NAD(+)-dependent (S)-specific secondary alcohol dehydrogenase involved in stereoinversion of 3-pentyn-2-ol catalyzed by Nocardia fusca AKU 2123

An NAD(+)-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The K(m) and Vmax for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 mumol/min/mg, and 0.33 mM and 130 mumol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The K(m) and Vmax for 2-hexanone reduction were 2.5 mM and 260 mumol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH2-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.     

Production of (R )-3-pentyn-2-ol through stereospecific hydrolysis of racemic 3-pentyn-2-ol esters with microbial enzymes

Microorganisms and commercial enzymes were screened for their ability to produce (R)-3-pentyn-2-ol from racemic 3-pentyn-2-ol esters through stereospecific hydrolysis. Among the esters formed with acetic acid, propionic acid, hexanoic acid and benzoic acid, the acetate was most effectively hydrolyzed by microbial cells and commercial lipases with high stereospecificity. Rhodococcus rubropertinctus AKU NOC082 was a good catalyst for (R)-3-pentyn-2-ol production through the hydrolytic resolution of racemic 3-pentyn-2-yl acetate. With 15%, 25% and 50% (v/v) racemic 3-pentyn-2-yl acetate as the substrate, 42.6%, 40.8% and 40.0% was hydrolyzed in 5 h, 10 h and 98 h respectively, under the optimized conditions (pH 7.0, 30 °C, 7.5% wet cell concentration), the (R) enantiomer of 3-pentyn-2-ol being formed with an optical purity of 97.8%, 98.0% and 94.2% respectively. Received: 2 June 1998 / Received revision: 3 August 1998 / Accepted: 3 September 1998     

NAD+-Dependent (S)-Specific Secondary Alcohol Dehydrogenase Involved in Stereoinversion of 3-Pentyn-2-ol Catalyzed by Nocardia fusca AKU 2123

An NAD⁺-dependent alcohol dehydrogenase was purified to homogeneity from Nocardia fusca AKU 2123. The enzyme catalyzed (S)-specific oxidation of 3-pentyn-2-ol (PYOH), i.e., part of the stereoinversion reaction for the production of (R)-PYOH, which is a valuable chiral building block for pharmaceuticals, from the racemate. The enzyme used a broad variety of secondary alcohols including alkyl alcohols, alkenyl alcohols, acetylenic alcohols, and aromatic alcohols as substrates. The oxidation was (S)-isomer specific in every case. The K _m and V _max for (S)-PYOH and (S)-2-hexanol oxidation were 1.6 mM and 53 μmol/min/mg, and 0.33 mM and 130 μmol/min/mg, respectively. The enzyme also catalyzed stereoselective reduction of carbonyl compounds. (S)-2-Hexanol and ethyl (R)-4-chloro-3-hydroxybutanoate in high optical purity were produced from 2-hexanone and ethyl 4-chloro-3-oxobutanoate by the purified enzyme, respectively. The K _m and V _max for 2-hexanone reduction were 2.5 mM and 260 μmol/min/mg. The enzyme has a relative molecular mass of 150,000 and consists of four identical subunits. The NH₂-terminal amino acid sequence of the enzyme shows similarity with those of the carbonyl reductase from Rhodococcus erythropolis and phenylacetaldehyde reductase from Corynebacterium sp.     

(S)-3-Pentyn-2-ol production through microbial enzyme-catalyzed, highly enantioselective hydrolysis of racemic 3-pentyn-2-ol esters

Air-dried cells of Hansenula nonfermentans AKU 4332 catalyzed the production of (S)-3-pentyn-2-ol from (RS)-3- pentyn-2-ol acetate ester at 10% (v/v). The product was formed at 96.6% e.e. with a molar yield of 45% in 24 h. © Rapid Science Ltd. 1998     

Stereoinversion of optically active 3-pentyn-2-ol by Nocardia species

Nocardia fusca and Nocardia pseudosporangifera produced (R)-3-pentyn-2-ol and (S)-3-pentyn-2-ol in 24 and 72 h reaction, respectively, from (RS)-3-pentyn-2-ol, with greater than 70% molar yields through a stereoinversion reaction involving stereoselective oxidation and reduction with 3-pentyn-2-one as an intermediate.     

Synthesis of (R)-2-phenylpropanoic acid from its racemate through an isomerase-involving reaction by Nocardia diaphanozonaria

(R)-2-Phenylpropanoic acid was synthesized from the racemic acid through an isomerization reaction involving resting cells of Nocardia diaphanozonaria JCM3208. The isomerization activity of the cells was enhanced 25-fold by adding 5.5 mM racemic 2-phenylpropanoic acid to the culture medium. When 5 mM racemic 2-phenylpropanoic acid was included in the reaction mixture (4 ml) containing resting cells (100 mg dry cell wt) in 25 mM K₂HPO₄/KH₂PO₄ buffer (pH 7.0) at 30 °C for 8 h, 4.56 mM (R)-2-phenylpropanoic acid (95.8% e.e.) was formed with a 91% molar conversion yield.     

Total synthesis and antifungal activity of (2S,3R)-2-aminododecan-3-ol

We report the total synthesis of (2S,3R)-2-aminododecan-3-ol has been achieved starting from commercially available 10-undecenoic acid. The key steps involved are Sharpless asymmetric epoxidation, Miyashita's boron-directed C-2 regioselective azidolysis, generated the asymmetric centers and in situ detosylation and reduction of azido tosylate. The antifungal activity of the synthesized (2S,3R)-2-aminododecan-3-ol was evaluated on several Candida strains and was comparable to miconazole, a standard drug.     

Production of (R)-1-phenylethanol through enantioselective oxidation of (S)-isomer from their racemic mixture by Pachysolen tannophilus IFO 1007

Summary Stereoselective oxidation of (S)-isomer of rac-1-phenylethanol (1-PA) by the yeast Pachysolen tannophilus IFO 1007 immobilized into calcium-alginate gels was investigated to produce (R)-isomer. Continuous production of (R)-isomer was accomplished for more than 80 h with an enantiomeric excess of > 90% using a bioreactor of a fluidized-bed type.     

Microbial production of (2R, 4R)-2,4-pentanediol by enatioselective reduction of acetylacetone and stereoinversion of 2,4-pentanediol

Summary (2R, 4R)-2,4-Pentanediol was obtained by the enatioselective reduction of acetylacetone (2,4-pentanedione) with the resting cells of methanol yeast,Candida boidinii KK912 (IFO 10574). (2R, 4R)-2,4-Pentanediol was also obtained by the stereoinversion of the isomeric mixture of 2,4-pentanediol.     

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.     

Preparation of alkyl (R)-(-)-3-hydroxybutyrate by acidic alcoholysis of poly-(R)-(-)-3-hydroxybutyrate

An efficient method for the preparation of optically active alkyl (R)-(-)-3-hydroxybutyrates by chemical depolymerization of biopolymer, poly-(R)-(-)-(3-hydroxybutyrate), was established. This method consists of simple recovery of poly-(R)-(-)-(3-hydroxybutyrate) from bacterial cells followed by acidic alcoholysis. When poly-(R)-(-)-(3-hydroxybutyrate) was purified by a simple digestion method that used 0.2 N sodium hydroxide, alkyl (R)-(-)-hydroxybutyrates were most efficiently produced by alcoholysis with anhydrous hydrochloric acid.     

Syntheses of 2-Deoxy-2-[(2R,3S)-2-fluoro-3-hydroxytetradecanamido]-3-O-[(3R)-3-hydroxytetradecanoyl]-4-O-phosphono-D-glucopyranose and Its (2S,3R)-Isomer

2-Deoxy-2-[(2R,3S)-2-fluoro-3-hydroxytetradecanamido]-3-O-[(3R)-3-hydroxytetradecanoyl]-4-O-phosphono-D-glucopyranose and its (2S,3R)-isomer were respectively synthesized from allyl 2-[(2R,3S)-3-(benzyloxycarbonyloxy)-2-fluorotetradecanamido]-2-deoxy-4,6-O-isopropylidene-β-D-glucopyranoside and its corresponding (2S,3R)-isomer. Both target compounds did not activate macrophage, but the (2S,3R)-analogue strongly inhibited the binding of LPS to macrophage.     

Production of (R)-3-quinuclidinol by a whole-cell biocatalyst with high efficiency

Optically pure (R)-3-quinuclidinol [(R)-3-Qui] is widely used as a chiral building block for producing various antimuscarinic agents. An asymmetric bioreduction approach using 3-quinuclidinone reductases is an effective way to produce (R)-3-Qui. In this study, a biocatalyst for producing (R)-3-Qui was developed by using Escherichia coli that coexpressed Kaistia granuli (KgQR) and mutant glucose dehydrogenase (GDH). KgQR catalyses the synthesis of (R)-3-Qui through the efficient reduction of 3-quinuclidinone. The specific activity of recombinant KgQR was 254?U/mg, and the Michaelis–Menten constant (K_m) for 3-quinuclidinone was 0.51?mM. The thermal stability of KgQR was relatively high compared with ArQR. Approximately 73% of the residual activity remained after incubation in 0.2 M potassium phosphate buffer (KPB) (pH 7.0) for 8?h at 30?°C. In addition, 80% residual activity remained for the double-mutant GDH (Q252L and E170K) after incubation in a buffer (pH 7.0) for 8?h at 30 and 40?°C. 3-Quinuclidinone (242?g/L) can be reduced to (R)-3-Qui in 3?h by coexpressing KgQR and mutant GDH in E. coli. The conversion rate reached 80.6?g/L/h, which is the highest reported to date. The results demonstrates that this whole-cell biocatalyst will have a great potential in industrial manufacturing.     

Novel substituted (Z)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-one and (Z)-(+/-)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol derivatives as potent thermal sensitizing agents

Use of ionizing radiation is essential for the management of many human cancers, and therapeutic hyperthermia has been identified as a potent radiosensitizer. Radiation therapy combined with adjuvant hyperthermia represents a potential tool to provide outstanding local-regional control for refractory disease. (Z)-(±)-2-(N-Benzylindol-3-ylmethylene)quinuclidin-3-ol (2) and (Z)-(±)-2-(N-benzenesulfonylindol-3-ylmethylene)quinuclidin-3-ol (4) were initially identified as potent thermal sensitizers that could lower the threshold needed for thermal sensitivity to radiation treatment. To define the structural requirements of the molecule that are essential for thermal sensitization, we have synthesized and evaluated a series of (Z)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-one (9), and (Z)-(±)-2-(N-benzylindol-3-ylmethylene)quinuclidin-3-ol (10) analogs that incorporate a variety of substituents in both the indole and N-benzyl moieties. These systematic structure–activity relationship (SAR) studies were designed to further the development and optimization of potential clinically useful thermal sensitizing agents. The most potent analog was compound 10 (R¹ = H, R² = 4-Cl), which potently inhibited (93% inhibition at 50 μM) the growth of HT-29 cells after a 41 °C/2 h exposure.     

Novel biosynthesis of (R)-ethyl-3-hydroxyglutarate with (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate by Rhodococcus erythropolis

(R)-ethyl-3-hydroxyglutarate with highly optical purity (≥99%) can be used as a novel precursor for synthesis of chiral side chain of rosuvastatin. In this study, a novel synthesis route of (R)-ethyl-3-hydroxyglutarate by whole microorganism cells from racemic ethyl 4-cyano-3-hydroxybutyate was created. A strain ZJB-0910 capable of transforming racemic β-hydroxy aliphatic nitrile was isolated by employing a screening method based on a colorimetric reaction of Co²⁺ ion with ammonia, and identified as Rhodococcus erythropolis based on its morphology, physiological tests, Biolog, and the 16S rDNA sequence. After cultivation in a sterilized medium with composition of 20 g glucose, 5 g yeast extract, 0.5 g KH₂PO₄, 0.5 g K₂HPO₄, 0.2 g MgSO₄·7H₂O per liter at 30°C and 150 rpm for 48 h, the whole cells of R. erythropolis ZJB-0910 were prepared as a catalyst in (R)-enantioselective hydrolysis of racemic ethyl 4-cyano-3-hydroxybutyate for synthesis of (R)-ethyl-3-hydroxyglutarate, without bearing hydrolase activity for the ester bond of ethyl 4-cyano-3-hydroxybutyate. Under the optimized biotransformation conditions of pH 7.5, 30°C, and 20 mM substrate concentration, (R)-ethyl-3-hydroxyglutarate with 46.2% yield (ee > 99%) was afforded, and its chemical structure was determined by ESI-MS, NMR, and IR. The apparent Michaelis constant K _m and maximum rate V _max for this biocatalytic reaction were 0.01 M and 85.6 μmol min⁻¹ g⁻¹, respectively.     

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

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

固定化诺卡氏菌细胞生产L(+)酒石酸的研究

用明胶包埋酒石酸诺卡氏菌(Nocardia tartaricans SWl3—57)得到顺式环氧琥珀酸开环水解酶活力较高的固定化细胞。固定化细胞的最适温度为30～45℃,而游离细胞的最适温度为35～45℃。两者的最适pH均为8．0～9．0,固定化细胞的表观米氏常数Km为0．256mol／L,而游离细胞有底物抑制作用,在底物浓度小于0．45mol／L时Km为0．246mol／L。用固定化细胞装柱(y=100ml),pH8．S,温度37℃,稀释速率D=0．25h^-1,以0．5mol／L浓度顺式环氧琥珀酸钠溶液为底物,连续运转53d,平均产L(+)酒石酸66．95g／L,克分子转化率为92．09％,反应器生产能力达到16．58g／L·h。     

Muscarinic M(3) receptor antagonists with (2R)-2-[(1R)-3,3-difluorocyclopentyl]-2-hydroxyphenylacetamide Structures. Part 2

Optimization of the amine part of our original muscarinic M(3) receptor antagonist 1 was performed to identify M(3) receptor antagonists that are superior to 1. Compounds carrying a variety of diamine moieties without hydrophobic substituent on the nitrogen atom were screened against the binding affinity for the M(3) receptor and the selectivity for M(3) over the M(1) and M(2) receptors. This process led to a 4-aminopiperidinamide (2l) with a K(i) value of 5.1 nM and with a selectivity of the M(3) receptor that was 46-fold greater than that of the M(2) receptor. Further derivatization of 2l by inserting a spacer group or by incorporating alkyl group(s) into the amine part resulted in the identification of an 4-(aminoethyl)piperidinamide 2l-b with a K(i) value of 3.7 nM for the M(3) receptor and a selectivity for the M(3) receptor that was 170-fold greater than that of the M(2) receptor.     

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.