Highly enantioselective reduction of 4-(trimethylsilyl)-3-butyn-2-one to enantiopure (R)-4-(trimethylsilyl)-3-butyn-2-ol using a novel strain Acetobacter sp. CCTCC M209061

The biocatalytic reduction of 4-(trimethylsilyl)-3-butyn-2-one to enantiopure (R)-4-(trimethylsilyl)-3-butyn-2-ol was successfully conducted with high enantioselectivity using immobilized whole cells of a novel strain Acetobacter sp. CCTCC M209061, newly isolated from kefir. Compared with other microorganisms that were investigated, Acetobacter sp. CCTCC M209061 was shown to be more effective for the bioreduction reaction, and afforded much higher yield and product enantiomeric excess (e.e.). The optimal buffer pH, co-substrate concentration, reaction temperature, substrate concentration and shaking rate were 5.0, 130.6 mM, 30 °C, 6.0 mM and 180 r/min, respectively. Under the optimized conditions, the maximum yield and the product e.e. were 71% and >99%, respectively, which are much higher than those reported previously. Additionally, the established biocatalytic system proved to be efficient for the bioreduction of acetyltrimethylsilane to (R)-1-trimethylsilylethanol with excellent yield and product e.e. The immobilized cells manifested a good operational stability under the above reaction conditions since they retained 70% of their catalytic activity after ten cycles of use.     

Steady-state kinetics of the oxidation of (S)-1-phenyl-1,2-ethanediol catalyzed by alcohol dehydrogenase from Candida parapsilosis CCTCC M203011

Kinetic studies of oxidation reaction of (S)-1-phenyl-1,2-ethanediol (PED) catalyzed by a NAD⁺-dependent alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 obtained from China Center for Type Culture Collection (CPADH) were observed for getting insight into the deracemization redox reaction. The data of initial velocity experiments in the absence of product, product (β-hydroxy-hypnone) inhibition experiments and dead-end (pyrazole) inhibition experiments strongly suggest that the reaction follows Theorell-Chance BiBi mechanism in which the coenzymes bind to the free form of the enzyme firstly. The kinetic parameters of this model were estimated by using non-linear regression analysis software.     

Using a water-immiscible ionic liquid to improve asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one catalyzed by immobilized Candida parapsilosis CCTCC M203011 cells

Background  

Whole cells are usually employed for biocatalytic reduction reactions to ensure efficient coenzyme regeneration and to avoid problems with enzyme purification and stability. The efficiency of whole cell-catalyzed bioreduction is frequently restricted by pronounced toxicity of substrate and/or product to the microbial cells and in many instances the use of two-phase reaction systems can solve such problems. Therefore, we developed new, biphasic reaction systems with biocompatible water-immiscible ionic liquids (ILs) as alternatives to conventional organic solvents, in order to improve the asymmetric reduction of 4-(trimethylsilyl)-3-butyn-2-one (TMSB) to (S)-4-(trimethylsilyl)-3-butyn-2-ol {(S)-TMSBOL}, a key intermediate for synthesis of 5-lipoxygenase inhibitors, using immobilized Candida parapsilosis CCTCC M203011 cells as the biocatalyst.     

Cloning and expression of the gene encoding (R)-specific carbonyl reductase from Candida parapsilosis CCTCC M203011

The gene which encodes (R)-specific carbonyl reductase (rCR) from Candida parapsilosis CCTCC M203011 was cloned, sequenced and compared with genes from the GenBank. The results indicated that rCR gene was 1011 bp, encoding a protein of 336 amino acids with a molecular weight of 35.9 kDa, and its nucleotide sequence showed 99% similarity to those of other members of the alcohol dehydrogenase superfamily. The rCR gene could express in recombinant strain Escherichia coli JM109, and the expression plasmid could produce (R)-1-pheny-1,2-ethanediol (100% e.e., 80.14% yield) from β-hydroxyacetophenone without any additive to regenerate NAD⁺ from NADH. __________ Translated from Microbiology, 2006, 33(4): 112–118 [译自: 微生物学通报]     

Asymmetric reduction of 3-chloropropiophenone to (S)-3-chloro-1-phenylpropanol using immobilized Saccharomyces cerevisiae CGMCC 2266 cells

(S)-3-Chloro-1-phenylpropanol is an important chiral precursor for numerous antidepressants such as tomoxetine. A high enantiomeric excess (e.e.) of (S)-3-chloro-1-phenylpropanol can be achieved by asymmetric reduction of 3-chloropropiophenone using Saccharomyces cerevisiae CGMCC 2266 cells immobilized in calcium alginate. Thermal pretreatment of the immobilized cells at 50 °C for 30 min resulted in high enantioselectivity (99% e.e.) and good percent conversion (80%). The effects of various conditions on the reduction reaction were investigated. The optimal conditions were found to be as follows: sodium alginate concentration, 2%; bead diameter, 2 mm; temperature, 30 °C; re-culture time, 24 h; and batch addition of the substrate. After reusing these three times, the immobilized cells retained approximately 60% of their original catalytic activity with their enantioselectivity intact.     

Microbial production of (S)-1-phenyl-1,3-propanediol by stereospecific reduction of 3-hydroxy-1-phenylpropane-1-one

A novel microbial method of synthesizing (S)-1-phenyl-1,3-propanediol [(S)-PPD] was developed in this study. Our laboratory stock cultures were screened for microorganisms that stereospecifically produced (S)-PPD from 3-hydroxy-1-phenylpropane-1-one (HPPO) using an intact cell system. Of the 828 strains examined (321 bacteria, 233 yeasts and 274 molds), certain strains of Williopsis saturnus var. mrakii and Cryptococcus albidus were found to produce (S)-PPD with over 99% enantiomeric excess (e.e.). Screening identified W. saturnus var. mrakii AJ-5620 as the most productive strain, and this strain was used for further experiments. The (S)-PPD-producing reaction using intact W. saturnus var. mrakii AJ-5620 cells was carried out by successive feeding of HPPO. A total (S)-PPD yield of 9.9 g/l was produced in 20 h. The molar yield was 81% and the optical purity of the (S)-PPD produced was over 99% e.e.     

Biotransformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one by four filamentous fungi

Microbial transformation of (20S)-20-hydroxymethylpregna-1,4-dien-3-one (1) by four filamentous fungi, Cunninghamella elegans, Macrophomina phaseolina, Rhizopus stolonifer, and Gibberella fujikuroi, afforded nine new, and two known metabolites 2–12. The structures of these metabolites were characterized through detailed spectroscopic analysis. These metabolites were obtained as a result of biohydroxylation of 1 at C-6β, -7β, -11α, -14α, -15β, -16β, and -17α positions, except metabolite 2 which contain an O-acetyl group at C-22. These fungal strains demonstrated to be efficient biocatalysts for 11α-hydroxylation. Compound 1, and its metabolites were evaluated for the first time for their cytotoxicity against the HeLa cancer cell lines, and some interesting results were obtained.     

Stereoselective total synthesis of a potent natural antifungal compound (6S)-5,6,dihydro-6-[(2R)-2-hydroxy-6-phenyl hexyl]-2H-pyran-2-one

A practical stereoselective synthesis of (6S)-5,6,dihydro-6-[(2R)-2-hydroxy-6-phenyl hexyl]-2H-pyran-2-one (1), a potent natural antifungal compound, is described. The sequence involves diastereoselective iodine-induced electrophilic cyclization, epoxide ring opening with a vinyl Grignard reagent and ring closing metathesis (RCM) as the key steps.     

Measurement of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood by high-performance liquid chromatography

A high-performance liquid chromatographic method for automated analysis of both protein-bound and total S-2-(3-aminopropylamino)ethanethiol (WR-1065) in blood has been developed in our laboratory. WR-1065 is the active thiol metabolite of the radio- and chemo-protector drug amifostine (WR-2721). Using WR-1065 quality control levels over the experimental range: 7.0, 45.0 and 85.0 μmol/l spiked into plasma, method validation for total WR-1065 included between-run assessment of imprecision (SD/C.V.%: 1.11/16.7%, 6.58/15.5% and 9.24/11.3%, respectively) and % accuracy (94.7, 106.0 and 97.2%).     

Synthesis of (E)-9-(1-pyrenyl)-4-hydroxynon-2-enal, a fluorescent probe of the (E)-4-hydroxynon-2-enal with retained biological properties

(E)-9-(1-pyrenyl)-4-hydroxynon-2-enal (FHNE), a fluorescent probe of (E)-4-hydroxynon-2-enal (HNE) is synthesised in seven steps and in 35% overall yield, starting from commercially available 1-pyrencarboxyaldehyde. When incubated with cultured HeLa cells this fluorescent probe penetrates cells and particularly concentrates in the region surrounding the nucleus. As the parent compound, HNE it is able to induce the activation of heat shock factor (HSF) and it is able to induce the binding of HSF to heat shock element (HSE).     

Synthesis of enantiopure (S)-6-chlorochroman-4-ol using whole-cell Lactobacillus paracasei biotransformation

Chromane, which has a fused cyclic structure, is a significant molecule that can be found in the structure of many important compounds. Lactobacillus paracasei BD101 was demonstrated as whole-cell biocatalyst for the synthesis of (S)-6-chlorochroman-4-ol with immense enantioselectivity. The conditions of asymmetric reduction were optimized one factor by one factor using L paracasei BD101 to achieve enantiomerically pure product and complete conversion. Using these obtained optimization conditions, asymmetric reduction of 6-chlorochroman-4-one was performed under environmentally friendly conditions; 6-chlorochroman-4-one, having a fused cyclic structure as previously noted to be difficult to asymmetric reduction with biocatalysts, was enantiomerically reduced to (S)-6-chlorochroman-4-ol with an enantiomeric excess >99% on a high gram scale. This study is the first example in the literature for the enantiopure synthesis of (S)-6-chlorochroman-4-ol using a biocatalyst. Also notably, the optical purity of (S)-6-chlorochroman-4-ol obtained in this study through asymmetric bioreduction using whole-cell biocatalyst is the highest value in the literature. In this study, (S)-6-chlorochroman-4-ol was produced on a gram scale by an easy, inexpensive, and environmentally friendly method, which has shown the production of valuable chiral precursors for drug synthesis and other industrial applications. This study provides a convenient method for the production of (S)-6-chlorochroman-4-ol, which can meet the industrial green production demand of this chiral secondary alcohol.     

Enantioselective synthesis of ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE) from ethyl-3-oxo-3-phenylpropanoate using recombinant fatty acid synthase (FAS2) from Kluyveromyces lactis KCTC 7133 in Pichia pastoris GS115

Various yeast strains were examined for the microbial reduction of ethyl-3-oxo-3-phenylpropanoate (OPPE) to ethyl-(S)-3-hydroxy-3-phenylpropanoate (S-HPPE), which is the chiral intermediate for the synthesis of a serotonin uptake inhibitor, Fluoxetine. Kluyveromyces lactis KCTC 7133 was found as the most efficient strain in terms of high yield (83% at 50 mM) and high optical purity ee > 99% of S-HPPE. Based on the protein purification, activity analysis and the genomic analysis, a fatty acid synthase (FAS) was identified as the responsible β-ketoreductase. To increase the productivity, a recombinant Pichia pastoris GS115 over-expressing FAS2 (α-subunit of FAS) of K. lactis KCTC7133 was constructed. In the optimized media condition, the recombinant P. pastoris functionally over-expressed the FAS2. Recombinant P. pastoris showed 2.3-fold higher reductase activity compared with wild type P. pastoris. With the recombinant P. pastoris, the 91% yield of S-HPPE was achieved at 50 mM OPPE maintaining the high optical purity of the product (ee > 99%).     

Mutations induced by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the lacZ and cII genes of Muta™ Mouse

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) found in chewing tobacco, snuff, cigarettes, and cigars is a tobacco-specific nitrosamine and classified as a possible human carcinogen (Class 2B) by the International Agency for Research on Cancer (IARC). NNK given intraperitoneally was seen to induce lung and liver adenomas.To evaluate the genotoxicity of NNK in vivo, NNK was intraperitoneally administered to Muta™ Mouse at two concentrations (125 and 250 mg/kg, once a week for 4 weeks) followed by the measurement of mutant frequencies in the lacZ and cII genes from lung and liver in the same mice. Characterization of the types of the mutation was determined by sequencing the cII genes from mutant plaques. The mutant frequencies in both target genes from both organs dose-dependently increased up to 10 times compared to those of the control group. For the types of mutations, the ratio of the G:C to A:T mutation in the total number of mutants was less than the ratio of A:T to T:A and A:T to C:G transversion, contrary to a previous report [Cancer Res, 49 (1989) 5305]. The A:T to T:A transversion was the most highly induced mutation both in the lung and liver cII genes. The increasing rate of mutant frequencies in lung and liver over the vehicle control was 55 and 56 times, respectively, while the increasing rate of G:C to A:T transition was only 1.9 and 2.8 times, respectively.These observations show that NNK predominantly induces DNA adducts leading to A:T to T:A and/or A:T to C:G mutations in the transgene.     

Biocatalytic production of (S)-4-bromo-3-hydroxybutyrate and structurally related chemicals and their applications

The enzymatic production of (S)-4-bromo-3-hydroxybutyrate has been poorly studied compared with (S)-4-chloro-3-hydroxybutyrate. This can be attributed to the toxicity of bromide for biocatalysis. Recently, we isolated cDNA that encodes Penicillium citrinum β-keto ester reductase (KER) and the gene that encodes Leifsonia sp. alcohol dehydrogenase, which catalyzes the reduction of methyl 4-bromo-3-oxobutyrate to methyl (S)-4-bromo-3-hydroxybutyrate with high optical purity and productivity and expressed them in Escherichia coli. Moreover, protein engineering was performed using error-prone PCR-based random mutagenesis to improve the thermostability and enantioselectivity of KER. This review focuses on the establishment of a novel biotechnological process for the production of (S)-4-bromo-3-hydroxybutyrate using E. coli transformants. This process is suitable for industrial production of (S)-4-bromo-3-hydroxybutyrate, an intermediate for statin compounds.     

Convenient and Efficient Syntheses of Oligodeoxyribonucleotides Containing O 6-(Carboxymethyl)Guanine and O 6-(4-Oxo-4-(3-Pyridyl)Butyl)Guanine

O ⁶-(carboxymethyl)guanine (O ⁶-CMG) and O ⁶-(4-oxo-4-(3-pyridyl)butyl)guanine (O ⁶-pobG) are toxic lesions formed in DNA following exposure to alkylating agents. O ⁶-CMG results from exposure to nitrosated glycine or nitrosated bile acid conjugates and may be associated with diets rich in red meat. O ⁶-pobG lesions are derived from alkylating agents found in tobacco smoke. Efficient syntheses of oligodeoxyribonucleotides (ODNs) containing O ⁶-CMG and O ⁶-pobG are described that involve nucleophilic displacement by the appropriate alcohol on a common synthetic ODN containing the reactive base 2-amino-6-methylsulfonylpurine. ODNs containing O ⁶-pobG and O ⁶-CMG were found to be good substrates for the S. pombe alkyltransferase-like protein Atl1.

[Supplemental materials are available for this article. Go to the publisher's online edition of Nucleosides, Nucleotides & Nucleic Acids to view the free supplemental file.]     

Chemopreventive potential of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one on 7,12-dimethylbenz[a]anthracene (DMBA) induced hamster buccal pouch carcinogenesis

In the present work, a new bis heterocyclic compound comprising both the piperidone and thiohydantoin nuclei namely 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one was synthesised and characterised with the help of mp, elemental analysis, FT-IR, MS and one-dimensional NMR (¹H and ¹³C) spectra. The inhibitory effect of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one on 7,12-dimethylbenz[a]anthracene (DMBA) induced buccal pouch carcinogenesis was investigated in Syrian male hamsters. All the hamsters that were painted with DMBA on their buccal pouches for 14 weeks developed squamous cell carcinoma. Administration of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one effectively suppressed the oral carcinogenesis initiated with the DMBA as revealed by a reduced incidence of neoplasms. Lipid peroxidation, glutathione (GSH) content and the activities of glutathione peroxidase (GPx), glutathione S-transferase (GST) were used to biomonitor the chemopreventive potential of 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one. Lipid peroxidation was found to be significantly decreased, whereas GSH, GPx, GST and GGT were elevated in the oral mucosa of tumour bearing animals. Our data suggest that 3-[2,6-bis(4-fluorophenyl)-3-methylpiperidin-4-ylideneamino]-2-thioxoimidazolidin-4-one may exert its chemopreventive effects in the oral mucosa by modulation of lipid peroxidation, antioxidants and detoxification systems.     

Enantioselective resolution of 2-(1-hydroxy-3-butenyl)-5-methylfuran by immobilized lipase

An efficient and convenient strategy for synthesis of enantiomerically pure S-2-(1-hydroxy-3-butenyl)-5-methylfuran was for the first time described utilizing a lipase-mediated asymmetric acylation in organic solvents. Rhizopus arrhizus lipase was chosen as the biocatalyst, and different immobilization methods including sol–gel encapsulation and covalent attachment were adopted to improve its catalytic characteristics. Various influential factors of the reaction were also investigated. Finally, the results showed that the lipase covalently attached onto epoxy resin exhibited the highest enantioselectivity and operational stability. Under optimized reaction conditions, i.e., n-hexane as the solvent, 5/1 (mol/mol) of vinyl acetate to 2-(1-hydroxy-3-butenyl)-5-methylfuran and 30 °C, the ee value of S-1 reached up to above 98% at 52% conversion with an E value of 99.     

Design,synthesis, and mechanism of action of 2-(3-hydroxy-5-methoxyphenyl)-6-pyrrolidinylquinolin-4-one as a potent anticancer lead

New 6- (or 6,7-) substituted 2-(hydroxyl substituted phenyl)quinolin-4-one derivatives were synthesized and screened for antiproliferative effects against cancer cell lines. Structure–activity relationship correlations were established and the most promising compound 2-(3-hydroxy-5-methoxyphenyl)-6-pyrrolidin-1-ylquinolin-4-one (6h) exhibited strong inhibitory activity against various human cancer cell lines, particularly non-small cell lung cancer NCI-H522. Additional studies suggested a mechanism of action resembling that of the antimitotic drug vincristine. The presence of a C-ring OH group in 6h will allow this compound to be converted readily to a water soluble and physicochemically stable hydrophilic prodrug. Compound 6h is proposed as a new anticancer lead compound.     

One pot three components microwave assisted and conventional synthesis of new 3-(4-chloro-2-hydroxyphenyl)-2-(substituted) thiazolidin-4-one as antimicrobial agents

A one-pot, three-component, microwave assisted and conventional synthesis of new 3-(4-chloro-2-hydroxyphenyl)-2-(substituted) thiazolidin-4-one (4a–n) was carried out by using N,N-dimethylformamide as a solvent with high product yield. Among these synthesized compounds (4f, 4g, 4l and 4m) were found to be a broad spectrum molecule active against all bacterial and fungus strains tested, except fungus Aspergillus niger. Amongst the compounds (4g, 4l and 4m) were found to be more potent than respective standard drugs used in the experiment against Candida albicans, Staphylococcus aureus and Aspergillus flavus, respectively. All synthesized compounds were also tested for their cytotoxic activity against HeLa and MCF-7 cell lines by the sulforhodamine B (SRB) assay. This study shows that all compounds were non-cytotoxic in nature, and confirmed their antimicrobial specificity apart from any general cytotoxicity.     

羰基还原酶产生菌Candida ontarioensis制备(R)-2-氯-1-(3-氯苯基)乙醇

从实验室保藏的菌株中筛选获得Candida sp.PT2A,并通过18S rRNA鉴定为安大略假单胞菌Candida on-tarioensis。对C.ontarioensis不对称还原合成(R)-2-氯-1-(3-氯苯基)乙醇的发酵产酶条件和转化条件进行优化,确定了最适的发酵产酶条件和转化条件:温度30℃,初始pH 6.5,摇床转速180 r/min,菌体质量浓度200 g/L。采用2-氯-1-(3-氯苯基)乙酮质量浓度为10 g/L时,还原反应72 h,(R)-2-氯-1-(3-氯苯基)乙醇的e.e.值为99.9%,产率为99%;底物质量浓度提高至30 g/L时,产率下降为84.3%。采用十六烷基三甲基溴化铵(CTAB)对C.ontarioensis细胞进行通透性处理(CTAB g/L,4℃下处理20 min),在30 g/L底物下反应24 h,产物的e.e.和产率分别达到99.9%和97.5%。