Sequential two-step conversion of 4-oxoisophorone to 4-hydroxy-2,2,6-trimethylcyclohexanone by thermophilic bacteria

Summary The sequential two-step conversion of 4-oxoisophorone (OIP) to 4-hydroxy-2,2,6-trimethylcyclohexanone (4-HTMCH) via diyhdrooxoisophorone (DOIP) was achieved using two kinds of thermophiles, Thermomonospora curvata and Bacillus stearothermophilus. In the first step, 83% OIP was converted to DOIP by T. curvata during 12 h incubation at 50° C. The resulting reaction mixture containing thermophile cells, DOIP (2.5 mg/ml), and OIP (0.5 mg/ml) was used directly in the second step after adjusting to pH 7 and adding glycerol. In the second step, DOIP in the reaction mixture was converted to 4-HTMCH by B. stearothermophilus. The final concentration of 4-HTMCH and DOIP after 24 h of incubation was 2.5 mg/ml and 0.5 mg/ml respectively; OIP was not detected. The total conversion yield of 4-HTMCH from OIP was 83% through the two-step conversion. The two-step conversion by a sequential culture system using T. curvata and B. stearothermophilus was found to be suitable for 4-HTMCH production.Offprint requests to: I. Karube     

Synthesis and polymerization of benzyl (3R,4R)-3-methylmalolactonate via enzymatic preparation of the chiral precursor

β-methylaspartate ammonia-lyase, EC 4.3.1.2, (β-methylaspartase) from Clostridium tetanomorphum was used to produce a 40/60 molar ratio of (2S,3R) and (2S,3S)-3-methylaspartic acids, 2a and 2b , respectively, from mesaconic acid 1 as substrate, on a large scale. To prepare (3R,4R)-3-methyl-4-(benzyloxycarbonyl)-2-oxetanone (benzyl 3-methylmalolactonate) 6, 2a and 2b were transformed, in the first step, into 2-bromo-3-methylsuccinic acids 3a and 3b and separated. After three further steps, (2S,3S)- 3a yielded the α,β-substituted β-lactone (3R,4R) 6 with a very high diastereoisomeric excess (>95% by chiral gas chromatography). The corresponding crystalline polymer, poly[benzyl β-(2R,3S)-3-methylmalate] 8 , prepared by an anionic ring opening polymerization, was highly isotactic as determined by ¹³C NMR. Catalytic hydrogenolysis of lactone 6 yielded (3R,4R)-3-methyl-4-carboxy-2-oxetanone (3-methylmalolactonic acid) 7 , to which reactive, chiral, or bioactive molecules can be attached through ester bonds leading to polymers with possible therapeutic applications. Because of the ability of β-methylaspartase to catalyse both syn- and anti-elimination of ammonia from (2S,3RS)-3-methylaspartic acid 2ab at different rates, the (2S,3R)-stereoisomer 2a was retained and isolated for further reactions. These results permit the use of the chemoenzymatic route for the preparation of both optically active and racemic polymers of 3-methylmalic acid with well-defined enantiomeric and diastereoisomeric compositions. Chirality 10:727–733, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis of (4R,6S,7R)-7-hydroxy-4,6-dimethyl-3-nonanone and (3R,5S,6R)- 6-hydroxy-3,5-dimethyl-2-octanone,the pheromone components of the bostrychid beetle,Dinoderus bifoveolatus

(4R,6S,7R)-7-Hydroxy-4,6-dimethyl-3-nonanone and (3R,5S,6R)-6-hydroxy-3,5-dimethyl-2-octanone, the pheromone components of the bostrychid beetle, Dinoderus bifoveolatus, as well as their (4R,6S,7S)- and (3R,5S,6S)-isomers were synthesized from (2R,4S,5R)- and (2R,4S,5S)-2,4-dimethyl-5-heptanolide, respectively.     

Continuous two step bioconversion of 4-oxoiso-phorone (OIP) to 4-hydroxy-2,2,6-trimethylcyclo-hexanone (4-HTMCH) by thermophilic bacteria

Summary The continuous two step conversion of 4-oxoisophorone(OIP) to 4-hydroxy-2,2,6-trimethylcyclohexanone(4-HTMCH) via dihydrooxoisophorone(DOIP) was carried out using two types of thermophilic growing cells, Thermomonospora curvata and Bacillus stearothermophilus, in sequential connected continuous stirred tank reactor(CSTR) and hollow fiber reactor system. For more than 80 hours operation, 46% of OIP was converted to 4-HTMCH with productivity of 179 mg/h/l.     

Synthesis of (-)-methyl shikimate via enzymatic resolution of (1S*, 4R*, 5R*)-4-hydroxy-6-oxabicyclo[3.2.1]oct-2-en-7-one.

The synthesis of methyl (-)-shikimate [(-)-2] was achieved via lipase-catalyzed optical resolution of (1S*, 4R*, 5R*)-4-hydroxy-6-oxabicyclo[3.2.1]oct-2-en-7-one (3). Transesterification of (+/-)-3 and vinyl acetate with lipase MY and subsequent hydrolysis gave optically pure (-)-3. This compound was converted to (-)-2 in two steps.     

Production of chiral alcohols from prochiral ketones by microalgal photo-biocatalytic asymmetric reduction reaction

Microalgal photo-biocatalysis is a green technique for asymmetric synthesis. Asymmetric reduction of nonnatural prochiral ketones to produce chiral alcohols by microalgal photo-biocatalysis was studied in this work. Acetophenone (ACP) and ethyl acetoacetate (EAA) were chosen as model substrates for aromatic ketones and β-ketoesters, respectively. Two prokaryotic cyanophyta and two eukaryotic chlorophyta were selected as photo-biocatalysts. The results proved that nonnatural prochiral ketones can be reduced by microalgal photo-biocatalysis with high enantioselectivity. Illumination is indispensable to the photo-biocatalysis. For aromatic ketone, cyanophyta are eligible biocatalysts. For ACP asymmetric reduction reaction, about 45% yield and 97% e.e. can be achieved by the photo-biocatalysis reaction with Spirulina platensis as biocatalyst. On the contrary, chlorophyta are efficient biocatalysts for β-ketoester asymmetric reduction reaction among the four tested algae. For EAA asymmetric reduction reaction, about 70% yield and 90% e.e. can be achieved with Scenedesmus obliquus as biocatalyst. The microalgae used in this study outperformed other characterized biocatalysts such as microbial and plant cells.     

酵母不对称催化制备R-1,2-丙二醇

研究了采用面包酵母还原丙酮醇制备R-1,2-丙二醇的工艺。采用摇瓶对转化条件进行单因素实验,确定最优转化条件：丙酮醇浓度0.3mmol/mL,pH7.0,酵母质量浓度150g/L,乙醇浓度为0.3mmol/mL,转化时间25h。在此条件下,采用分批流加策略进行1.5L规模发酵罐转化试验,转化25h后,发酵液的R-1,2-丙二醇浓度为0.27mmol/mL。     

A two-step enzymatic resolution process for large-scale production of (S)- and (R)-ethyl-3-hydroxybutyrate

An efficient two-step enzymatic process for production of (R)- and (S)-ethyl-3-hydroxybutyrate (HEB), two important chiral intermediates for the pharmaceutical market, was developed and scaled-up to a multikilogram scale. Both enantiomers were obtained at 99% chemical purity and over 96% enantiomeric excess, with a total process yield of 73%. The first reaction involved a solvent-free acetylation of racemic HEB with vinylacetate for the production of (S)-HEB. In the second reaction, (R)-enriched ethyl-3-acetoxybutyrate (AEB) was subjected to alcoholysis with ethanol to derive optically pure (R)-HEB. Immobilized Candida antarctica lipase B (CALB) was employed in both stages, with high productivity and selectivity. The type of butyric acid ester influenced the enantioselectivity of the enzyme. Thus, extending the ester alkyl chain from ethyl to octyl resulted in a decrease in enantiomeric excess, whereas using bulky groups such as benzyl or t-butyl, improved the enantioselectivity of the enzyme. A stirred reactor was found unsuitable for large-scale production due to attrition of the enzyme particles and, therefore, a batchwise loop reactor system was used for bench-scale production. The immobilized enzyme was confined to a column and the reactants were circulated through the enzyme bed until the targeted conversion was reached. The desired products were separated from the reaction mixture in each of the two stages by fractional distillation. The main features of the process are the exclusion of solvent (thus ensuring high process throughput), and the use of the same enzyme for both the acetylation and the alcoholysis steps. Kilogram quantities of (S)-HEB and (R)-HEB were effectively prepared using this unit, which can be easily scaled-up to produce industrial quantities.     

An efficient enzymatic preparation of 4(S)-hydroxy-1 (R)-acetoxy-cyclopent-2-ene by using new yeast isolate

Summary The Enzymatic enantioselective hydrolysis of prochiral 1,4-cyclopent-2-ene diacetate (3) was carried out using yeast and fungal cultures from inhouse culture collection. Of all the cultures tested, the yeast sp. NCIM 3574 gave 4 (S)-hydroxy-1 (R)- acetoxy-cyclopent-2-ene (4b) in high optical yields (99% ee).     

Modulation of the immune response to tumors by a novel synthetic compound, (4R)-3-benzoyl-N-[(1R)-1-phenylethyl]-4-thiazolidinecarboxamide (RS-0481)

Summary RS-0481, (4R)-3-benzoyl-N-[(1R)-phenylethyl]-4-thiazolidinecarboxamide, is a compound that can re-establish the function of certain lymphoid cell populations impaired by the presence of a growing tumor in an animal. The compound markedly augmented the tumorspecific cytotoxic T lymphocytes,Tdth (delayed-type hypersensitivity T cells), and the nonspecific lymphokine-activated-killer-cell-like cell responses. It also enhanced the tumor-inhibitory effect of macrophages in tumor-bearing mice, but not in normal mice, indicating that it enhances the antitumor immune responses. Lymphocytes from RS-0481-treated tumor-bearing mice released significantly higher amounts of macrophage-activating factor(s) (MAF) and interleukin-2(IL-2)-like factors in culture compared with lymphocytes from untreated animals. Also, sera from treated tumor bearers showed elevated colony-stimulating factor (CSF) activity. Although the compound did not influence the factor-producing activity in mice without tumor, it enhanced the responsiveness of their bone marrow cells, T cells, and macrophages to CSF, IL-2, and MAF. It seems therefore possible that the compound enhances the responsiveness of immunocompetent cells to cytokines, resulting in a marked augmentation of antitumor T cell responses in tumor-bearing mice. Consistently it inhibited the development of lymph node metastasis of transplanted X5563 plasmacytoma, and we showed that T cells play a decisive role in this inhibition. The compound also counteracted the development of suppressor T cell activity in the spleen of tumor-bearing mice.     

Novel potato micro-tuber-inducing compound, (3R,6S)-6-hydroxylasiodiplodin, from a strain of Lasiodiplodia theobromae

A novel potato micro-tuber-inducing compound was isolated from the culture broth of Lasiodiplodia theobromae Shimokita 2. The structure of the isolated compound was determined as (3R,6S)-6-hydroxylasiodiplodin by means of spectroscopic analyses, the modified Mosher method, and chemical conversion. The compound showed potato micro-tuber-inducing activity at a concentration of 10(-4) M, using the culture of single-node segments of potato stems in vitro.     

Enantioselective metabolism of (R)- and (S)-4-hydroxy-2-nonenal in rat

4-Hydroxy-2-nonenal (HNE) is an endogenous product of lipid peroxidation, which is believed to play a biological role in the pathogenesis of various diseases. HNE is formed as a racemic mixture of (R)- and (S)- enantiomers. These enantiomers differ in their biological properties. The aim of this study was to investigate separately the in vivo metabolism of the two HNE enantiomers in male rats after intravenous administration of the corresponding radiolabeled compounds and to compare the results with those obtained with the racemic mixture. Although the difference in the excretion rates was not statistically significant, the HPLC profiles of urinary metabolites showed qualitative and quantitative differences between the two enantiomers. The level of 3-mercapturic acid-1,4-dihydroxynonane, which is considered as the major urinary metabolite of HNE, was significantly lower in the case of (S)-HNE injected rats. In vitro studies using rat liver cytosolic incubations and HNE-glutathione conjugate as substrate were performed to clarify the intermediate pathways involved in their metabolism. Large differences were obtained in the reduction and retro-Michael conversion steps of the metabolism between the conjugates originating from the two enantiomers.     

A novel enantioselective synthesis of (S)-(-)- and (R)-(+)-nornicotine via alkylation of a chiral 2-hydroxy-3-pinanone ketimine template

An asymmetric synthesis of the optically pure isomers of the minor tobacco alkaloid and CNS nicotine metabolite, nornicotine, has been achieved with moderately high optical purity. The synthetic pathway involves alkylation of a chiral ketimine, prepared from either 1R,2R,5R-(+)- or 1S,2S,5S-(-)-2-hydroxy-3-pinanone and 3-(aminomethyl)pyridine with 3-bromopropan-1-ol. After cleavage of the respective C-alkylated ketimines with NH2OH.HCl, and treatment of the resulting amino alcohols with HBr, followed by base-catalyzed intramolecular ring closure, (S)-(-)-nornicotine and (R)-(+)-nornicotine were obtained with ee values of 91% and 81%, respectively.     

A novel synthetic compound, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (MHY773) inhibits mushroom tyrosinase

As part of continued efforts for the development of new tyrosinase inhibitors, (Z)-5-(substituted benzylidene)-2-iminothiazolidin-4-one derivatives (1a – 1l) were rationally synthesized and evaluated for their inhibitory potential in vitro. These compounds were designed and synthesized based on the structural attributes of a β-phenyl-α,β-unsaturated carbonyl scaffold template. Among these compounds, (Z)-5-(3-hydroxy-4-methoxybenzylidene)-2-iminothiazolidin-4-one (1e, MHY773) exhibited the greatest tyrosinase inhibition (IC₅₀ = 2.87 μM and 8.06 μM for monophenolase and diphenolase), and outperformed the positive control, kojic acid (IC₅₀ = 15.59 and 31.61 μM). The kinetic and docking studies demonstrated that MHY773 interacted with active site of tyrosinase. Moreover, a melanin quantification assay demonstrated that MHY773 attenuates α-melanocyte-stimulating hormone (α-MSH) and 3-isobutyl-1-methylxanthine (IBMX)-induced melanin contents in B16F10 melanoma cells. Taken together, these data suggest that MHY773 suppressed the melanin production via the inhibition of tyrosinase activity. MHY773 is a promising for the development of effective pharmacological and cosmetic agents for skin-whitening.     

Production of a chiral alcohol, 1-(3,4-dihydroxyphenyl) ethanol, by mushroom tyrosinase

1-(3,4-Dihydroxyphenyl) ethanol was produced biocatalytically for the first time using mushroom tyrosinase. 4-Ethylphenol at 1 mM was consumed over 12 min giving 0.23 mM 4-ethylcatechol and 0.36 mM (R/S)-1-(3,4-dihydroxyphenyl) ethanol (ee 0.5 %). Mushroom tyrosinase consumed 4-ethylphenol at 6.7 μmol min^?1 mg protein^?1 while the rates of formation of 4-ethylcatechol and 1-(3,4-dihydroxyphenyl) ethanol were 1.1 and 1.9 μmol min^?1 mg protein^?1. Addition of the ascorbic acid, as a reducing agent to biotransformation reactions, increased 4-ethylcatechol formation by 340 %. However, accumulation of 1-(3,4-dihydroxyphenyl) ethanol was not observed in the presence of ascorbic acid. While the 1-(3,4-dihydroxyphenyl) ethanol was racemic, it is the first chiral product produced by tyrosinase starting from a non-chiral substrate.     

Asymmetric reduction of 4-hydroxy-2-butanone to (R)-1,3-butanediol with absolute stereochemical selectivity by a newly isolated strain of Pichia jadinii

In this study, a novel strain of Pichia jadinii, HBY61, capable of the biocatalysis of 4-hydroxy-2-butanone (4H2B) to (R)-1,3-BD was isolated. HBY61 produced (R)-1,3-BD with high activity and absolute stereochemical selectivity (100 % e.e). Glucose and beef extract were found to be the key factors governing the fermentation, and their optimal concentrations were determined to be 84.2 and 43.7 g/L, respectively. The optimal bioconversion conditions of 4H2B catalyzed by HBY61 were pH 7.4, 30 °C, and 250 rpm with 6 % (v/v) glucose as the co-substrate. Accordingly, when 45 g/L of 4H2B was divided into three equal parts and added successively into the system at set time intervals, the maximum (R)-1,3-BD concentration reached 38.3 g/L with high yield (85.1 %) and strict 100 % enantioselectivity. Compared with previously reported yields for the biocatalytic production of (R)-1,3-BD, the use of strain HBY61 provided a high yield with excellent stereoselectivity.     

Synthesis of methyl (R)-3-(4-fluorophenyl)glutarate via enzymatic desymmetrization of a prochiral diester

An efficient procedure for enzymatic desymmetrization of the prochiral dimethyl 3-(4-fluorophenyl)glutarate (3-DFG) in an aqueous–organic phase was successfully developed to prepare methyl (R)-3-(4-fluorophenyl)glutarate ((R)-3-MFG). Novozym 435 was selected as a highly efficient biocatalyst through lipase screening. The effects of various parameters in terms of co-solvent and its concentration, buffer pH, ionic strength and reaction temperature, on the reaction were investigated. It was found that 0.2 M phosphate buffer (pH 8.0) containing 20% MTBE (v/v) was the optimum reaction medium, and the optimum reaction temperature was 30 °C. Under the optimized reaction conditions, (R)-3-MFG was obtained in 95.6% ee value and 92.6% yield after 64 h when the concentration of 3-DFG and Novozym 435 were 200 mmol/l and 20 g/l respectively. Furthermore, Novozym 435 showed an excellent operational stability, retaining above 95% of the initial activity and enantioselectivity after 10 cycles of reaction. The developed method has a potential to be used for efficient enzymatic production of (R)-3-MFG.     

Efficient synthesis of (R)-3-hydroxypentanenitrile in high enantiomeric excess by enzymatic reduction of 3-oxopentanenitrile

(R)-3-Hydroxypentanenitrile (HPN) is an important intermediate in the synthesis of an immunosuppressive inosine 5′-monophosphate dehydrogenase inhibitor. An efficient enzymatic procedure for the synthesis of (R)-HPN with over 99 % enantiomeric excess using a novel acetoacetyl-CoA reductase (AdKR) from Achromobacter denitrificans was successfully established. Many microorganisms are known to reduce 3-oxopentannitrile (KPN) to (R)-HPN. An enzyme from A. denitrificans partially purified using ion exchange chromatography reduced KPN to (R)-HPN with high enantioselectivity. The AdKR gene was cloned and sequenced and found to comprise 738 bp and encode a polypeptide of 26,399 Da. The deduced amino acid sequence showed a high degree of similarity to those of other putative acetoacetyl-CoA reductases and putative 3-ketoacyl-ACP reductases. The AdKR gene was singly expressed and coexpressed together with a glucose dehydrogenase (GDH) as a coenzyme regenerator in Escherichia coli under the control of the lac promoter. (R)-HPN was synthesized with over 99 % e.e. using a cell-free extract of recombinant E. coli cells coexpressing AdKR and GDH.     

Design and synthesis of BACE1 inhibitors containing a novel norstatine derivative (2R,3R)-3-amino-2-hydroxy-4-(phenylthio)butyric acid

A novel norstatine derivative, phenylthionorstatine [(2R,3R)-3-amino-2-hydroxy-4-(phenylthio)butyric acid; Ptns], containing a hydroxymethylcarbonyl (HMC) isostere was designed, synthesized, and stereochemically determined. Then, Ptns was introduced into the structure of BACE1 inhibitors at the P(1) position. Finally, Ptns was found as a suitable P(1) moiety for potent BACE1 inhibitor design.     

Analogues of 2-crotonyloxymethyl-(4R,5R,6R)-4,5,6-trihydroxycyclohex-2-enone (COTC) with anti-tumor properties

The syntheses of three novel analogues of the naturally occurring cytotoxic agent COTC are described and the results of bioassays of the target compounds against two lung cancer cell lines are presented.