Syntheses of poly[beta-(l)-menthyl D- and L-aspartates] and their secondary structures

β-(l)-Menthyl D - and L -aspartates were prepared by a fusion reaction of N-phthalyl D - and L -aspartic anhydrides with l-menthol, followed by hydrazinolysis. The monomers were then polymerized to poly[β-(l)-menthyl D - and L -aspartates] by the N-carboxyanhydride method. These polymers were soluble in many organic solvents, such as diethyl ether, tetrahydrofuran, chloroform, n-bexane, and dioxane. From the results obtained by a study of the optical rotatory dispersions and circular dichroisms, poly [β-(l)-menthyl D -aspartate] was found to be a β form structure in solution. On the other hand, poly[β-(l)-menthyl L -aspartate] was a random-coil structure. These results suggest that the asymmetry of the l-menthyl chromophore in the side chain interacts with the polypeptide main chain and causes an extraordinary optical rotation.     

Reduction and cyclization in biotransformation of carbonyl compounds by cultured cells of Taxus species

Cultured plant cells from Taxus brevifolia Nutt and Taxus globosa Schltdl were investigated as biocatalysts using exogenous substrates. Production of highly specific metabolites by these species prompted us to analyse their synthetic potential. Whole cells suspensions have the capacity to chemoselectively reduce ethyl acetoacetate to ethyl 3-hydroxybutyrate chemo- and stereoselectively reduce rac-2-benzoylcyclohexanone to (1R, 2S)- and (1S, 2S)-2-hydroxycyclohexylphenylmethanones, and to cyclize N-phthaloyl-L-glutamine to thalidomide.     

Syntheses of poly[γ-(l)-menthyl L- and D-glutamates] and their secondary structures

γ-(l)-Menthyl L - and D -glutamates were prepared by a fusion reaction of N-phthalyl-L - and D -glutamic anhydrides with l-menthol, followed by hydrazinolysis. The monomers were polymerized to poly[γ-(l)-menthyl L - and D -glutamates] by the N-carboxyanhydride method. These polymers were soluble in many organic solvents, such as ethyl ether, chloroform, tetrahydrofuran, and n-hexane. From the results obtained by a study of the infrared absorption spectra, the x-ray photographs, the optical rotatory dispersions and the circular dichroisms, poly[γ-(l)-menthyl L -glutamate] was found to be a right-handed α-helix in the solid state and in solution. Similarly, poly[γ-(l)-menthyl D -glutamate] was a left-handed α-helix. The helix-coil transition of these polymers was observed in the vicinity of 40% dichloroacetic acid in a chloroform–dichloroacetic acid mixture.     

Synthesis and characterization of pyridoxine,nicotine and nicotinamide salts of dithiophosphoric acids as antibacterial agents against resistant wound infection

The pyridine-derived biomolecules are of considerable interest in developing medicinal compounds with various specific activities. Novel ammonium salts of pyridoxine, (S)-(–)-nicotine and nicotinamide with O,O-diorganyl dithiophosphoric acids (DTPA) were synthesized and characterized. The complexation of chiral monoterpenyl DTPA, including (S)-(–)-menthyl, (R)-(+)-menthyl, (1R)-endo-(+)-fenchyl, (1S,2S,3S,5R)-(+)-isopinocampheolyl derivatives, with pyridoxine and nicotine provided effective antibacterial compounds 3a,b,e,f, and 5a,b,d,f with MIC values against Gram-positive bacteria as low as 10?µM (6?µg/mL). Two selected pyridoxine and nicotine salts based on menthyl DTPA 3a and 5a were similarly active against antibiotic-resistant bacteria from burn wounds including MRSA. The compounds had enhanced amphiphilic and hemolytic properties and effectively altered surface characteristics and matrix-secreting ability of P. aeroginosa and S. aureus. MBC/MIC ratios of 3a and 5a suggested the bactericidal mode of their action. Furthermore, the compounds exhibited moderate cytotoxicity towards human skin fibroblasts (IC₅₀?=?48.6 and 57.6?µM, respectively, 72?h), encouraging their further investigation as potential antimicrobials against skin and wound infections.     

Effect of ionic liquid [BMIM][PF<Subscript>6</Subscript>] on asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

The effect of ionic liquid 1-butyl-3-methylimidazolium hexafluorophosphate ([BMIM][PF₆]) on the asymmetric reduction of ethyl 2-oxo-4-phenylbutyrate (EOPB) to synthesize optical active ethyl 2-hydroxy-4-phenylbutyrate (EHPB) catalyzed by Saccharomyces cerevisiae was investigated. (R)-EHPB [70.4%, e.e.(R)] is obtained using ethyl ether or benzene as the solvent. The main product is (S)-EHPB [27.7%, e.e.(S)] in [BMIM][PF₆]. However, in ionic liquid-water (10:1, v/v) biphasic system, the enantioselectivity of the reduction is shifted towards (R)-side, and e.e.(R) is increased from 6.6 to 82.5% with the addition of ethanol (1%, v/v). The effect of the use of [BMIM][PF₆] as an additive in relatively small amounts on the reduction was also studied. We find that there is a decline in the enantioselectivity of the reduction in benzene. In addition, a decrease in the conversion of EOPB and the yield of EHPB with increasing [BMIM][PF₆] concentrations occurs in either organic solvent–water biphasic systems or benzene.     

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.     

6-Deoxotyphasterol and 3-Dehydro-6-deoxoteasterone,Possible Precursors to Brassinosteroidsin the Pollen of Cupressus arizonica

Two new congeners (22R,23R,24S)-22,23-dihydroxy-24-methyl-5α-cholestan-3α-ol 2 and (22R,23R,24S)-22,23-dihydroxy-24-methyl-5α-cholestan-3-one 4 that are termed 6-deoxotyphasterol and 3-dehydro-6-eoxoteasterone, respectively, occur in relatively large amounts in the mature pollen of Cupressus arizonica. GC-MS, NMR spectroscopy, the reduction of 4 to 2, and the independent formation of 2 by the reduction of typhasterol were used to identify the new compounds. In the rice lamina bioassay, 2 showed weak activity. 6-Deoxocastasterone, castasterone, typha sterol, an epicastasterone-like compound, teasterone, 28-homocastasterone, 3-dehydroteasterone, brassinolide, and dolichosterone (or 24-epibrassinolide) were also present. These brassinosteroids were identified by co-chromatography with standards after being converted for an HPLC analysis of bioactive fractions. Six other peaks have not yet been assigned. 6-Deoxotyphasterol and 3-dehydro-6-deoxoteasterone should prove useful for exploring the early stages of the biosynthetic pathway(s) to brassinosteroids.     

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.     

Synthesis of ethyl-(3R,5S)-dihydroxy-6-benzyloxyhexanoates via diastereo- and enantioselective microbial reduction: Cloning and expression of ketoreductase III from Acinetobacter sp. SC 13874

Previously we have demonstrated the reduction of ethyl and t-butyl diketoesters 1 to the corresponding syn-(3R,5S)-dihydroxy esters 2a by Acinetobacter sp. 13874. The syn-(3R,5S)-dihydroxy ester 2a was obtained with an enantiomeric excess (e.e.) of 99% and a diastereomeric excess (de) of 63%. In this report, we identified a gene encoding desired ketoreductase III which catalyzed the diastereoselective reduction of diketoesters 1 to syn-(3R,5S)-dihydroxy esters 2a and describe cloning and expression of ketoreductase III into Escherichia coli. Cells or extracts of recombinant E. coli efficiently reduced the diketoester 1 to the corresponding syn-(3R,5S)-dihydroxy ester 2a in 99.3% yield, 100% e.e., and 99.8% de.     

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.     

Production of tert-butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate using carbonyl reductase coupled with glucose dehydrogenase with high space–time yield

tert-Butyl (3R,5S)-6-chloro-3,5-dihydroxyhexanoate ((3R,5S)-CDHH) is an important chiral intermediate for the synthesis of rosuvastatin. The biotechnological production of (3R,5S)-CDHH is catalyzed from tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate ((S)-CHOH) by a carbonyl reductase, and this synthetic pathway is becoming a primary route for (3R,5S)-CDHH production due to its high enantioselectivity, mild reaction conditions, low cost, process safety, and environmental friendship. However, the requirement of the pyridine nucleotide cofactors, reduced nicotinamide adenine dinucleotide (NADH) or reduced nicotinamide adenine dinucleotide phosphate (NADPH) limits its economic flexibility. In the present study, a recombinant Escherichia coli strain harboring carbonyl reductase R9M and glucose dehydrogenase (GDH) was constructed with high carbonyl reduction activity and cofactor regeneration efficiency. The recombinant E. coli cells were applied for the efficient production of (3R,5S)-CDHH with a substrate conversion of 98.8%, a yield of 95.6% and an enantiomeric excess (e.e.) of >99.0% under 350 g/L of (S)-CHOH after 12 hr reaction. A substrate fed-batch strategy was further employed to increase the substrate concentration to 400 g/L resulting in an enhanced product yield to 98.5% after 12 hr reaction in a 1 L bioreactor. Meanwhile, the space–time yield was 1,182.3 g L⁻¹ day⁻¹, which was the highest value ever reported by a coupled system of carbonyl reductase and glucose dehydrogenase.     

Two New Ergosterol Derivatives from the Basidiomycete Cortinarius glaucopus

Two new sterols 1 and 2 and five known ones 3 – 7 were isolated for the first time from the fruiting bodies of Cortinarius glaucopus. Their structures were established by 1‐ and 2D‐NMR spectra and HR‐FABS‐MS. The relative configuration of 1 was firmly determined by comparison of the observed ¹H–¹H couplings and NOESY correlations, with those predicted for the computed geometries of the conformers. Calculations were performed by means of DFT with the B3LYP functional at 6‐31 + G(d,p) level of theory, in CHCl₃ as the solvent. The structures of the new ergosterol derivatives, called glaucoposterol A ( 1 ) and B ( 2 ), were thus established as (3S,5R,7R,10R,13R,17R,20S,22R,23R,24R)‐5,6‐epoxy‐3,7,23‐trihydroxystrophast‐8‐en‐14‐one and (22E,3S,5S,9S,10R,13R,17R,20R,24R)‐3,5‐dihydroxyergosta‐6,8(14),22‐trien‐15‐one, respectively. Moreover, the configuration of known strophasterol C ( 3 ) was determined as (3S,5R,6S,7R,10R,13R,17R,20S,22S,24R). Glaucoposterol A ( 1 ) and strophasterol C ( 3 ) represent the second finding in nature of steroids with the rare strophastane skeleton.     

Impact of rainfall manipulations and biotic controls on soil respiration in Mediterranean and desert ecosystems along an aridity gradient

Spatially heterogeneous ecosystems form a majority of land types in the vast drylands of the globe. To evaluate climate‐change effects on CO₂ fluxes in such ecosystems, it is critical to understand the relative responses of each ecosystem component (microsite). We investigated soil respiration (R_s) at four sites along an aridity gradient (90–780 mm mean annual precipitation, MAP) during almost 2 years. In addition, R_s was measured in rainfall manipulations plots at the two central sites where ～30% droughting and ～30% water supplementation treatments were used over 5 years. Annual R_s was higher by 23% under shrub canopies compared with herbaceous gaps between shrubs, but R_s at both microsites responded similarly to rainfall reduction. Decreasing precipitation and soil water content along the aridity gradient and across rainfall manipulations resulted in a progressive decline in R_s at both microsites, i.e. the drier the conditions, the larger was the effect of reduction in water availability on R_s. Annual R_s on the ecosystem scale decreased at a slope of 256/MAP g C m^?2 yr^?1 mm^?1 (r²=0.97). The reduction in R_s amounted to 77% along the aridity gradient and to 16% across rainfall manipulations. Soil organic carbon (SOC) decreased with declining precipitation, and variation in SOC stocks explained 77% of the variation in annual R_s across sites, rainfall manipulations and microsites. This study shows that rainfall manipulations over several years are a useful tool for experimentally predicting climate‐change effects on CO₂ fluxes for time scales (such as approximated by aridity gradients) that are beyond common research periods. Rainfall reduction decreases rates of R_s not only by lowering biological activity, but also by drastically reducing shrub cover. We postulate that future climate change in heterogeneous ecosystems, such as Mediterranean and deserts shrublands will have a major impact on R_s by feedbacks through changes in vegetation structure.     

Optimization of immobilized Candida rugosa lipase LIP2-catalyzed resolution to produce l-menthyl acetate

Esterification of l-menthol by lipase is a highly selective method for the resolution of dl-menthol. The present work focuses on the reaction parameters that affect lipase-catalyzed synthesis of l-menthyl acetate in n-hexane using triacetin as acyl donor. Genetically engineered LIP2, an isoform of Candida rugosa lipase, was used as a biocatalyst in the present study. The main objectives of the work were to develop an approach that would enable a better understanding of relationships between the variables (reaction time, temperature, enzyme amount, substrate molar ratio) and the response (molar conversion) for l-menthyl acetate synthesis, and to obtain the optimum conditions for synthesis. By using central composite rotatable design (CCRD) and response surface methodology (RSM) analysis, we found that substrate molar ratio and enzyme amount were the most important variables for the reaction. Based on ridge max analysis, the optimum synthesis conditions were found to be: reaction time 2.2 days, temperature 34.3°C, enzyme amount 0.09 g and substrate molar ratio (dl-menthol:triacetin) 1:1.9, and molar conversion of dl-menthol to l-menthyl acetate was calculated to be 50%. An experiment under optimum conditions was carried out and molar conversion of 48.3% was obtained.     

Complementary selectivity to (S)-1-phenyl-1,2-ethanediol-forming Candida parapsilosis by expressing its carbonyl reductase in Escherichia coli for (R)-specific reduction of 2-hydroxyacetophenone

An (R)-specific carbonyl reductase from Candida parapsilosis CCTCCM203011 (CprCR) was shown to catalyze the asymmetric reduction of 2-hydroxyacetophenone to (R)-1-phenyl-1,2-ethanediol (PED), which is a critical chiral building block in organic synthesis. The gene (rcr) encoding CprCR was cloned based on the amino acid sequences of tryptic fragments of the enzyme. Sequence analysis revealed that rcr is comprised of 1008 nucleotides encoding a 35 977 Da polypeptide, and shares similarity to proteins of the medium-chain dehydrogenase/reductase (MDR) superfamily. Recombinant rcr expressed in Escherichia coli showed a specific 2-hydroxyacetophenone-reducing activity. Using rcr expressing cells, (R)-PED was obtained by asymmetric reduction, which is complementary in enantiomeric configuration to (S)-PED obtained by using whole cells of C. parapsilosis. After optimization of reaction conditions, (R)-PED was produced at 95.5% enantiomeric excess with a yield of 92.6% when isopropanol was used for cofactor regeneration.     

Enzymatic production of ethyl (R )-4-chloro-3-hydroxybutanoate: asymmetric reduction of ethyl 4-chloro-3-oxobutanoate by an Escherichia coli transformant expressing the aldehyde reductase gene from yeast

The asymmetric reduction of ethyl 4-chloro-3-oxobutanoate (COBE) to ethyl (R)-4-chloro-3-hydroxybutanoate (CHBE) using Escherichia coli JM109 (pKAR) cells expressing the aldehyde reductase gene from Sporobolomyces salmonicolor AKU4429 as a catalyst was studied. The reduction required NADP⁺, glucose and glucose dehydrogenase for NADPH regeneration. In an aqueous system, the substrate was unstable, and inhibition of the reaction by the substrate was also observed. Efficient conversion of COBE to (R)-CHBE with a satisfactory enantiomeric excess (ee) was attained on incubation with transformant cells in an n-butyl acetate/water two-phase system containing the above NADPH-regeneration system. Under the optimized conditions, with the periodical addition of COBE, glucose and glucose dehydrogenase, the (R)-CHBE yield reached 1530 mM (255 mg/ml) in the organic phase, with a molar conversion yield of 91.1% and an optical purity of 91% ee. The calculated turnover of NADP⁺, based on the amounts of NADP⁺ added and CHBE formed, was about 5100 mol/mol. Received: 26 May 1997 / Received revision: 16 July 1997 / Accepted: 29 August 1997     

Isolation of 4-Hydroxy-5-methyl-3(2H)-furanone,a Flavor Component in Shoyu (Soy Sauce)

An analytical method was developed for determining the four optical isomers of a new synthetic pyrethroidal insecticide, α-cyano-3-phenoxy-benzyl 2-(4-chlorophenyl)isovalerate (Fenvalerate, S–5602). The cyano group in S–5602 molecule was converted into l-menthyloxycarbonyl group by heating S–5602 with l-menthol and hydrochloric acid. The obtained diastereoisomer derivative (l-menthyl ester deriv.) was chromatographed on a μPorasil column (4 mm i.d. × 0.3 m) using a solvent system of hexane-ethyl acetate (500+7, v/v) as a mobile phase, and the four isomers of S–5602 were separated from one another within 20 min. The compositions of S–5602 isomers were determined from their peak areas.     

Expression and characterization of chimeric spidroins from flagelliform-aciniform repetitive modules

Spiders can produce up to seven different types of silks or glues with different mechanical properties. Of these, flagelliform (Flag) silk is the most elastic, and aciniform (AcSp1) silk is the toughest. To produce a chimeric spider silk (spidroin) Flag_R-AcSp1_R, we fused one repetitive module of flagelliform silk from Araneus ventricosus and one repetitive module of aciniform silk from Argiope trifasciata. The recombinant protein expressed in E. coli formed silk-like fibers by manual-drawing. CD analysis showed that the secondary structure of Flag_R-AcSp1_R spidroin remained stable during the gradual reduction of pH from 7.0 to 5.5. The spectrum of FTIR indicated that the secondary structure of Flag_R-AcSp1_R changed from α-helix to β-sheet. The conformation change of Flag_R-AcSp1_R was similar to other spidroins in the fiber formation process. SEM analysis revealed that the mean diameter of the fibers was around 1 ~ 2 μm, and the surface was smooth and uniform. The chimeric fibers exhibited superior toughness (~33.1 MJ/m³) and tensile strength (~261.4 MPa). This study provides new insight into design of chimeric spider silks with high mechanical properties.     

The comparative effects of short-term DNA inhibition on replicon synthesis in mammalian cells

The inter-replicon distance (ID) and rate (R) of DNA chain growth along the replicon were investigated with a [³H]TdR pulse-chase protocol in DNA autoradiographs of cells from seven different cultures of mammalian cells from various species. Asynchronous cultures were labelled with or without a 4 h pretreatment with the DNA inhibitor 5-fluorodeoxyuridine (FUdR). DNA inhibition was found to reduce both the mean ID and R by different amounts in the different cultures. This reduction appeared to correlate with the effectiveness of the inhibition in reducing cell viability. These findings generally could account for the considerable variability found in published data where FUdR pretreatment has been used. When individual values of ID and R in units of μm are plotted against each other, their relationship is given by the mean linear regressions: R = 0.26 ± 0.04 + (0.88 ± 0.05) 10⁻²ID for control, and R = 0.16 ± 0.04 + (1.04 ± 0.06) 10⁻²ID for FUdR-pretreated cultures.The relationship between ID and R in both sets of cultures suggests the presence of a regulating mechanism within a cell which maintains a relatively constant overall rate of chain growth over long stretches of DNA. A mechanism involving changes in the levels of various DNA replication complexes is suggested as one explanation for this relationship.     

The dynamic response of soil respiration to land‐use changes in subtropical China

Assessing the impact of land‐use changes on soil respiration (R_S) is of vital significance to understand the interactions between belowground metabolism and regional carbon budgets. In this study, the monthly in situ R_S was examined between 09:00 and 12:00 hours over a 3‐year period within a representative land‐use sequence in the subtropical region of China. The land‐use sequence contained natural forest (control treatment), secondary forest, two plantations, citrus orchard and sloping tillage land. Results showed that the R_S exhibited a distinct seasonal pattern, and it was dominantly controlled by the soil temperature. After the land‐use conversion, the apparent temperature sensitivity of R_S (Q₁₀) was increased from 2.10 in natural forest to 2.71 in sloping tillage land except for an abnormal decrease to 1.66 in citrus orchard. Contrarily, the annual R_S was reduced by 32% following the conversion of natural forest to secondary forest, 46–48% to plantations, 63% to citrus orchard and 50% to sloping tillage land, with the average reduction of 48%. Such reduction of annual R_S could be explained by the decrease of topsoil organic carbon and light‐fraction organic carbon storages, live biomass of fine root (<2 mm) and annual litter input, which indirectly/directly correlated with plant productivity. Our results suggest that substrate availability (e.g., soil organic carbon and nutrients) and soil carbon input (e.g., fine root turnover and litterfall) through plant productivity may drive the R_S both in natural and managed ecosystems following strong disturbance events.