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To design a large-scale pyrolysis reactor, a complete particle level model was developed using pyrolysis of babul wood. Babul wood, the abundant woody biomass in India, is a highly reactive and promising feedstock for pyrolysis and considered as a case study in the present work. Thermo-balance was used to devolatilize the wood particles at different heating rates from (10 to 20 K min−1), thereby estimate the intrinsic kinetics parameters. Non-condensable gases released from wood pyrolysis were also modeled and derived the kinetic parameters. Large wood single particle dimension of L/D ∼3.5 was chosen to investigate the pyrolysis reaction and developed a particle level model which involves both heat transfer and chemical reactions phenomena. Particle model developed was simulated with intrinsic kinetic parameters generated from kinetic models. Two-step consecutive model with different reaction orders (m = 1, n = 2) was found to explain the experimental data very well. Additionally, optimum conditions for maximum product yields and detail characterization of bio-oil was carried out using different analytical techniques.

Chirality is a key factor in the safety and efficacy of many drug products and thus the production of single enantiomers of drug intermediates and drugs has become important and state of the art in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes (biocatalysts) for the transformation of synthetic chemicals with high chemo-, regio- and enatioselectivities providing products in high yields and purity. In this article, biocatalytic processes are described for the synthesis of key chiral intermediates for development pharmaceuticals.

The production of single enantiomers of key chiral intermediates has become increasingly important for the development of drugs. Demand for chiral compounds continues to rise in the pharmaceutical industry due to federal regulations covering active pharmaceutical ingredients (APIs), and the recognition that enantiomers of a chiral compound could have dramatically different biological activities and toxicity. In this area, there are a number of advantages in using biocatalysis over chemical synthesis. Typically, enzyme-catalyzed reactions are highly enantioselective, regioselective and do not require protection and deprotection steps during synthesis. They can be carried out at ambient temperature, atmospheric pressure in aqueous systems, and are often regarded as green processes that avoid the use of more extreme conditions used in chemical reactions which could cause undesirable side reactions. Microbial cells and enzymes (biocatalysts) can be immobilized and reused for many cycles, and enzymes are generally over-expressed in suitable hosts such as Escherichia coli to make enzymatic processes economically efficient, safer and cheaper. The preparation of thermostable, pH stable, organic solvent tolerant and highly active biocatalysts towards substrates by random and site-directed mutagenesis by directed evolution has led to the production of novel biocatalysts to be used under desired process conditions. This chapter describes the synthesis of key intermediates for the synthesis of APIS by biocatalysis at Bristol-Myers Squibb to provide sustainable, energy efficient and economical processes.

An enzymatic reduction process was developed to convert the ketone 2-(6-oxo-2-phenyladamantan-2-yl)acetic acid to the chiral alcohol 2-((2S, 6S)-6-hydroxy-2-phenyladamantan-2-yl)acetic acid and to ...

Chirality is a key factor in the safety and efficacy of many drug products and thus producing single enantiomers of drug intermediates and drugs has become increasingly important in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes derived from them for the transformation of synthetic chemicals with high chemo-, regio-, and enatioselectivities. In this article, biocatalytic processes are described for the synthesis of key chiral intermediates for development of pharmaceuticals and fine chemicals.

Chirality is a key factor for the safety and efficacy of many drug products. The production of single enantiomers of drug intermediates has become increasingly important in the pharmaceutical industry. There has been an enormous potential of microorganisms and enzymes derived from there for the transformation of synthetic chemicals with high chemo-, regio-, and enatioselectivities. Recent development in the area of directed evolution has led screen mutants under process conditions to increase activity and selectivity of biocatalysts, thus making the enzymatic process highly efficient and economically feasible. In this review, chemoenzymatic processes are described for the synthesis of chiral intermediates for the development of pharmaceuticals.

The (S)-amino acid, (S)-2-amino-3-(6-o-tolylpyridin-3-yl)propanoic acid (3), is a key intermediate needed for synthesis of an antidiabetic drug candidate. Three enzymatic routes to 3 were explored. (S)-Amino acid 3 could be prepared in 73% isolated yield with 99.9% ee from racemic amino acid 1 using (R)-amino acid oxidase from Trigonopsis variabilis expressed in Escherichia coli in combination with an (S)-aminotransferase using (S)-aspartate as amino donor. The (S)-aminotransferase was purified from a soil organism identified as Burkholderia sp. and cloned and expressed in E. coli. (S)-Amino acid 3 with 100% ee was also prepared in 68% solution yield and 54% isolated yield from 1 using recombinant (R)-amino acid oxidase from T. variabilis and an (S)-amino acid dehydrogenase from Sporosarcina ureae. The cofactor NADH required for the reductive amination reaction was regenerated using formate and formate dehydrogenase. The chemoenzymatic dynamic resolution of 1 by (R)-selective oxidation with Celite-immobil...

The d-amino acid (R)-2-amino-3-(7-methyl-1 H-indazol-5-yl)propanoic acid (3) is a key intermediate needed for synthesis of a drug candidate compound. Enzymatic routes to 3 were explored. d-Amino acid 3 was prepared in 68% isolated yield with >99% ee from racemic amino acid 1 using l-amino acid deaminase from Proteus mirabilis expressed in Escherichia coli in combination with a commercially available d-transaminase using d-alanine as amino donor. The d-enantiomer was also prepared in 79% isolated yield with >99% ee from the corresponding keto acid 2 using the d-transaminase with racemic alanine as the amino donor. The rate and yield of this reaction could be accelerated by addition of lactate dehydrogenase (with NAD, formate and formate dehydrogenase to regenerate NADH) to remove the inhibitory pyruvate produced during the reaction. A d-transaminase was purified from a soil organism identified as Bacillus thuringiensis and cloned and expressed in E. coli. The d-transaminase was very effective for the prepa...

Chiral intermediates were prepared by biocatalytic processes for the chemical synthesis of four pharmaceutical drug candidates. These include: (i) the microbial reduction of 3,5-dioxo-6-(benzyloxy) hexanoic ethyl ester to (3S,5R)-dihydroxy-6-(benzyloxy) hexanoic acid ethyl ester, an intermediate for a new anticholesterol drug; (ii) synthesis of (2R,3S)-(-)-N-benzoyl-3-phenyl isoserine ethyl ester, a taxol side-chain synthon; (iii) the microbial oxygenation of 2,2-dimethyl-2H-1-benzopyran-6-carbonitrile to the corresponding (3S,4S) epoxide and (3S,4R)-trans diol, intermediates for synthesis of potassium channel opener; (iv) the biotransformation of (exo,exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimethanol to the corresponding chiral lactol and lactone, intermediates for thromboxane A2 antagonist.

Chirality is a key factor in the safety and efficacy of many drug products and, thus, the production of single enantiomers of drug intermediates has become increasingly important in the pharmaceutical industry. There has been an increasing awareness of the enormous potential of microorganisms and enzymes derived therefrom for the transformation of synthetic chemicals with high chemo-, regio- and enantioselectivities. In this article, some selected enzymatic processes are described for the synthesis of chiral intermediates for pharmaceuticals.

An efficient biocatalytic process has been developed for the resolution of methyl (1 RS )- N - t Boc-6-hydroxy-3,4-dihydro-1 H -isoquinoline-1-carboxylate rac - 1 by means of Seaprose S-mediated enantioselective hydrolysis to afford (1 S )- 2 and (1 R )- 1 in 87% and 93% isolated yield, 101% and 96% potency, and ee >99.8% and >99.5%, respectively.

An efficient biocatalytic process has been developed for the resolution of 1-(3′-bromophenyl)ethylamine ( RS )- 1 by way of enantioselective lipase-mediated ( R )-selective acylation with ethyl 2-methoxyacetate to afford ( S )-amine ( S )- 1 and ( R )-2″-methoxyacetamide (( R )- 2 ) in 91–95% and 90–92% isolated yield, respectively, and both with >99% ee.

Microbial reduction of ethyl 1-benzyl-3-oxo-piperidine-4-carboxylate by the majority of evaluated microorganisms gave the ethyl cis-(3R,4R)-1-benzyl-3R-hydroxy-piperidine-4R-carboxylate as the major product in high diastereo- and enantioselectivities. The 3R,4R-hydroxy ester was produced in 97.4% diastereomeric excess (de) and 99.8% enantiomeric excess (ee) by Candida parapsilosis SC16347, while 99.5% de and 98.2% ee were obtained from reduction by Pichia methanolica SC16415. A few of the evaluated microorganisms gave 10–40% of the ethyl trans-(3R,4S)-1-benzyl-3R-hydroxy-piperidine-4S-carboxylate as the minor product.

Previously we have demonstrated the reduction of ethyl diketoester 4 to the corresponding dihydroxy ester 6a by Acinetobacter sp. SC13874. Recently we screened more than 100 cultures for microbial reduction of both the ethyl and t-butyl diketoesters 4 and 5. Most yeast cultures showed a preference for reduction at the C-3 with low enantioselectivity. Among the three Acinetobacter strains screened, Acinetobacter sp. SC13874 reduced both compounds 4 and 5 to the corresponding (3R)- and (5S)-monohydroxy compounds. Monohydroxy compounds were isolated and their absolute configurations determined. (3R)- and (5S)-Monohydroxy compounds were reduced further to the corresponding dihydroxy esters 6a and 8a to provide alternate routes for the synthesis of compounds 14a and 16a, potential intermediates for the synthesis of HMG-CoA reductase inhibitors. Cell suspensions of Acinetobacter sp. SC13874 reduced the ethyl diketoester 4 to a mixture of desired syn and undesired anti diastereomers. The desired syn-(3R,5S)-dihydroxy ester 6a was obtained with an enantiomeric excess (ee) of 99% and a diastereomeric excess (de) of 63%. Cell suspensions reduced the t-butyl diketoester 5 to a mixture of mono- and dihydroxy esters with the dihydroxy ester showing an ee of 87% and de of 51% for the desired syn-(3R,5S)-dihydroxy ester 8a. Three different ketoreductases were purified to homogeneity, and their biochemical properties compared. Reductase I only catalyzes the reduction of ethyl diketoester 4 to its monohydroxy products 10 and 11, whereas reductase II catalyzes the formation of dihydroxy products 6 and 7 from monohydroxy substrates 10 and 11. A third reductase (III) was identified, which catalyzes the reduction of diketoester 4 to syn-(3R,5S)-dihydroxy ester 6a.

An efficient biocatalytic method has been developed for the conversion of (5S)-4,5-dihydro-1H-pyrrole-1,5-dicarboxylic acid, 1-(1,1-dimethylethyl)-5-ethyl ester (1) into the corresponding amide (5S)-5-aminocarbonyl-4,5-dihydro-1H-pyrrole-1-carboxylic acid, 1-(1,1-dimethylethyl)ester (2), which is a critical intermediate in the synthesis of the dipeptidyl peptidase IV (DPP4) inhibitor Saxagliptin (3). Candida antartica lipase B mediates ammonolysis of the ester with ammonium carbamate as ammonia donor to yield up to 71% of the amide. The inclusion of Ascarite and calcium chloride as adsorbents for carbon dioxide and ethanol byproducts, respectively, increases the yield to 98%, thereby offering an efficient and practical alternative to chemical routes which yield 57-64%.

Enantioselective asymmetric hydrolysis of (1α,2β,3α)-2-(benzyloxymethyl)-cyclopent-4-ene-1,3-diol diacetate 1 to the corresponding (+)-monoacetate 2 was carried out using lipase PS-30 from Pseudomonas cepacia or pancreatin. A reaction yield of 85 M % with an enantiomeric excess (ee) of 98% was obtained. Using pancreatin, a reaction yield of 75 M % with an ee of 98.5% was obtained. Asymmetric acetylation of (1α,2β,3α)-2-(benzyloxymethyl)-cyclopent-4-ene-1,3-diol 3 to the corresponding (−)-monoacetate 4 was carried out using lipase PS-30 with isopropenyl acetate as the acylating agent. A reaction yield of 80 M % with an ee of 98% was obtained for (−)-monoacetate 4 .

The enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione 1 to either of the corresponding (R)- or (S)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones 2 and 3 is described.

6-Hydroxybuspirone is an active metabolite of the antianxiety drug buspirone. The (R)- and (S)-enantiomers of 6-hydroxybuspirone were prepared using an enzymatic resolution process. l -Amino acid acylase from Aspergillus melleus (Amano Acylase 30000) was used to hydrolyze racemic 6-acetoxybuspirone to (S)-6-hydroxybuspirone in 95% ee after 45% conversion. The remaining (R)-6-acetoxybuspirone with 88% ee was converted to (R)-6-hydroxybuspirone by acid hydrolysis. The ee of both enantiomers could be improved to 99% by crystallization as a metastable polymorph. (S)-6-Hydroxybuspirone was also obtained in 88% ee and 14.5% yield by hydroxylation of buspirone using Streptomyces antibioticus ATCC 14890.

The synthesis of the leading candidate compound in an anticancer program required (S)-2-chloro-1-(3-chlorophenyl)-ethanol as an intermediate. Other possible candidate compounds used analogues of the S-alcohol. Of 119 microbial cultures screened for reduction of the corresponding ketone to the S-alcohol, Hansenula polymorpha ATCC 58401 (73.8% ee) and Rhodococcus globerulus ATCC 21505 (71.8% ee) had the highest enantioselectivity for producing the desired alcohol. A ketoreductase from Hansenula polymorpha, after purification to homogeneity, gave the S-alcohol with 100% ee. Amino acid sequences from the purified enzyme were used to design PCR primers for cloning the ketoreductase. The cloned ketoreductase required NADP(H), had a subunit molecular weight of 29,220 and a native molecular weight of 88,000. The cloned ketoreductase was expressed in E. coli together with a cloned glucose 6-phosphate dehydrogenase from Saccharomyces cerevisiae to allow regeneration of the NADPH required by the ketoreductase. An extract of E. coli containing the two recombinant enzymes was used to reduce 2-chloro-1-(3-chloro-4-fluorophenyl)-ethanone and two related ketones to the corresponding S-alcohols. Intact E. coli cells provided with glucose were used to prepare (S)-2-chloro-1-(3-chloro-4-fluorophenyl)-ethanol in 89% yield with 100% ee.

Resolution of (±)- sec -butylamine by Candida antarctica lipase provided a very low enantiomeric excess of the residual amine when either ethyl or vinyl butyrate was used as the acylating agent. The enantiomeric excess was increased by using ethyl esters of long chain fatty acids. The rate of the reaction was increased by using methyl t -butyl ether as a solvent. ( S )- sec -Butylamine of very high enantiomeric excess was obtained by C. antarctica lipase catalyzed acylation with ethyl decanoate in methyl t -butyl ether.

The chiral intermediate ( S )-1-(2′-bromo-4′-fluoro phenyl)ethanol 2 was prepared by the enantioselective microbial reduction of 2-bromo-4-fluoro acetophenone 1 . Organisms from genus Candida , Hansenula , Pichia , Rhodotorula , Saccharomyces , Sphingomonas and Baker's yeast reduced 1 to 2 in >90% yield and 99% enantiomeric excess (ee). In an alternative approach, the enantioselective microbial reductions of methyl, ethyl, and tert -butyl 4-(2′-acetyl-5′-fluorophenyl) butanoates 3 , 5 , and 7 , respectively, were demonstrated using strains of Candida and Pichia . Reaction yields of 40–53% and ee's of 90–99% were obtained for the corresponding ( S )-hydroxy esters 4 , 6 , and 8 . The reductase, which catalyzed the enantioselective reduction of ketoesters was purified to homogeneity from cell extracts of Pichia methanolica SC 13825. It was cloned and expressed in Escherichia coli with recombinant cultures used for the enantioselective reduction of keto methyl ester 3 to the corresponding ( S )-hydroxy methyl ester 4 . On a preparative scale, a reaction yield of 98% and an ee of 99% was obtained.

The series of compounds (o-CH3SC6H4)CH2EPh3(E = Si (1), Ge (2), Sn (3), and Pb (4)) have been synthesized and characterized by NMR spectroscopy and by single crystal X-ray diffraction. Compounds 1 and 2 are isostructural with a triclinic crystal system and P-1 space group; however, morphotropic steps occur between Ge and Sn, and Sn and Pb. While the E-S distances in 1 and 2 are 3.985 and 3.974 Å, respectively, ~100% of the sum of the respective van der Waals (vdW) radii, there is a notable distortion from tetrahedral geometry about E. Compound 3 is also triclinic with P-1 symmetry, but has two molecules in the unit cell that demonstrate a distorted tetrahedral geometry and intramolecular Sn-S distances of 3.699 and 3.829 Å, 88% and 91% of the sum of the vdW radii. Compound 4 has a Pb-S distance of 3.953 Å (91% of Σ vdW radii). The structure of the Grignard coupling product [o-(SCH3)C6H4CH2]2is also reported.Key words: intramolecular self-assembly, silicon, germanium, tin, lead, sulfur.

The chiral intermediate (3R)-cis-3-acetyloxy-4-(1,1-dimethylethyl)-2-azetidinone 2 was prepared for the semi-synthesis of the new taxane 5, an orally active anticancer compound. The enantioselective enzymatic hydrolysis of cis-3-acetyloxy-4-(1,1-dimethylethyl)-2-azetidinone 1 to the corresponding undesired (S)-alcohol 3 and unreacted desired 2 was carried out using immobilized lipase PS-30 or BMS lipase. Reaction yields of >48% and enantiomeric excesses of >99% were obtained for the desired 2. Acetoxy β-lactam 2 was converted to hydroxy β-lactam 4 for use in the semisynthesis of 5.

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 the HIV protease inhibitor Atazanavir. The diastereoselective 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 Rhodococcus, Brevibacterium, and Hansenula strains reduced 1 to 2a. Three strains of Rhodococcus gave >90% yield. A diastereomeric purity of >98% and enantiomeric excess of >99.3% were obtained for alcohol 2a.

There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enatioselective manner. Chiral intermediates are in high demand by pharmaceutical industries for the preparation bulk drug substances. In this review article, microbial/enzymatic processes for the synthesis of chiral intermediates for antihypertensive drugs, melatonin receptor agonists, and beta3-receptor receptor agonists are described.

There has been an increasing awareness of the enormous potential of microorganisms and enzymes for the transformation of synthetic chemicals with high chemo-, regio- and enatioselective manner. Chiral intermediates and fine chemicals are in high demand both from pharmaceutical and agrochemical industries for the preparation bulk drug substances and agricultural products. In this review article, microbial/enzymatic processes have been described for the synthesis of chiral intermediates for anticancer drugs, antiviral agents, β3-receptor agonists, antihypertensive drugs, melatonin receptor agonists, anticholesterol drugs, and anti-Alzheimer’s drugs.

Biotransformation of mutilin and pleuromutilin by microbial cultures was investigated to provide a source of 8-hydroxymutilin or 8-hydroxypleuromutilin. LC/MS analysis of culture broths showed that...

The chiral ester ethyl (2 R )-hydroxy-2-(1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′-naphthalenyl) acetate 2 and the corresponding acid 4 were prepared as intermediates in the synthesis of the retinoic acid receptor gamma-specific agonist ( R )-3-fluoro-4-[[hydroxy(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)acetyl]amino]benzoic acid 7 . Enantioselective reduction of ethyl 2-oxo-2-(1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′naphthalenyl)acetate 1 to alcohol 2 was carried out using Aureobasidium pullulans SC 13849 in 98% yield and with an enantiomeric excess (e.e.) of 96%. Among microorganisms screened for the reduction of 2-oxo-2-(1′,2′,3′,4′-tetrahydro-1′,1′,4′,4′-tetramethyl-6′-naphthalenyl)acetic acid 3 to hydroxy acid 4 , Candida maltosa SC 16112 and two strains of Candida utilis (SC 13983, SC 13984) gave reaction yields of >53% with e.e.s of >96%.

(S)-N-(tert-Butoxycarbonyl)-3-hydroxymethylpiperidine 1 was made from (R,S)-3-hydroxymethylpiperidine 2 via fractional crystallization of the corresponding l(−)-dibenzoyl tartarate salt 3 followed by hydrolysis and acylation. Lipase from Pseudomonas cepacia was found to be the best enzyme for the stereospecific resolution of (R,S)-N-(tert-butoxycarbonyl)-3-hydroxymethylpiperidine 4. (S)-N-(tert-Butoxycarbonyl)-3-hydroxymethylpiperidine 1 was obtained in 16% yield and >95% enantiomeric excess (ee) by hydrolysis of (R,S)-acetate 5 by lipase PS from Pseudomonas cepacia. Lipase PS-catalyzed esterification of the (R,S)-N-(tert-butoxycarbonyl)-3-hydroxymethylpiperidine 4 with succinic anhydride provided the S-hemisuccinate ester 6, which could be easily separated and hydrolyzed by base to the (S)-N-(tert-butoxycarbonyl)-3-hydroxymethylpiperidine 1. The yield and ee could be improved greatly by repetition of the process. Using the repeated esterification procedure (S)-N-(tert-butoxycarbonyl)-3-hydroxymethylpiper...

Biocatalytic processes were used to prepare chiral intermediates required for the synthesis of Omapatrilat 1 by three different routes. The synthesis and enzymatic conversion of 2-keto-6-hydroxyhexanoic acid 3 to L-6-hydroxynorleucine 2 was demonstrated by reductive amination using beef liver glutamate dehydrogenase. To avoid the lengthy chemical synthesis of the ketoacid 3, a second route was developed to prepare the ketoacid by treatment of racemic 6-hydroxy norleucine [readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin 4] with D-amino acid oxidase from porcine kidney or Trigonopsis variabilis followed by reductive amination to convert the mixture completely to L-6-hydroxynorleucine in 98% yield and 99% enantiomeric excess (e.e.). The enzymatic synthesis of (S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (allysine ethylene acetal, 5) was demonstrated using phenylalanine dehydrogenase (PDH) from T. intermedius. Phenylalanine dehydrogenase was cloned and overexpressed in Escherichia coli and Pichia pastoris. Using PDH from E. coli or P. pastoris, the enzymatic process was scale-up to prepare kg quantity of allysine ethylene acetal 5. The reaction yields of >94% and e.e. of >98% were obtained for allysine ethylene acetal 5. An enzymatic process was developed for the synthesis of [4S-(4a,7a,10ab)]1-octahydro-5-oxo-4 [[(phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid [BMS-199541-01]. The enzymatic oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[[(phenyl-methoxy)carbonyl] L-homocysteinyl] L-lysine)-1,1-disulphide [BMS-201391-01] to produce BMS-199541-01 using a novel L-lysine epsilon-aminotransferase (LAT) from Sphingomonas paucimobilis SC 16113 was demonstrated. This enzyme was overexpressed in E. coli and a process was developed using the recombinant enzyme.

Phenylpyruvate decarboxylase from Achromobacter eurydice was used to catalyse the asymmetric acyloin condensation of phenylpyruvate 1 with various aldehydes 2 to produce optically active acyloins PhCH2COCH(OH)R 3. The specific activity of the phenylpyruvate decarboxylase enzyme was increased by a factor of 332 after its purification. The molecular weight of the purified enzyme was shown to be 150 kDa by gel filtration chromatography, while SDS gel electrophoresis showed two sub-units with molecular weights of 90 and 40 kDa. The acyloin condensation yield decreased with increasing chain length for straight chain aliphatic aldehydes from 76% for acetaldehyde to 24% for valeraldehyde. The e.e.s of the acyloin products were 87–98%. Low yields of acyloin products were obtained with chloroacetaldehyde (13%) and glycoaldehyde (16%). Indole-3-pyruvate was a substrate of the enzyme and provided acyloin condensation product 3-hydroxy-1-(3-indolyl)-2-butanone 5 with acetaldehyde in 19% yield, while benzoylformate was not a substrate for the enzyme.

Chiral intermediates S-(+)-2-pentanol and S-(+)-2-heptanol were prepared by a lipase-catalyzed enzymatic resolution proces. Among various lipases evaluated for the stereoselective acylation of racemic alcohols, lipase B from Candida antarctica catalyzed the acylation of the undesired enantiomer of racemic alcohols leaving the desired S-(+)-alcohols unreacted. A reaction yield of 43–45% and an enantiomeric excess (e.e.) of >99% were obtained for S-(+)-2-pentanol or S-(+)-2-heptanol when the reaction was carried out using vinyl acetate or succnic anhydride as acylating agent. In an alternative process, an enantioselective hydrolysis of 2-pentyl acetate was demonstrated using lipase B giving S-(+)-2-pentyl acetate and R-(−)-2-pentanol. A reaction yield of 45% and an e.e. of 98.6% were obtained for S-(+)-2-pentyl acetate.

Two systems were analyzed that make it possible to reuse enzymes and cofactors in the reductive amination of the lithium salt of 6-(1′,3′-dioxolan-2′-yl)-2-keto-hexanoic acid (compound 1) to 6-(1′,3′-dioxolan-2′-yl)-2S-aminohexanoic acid, lithium salt (compound 2) catalyzed by phenylalanine dehydrogenase (PDH) with in situ NADH regeneration catalyzed by formate dehydrogenase (FDH). First, both enzymes and PEG-NADH, a mass-enlarged cofactor, were encapsulated within semipermeable microcapsules. These microcapsules catalyzed the biotransformation in the absence of external NAD. About 20 mg of compound 2 was formed per mL microcapsule per day. Substrate diffusion into the microcapsules was not rate-limiting. PEG(35,000)—NADH, but not PEG(20,000)—NADH, was retained within the microcapsules. The activity of the encapsulated enzyme was about 15–25% of the activity of the free enzyme. If saturating free NAD was added to microcapsules, the activity increased about threefold. The microcapsules could be reused, and...

Cells obtained from growth of Achromobacter eurydice , Pseudomonas aromatica and Pseudomonas putida on l -phenylalanine containing medium catalyzed the enzymatic acyloin condensation of phenylpyruvic acid 1 and acetaldehyde 2 by phenylpyruvate decarboxylase to produce 3-hydroxy-1-phenyl-2-butanone 3 . The acyloin condensation by Achromobacter eurydice and P. aromatica was stereoselective, providing the 3 R enantiomer 3a with enantiomeric excess (ee) of 95% and 84%, respectively. A partially purified enzyme was prepared from the cell free extract of Achromobacter eurydice. The acyloin product 3a was obtained in 45% yield with an ee of 91% by using this partially purified preparation of phenylpyruvate decarboxylase.

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 a human immunodeficiency virus protease inhibitor, BMS-186318. The stereoselective reduction of (1S) [3-chloro-2-oxo-1(phenylmethyl)propyl] carbamic acid, 1,1-dimethylethyl ester 1 was carried out using microbial cultures, among which Streptomyces nodosus SC 13149 efficiently reduced 1 to 2a. A reaction yield of 80%, enantiomeric excess (e.e.) of 99.8%, and diastereomeric purity of 99% were obtained for chiral alcohol 2a. Chiral l-6-hydroxy norleucine 3, an intermediate in the synthesis of antihypertensive drug, was prepared by reductive amination of 2-keto-6-hydroxyhexanoic acid 4 using beef liver glutamate dehydrogenase. The cofactor NADH required for this reaction was regenerated using glucose dehydrogenase from Bacillus sp. A reaction yield of 80% and e.e. of 99.5% were obtained for l-6-hydroxynorleucine 3. To avoid the lengthy chemical synthesis of the ketoacid, a second route was developed in which racemic 6-hydroxynorleucine [readily available from hydrolysis of 5-(4-hydroxybutyl) hydantoin 5] was treated with d-amino acid oxidase from Trigonopsis variabilis to selectively convert the d-isomer of racemic 6-hydroxynorleucine to 2-keto-6-hydroxyhexanoic acid 4 and l-6-hydroxynorleucine 3. Subsequently, the 2-keto-6-hydroxyhexanoic acid 4 was converted to l-6-hydroxynorleucine by reductive amination using glutamate dehydrogenase. A reaction yield of 98% and an e.e. of 99.5% were obtained.

Deracemization of racemic 1,2-diol, (RS)-1-{2′,3′-dihydrobenzo[b]furan-4′-yl}-ethane-1,2-diol (1), by stereoinversion by several microorganisms belonging to the genera Candida and Pichia provided the corresponding chiral (S)-diol, (S)-1-{2′,3′-dihydrobenzo[b]furan-4′-yl}-ethane-1,2-diol (2). (S)-Diol 2 was obtained in yields of 60–70% with 90–100% enantiomeric excess.

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%.

Among various microbial cultures evaluated, Rhodotorula glutinis SC 16293 and Aspergillus niger SC 16311 catalyzed the stereospecific hydrolysis of the racemic epoxide, RS -1-{2′,3′-dihydrobenzo[ b ]furan-4′-yl}-1,2-oxirane, 1 to the corresponding R -diol, R -1-{2′,3′-dihydrobenzo[ b ]furan-4′-yl}-ethane-1,2-diol, 3 . The S -epoxide, S -1-{2′,3′-dihydrobenzo[ b ]furan-4′-yl}-1,2-oxirane, 2 remained unreacted in the reaction mixture. A reaction yield of 45–50% (theoretical maximum yield is 50%) and an enantiomeric excess (ee) of >95% were obtained for unreacted S -epoxide 2 using each culture. Addition of 10% methyl tert -butyl ether to an aqueous reaction mixture during hydrolysis by R. glutinis improved the ee of the unreacted S -epoxide 2 to >99% (yield 48%) and that of the R -diol 3 to 79%. Unlike R. glutinis , hydrolysis of racemic epoxide 1 in the presence of 10% methyl tert -butyl ether by A. niger showed an adverse effect and gave S -epoxide 2 in 54% yield and 49% ee.

The chiral intermediates (S)-3,4-dihydro-1,2(2H)-pyridinedicarboxylic acid, 1-(phenylmethyl)ester [BMS 202665-01] and (S)-3,4-dihydro-1,2(2H)-pyridinedicarboxylic acid, 1,1-dimethylethyl ester [BMS 264406-01] were prepared by oxidation of Nα-carbobenzoxy- l -lysine (Nα-CBZ- l -lysine) and Nα-t-butoxycarbonyl- l -lysine (Nα-t-BOC- l -lysine), respectively, by cell suspensions of Rhodotorula graminis SC 16005.

A dimolybdenum derivative of decamethylpentasilacycloheptyne (1) was synthesized by direct reaction of the heptyne with Mo2(CO)4(η5-C5H5)2. 1 crystallized in the space group P 1 , a=9.386(2), b=9.866(3), c=20.178(4) A, α=92.17(2), β=97.17(2), γ=115.71(2)°. The acetylenic bond is lengthened from 1.213 A in the free ligand to 1.359(4) A and all the Si–Si bond lengths in 1 are significantly lengthened upon complexation. This is due to relaxation of the ring strain as evidenced by the Si–C–C bond angles in 1 of 132.7 and 140.9° compared to 159.2 and 162.6° in the uncomplexed ring. 29Si-NMR data exhibit significant downfield chemical shifts upon complexation for the Si atoms adjacent to the triple bond, with moderate upfield shifts for the other Si atoms. The related cycloheptyne·Mo2(CO)4(η5-C5H5)2 (2) was synthesized by the reaction of cyclohepteno-1,2,3-selenadiazole with Mo2(CO)4(η5-C5H5)2. 2 crystallized in the space group C21/c, a=30.396(10), b=8.9093(3), c=16.156(4) A, β=115.39(2)°. The acetylenic bond in 2 is 1.345 A, compared with a calculated value (ab initio 3-21 G*) of 1.190 A for the free cycloheptyne.

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 .

Chiral intermediate (−)-7-[ N , N ′-Bis(benzyloxycarbonyl)- N -(guanidinoheptanoyl)]-α-acetoxyglycine 2 was prepared for the total synthesis of a (−)-15 deoxyspergualin, an antitumor antibiotic and immunosuppressive agent. The stereoselective acetylation of racemic 7-[ N , N ′-Bis(benzyloxycarbonyl)- N -(guanidinoheptanoyl)]-α-hydroxyglycine 1 was carried out in methyl ethyl ketone (MEK) using lipase from Pseudomonas sp. (lipase AK). Vinyl acetate was used as an acylating agent. A reaction yield of 48% (theoretical max 50%) and an optical purity of 98% were obtained for S-(−)-acetate 2 . The unreacted alcohol (+)- 1 was obtained in 41% yield and 98.5% optical purity.

The chiral intermediate (S) [1-(acetoxyl)-4-(3-phenyl)butyl]phosphonic acid, diethyl ester 2 was prepared for the total synthesis of a squalene synthase inhibitor, BMS-188494. The stereoselective acetylation of racemic [1-(hydroxy)-4-(3-phenyl)butyl] phosphonic acid, diethyl ester 1 was carried out in toluene using lipase from Geotrichum candidum. Isopropenyl acetate was used as an acylating agent. A reaction yield of 38% and an optical purity of 95% were obtained for chiral 2.

Chiral intermediates were prepared by biocatalytic processes for the chemical synthesis of three pharmaceutical drug candidates. These include (i) the synthesis of [(3R-cis)-3-(acetyloxy)-4-phenyl-2-azetidinone2 for the semi-synthesis of paclitaxel (taxol)5, an anticancer compound; (ii) synthesis of chiral (exo,exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimenthanol monoacetate ester9 for the chemoenzymatic preparation of a thromboxane A2 antagonist; (iii) the enzymatic synthesis ofS-(−) 3-benzylthio-2-methylpropanoic acid, a key chiral intermediate for the synthesis of antihypertensive drugs captopril10 or zofenopril13.