Complementary microbial approaches for the preparation of optically pure aromatic molecules

Different strategies for stereoselective microbial preparation of various chiral aromatic compounds are described. Optically pure 2-methyl-3-phenyl-1-propanol, ethyl 2-methyl-3-phenylpropanoate, 2-methyl-3-phenylpropanal, 2-methyl-3-phenylpropionic acid and 2-methyl-3-phenylpropyl acetate have been prepared using different microbial biotransformations starting from different prochiral and/or racemic substrates. (S)-2-Methyl-3-phenyl-1-propanol and (S)-2-methyl-3-phenylpropanal were prepared by biotransformation of 2-methyl cinnamaldehyde using the recombinant strain Saccharomyces cerevisiae BY4741ΔOye2Ks carrying a heterologous OYE gene from Kazachstania spencerorum. (R)-2-Methyl-3-phenylpropionic acid was obtained by oxidation of racemic 2-methyl-3-phenyl-1-propanol with acetic acid bacteria. Kinetic resolution of racemic 2-methyl-3-phenylpropionic acid was carried out by direct esterification with ethanol using dry mycelia of Rhizopus oryzae CBS 112.07 in organic solvent, giving (R)-ethyl 2-methyl-3-phenylpropanoate as major enantiomer. Finally, (R,S)-2-methyl-3-phenylpropyl acetate was enantioselectively hydrolysed employing different bacteria and yeasts having cell-bound carboxylesterases with prevalent formation of (R)- or (S)-2-methyl-3-phenyl-1-propanol, depending on the strain employed.     

1,11-diarylundecan-1-one and 4-aryltetralone neolignans from Virola sebifera

The seed of Virola sebifera contains besides the polyketide 1 - (2′,6′ - dihydroxyphenyl) - 11 - henylundecan - 1 - one, four neolignans: (2S, 3S, 4R) - 4 - hydroxy - 2,3 - dimethyl - 5,6 - methylenedioxy - 4 - piperonyl - 1 - tetralone and its 2-epimer, as well as (2R, 3R, 4S) - 4 - hydroxy - 6,7 - dimethoxy - 2,3 - dimethyl 4 - piperonyl - 1 - tetralone and its (2R, 3S, 4R) - dehydroxy analogue.     

Synthesis and biological evaluation of 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide stereoisomers as novel positive allosteric modulators of sigma-1 receptor

Novel positive allosteric modulators of sigma-1 receptor represented by 2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamide enantiomers were synthesised using an asymmetric Michael addition of 2-nitroprop-1-enylbenzene to diethyl malonate. Following the chromatographic separation of the methyl erythro- and threo-4-nitro-3R- and 3S-phenylpentanoate diastereoisomers, target compounds were obtained by their reductive cyclisation into 5-methyl-4-phenylpyrrolidin-2-one enantiomers and the attachment of the acetamide group to the heterocyclic nitrogen. Experiments with electrically stimulated rat vas deference contractions induced by the PRE-084, an agonist of sigma-1 receptor, showed that (4R,5S)- and (4R,5R)-2-(5-methyl-4-phenyl-2-oxopyrrolidin-1-yl)-acetamides with an R-configuration at the C-4 chiral centre in the 2-pyrrolidone ring were more effective positive allosteric modulators of sigma-1 receptor than were their optical antipodes.     

Facile route for the synthesis of the iminosugar nucleoside (3R,4R)-1-(pyren-1-yl)-4-(hydroxymethyl)pyrrolidin-3-ol

N-(Pyren-1-yl)-(3R,4S)-4-[(1S,2R)-1,2,3-trihydroxypropyl]pyrrolidin-3-ol (4) was obtained in 36% yield from 3-deoxy-3-C-formyl-1,2:5,6-di-O-isopropylidene-α-d-allofuranose (3) by combined hydrolysis and aminoalkylation reactions with 1-aminopyrene in a one-pot reaction. Cleavage reactions of the exocyclic triol chain in 4 with NaIO₄ and NaBH₄ resulted in iminosugars 7 and 8, which are analogues of the furanose forms of 2-deoxy-d-allose and of 2-deoxy-d-ribose, the latter analogue N-(pyren-1-yl)-(3R,4R)-4-(hydroxymethyl)pyrrolidin-3-ol (8) being formed in 83% yield.     

Synthesis of Optically Active 1,4-Thiazane-3-carboxylic Acid via Optical Resolution by Preferential Crystallization of (RS)-2-Amino-3-[(2-chloroethyl)sulfanyl]propanoic Acid Hydrochloride

Optically active 1,4-thiazane-3-carboxylic acid [TCA] was synthesized from cysteine via optical resolution by preferential crystallization. The intermediate (RS)-2-amino-3-[(2-chloroethyl)sulfanyl]propanoic acid hydrochlo-ride [(RS)-ACS?HCl] was found to exist as a conglomerate based on its melting point, solubility and IR spectrum. (RS)-ACS?HCl was optically resolved by preferential crystallization to yield (R)- and (S)-ACS?HCl. (R)- and (S)-ACS?HCl thus obtained were recrystallized from a mixture of hydrochloric acid and 2-propanol, taking account of the solubility of (RS)-ACS?HCl, efficiently yielding both enantiomers in optically pure forms. (R)- and (S)-TCA were then respectively synthesized by the cyclization of (R)- and (S)-ACS?HCl in ethanol in the presence of triethylamine.     

Improvement of carbonyl reductase activity for the bioproduction of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is a key chiral intermediate for the side chain synthesis of rosuvastatin. In this study, random mutagenesis, site-saturation mutagenesis and combinatorial mutagenesis methods were applied to improve the activity of a synthesized stereoselective short chain carbonyl reductase (SCR) to prepare (3R,5S)-CDHH. After screened by high-throughput screening method and high-performance liquid chromatography, mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser, were obtained, and the enzyme activities of mutants were improved by 1.60- and 1.91-fold compared with parent enzyme, respectively. The catalytically efficiencies (kcat/Km) of mut-Phe145Met/Thr152Ser and mut-Phe145Tyr/Thr152Ser exhibited 5.11- and 8.07-fold improvements in initial activity toward (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH), respectively. In the asymmetric reduction, mut-Phe145Tyr/Thr152Ser catalyzed 500 g L⁻¹ of (S)-CHOH to produce (3R,5S)-CDHH with >99% yield and >99% e.e., and the highest space-time yield achieved at 752.76 mmol L⁻¹ h⁻¹ g⁻¹ wet cell weight within 8 h bioconversion. This study provides a foundation for the preparation of (3R,5S)-CDHH by carbonyl reductase.     

Determination of (R)- and (S)-Disophyramide in human plasma using a chiral α1-acid glycoprotein column

The direct resolution and quantitation of (R)- and (S)-disopyramide, isolated from human plasma, was accomplished using a chiral α₁-acid glycoprotein column. A LiChrosorb RP-2 column (50 × 3.0 mm I.D.) was used as a precolumn. Phosphate buffer, pH 6.20, containing 2-propanol and N,N-dimethyloctylamine was used as mobile phase, expressed as the relative standard deviation, was 1.8% and 3.3% for (R)- and (S)-disopyramide, respectively, at a drug level of 0.5 μg/ml. In two subjects who received a single capsule of racemic disopyramide (150 mg), the plasma levels of the (R) isomer were about half those of the (S) isomer. The half-lives of (R)- and (S)-disopyramide were similar.     

谷氨酸脱氢酶与醇脱氢酶的共表达及其在制备(R)-2-氯-1-苯乙醇中的应用

【背景】醇脱氢酶AdhS能催化不对称还原反应制备(R)-2-氯-1-苯乙醇,但由于自身再生辅酶NADH的能力不足,需要辅酶再生酶协助其再生NADH。谷氨酸脱氢酶能以谷氨酸为底物,再生辅酶NAD(P)H,具有辅酶再生酶的潜力。【目的】克隆表达谷氨酸脱氢酶基因gdhA,构建谷氨酸脱氢酶GdhA与醇脱氢酶AdhS的大肠杆菌共表达体系,提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。【方法】从枯草芽孢杆菌(Bacillus subtilis) 168中克隆基因gdhA,并在大肠杆菌(Escherichia coli) BL21(DE3)中表达,分析辅酶再生活力;再与醇脱氢酶AdhS共表达,优化表达条件;分析不同辅酶再生方案对制备(R)-2-氯-1-苯乙醇的转化效率的影响。【结果】谷氨酸脱氢酶GdhA再生NADH的比活力为694 U/g。经GdhA与AdhS的共表达及表达条件优化后,制备(R)-2-氯-1-苯乙醇的转化效率达465 U/L。经比较,GdhA协助再生辅酶NADH,可使AdhS制备(R)-2-氯-1-苯乙醇的转化效率提高到约3倍。【结论】谷氨酸脱氢酶GdhA为NADH高效再生酶,与醇脱氢酶AdhS共表达可显著提高AdhS制备(R)-2-氯-1-苯乙醇的转化效率。     

Production of (R)-3-Chloro-1,2-Propanediol from Prochiral 1,3-Dichloro-2-Propanol by Corynebacterium sp. Strain N-1074

The production of (R)-3-chloro-1,2-propanediol [(R)-MCP] from prochiral 1,3-dichloro-2-propanol (DCP) was examined with a bacterial strain identified as a Corynebacterium strain. The addition of glycerol as a carbon source or some chlorinated alcohols to a medium was effective for the induction of activity catalyzing the transformation of DCP into MCP. The optimum pH for (R)-MCP production by the resting cell reaction was around 8.0. The optical purity of (R)-MCP formed was improved by keeping the level of DCP in the reaction mixture at a low concentration. (R)-MCP was obtained from 77.5 mM DCP with a 97.3% molar conversion yield and an 83.8% enantiomeric excess of its optical purity by periodic feeding of the substrate.     

Stereoselective synthesis of (R)-1-chloro-3(3,4-difluorophenoxy)-2-propanol using lipases from Pseudomonas aeruginosa in ionic liquid-containing system

Direct transesterification of (R,S)-1-chloro-3-(3,4-difluorophenoxy)-2-propanol (rac-CDPP) (a key intermediate in the synthesis of the chiral drug (S)-lubeluzole) with vinyl butyrate by lipases from Pseudomonas aeruginosa (P. aeruginosa) MTCC 5113 was performed in hexane with ionic liquids (ILs) 1-butyl-3-methyl imidazolium hexafluorophosphate [BMIm][PF₆] and 1-butyl-3-methyl imidazolium tetrafluoroborate [BMIm][BF₄] as co-solvents. The maximum conversion (>49%) and enantiomeric excess (ee > 99.9%) was achieved in 6 h of incubation at 30 °C with [BMIm][PF₆] as co-solvent in a two-phase system. The enzyme was able to perform with the same specificity even at 60 °C in the presence of ILs. It was possible to use lipases repeatedly for more than 10 times while still maintaining absolute enantioselectivity and reactivity. Stability studies on lipases from P. aeruginosa in ILs revealed the fact that the enzyme constancy and the reactivity in catalyzing transesterification of rac-CDPP into (S)-1-chloro-3-(3,4-difluorophenoxy)-2-butanoate was of the order of [BMIm][PF₆] > [BMIm][BF₄] in two-phase system.     

Enantioselective microbial reduction of 6-oxo-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-dione: Cloning and expression of reductases

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 (S)- and (R)-6-hydroxy-8-[4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl]-8-azaspiro[4.5]decane-7,9-diones (2 and 3, respectively) is described. The NADP⁺-dependent (R)-reductase (RHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (R)-6-hydroxybuspirone (3) was purified to homogeneity from cell extracts of Hansenula polymorpha SC 13845. The subunit molecular weight of the enzyme is 35,000 kDa based on sodium dodecyl sulfate gel electrophoresis and the molecular weight of the enzyme is 37,000 kDa as estimated by gel filtration chromatography. (R)-reductase from H. polymorpha was cloned and expressed in Escherichia coli. To regenerate the cofactor NADPH required for reduction we have cloned and expressed the glucose-6-phosphate dehydrogenase gene from Saccharomyces cerevisiae in E. coli. The NAD⁺-dependent (S)-reductase (SHBR) which catalyzes the reduction of 6-ketobuspirone (1) to (S)-6-hydroxybuspirone (2) was purified to homogeneity from cell extracts of Pseudomonas putida SC 16269. The subunit molecular weight of the enzyme is 25,000 kDa based on sodium dodecyl sulfate gel electrophoresis. The (S)-reductase from P. putida was cloned and expressed in E. coli. To regenerate the cofactor NADH required for reduction we have cloned and expressed the formate dehydrogenase gene from Pichia pastoris in E. coli. Recombinant E. coli expressing (S)-reductase and (R)-reductase catalyzed the reduction of 1 to (S)-6-hyroxybuspirone (2) and (R)-6-hyroxybuspirone (3), respectively, in >98% yield and >99.9% e.e.     

Resolution and Synthesis of (S)-1-(2-Naphthyl)ethanol with Immobilized Pea Protein: As a New Biocatalyst

(S)-1-(2-Naphthyl)ethanol was yielded by immobilized pea (Pisum sativum L.) protein (IPP) from (R, S) 2-naphthyl ethanol (>99% ee, yield; about 50%), in which the (R)-enantiomer was selectively oxidized to 2-acetonaphthone. IPP could be reused consecutively at least three times without any decrease of yield and optical purity.     

Identification of a plausible biosynthetic enzyme for the IM-2-type autoregulator in Streptomyces antibioticus

Virginiae butanolides (VBs) and IM-2 are members of Streptomyces hormones called ‘butyrolactone autoregulators’ which regulate the antibiotic production in Streptomyces species at nanomolar concentrations. Cell-free extract of a VB-A overproducer, Streptomyces antibioticus NF-18, is capable of catalyzing the final step of the autoregulator biosynthesis, namely, the NADPH-dependent reduction of 6-dehydroVB-A. However, physico-chemical analyses of the purified enzymatic products revealed that, in addition to the VB-type isomer [(2R,3R,6S)-enantiomer], IM-2-type isomers [(2R,3R,6R)- and (2S,3S,6S)-enantiomers] were also produced from (±)-6-dehydroVB-A, suggesting the existence of several 6-dehydroVB-A reductases with respective stereoselectivities. The reductase activity of the crude extracts was separated into two activity peaks, peak I (major) and peak II (minor), by DEAE-5PW HPLC. Chiral HPLC analyses demonstrated that peak I enzyme and peak II enzyme catalyzed the production of (2R,3R,6S), (2R,3R,6R) and (2S,3S,6S) isomers at ratios of 46:1:3.2 and 4.9:1:1.5, respectively, indicating clearly that S. antibioticus NF-18 possesses at least two 6-dehydroVB-A reductases: one much favored toward VB-A biosynthesis, the other with relaxed stereoselectivity capable of synthesizing both VB-type and IM-2-type autoregulators.     

Preparation of Optically Active 2-Aminobutanoic Acid via Optical Resolution by Replacing Crystallization

An attempt was made to use a simple procedure to obtain (R)- and (S)-2-aminobutanoic acids [(R)- and (S)-1] which are non-proteinogenic α-amino acids and are useful as chiral reagents in asymmetric syntheses. Compound (RS)-1 p-toluenesulfonate [(RS)-2], which is known to exist as a conglomerate, was optically resolved by replacing crystallization with (R)- and (S)-methionine p-toluenesulfonate [(R)- and (S)-3] as optically active co-solutes. When (S)-3 was employed as the co-solute, (R)-2 was preferentially crystallized from a supersaturated solution of (RS)-2 in 1-propanol, as was (S)-2 in the presence of (R)-3. (R)- and (S)-2 recrystallized from 1-propanol were treated with triethylamine in methanol to give (R)- and (S)-1 in optically pure forms.     

Asymmetric synthesis of duloxetine intermediate (S)-(-)-3-N-methylamino-1-(2-thienyl)-1-propanol using immobilized Saccharomyces cerevisiae in liquid-core sodium alginate/chitosan/sodium alginate microcapsules

Duloxetine intermediate (S)-(-)-3-N-methylamino-1-(2-thienyl)-1-propanol was synthesized using ACA liquid-core immobilized Saccharomyces cerevisiae CGMCC No. 2230. The optimum culture time for ACA liquid-core immobilized cells was found to be 28 h. The optimum ACA liquid-core capsule formation conditions were found to be 90 % chitosan deacetylation, 30,000–50,000 chitosan molecular weight, 5.0 g/L chitosan, and pH 6.0 citrate buffer solution. The highest activity was found when reduction conditions were pH 6.0, 30 °C and 180 rpm. The ACA-immobilized cells can be reused nine times and only 40 % of the activity is retained after nine cycles. Product inhibition of reduction was observed in batch reduction. Continuous reduction in the membrane reactor was found to remove the product inhibition on reduction and improve production capacity. Conversion reached 100 % and enantiometric excess of (S)-(-)-3-N-methylamino-1-(2-thienyl)-1-propanol exceeded 99.0 % in continuous reduction of 5 g/L 3-N-methylamino-1-(2-thienyl)-1-propanone in the membrane reactor.     

Neuroprotective Activity of (+)-(S)-2-[(1S,5R)-(3,7-Dioxo-2,4,6,8-Tetraazabicyclo[3.3.0]oct-2-yl)]-4-methylthiobutanoic acid

A study of the neurotropic, neuroprotective, and antioxidant action of the enantiomers and racemate of 2-[(3,7-dioxo-2,4,6,8-tetraazabicyclo[3.3.0]oct-2-yl)]-4-methylthiobutanoic acid synthesized in a stereoselective reaction of (R)-, (S)-, or (R,S)-N-carbamoylmethionine with 4,5-dihydroxyimidazolidine-2-one showed that only (+)-(S)-2-[(1S,5R)-(3,7-dioxo-2,4,6,8-tetraazabicyclo[3.3.0]oct-2-yl)]-4-methylthiobutanoic acid had neuroprotective properties. X-ray structure analysis showed that the predominating racemate of glycolurils is crystallized from aqueous solutions as a conglomerate. Antioxidant activity was not detected.     

Stereochemical Control in the Still-Wittig Rearrangement Synthesis of Cyclohexyl (Z)-Alkene Inhibitors of Pin1

Three stereoisomeric inhibitors of Pin1: (2R,5S)-, (2S,5R)- and (2S,5S)-Ac–pSer–Ψ[(Z)CH = C]–pipecolyl(Pip)–2-(2-naphthyl)ethylamine 1, that mimic L-pSer–D-Pro, D-pSer–L-Pro, and D-pSer–D-Pro amides respectively, were synthesized by a 13-step route. The newly formed stereogenic centers in the pipecolyl ring were introduced by Luche reduction, followed by stereospecific [2,3]-Still-Wittig rearrangement. The (Z)- to (E)-alkene ratio in the rearrangements were consistently 5.5 to 1. The stereochemistry at the original Ser α-carbon controlled the stereochemistry of the Luche reduction, but it did not affect the stereochemical outcome of the rearrangement, which consistently gave the (Z)-alkene. The epimerized by-product, (2S,5S)-10, resulting from the work-up after Na/NH₃ debenzylation of (2S,5R)-9, was carried on to the (2S,5S)-1 isomer. Compound (2S,5S)-10 was resynthesized from the Luche reduction by-product, (2R,3R)-3, and the stereochemistry was confirmed by comparison of the optical rotations. The IC₅₀ values for (2R,5S)-1, (2S,5R)-1 and (2S,5S)-1 Pin1 inhibition were: 52, 85, and 140 μM, respectively.     

Enantioselective synthesis of (R)-(+)- and (S)-(-)-higenamine and their analogues with effects on platelet aggregation and experimental animal model of disseminated intravascular coagulation

Optically active tetrahydroisoquinoline alkaloids, (R)-(+)-higenamine (1R) and (S)-(−)-higenamine (1 S), and their optically active 1-naphthylmethyl analogues (2 and 3), were synthesized by enantioselective hydrogenation of the corresponding dihydroisoquinoline intermediates 7 as a key step. The evaluation of the platelet anti-aggregation effect demonstrated clearly that the (S)-(−)-enantiomers, 1S, 2S, and 3S, had higher inhibitory potency than the corresponding (R)-(+)-antipodes, 1R, 2R, and 3R, respectively, to platelet aggregation induced by epinephrine. 1S enantiomer was superior to the corresponding 1R enantiomer in attenuating all of the disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) parameters tested, while the S enantiomers 2S and 3S ameliorated some of the DIC and MOF parameters more effectively than the corresponding antipodes 2R and 3R.     

Production of (R)-3-hydroxybutyric acid by fermentation and bioconversion processes with Azohydromonas lata

Feasibility of producing (R)-3-hydroxybutyric acid ((R)-3-HB) using wild type Azohydromonas lata and its mutants (derived by UV mutation) was investigated. A. lata mutant (M5) produced 780 mg/l in the culture broth when sucrose was used as the carbon source. M5 was further studied in terms of its specificity with various bioconversion substrates for production of (R)-3-HB. (R)-3-HB concentration produced in the culture broth by M5 mutant was 2.7-fold higher than that of the wild type strain when sucrose (3% w/v) and (R,S)-1,3-butanediol (3% v/v) were used as carbon source and bioconversion substrate, respectively. Bioconversion of resting cells (M5) with glucose (1% v/w), ethylacetoacetate (2% v/v), and (R,S)-1,3-butanediol (3% v/v), resulted in (R)-3-HB concentrations of 6.5 g/l, 7.3 g/l and 8.7 g/l, respectively.     

Two sesquiterpene lactones from Trichogonia gardneri

The structures and stereochemistries of two sesquiterpene lactones from Trichogonia gardneri were established as (6R,7S,8S,9S,IOR)-4E-9,10-dihydroxy-8-tigloxygermacr-4-en-6,12-olide) and (5R*,6R*,7S*,8S*,9R*)-14-acetoxy-3-chloro-9-hydroxy-2-oxo-8-tigloxyguia-1(10),3-dien-6,12-of olide by a combination of NMR spectrometry and X-ray diffraction. The results show that the structures of several sesquiterpene lactones which were isolated previously from related species require revision.