Obtusastyrene and obtustyrene,cinnamylphenols from Dalbergia retusa

The heartwood of Dalbergia retusa contains, in addition to 8-O-methylretusin, (R)-4-methoxydalbergione, (R)-obtusaquinol and (+)-obtusafuran [(2R,3R)-2-phenyl-3-methyl-2,3-dihydro-5-hydroxy-6-methoxybenzofuran], the cinnamylphenols obtusastyrene [E-1-(4-hydroxybenzyl)-2-phenylethylenel, obtustyrene [E-1-(4-hydroxy-2-methoxybenzyl)-2-phenylethylene] and obtusaquinone [styryl-5-hydroxy-2-methoxy-4-quinonemethide]. The structural determination of compounds relied on spectra, degradations and syntheses.     

Purification and Characterization of a (R)-1-Phenyl-1,3-propanediol-producing Enzyme from Trichosporon fermentans AJ-5152 and Enzymatic (R)-1-Phenyl-1,3-propanediol Production

An (R)-1-phenyl-1,3-propanediol-producing enzyme was purified from Trichosporon fermentans AJ-5152. It was NADPH-dependent and converted 3-hydroxy-1-phenylpropane-1-one (HPPO) to (R)-1-phenyl-1,3-propanediol [(R)-PPD] with anti-Prelog’s specificity. It showed maximum activity at pH 7.0 and 40 °C. Its K _m and V _max values toward HPPO were 20.1 mM and 3.4 μmol min^?1 mg protein^?1 respectively. The relative molecular weight of the enzyme was estimated to be 68,000 on gel filtration and 32,000 on SDS-polyacrylamide gel electrophoresis. An (R)-PPD-producing reaction using the (R)-PPD-producing enzyme and an NADPH recycling system was carried out by successive feeding of HPPO. A total (R)-PPD yield of 8.9 g/l was produced in 16 h. The molar yield was 76%, and the optical purity of the (R)-PPD produced was over 99% e.e.     

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.     

Continuous production of chiral 1,3-butanediol using immobilized biocatalysts in a packed bed reactor: promising biocatalysis method with an asymmetric hydrogen-transfer bioreduction

An asymmetric hydrogen-transfer biocatalyst consisting of mutated Rhodococcus phenylacetaldehyde reductase (PAR) or Leifsonia alcohol dehydrogenase (LSADH) was applied for some water-soluble ketone substrates. Among them, 4-hydroxy-2-butanone was reduced to (S)/(R)-1,3-butanediol, a useful intermediate for pharmaceuticals, with a high yield and stereoselectivity. Intact Escherichia coli cells overexpressing mutated PAR (Sar268) or LSADH were directly immobilized with polyethyleneimine or 1,6-diaminehexane and glutaraldehyde and evaluated in a batch reaction. This system produced (S)-1,3-butanediol [87% enantiomeric excess (e.e.)] with a space time yield (STY) of 12.5 mg h⁻¹ ml⁻¹ catalyst or (R)-1,3-butanediol (99% e.e.) with an STY of 60.3 mg h⁻¹ ml⁻¹ catalyst, respectively. The immobilized cells in a packed bed reactor continuously produced (R)-1,3-butanediol with a yield of 99% (about 49.5 g/l) from 5% (w/v) 4-hydroxy-2-butanoate over 500 h.     

Synthesis of regioisomeric 17β-N-phenylpyrazolyl steroid derivatives and their inhibitory effect on 17α-hydroxylase/C17,20-lyase

The reaction of 3β-hydroxy-21-hydroxymethylidenepregn-5-en-3β-ol-20-one (1) with phenylhydrazine (2a) affords two regioisomers, 17β-(1-phenyl-3-pyrazolyl)androst-3-en-3β-ol (5a) and 17β-(1-phenyl-5-pyrazolyl)androst-5-en-3β-ol (6a). The direction of the ring-closure reactions of 1 with p-substituted phenylhydrazines (2b-e) depends strongly on the electronic features of the substituents. Oppenauer oxidation of 3β-hydroxy-17β-exo-heterocyclic steroids 5a-e and 6a-e yielded the corresponding Δ⁴-3-ketosteroids 9a-e and 10a-e. The inhibitory effects (IC₅₀) of these compounds on rat testicular C_17,20-lyase were investigated by means of an in vitro radioligand incubation technique.     

Stereoselective Synthesis of Ectocarpene and Its Antipode via Microbiological Asymmetric Hydrolysis

Biological asymmetric hydrolysis of ethyl (±)-cycloheptadienecarboxylate with Rhodotorula minuta var. texensis IFO 1102 and chemical resolution of the corresponding carboxylic acid with (?)-quinine provided (R)-(+)-ethyl 2,5-cycloheptadienecarboxylate (78% e.e.) and (S)-(+)-2,5-cycloheptadienecarboxylic acid (95% e.e.), respectively. The (R)-(+)-carboxylate was converted to (R)-(?)-2,5-cycloheptadienylcarbaldehyde and the (S)-(+)-carboxylic acid to (S)-(+)-2,5-cycloheptadienylcarbaldehyde. Ectocarpene (78% e.e.), male-gamete attractant of marine brown alga, and its antipode (95% e.e.) were synthesized by stereoselective Wittig reaction between the (R)-(?)- and (S)-(+)-aldehydes and propyltriphenylphosphonium bromide in a liquid-solid two phase system using 18-crown ether-t-BuOK, respectively.     

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.     

Comparison of Soybean Tissue Extracted with Different Solvents

The enantioselective hydrolysis of (R,S)-3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4H-[1,4]benzoxazine (I) with enzymes was investigated. Optically active I and its hydrolyzate, 7,8-difluoro-2,3-dihydro-3-hydroxymethyl-4H-[1,4]benzoxazine (II), are the intermediates for preparing optically active ofloxacins, whose racemate is known to be an excellent antibacterial agent. Lipoprotein lipase from Pseudomonas fluorescens (LPL Amano 3) was found to predominantly hydrolyze (S)-I, giving (R)-I in 54% e.e. and (R)-II in 44% e.e. On the other hand, lipase from Candida cylindracea was found to predominantly hydrolyze (R)-I, giving (S)-I in 24% e.e. and (S)-II in 20% e.e. Since, the optical purities of I and II thus obtained were not particularly high, these optically active I and II were converted into 3-acetoxymethyl-7,8-difluoro-2,3-dihydro-4-(3,5-dinitrobenzoyl)-4H-[1,4]benzoxazine (IV). After recrystallizing IV from ethyl acetate-hexane, (S)- and (R)-II were obtained with high enantiomeric excess by removing the crystallized racemic IV and subsequently hydrolyzing the resulting optically active IV with alkali. The reduction of II afforded 7,8-difluoro-2,3-dihydro-3-methyl-4H-[1,4]benzoxazine (III), for which the optical purity was estimated to be >96%e.e. by HPLC analysis. (R)- and (S)-ofloxacin were prepared from (R)- and (S)-III with retention of their configuration.     

Synthesis of (+)-Turmeronol A,an Inhibitor of Soybean Lipoxygenase,and (+)-ar-Turmerone

Syntheses of optically pure turmeronol A and turmerone were achieved in a simple manner starting from ethyl (R)-3-hydroxybutanoate (4) of 100% e.e. The key step was the displacement of the chiral tosylate (6) with an organocopper reagent.     

Screening for Antioxidants of Microbial Origin

Asymmetric hydrolysis of (dl)-1-acyloxy-2-halo-1-phenylethanes by lipoprotein lipase Amano P from Pseudomonas fluorescens and the lipase from Chromobacterium viscosum afforded the optically active (R) residual substrates and (S)-2-halo-1-hydroxy-1-phenylethanes in 100% enantiomeric excess (e.e.). The length of acyl residues from acetyl to octanoyl in the substrates did not influence the enantioselectivity.

Both enantiomers of optically active styrene oxides were synthesized from the enzymatic products.     

Degradation Mechanism of Poly(vinyl alcohol) by Successive Reactions of Secondary Alcohol Oxidase and β-Diketone Hydrolase from Pseudomonas sp.

The secondary alcohol oxidase from Pseudomonas sp. catalyzed the oxidation of various vinyl alcohol oligomers with the molecular weight of 220 to 1500 and of β-ketols such as 5-hydroxy-3-heptanone, 4-hydroxy-2-nonanone, 3-hydroxy-5-nonanone, 6-hydroxy-4-nonanone, 7-hydroxy-5-dodecanone, and 8-hydroxy-6-tridecanone. β-Diketone hydrolase from the same strain catalyzed the hydrolysis of various aliphatic β-diketones and some aromatic β-diketones such as 1-phenyl-1,3-butanedione and 1-phenyl-2,4-pentanedione. 4,6-Nonanediol, used as a low molecular weight model of poly(vinyl alcohol) (PVA), was oxidized to 4,6-nonanedione by way of 6-hydroxy-4-nonanone by secondary alcohol oxidase. 4,6-Nonanedione was hydrolyzed to 2-pentanone and n-butyric acid by β-diketone hydrolase. These reactions were stoichiometric.

The presence of the β-diketone structure in PVA oxidized by secondary alcohol oxidase was confirmed by spectral experiments. The absorption due to β-diketone structure in the oxidized PVA decreased as it was hydrolyzed by β-diketone hydrolase. The ratio of the amount of carboxyl groups in the degraded PVA to that of carbonyl groups in the oxidized PVA became more than 0.5. A pathway for the enzymatic degradation of PVA was proposed.     

Green and scalable synthesis of chiral aromatic alcohols through an efficient biocatalytic system

Chiral aromatic alcohols have received much attention due to their widespread use in pharmaceutical industries. In the asymmetric synthesis processes, the excellent performance of alcohol dehydrogenase makes it a good choice for biocatalysts. In this study, a novel and robust medium-chain alcohol dehydrogenase RhADH from Rhodococcus R6 was discovered and used to catalyse the asymmetric reduction of aromatic ketones to chiral aromatic alcohols. The reduction of 2-hydroxyacetophenone (2-HAP) to (R)-(-)-1-phenyl-1,2-ethanediol ((R)-PED) was chosen as a template to evaluate its catalytic activity. A specific activity of 110 U mg⁻¹ and a 99% purity of e.e. was achieved in the presence of NADH. An efficient bienzyme-coupled catalytic system (RhADH and formate dehydrogenase, CpFDH) was established using a two-phase strategy (dibutyl phthalate and buffer), which highly raised the tolerated substrate concentration (60 g l⁻¹). Besides, a broad range of aromatic ketones were enantioselectively reduced to the corresponding chiral alcohols by this enzyme system with highly enantioselectivity. This system is of the potential to be applied at a commercial scale.     

Evaluation of the Suppressive Effect on Bitter Taste of Gluconate

Improved conditions for the production and characterization of 1-arylpropane-1, 2-diols and related compounds were developed. Experimental conditions providing highly enhanced activity of pyruvate decarboxylase in bakers’ yeast in the presence of pyruvate, thiamine pyrophosphate, and magnesium(II) salt were applied to the preparation of (R)-1-hydroxy-1-phenyl-2-propanone from benzaldehyde. Subsequent reduction with bakers’ yeast efficiently afforded 1-phenyl-1, 2-propanediol (35%). The composition of its stereoisomers was precisely determined, and the major (1R, 2S)-isomer (89% of the total mixture) could be isolated by recrystallizing the corresponding benzoate. The analytical method for identifying the stereoisomeric composition was also effective for the determination of 5-phenyl-4-pentene-2, 3-diol, the biotransformation product from cinnamaldehyde, the vinylogous substrate of benzaldehyde. Furthermore, the structural characterization of 1-(2-furyl)propane-1, 2-diol, which was obtained from furfural (28%) by the action of brewers’ yeast Saccharomyces cerevisiae (carlsbergensis), is described. The major (1S, 2S)-isomer could be isolated by recrystallizing the crude product.     

Biosynthesis of (R)-phenyl-1,2-ethanediol from racemic styrene oxide by using bacterial and marine fish epoxide hydrolases

Enantio-convergent hydrolysis of racemic styrene oxides was achieved to prepare enantiopure (R)-phenyl-1,2-ethanediol by using two recombinant epoxide hydrolases (EHs) of a bacterium, Caulobacter crescentus, and a marine fish, Mugil cephalus. The recombinant C. crescentus EH primarily attacked the benzylic carbon of (S)-styrene oxide, while the M. cephalus EH preferentially attacked the terminal carbon of (R)-styrene oxide, thus leading to the formation of (R)-phenyl-1,2-ethanediol as the main product. (R)-Phenyl-1,2-ethanediol was obtained with 90% enantiomeric excess and yield as high as 94% from 50 mM racemic styrene oxides in a one-pot process.     

Neoflavonoids and the cinnamylphenol kuhlmannistyrene from Machaerium kuhlmannii and M. Nictitans

The trunkwood of Machaerium kuhlmannii contains methyl palmitate, 3-O-acetyloleanolic acid and sitosterol; the benzene derivatives 2,3-dimethoxyphenol, 2,6-dimethoxyphenol, 2-hydroxy-3-methoxyphenol, 2,3-dimethoxybenzaldehyde and methyl 3-(2-hydroxy-4-methoxyphenyl)-propionate; the isoflavonoids formononetin and (6aS,11aS)-medicarpin; the neoflavonoids (R)-3,4-dimethoxydalbergione, (R)-3,4-dimethoxydalbergiquinol, kuhlmanniquinol [(R)-3-(4-hydroxyphenyl)-3-(5-hydroxy-2,3,4-trimethoxyphenyl)-propene], dalbergin, kuhlmannin (6-hydroxy-7,8-dimethoxy-4-phenylcoumarin) and kuhlmannene (6-hydroxy-7,8-dimethoxy-4-phenylchrom-3-ene), as well as the cinnamylphenol kuhlmannistyrene [Z-1-(5-hydroxy-2,3,4-trimethoxybenzyl)-2-(2-hydroxyphenyl)-ethylene]. Five of these compounds, in addition to (R)-4′-hydroxy-3,4-dimethoxydalbergione, were also isolated from a trunkwood extract of M. nictitans. Structural assignments were confirmed by chemical interconversion and by the synthesis of (±)-kuhlmanniquinol.     

Residue of Kitazin P Metabolite in Rice Plants and Soil

Debutenoyl-aspertetronin A was synthesized from γ-valerolactone-γ-carboxylic acid (4) via 2, 5-dihydro-3-hydroxy-2-methyl-5-oxo-2-furanpropanoic acid. Starting from (?)-(S)-4, (+)-(S)-5-hexyl-4-hydroxy-5-methyl-2(5H)furanone (19) was synthesized, and by comparison of its optical rotation with that of an authentic sample it was proved that aspertetronin A had (R) configuration, and gregatin A had (S) configuration at their respective chiral carbon.     

Chemical constituents from the fresh flower buds of Musa nana and their chemotaxonomic significance

Phytochemical study on the fresh flower of Musa nana Lour. provided seventeen known compounds including two alkaloids, 3-(hydroxyacetyl)-indole (1), bi-indol-3-yl (2), two terpenoids, 5-[(1R)-1-hydroxy-2,6,6-trimethyl-4-oxo-2-cyclohexen-1-yl]-3-methyl-, (2Z, 4E) −2, 4-pentadienoic acid (Valdes), 5, 6(S), 7, 7a(R)-tetrahydro-6-hydroxy-4,4-dimethyl-2(4H)-benzofuranone (4), seven phenols (5–11), three phenylphenalenones, 2-hydroxy-4-(4-methoxyphenyl)-1H-phenalen-1-one (12), 2-methoxy-9-phenyl-1H-phenalen-1-one (13), 2-methoxy-9-(4-methoxyphenyl)-1H-phenalen-1-one (14), and three lipids (15–17). In the present study, all the compounds were isolated for the first time from the species M. nana. Ten compounds including 1-8 and 15-16 have never been previously encountered in the Musaceae family. Furthermore, the chemotaxonomic significance of these isolates was also discussed.     

Low Resolution Mass Spectra of Some Unsaturated δ-Lactones

Mass spectra of the δ-lactones of the following 5-hydroxy-2-enoic acids were determined: 5-hydroxyhex-2-enoic acid (I), 5-hydroxyoct-2-enoic acid (II), 5-hydroxydec-2-enoic acid (III), 5-hydroxydodec-2-enoic acid (IV), 5-hydroxy-8-methylnon-2-enoic acid (V), 5-hydroxy-6-ethyloct-2-enoic acid (VI), 5-hydroxy-5, 6, 6-trimethylhept-2-enoic acid (VII), and 5-hydroxy-5-methylnon-2-enoic acid (VIII). The following modes of fragmentation are consistent with observed m/e values, metastable peaks, and established modes of breakdown in compounds containing similar atomic groupings:—1. Loss of side chain, resulting in ions at m/e 97 for I-VI and at m/e 111 and 153 for VII and VIII (diagnostic peaks); 2. 1,4-Rupture of the ring giving an ion at m/e 68 (diagnostic peak) which loses CO to give m/e 40; 3. Loss of CO from m/e 97 fragment to give m/e 69 which breaks down further to m/e 41→m/e 39; 4. 1, 4-Rupture of m/e 111 and m/e 153 fragments to give m/e 43 and 85, further breakdown of m/e 85→57→41→39; 5. Loss of H₂O from the molecular ion providing there is a hydrogen atom on C₅ and the side chain is at least 3 carbon atoms in length, further loss of H₂O when the side chain is equal to C5 or C7; 6. Loss of CO₂ from the molecular ion of I, IV-VIII; 7. Loss of CO from all molecular ions; 8. Loss of 2×28 from the molecular ions of III, IV, V, VI; 9. Loss of (18 + 28) from the molecular ion of III, IV, V, VI; 10. Loss of 60 from the molecular ion of II, III, IV, V, VI; 11. Formation of M + 1 ion (169) of VII and VIII; 12. Formation of M + 1 ion (143) of saturated δ-octalactone and loss of H₂O from this M + 1 ion.     

Transformation of menthane monoterpenes by Mentha piperita cell culture

(+)-Isopiperitenone (100 mg l^–1) was converted into (4S,6R)-6-hydroxy- and (4S,8R)-8,9-epoxyisopiperitenone, aside from the already known (+)-7-hydroxyisopiperitenone, by suspension cell culture of Mentha piperita. As (–)-isopiperitenone was hydroxylated similarly, this implies that the hydroxylating enzyme(s) have a broad substrate stereospecificity in regards to the stereochemistry at C4. (–)-(4R)-Carvone was reduced by the Mentha cells both at carbonyl and C1-C6 double bond to give (1R,2S,4R)-neodihydrocarveol and (1R,2R,4R)-dihydrocarveol with the former being the major product. (+)-(4S)-Carvone had a similar reduction pattern, producing (1S,2R,4S)-neodihydrocarveol and (1S,4S)-dihydrocarvone. Formation of these compounds indicates that the peppermint cell culture cannot only hydroxylate the allylic position but also reduce the ,-unsaturated carbonyl system.     

Effects of Trialkyltin Chlorides on Escherichia coli Spheroplasts

Valsa ceratosperma, which is the pathogenic fungus of apple canker, was grown in a synthetic medium. The neutral extract from the culture filtrate was chromatographed on a silica gel column to give five isocoumarins. Their structures were determined by MS, UV, IR, ¹H and ¹³C NMR, and CD spectra. Three of them were known compounds; ( ? )-5-methylmellein (1), ( ? )-5-carboxylmellein (2) and ( ? )-5-hydroxylmethylmellein (3). Since the absolute configurations at C-3 in 2 and 3 were not known until now, both were determined to be R by chemical correlations. The two were new compounds; ( + )-(3R,4S)-trans-4-hydroxy-5-methylmellein (4) and ( ? )-(3R,4R)-cis-4-hydroxy-5-methylmellein (5). All the five compounds showed phytotoxicity in a bioassay using detached apple shoots and lettuce seedlings.