Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.     

Stereospecific induction of rat liver bilirubin UDPglucuronosyltransferase and lauric acid 12-hydroxylation by the isomers of 2-phenylpropionic acid

The inductive effects of racemic 2-phenylpropionic acid and its isomers on rat liver bilirubin UDP-glucuronosyltransferase activity and lauric acid 12-hydroxylation (cytochrome P-452-dependent) were compared. The (S)-(+)-enantiomer and the racemic mixture gave the greatest induction of both enzyme activities, whereas (R)-(-)-2-phenylpropionic acid produced increases of only one-third of those of its antipode. The determination of the enantiomeric composition of the excreted 2-phenylpropionic acid after a single oral dose indicated that the (R)-(-)-enantiomer given as such or in the racemate was inverted to its antipode, which strongly suggests that (S)-(+)-2-phenylpropionic acid is responsible for the inductive effects observed. The demonstration of the same stereospecificity for the induction of bilirubin UDPglucuronosyltransferase and lauric acid 12-hydroxylation further indicates a close mechanistic link between these two processes.     

Enantioconvergent synthesis of (-)-(S)- and (+)-(R)-2-acetyl-3,6-dihydroxycyclohex-2-enone starting from rac-6-hydroxy-3-methoxycyclohex-2-enone

The synthesis of enantiomerically pure (-)-(S)- and (+)-(R)-2-acyl-3,6-dihydroxycyclohex-2-enone starting from diastereomerically pure N-tosyl-(S)-proline esters 3-methoxy-6-hydroxycyclohex-2-enone 1 is presented. An enantioconvergent synthesis of either (-)-(S)- and (+)-(R)-2-acyl-3,6-dihydroxycyclohex-2-enone starting with the racemic alpha-ketol 1 through a conversion of ( approximately 1:1) mixture of diastereomeric esters into one diastereomer by a repeated crystallization, followed by dimethylaminopyridine-catalyzed equilibration as key steps is described.     

Production of highly optically active (R)-3-chloro-1,2-propanediol using a bacterium assimilating the (S)-isomer

A bacterium that assimilates (S)-3-chloro-1,2-propanediol [monochlorohydrin (MCH)] was isolated from soil by enrichment culture. The bacterium was identified as Pseudomonas sp. by taxonomic studies. The strain grew in a medium containing racemic MCH as a source of carbon and degraded (S)-MCH stereoselectively, liberating chloride ions. The residual isomer was the (R)-form [99.5% enantiomeric excess (ee)], which was obtained from the racemate in a final yield of 36% by using this strain. Subsequently, highly optically active (R)-glycidol (GLD) (99.3% ee) was prepared from the (R)-MCH obtained by reaction in alkaline solution. The cell-free extracts of the cells had both dehalogenating and epoxide-opening activities, which converted various halohydrins to the corresponding epoxides and epoxides to the corresponding diols, respectively. Correspondence to: T. Suzuki     

Asymmetric biocatalysis of S-3-amino-3-phenylpropionic acid with new isolated Methylobacterium Y1-6

β-amino acids are widely used in drug research, and S-3-amino-3-phenylpropionic acid (S-APA) is an important pharmaceutical intermediate of S-dapoxetine, which has been approved for the treatment of premature ejaculation. Chiral catalysis is an excellent method for the preparation of enantiopure compounds. In this study, we used (±)-ethyl-3-amino-3-phenylpropanoate (EAP) as the sole carbon source. Three hundred thirty one microorganisms were isolated from 30 soil samples, and 17 strains could produce S-APA. After three rounds of cultivation and identification, the strain Y1-6 exhibiting the highest enantioselective activity of S-APA was identified as Methylobacterium oryzae. The optimal medium composition contained methanol (2.5 g/L), 1,2-propanediol (7.5 g/L), soluble starch (2.5 g/L), and peptone (10 g/L); it was shaken at 220 rpm for 4–5 days at 30 °C. The optimum condition for biotransformation of EAP involved cultivation at 37 °C for 48 h with 120 mg of wet cells and 0.64 mg of EAP in 1 ml of transfer solution. Under this condition, substrate ee was 92.1% and yield was 48.6%. We then attempted to use Methylobacterium Y1-6 to catalyze the hydrolytic reaction with substrates containing 3-amino-3-phenyl-propanoate ester, N-substituted-β-ethyl-3-amino-3-phenyl-propanoate, and γ-lactam. It was found that 5 compounds with ester bonds could be stereoselectively hydrolyzed to S-acid, and 2 compounds with γ-lactam bonds could be stereoselectively hydrolyzed to (-)-γ-lactam.     

产碱杆菌ECU0401催化拆分扁桃酸及其衍生物

从土壤中分离的1株产碱杆菌Alcaligenes sp.ECU0401具有扁桃酸脱氢酶活性,可以以扁桃酸、苯甲酰甲酸或苯甲酸为唯一C源生长,并且具有较高的脱氢酶活力。以外消旋扁桃酸为C源,采用分批补料策略培养（或反应）99h,扁桃酸累计投入量为30.4g/L,（S）-（＋）-扁桃酸被完全降解,（R）-（-）-扁桃酸回收产率为32.8%,对映体过量值（e.e.）〉99.9%。利用静息细胞作为催化剂不对称降解外消旋扁桃酸的氯代衍生物,制备获得光学活性的（R）-（-）-邻氯扁桃酸、（S）-（＋）-间氯扁桃酸和（S）-（＋）-对氯扁桃酸,光学纯度均超过99.9%e.e.。     

Synthesis, enantiomeric resolution, and selective C-11 methylation of a highly selective radioligand for imaging the norepinephrine transporter with positron emission tomography

Reboxetine, 2-[alpha-(2-ethoxyphenoxy)benzyl]morpholine, is a highly selective norepinephrine transporter (NET) blocker that has been used for the treatment of depression. Its methyl analogue, 2-[alpha-(2-methoxyphenoxy)benzyl]morpholine (MRB), has been radiolabeled with C-11 for studies of the NET system with positron emission tomography (PET). The normethyl precursor, 2-[alpha-(2-hydroxyphenoxy)benzyl]morpholine (desethylreboxetine), was synthesized in 6% overall yield via a multi-step regio- and stereo-specific synthesis, starting from a mono-O-protected catechol. The resulting racemic mixture of desethylreboxetine was resolved by chiral HPLC to provide the (2S,3S) and (2R,3R) enantiomers in >98% enantiomeric excess. These enantiomers were then used as precursors for radiosynthesis to prepare enantiomerically pure individual 11C-labeled MRB enantiomers for comparative PET studies in baboons. Selective C-11 methylation at the phenolic oxygen with [11C]CH3I was achieved in the presence of excess base. After HPLC purification, racemic ((2S,3S)/(2R,3R)) or enantiomerically pure ((2S,3S) or (2R,3R)) [11C]MRB was obtained in 61-74% decay-corrected radiochemical yields from [11C]CH3I in a synthesis time of 40 min with a radiochemical purity of >96% and a specific activity of 1.7-2.3 Ci/micromol (63-85 GBq/micromol) corrected from the end of bombardment (EOB).     

环氧丙醇酯立体专-性水解酶产生菌的筛选

从土壤中筛选出一株能拆分（Ｒ,Ｓ）－环氧丙醇丁酸酯的根霉（Ｒｈｉｚｏｐｕｓｓｐ．Ｂｃ０－０９）,该菌株所产胞外脂肪酶在水解环氧丙醇丁酸酯的反应中具有良好的立体专一性。在ｐＨ恒定７．０的条件下,以其发酵液水解底物,当转化率为５８％时,残留的（Ｒ）－环氧丙醇丁酸酯的光学纯度（对映体过量值）达９６．１％。     

Efficient route to (S)-azetidine-2-carboxylic acid

A new and efficient route to (S)-azetidine-2-carboxylic acid (>99.9% ee) in five steps and total yield of 48% via malonic ester intermediates was established. As the key step, efficient four-membered ring formation (99%) was achieved from dimethyl (S)-(1'-methyl)benzylaminomalonate by treating with 1,2-dibromoethane (1.5 eq) and cesium carbonate (2 eq) in DMF. Krapcho dealkoxycarbonylation of dimethyl (1'S)-1-(1'-methyl)benzylazetidine-2,2-dicarboxylate, the product of this cyclization procedure, proceeded with preferential formation (2.7:1, 78% total yield) of the desired (2S,1'S)-monoester, with the help of a chiral auxiliary which was introduced on the nitrogen atom. The undesired (2R,1'S)-isomer could be converted to that with proper stereochemistry, by a deprotonation and subsequent re-protonation step. Finally, lipase-catalyzed preferential hydrolysis of the (2S,1'S)-monoester and subsequent deprotection provided enantiomerically pure (S)-azetidine-2-carboxylic acid in a 91% yield from the mixture of (2S,1'S)- and (2R,1'S)-isomers.     

Production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile by the combination of nitrile hydratase and stereoselective amidase in Rhodococcus equi TG328

A new soil isolate, tentatively identified as Rhodococcus equi TG328, was found to be effective in the production of S-(+)-2-phenylpropionic acid from (R,S)-2-phenylpropionitrile. The conversion is catalysed by two enzymes. First, a nitrile hydratase converts the (R,S)-nitrile to (R,S)-2-phenylpropionamide. Second, a stereoselective amidase converts the S-(+)-amide to S-(+)-2-phenylpropionic acid. Conditions for optimal enzyme production and accumulation of S-(+)-2-phenylpropionic acid by resting cells were studied. The reaction of resting cells for 30 h at 10° C with (R,S)-2-phenylpropionitrile resulted in the production of 100 g of S-(+)-2-phenylpropionic acid per litre of reaction mixture. The enantiometric excess of the purified S-(+)-2-phenylpropionic acid was 99.4%. The amount of S-(+)-2-phenylpropionic acid accumulated was enhanced by lower reaction temperatures. In addition, unreacted R-(–)-2-phenylpropionamide with 99.0% enantiometric excess was isolated. Correspondence to: T. Nagasawa     

Chiral inversion of 2-phenylpropionic acid by Cordyceps militaris

Y.-F. HUNG, M.J. THOMASON, W. RHYS-WILLIAMS, A.W. LLOYD AND G.W. HANLON. 1996. Fifty-nine microbial cultures were screened for their ability to carry out the chiral inversion of (R,S) and (R)-2-phenylpropionic acid under both growing and non-growing conditions. While a number of these micro-organisms metabolized the substrate, only Verticillium lecanii IMI 68689 and Cordyceps militaris CBS 128.25 were able to invert the (R)-enantiomer to its (S)-antipode with no other biotransformation occurring. The rate of inversion was faster for C. militaris but in both cases the reaction was independent of growth and reached an equilibrium after 5 d incubation. Evidence suggests that the (R) to (S) inversion is inhibited in the presence of excess (S)-enantiomer which may account for the equilibrium enantiomeric ratios (S:R) of 90:10 and 70:30 for C. militaris and V. lecanii , respectively. Optimization studies revealed that the fastest rates of reaction for C. militaris were found at lower substrate concentrations (100 μg ml^-1) whilst the greatest yield of (S)-2-PPA was obtained at 250 μg ml^-1. Optimum inversion was found to occur using Sørensen's phosphate buffer at pH 5.5. These results may have implications for the use of micro-organisms in the production of pure enantiomers from racemic mixtures and, in addition, provide an in vitro model system for the study of a unique and interesting biotransformation.     

Cloning and characterization of a novel beta-transaminase from Mesorhizobium sp. strain LUK: a new biocatalyst for the synthesis of enantiomerically pure beta-amino acids

A novel beta-transaminase gene was cloned from Mesorhizobium sp. strain LUK. By using N-terminal sequence and an internal protein sequence, a digoxigenin-labeled probe was made for nonradioactive hybridization, and a 2.5-kb gene fragment was obtained by colony hybridization of a cosmid library. Through Southern blotting and sequence analysis of the selected cosmid clone, the structural gene of the enzyme (1,335 bp) was identified, which encodes a protein of 47,244 Da with a theoretical pI of 6.2. The deduced amino acid sequence of the beta-transaminase showed the highest sequence similarity with glutamate-1-semialdehyde aminomutase of transaminase subgroup II. The beta-transaminase showed higher activities toward d-beta-aminocarboxylic acids such as 3-aminobutyric acid, 3-amino-5-methylhexanoic acid, and 3-amino-3-phenylpropionic acid. The beta-transaminase has an unusually broad specificity for amino acceptors such as pyruvate and alpha-ketoglutarate/oxaloacetate. The enantioselectivity of the enzyme suggested that the recognition mode of beta-aminocarboxylic acids in the active site is reversed relative to that of alpha-amino acids. After comparison of its primary structure with transaminase subgroup II enzymes, it was proposed that R43 interacts with the carboxylate group of the beta-aminocarboxylic acids and the carboxylate group on the side chain of dicarboxylic alpha-keto acids such as alpha-ketoglutarate and oxaloacetate. R404 is another conserved residue, which interacts with the alpha-carboxylate group of the alpha-amino acids and alpha-keto acids. The beta-transaminase was used for the asymmetric synthesis of enantiomerically pure beta-aminocarboxylic acids. (3S)-Amino-3-phenylpropionic acid was produced from the ketocarboxylic acid ester substrate by coupled reaction with a lipase using 3-aminobutyric acid as amino donor.     

AMPA receptor agonists: Resolution,configurational assignment,and pharmacology of (+)-(S)- and (-)-(R)-2-amino-3-[3-hydroxy-5-(2-pyridyl)-isoxazol-4-yl]-propionic acid (2-Py-AMPA)

We have previously shown that whereas (RS)-2-amino-3-(3-hydroxy-5-phenylisoxazol-4-yl)propionic acid (APPA) shows the characteristics of a partial agonist at (RS)-2-amino-3-(3-hydroxy-5-methylisoxazol-4-yl)propionic acid (AMPA) receptors, (S)-APPA is a full AMPA receptor agonist and (R)-APPA a weak competitive AMPA receptor antagonist. This observation led us to introduce the new pharmacological concept, functional partial agonism. Recently we have shown that the 2-pyridyl analogue of APPA, (RS)-2-amino-3-[3-hydroxy-5-(2-pyridyl)isoxazol-4-yl]propionic acid (2-Py-AMPA), is a potent and apparently full AMPA receptor agonist, and this compound has now been resolved into (+)- and (-)-2-Py-AMPA (ee ≥ 99.0%) by chiral HPLC using a Chirobiotic T column. The absolute stereochemistry of the enantiomers of APPA has previously been established by X-ray analysis, and on the basis of comparative studies of the circular dichroism spectra of the enantiomers of APPA and 2-Py-AMPA, (+)- and (-)-2-Py-AMPA were assigned the (S)- and (R)-configuration, respectively. In a series of receptor binding studies, neither enantiomer of 2-Py-AMPA showed detectable affinity for kainic acid receptor sites or different sites at the N-methyl-D-aspartic acid (NMDA) receptor complex. (+)-(S)-2-Py-AMPA was an effective inhibitor of [³H]AMPA binding (IC⁵⁰ = 0.19 ± 0.06 μM) and a potent AMPA receptor agonist in the rat cortical wedge preparation (EC₅₀ = 4.5 ± 0.3 μM) comparable with AMPA (IC₅₀ = 0.040 ± 0.01 μM; EC₅₀ = 3.5 ± 0.2 μM), but much more potent than (+)-(S)-APPA (IC₅₀ = 5.5 ± 2.2 μM; EC₅₀ = 230 ± 12 μM). Like (-)-(R)-APPA (IC₅₀ > 100 μM), (-)-(R)-2-Py-AMPA (IC₅₀ > 100 μM) did not significantly affect [³H]AMPA binding, and both compounds were week AMPA receptor antagonists (K_i = 270 ± 50 and 290 ± 20 μM, respectively). Chirality 9:274–280, 1997. © 1997 Wiley-Liss, Inc.     

Enantioselective hydrolysis of racemic 2-phenylpropionitrile and other (R,S)-2-arylpropionitriles by a new bacterial isolate,Agrobacterium tumefaciens strain d3

Bacteria were enriched from soil samples with succinate as carbon source and racemic 2-phenylpropionitrile as sole source of nitrogen. One of the isolates, strain d3, converted (R,S)-2-phenylpropionitrile with high enantioselectivity to (S)-2-phenylpropionic acid. Strain d3 was identified as Agrobacterium tumefaciens. Resting cells hydrolysed 2-phenylpropionitrile via 2-phenylpropionamide to 2-phenylpropionic acid. Racemic 2-phenylpropionitrile as well as 2-phenylpropionamide were converted to (S)-2-phenylpropionic acid with an enantiometric excess above 96%. The nitrile hydratase and the amidase were both shown to convert preferentially the S enantiomer of their respective substrate. These two enzymes were induced in the presence of (R,S)-2-phenylpropionitrile but only in the absence of ammonia. In addition to 2-phenylpropionitrile strain d3 could utilize various aliphatic and aromatic nitriles as nitrogen sources. Resting cells of strain d3 also converted (R,S)-2-phenylbutyronitrile, ibuprofen nitrile, ketoprofen nitrile and -aminophenylacetonitrile with high enantioselectivity. The nitrile- and amide-converting enzyme activities were also found in cell-free extracts.     

Use of enzyme penicillin acylase in selective amidation/amide hydrolysis to resolve ethyl 3-amino-4-pentynoate isomers

The beta-amino acid, (S)-ethyl-3-amino-4-pentynoate, is a chiral synthon used in the synthesis of xemilofiban hydrochloride, an anti-platelet agent. A biocatalytic approach was developed to resolve (R)- and (S)-enantiomers of ethyl 3-amino-4-pentynoate in enantiomerically pure form employing the enzyme Penicillin acylase. In the acylation, phenylacetic acid was used as an acylating agent. We have shown that both the acylation and deacylation can be employed and that the activity of the enzyme Penicillin acylase can be controlled by maintaining an appropriate pH of the reaction medium.     

A novel enantioselective synthesis of (S)-(-)- and (R)-(+)-nornicotine via alkylation of a chiral 2-hydroxy-3-pinanone ketimine template

An asymmetric synthesis of the optically pure isomers of the minor tobacco alkaloid and CNS nicotine metabolite, nornicotine, has been achieved with moderately high optical purity. The synthetic pathway involves alkylation of a chiral ketimine, prepared from either 1R,2R,5R-(+)- or 1S,2S,5S-(-)-2-hydroxy-3-pinanone and 3-(aminomethyl)pyridine with 3-bromopropan-1-ol. After cleavage of the respective C-alkylated ketimines with NH2OH.HCl, and treatment of the resulting amino alcohols with HBr, followed by base-catalyzed intramolecular ring closure, (S)-(-)-nornicotine and (R)-(+)-nornicotine were obtained with ee values of 91% and 81%, respectively.     

Efficient synthesis of the ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer

The ketone body ester (R)-3-hydroxybutyryl-(R)-3-hydroxybutyrate and its (S,S) enantiomer were prepared in a short, operationally simple synthetic sequence from racemic β-butyrolactone. Enantioselective hydrolysis of β-butyrolactone with immobilized Candida antarctica lipase-B (CAL-B) results in (R)-β-butyrolactone and (S)-β-hydroxybutyric acid, which are easily converted to (R) or (S)-ethyl-3-hydroxybutyrate and reduced to (R) or (S)-1,3 butanediol. Either enantiomer of ethyl-3-hydroxybutyrate and 1,3 butanediol are then coupled, again using CAL-B, to produce the ketone body ester product. This is an efficient, scalable, atom-economic, chromatography-free, and low cost synthetic method to produce the ketone body esters.     

Synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate with recombinant Escherichia coli cells expressing (S)-specific secondary alcohol dehydrogenase

The synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate ((R)-ECHB) from ethyl 4-chloroacetoacetate was studied using whole recombinant cells of Escherichia coli expressing a secondary alcohol dehydrogenase of Candida parapsilosis. Using 2-propanol as an energy source to regenerate NADH, the yield of (R)-ECHB reached 36.6 g/l (more than 99% ee, 95.2% conversion yield) without addition of NADH to the reaction mixture.     

Preparation of (S)-2,3-dichloro-1-propanol byPseudomonas sp. and its use in the synthesis of (R)-epichlorohydrin

Summary Pseudomonas sp. OS-K-29 assimilated (R)-2,3-dichloro-1-propanol preferentially as the sole source of carbon. Isolation of optically pure (S)-2,3-dichloro-1-propanol with 100% enantiomer excess (e.e.) from the racemate was done based on this bacterial assimilation using immobilized-cells of OS-K-29 with calcium-alginate. The overall examination of the reactor involved 19 batches for 50 days without loss of its activity. Highly pure (R)-epichlorohydrin with 99.5% e.e. was prepared from the (S)-2,3-dichloro-1-propanol with treatment of aqueous NaOH. This new method is simple and useful for manufacturing optically active (S)-2,3-dichloro-1-propanol and (R)-epichlorohydrin.     

高对映选择性环氧化物水解酶产生菌的筛选及特性研究

从土壤中分离的芽孢杆菌Bacillus megaterium ECU1001所产五氧化物水解酶能高对映选择性水解缩水甘油苯基醚（对映选择率E值可达47.8）,当转化率为55.9%时,剩余的（S）－缩水甘油苯基醚的光学纯度（对映体过量值,ee）可达99.5%;当底物浓度提高到60mmol/L时,光学纯（S）－缩水基油苯基醚的收率达到25.6%。