Clyde G. McNamee

Stereoselective reduction of β-keto esters by Geotrichum candidum

Abstract A key chiral intermediate S (−)-4-chloro-3-hydroxybutanoic acid, methyl ester 2 was made in high optical purity by the stereoselective reduction of 4-chloro-3-oxobutanoic acid methyl ester 1 by cell suspensions of Geotrichum candidum SC 5469. A reaction yield of 95% and optical purity of 96% was obtained for 2 by glucose-, acetate-, or glycerol-grown cells (10% w/v) of G. candidum SC 5469. Substrate was used at 10 mg ml −1 concentration. The optical purity of 2 was increased to 99% by heat treatment of cell suspensions (55°C for 30 min) prior to conducting bioreduction of 1 at 28°C. Glucose-grown cells of G. candidum SC 5469 have also catalyzed the stereoselective reduction of ethyl-, isopropyl-, and tertiary-butyl esters of 4-chloro-3-oxobutanoic acid and methyl- and ethyl esters of 4-bromo-3-oxobutanoic acid. A reaction yield of > 85% and optical purity of > 94% were obtained. NADP-dependent oxidoreductase responsible for the stereoselective reduction of β-keto esters of 4-chloro- and 4-bromo-3-oxobutanoic acid was purified 100-fold. The molecular weight of the purified enzyme is 950,000. The purified oxidoreductase was immobilized on Eupergit C and used to catalyze the reduction of 1 to 2. The cofactor NADP required for the reduction reaction was regenerated by glucose dehydrogenase.

Enantioselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester

The key chiral intermediate 3,5-dihydroxy-6-(benzyloxy) hexanoic acid, ethyl ester 2a, was made by the stereoselective microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic acid, ethyl ester 1. Among various microbial cultures evaluated, cell suspensions of Acinetobacter calcoaceticus SC 13876 reduced 1 to 2a. The reaction yield of 85% and optical purity of 97% was obtained using glycerol-grown cells. The substrate was used at 2 g l−1 and cells were used at 20% (w/v, wet cells) concentrations. The optimum pH for the reduction of 1 to 2a was 5.5 and the optimum temperature was 32°C. Cell extracts of A. calcoaceticus SC 13876 in the presence of NAD+, glucose, and glucose dehydrogenase reduced 1 to the corresponding monohydroxy compounds 3 and 4 [3-hydroxy-5-oxo-6-(benzyloxy) hexanoic acid ethyl ester 3, and 5-hydroxy-3-oxo-6-(benzyloxy) hexanoic acid ethyl ester 4]. Both 3 and 4 were further reduced to 2a by cell extracts. Reaction yield of 92% and optical purity of 99% were obtained when the reaction was carried out in a 1-l batch using cell extracts. The substrate was used at 10 g l−1. Product 2a was isolated from the reaction mixture in 72% overall yield. The GC and HPLC area % purity of the isolated product was 99% and the optical purity was 99.5%. The reductase which converted 1 to 2a was purified about 200-fold from cell extracts of A. calcoaceticus SC 13876. The purified enzyme gave a single protein band on SDS-PAGE corresponding to 35,000 daltons.

Enzymatic synthesis of L-6-hydroxynorleucine.

L-6-Hydroxynorleucine, a key chiral intermediate used for synthesis of a vasopeptidase inhibitor, was prepared in 89% yield and > 99% optical purity by reductive amination of 2-keto-6-hydroxyhexanoic acid using glutamate dehydrogenase from beef liver. In an alternate process, racemic 6-hydroxynorleucine produced by hydrolysis of 5-(4-hydroxybutyl)hydantoin was treated with D-amino acid oxidase to prepare a mixture containing 2-keto-6-hydroxyhexanoic acid and L-6-hydroxynorleucine followed by the reductive amination procedure to convert the mixture entirely to L-6-hydroxynorleucine, with yields of 91 to 97% and optical purities of > 99%.

Microbial synthesis of (2R,3S)-(−)-N-benzoyl-3-phenyl isoserine ethyl ester-a taxol side-chain synthon

Abstract The chiral intermediate (2R,3S)-(−)-N-benzoyl-3-phenyl isoserine ethyl ester 2a , a potential taxol 5 side-chain synthon, was prepared by microbial and enzymatic processes. Taxol 5 is an anticancer compound recently approved by FDA for the treatment of ovarian cancer. The stereoselective reduction of racemic 2-keto-3-(N-benzoylamino)-3-phenylpropionic acid ethyl ester 1 to the corresponding alcohol 2 was carried out using microbial cultures. Among microorganisms evaluated, Hansenula polymorpha SC 13865 and Hansenula fabianii SC 13894 effectively reduced compound 1 to the desired syn diastereomer 2a . Reaction yields of >80% and enantiomeric excesses of >98% were observed for these bioreduction process. About 10–20% of anti diastereomers ( 2c,2d ) were produced during bioreduction.

Biocatalytic preparation of a chiral synthon for a vasopeptidase inhibitor: enzymatic conversion of N2-[N-Phenylmethoxy)carbonyl] L-homocysteinyl]- L-lysine (1- > 1′)-disulfide to [4S-(4I,7I,10aJ)] 1-octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester by a novel L-lysine ϵ-aminotransferase

[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine ϵ-aminotransferase. This enzyme catalyzed the oxidation of the ϵ-amino group of lysine in the dipeptide dimer N2-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required α-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to α-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine ϵ-amino transferase and glutamate oxidase, respectively. L-lysine ϵ-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mol wt of L-lysine ϵ-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine ϵ-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS-201391-01 to BMS-199541-01 by using L-lysine ϵ-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65–70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine ϵ-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine ϵ-aminotransferase. N-α-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine ϵ-aminotransferase.

Preparation of chiral synthon for HIV protease inhibitor: stereoselective microbial reduction of N-protected α-aminochloroketone

Abstract The chiral intermediate (1S,2R) [3-chloro-2-hydroxy-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethylethyl ester 2a was prepared for the total synthesis of an HIV protease inhibitor, BMS-186318. The stereoselective reduction of (1S) [3-chloro-2-oxo-1-(phenylmethyl)propyl] carbamic acid, 1,1-dimethyl-ethyl ester 1 was carried out using microbial cultures among which Streptomyces nodosus SC 13149 efficiently reduced 1 to 2a . A reaction yield of 80% was obtained. The optical purity of 99.8% and the diastereomeric purity of 99% were obtained for chiral alcohol 2a .

Stereoselective epoxidation of 2,2-dimethyl-2H-1- benzopyran-6-carbonitrile

The chiral intermediate (3S,4R)-trans-3,4-dihydro-3,4-dihydroxy-2,2-dimethyl- 2H-1-benzopyran-6-carbonitrile [(+)-trans diol 3] was made by the stereoselective microbial epoxidation of 2,2-dimethyl-2H-1-benzopyran-6-carbonitrile 1. This compound is a potential intermediate for the total synthesis of potassium-channel openers. Several microbial cultures were found which catalyzed the transformation of 1 to the corresponding (3S,4S)-epoxide 2 and (+)-trans diol 3. The two best cultures, Corynebacterium sp. SC 13876 and Mortierella ramanniana SC 13840 gave reaction yields of 32 M% and 67.5 M% and optical purities of 88 and 96%, respectively, for (+)-trans diol 3. A single-stage process (fermentation-epoxidation) for the biotransformation of 1 was developed using Corynebacterium sp. SC 13876 and M. ramanniana SC 13840. In a 25-L fermentor, the (+)-trans diol 3 was obtained in 38.6 M% yield with an optical purity of 90% using Corynebacterium SC 13876. The reaction yield of 60.7 M% and optical purity of 92.5% were obtained for (+)-trans diol 3 using M. ramanniana SC 13840. A two-stage process for the preparation of (+)-trans diol 3 was also developed using a 3 L cell-suspension (10% w/v, wet cells) of M. ramanniana SC 13840. The reaction was carried out in a 5-L Bioflo fermentor. The concentration of substrate 1 was 2 g L-1 with glucose present at 10 g L-1. After 48 h, (+)-trans diol 3 was obtained in 76 M% yield with an optical purity of 96%.(ABSTRACT TRUNCATED AT 250 WORDS)

Stereoselective acetylation of 3,4-dihydro-3,4-dihydroxy-2,2-dimethyl-2H-1-benzopyran-6-carbonitrile

Abstract The chiral intermediate diol (3 S ,4 R )-trans-3,4-dihydro-3,4-dihydroxy-2,2-dimethyl-2 H -1-benzopyran-6-carbonitrile 2 . was prepared for the total synthesis of a potassium channel opener drug candidate. The stereoselective acetylation of racemic 1 was carried out with various lipases among which the lipases from Candida cylindraceae and Pseudomonas cepacia catalyzed the acetylation of the undesired enantiomer of racemic 1 to yield monoacetylated product and unreacted desired (+)-trans diol 2 . A reaction yield of >40% and an optical purity >90% were obtained using each lipase.

Stereoselective microbial reduction of N-(4-(1-oxo-2-chloroacetyl ethyl) phenyl methane sulfonamide

Several microbial cultures were screened for the ability to catalyse the reduction of N-(4-(1-oxo-2-chloroacetyl ethyl) phenyl methane sulfonamide (1). The chiral intermediate (+)N-(4-(1-hydroxy-2-chloroethyl) phenyl methane sulfonamide (2) was prepared by the stereoselective microbial reduction of the parent ketone 1. Compound 2 is a potential chiral intermediate for synthesis of 4-(2-isopropylamino-1-hydroxyethyl) phenyl methanesulfonanilide (d-sotalol), a beta-receptor antagonist. Microorganisms from the genera Rhodococcus, Nocardia, and Hansenula reduced 1 to 2. A reaction yield of >50% and optical purities of >90% were obtained. The best strain (H.polymorpha ATCC 26012) effectively reduced compound 1 to compound 2 in 95% reaction yield and 99% optical purity. Compound 2 (8.2 g) was isolated from a 3-1 preparative batch in 68% overall yield. Isolated compound 2 had a specific rotation of +20° (CH2Cl2, C-1), an optical purity of 99.5%, and a chemical purity of 97% as analyzed by gas chromatography and HPLC. The nuclear magnetic resonance and mass spectra of compound 2 prepared by bioreduction and a standard chemical sample of 2 were virtually identical. Cell extracts of H. polymorpha in the presence of glucose dehydrogenase, glucose and nicotinamide adenine dinucleotide (NAD +) catalyzed the reduction of 1 to 2 with 98% reaction yield and resulted in an optical purity of 99.4%.