles (**94**) were prepared by the reaction of *α*-bromoacetophenones (**92**) with *N*-aryl substituted thioureas (**93**), as in the classical Hantzsch synthesis, but using ultrasonic irradiation (Scheme 26) (Gupta et al., 2010). This further confirmed that thiazole heterocycles can be conveniently synthesized in good yields (88-97%) by the application of sonochemistry. The insecticidal activity of these 1,3-thiazoles was evaluated.

Scheme 26.

Recently, we reported an ultrasound-based procedure for the synthesis of pyrazolylsubstituted thiazoles (**97**) by the cyclization reaction between thiocarbamoyl-pyrazoles (**95**) and *α*-bromoacetophenone (**96**) (Venzke et al., 2011). The reactions occurred in only 15

Recent Advances in the Ultrasound-Assisted Synthesis of Azoles 93

A library of spiro[indole-thiazolidinones] (**112**) was prepared sonochemically by a three component reaction in aqueous medium in the presence of cetyltrimethylammonium bromide (CTAB) as a phase transfer catalyst (Dandia et al., 2011). The reaction of indole-2,3 diones (**109**), aryl- or heteroaryl-amines (**110**), and *α*-mercaptocarboxylic acids (**111**) under ultrasound for 40-50 minutes afforded the target molecules in good to excellent yields (80-

)))), 45 C, 40-50 min

An ultrasound-mediated preparation of 1,3-selenazoles (**115**) was reported by Lalithamba and co-workers in 2010 (Lalithamba et al., 2010). The products were efficiently prepared by treatment of bromomethyl ketones (**113**) with selenourea (**114**) in acetone under ultrasonic

Ultrasonic activation of metal catalysts due to mechanical depassivation has been extensively exploited in organic synthesis. In this context, Cravotto and co-workers (Cravotto et al., 2010) reported an efficient copper-catalyzed azide-alkyne cycloaddition reaction for producing 1,2,3-triazole derivatives (**118**) using ultrasound or ultrasound and microwave simultaneously. Other activation methods were tested, but the best results were obtained when azides (**116**) and terminal alkynes (**117**) were sonicated in the presence of Cu turnings in dioxane/H2O at 70 °C or DMF at 100 °C (Scheme 32). Substitution of water by DMF was required to prevent the formation of copper complexes that made the purification of the products difficult when 6-monoazido-*β*-cyclodextrin derivatives were utilized as starting materials. No particularly significant differences were observed between the efficiencies of the reactions performed using ultrasound or ultrasound/microwave

H2N NH2 Se

**114**

acetone )))), 5-10 min <sup>N</sup>

HS CO2H Me

CTAB, H2O

**<sup>111</sup> <sup>112</sup>**

47 examples 80-98%

N H

<sup>N</sup> <sup>S</sup>

Me

R

11 examples 80-90%

R2

R1HN

**115**

Se

NH2

O

Ar

O

98%) (Scheme 30).

R

Scheme 30.

Scheme 31.

irradiation.

N H

**3.6 Selenazole derivatives** 

irradiation for 5-10 minutes (Scheme 31).

R1HN Br O

**113**

**4. Azoles with three heteroatoms** 

**4.1 Triazole derivatives** 

R2

**109**

O

O

Ar NH2

**110**

minutes in ethanol at room temperature, affording the pure products in 47-93% yields by simple filtration of the reaction mixture (Scheme 27).

Scheme 27.

Recently, Mamaghani and co-workers described a sonochemical method for the preparation of iminothiazolidinones (**102** and **103**) (Mamaghani et al., 2011). Thioureas (**100**) were generated *in situ* and treated with a mixture of a suitable aldehyde (**101**), chloroform and 1,8 diazabicyclo[5.4.0]undec-7-ene (DBU) in dimethyl ether (DME) under an inert atmosphere. Subsequent addition of aqueous NaOH at 0 °C and sonication furnished the products (**102** and **103**) in 75-91% yields (Scheme 28). A 1:1 mixture of regioisomers was observed when *N*cyclohexyl-*N'*-ethylthiourea was employed. However, a regiosselective reaction took place with other substituents in the thiourea. The target molecules were obtained in better yields and much shorter reaction times using ultrasound than with conventional methodology.

Scheme 28.

Neuenfeldt and co-workers used ultrasonic power to promote the synthesis of thiazolidinones (**108**) (Neuenfeldt et al., 2011). The products were obtained in good yields from the reaction of *in situ* generated imines (**106**) with one equivalent of mercaptoacetic acid (**107**) in toluene, under sonication for 5 minutes (Scheme 29). The corresponding conventional thermal reactions in the absence of ultrasound also furnished similar yields of these heterocycles, but required much longer times (16 h).

Scheme 29.

A library of spiro[indole-thiazolidinones] (**112**) was prepared sonochemically by a three component reaction in aqueous medium in the presence of cetyltrimethylammonium bromide (CTAB) as a phase transfer catalyst (Dandia et al., 2011). The reaction of indole-2,3 diones (**109**), aryl- or heteroaryl-amines (**110**), and *α*-mercaptocarboxylic acids (**111**) under ultrasound for 40-50 minutes afforded the target molecules in good to excellent yields (80- 98%) (Scheme 30).

Scheme 30.

92 Green Chemistry – Environmentally Benign Approaches

minutes in ethanol at room temperature, affording the pure products in 47-93% yields by

Recently, Mamaghani and co-workers described a sonochemical method for the preparation of iminothiazolidinones (**102** and **103**) (Mamaghani et al., 2011). Thioureas (**100**) were generated *in situ* and treated with a mixture of a suitable aldehyde (**101**), chloroform and 1,8 diazabicyclo[5.4.0]undec-7-ene (DBU) in dimethyl ether (DME) under an inert atmosphere. Subsequent addition of aqueous NaOH at 0 °C and sonication furnished the products (**102** and **103**) in 75-91% yields (Scheme 28). A 1:1 mixture of regioisomers was observed when *N*cyclohexyl-*N'*-ethylthiourea was employed. However, a regiosselective reaction took place with other substituents in the thiourea. The target molecules were obtained in better yields and

R3CHO (**101**), CHCl3, DBU, DME

Neuenfeldt and co-workers used ultrasonic power to promote the synthesis of thiazolidinones (**108**) (Neuenfeldt et al., 2011). The products were obtained in good yields from the reaction of *in situ* generated imines (**106**) with one equivalent of mercaptoacetic acid (**107**) in toluene, under sonication for 5 minutes (Scheme 29). The corresponding conventional thermal reactions in the absence of ultrasound also furnished similar yields of

N

**104** 60-92%

**106**

Z

NaOH, H2O, )))), rt, 12-15 min NR<sup>1</sup> <sup>S</sup>

**<sup>100</sup> <sup>102</sup> <sup>103</sup>**

O Br

much shorter reaction times using ultrasound than with conventional methodology.

EtOH )))), rt, 15 min

**<sup>96</sup> <sup>97</sup>**

11 examples 47-93%

R<sup>3</sup> O

NR<sup>2</sup>

10 examples 75-91%

R N S O O

HSCH2CO2H (**107**) toluene, )))), 2.5 min

NR<sup>2</sup> S R<sup>3</sup> O

O

Z R

11 examples

**108**

NR1

N <sup>N</sup> <sup>R</sup>

S N

simple filtration of the reaction mixture (Scheme 27).

N N

R

Scheme 27.

**98**

Scheme 28.

Z

R

Scheme 29.

H O

O O

**105**

H2N

**95**

S NH2

N H N H

these heterocycles, but required much longer times (16 h).

toluene )))), 2.5 min O O

<sup>S</sup> <sup>R</sup><sup>2</sup> NCS (**99**) CH2Cl2 R2 <sup>R</sup><sup>1</sup> R1NH2

#### **3.6 Selenazole derivatives**

An ultrasound-mediated preparation of 1,3-selenazoles (**115**) was reported by Lalithamba and co-workers in 2010 (Lalithamba et al., 2010). The products were efficiently prepared by treatment of bromomethyl ketones (**113**) with selenourea (**114**) in acetone under ultrasonic irradiation for 5-10 minutes (Scheme 31).

Scheme 31.

## **4. Azoles with three heteroatoms**

#### **4.1 Triazole derivatives**

Ultrasonic activation of metal catalysts due to mechanical depassivation has been extensively exploited in organic synthesis. In this context, Cravotto and co-workers (Cravotto et al., 2010) reported an efficient copper-catalyzed azide-alkyne cycloaddition reaction for producing 1,2,3-triazole derivatives (**118**) using ultrasound or ultrasound and microwave simultaneously. Other activation methods were tested, but the best results were obtained when azides (**116**) and terminal alkynes (**117**) were sonicated in the presence of Cu turnings in dioxane/H2O at 70 °C or DMF at 100 °C (Scheme 32). Substitution of water by DMF was required to prevent the formation of copper complexes that made the purification of the products difficult when 6-monoazido-*β*-cyclodextrin derivatives were utilized as starting materials. No particularly significant differences were observed between the efficiencies of the reactions performed using ultrasound or ultrasound/microwave irradiation.

Recent Advances in the Ultrasound-Assisted Synthesis of Azoles 95

)))), 70 C, 40-320 min Ar CN Ar

**125 126 127**

4-Sulphonyl-substituted pyrazoles (**130**) and isoxazoles (**132**) were synthesized *via* the onepot reaction of the carbanions of 1-aryl-2-(phenylsulphonyl)-ethanone (**128**) with several different hydrazonyl halides (**129**) or 1-aryl-2-bromo-2-hydroximinoethanones (**131**) in ethanol, respectively (Scheme 36) (Saleh et al., 2009). These reactions were accelerated by

> N R2 X

**129**

)))), 15-30 min

NH Ph

O

**131**

In 2009, Al-Zaydi (Al-Zaydi, 2009) reported the synthesis of triazole (**135**) and pyrazole (**138**) derivatives starting from arylhydrazononitriles (**133**) under ultrasonic irradiation (Scheme 37). The triazoles (**135**) were obtained *via* amidoxime intermediates (**134**) followed by cyclization with elimination of water. The pyrazoles (**138**) were prepared directly by reaction with chloroacetonitrile (**136**). This latter reaction involves the formation of a non-isolable intermediate (**137**) that undergoes intramolecular cyclization to give the final products (**138**).

> O N H

O N H

N N Ar

Ph

**137**

N NH Ar

CN

NOH

CN

Ph

N Br

OH

Br

ultrasonic irradiation and the products were isolated in high yields (90-97%).

Clay = Montmorillonite K-10, Kaolin

Scheme 35.

**6. Miscellaneous** 

<sup>S</sup> Ph

EtONa, EtOH, 10 min

NH2OHHCl, NaOAc EtOH, )))), 40 C, 30 min

ClCH2CN (**136**), TEA )))), 40 C, 30 min

O O

O

**128**

R1

Scheme 36.

O N H

CN

N NH Ar

Ph

**133**

Scheme 37.

NaN3

clay, DMF or H2O

10 examples

N N Ph

R<sup>2</sup> S Ph <sup>O</sup> <sup>O</sup>

Br 3 examples )))), 15-20 min 90-93%

**132**

NH2 O

TEA, DMF )))), 40C, 60 min

**134 135**

**130**

O

R1

S Ph <sup>O</sup> <sup>O</sup>

O N

2 examples 75-79%

O N H

Ph NH2

N N Ar

15 examples 90-97%

R1

2 examples 78-80%

CN

**138**

Ph NH2

N N N Ar

N H

N H N <sup>N</sup> <sup>N</sup>

$$\begin{array}{ccccc} \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } \\ \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } \\ \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } \\ \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } \\ \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } & \text{ $ \mathfrak{sl}$ } \\ \end{array}$$

Scheme 32.

#### **4.2 Oxadiazole derivatives**

Azoles containing polyhaloalkyl groups are of considerable interest due to their potential herbicidal, fungicidal, insecticidal, analgesic, antipyretic, and anti-inflammatory properties. In addition, 1,2,4-oxadiazoles are reported to posses various types of biological activities (Elguero et al., 2002). Very recently, the rapid preparation of 1,2,4-oxadiazoles (**121**) under ultrasound irradiation was reported (Bretanha et al., 2011). The products were obtained with short reaction times (15 minutes) and in excellent yields (84-98%) (Scheme 33).

Scheme 33.

#### **4.3 Thiadiazole derivatives**

1,3,4-Thiadiazole derivatives (**124**) were synthesized by the reaction of 1-methyl-5-oxo-3 phenyl-2-pyrazolin-4-thiocarboxanilide (**122**) with a series of hydrazonyl halides or *N*,*N'* diphenyl-oxalodihydrazonoyl dichloride (**123**) in the presence of triethylamine (TEA) under ultrasonic irradiation (Scheme 34) (El-Rahman et al., 2009). The products were obtained in excellent yields in short reaction times.

Scheme 34.

#### **5. Azoles with four heteroatoms**

#### **5.1 Tetrazole derivatives**

In 2010, Chermahini and co-workers reported the clay-catalyzed preparation of tetrazoles (**127**) under ultrasound (Scheme 35) (Chermahini et al., 2010). Compared to conventional heating, ultrasonic irradiation reduced the reaction times and increased the catalyst activity. Unfortunately, the yields obtained by this methodology were not specified by the authors.

Scheme 35.

94 Green Chemistry – Environmentally Benign Approaches

Cu(0) dioxane/H2O (70 C) or DMF (100 C) )))) or ))))/MW, 2 h

Azoles containing polyhaloalkyl groups are of considerable interest due to their potential herbicidal, fungicidal, insecticidal, analgesic, antipyretic, and anti-inflammatory properties. In addition, 1,2,4-oxadiazoles are reported to posses various types of biological activities (Elguero et al., 2002). Very recently, the rapid preparation of 1,2,4-oxadiazoles (**121**) under ultrasound irradiation was reported (Bretanha et al., 2011). The products were obtained with

> AcOEt )))), 15 min <sup>N</sup> <sup>O</sup> R Cl N

1,3,4-Thiadiazole derivatives (**124**) were synthesized by the reaction of 1-methyl-5-oxo-3 phenyl-2-pyrazolin-4-thiocarboxanilide (**122**) with a series of hydrazonyl halides or *N*,*N'* diphenyl-oxalodihydrazonoyl dichloride (**123**) in the presence of triethylamine (TEA) under ultrasonic irradiation (Scheme 34) (El-Rahman et al., 2009). The products were obtained in

Me N N

<sup>R</sup> **<sup>122</sup> <sup>123</sup>**

In 2010, Chermahini and co-workers reported the clay-catalyzed preparation of tetrazoles (**127**) under ultrasound (Scheme 35) (Chermahini et al., 2010). Compared to conventional heating, ultrasonic irradiation reduced the reaction times and increased the catalyst activity. Unfortunately, the yields obtained by this methodology were not specified by the authors.

TEA, EtOH )))), rt, 3-15 min

<sup>R</sup><sup>1</sup> <sup>N</sup> <sup>3</sup> <sup>R</sup><sup>2</sup> <sup>N</sup> <sup>N</sup>

short reaction times (15 minutes) and in excellent yields (84-98%) (Scheme 33).

O

**116 117**

**4.2 Oxadiazole derivatives** 

**4.3 Thiadiazole derivatives** 

excellent yields in short reaction times.

N N

PhHN SH Ph O

**5. Azoles with four heteroatoms** 

**5.1 Tetrazole derivatives** 

N Cl3C NH2

OH

**119 120**

N R X

NH Ar

Scheme 32.

Scheme 33.

Scheme 34.

6 examples 80-89% under ultrasound 80-95% under ultrasound/microwave

12 examples 84-98%

Cl3C

R

8 examples 90-95%

Ph O

N N S

Ar

Me

**124**

**121**

<sup>N</sup> <sup>R</sup><sup>1</sup> <sup>R</sup><sup>2</sup>

**118**

#### **6. Miscellaneous**

4-Sulphonyl-substituted pyrazoles (**130**) and isoxazoles (**132**) were synthesized *via* the onepot reaction of the carbanions of 1-aryl-2-(phenylsulphonyl)-ethanone (**128**) with several different hydrazonyl halides (**129**) or 1-aryl-2-bromo-2-hydroximinoethanones (**131**) in ethanol, respectively (Scheme 36) (Saleh et al., 2009). These reactions were accelerated by ultrasonic irradiation and the products were isolated in high yields (90-97%).

Scheme 36.

In 2009, Al-Zaydi (Al-Zaydi, 2009) reported the synthesis of triazole (**135**) and pyrazole (**138**) derivatives starting from arylhydrazononitriles (**133**) under ultrasonic irradiation (Scheme 37). The triazoles (**135**) were obtained *via* amidoxime intermediates (**134**) followed by cyclization with elimination of water. The pyrazoles (**138**) were prepared directly by reaction with chloroacetonitrile (**136**). This latter reaction involves the formation of a non-isolable intermediate (**137**) that undergoes intramolecular cyclization to give the final products (**138**).

Scheme 37.

Recent Advances in the Ultrasound-Assisted Synthesis of Azoles 97

acidic or basic media, respectively, has been reported (Shinde et al., 2010). The reactions were carried out employing ultrasound, microwaves and conventional conditions. Ultrasound afforded the best yields. The scope of these reactions was subsequently

> conc. H2SO4 )))), 20 min

aq. NaOH )))), 30-35 min

aq. NaOH )))), rt, 26-30 min

conc. H2SO4 )))), rt, 25-35 min

Yuan and Guo (Yuan & Guo, 2011) reported the one-pot cyclocondensation of *o*aminothiophenol (**155**) or aromatic *o*-diamines (**157**) with aromatic aldehydes (**154**) in the presence of chlorotrimethylsilane [TMSCl/Fe(NO3)3] in dimethylformamide under ultrasonic irradiation for the preparation of benzothiazoles (**156**) and benzimidazoles (**158**)

71-85% <sup>R</sup>

6 examples 70-78%

5 examples 72-83%

OMe

5 examples 80-88%

OMe

N

O

N

O

N NH N S R1

N

HN

R1 R2

R3

S N

MeO

F

F

F

F

**153**

MeO

O

N N N

N S N

9 examples HN

O

SH

**150**

R2

**152**

R3

R

R

**149**

expanded by Shelke and co-workers (Scheme 41) (Shelke et al., 2010).

H <sup>N</sup> <sup>N</sup> H

**148**

MeO O

Scheme 40.

N

OMe

Scheme 41.

F

F

H <sup>N</sup> <sup>N</sup> H

**151**

in 84-97% yields (Scheme 42).

<sup>H</sup> <sup>O</sup>

N

R1

R2

R3

S

O

<sup>H</sup> <sup>O</sup>

N

S

In 2009, we described a scaled-up sonochemical method to convert acetylacetone (**139**) into structurally simple pyrazoles (**140**) or an isoxazole (**141**) in aqueous media (Scheme 38) (Silva et al., 2009). The products were obtained after sonication for only 10 minutes, as compared to 12 hours in the thermal reaction without ultrasound.

Scheme 38.

As shown in Scheme 39 (Shinde et al., 2010), 2-ethyl-2-methyl-4*H*-chromen-4-ones (**142**) were transformed into semicarbazones (**143**) and thiosemicarbazones (**146**). The semicarbazones (**143**) could be sonochemically converted into selenadiazole derivatives (**144**) in 30 minutes by treatment with SeO2 in acetic anhydride. The same semicarbazones (**143**) afforded 1,2,3-thiadiazoles (**145**) under ultrasonic irradiation in 20 minutes in the presence of thionyl chloride. Similarly, sonication of the thiosemicarbazones (**146**) for 45 minutes in acetic anhydride produced the thiadiazolines (**147**) in good yields. Comparison of these results with those for the same reactions under microwave irradiation showed that the time required was longer and yields lower.

Scheme 39.

The syntheses of 1,3,4-thiadiazoles (**149**) and 1,3,4-triazoles (**150**) (Scheme 40) *via* the intramolecular cyclocondensation of benzofuran-substituted thiosemicarbazides (**148**) in

In 2009, we described a scaled-up sonochemical method to convert acetylacetone (**139**) into structurally simple pyrazoles (**140**) or an isoxazole (**141**) in aqueous media (Scheme 38) (Silva et al., 2009). The products were obtained after sonication for only 10 minutes, as

> NH2NHR, H2O )))), rt, 10 min

NH2OH, H2O )))), rt, 10 min

As shown in Scheme 39 (Shinde et al., 2010), 2-ethyl-2-methyl-4*H*-chromen-4-ones (**142**) were transformed into semicarbazones (**143**) and thiosemicarbazones (**146**). The semicarbazones (**143**) could be sonochemically converted into selenadiazole derivatives (**144**) in 30 minutes by treatment with SeO2 in acetic anhydride. The same semicarbazones (**143**) afforded 1,2,3-thiadiazoles (**145**) under ultrasonic irradiation in 20 minutes in the presence of thionyl chloride. Similarly, sonication of the thiosemicarbazones (**146**) for 45 minutes in acetic anhydride produced the thiadiazolines (**147**) in good yields. Comparison of these results with those for the same reactions under microwave irradiation showed that

R O <sup>1</sup>

R O <sup>1</sup>

R O <sup>1</sup>

**145**

The syntheses of 1,3,4-thiadiazoles (**149**) and 1,3,4-triazoles (**150**) (Scheme 40) *via* the intramolecular cyclocondensation of benzofuran-substituted thiosemicarbazides (**148**) in

**144**

NNHCSNH2

N N Se

N N S

R2

**142 146 147**

R2

R2

2 examples 76-99%

> O N

Ac2O )))), 45 min

5 examples 78-84%

5 examples 80-88%

1 example 70%

N N R

**140**

**141**

5 examples 70-89%

NHCONH2

N H3COC S

R O <sup>1</sup>

R2

compared to 12 hours in the thermal reaction without ultrasound.

O O

**139**

the time required was longer and yields lower.

NH2NHCSNH2 NaOAc, HCl, EtOH

> SeO2, Ac2O )))), 30 min

SOCl2 )))), 20 min

Scheme 38.

R O <sup>1</sup>

R O <sup>1</sup>

**143**

Scheme 39.

R2

O

NNHCONH2

NH2NHCONH2HCl NaOAc, H2O/EtOH

R2

acidic or basic media, respectively, has been reported (Shinde et al., 2010). The reactions were carried out employing ultrasound, microwaves and conventional conditions. Ultrasound afforded the best yields. The scope of these reactions was subsequently expanded by Shelke and co-workers (Scheme 41) (Shelke et al., 2010).

Scheme 41.

Yuan and Guo (Yuan & Guo, 2011) reported the one-pot cyclocondensation of *o*aminothiophenol (**155**) or aromatic *o*-diamines (**157**) with aromatic aldehydes (**154**) in the presence of chlorotrimethylsilane [TMSCl/Fe(NO3)3] in dimethylformamide under ultrasonic irradiation for the preparation of benzothiazoles (**156**) and benzimidazoles (**158**) in 84-97% yields (Scheme 42).

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Scheme 42.

### **7. Conclusion**

As we have shown in this chapter, several convenient ultrasound-promoted synthetic methodologies have been established for the preparation of the title class of compounds. The main advantages of the use of ultrasound in azole synthesis are evident when compared with classical methodologies i.e., a reduction in the reaction times and an improvement in yields. Most of the papers covered by this review employed simple ultrasonic cleaning baths as energy sources. Although these low potency sources of ultrasonic radiation are usually less efficient than immersion sonication probes, requiring longer reaction times, cleaning baths are relatively inexpensive and widely available in chemistry laboratories.

## **8. Acknowledgement**

The authors acknowledge CAPES and the CNPq (INCT Estudos do Meio Ambiente, grant 573.667/2008-0) for the financial support. FHQ is affiliated with INCT-Catalysis and NAP-PhotoTech (the USP Research Consortium for Photochemical Technology) and thanks the CNPq for fellowship support.

#### **9. References**


SH

NH2

**155**

NH2

NH2

As we have shown in this chapter, several convenient ultrasound-promoted synthetic methodologies have been established for the preparation of the title class of compounds. The main advantages of the use of ultrasound in azole synthesis are evident when compared with classical methodologies i.e., a reduction in the reaction times and an improvement in yields. Most of the papers covered by this review employed simple ultrasonic cleaning baths as energy sources. Although these low potency sources of ultrasonic radiation are usually less efficient than immersion sonication probes, requiring longer reaction times, cleaning

The authors acknowledge CAPES and the CNPq (INCT Estudos do Meio Ambiente, grant 573.667/2008-0) for the financial support. FHQ is affiliated with INCT-Catalysis and NAP-PhotoTech (the USP Research Consortium for Photochemical Technology) and thanks the

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TMSCl, Fe(NO3)3, DMF )))), 60 C, 30-150 min

TMSCl, Fe(NO3)3, DMF )))), 60 C, 30-150 min

O2N

**157 154**

S CHO

R

Scheme 42.

**7. Conclusion** 

**8. Acknowledgement** 

CNPq for fellowship support.

**9. References** 

8 examples 84-97%

S

S

R

R

N S

N

8 examples 89-96%

H N

NO2

**156**

**158**


Recent Advances in the Ultrasound-Assisted Synthesis of Azoles 101

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**6** 

*I. R. Iran* 

Mona Hosseini-Sarvari

**Greener Solvent-Free Reactions on ZnO** 

*Department of Chemistry, Faculty of Science, Shiraz University, Shiraz,* 

Due to the growing concern for the influence of the organic solvent on the environment as well as on human body, organic reactions without use of conventional organic solvents have attracted the attention of synthetic organic chemists. Although a number of modern solvents, such as fluorous media, ionic liquids and water have been extensively studied recently, not using a solvent at all is definitely the best option. Development of solvent-free

During the last decades, a central objective in synthetic organic chemistry has been to develop greener and more economically competitive processes for the efficient synthesis of biologically active compounds with potential application in the pharmaceutical or agrochemical industries. In this context, the solventless approach is simple with amazing versatility. It reduces the use of organic solvents and minimizes the formation of other waste. The reactions occur under mild conditions and usually require easier workup procedures and simpler equipment. Moreover, it may allow access to compounds that require harsh reaction conditions under traditional approaches or when the yields are too low to be of practical convenience. Because of economy and pollution, solvent-free reactions are of great interest in order to modernize classical procedures making them more clean, safe and easy to perform. Reactions on solid mineral supports, reactions without any solvent/support or catalyst, and solid-liquid phase transfer catalysis can be thus employed with noticeable increases in reactivity and selectivity. Therefore the following benefits could

1. Avoid of large volumes of solvent reduces emission and needs for distillation.

Zinc oxide was known for a long time, since it was a by-product of copper smelting. ZnO has been intensively studied since 1935 (Bunn, 1935) and the theory of semiconductors got a firm start. The interest in ZnO is fueled and fanned by its direct wide band gap (Eg ~ 3.3 eV at 300 K), (Kakiuchi et al., 2006). ZnO also has much simpler crystal-growth technology, resulting in a potentially lower cost for ZnO base devices. ZnO has wide applications in the field of optoelectronics, (Kong & Wang, 2004) spintronics, (Sharma et al., 2003) piezoelectric

5. Recyclable solid supports can be used instead of polluting mineral acids. 6. Safety is enhanced by reducing risks of overpressure and explosions.

**1. Introduction** 

organic reactions is thus gaining prominence.

be mentioned for solvent-free conditions:

2. Simple work-up, by extraction or distillation. 3. The absence of solvents facilitates scale-up.

4. Reactions are often cleaner, faster, and higher yielding.

aqueous solution under ultrasound irradiation. *Ultrasonics Sonochemistry*, Vol. 18, pp. 911-916.

