**2. Glyphosate and its biochemistry**

#### **2.1 The discovery of glyphosate**

The molecule *N*-(phosphonomethyl)glycine was first synthesised in 1950 by a researcher of the small Swiss pharmaceutical firm Cilag, Henri Martin (Franz et al. 1997). Yet, showing no pharmaceutical perspective, the compound has not been investigated any further. A decade later through the acquisition of the company, it was transferred to the distributor of laboratory research chemicals, Aldrich Chemical Co., along with research samples of Cilag. This is how it came to the attention of Monsanto Company (St. Louis, MO) in the course of its research to develop phosphonic acid type water-softening agents, through testing over 100 chemical substances related to aminomethylphosphonic acid (AMPA). Monsanto later extended the study of these compounds to herbicide activity testing, and observed their potential against perennial weeds (Dill et al., 2010). *N*-(phosphonomethyl)glycine (later termed glyphosate) was first re-synthesised and tested by Monsanto in 1970. Its herbicidal effect was described by Baird and co-workers in 1971, the subsequent patent (US 3799758), followed by numerous others, was claimed and obtained by Monsanto, and was introduced as a herbicide product Roundup® (formulation of the isopropylamine salt of glyphosate with a surfactant). Upon its introduction in the mid seventies, glyphosate jumped to a leading position on the pesticide market, became the most marketed herbicide active ingredient by the nineties, and more or less holds that position ever since. A great change came about, when the original patent protection expired in many parts of the world outside the United States in 1991. As a result, an almost immediate price decline occurred (by 30% in one year, 40% in two years and about 50% in two decades (Cox, 1998). Upon the expiration of the patent protection also in the United States in 2000, sales of generic preparations intensively expanded (main international producers include Dow, Syngenta, NuFarm, etc.), but the leading preparation producer remained Monsanto (Duke & Powles, 2008).

The current situation of the international active ingredient producers shows a rather different picture. Recently, Chinese chemical factories (e.g., Zhejiang Wynca Chemical Co., Zeijang Jinfanda Biochemical Co. and Hubei Xingta Chemical Group., Nantong Jiangshan Agrochemical and Chemical Co., Sichuan Fuhua Agricultural Investment Group, Jiangsu Yangnong Chemical Group, Jiangshu Good Harvest-Welen, etc.) gained leading parts of this business. At present, the global glyphosate production capacity is 1.1 million tonnes, while the global demand is only 0.5 million tonnes. The overall glyphosate production capacity of Chinese companies rose from 323,400 tonnes in 2007 to 835,900 tonnes in 2010, by a compounded annual growth rate of 37 percent (Yin, 2011). China has enough glyphosate capacity to satisfy the global demand even if all other glyphosate manufacturers cease production. The domestic demand of China is only 30-40 thousand tonnes, about 0.3 million tonnes of glyphosate is produced for export. Presently Chinese glyphosate production

by the recognition of the water-polluting feature of the parent compound, nor by the

It is not a simple task to predict whether glyphosate continues to rise in the near future, or its application will be abating. To facilitate better assessment of these two possibilities, the present work attempts to provide a summary of the utility and the environmental health

The molecule *N*-(phosphonomethyl)glycine was first synthesised in 1950 by a researcher of the small Swiss pharmaceutical firm Cilag, Henri Martin (Franz et al. 1997). Yet, showing no pharmaceutical perspective, the compound has not been investigated any further. A decade later through the acquisition of the company, it was transferred to the distributor of laboratory research chemicals, Aldrich Chemical Co., along with research samples of Cilag. This is how it came to the attention of Monsanto Company (St. Louis, MO) in the course of its research to develop phosphonic acid type water-softening agents, through testing over 100 chemical substances related to aminomethylphosphonic acid (AMPA). Monsanto later extended the study of these compounds to herbicide activity testing, and observed their potential against perennial weeds (Dill et al., 2010). *N*-(phosphonomethyl)glycine (later termed glyphosate) was first re-synthesised and tested by Monsanto in 1970. Its herbicidal effect was described by Baird and co-workers in 1971, the subsequent patent (US 3799758), followed by numerous others, was claimed and obtained by Monsanto, and was introduced as a herbicide product Roundup® (formulation of the isopropylamine salt of glyphosate with a surfactant). Upon its introduction in the mid seventies, glyphosate jumped to a leading position on the pesticide market, became the most marketed herbicide active ingredient by the nineties, and more or less holds that position ever since. A great change came about, when the original patent protection expired in many parts of the world outside the United States in 1991. As a result, an almost immediate price decline occurred (by 30% in one year, 40% in two years and about 50% in two decades (Cox, 1998). Upon the expiration of the patent protection also in the United States in 2000, sales of generic preparations intensively expanded (main international producers include Dow, Syngenta, NuFarm, etc.),

but the leading preparation producer remained Monsanto (Duke & Powles, 2008).

The current situation of the international active ingredient producers shows a rather different picture. Recently, Chinese chemical factories (e.g., Zhejiang Wynca Chemical Co., Zeijang Jinfanda Biochemical Co. and Hubei Xingta Chemical Group., Nantong Jiangshan Agrochemical and Chemical Co., Sichuan Fuhua Agricultural Investment Group, Jiangsu Yangnong Chemical Group, Jiangshu Good Harvest-Welen, etc.) gained leading parts of this business. At present, the global glyphosate production capacity is 1.1 million tonnes, while the global demand is only 0.5 million tonnes. The overall glyphosate production capacity of Chinese companies rose from 323,400 tonnes in 2007 to 835,900 tonnes in 2010, by a compounded annual growth rate of 37 percent (Yin, 2011). China has enough glyphosate capacity to satisfy the global demand even if all other glyphosate manufacturers cease production. The domestic demand of China is only 30-40 thousand tonnes, about 0.3 million tonnes of glyphosate is produced for export. Presently Chinese glyphosate production

emerging weed resistance worldwide.

problems of glyphosate applications.

**2.1 The discovery of glyphosate** 

**2. Glyphosate and its biochemistry** 

facilities have been suspended being limited by the market demand. Extended use of GT plants in the Word would help on this problem, even if Europe is hesitant to allow commercial cultivation of this kind of GM plants. The overall situation has led to continously decreasing glyphosate prices on the Word market, and has significant effects on dispread of GT plants.

#### **2.2 Mode of action**

Glyphosate is a phosphonomethyl derivative of the amino acid glycine. It is an amphoteric chemical substance containing a basic secondary amino function in the middle of the molecule and monobasic (carboxylic) and dibasic (phosphonic) acidic sites at both ends (Fig. 1). Containing both hydrogen cation (H+) donor (acidic) and acceptor (basic) functional groups, it can form cationic and anionic sites within the small molecule, the dissociation constants (pKa) of these three functional groups are 10.9, 5.9 and 2.3, and therefore, similarly to amino acids, glyphosate can form a zwitterionic structure (Knuuttila & Knuuttila, 1979). This is reflected in excellent water solubility (11.6 g/l at 25 oC). Consequently, its lipophilicity is very low (logP < -3.2 at 20 oC, pH 2-5), and is insoluble in organic solvents e.g., ethanol, acetone or xylene (Tomlin, 2000). To further increase its already good water solubility it is often formulated in form of its ammonium, isopropylammonium, potassium, sodium or trimethylsulphonium (trimesium) salts. The order of water solubility is glyphosate << ammonium salt < sodium salt < potassium salt < isopropylammonium salt < trimesium salt, the solubility of the trimesium salt being two orders of magnitude higher than that of glyphosate.

Fig. 1. The chemical structure of *N*-(phosphonomethyl)glycine, glyphosate, containing a basic function (amine) in the middle of the molecule and two acidic moieties (carboxylic and phosphonic acids) at both ends.

It has been known since the early seventies that glyphosate acts by inhibiting aromatic amino acid biosynthesis in plants (Jaworski, 1972; Amrhein et al., 1980), and elaborate research has revealed that the responsible mechanism is blocking a key step in the so-called shikimate pathway (Herman & Weaver, 1999), responsible for the synthesis of aromatic amino acids and critical plant metabolites. Glyphosate exerts this effect by inhibiting the activity of the enzyme 5-enolpyruvyl shikimate 3-phosphate synthase (EPSPS) catalyzing the transformation of phosphoenol pyruvate (PEP) to shikimate-3-phosphate (S3P) (Amrhein et al., 1980). This metabolic pathway exists in plants, fungi, and bacteria, but not in animals (Kishore & Shah 1988). Although higher order living organisms lack this metabolic route, therefore, are not expected to be directly affected by this herbicide, the environmental consequences of the widespread use of glyphosate have been reported (Cox, 2000; Santillo et al., 1989).

Being an amino acid (glycine) derivative itself, glyphosate inhibits the formation of the main intermediate, by binding as an analogue of the substrate PEP to its catalytic site on the enzyme. The inhibition of this catabolic pathway blocks the synthesis of triptophan, phenylalanine and tyrosine, and in consequence, the synthesis of proteins. The lack of the

Forty Years with Glyphosate 251

(Samland et al., 1999), structurally and mechanistically closely related EPSPS, and playing a

Various biochemical interactions of glyphosate, besides its identified mode of action, in plants and microorganisms were summarised by Hoagland and Duke (1982). The authors refer to numerous secondary or more complex indirect effects of glyphosate, and point out that a compound with such a powerful growth retardant effect or strong phytotoxicity will

The effects of glyphosate in the plant possibly include influences on the regulation of hormonal processes. Methionine levels are greatly reduced by glyphosate (Duke et al., 1979), which suggests that this herbicide may alter ethylene biosynthesis. Results of Baur (1979) suggest that glyphosate may inhibit auxin transport by increasing ethylene biosynthesis. Glyphosate may also affect the biosynthesis of non-aromatic amino acids. Nilsson (1977) suggested that the build-up of glutamate and glutamine in glyphosate-treated tissue might

It has been hypothesised that glyphosate lower phenylalanine and tyrosine pools not only by its primary mode of action, but possibly also by induction of phenylalanine ammonialyase (PAL) activity. Indeed, pronounced PAL activity has been detected in glyphosatetreated maize and soy (Duke et al., 1979; Cole et al., 1980), yet not by direct effect according to *in vitro* tests. Therefore, although glyphosate has been evidenced to cause profound effects on extractable PAL, substrate(s) and end products, increased PAL activity has been

Glyphosate did not appear to cause direct effects on photosynthesis, but its possible effect on chlorophyll biosynthesis has been considered, and its strong inhibitory effect on chlorophyll accumulation has been shown (Kitchen et al., 1981). Experimental result indicated that the effect of glyphosate on chlorophyll may be indirect through

Glyphosate has been shown to significantly affect the membrane transport of cellular contents only at very high concentrations (Brecke & Duke, 1980; Fletcher et al., 1980). Phosphorous uptake was retarded (Brecke & Duke, 1980), but loss of membrane integrity, decrease in energy supply or external ion chelation were excluded as causes. Moreover, uptake of amino acids, nucleotides and glucose were also found to be retarded by glyphosate in isolated cells (Brecke & Duke, 1980). Other studies (Cole et al., 1980; Duke & Hoagland, 1981) found inhibition of amino acid uptake by glyphosate not severe. Glyphosate has been reported to uncouple oxidative phosphorylation in plant (Olorunsogo et al., 1979) and mammalian (Olorunsogo & Bababunmi, 1980) mitochondria, the latter is likely to be due to altered membrane transport processes, as glyphosate was found to enhance proton permeability of mitochondrial membranes in a concentration-dependent

Glyphosate, exerting global herbicidal action, has originally been intended to pre-emergent weed control treatments of field vegetation and weed control of orchards and ruderal areas.

ultimately affect virtually all biochemical processes in the affected cells.

key role in the biosynthesis of UDP-muramic acid.

**2.4 Other biochemical effects of glyphosate** 

be due to blocked transamination reactions.

evaluated as a secondary effect (Hoagland & Duke, 1982).

photobleaching and/or peroxidation of chlorophyll.

**3. Pre-emergent application technology of glyphosate** 

manner (Olorunsogo, 1990).

synthesis of these essential amino acids and the proteins that contain them leads to rapid necrosis of the plant. Because this metabolic pathway is present in all higher order plants, and because the amino acid sequence of the active site of the EPSPS is a very conservative region in higher plants, the herbicidal effect is global among plant species.

Moreover, through its excellent solubility features glyphosate is a systemically active herbicide ingredient. As it is capable to be transported in the plant from the leaves towards the roots, it belongs to the relatively uncommon group of basipetally translocated herbicides (Ashton & Crafts, 1981). Its uptake and translocation is relatively rapid in diverse species (Sprankle et al., 1975).

#### **2.3 Transition state analogue theory of enzyme inhibition**

A unique feature of the mechanism of the inhibition of EPSPS by glyphosate is that glyphosate is reported to show close similarity in its structure to the tetrahedral phosphoenolpyruvoyl oxonium ion derivative of PEP, formed during its catalytic conversion to S3P, and the adduct formation with EPSPS has been verified by nuclear magnetic resonance spectroscopy (Christensen & Schaefer, 1993). Therefore, it has been proposed that glyphosate exerts its inhibitory activity as transition-state analogue (TSA) of the putative phosphoenolpyruvoyl oxonium ion derivative of PEP from plants (Anton et al., 1983; Steinrücken & Amrhein, 1984; Kishore & Shah, 1988) and bacteria (Du et al., 2000; Arcuri et al., 2004).

The so-called transition state theory has been advanced by Pauling (1948) to explain the mechanism of enzymatic reactions. Enyzmes are catalysts therefore they accelerate a reaction without influencing its equilibrium constant. One way to achieve that is to diminish the energy barrier of the reaction by lowering the energy of the transition state, transient, unstable intermediate of the reaction. This may be accomplished through stabilizing the transition state by binding to it as soon as it has occurred, and thus facilitating its formation. This results in the enzymatic effect that lowers the activation energy of the catalyzed reaction. Based on this idea, extremely potent inhibitors can be developed for a given enzymatic reaction if one can synthesize "transition state analogues" or "transition state mimics": stable chemical compounds resembling the transition state (Wolfenden, 1969). The TSA theory has therefore been successfully applied to the development of various biologically active substances, including insect control agents (Hammock et al., 1988), sulfonylurea microherbicides (Schloss & Aulabaugh, 1990) or compounds relatd to glyphosate (Marzabadi et al., 1992; Anderson et al., 1995).

The TSA hypothesis as it applies to the mechanism of the inhibition of EPSPS by glyphosate, became widely accepted as it has been evidenced in numerous studies that glyphosate forms a tight ternary complex with EPSPS (Herman & Weaver, 1999). It is easy to understand, however, that a classical TSA inhibitor would cause irreversible inhibition of the enzyme, competeable (although possibly with a low affinity) by the natural substrate of the enzyme. In later studies, it has been evidenced by biochemist researchers of Monsanto that glyphosate was an inhibitor of EPSPS uncompetitive with EPSP, and therefore, the TSA hypothesis has been reconsidered (Sammons et al., 1995; Schönbrunn et al., 2001; Alibhai & Stallings, 2001; Funke et al., 2006). The effects of glyphosate on aromatic amino acid synthesis in *Escherychia coli* have been attributed to chelation of Co2+ and Mg2+ (Roisch & Lingens, 1980), cofactors for enzymes in this pathway. Moreover, it is interesting, that glyphosate does not inhibit the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase

synthesis of these essential amino acids and the proteins that contain them leads to rapid necrosis of the plant. Because this metabolic pathway is present in all higher order plants, and because the amino acid sequence of the active site of the EPSPS is a very conservative

Moreover, through its excellent solubility features glyphosate is a systemically active herbicide ingredient. As it is capable to be transported in the plant from the leaves towards the roots, it belongs to the relatively uncommon group of basipetally translocated herbicides (Ashton & Crafts, 1981). Its uptake and translocation is relatively rapid in diverse species

A unique feature of the mechanism of the inhibition of EPSPS by glyphosate is that glyphosate is reported to show close similarity in its structure to the tetrahedral phosphoenolpyruvoyl oxonium ion derivative of PEP, formed during its catalytic conversion to S3P, and the adduct formation with EPSPS has been verified by nuclear magnetic resonance spectroscopy (Christensen & Schaefer, 1993). Therefore, it has been proposed that glyphosate exerts its inhibitory activity as transition-state analogue (TSA) of the putative phosphoenolpyruvoyl oxonium ion derivative of PEP from plants (Anton et al., 1983; Steinrücken & Amrhein, 1984;

The so-called transition state theory has been advanced by Pauling (1948) to explain the mechanism of enzymatic reactions. Enyzmes are catalysts therefore they accelerate a reaction without influencing its equilibrium constant. One way to achieve that is to diminish the energy barrier of the reaction by lowering the energy of the transition state, transient, unstable intermediate of the reaction. This may be accomplished through stabilizing the transition state by binding to it as soon as it has occurred, and thus facilitating its formation. This results in the enzymatic effect that lowers the activation energy of the catalyzed reaction. Based on this idea, extremely potent inhibitors can be developed for a given enzymatic reaction if one can synthesize "transition state analogues" or "transition state mimics": stable chemical compounds resembling the transition state (Wolfenden, 1969). The TSA theory has therefore been successfully applied to the development of various biologically active substances, including insect control agents (Hammock et al., 1988), sulfonylurea microherbicides (Schloss & Aulabaugh, 1990) or compounds relatd to

The TSA hypothesis as it applies to the mechanism of the inhibition of EPSPS by glyphosate, became widely accepted as it has been evidenced in numerous studies that glyphosate forms a tight ternary complex with EPSPS (Herman & Weaver, 1999). It is easy to understand, however, that a classical TSA inhibitor would cause irreversible inhibition of the enzyme, competeable (although possibly with a low affinity) by the natural substrate of the enzyme. In later studies, it has been evidenced by biochemist researchers of Monsanto that glyphosate was an inhibitor of EPSPS uncompetitive with EPSP, and therefore, the TSA hypothesis has been reconsidered (Sammons et al., 1995; Schönbrunn et al., 2001; Alibhai & Stallings, 2001; Funke et al., 2006). The effects of glyphosate on aromatic amino acid synthesis in *Escherychia coli* have been attributed to chelation of Co2+ and Mg2+ (Roisch & Lingens, 1980), cofactors for enzymes in this pathway. Moreover, it is interesting, that glyphosate does not inhibit the enzyme UDP-N-acetylglucosamine enolpyruvyl transferase

region in higher plants, the herbicidal effect is global among plant species.

**2.3 Transition state analogue theory of enzyme inhibition** 

Kishore & Shah, 1988) and bacteria (Du et al., 2000; Arcuri et al., 2004).

glyphosate (Marzabadi et al., 1992; Anderson et al., 1995).

(Sprankle et al., 1975).

(Samland et al., 1999), structurally and mechanistically closely related EPSPS, and playing a key role in the biosynthesis of UDP-muramic acid.

#### **2.4 Other biochemical effects of glyphosate**

Various biochemical interactions of glyphosate, besides its identified mode of action, in plants and microorganisms were summarised by Hoagland and Duke (1982). The authors refer to numerous secondary or more complex indirect effects of glyphosate, and point out that a compound with such a powerful growth retardant effect or strong phytotoxicity will ultimately affect virtually all biochemical processes in the affected cells.

The effects of glyphosate in the plant possibly include influences on the regulation of hormonal processes. Methionine levels are greatly reduced by glyphosate (Duke et al., 1979), which suggests that this herbicide may alter ethylene biosynthesis. Results of Baur (1979) suggest that glyphosate may inhibit auxin transport by increasing ethylene biosynthesis. Glyphosate may also affect the biosynthesis of non-aromatic amino acids. Nilsson (1977) suggested that the build-up of glutamate and glutamine in glyphosate-treated tissue might be due to blocked transamination reactions.

It has been hypothesised that glyphosate lower phenylalanine and tyrosine pools not only by its primary mode of action, but possibly also by induction of phenylalanine ammonialyase (PAL) activity. Indeed, pronounced PAL activity has been detected in glyphosatetreated maize and soy (Duke et al., 1979; Cole et al., 1980), yet not by direct effect according to *in vitro* tests. Therefore, although glyphosate has been evidenced to cause profound effects on extractable PAL, substrate(s) and end products, increased PAL activity has been evaluated as a secondary effect (Hoagland & Duke, 1982).

Glyphosate did not appear to cause direct effects on photosynthesis, but its possible effect on chlorophyll biosynthesis has been considered, and its strong inhibitory effect on chlorophyll accumulation has been shown (Kitchen et al., 1981). Experimental result indicated that the effect of glyphosate on chlorophyll may be indirect through photobleaching and/or peroxidation of chlorophyll.

Glyphosate has been shown to significantly affect the membrane transport of cellular contents only at very high concentrations (Brecke & Duke, 1980; Fletcher et al., 1980). Phosphorous uptake was retarded (Brecke & Duke, 1980), but loss of membrane integrity, decrease in energy supply or external ion chelation were excluded as causes. Moreover, uptake of amino acids, nucleotides and glucose were also found to be retarded by glyphosate in isolated cells (Brecke & Duke, 1980). Other studies (Cole et al., 1980; Duke & Hoagland, 1981) found inhibition of amino acid uptake by glyphosate not severe. Glyphosate has been reported to uncouple oxidative phosphorylation in plant (Olorunsogo et al., 1979) and mammalian (Olorunsogo & Bababunmi, 1980) mitochondria, the latter is likely to be due to altered membrane transport processes, as glyphosate was found to enhance proton permeability of mitochondrial membranes in a concentration-dependent manner (Olorunsogo, 1990).
