Production of a Doubly Chiral Compound, (4R,6R)-4-Hydroxy-2,2,6-Trimethylcyclohexanone, by Two-Step Enzymatic Asymmetric Reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD⁺, and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.     

Production of Ethyl (R)-2-Hydroxy-4-phenylbutanoate via Reduction of Ethyl 2-Oxo-4-phenylbutanoate in an Interface Bioreactor

Ethyl (R)-2-hydroxy-4-phenylbutanoate [(R)-EHPB], a useful intermediate for the synthesis of various anti-hypertension drugs, was produced via microbial reduction of ethyl 2-oxo-4-phenylbutanoate [EOPB] in an interface bioreactor. Rhodotorula minuta IFO 0920 and Candida holmii KPY 12402 were selected as the best type culture and isolated yeasts, respectively. The highest enantiomeric excess of (R)-EHPB produced by R. minuta and C. holmii were 95 and 94%, respectively. C. holmii was used for the reduction of EOPB in a pad-packed interface bioreactor (inner volume, 3 liter). After incubation for 4 days, 4.4 g of (R)-EHPB was obtained via extraction with methanol followed by column chromatography. The overall yield, chemical purity, and enantiomeric excess of (R)-EHPB were 58%, 99.1%, and 90%, respectively.     

Efficient CO2-Reducing Activity of NAD-Dependent Formate Dehydrogenase from Thiobacillus sp. KNK65MA for Formate Production from CO2 Gas

NAD-dependent formate dehydrogenase (FDH) from Candida boidinii (CbFDH) has been widely used in various CO₂-reduction systems but its practical applications are often impeded due to low CO₂-reducing activity. In this study, we demonstrated superior CO₂-reducing properties of FDH from Thiobacillus sp. KNK65MA (TsFDH) for production of formate from CO₂ gas. To discover more efficient CO₂-reducing FDHs than a reference enzyme, i.e. CbFDH, five FDHs were selected with biochemical properties and then, their CO₂-reducing activities were evaluated. All FDHs including CbFDH showed better CO₂-reducing activities at acidic pHs than at neutral pHs and four FDHs were more active than CbFDH in the CO₂ reduction reaction. In particular, the FDH from Thiobacillus sp. KNK65MA (TsFDH) exhibited the highest CO₂-reducing activity and had a dramatic preference for the reduction reaction, i.e., a 84.2-fold higher ratio of CO₂ reduction to formate oxidation in catalytic efficiency (k _cat/K _B) compared to CbFDH. Formate was produced from CO₂ gas using TsFDH and CbFDH, and TsFDH showed a 5.8-fold higher formate production rate than CbFDH. A sequence and structural comparison showed that FDHs with relatively high CO₂-reducing activities had elongated N- and C-terminal loops. The experimental results demonstrate that TsFDH can be an alternative to CbFDH as a biocatalyst in CO₂ reduction systems.     

Stereospecific Reduction of 2-Oxo-4-phenylbutanoate to (R)-2-Hydroxy-4-phenylbutanoate with Microbial Cells

A method for analyzing the EGCg concentration in human serum was developed by using high-performance liquid chromatography with electrochemical detection. EGCg was detected in human serum after the ingestion of 5 g of green tea powder (matsu-cha) dissolved in 200 ml of hot water. The concentration of EGCg in the serum reached the highest level about 2 h after ingesting the green tea, and then decreased.     

羟基蛋氨酸及其钙盐的合成研究进展

羟基蛋氨酸钙是复方α-酮酸片中一个重要成分,可防止氨基酸缺乏和改善代谢紊乱,对肾功能衰竭具有一定的疗效,并对钙代谢和继发性甲状旁腺功能亢进都具有益作用,同时也是重要的有机合成、药物合成及生物合成中间体.本文对近年来羟基蛋氨酸及其钙盐的合成方法进行了综述.重点介绍了氰醇水解法、蛋氨酸转化法、酮酸转化法、酮醇氧化法、氰代磷酸二乙酯法和α-羟基-γ-丁内酯法等在羟基蛋氨酸及其钙盐合成中的应用.     

Identification and Characterization of a Mycobacterial (2R,3R)-2,3-Butanediol Dehydrogenase

Bacterial strain B-009, capable of using racemic 1,2-propanediol (PD), was identified as a rapid-growing member of the genus Mycobacterium. The strain is phylogenetically related to M. gilvum, but has slightly different physiological characteristics. An NAD⁺-dependent enantioselective alcohol dehydrogenase, which acts on R-PD, was purified from the strain. The enzyme was a homodimer of a peptide coded by a 1047-bp gene (mbd1). A highly conserved sequence for medium-chain dehydrogenase/reductases with a preference for secondary alcohols was found in the gene. Hydroxyacetone was produced from R-PD by an enzymatic reaction, indicating that position 2 of the substrate was oxidized. The enzyme activity was highest for (2R,3R)-2,3-butanediol (R,R-BD), enabling the enzyme to be identified as (2R,3R)-2,3-butanediol dehydrogenase (R,R-BD-DH). A homology search revealed M. gilvum, M. vanbaalenii, and M. semegmatis to have ORFs similar to mbd1, suggesting the widespread distribution of genes encoding R,R-BD-DH among mycobacterial strains.     

Light Driven CO2 Fixation by Using Cyanobacterial Photosystem I and NADPH-Dependent Formate Dehydrogenase

The ultimate goal of this research is to construct a new direct CO₂ fixation system using photosystems in living algae. Here, we report light-driven formate production from CO₂ by using cyanobacterial photosystem I (PS I). Formate, a chemical hydrogen carrier and important industrial material, can be produced from CO₂ by using the reducing power and the catalytic function of formate dehydrogenase (FDH). We created a bacterial FDH mutant that experimentally switched the cofactor specificity from NADH to NADPH, and combined it with an in vitro-reconstituted cyanobacterial light-driven NADPH production system consisting of PS I, ferredoxin (Fd), and ferredoxin-NADP⁺-reductase (FNR). Consequently, light-dependent formate production under a CO₂ atmosphere was successfully achieved. In addition, we introduced the NADPH-dependent FDH mutant into heterocysts of the cyanobacterium Anabaena sp. PCC 7120 and demonstrated an increased formate concentration in the cells. These results provide a new possibility for photo-biological CO₂ fixation.     

Enzymatic Preparation of (R)-3-Hydroxy-2-Methylpropyl Butyrate by Asymmetric Hydrolysis

(R)-3-Hydroxy-2-methylpropyl butyrate was formed by asymmetric hydrolysis of the corresponding prochiral diester with lipase P (Amano) in high enantiomeric excess. Various physical and chemical reaction parameters were altered in order to optimize the stereoselectivity of the enzymatic reaction; low temperature (0d`C) combined with the application of salting-in salts or (polyhydric) alcohols turned out to be the most suitable systems providing the monobutyrate in 96% ee. Attempts towards chiral monobutyrate by enzymatic esterification of the corresponding prochiral diol were unsuccessful.     

CO(2) Incorporation and 4-Hydroxy-2-Methylbenzoic Acid Formation during Anaerobic Metabolism of m-Cresol by a Methanogenic Consortium

The metabolism of m-cresol by methanogenic cultures enriched from domestic sewage sludge was investigated. In the initial studies, bromoethanesulfonic acid was used to inhibit methane production. This led to the accumulation of 4.0 +/- 0.8 mol of acetate per mol of m-cresol metabolized. These results suggested that CO(2) incorporation occurred because each molecule of m-cresol contained seven carbon atoms, whereas four molecules of acetate product contained a total of eight carbon atoms. To verify this, [C]bicarbonate was added to bromoethanesulfonic acid-inhibited cultures, and those cultures yielded [C]acetate. Of the label recovered as acetate, 89% was found in the carboxyl position. Similar cultures fed [methyl-C]m-cresol yielded methyl-labeled acetate. A C-labeled transient intermediate was detected in cultures given either m-cresol and [C]bicarbonate or bicarbonate and [methyl-C]m-cresol. The intermediate was identified as 4-hydroxy-2-methylbenzoic acid. In addition, another metabolite was detected and identified as 2-methylbenzoic acid. This compound appeared to be produced only sporadically, and it accumulated in the medium, suggesting that the dehydroxylation of 4-hydroxy-2-methylbenzoic acid led to an apparent dead-end product.     

Chemical Chaperones Curcumin and 4-Phenylbutyric Acid Improve Secretion of Mutant Factor H R127H by Fibroblasts from a Factor H-Deficient Patient

Factor H (FH) is one of the most important regulatory proteins of the alternative pathway of the complement system. Patients with FH deficiency have a higher risk for development of infections and kidney diseases because of the uncontrolled activation and subsequent depletion of the central regulatory component C3 of the complement system. In this study, we investigated the consequences of the Arg(127)His mutation in FH (FH(R127H)) previously described in an FH-deficient patient, on the secretion of this protein by skin fibroblasts in vitro. We observed that, although the patient cells stimulated with IFN-γ were able to synthesize FH(R127H), the mutant protein was largely retained within the endoplasmic reticulum (ER), whereas normal human fibroblasts stimulated with IFN-γ secrete FH without retention in the ER. Moreover, the retention of FH(R127H) provoked enlargement of ER cisterns after treatment with IFN-γ. A similar ER retention was observed in Cos-7 cells expressing the mutant FH(R127H) protein. Despite this deficiency in secretion, we show that the FH(R127H) mutant is capable of functioning as a cofactor in the Factor I-mediated cleavage of C3. We then evaluated whether a treatment could increase the secretion of FH, and observed that the patient's fibroblasts treated with the chemical chaperones 4-phenylbutiric acid or curcumin increased the secretion rate of FH. We propose that these chemical chaperones could be used as alternative therapeutic agents to increase FH plasma levels in FH-deficient patients caused by secretion delay of this regulatory protein.     

Selective inhibition of trypsin by (2R,4R)-4-phenyl-1-[Nalpha-(7- methoxy-2-naphthalenesulfonyl)-L-arginyl]-2-piperidinecarboxylic acid

Evidence is accumulating indicating that trypsin stimulates divergent cellular reactions through the proteinase-activated receptor, in addition to its role as the digestive enzyme. In this report, we introduce (2R,4R)- 4-phenyl-1-[N(alpha)-(7-methoxy-2-naphthalenesulfonyl)-l-arginyl]- 2-p iperidinecarboxylic acid as a potent and selective trypsin inhibitor. The agent inhibited trypsin competitively with the K(i) value of 0. 1 micrometer. It inhibited thrombin weakly (K(i) = 2 micrometer) and did not inhibit plasmin, plasma kallikrein, urokinase, and mast cell tryptase (K(i) values for these enzymes are >60 micrometer). Comparative studies with several established proteinase inhibitors revealed that the compound was the first small molecular weight trypsin inhibitor without tryptase inhibitory activity. A docking study has provided a plausible explanation for the molecular mechanism of the selective inhibition showing that the agent fits into the active site of trypsin without any severe collision but that it comes into clash at the 4-phenyl group of piperidine ring against the "60-insertion loop" of thrombin and at the 7-methoxy-2-naphthalenesulfonyl group against Gln(98) of tryptase.     

Production of 10-Hydroxy-8(E)-Octadecenoic Acid from Oleic Acid Conversion by Strains of Pseudomonas aeruginosa

Eighteen Pseudomonas aeruginosa strains were examined for their ability to convert oleic acid to produce 10-hydroxy-8(E)-octadecenoic acid (HOD), which was structurally confirmed by GC-MS, NMR, and FTIR. There were no substantial amounts of other new compounds found in the fermentation broths in addition to HOD and 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). The results demonstrated that P. aeruginosa strains possessed varying levels of activity for producing HOD. Under the experimental conditions, strain NRRL B-14938 isolated from sheep manure was the best HOD producer exhibiting the highest HOD to DOD product ratio in the medium most suitable for purifying HOD. Using strain B-14938 as a model system for further characterization, optimum conditions for producing HOD were found to be at 26°C and pH 7.0 after 60 h of reaction time using a medium containing EDTA as a chelating agent. This study has identified a high-yielding P. aeruginosa strain and provided the reaction characteristics needed to develop a scale-up production process of HOD for testing its properties and potential new uses.     

Characterisation of (R)-2-(2-Fluorobiphenyl-4-yl)-N-(3-Methylpyridin-2-yl)Propanamide as a Dual Fatty Acid Amide Hydrolase: Cyclooxygenase Inhibitor

Background

Increased endocannabinoid tonus by dual-action fatty acid amide hydrolase (FAAH) and substrate selective cyclooxygenase (COX-2) inhibitors is a promising approach for pain-relief. One such compound with this profile is 2-(2-fluorobiphenyl-4-yl)-N-(3-methylpyridin-2-yl)propanamide (Flu-AM1). These activities are shown by Flu-AM1 racemate, but it is not known whether its two single enantiomers behave differently, as is the case towards COX-2 for the parent flurbiprofen enantiomers. Further, the effects of the compound upon COX-2-derived lipids in intact cells are not known.

Methodology/Principal Findings

COX inhibition was determined using an oxygraphic method with arachidonic acid and 2-arachidonoylglycerol (2-AG) as substrates. FAAH was assayed in mouse brain homogenates using anandamide (AEA) as substrate. Lipidomic analysis was conducted in unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Both enantiomers inhibited COX-2 in a substrate-selective and time-dependent manner, with IC₅₀ values in the absence of a preincubation phase of: (R)-Flu-AM1, COX-1 (arachidonic acid) 6 μM; COX-2 (arachidonic acid) 20 μM; COX-2 (2-AG) 1 μM; (S)-Flu-AM1, COX-1 (arachidonic acid) 3 μM; COX-2 (arachidonic acid) 10 μM; COX-2 (2-AG) 0.7 μM. The compounds showed no enantiomeric selectivity in their FAAH inhibitory properties. (R)-Flu-AM1 (10 μM) greatly inhibited the production of prostaglandin D₂ and E₂ in both unstimulated and lipopolysaccharide + interferon γ- stimulated RAW 264.7 macrophage cells. Levels of 2-AG were not affected either by (R)-Flu-AM1 or by 10 μM flurbiprofen, either alone or in combination with the FAAH inhibitor URB597 (1 μM).

Conclusions/Significance

Both enantiomers of Flu-AM1 are more potent inhibitors of 2-AG compared to arachidonic acid oxygenation by COX-2. Inhibition of COX in lipopolysaccharide + interferon γ- stimulated RAW 264.7 cells is insufficient to affect 2-AG levels despite the large induction of COX-2 produced by this treatment.     

Determination of Site-Specific Modifications of Glucose-6-Phosphate Dehydrogenase by 4-Hydroxy-2-Nonenal Using Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry

Products of the reaction of 4-hydroxy-2-nonenal (4HNE) with native and heat-denatured Leuconostoc mesenteroides glucose-6-phosphate dehydrogenase (G6PDH) were analyzed to determine the structure and position of the protein modifications. Matrix assisted laser desorption time-of-flight mass spectrometry was used to measure molecular weights of the modified proteins and determine mass maps of peptides formed by digestion with cyanogen bromide. The molecular weight data show that one to two 4HNE molecules add to each subunit of native enzyme while approximately nineteen 4HNE molecules add to each subunit of heat-denatured enzyme. Peptides are observed in the cyanogen bromide mass map of modified native G6PDH that are consistent with selective modification of two segments of the amino acid sequence. One modified segment contains Lysine-182 that has been found to be part of the enzyme active site. Peptides are observed in the cyanogen bromide mass map of modified heat-denatured enzyme that are consistent with extensive modification of several segments of the amino acid sequence. The magnitude of the mass differences between modified and unmodified peptides were approximately 156 Da, consistent with a 1, 4-addition of 4HNE. These results support the conclusion that 4HNE inactivates G6PDH by selectively modifying only two or three sites in the protein by a 1, 4-addition reaction and that some aspect of the tertiary structure of the enzyme directs those modification reactions.     

Protein Engineering of a Nitrilase from Burkholderia cenocepacia J2315 for Efficient and Enantioselective Production of (R)-o-Chloromandelic Acid

The nitrilase-mediated pathway has significant advantages in the production of optically pure aromatic α-hydroxy carboxylic acids. However, low enantioselectivity and activity are observed on hydrolyzing o-chloromandelonitrile to produce optically pure (R)-o-chloromandelic acid. In the present study, a protein engineering approach was successfully used to enhance the performance of nitrilase obtained from Burkholderia cenocepacia strain J2315 (BCJ2315) in hydrolyzing o-chloromandelonitrile. Four hot spots (T49, I113, Y199, and T310) responsible for the enantioselectivity and activity of BCJ2315 were identified by random mutagenesis. An effective double mutant (I113M/Y199G [encoding the replacement of I with M at position 113 and Y with G at position 199]), which demonstrated remarkably enhanced enantioselectivity (99.1% enantiomeric excess [ee] compared to 89.2% ee for the wild type) and relative activity (360% of the wild type), was created by two rounds of site saturation mutagenesis, first at each of the four hot spots and subsequently at position 199 for combination with the selected beneficial mutation I113M. Notably, this mutant also demonstrated dramatically enhanced enantioselectivity and activity toward other mandelonitrile derivatives and, thus, broadened the substrate scope of this nitrilase. Using an ethyl acetate-water (1:9) biphasic system, o-chloromandelonitrile (500 mM) was completely hydrolyzed in 3 h by this mutant with a small amount of biocatalyst (10 g/liter wet cells), resulting in a high concentration of (R)-o-chloromandelic acid with 98.7% ee, to our knowledge the highest ever reported. This result highlights a promising method for industrial production of optically pure (R)-o-chloromandelic acid. Insight into the source of enantioselectivity and activity was gained by homology modeling and molecular docking experiments.     

Production of (R)-2-(4-hydroxyphenoxy) propionic acid by Beauveria bassiana ZJB16007 in solid state fermentation using rice bran

Abstract

R-2-(4-hydroxyphenoxy)propionic acid (R-HPPA) is a key chiral intermediate for phenoxypropionic acid herbicide synthesis. In this study, to improve the production of R-HPPA with B. bassiana ZJB16007, the cultivation conditions in solid-state fermentation (SSF) were investigated. The effects of various substrates on R-HPPA production were evaluated and the process parameters were also optimized. The results showed that rice bran was the optimal substrate for R-HPPA production. The optimal medium components and cultivation conditions were: rice bran: silkworm chrysalis powder = 5.25: 2.25 (g: g), nutrient salts solution 12?mL which contained 50?g/L R-PPA, pH 5.0, and cultivated at 28?°C for 11 days. Under the optimized conditions, the transformation of R-HPPA was significantly improved and the yield of R-HPPA reached 77.78%, which was 15.14% higher than that of the control (67.55%). Therefore, SSF may serve as an alternative for R-HPPA production by B. bassiana ZJB16007.     

Production of a doubly chiral compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone,by two-step enzymatic asymmetric reduction

A practical enzymatic synthesis of a doubly chiral key compound, (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone, starting from the readily available 2,6,6-trimethyl-2-cyclohexen-1,4-dione is described. Chirality is first introduced at the C-6 position by a stereoselective enzymatic hydrogenation of the double bond using old yellow enzyme 2 of Saccharomyces cerevisiae, expressed in Escherichia coli, as a biocatalyst. Thereafter, the carbonyl group at the C-4 position is reduced selectively and stereospecifically by levodione reductase of Corynebacterium aquaticum M-13, expressed in E. coli, to the corresponding alcohol. Commercially available glucose dehydrogenase was also used for cofactor regeneration in both steps. Using this two-step enzymatic asymmetric reduction system, 9.5 mg of (4R,6R)-4-hydroxy-2,2,6-trimethylcyclohexanone/ml was produced almost stoichiometrically, with 94% enantiomeric excess in the presence of glucose, NAD(+), and glucose dehydrogenase. To our knowledge, this is the first report of the application of S. cerevisiae old yellow enzyme for the production of a useful compound.