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With various recombinant DNA and protein engineering techniques now available, enzyme-bad technologies are emerging as practical methods for large-scale synthesis of chiral intermediates and bioactive molecules, especially carbohydrates, oligosaccharides, their conjugates and related substances. This paper describes recent developments in the synthesis of novel monosaccharides and aza sugars based on aldolases, and the synthesis of oligosaccharides and analogs based on glycosyltransferases coupled with in sins regeneration of sugar nucleotides. As many enzymes are available for the stereocontrolled synthesis of chiral synthons (l), attention has been extended to the development of more effective and stable enzymes for the synthesis of molecules with increasing complexity (2). One class of such complex molecules are carbohydrates and their conjugates, especially those that exist on cell surfaces (3). These molecules are involved in many types of recognition phenomena (3-6); however, most of their precise functions have not been clearly identified at the molecular level. Part of the reason is that these molecules have been difficult to isolate, characterize and synthesize. Enzyme-based technology seems to be well suited for the synthesis of glycoconjugates and related substances for the study of their functions as these molecules are multifunctional and highly soluble in polar solvents, and many enzymes are available for the transformation of these molecules (7). The following describes some new technologies developed for the synthesis of sugar- and peptide- related substances based on recombinant or engineered enzymes.

New practical procedures have been developed for the enzyme-catalyzed synthesis of carbohydrates and peptides. Aldolases have been shown to be effective catalysts for the synthesis of uncommon sugars, particularly azasugars. Enzymatic methods for the large-scale synthesis of oligosaccharides have been developed with the use of glycosyltransferases coupled with the regeneration of sugar nucleotides. Engineered subtilisin variants that are stable and active in anhydrous dimethylformamide and in aqueous solution have been developed for peptide segment coupling.

Sialyl Lewis x and derivatives have been synthesized using β-1,4-galactosyltransferase and recombinant α-2,3-sialyltransferase and α-1,3-fucosyltransferase. The enzymatic glycosylations have been achieved on preparative scales with in situ regeneration of UDP-galactose, CMP-N-acetylneuraminic acid, and GDP-fucose. Additionally, galactosyltransferase and fucosyltransferases have been studied with respect to their substrate specificity and inhibition

With various recombinant DNA and protein engineering techniques now available, enzyme-bad technologies are emerging as practical methods for large-scale synthesis of chiral intermediates and bioactive molecules, especially carbohydrates, oligosaccharides, their conjugates and related substances. This paper describes recent developments in the synthesis of novel monosaccharides and aza sugars based on aldolases, and the synthesis of oligosaccharides and analogs based on glycosyltransferases coupled with in sins regeneration of sugar nucleotides. As many enzymes are available for the stereocontrolled synthesis of chiral synthons (l), attention has been extended to the development of more effective and stable enzymes for the synthesis of molecules with increasing complexity (2). One class of such complex molecules are carbohydrates and their conjugates, especially those that exist on cell surfaces (3). These molecules are involved in many types of recognition phenomena (3-6); however, most of their precise functions have not been clearly identified at the molecular level. Part of the reason is that these molecules have been difficult to isolate, characterize and synthesize. Enzyme-based technology seems to be well suited for the synthesis of glycoconjugates and related substances for the study of their functions as these molecules are multifunctional and highly soluble in polar solvents, and many enzymes are available for the transformation of these molecules (7). The following describes some new technologies developed for the synthesis of sugar- and peptide- related substances based on recombinant or engineered enzymes.

Sialyl Lewis x and derivatives have been synthesized using β-1,4-galactosyltransferase and recombinant α-2,3-sialyltransferase and α-1,3-fucosyltransferase. The enzymatic glycosylations have been achieved on preparative scales with in situ regeneration of UDP-galactose, CMP-N-acetylneuraminic acid, and GDP-fucose. Additionally, galactosyltransferase and fucosyltransferases have been studied with respect to their substrate specificity and inhibition

Several azasugar and glycal inhibitors were studied to map the active site of α-L-fucosidase enzyme.

New practical procedures have been developed for the enzyme-catalyzed synthesis of carbohydrates and peptides. Aldolases have been shown to be effective catalysts for the synthesis of uncommon sugars, particularly azasugars. Enzymatic methods for the large-scale synthesis of oligosaccharides have been developed with the use of glycosyltransferases coupled with the regeneration of sugar nucleotides. Engineered subtilisin variants that are stable and active in anhydrous dimethylformamide and in aqueous solution have been developed for peptide segment coupling.

A recombinant human Lewis α(1,3/1,4)fucosyltranferase has been studied for its acceptor substrate specificity and used in the synthesis of sialyl Lex and derivatives.

The pH rate profile for the hydrolysis of diethyl-p-nitrophenyl phosphate catalyzed by the phosphotriesterase from Pseudomonas diminuta shows a requirement for the deprotonation of an ionizable group for full catalytic activity. This functional group has an apparent pKa of 6.1 +/- 0.1 at 25 degrees C, delta Hion of 7.9 kcal/mol, and delta Sion of -1.4 cal/K.mol. The enzyme is not inactivated in the presence of the chemical modification reagents dithiobis-(2-nitrobenzoate), methyl methane thiosulfonate, carbodiimide, pyridoxal, butanedione, or iodoacetic acid and thus cysteine, asparate, glutamate, lysine, and arginine do not appear to be critical for catalytic activity. However, the phosphotriesterase is inactivated completely with methylene blue, Rose Bengal, or diethyl pyrocarbonate. The enzyme is not inactivated by diethyl pyrocarbonate in the presence of bound substrate analogs, and inactivation with diethyl pyrocarbonate is reversible upon addition of neutralized hydroxylamine. The modification of a single histidine residue by diethyl pyrocarbonate, as shown by spectrophotometric analysis, is responsible for the loss of catalytic activity. The pKinact for diethyl pyrocarbonate modification is 6.1 +/- 0.1 at 25 degrees C. These results have been interpreted to suggest that a histidine residue at the active site of phosphotriesterase is facilitating the reaction by general base catalysis.

Adenosine levels increase at seizure foci as part of a postulated endogenous negative feedback mechanism that controls seizure activity through activation of A1 adenosine receptors. Agents that amplify this site- and event-specific surge of adenosine could provide antiseizure activity similar to that of adenosine receptor agonists but with fewer dose-limiting side effects. Inhibitors of adenosine kinase (AK) were examined because AK is normally the primary route of adenosine metabolism. The AK inhibitors 5'-amino-5'-deoxyadenosine, 5-iodotubercidin, and 5'-deoxy-5-iodotubercidin inhibited maximal electroshock (MES) seizures in rats. Several structural classes of novel AK inhibitors were identified and shown to exhibit similar activity, including a prototype inhibitor, 4-(N-phenylamino)-5-phenyl-7-(5'-deoxyribofuranosyl)pyrrolo[2, 3-d]pyrimidine (GP683; MES ED50 = 1.1 mg/kg). AK inhibitors also reduced epileptiform discharges induced by removal of Mg2+ in a rat neocortical preparation. Overall, inhibitors of adenosine deaminase or of adenosine transport were less effective. The antiseizure activities of GP683 in the in vivo and in vitro preparations were reversed by the adenosine receptor antagonists theophylline and 8-(p-sulfophenyl)theophylline. GP683 showed little or no hypotension or bradycardia and minimal hypothermic effect at anticonvulsant doses. This improved side effect profile contrasts markedly with the profound hypotension, bradycardia, and hypothermia and greater inhibition of motor function observed with the adenosine receptor agonist N6-cyclopentyladenosine and opens the way to clinical evaluation of AK inhibitors as a novel, adenosine-based approach to anticonvulsant therapy.

This paper describes the use of three types of enzymes in carbohydrate synthesis. Aldolases are a useful class of catalysts for the synthesis of aza sugars and related compounds. Glycosyltransferases have been developed for the synthesis of oligosaccharides. The enzymatic oligosaccharide synthesis has been proven effective and practical when coupled with in situ regeneration of sugar nucleotides. A subtilisin variant prepared via site-directed mutagenesis is stable and active in dimethylformamide and is useful for regioselective acylation of sugars.

The pH rate profile for the hydrolysis of diethyl-p-nitrophenyl phosphate catalyzed by the phosphotriesterase from Pseudomonas diminuta shows a requirement for the deprotonation of an ionizable group for full catalytic activity. This functional group has an apparent pKa of 6.1 +/- 0.1 at 25 degrees C, delta Hion of 7.9 kcal/mol, and delta Sion of -1.4 cal/K.mol. The enzyme is not inactivated in the presence of the chemical modification reagents dithiobis-(2-nitrobenzoate), methyl methane thiosulfonate, carbodiimide, pyridoxal, butanedione, or iodoacetic acid and thus cysteine, asparate, glutamate, lysine, and arginine do not appear to be critical for catalytic activity. However, the phosphotriesterase is inactivated completely with methylene blue, Rose Bengal, or diethyl pyrocarbonate. The enzyme is not inactivated by diethyl pyrocarbonate in the presence of bound substrate analogs, and inactivation with diethyl pyrocarbonate is reversible upon addition of neutralized hydroxylamine. The modification of a single histidine residue by diethyl pyrocarbonate, as shown by spectrophotometric analysis, is responsible for the loss of catalytic activity. The pKinact for diethyl pyrocarbonate modification is 6.1 +/- 0.1 at 25 degrees C. These results have been interpreted to suggest that a histidine residue at the active site of phosphotriesterase is facilitating the reaction by general base catalysis.

The gene encoding for the phosphotriesterase (opd) from Pseudomonas diminuta has been subcloned into a baculovirus expression system. Functional enzyme is produced when the recombinant baculovirus is used to infect either cultured Spodoptera frugiperda sf9 cells or the larval stage of the fall armyworm. The LD50 for paraoxon toxicity was found to increase 280-fold in the larvae after infection with the recombinant baculovirus and expression of the functional phosphotriesterase.

The phosphotriesterase from Pseudomonas diminuta was tested as a catalyst for the hydrolysis of phosphofluoridates. The purified enzyme has been shown to hydrolyze the phosphorus-fluorine bond of diisopropyl fluorophosphate, isopropyl methylphosphonofluoridate, and 1,2,2-trimethylpropylmethylphosphonofluoridate at pH 7.0, 25 degrees C, with turnover numbers of 41, 56, and 5 s-1, respectively. The enzymatic rate enhancement for the hydrolysis of sarin at pH 7.0 is 2.2 X 10(7). The turnover number for paraoxon hydrolysis is 2100 s-1. The enzyme does not hydrolyze methanesulfonyl fluoride, phenylmethylsulfonyl fluoride, or O-p-nitrophenyl phenylsulfonate nor do these compounds inactivate or inhibit the ability of the enzyme to hydrolyze diethyl p-nitrophenyl phosphate. The breadth of substrate utility and the efficiency of the hydrolytic reaction exceed the more limited abilities of other prokaryotic and eukaryotic enzymes that catalyze similar reactions. The substantial rate enhancement exhibited by this enzyme for the hydrolysis of a wide variety of organophosphorus nerve agents make this enzyme the prime candidate for the biological detoxification of insecticide and mammalian acetylcholinesterase inhibitors.

The mechanism and substrate specificity of the phosphotriesterase from Pseudomonas diminuta have been examined. The enzyme hydrolyzes a large number of phosphotriester substrates in addition to paraoxon (diethyl p-nitrophenyl phosphate) and its thiophosphate analogue, parathion. The two ethyl groups in paraoxon can be changed to propyl and butyl groups, but the maximal velocity and Km values decrease substantially. The enzyme will not hydrolyze phosphomonoesters or -diesters. There is a linear correlation between enzymatic activity and the pKa of the phenolic leaving group for 16 paraoxon analogues. The beta value in the corresponding Brønsted plot is -0.8. No effect on either Vmax or Vmax/Km is observed when sucrose is used to increase the relative solvent viscosity by 3-fold. These results are consistent with rate-limiting phosphorus-oxygen bond cleavage. A plot of log V versus pH for the hydrolysis of paraoxon shows one enzymatic group that must be unprotonated for activity with a pKa of 6.1. The deuterium isotope effect by D2O on Vmax and Vmax/Km is 2.4 and 1.2, respectively, and the proton inventory is linear, which indicates that only one proton is "in flight" during the transition state. The inhibition patterns by the products are consistent with a random kinetic mechanism.

The phosphotriesterase produced from the opd cistron of Pseudomonas diminuta was purified 1500-fold to homogeneity using a combination of gel filtration, ion exchange, hydrophobic, and dye matrix chromatographic steps. This is the first organophosphate triesterase or organophosphofluoridate hydrolyzing enzyme to be purified to homogeneity. The enzyme is a monomeric, spherical protein having a molecular weight of 39,000. A single zinc atom is bound to the enzyme and is required for catalytic activity. Incubation with metal chelating compounds, o-phenanthroline, EDTA, or 2,6-pyridine dicarboxylate inactivate the enzyme. The kinetic rate constants, kcat and kcat/Km, for the hydrolysis of paraoxon are 2100 s-1 and 4 x 10(7) M-1 s-1, respectively. The enzyme is inhibited competitively by dithiothreitol, dithioerithritol, and beta-mercaptoethanol. In addition to paraoxon the phosphotriesterase was found to hydrolyze the commonly used organophosphorus insecticides, dursban, parathion, coumaphos, diazinon, fensulfothion, methyl parathion, and cyanophos.

The phosphotriesterase produced from the opd cistron of Pseudomonas diminuta was purified 1500-fold to homogeneity using a combination of gel filtration, ion exchange, hydrophobic, and dye matrix chromatographic steps. This is the first organophosphate triesterase or organophosphofluoridate hydrolyzing enzyme to be purified to homogeneity. The enzyme is a monomeric, spherical protein having a molecular weight of 39,000. A single zinc atom is bound to the enzyme and is required for catalytic activity. Incubation with metal chelating compounds, o-phenanthroline, EDTA, or 2,6-pyridine dicarboxylate inactivate the enzyme. The kinetic rate constants, kcat and kcat/Km, for the hydrolysis of paraoxon are 2100 s-1 and 4 x 10(7) M-1 s-1, respectively. The enzyme is inhibited competitively by dithiothreitol, dithioerithritol, and beta-mercaptoethanol. In addition to paraoxon the phosphotriesterase was found to hydrolyze the commonly used organophosphorus insecticides, dursban, parathion, coumaphos, diazinon, fensulfothion, methyl parathion, and cyanophos.