Newbouldiosides A-C, phenylethanoid glycosides from the stem bark of Newbouldia laevis

From the stem bark of Newbouldia laevis three phenylethanoid glycosides, designated as newbouldioside A-C, were isolated together with a sodium salt of analogue B and the known compounds, verbascoside, 5-hydroxydehydro-iso-alpha-lapachone, 3,8-dihydroxydehydro-iso-alpha-lapachone, apigenin and luteolin. The structures of the phenylethanoid glycosides were elucidated by spectroscopic methods as beta-(3,4-dihydroxyphenyl)ethyl 5-O-syringoyl-beta-D-apiofuranosyloxy-(1-->2)-O-[alpha-L-rhamnopyranosyl-(1-->3)]-beta-D-glucopyranoside, ss-(3,4-dihydroxyphenyl)ethyl 5-O-syringoyl-beta-D-apiofuranosyloxy-(1-->2)-O-[alpha-L-rhamnopyranosyl-(1-->3)]-6-O-E-feruloyl-beta-D-glucopyranoside, and beta-(3,4-dihydroxyphenyl)ethyl 3-O-E-feruloyl-beta-D-apiofuranosyloxy-(1-->2)-O-alpha-L-rhamnopyranosyl-(1-->2)-6-O-E-sinapoyl-beta-D-glucopyranoside, respectively.     

Carbohydrates from Cynanchum otophyllum

Four new carbohydrates were isolated from the acidic hydrolysis part of the ethyl acetate extract of Cynanchum otophyllum Schneid (Asclepiadaceae). Their structures were determined as methyl 2,6-dideoxy-3-O-methyl-beta-D-arabino-hexopyranosyl-(1-->4)-6-deoxy-3-O-methyl-beta-D-ribo-hexopyranosyl-(1-->4)-6-deoxy-3-O-methyl-alpha-L-ribo-hexopyranoside (1), methyl 6-deoxy-1,3-di-O-methyl-beta-D-ribo-hexosyl-(1-->4)-2,6-dideoxy-3-O-methyl-alpha-D-arabino-hexopyranoside (2), methyl 2,6-dideoxy-3-O-methyl-beta-D-arabino-hexopyranosyl-(1-->4)-6-deoxy-3-O-methyl-alpha-L-ribo-hexopyranoside (3), and 2,6-dideoxy-3-O-methyl-beta-D-arabino-hexopyranosyl-(1-->4)-2,6-dideoxy-3-O-methyl-alpha-D-arabino-hexopyranosyl-(1-->4)-2,6-dideoxy-3-O-methyl-beta-D-lyxo-hexopyranose (4), respectively, by spectral methods.     

Synthesis of the pentasaccharide related to the repeating unit of the antigen from Shigella dysenteriae type 4 in the form of its methyl ester 2-(trimethylsilyl)ethyl glycoside

Starting from D-mannose, D-glucose and L-fucose, the pentasaccharide derivative methyl 2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl-(1-->3)-2-O-acetyl-4,6-O-benzylidene-alpha-D-mannopyranosyl-(1-->3)-2-O-acetyl-6-O-benzyl-4-O-(2,3,4-tri-O-benzyl-alpha-L-fucopyranosyl)-alpha-D-mannopyranosyl-(1-->4)-[2-(trimethylsilyl)ethyl 2,3-di-O-benzyl-beta-D-glucopyranosid]uronate was synthesized. This compound with two alpha-mannopyranosyl units was transformed, via Walden inversion and subsequent deprotection, into the alpha-D-glucosamine-type target compound, namely methyl alpha-L-fucopyranosyl-(1-->3)-2-acetamido-2-deoxy-alpha-D-glucopyranosyl-(1-->3)-2-acetamido-2-deoxy-4-O-(alpha-L-fucopyranosyl)-alpha-D-glucopyranosyl-(1-->4)-[2-(trimethylsilyl)ethyl beta-D-glucopyranosid]uronate which is related to the repeating unit of the O-antigen from Shigella dysenteriae type 4.     

Microbial asymmetric reduction of ethyl 4-chloro-3-oxobutanoate to optically active ethyl-4-chloro-3-hydroxybutanoate

Summary The synthesis of ethyl (R)-4-chloro-3-hydroxybutanoate through the asymmetric reduction of ethyl 4-chloro-3-oxobutanoate with the NADPH-dependent aldehyde reductase ofSporobolomyces salmonicolor AKU 4429 is described. Under preparative scale reaction conditions with the acetone-fractionated aldehyde reductase, the amount of ethyl-4-chloro-3-hydroxybutanoate reached 33.1 mg/ml (85%ee; molar yield, 74.0%). Furthermore, conversion to ethyl (S)-4-chloro-3-hydroxybutanoate occurred on incubation with washed cells ofTrichosporon cutaneum AKU 4864 as the catalyst.     

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.     

Influence of sugars on enantioselective reduction using Saccharomyces cerevisiae in organic solvent

Haploid Saccharomyces cerevisiae W303-1A cells grown on different carbon sources were employed as the biocatalyst for ethyl acetoacetate reduction in n-hexane. The effects of cell immobilization on montmorillonite, as well as the addition of trehalose or sucrose solutions, were also tested. Best conversions (∼50%) to the chiral alcohol ethyl (S)-(+)-3-hydroxybutanoate (ee > 99%) were obtained with cells grown under respiratory metabolism with glycerol–ethanol, and higher yields were observed when trehalose was added to the reaction media. Although cells with fermentative metabolism grown on glucose were able to reduce the substrate when sucrose was added, the disaccharide was consumed by the cells during the course of the reaction, and no enantioselective product was obtained. Immobilized cells also required the addition of trehalose in order to reduce the substrate with high yield. Thus, our results indicate that trehalose may be an efficient protector of immobilized or free yeast cells during enantioselective reductions in organic solvent.     

Synthesis of (S,E)-1-Methyl-9-dodecenyl Acetate,the Sex Pheromone of the Hessian Fly,Mayetiola destructor

(S,E)-1-Methyl-9-dodecenyl acetate (1), the sex pheromone of the Hessian fly, was synthesized by starting from ethyl (S)-3-hydroxybutanoate (2).     

Effect of Cycloheximide on Protein Breakdown in Germinating Rice Seeds

1′-Epi-stegobinone [(2S,3R,1′S)-2,3-dihydro-2,3,5-trimethyl-6-(1′-methyl-2′-oxobutyl)-4H-pyran-4-one], an inhibitor of stegobinone, which is the sex pheromone of drugstore beetle (Stegobium paniceum L.), was synthesized by stereocontrol at C-2 and C-1′ starting from ethyl (R)-3-hydroxybutanoate and methyl (R)-3-hydroxypentanoate.     

Triterpenoid saponins from Oreopanax guatemalensis

Seven oleanane-type saponins were isolated from the leaves and stems of Oreopanax guatemalensis, together with ten known saponins of lupane and oleanane types. The new saponins were respectively characterized as 3-O-alpha-L-arabinopyranosyl echinocystic acid 28-O-[alpha- L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester, 3-O-beta-D-glucopyranosyl 3beta-hydroxy olean-11,13(18)-dien-28-oic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta- D-glucopyranosyl]ester, 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-beta-D-glucopyranosyl]3beta-hydroxy olean-11,13(18)-dien-28-oic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester, 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl]3beta, 23 dihydroxy olean-18-en-28-oic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-6-O-acetyl glucopyranosyl-(1-->6)-beta-D-glucopyranosyl]ester, 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl] hederagenin 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-[beta-D-xylopyranosyl-(1-->2 )-]beta-D-glucopyranosyl]ester, 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl]hederagenin 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-[beta-D-glucopyranosyl-(1-->2)-]beta-D-glucopyranosyl] ester and 3-O-[alpha-L-rhamnopyranosyl-(1-->2)-alpha-L-arabinopyranosyl] hederagenin 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-[alpha-L-arabinofuranosyl-(1-->2)]-beta-D-glucopyranosyl] ester. The structures were determined by spectral analyses. The NMR assignments were made by means of HOHAHA, 1H-1H COSY, HMQC, HMBC spectra and NOE difference studies.     

Further saponins from Meryta lanceolata

Five new oleanane-type saponins along with 11 known ones were isolated from the leaves and stems of Meryta lanceolata. The new saponins were characterised by spectroscopic analysis including FAMS, 1 and 2D NMR experiments and the results of hydrolysis as 3-O-[beta-d-glucopyranosyl-(1-->2)-beta-d-glucuronopyranosyl] hederagenin 28-O-[alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranosyl-(1-->6)-beta-d-glucopyranosyl] ester, 3-O-[beta-d-glucopyranosyl-(1-->2)-beta-d-glucuronopyranosyl] oleanolic acid 28-O-[alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranosyl-(1-->6)-beta-d-glucopyranosyl]ester, 3-O-[beta-d-glucopyranosyl-(1-->2)-beta-d-glucuronopyranosyl] oleanolic acid 28-O-[alpha-l-rhamnopyranosyl-(1-->4)-beta-d-6-O-acetyl glucopyranosyl-(1-->6)-beta-d-glucopyranosyl]ester, 3-O-[beta-d-glucopyranosyl-(1-->3)-beta-d-glucopyranosyl-(1-->3)-alpha-l-arabinopyranosyl] oleanolic acid 28-O-[alpha-l-rhamnopyranosyl-(1-->4)-beta-d-glucopyranosyl-(1-->6)-beta-d-glucopyranosyl] ester and 3-O-[beta-d-glucopyranosyl-(1-->2)-beta-d-glucuronopyranosyl] hederagenin, respectively.     

Chemo-enzymatic synthesis of tetra-, penta-, and hexasaccharide fragments of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->O(CH(2))(6)NH(2) (1), beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (2), beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->O(CH(2))(6)NH(2) (3), and beta-D-Galp-(1-->4)-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (4), representing fragments of the repeating unit of the Streptococcus pneumoniae serotype 14 capsular polysaccharide. Linear intermediate oligosaccharides 5-8 were synthesized via chemical synthesis, followed by enzymatic galactosylation using bovine milk beta-1,4-galactosyltransferase as a catalyst. The title oligosaccharides form suitable compounds for conjugation with carrier proteins, to be tested as potential vaccines in animal models.     

Synthesis of alpha-series ganglioside GM1alpha containing C20-sphingosine

A synthesis of alpha-series ganglioside GM1alpha (III(6)Neu5AcGgOse4Cer) containing C20-sphingosine(d20:1) is described. Glycosylation of 2-(trimethylsilyl)ethyl 2,3,6-tri-O-benzyl-beta-D-galactopyranosyl-(1-->4)-2,3,6-tri-O-benzyl-beta-D-glucopyranoside with the glucosamine donor ethyl 3-O-acetyl-2-deoxy-4,6-O-[(4-methoxyphenyl)methylene]-2-phthalimido-1-thio-beta-D-glucopyranoside furnished a beta-(1-->4)-linked trisaccharide. Reductive cleavage of the p-methoxybenzylidene group followed by intramolecular inversion of its triflate afforded the desired trisaccharide, which was transformed into a trisaccharide acceptor via removal of the phthaloyl and O-acetyl groups followed by N-acetylation. A tetrasaccharide acceptor was obtained by glycosylation of the trisaccharide acceptor with dodecyl 2,3,4,6-tetra-O-benzoyl-1-thio-beta-D-galactopyranoside, followed by removal of the p-methoxybenzyl group. Coupling of the tetrasaccharide acceptor with ethyl (methyl 4,7,8,9-tetra-O-acetyl-3,5-dideoxy-1-thio-5-trichloroacetamido-D-glycero-D-galacto-2-nonulopyranosid)onate and subsequent radical reduction gave the desired GM1alpha saccharide derivative, which was coupled with (2S,3R,4E)-2-azido-3-O-benzoyl-4-eicosene-1,3-diol after conversion into the imidate.     

Methanolysis of ethyl esters of N-acetyl amino acids catalyzed by cyclosophoraoses isolated from Rhizobium meliloti

Methanolysis of four ethyl esters, N-acetyl-L-phenylalanine ethyl ester, N-acetyl-l-tyrosine ethyl ester, N-acetyl-l-tryptophan ethyl ester, and ethyl phenylacetate was catalyzed by a mixture of microbial cyclooligosaccharides termed cyclosophoraoses isolated from Rhizobium meliloti. Cyclosophoraoses [cyclic-(1-->2)-beta-d-glucans, collectively 'Cys'] are a mixture of large-ring molecules consisting of various numbers of glucose residues (17-27) linked by beta-(1-->2)-glycosidic bonds. Cys as a catalytic carbohydrate enhanced the methanolysis about 233-fold for N-acetyl-L-tyrosine ethyl ester in comparison with a control. The effect of dry organic solvents on the methanolysis of N-acetyl-L-tyrosine ethyl ester was investigated by high-performance liquid chromatography (HPLC), and it was found that the rate enhancement correlated closely with the hydrophobicity of the solvent.     

Saponins and acylated saponins from Dizygotheca kerchoveana

Four new triterpenoid saponins were isolated from the leaves and stem of branches of Dizygotheca kerchoveana along with seven known ones. The new saponins were respectively characterized as 3-O-[beta-D-glucopyranosyl-(1-->3)]-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl echinocystic acid, 3-O-[beta-D-glucopyranosyl-(1-->3)]-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl echinocystic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester, 3-O-[beta-D-3-O-trans-p-coumaroyl-glucopyranosyl-(1-->3)]-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl echinocystic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester and 3-O-[beta-d-3-O-cis-p-coumaroyl-glucopyranosyl-(1-->3)]-[beta-D-glucopyranosyl-(1-->2)]-alpha-L-arabinopyranosyl echinocystic acid 28-O-[alpha-L-rhamnopyranosyl-(1-->4)-beta-D-glucopyranosyl-(1-->6)-beta-D-glucopyranosyl] ester. Their structures were elucidated by 1D and 2D NMR experiments, FAB-MS as well as chemical means.     

Yeast Catalysed Reduction of β-Keto Esters (1): Factors Affecting Whole-Cell Catalytic Activity and Stereoselectivity

Six yeasts were studied for their ability to reduce ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) stereoselectively. Five species reduced the substrate to ethyl (S)-4-chloro-3-hydroxybutanoate of high (92–99%) optical purity. With glucose-grown cells, substrate reduction could only be demonstrated when growth was oxygen-limited, whereas xylose-grown Pichia capsulata could be grown under conditions of oxygen excess without losing its reducing ability. Zygosaccha-romyces rouxii exhibited high enantioselectivity (≥98% ee (S)-enantiomer) under all conditions tested, whilst in P. capsulata, a novel switch was observed from producing mainly the (R)-enantiomer using glucose as co-substrate to producing mainly the (R)-enantiomer using 2-propanol as co-substrate. This switch was correlated with a change in reduction predominantly from an NADPH-dependent dehydrogenase system to an NADH-dependent system. In the production of ethyl (R)-4-chloro-3-hydroxybutanoate with P. capsulata, the enantioselectivity was also found to depend upon growth conditions. With glucose-grown cells, higher enantioselectivity was observed using cells harvested in stationary phase (93–94% ee) compared with cells harvested in exponential phase (43–60% ee). Growing P. capsulata with xylose rather than glucose as the major source of carbon for growth resulted in an eight-fold increase in the specific rate of ethyl (R)-4-chloro-3-hydroxybutanoate production using 2-propanol as co-substrate, although enantioselectivity was slightly reduced (65–81% ee) compared with the maximum achieved with glucose-grown cells. The effect of growth on xylose could also be correlated with enhanced activity of an NADH-dependent (R)-selective dehydrogenase system.     

Refinement of the structures of cell-wall glucans of Schizosaccharomyces pombe by chemical modification and NMR spectroscopy

Alkali extraction and methylation analyses in the 1970s revealed that the cell walls of the yeast Schizosaccharomyces pombe contain a (1-->3)-alpha-d-glucan, a (1-->3)-beta-d-glucan, a (1-->6)-beta-d-glucan, and a alpha-galactomannan. To refine the structures of these polysaccharides, cell-wall glucans of S. pombe were extracted, fractionated, and analyzed by NMR spectroscopy. S. pombe cells were treated with 3% NaOH, and alkali-soluble and insoluble fractions were prepared. The alkali-insoluble fraction was treated with 0.5M acetic acid or Zymolyase 100T to yield an alkali-insoluble, acetic acid-insoluble fraction, an alkali-insoluble, Zymolyase-insoluble fraction, and an alkali-insoluble, Zymolyase-soluble fraction. (13)C NMR and 2D-NMR spectra disclosed that the cell wall of S. pombe is composed of three types of glucans, specifically, a (1-->3)-alpha-d-glucan, a (1-->3)-beta-d-glucan, which may either be linear or slightly branched, and a highly branched (1-->6)-beta-d-glucan, in addition to alpha-galactomannan. The highly branched (1-->6)-beta-d-glucan was identified by selective periodate degradation of side-chain glucose as a highly (1-->3)-beta-branched (1-->6)-beta-d-glucan with more branches than that of Saccharomyces cerevisiae. Flexibility of these polysaccharides in the cell wall was analyzed by (13)C NMR spectra in D(2)O. The data collectively indicate that (1-->3)-alpha- and (1-->3)-beta-d-glucans are rigid and contribute to the cell shape, while the highly branched (1-->6)-beta-d-glucan and alpha-galactomannan are flexible.     

Chemo-enzymatic synthesis of a tetra- and octasaccharide fragment of the capsular polysaccharide of Streptococcus pneumoniae type 14

The chemo-enzymatic synthesis is described of tetrasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (1) and octasaccharide beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->3)-beta-D-Galp-(1-->4)-beta-D-Glcp-(1-->6)-[beta-D-Galp-(1-->4)]-beta-D-GlcpNAc-(1-->O(CH(2))(6)NH(2) (2), representing one and two tetrasaccharide repeating units of Streptococcus pneumoniae serotype 14 capsular polysaccharide. In a chemical approach, the intermediate linear trisaccharide 3 and hexasaccharide 4 were synthesized. Galactose residues were beta-(1-->4)-connected to the internal N-acetyl-beta-D-glucosamine residues by using bovine milk beta-1,4-galactosyltransferase. Both title oligosaccharides will be conjugated to carrier proteins to be tested as potential vaccines in animal models.     

Structural investigation of a fucoidan containing a fucose-free core from the brown seaweed, Hizikia fusiforme

A fucoidan, obtained from the hot-water extract of the brown seaweed, Hizikia fusiforme, was separated into five fractions by DEAE Sepharose CL-6B and Sepharose CL-6B column chromatography. All five fractions contained predominantly fucose, mannose and galactose and also contained sulfate groups and uronic acid. The fucoidans had MWs from 25 to 950 kDa. The structure of fraction F32 was investigated by desulfation, carboxyl-group reduction, partial hydrolysis, methylation analysis and NMR spectroscopy. The results showed that the sugar composition of F32 was mainly fucose, galactose, mannose, xylose and glucuronic acid; sulfate was 21.8%, and the MW was 92.7 kDa. The core of F32 was mainly composed of alternating units of -->2)-alpha-D-Man(1--> and -->4)-beta-D-GlcA(1-->, with a minor portion of -->4)-beta-D-Gal(1--> units. The branch points were at C-3 of -->2)-Man-(1-->, C-2 of -->4)-Gal-(1--> and C-2 of -->6)-Gal-(1-->. About two-thirds of the fucose units were at the nonreducing ends, and the remainder were (1-->4)-, (1-->3)- and (1-->2)-linked. About two-thirds of xylose units were at the nonreducing ends, and the remainder were (1-->4)-linked. Most of the mannose units were (1-->2)-linked, and two-thirds of them had a branch at C-3. Galactose was mainly (1-->6)-linked. The absolute configurations of the sugar residues were alpha-D-Manp, alpha-L-Fucp, alpha-D-Xylp, beta-D-Galp and beta-D-GlcpA. Sulfate groups in F32 were at C-6 of -->2,3)-Man-(1-->, C-4 and C-6 of -->2)-Man-(1-->, C-3 of -->6)-Gal-(1-->, C-2, C-3 or C-4 of fucose, while some fucose had two sulfate groups. There were no sulfate groups in either the GlcA or xylose residues.     

Yeast Catalysed Reduction of β-Keto Esters (1): Factors Affecting Whole-Cell Catalytic Activity and Stereoselectivity

Six yeasts were studied for their ability to reduce ethyl 4-chloroacetoacetate (ethyl 4-chloro-3-oxobutanoate) stereoselectively. Five species reduced the substrate to ethyl (S)-4-chloro-3-hydroxybutanoate of high (92-99%) optical purity. With glucose-grown cells, substrate reduction could only be demonstrated when growth was oxygen-limited, whereas xylose-grown Pichia capsulata could be grown under conditions of oxygen excess without losing its reducing ability. Zygosaccha-romyces rouxii exhibited high enantioselectivity (≥98% ee (S)-enantiomer) under all conditions tested, whilst in P. capsulata, a novel switch was observed from producing mainly the (R)-enantiomer using glucose as co-substrate to producing mainly the (R)-enantiomer using 2-propanol as co-substrate. This switch was correlated with a change in reduction predominantly from an NADPH-dependent dehydrogenase system to an NADH-dependent system. In the production of ethyl (R)-4-chloro-3-hydroxybutanoate with P. capsulata, the enantioselectivity was also found to depend upon growth conditions. With glucose-grown cells, higher enantioselectivity was observed using cells harvested in stationary phase (93-94% ee) compared with cells harvested in exponential phase (43-60% ee). Growing P. capsulata with xylose rather than glucose as the major source of carbon for growth resulted in an eight-fold increase in the specific rate of ethyl (R)-4-chloro-3-hydroxybutanoate production using 2-propanol as co-substrate, although enantioselectivity was slightly reduced (65-81% ee) compared with the maximum achieved with glucose-grown cells. The effect of growth on xylose could also be correlated with enhanced activity of an NADH-dependent (R)-selective dehydrogenase system.