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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 the production of 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 there from for the transformation of synthetic chemicals with high chemo-, regio- and enatioselectivities. In this article, biocatalytic processes are described for the synthesis of chiral alcohols and unntural aminoacids for pharmaceuticals.

Biocatalytic processes to selectively hydrolyze the N-carbobenzyloxy (CBz) group from CBz-protected d - or l -amino acids have been developed. The substrate specificities of the CBz-deprotecting enzymes from Burkholderia phenazinium SC 16530, and Sphingomonas paucimobilis expressed into Escherichia coli SC 16501 were evaluated on CBz-protected amino acids and structurally related compounds. Modifications of various structural components and their effects on enzyme activity and enantioselectivity provided a greater understanding of the two CBz-deprotecting enzymes.

Enzymes are widely used for peptide synthesis, both for coupling and for terminal activation and modification. Broad-specificity proteases of different mechanistic classes can be used for C-terminal activation and peptide coupling reactions, whereas enzymes that recognize specific sequences are increasingly used for selective modification and tagging. Protein engineering can tailor hydrolases for improved synthesis, better reaction medium tolerance, and desired substrate selectivity. This chapter highlights properties of natural and engineered peptidases, amidases, and peptide transferases for chemoenzymatic peptide synthesis and terminal modification, with an emphasis on basic principles and innovations leading to new applications.

The synthesis of optically active sulfoxides, compounds due to their unique properties, has been a main target for synthetic organic chemistry. Recent efforts in the field of biocatalysis have allowed the preparation of enantiopure sulfoxides starting from the corresponding sulfides while using relatively mild conditions. In fact, several different types of redox biocatalysts have been found that can catalyze enantio- and/or regioselective sulfoxidations. The most promising group of enzymes able to perform selective sulfoxidations is the flavin-containing monooxygenases (FMOs). Enzymes containing a flavin cofactor have already been widely studied and used in organic synthesis, especially in reduction and/or oxidation processes. This chapter highlights the recent efforts in the preparation of chiral sulfoxides catalyzed by different types of flavoenzymes, with special attention to the parameters that can improve their catalytic properties. Novel approaches that allow performing selective sulfoxidations in which modified flavin systems are used are also discussed.

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...

A synthetic gene encoding a Streptomyces l -proline-3-hydroxylase was constructed and used to produce the hydroxylase protein in recombinant Escherichia coli. A fermentation process for growth of this recombinant E. coli for enzyme production was scaled-up to 250 L. A biotransformation process was developed using cell suspensions of the recombinant E. coli and subsequently scaled-up to 10 L for conversion of l -proline to cis-3-hydroxy- l -proline. A reaction yield of 85 M% and d.e. of 99.9% was obtained for cis-3-hydroxy- l -proline.

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...

Screening was carried out to identify strains useful for the preparation of (R)-1-cyclopropylethylamine and (R)-sec-butylamine by resolution of the racemic amines with an (S)-specific transaminase. Several Bacillus megaterium strains from our culture collection as well as several soil isolates were found to have the desired activity for the resolution of the racemic amines to give the (R)-enantiomers. Using an extract of the best strain, Bacillus megaterium SC6394, the reaction was shown to be a transamination requiring pyruvate as amino acceptor and pyridoxal phosphate as a cofactor. Initial batches of both amines were produced using whole cells of Bacillus megaterium SC6394. The transaminase was purified to homogeneity to obtain N-terminal as well as internal amino acid sequences. The sequences were used to design polymerase chain reaction (PCR) primers to enable cloning and expression of the transaminase in E. coli SC16578. In contrast to the original B. megaterium process, pH control and aeration were not required for the resolution of sec-butylamine and an excess of pyruvate was not consumed by the recombinant cells. The resolution of sec-butylamine (0.68 M) using whole cells of E. coli SC16578 was scaled up to give (R)-sec-butylamine⋅1/2 H2SO4 in 46.6% isolated yield with 99.2% ee. An alternative isolation procedure was also used to isolate (R)-sec-butylamine as the free base.

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.

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.

The non-proteinogenic amino acid 2-(3-hydroxy-1-adamantyl)-(2S)-aminoethanoic acid [2, (S)-3-hydroxyadamantylglycine], is a key intermediate required for the synthesis of Saxagliptin, a dipeptidyl peptidase IV inhibitor under development for treatment of type 2 diabetes mellitus. Keto acid 2-(3-hydroxy-1-adamantyl)-2-oxoethanoic acid (1) was converted to (S)-3-hydroxyadamantylglycine by reductive amination using a phenylalanine dehydrogenase from Thermoactinomyces intermedius expressed in a modified form in Pichia pastoris or Escherichia coli. NAD (nicotinamide adenine dinucleotide) produced during the reaction was recycled to NADH (reduced form of nicotinamide adenine dinucleotide) using formate dehydrogenase. Pichia pastoris produces an endogenous formate dehydrogenase when grown on methanol, and the corresponding gene was cloned and expressed in E. coli. The modified phenylalanine dehydrogenase contains two amino acid changes at the C-terminus and a 12-amino acid extension of the C-terminus. The modified enzyme is more effective with keto acid 1 than the wild-type enzyme, but less effective with the natural substrate, phenylpyruvate. Production of multi-kg batches was originally carried out with extracts of Pichia pastoris expressing the modified phenylalanine dehydrogenase from Thermoactinomyces intermedius and endogenous formate dehydrogenase, and further scaled up using a preparation of the two enzymes expressed in E. coli.

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.

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%.

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.

The synthesis of a new series of arylmethyltin chlorides is reported, ArnMeSnCl3−n (Ar = RC6H4, R = H, ethyl, i-propyl, t-butyl; hexyl, octyl). The synthesis involves initial formation of triarylmethyltin compounds, Ar3MeSn, via Grignard techniques followed by HCl–Et2O aryl group cleavage, preferably in a stepwise manner. Preliminary biological activity against Staphylococcus aureus illustrates the importance of the para-alkyl substituents and reinforces that an optimal hydrophobic character is needed for maximum efficacy. Copyright © 2005 John Wiley & Sons, Ltd.

A novel enantioselective N-carbobenzyloxy (Cbz)-cleaving enzyme was purified to homogeneity from Sphingomonas paucimobilis SC 16113. The gene encoding the protein has been identified and its complete DNA sequence was determined. The Cbz-cleaving enzyme was expressed in Escherichia coli.

A new enzymatic process for the enantioselective cleavage of N-benzyloxycarbonyl (Cbz) groups from protected amino acids and related compounds has been developed. The Cbz-deprotecting enzyme was isolated from cell extracts of Sphingomonas paucimobilis SC 16113 and purified to homogeneity. The purified protein has a molecular weight of 155,000 daltons and a subunit size of 44,000 daltons.

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.

(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...

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.

Currently, much attention has been focused on the interaction of small molecules with biological macromolecules. The search for selective enzyme inhibitors and receptor agonists or antagonists is o...

[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.

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...

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.

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.

10-Deacetylabaccatin III (10 DAB), an important precursor for paclitaxel semisynthesis, is enhanced in yew extracts using C10-deacetylase and C13-deacylase enzymes.(4) C10-deacetylase is an intracellular enzyme produced by the fermentation of a soil microorganism, Nocardioides luteus (SC 13912). During the fermentation of Nocardioides luteus, the growth of cells reaches a maximum growth at 28 h. C10-deacetylase enzyme activity starts at 26 h and peaks at 38 h of the fermentation. The cells are recovered by centrifugation. The C10-deacetylase enzyme was purified from the Nocardioides luteus cells. The enzyme was purified 190-fold to near homogeneity. The purified enzyme appeared as a single band on 12.5% SDS-PAGE analysis with a molecular weight of 40,000 daltons. (c) 1995 John WileySons, Inc.

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.

A C-8 keto pleuromutilin derivative has been synthesized from the biotransformation product 8-hydroxy mutilin. A key step in the process was the selective oxidation at C-8 of 8-hydroxy mutilin using tetrapropylammonium perruthenate. The presence of the C-8 keto group precipitated interesting intramolecular chemistry to afford a compound (10) with a novel pleuromutilin-derived ring system.

Treatment of ferrocenecarboxaldehyde with base at 0°C, lithium diisopropylamide (LDA) or n-butyllithium, followed by the addition of trimethylsilylcyanide, produced 1-(diisopropylamino)cyanomethylferrocene, FcCH(N{iPr}2(CN) (II) or 1-trimethyl-siloxy-n-pentylferrocene, FcCH(nBu)OSiMe3 (IIIa). The reaction of naphthaldehyde, NpCHO, with LDA/Me3SiCN led to formation of 1-(diisopropylamino)cyanomethylnaphthalene, NpCH{N(iPr)2}(CN) suggesting a general new synthesis for cyanoamines. The reaction of ferrocenecarboxaldehyde with Me3SiCN led to the expected silylether FcCH(CN)OSiMe3 (I). An alternative synthesis of ferrocenylmethylsilylethers, FcCH2OSiR3 (IV), involved the reaction of 1-ferrocenylmethanol with R3SiCl in the presence of pyridine. The basicity of the new silylethers III and IV is reported.

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.

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.

A key chiral intermediate, lactol 2[3aS(3aα, 4α, 7α, 7aα)]-hexahydro-4,7-epoxy-isobenzofuran-1 (3H)-one, and the corresponding chiral lactone 3 were made in high optical purity by stereoselective enzymatic and microbial oxidation of the parent diol 1 . Horse liver alcohol dehydrogenase (HLADH) in the presence of nicotinamide adenine dinucleotide (NAD + ) and riboflavin oxidized diol 1 (exo, exo)-7-oxabicyclo [2.2.1] heptane-2,3-dimethanol to the corresponding lactol 2 and lactone 3 . Regeneration of NAD + required for the oxidation of diol 1 was carried out by the NADH-dependent alanine dehydrogenase and leucine dehydrogenase in the presence of alanine and leucine, respectively. Since expensive enzyme (HLADH) and cofactors (NAD + ) were required for the oxidation reaction, various microorganisms were screened for the ability to catalyze the stereoselective oxidation of diol 1 . Two organisms, Nocardia globerula ATCC 21505 and Rhodococcus sp. ATCC 15592, catalyzed the efficient oxidation of diol 1 to the corresponding chiral lactol 2 and lactone 3 . The reaction yield of 70% and optical purity of 96% was obtained for lactone 3 prepared from the oxidation of diol 1 by cell suspensions (10% w/v, wet cells) of N. globerula ATCC 21505. Substrate was used at 5 gl −1 concentration. A 10% (w/v, wet cells) cell suspension of Rhodococcus sp. ATCC 15592, after 120 h reaction period, produced lactol 2 in 12% yield and lactone 3 in 34% yield. An overall 46% reaction yield was obtained based on diol 1 oxidation. Substrate was used at 5 gl −1 concentration. Optical purity of 96.7% and 98.4% was obtained for lactol 2 and lactone 3 , respectively. Lactone 3 purified by slilica gel chromatography gave a specific rotation of +117° and enantiomeric excess of >99%.

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.

A key intermediate, S-(−)-3-benzoylthio-2-methylpropanoic acid (1) was made in high optical purity by the lipase-catalyzed stereoselective esterification of racemic 1 with methanol in an organic solvent system. Among various lipases evaluated, Amano P-30 lipase from Pseudomonas sp. efficiently catalyzed the esterification of 1 to yield R-(+) methyl ester and unreacted S-(−) 1. A reaction yield of 40 mol% and an optical purity of 97.2% were obtained for compound 1 at a substrate concentration of 0.1 m (22 mg/ml). Lipase P-30 was immobilized on Accurel polypropylene (PP) and the immobilized enzyme was reused (23 cycles) in the esterification reaction without loss of enzyme acitivity, productivity or optical purity. Among various solvents evaluated, toluene was found to be the most suitable organic solvent and methanol was the best alcohol for the esterification of racemic 1 by immobilized lipase. Substrate concentrations as high as 1.0 m were used in the esterification reaction. When the temperature was increased from 28° C to 60° C, the reaction time required for the esterification of 0.1 m substrate decreased from 16 h to 2 h. On increasing the methanol to substrate molar ratio from 1:1 to 4:1, the rate of esterification decreased. A lipase fermentation using Pseudomonas sp. ATCC 21 808 was developed. In the batch-fermentation process, 56 units/ml of extracellular lipase activity was obtained. A fed-batch process using soybean oil gave a significant increase in the lipase activity (126 units/ml). Crude lipase recovered from the filtrate by ethanol precipitation and immobilized on Accurel PP was also effective: S-(−) compound 1 was obtained in 35 mol% yield and 95% optical purity.

α-Keto-β-bromoisovaleric acid or its ethyl ester was hydrolyzed with sodium hydroxide to α-keto-β-hydroxyisovalerate and converted in situ to l -β-hydroxyvaline by reaction with NADH and NH3 catalyzed by leucine dehydrogenase from Bacillus species. Methyl 2-chloro-3,3-dimethyloxiranecarboxylate and the corresponding isopropyl or 1,1-dimethylethyl esters were prepared by Darzens condensation. These glycidic esters, after hydrolysis by sodium bicarbonate and sodium hydroxide to α-keto-β-hydroxyisovalerate, were also converted to l -β-hydroxyvaline by leucine dehydrogenase. NAD was recycled to NADH with either formate dehydrogenase from Candida boidinii or glucose dehydrogenase from Bacillus megaterium. Polyethylene glycol-NADH was an effective reductant with formate dehydrogenase and dextran-NAD was effective with glucose dehydrogenase. Reductive amination activity for α-keto-β-hydroxyisovalerate was found in most Bacillus strains screened, including megaterium, subtilis, cereus, pumilus, licheniformis, thuringiensis, and brevis. Highest specific activity was found in B. sphaericus ATCC 4525. pH 8.5 was optimum for both glucose dehydrogenase and reductive amination of α-keto-β-hydroxyisovalerate by the B. sphaericus enzyme. The apparent Km for α-keto-β-hydroxyisovalerate was 11.5 m m compared to 1.06 m m for α-ketoisovalerate. The apparent Vmax with α-keto-β-hydroxyisovalerate was 41% of the value with α-ketoisovalerate, making the enzyme very suitable for the preparation of l -β-hydroxyvaline.

▪ Abstract In collaboration with the National Cancer Institute, Bristol-Myers Squibb has developed paclitaxel for treatment of various cancers; it has been approved by the Food and Drug Administration for the treatment of ovarian and metastatic breast cancer. Originally paclitaxel was isolated and purified from the bark of Pacific yew trees. This source of paclitaxel was considered to be economically and ecologically unsuitable as it required the destruction of the yew trees. This review article describes alternate methods for the production of paclitaxel, specifically, a semisynthetic approach and the application of biocatalysis in enabling the semisynthesis of paclitaxel. Three novel enzymes were discovered in our laboratory that converted the variety of taxanes to a single molecule, namely 10-deacetylbaccatin III (paclitaxel without C-13 side chain and C-10 acetate), a precursor for paclitaxel semisynthesis. These enzymes are C-13 taxolase (catalyzes the cleavage of C-13 side chain of various taxanes), C-10 deacetylase (catalyzes the cleavage of C-10 acetate of various taxanes), and C-7 xylosidase (catalyzes the cleavage of C-7 xylose from various xylosyltaxanes). Using a biocatalytic approach, paclitaxel and a variety of taxane in extracts of a variety of Taxus cultivars were converted to a 10-deacetylbaccatin III. The concentration of 10-deacetylbaccatin III was increased by 5.5- to 24-fold in the extracts treated with the enzymes, depending upon the type of Taxus cultivars used. Biocatalytic processes have also been described for the preparation of C-13 paclitaxel side chain synthons. The chemical coupling of 10-deacetylbaccatin III or baccatin III to C-13 paclitaxel side chain has been summarized to prepare paclitaxel by semisynthesis.

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%.

Biotransformations were developed to oxidize N epsilon-carbobenzoxy(CBZ)-L-lysine and to reduce the product keto acid to L-CBZ-oxylysine. Lysyl oxidase (L-lysine: O2 oxidoreductase, EC 1.4.3.14) from Trichoderma viride was relatively specific for L-lysine and had very low activity with N epsilon-substituted derivatives. L-Amino acid oxidase (L-amino acid: O2 oxidoreductase [deaminating], EC 1.4.3.2) from Crotalus adamanteus venom had low activity with L-lysine but high activity with N epsilon-formyl-, t-butyoxycarbonyl(BOC)-, acetyl-, trifluoroacetyl-, or CBZ-L-lysine. L-2-Hydroxyisocaproate dehydrogenase (EC 1.1.1.-) from Lactobacillus confusus catalyzed the reduction by NADH of the keto acids from N epsilon-acetyl-, trifluoroacetyl-, formyl- and CBZ-L-lysine but was inactive with the products from oxidation of L-lysine, L-lysine methyl ester, L-lysine ethyl ester or N epsilon-t-BOC-L-lysine. Providencia alcalifaciens (SC9036, ATCC 13159) was a good microbial substitute for the snake venom oxidase and also provided catalase (H2O2:H2O2 oxidoreductase EC 1.11.1.6). N epsilon-CBZ-L-Lysine was converted to CBZ-L-oxylysine in 95% yield with 98.5% optical purity by oxidation using P. alcalifaciens cells followed by reduction of the keto acid using L-2-hydroxyisocaproate dehydrogenase. NADH was regenerated using formate dehydrogenase (formate: NAD oxidoreductase, EC 1.2.1.2) from Candida boidinii. The Providencia oxidase was localized in the particulate fraction and catalase activity was predominantly in the soluble fraction of sonicated cells. The pH optima and kinetic constants were determined for the reactions.

A key intermediate, (3R-cis)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluorome thyl)- 2H-1-benzazepin-2-one (compound II or SQ32191), with high optical purity was made by the stereoselective microbial reduction of the parent ketone 1. Several strains of bacterial and yeast cultures were screened for the ability to catalyse the stereoselective reduction of 4,5-dihydro-4-(4-methoxyphenyl)-6-(trifluoromethyl)-1H-1-benzazepin++ +-2,3-dione [compound I or SQ32425]. Microorganisms from the genera Nocardia, Rhodococcus, Alkaligenes, Corynebacterium, Arthrobacter, Hansenula, and Candida reduced compound I to compound II with 60-70% conversion yield. In contrast, microorganisms from the genera Pseudomonas and Acinetobacter reduced compound I stereospecifically to (trans)-1,3,4,5-tetrahydro-3-hydroxy-4-(4-methoxyphenyl)-6-(trifluoromet hyl-2H- 1-benzazepin-2-one (compound III or SQ32408). Among various cultures evaluated, N. salmonicolor SC6310 effectively catalysed the transformation of compound I to compound II with 96% conversion yield at 1.5-2.0 gl-1 concentration. Compound II was isolated and identified by NMR analysis, mass spectrometry, and comparison to an authentic sample. Preparative scale fermentation process and transformation process were developed using cell suspensions of N. salmonicolor SC6310 to catalyse the transformation of compound I to compound II. The isolated compound II had a melting point of 222 degrees C (reference 221-223 degrees C), optical rotation of +130.4 (reference +128 degrees C), and optical purity of greater than 99.9% as analyzed by NMR and chiral HPLC.

Second-generation paclitaxel analogues that require replacement of the C-4 acetate by other substituents are in development. An enzyme able to specifically remove the C-4 acetate from paclitaxel could simplify preparation of the analogues. Several strains were isolated from soil samples that contain enzyme activities able to 4-deacetylate 10-DAB (10-deacetylbaccatin III). Selection was made using plates containing 10-DAB as the sole carbon source and screening colonies for deacetylation of 10-DAB. Two strains initially isolated were identified as Rhodococcus sp. and deposited with the A.T.C.C. (Manassas, VA, U.S.A.) as strains 202191 and 202192. Whole cells were able to convert 10-DAB into 4,10-DDAB (4-deacetyl-10-deacetylbaccatin III) in 90% yield. The enzyme activity in these strains was not effective with paclitaxel and 10-deacetylpaclitaxel, although 4,10-DDAB was produced from baccatin III. The activity in these strains was associated with an insoluble fraction of cell extracts. Several additional isolates were obtained that were identified as variants of Stenotrophomonas maltophilia, and a soluble C-4 deacetylase was purified approx. 218-fold from one of them. The activity of this enzyme was limited to 10-DAB, and the enzyme was not effective with paclitaxel or baccatin III.

An anaerobic procedure was developed for the purification of the flavin:NADH oxidoreductase (flavoprotein) component of methane monooxygenase to homogeneity. The molecular weight of the flavoprotein determined by gel filtration was about 40,000, and by sedimentation equilibrium analysis, about 38,000. The purified flavoprotein is a monomeric protein with a sedimentation constant ( S 20W ) value of about 2.1 S. The absorption spectrum of the flavoprotein has a peak at 460 nm and shoulder at 395 nm. The fluorescent excitation and emission spectra of the fluorescent component of flavoprotein had peaks at 450, 370, and 530 nm, respectively. A FAD was identified as a prosthetic group of flavoprotein by thin-layer chromatography. The flavoprotein contained about 1 mol of FAD and 2 mol each of iron and acid-labile sulfide per mole of protein. The flavoprotein was directly reduced by NADH under anaerobic conditions. The formation of neutral flavin semiquinone was detected during anaerobic titration of flavoprotein by NADH and also as a free radical signal at a g value of 2.004 by EPR spectroscopy. The iron sulfur cluster has g values of 2.04, 1.96, and 1.87, yielding a g average of 1.96, characteristic of a Fe 2 S 2 center. Antibody prepared against the flavoprotein reacted with flavoprotein and inhibited methane monooxygenase activity.

Trimethyl- and tri ethyllead derivatives (η 5 -C 5 H 5 )M(CO) 3 PbR 3 (M  Cr, Mo, and W) have been synthesized. The trimethyllead derivatives are photochemically, and to a lesser extent, thermally unstable with respect to methyl transfer reactions that yield the corresponding (η 5 -C 5 H 5 )M(CO) 3 CH 3 complex. The related triethyllead complexes behave in a distinctly different fashion exhibiting rearrangements to [(η 5 -C 5 H 5 )M(CO) 3 ] 3 PbEt 2 (M  W, Mo). The reactions of the Cr, Mo, and W complexes with SO 2 yielded the corresponding alkyl sulphinato complexes as did the reaction of the related Fe complex [(η 5 -C 5 H 5 )Fe(CO) 2 PbEt 3 ].

Reactions of Aluminium isopropoxide with monofunctional bidentateSchiff bases, such as salicylidene-o-toluidine, salicylidene-p-toluidine, 2-hydroxy-1-naphthylmethylidene-p-toluidine and 2-hydroxy-1-naphthylmethylidene-p-toluidine in different stoichiometric ratios have yielded products of the type Al(OPr i)2(SB), Al(OPr i)(SB)2, and Al(SB)3 (whereSB − is the anion of the correspondingSchiff base,SBH). Bifunctional tetradentateSchiff bases in 1∶1 and 2∶3 molar ratios [Al(OPr i)3:SBH2] yielded insoluble derivatives of the type Al(OPr i)SB and Al2(SB)3 (whereSB − represents the anion of theSchiff baseSBH2 andSBH2=bis-salicylaldehyde-o-phenylene-diamine or bis-salicylaldehyde-p-phenylenediamine). The newSchiff base derivatives have been characterized by elemental analyses, infrared spectroscopy and molecular weight determinations.