**1. Introduction**

Biocatalysis is the application of enzymes for chemical transformations of organic compounds. Enzymes as biocatalysts have many advantages [1–3]: (1) enzymes are very efficient catalysts. Typically the rates of enzyme-mediated processes are accelerated, compared to those of the corresponding nonenzymatic reaction, by a factor of 10<sup>8</sup> –1010. The acceleration may even exceed a value of 1012, which is far above the values that chemical catalysts are capable of achieving; (2) enzymes are environmentally acceptable. Unlike heavy metals, for instance, biocatalysts are completely degraded in the environment; (3) enzymes act under mild conditions. Enzymes act in a temperature range of 20–40°C, under neutral aqueous, and in the absence of substrate functional group protection. This minimizes problems of undesired side reactions such as decomposition, isomerization, racemization, and rearrangement, which often plague traditional methodology; (4) enzymes display high chemoselectivity, regioselectivity, and enantioselectivity. As a result, reactions that generally tend to be "cleaner" and laborious can largely be omitted; and (5) enzymes can catalyze a broad spectrum of reactions. There is an enzyme-catalyzed process equivalent to almost every type of organic reaction, such as oxidation, hydrolysis, addition, halogenation, alkylation, and isomerization. In addition, many enzymes accept unnatural substrates, and genetic engineering can further alter their stability, broaden their substrate specificity, and increase their specific activity. Thus, the application of enzymes in synthesis thus represents a remarkable opportunity for the development of industrial chemical and pharmaceutical processes [4–7].

organic solvents, not requiring cofactors, broad substrate tolerance, commercial availability, and high chemo-, regio-, and stereoselectivity. Hydrolases have demonstrated a great versatility in hydrolysis, transesterification, aminolysis reactions, etc. Some hydrolase-catalyzed promiscuous reactions have been done in the last decades (**Figure 1**). These research and other relevant reports encouraged us to believe that the catalytic activities for unconventional reactions rather than the well-known hydrolytic function may also have a natural role in hydrolase evolution. The aim of the present chapter is to give a brief overview of the hydrolasecatalyzed C▬C and C▬N reactions and present some of the most recent applications in different fields for recent decade. The main work in our group will be disclosed, highlighting the catalytic properties of hydrolases to catalyze not only single processes but also multicomponent and tandem reactions. Consequently, the promiscuous hydrolase-catalyzed reactions are outlined with focus on Michael

*Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis*

*DOI: http://dx.doi.org/10.5772/intechopen.89918*

Michael addition is a 1,4-addition of a nucleophile to α,β-unsaturated compounds, and it is one of the most fundamental and important reactions for the formation of carbon-carbon bonds and carbon-heteroatom bonds in organic synthesis. Michael addition reactions are traditionally catalyzed under strong basic or acidic conditions, which can cause unwanted side reactions such as further condensation or polymerization of α,β-unsaturated compounds. Thus, biocatalysis can afford a green and facile method for organic synthesis. Among different biocatalysts, hydrolases such as protease and lipase have been widely used as a

Michael addition is the early promiscuous reaction catalyzed by hydrolase. In 1986, Kitazume et al. reported the hydrolytic enzyme-catalyzed stereospecific Michael addition reactions in buffer solution (pH = 8.0) at 40–41°C (**Figure 2**) [16]. This discovery overthrows the long erroneous concept of enzymology that "biocatalysis must be carried out in aqueous solution," making many organic reactions that cannot be carried out in water be completed in organic solvents and greatly expanding the application scope of enzymes as catalysts. Moreover, enzymes are frequently more stable in organic solvents than in water. Thus, some research groups began to focus on enzyme-catalyzed Michael addition reactions in organic

Lin and Gotor et al. firstly reported the hydrolase-catalyzed Michael addition of

imidazole with acrylates catalyzed by alkaline protease from *Bacillus subtilis* in organic solvent in 2004 [17, 18]. Subsequently, other hydrolase-catalyzed Michael addition reactions were reported. In 2010, Bhanage et al. developed an efficient protocol for the regioselective aza-Michael addition of amines with acrylates using

addition, aldol reaction, Mannich reaction, Biginelli reaction, etc.

green and efficient catalyst for Michael addition.

*The first hydrolase-catalyzed Michael addition in buffer.*

**2.1 Carbon-heteroatom bond formation Michael addition**

**2. Michael addition**

solvents.

**Figure 2.**

**35**

Although it is well known that a given enzyme is able to catalyze a specific reaction efficiently, some unexpected experimental results have indicated that many enzymes have catalytic promiscuity [8–12]. Enzyme promiscuity is classified into three categories: (a) condition promiscuity, which is an enzyme's ability to work under unexpected condition; (b) substrate promiscuity, which is an enzyme's ability to work with unexpected substrates; and (c) catalytic promiscuity, which is an enzyme's ability to catalyze unexpected reactions. Among them, catalytic promiscuity has gained much attention as it opens a wide scope for the industrial application of enzymes.

During the past decade, biocatalytic promiscuity, as a new frontier extending the use of enzymes in organic synthesis, has received considerable attention and expanded rapidly. A classic example of promiscuous enzymatic behavior is pyruvate decarboxylase, which not only decarboxylates pyruvate but also links acetaldehyde and benzaldehyde to form R-phenylacetylcarbinol. The use of pyruvate decarboxylase to form carbon–carbon bonds, which does not occur in the natural reaction, was first studied in 1921 and was applied in industry today [13]. As one of the most rapidly growing areas in enzymology, multifunctional biocatalytic reactions not only highlights the existing catalysts but may provide novel and practical synthetic pathways which are not currently available. Miao et al. reviewed enzyme promiscuity for carbon-carbon bond-forming reactions like aldol couplings, Michael(-type) additions, Mannich reactions, Henry reactions, and Knoevenagel condensations [14]. Gotor-Fernández et al. also highlighted the hydrolase-catalyzed reactions for nonconventional transformations in the same year [15].

Hydrolases (such as lipase, protease, acylase) have received extensive attention as biocatalysts for a long time due to their many attractive properties like stability in

**Figure 1.** *Hydrolase-catalyzed promiscuous reactions.*

### *Hydrolase-Catalyzed Promiscuous Reactions and Applications in Organic Synthesis DOI: http://dx.doi.org/10.5772/intechopen.89918*

organic solvents, not requiring cofactors, broad substrate tolerance, commercial availability, and high chemo-, regio-, and stereoselectivity. Hydrolases have demonstrated a great versatility in hydrolysis, transesterification, aminolysis reactions, etc. Some hydrolase-catalyzed promiscuous reactions have been done in the last decades (**Figure 1**). These research and other relevant reports encouraged us to believe that the catalytic activities for unconventional reactions rather than the well-known hydrolytic function may also have a natural role in hydrolase evolution.

The aim of the present chapter is to give a brief overview of the hydrolasecatalyzed C▬C and C▬N reactions and present some of the most recent applications in different fields for recent decade. The main work in our group will be disclosed, highlighting the catalytic properties of hydrolases to catalyze not only single processes but also multicomponent and tandem reactions. Consequently, the promiscuous hydrolase-catalyzed reactions are outlined with focus on Michael addition, aldol reaction, Mannich reaction, Biginelli reaction, etc.
