Synthesis of l-muscone by asymmetric methylation via enol esters

An effective synthetic route of l-muscone (1) by asymmetric methylation, followed by enolate-trapping to generate enol esters as intermediates, was described. Interestingly, the enol esters can be used as substrates for enzymatic optical resolution to improve optical purity. Additionally, several excellent new chiral ligands were discovered for asymmetric methylation of (E)-cyclopentadec-2-enone to produce l-muscone with high optical purity.     

Metabolism of Monoterpenes : Early Steps in the Metabolism of d-Neomenthyl-beta-d-Glucoside in Peppermint (Mentha piperita) Rhizomes

Previous studies have shown that the monoterpene ketone l-[G-³H] menthone is reduced to the epimeric alcohols l-menthol and d-neomenthol in leaves of flowering peppermint (Mentha piperita L.), and that a portion of the menthol is converted to menthyl acetate while the bulk of the neomenthol is transformed to neomenthyl-β-d-glucoside which is then transported to the rhizome (Croteau, Martinkus 1979 Plant Physiol 64: 169-175). Analysis of the disposition of l-[G-³H]menthone applied to midstem leaves of intact flowering plants allowed the kinetics of synthesis and transport of the monoterpenyl glucoside to be determined, and gave strong indication that the glucoside was subsequently metabolized in the rhizome. Studies with d-[G-³H]neomenthyl-β-d-glucoside as substrate, using excised rhizomes or rhizome segments, confirmed the hydrolysis of the glucoside as an early step in metabolism at this site, and revealed that the terpenoid moiety was further converted to a series of ether-soluble, methanol-soluble, and water-soluble products. Studies with d-[G-³H]neomenthol as the substrate, using excised rhizomes, showed the subsequent metabolic steps to involve oxidation of the alcohol back to menthone, followed by an unusual lactonization reaction in which oxygen is inserted between the carbonyl carbon and the carbon bearing the isopropyl group, to afford 3,4-menthone lactone. The conversion of menthone to the lactone, and of the lactone to more polar products, were confirmed in vivo using l-[G-³H]menthone and l-[G-³H]-3,4-menthone lactone as substrates. Additional oxidation products were formed in vivo via the desaturation of labeled neomenthol and/or menthone, but none of these transformations appeared to lead to ring opening of the p-menthane skeleton. Each step in the main reaction sequence, from hydrolysis of neomenthyl glucoside to lactonization of menthone, was demonstrated in cell-free extracts from the rhizomes of flowering mint plants. The lactonization step is of particular significance in providing a means of cleaving the p-menthane ring to afford an acyclic carbon skeleton that can be further degraded by modifications of the well-known β-oxidation sequence.     

In vivo and in vitro preparation of divinyl-132,173-cyclopheophorbide-a enol

Divinyl-13²,17³-cyclopheophorbide-a enol was in vivo produced as a metabolite of divinyl-chlorophyll-a by protists and in vitro prepared by the intramolecular cyclization of methyl divinyl-pyropheophorbide-a, one of the divinyl-chlorophyll-a derivatives. The ¹H NMR spectra in CDCl₃ showed that the obtained product took exclusively its enol form in the solution. The intramolecular cyclization of chlorin π-system at the C13² and C17³ positions affected the optical properties of such chlorophyll derivatives including the non-fluorescent emission of the enol.     

Lipase-catalyzed Optical Resolution of 2,2,2-Trifluoro-1-(1-naphthyl)ethanol

The optical resolution of the title compound (TFNE) was achieved by lipase LIP-catalyzed enantioselective acetylation (E value > 100) with vinyl acetate in octane. S-TFNE acetate and R-TFNE were obtained with high optical purity.     

Chemogenetic evidence supporting multiple allele control of the biosynthesis of (−)-menthone and (+)-isomenthone stereoisomers in Mentha species

The essential oils of certain Mentha species and chemotypes have proportions of (?)-menthone and (+)-isomenthone which differ but show a high degree of heritability in clonal propagation. Oil from an F₂ individual (69–296), selected from numerous 4n M. longifolia (4n = 48) × M. crispa (2n = 48) hybrids for high isomenthone content, had 41.3% isomenthone; the associated but seldom observed alcohols, 1.6% isomenthol, 10.3% neoiso-menthol; and 13% of their esters; in contrast to 8% menthone with 0.1% menthol, 5.0% neo-menthol, and 1.7% esters. Self-pollination of strain 69–296 gave a 3:1 ratio of high isomenthone: high menthone. Crosses with a true breeding high menthone plant having 80% menthone and 3.2% isomenthone gave a 1:1 ratio of the parental phenotypes by GLC analyses and herbage odor. This and data from high isomenthone and high menthone crosses with tester strains lead us to postulate the involvement of a single locus having multiple alleles with true breeding menthone having the genotype P^s P^s, true breeding isomenthone P^r P^r, 69–296 P^r P^s, and high pulegone pp. The P^r allele is not completely dominant over the P^s allele in 69–296 as about 18% of the total ketone derived from pulegone is menthone. Both are dominant over the recessive allele p that largely prevents menthone development. The quantitative amounts of the two isomers are believed to be controlled by the six combinations of the three alleles in a diploid species with graded effects obtained in the more complex genotypes possible in double diploid and octoploid species. 69–296 has (?)-piperitone even though (+)-piperitone is believed to be the common isomer in Mentha.     

Enzymic Resolution of (±)-Acyclic Alcohols via Asymmetric Hydrolysis of Corresponding Acetates by Microorganisms

Asymmetric hydrolysis of (±)-1-pentyl-2-propynyl and 1-pentyl-2-propenyl acetates by selected microorganisms produced chiral 1-octyn-3-ol and 1-octen-3-ol, respectively, with high optical purities and acetates of their antipodes. Enantioselectivity of microbial hydrolysis changed with the microorganisms used. Also, (±)-1-ethylhexyl acetate was asymmetrically hydrolyzed by microorganisms to give (S)-3-octanol and (R)-1-ethylhexyl acetate of relatively low optical purity and hydrolytic ratio, compared with those of (±)-1-pentyl-2-propynyl acetate.     

An NMR technique for carbon-5 configurations in 6-keto-delta7-steroids

Enol acetates may be readily prepared from 6-keto-Δ⁷-steroids, The NMR examination of the C-7 proton allows assessment of the allylic coupling to the C-5 proton. If split into a doublet, then the C-5 configuration is α; if a singlet, then C-5 is β. The configuration at C-5 in the initial ketone is preserved in the enol acetate.     

Synthesis of (4-C)-labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one and 3β,15α-dihydroxy-5-androsten-17-one

The synthesis of labeled and non-labeled 3β,15α-dihydroxy-5-pregnen-20-one (V) and 3β, 15α-dihydroxy-5-androsten-17-one (XI) is described. Treatment of 15α-hydroxy-4-pregnene-3,20-dione (I) with acetic anhydride and acetyl chloride gave 3,15α-diacetoxy-3,5-pregnadien-20-one (II). The enol acetate (II) was ketalized by a modification of the general procedure to yield 3,15α-diacetoxy-3,5-pregnadien-20-one cyclic ethylene ketal (III) which was then reduced with NaBH₄ and LiAlH₄ to give 3β, 15α-dihydroxy-5-pregnen-20-one cyclic ethylene ketal (IV). Cleavage of the ketal group of IV gave V. Similarly, XI was prepared by starting with 15α-hydroxy-4-androstene-3,17-dione (VII). The (4-¹⁴C)-3β,15α-dihydroxy-5-pregnen-20-one was prepared by a modification of the above procedure in that the enol acetate (II)was directly reduced with NaBH₄ and LiAlH₄ to yield 5-pregnene-3β,15α,20β-triol (XIII) which was then oxidized enzymatically with 20β-hydroxysteroid dehydrogenase to V.     

Stereoselective hydrolysis of optically active isomeric isopulegyl acetates by rhodotorula mucilaginosa and bacillus sp.

A method for isolation of optically pure l-isopulegol from a mixture of its optically active isomers using the microorganisms Rhodotorula mucilaginosa and Bacillus sp. is described. Microorganisms hydrolyzed l-isopulegyl acetate (26–40%) and in a small degree d-isopulegol acetate whereas the d-neoisopulegol acetate remained non-hydrolyzed. The optical purity of the chromatographie pure l-isopulegol was 97.6%.     

Variations in composition of peppermint oil in relation to production areas

Compositional data obtained for peppermint oil fromMentha piperita L. produced in Tasmania as well as oils produced in other major production areas, were analysed by principal coordinate analysis. The 6 variates included in the analysis were the oil compounds limonene, cineole, menthone, menthofuran, menthyl acetate and menthol. In general, Tasmanian oils were characterised by low menthofuran, low limonene, low menthyl acetate and to a lesser extent high menthone, low cineole and high menthol concentrations relative to most major production areas. When the variation in composition of oil samples from within Tasmania was displayed in 3 dimensions (using the first 3 principal coordinates) it was not possible to achieve any pronounced separation of oils produced at different locations within southern Tasmania. Principal coordinates which were based mainly on cineole and menthol concentrations, respectively, did allow a degree of separation between oils produced in southern and northern Tasmania. Generally, oils produced in northern Tasmania had lower cineole and, in some cases, higher menthol than southern Tasmanian oils. Oil extracted from regrowth herb after commercial harvest was distinguished by having very high menthyl acetate, low menthone, high menthol, high menthofuran, low cineole and low limonene concentrations. The effect of some cultural and environmental factors on oil composition is also discussed.     

Cytotoxic Activity of Cunila angustifolia Essential Oil

The Cunila angustifolia essential oil was obtained from fresh leaves by hydrodistillation and analyzed by GC‐FID and GC‐MS to determine its chemical composition. The essential oil presented pulegone (29.5 %) and isomenthol (27.0 %) as major components, and other compounds such as menthone (8.6 %), neomenthol (7.2 %), menthyl acetate (2.5 %) and caryophyllene oxide (2.0 %) were identified. The cytotoxic activity of the essential oil was evaluated by MTS assay, with the human cancer cell lines of the lung (A549), breast (MCF‐7) and skin melanoma (SK‐Mel‐28). The assay showed the highest selectivity, to MCF‐7 cell lines, with IC₅₀ equal to 34.0 μg mL^?1, low selectivity for SK‐Mel‐28 cell lines, with IC₅₀ equal to 279.9 μg mL^?1, and no mortality to A549 cell lines.     

新颖深海微生物酯酶EstC11的酶学性质及其在手性拆分乙酸苏合香酯中的应用

【背景】手性乙酸苏合香酯是重要的手性香料产品,在食品及精细化工等领域都有重要的应用。酶催化不对称合成手性乙酸苏合香酯产品具有极好的工业应用前景。【目的】研究酯酶EstC11的基本酶学性质及其在制备手性乙酸苏合香酯中的应用。【方法】对来自西太平洋深海热液口芽孢杆菌Bacillus sp.CX01中的新颖微生物酯酶基因EstC11进行克隆、表达及酶学性质鉴定。通过对p H、温度、有机溶剂等反应条件的优化提高酯酶手性拆分乙酸苏合香酯的光学纯度。【结果】酯酶EstC11的最适反应p H为8.5,最适温度为25°C,一些金属离子和有机溶剂对酯酶EstC11的水解活性具有不同程度的抑制作用。通过对反应条件的优化,在最适反应条件下(p H 9.0 50 mmol/L Tris-HCl,20°C,50 mmol/L底物浓度)反应3 h后,(R)-乙酸苏合香酯的光学纯度达98%,得率为39%。【结论】通过对酯酶拆分条件的优化,手性拆分乙酸苏合香酯生成(R)-乙酸苏合香酯的光学纯度明显提高,为酯酶EstC11在工业化上的应用奠定了基础。     

Process for the Stereoselective Biotransformation of Acetoacetic Acid Esters Using Yeasts

A process for the stereospecific reduction of acetoacetic acid esters to the 3-(S)-hydroxy-butanoic acid esters by the yeasts Saccharomyces cerevisiae and Candida utilis grown on glucose and ethanol media was developed. A continuous single stage steady state production system was found to be superior to pulse-, batch- and fed-batch systems in terms of optical product purity, biomass concentration and production rates.

Optical purity of 3-(S)-hydroxybutanoic acid esters produced with Saccharomyces cerevisiae and Candida utilis was dependent on pH. A maximal optical purity was obtained at pH2.2 from S. cerevisiae growing on ethanol medium. The specific product formation rate of the chemostat cultures was 0.02…0.05 gg^?1 h^?1. C. utilis was more productive than S. cerevisiae but it reconsumed the product under carbon limited growth conditions.     

Concentration of bovine plasmalogen using phospholipase A1 in two-phase system

Plasmalogen that contains choline was concentrated from bovine heart to 95% purity from an initial value of 45%, and ethanolamine-containing plasmalogen from bovine brain to 99% purity from initial 64% using phospholipase A₁ from Serratia sp. MK1 in a two-phase system. In the two-phase system, Ca2⁺ was not required and Tris/HCl buffer and butyl acetate were used as an aqueous phase and a solvent phase of the two-phase system, respectively.     

A Novel Reductase from Candida albicans for the Production of Ethyl (S)-4-Chloro-3-hydroxybutanoate

A novel NADPH-dependent reductase (CaCR) from Candida albicans was cloned for the first time. It catalyzed asymmetric reduction to produce ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE). It contained an open reading frame of 843 bp encoding 281 amino acids. When co-expressed with a glucose dehydrogenase in Escherichia coli, recombinant CaCR exhibited an activity of 5.7 U/mg with ethyl 4-chloro-3-oxobutanoate (COBE) as substrate. In the biocatalysis of COBE to (S)-CHBE, 1320 mM (S)-CHBE was obtained without extra NADP⁺/NADPH in a water/butyl acetate system, and the optical purity of the (S)-isomer was higher than 99% enantiomeric excess.     

Microbial acetate oxidation in horizontal rotating tubular bioreactor

The aim of this work was to investigate the possibility of conducting a continuous aerobic bioprocess in a horizontal rotating tubular bioreactor (HRTB). Aerobic oxidation of acetate by the action of a mixed microbial culture was chosen as a model process. The microbial culture was not only grown in a suspension but also in the form of a biofilm on the interior surface of HRTB. Efficiency of the bioprocess was monitored by determination of the acetate concentration and chemical oxygen demand (COD). While acetate inlet concentration and feeding rate influenced efficiency of acetate oxidation, the bioreactor rotation speed did not influence the bioprocess dynamics significantly. Gradients of acetate concentration and pH along HRTB were more pronounced at lower feeding rates. Volumetric load of acetate was proved to be the most significant parameter. High volumetric loads (above 2 g acetate l⁻¹ h⁻¹) gave poor acetate oxidation efficiency (only 17 to 50%). When the volumetric load was in the range of 0.60–1.75 g acetate l⁻¹ h⁻¹, acetate oxidation efficiency was 50–75%. At lower volumetric loads (0.14–0.58 g acetate l⁻¹ h⁻¹), complete acetate consumption was achieved. On the basis of the obtained results, it can be concluded that HRTB is suitable for conducting aerobic continuous bioprocesses.     

Stereoselective microbial reduction of N-(4-(1-oxo-2-chloroacetyl ethyl) phenyl methane sulfonamide

Several microbial cultures were screened for the ability to catalyse the reduction of N-(4-(1-oxo-2-chloroacetyl ethyl) phenyl methane sulfonamide (1). The chiral intermediate (+)N-(4-(1-hydroxy-2-chloroethyl) phenyl methane sulfonamide (2) was prepared by the stereoselective microbial reduction of the parent ketone 1. Compound 2 is a potential chiral intermediate for synthesis of 4-(2-isopropylamino-1-hydroxyethyl) phenyl methanesulfonanilide (d-sotalol), a beta-receptor antagonist. Microorganisms from the genera Rhodococcus, Nocardia, and Hansenula reduced 1 to 2. A reaction yield of >50% and optical purities of >90% were obtained. The best strain (H.polymorpha ATCC 26012) effectively reduced compound 1 to compound 2 in 95% reaction yield and 99% optical purity. Compound 2 (8.2 g) was isolated from a 3-1 preparative batch in 68% overall yield. Isolated compound 2 had a specific rotation of +20° (CH₂Cl₂, C-1), an optical purity of 99.5%, and a chemical purity of 97% as analyzed by gas chromatography and HPLC. The nuclear magnetic resonance and mass spectra of compound 2 prepared by bioreduction and a standard chemical sample of 2 were virtually identical. Cell extracts of H. polymorpha in the presence of glucose dehydrogenase, glucose and nicotinamide adenine dinucleotide (NAD ⁺) catalyzed the reduction of 1 to 2 with 98% reaction yield and resulted in an optical purity of 99.4%. Correspondence to: R. N. Patel     

Heterotrophic cultivation of Paracoccus denitrificans in a horizontal rotating tubular bioreactor

In this work, the heterotrophic cultivation of bacterium Paracoccus denitrificans has been studied in a horizontal rotating tubular bioreactor (HRTB). After development of a microbial biofilm on the inner surface of the HRTB, conditions for one-step removal of acetate and ammonium ion were created. The effect of bioreactor process parameters [medium inflow rate (F) and bioreactor rotation speed (n)] on the bioprocess dynamics in the HRTB was studied. Nitrite and nitrogen oxides (NO and N₂O) were detected as intermediates of ammonium ion degradation. The biofilm thickness and the nitrite concentration were gradually reduced with increase of bioreactor rotation speed when the medium inflow rate was in the range of 0.5–1.5 l h⁻¹. Further increase of inflow rate (2.0–2.5 l h⁻¹) did not have a significant effect on the biofilm thickness and nitrite concentration along the HRTB. Complete acetate consumption was observed when the inflow rate was in the range of 0.5–1.5 l h⁻¹ at all bioreactor rotation speeds. Significant pH gradient (cca 1 pH unit) along the HRTB was only observed at the highest inflow rate (2.5 l h⁻¹). The results have clearly shown that acetate and ammonium ion removal by P. denitificans can be successfully conducted in a HRTB as a one-step process.     

D-Lactic acid fermentation performance and the enzyme activity of a novel bacterium Terrilactibacillus laevilacticus SK5–6

Purpose

The aim of this study was to prove that Terrilactibacillus laevilacticus SK5-6, a novel D-lactate producer, exhibited a good fermentation performance comparing to the reference D-lactate producer Sporolactobacillus sp.

Methods

Glucose bioconversion for D-lactate production and the activity of five key enzymes including phosphofructokinase (PFK), pyruvate kinase (PYK), D-lactate dehydrogenase (D-LDH), L-lactate dehydrogenase (L-LDH), and lactate isomerase (LI) were investigated in the cultivation of T. laevilacticus SK5–6 and S. laevolacticus 0361^T.

Results

T. laevilacticus SK5–6 produced D-lactate at higher yield, productivity, and optical purity compared with S. laevolacticus 0361^T. T. laevilacticus SK5–6, the catalase-positive isolate, simultaneously grew and produced D-lactate without lag phase while delayed growth and D-lactate production were observed in the culture of S. laevolacticus 0361^T. The higher production of D-lactate in T. laevilacticus SK5–6 was due to the higher growth rate and the higher specific activities of the key enzymes observed at the early stage of the fermentation. The low isomerization activity was responsible for the high optical purity of D-lactate in the cultivation of T. laevilacticus SK5–6.

Conclusion

The lowest specific activity of PFK following by PYK and D/L-LDHs, respectively, indicated that the conversion of fructose-6-phosphate was the rate limiting step. Under the well-optimized conditions, the activation of D/L-LDHs by fructose-1,6-phosphate and ATP regeneration by PYK drove glucose bioconversion toward D-lactate. The optical purity of D-lactate was controlled by D/L-LDHs and the activation of isomerases. High D-LDH with limited isomerase activity was preferable during the fermentation as it assured the high optical purity.