IN-VITRO SYNTHESIS OF CDP-D-ABEQUOSE USING SALMONELLA ENZYMES OF CLONED RFB GENES - PRODUCTION OF CDP-6-DEOXY-D-XYLO-4-HEXULOSE, CDP-3,6-DIDEOXY-D-XYLO-4-HEXULOSE AND CDP-3,6-DIDEOXY-D-GALACTOSE, AND ISOLATION BY HPLC

In vitro enzymic synthesis of CDP-D-abequose, CDP-D-[U-C-14]abequose, CDP-6-deoxy-D-xylo-4-hexulose and CDP-3,6-dideoxy-D-xylo-4-hexulose was achieved using enzymes from cell extracts of cultures of Escherichia coli strains harbouring and expressing genes of the rfb gene cluster of Salmonella enterica LT2. From an initial synthesis step, CDP-6-deoxy-D-xylo-4-hexulose was isolated after 30 min reaction, using CDP-D-glucose, NAD and CDP-glucose 4,6-dehydratase, followed by protein precipitation and desalting by gel chromatography (yield 90.6%). From that intermediate, CDP-3,6-dideoxy-D-xylo-4-hexulose was produced in a reaction using NADH and a crude extract containing the required enzymes. CDP-D-abequose synthesis was performed either in the presence of excess NADH and NADPH or using an enzymic system which regenerates low concentrations of the coenzymes. In a two-step reaction, CDP-D-glucose was first converted to CDP-6-deoxy-D-xylo-6-hexulose, then, following addition of the required coenzymes and enzymes, CDP-D-abequose was formed from this intermediary product in a 1-h incubation. Starting from 250 mg CDP-D-glucose, the molar yield of CDP-D-abequose after protein precipitation and HPLC was 82%, corresponding to more than 200 mg. CDP-D-[U-C-14]abequose was synthesised from alpha-D-[(UC)-C-14]glucose 1-phosphate and CTP using purified glucose-1-phosphate cytidylyltransferase in a reaction preceding the later steps. GC-MS and NMR revealed that the hexose part of the end product was 3,6-dideoxy-D-galactose (abequose) and that the corresponding intermediates were 4-keto-6-deoxy-D-xylo-hexose and 4-keto-3,6-dideoxy-D-xylo-hexose, respectively. The synthesized CDP-6-deoxy-D-xylo-4-hexulose exhibited the characteristic ultraviolet light absorption at 318 nm but no corresponding absorption was found for CDP-3,6-dideoxy-D-xylo-4-hexulose. A HPLC technique, where the four CDP-sugars were baseline separated, was developed and used for enzyme assays and for the analysis of synthesized products.