C.A.G.M. Weijers

Enantioselective hydrolysis of aryl, alicyclic and aliphatic epoxides by Rhodotorula glutinis

Abstract Enantioselective epoxide hydrolysis by yeasts has been demonstrated for the hydrolysis of several aryl, alicyclic and aliphatic epoxides by a strain of Rhodotorula glutinis. High enantioselectivity was obtained in the hydrolysis of methyl substituted arylryl and aliphatic epoxides whereas selectivity towards terminal epoxides in all cases was lower. Homochiral vicinal diols were formed from several methyl substituted epoxides and also from meso epoxides. Kinetic resolution of trans-1-phenyl-1,2-epoxypropane was studied in more detail.

Epoxide hydrolases from yeasts and other sources: versatile tools in biocatalysis

Abstract Major characteristics, substrate specificities and enantioselectivities of epoxide hydrolases from various sources are described. Epoxide hydrolase activity in yeasts is discussed in more detail and is compared with activities in other microorganisms. Constitutively produced bacterial epoxide hydrolases are highly enantioselective in the hydrolysis of 2,2- and 2,3-disubstituted epoxides. A novel bacterial limonene-1,2-epoxide hydrolase, induced by growth on monoterpenes, showed high activities and selectivities in the hydrolysis of several substituted alicyclic epoxides. Constitutively produced epoxide hydrolases are found in eukaryotic microorganisms. Enzymes from filamentous fungi are useful biocatalysts in the resolution of aryl- and substituted alicyclic epoxides. Yeast epoxide hydrolase activity has been demonstrated for the enantioselective hydrolysis of various aryl-, alicyclic- and aliphatic epoxides by a strain of Rhodotorula glutinis . The yeast enzyme, moreover, is capable of asymmetric hydrolysis of meso epoxides and performs highly enantioselective resolution of unbranched aliphatic 1,2-epoxides. Screening for other yeast epoxide hydrolases shows that high enantioselectivity is restricted to a few basidiomycetes genera only. Resolution of very high substrate concentrations is possible by using selected basidiomycetes yeast strains.

Glycosyltransferase-catalyzed synthesis of bioactive oligosaccharides

Mammalian cell surfaces are all covered with bioactive oligosaccharides which play an important role in molecular recognition events such as immune recognition, cell-cell communication and initiation of microbial pathogenesis. Consequently, bioactive oligosaccharides have been recognized as a medicinally relevant class of biomolecules for which the interest is growing. For the preparation of complex and highly pure oligosaccharides, methods based on the application of glycosyltransferases are currently recognized as being the most effective. The present paper reviews the potential of glycosyltransferases as synthetic tools in oligosaccharide synthesis. Reaction mechanisms and selected characteristics of these enzymes are described in relation to the stereochemistry of the transfer reaction and the requirements of sugar nucleotide donors. For the application of glycosyltransferases, accepted substrate profiles are summarized and the whole-cell approach versus isolated enzyme methodology is compared. Sialyltransferase-catalyzed syntheses of gangliosides and other sialylated oligosaccharides are described in more detail in view of the prominent role of these compounds in biological recognition.

Enantiomeric composition of lower epoxyalkanes produced by methane-, alkane-, and alkene-utilizing bacteria

Abstract The enantiomeric composition of 1,2-epoxypropane, 1-chloro-2,3-epoxypropane, 1,2-epoxybutane and trans-2,3-epoxybutane formed by 23 bacterial strains from the respective alkenes was determined using complexation gas chromatography. Bacteria grown on methane produced racemic epoxyalkanes and strains grown on other gaseous alkanes in general also formed racemic mixtures. Bacteria grown on 1-alkenes formed epoxyalkanes stereospecifically, but the enantiomeric composition depended on both the organism used and on the epoxyalkane formed.

ENANTIOSELECTIVITIES OF YEAST EPOXIDE HYDROLASES FOR 1,2-EPOXIDES

Abstract Kinetic resolution of homologous series of unbranched 1,2-epoxyalkanes (C-4 to C-12), 1,2-epoxyalkenes (C-4, C-6 and C-8), a 2,2-dialkylsubstituted epoxide (2-methyl-1,2-epoxyheptane) and a benzyloxy-substituted epoxide (benzyl glycidyl ether) was investigated using resting cells of 10 different yeast strains. Biocatalysts with excellent enantioselectivity (E>100) and high initial reaction rates (>300 nmol/min/mg dry weight) were found for the 2-monosubstituted aliphatic epoxides C-6 to C-8. Yeast strains belonging to the genera Rhodotorula , Rhodosporidium and Trichosporon all preferentially hydrolyzed ( R )-1,2-epoxides with retention of configuration. The epoxide hydrolases of all the yeast strains are membrane-associated.

ENANTIOSELECTIVE HYDROLYSIS OF UNBRANCHED ALIPHATIC 1,2-EPOXIDES BY RHODOTORULA GLUTINIS

Abstract Epoxide hydrolase catalysed resolution of aliphatic terminal epoxides has been demonstrated for the hydrolysis of a homologous range of unbranched 1,2-epoxyalkanes by the yeast Rhodotorula glutinis . Both enantioselectivity and reaction rate were strongly influenced by the chain length of the epoxide used. Enantioselectivity showed an optimum in the hydrolysis of 1,2-epoxyhexane (E=84). Resolution of (±)-1,2-epoxyhexane resulted in ( S )-1,2-epoxyhexane (e.e.>98%, yield=48%) and ( R )-1,2-hexanediol (e.e.=83%, yield=47%).

GM3, GM2 and GM1 mimics designed for biosensing : chemoenzymatic synthesis, target affinities and 900 MHz NMR analysis

Undec-10-enyl, undec-10-ynyl and 11-azidoundecyl glycoside analogues corresponding to the oligosaccharides of human gangliosides GM3, GM2 and GM1 were synthesized in high yields using glycosyltransferases from Campylobacter jejuni. Due to poor water solubility of the substrates, the reactions were carried out in methanol-water media, which for the first time were shown to be compatible with the C. jejuni alpha-(2-->3)-sialyltransferase (CST-06) and beta-(1-->4)-N-acetylgalactosaminyltransferase (CJL-30). Bioequivalence of our synthetic analogues and natural gangliosides was examined by binding to Vibrio cholerae toxin and to the B subunit of Escherichia coli heat-labile enterotoxin. This bioequivalence was confirmed by binding mouse and human monoclonal antibodies to GM1 and acute phase sera containing IgM and IgG antibodies to GM1 from patients with the immune-mediated polyneuropathy Guillain-Barré syndrome. The synthesized compounds were analyzed by 1D and 2D 900 MHz NMR spectroscopy. TOCSY and DQF-COSY experiments in combination with 13C-1H correlation measurements (HSQC, HMBC) were carried out for primary structural characterization, and a complete assignment of all 1H and 13C chemical shifts is presented.

Biocatalytic resolution of 1,2-epoxyoctane using resting cells of different yeast strains with novel epoxide hydrolase activities

Yeast strains (187) from 25 different genera were screened for the hydrolysis of 1,2-epoxyoctane. Epoxide hydrolase activity was found for 54 yeast strains. Asymmetric hydrolysis of 1,2-epoxyoctane was found for 8 yeast strains belonging to the genera Trichosporon, Rhodotorula, and Rhodosporidium. All these strains preferentially hydrolysed (R)-1,2-epoxyoctane to (R)-1,2-octanediol. Excellent enantioselectivity (E > 200) for 1,2-epoxyoctane is reported for the first time. Substrate concentration of 500 mM were used without any decrease in enzyme activity.

Continuous production of enantiopure 1,2-epoxyhexane by yeast epoxide hydrolase in a two-phase membrane bioreactor.

Abstract A two-phase membrane bioreactor was developed to continuously produce enantiopure epoxides using the epoxide hydrolase activity of Rhodotorula glutinis. An aqueous/organic cascade, hydrophilic, hollow-fiber membrane bioreactor was used: (1) to carry out large-scale resolution of epoxides, (2) to continuously extract residual enantiopure epoxides from the aqueous phase, and (3) to separate inhibitory formed diol from the yeast cells contained in the aqueous phase. Dodecane was employed to dissolve-feed epoxide as well as to extract residual epoxide. 1,2-Epoxyhexane was used as a model substrate. By use of this membrane bioreactor, enantiopure (S)-1,2-epoxyhexane (>98% enantiomeric excess) was obtained with a volumetric productivity of 3.8 g l−1 h−1. The continuous-production system was operated for 12 days and resulted in 38 g enantiopure (S)-1,2-epoxyhexane.

Chiral resolution of 2,3-epoxyalkanes by Xanthobacter Py2

SummaryWith propene-grown cells of Xanthobacter Py2 it was possible to resolve racemic mixtures of 2,3-epoxyalkanes. Only 2S-forms were metabolized by this organism, resulting in pure 2R-2,3-epoxyalkanes. Chiral resolution was obtained with trans-2,3-epoxybutane, trans-2,3-epoxypentane and cis-2,3-epoxypentane. Xanthobacter Py2 was however not able to discriminate between the enantiomeric forms of 1,2-epoxyalkanes, resulting in the complete degradation of both chiral forms of 1,2-epoxyalkanes.