Synthesis of a fluorescent acceptor substrate for glycosyltransferases involved in the assembly of O-antigens of enterohemorrhagic Escherichia coli O157 and O5.

The assembly of the repeating units of O-antigens in Gram negative bacteria is catalyzed by specific glycosyltransferases. Previously we used GlcNAc/GalNAcα-diphosphate-phenoxyundecyl as natural acceptor substrate analogs in assays of the transfer of radioactive sugars by bacterial glycosyltransferases. In order to develop new, fluorescence based assays we have synthesized a fluorescent acceptor P¹-[11-(anthracen-9-ylmethoxy)undecyl]-P²-(2-acetamido-2-deoxy-α-D-galactopyranosyl) diphosphate and have shown that the compound was an excellent acceptor for glucosyltransferase WbdN from Escherichia coli (E.

Biochemical characterization of WbdN, a β1,3-glucosyltransferase involved in O-antigen synthesis in enterohemorrhagic Escherichia coli O157.

The enterohemorrhagic O157 strain of Escherichia coli, which is one of the most well-known bacterial pathogens, has an O-antigen repeating unit structure with the sequence [-2-d-Rha4NAcα1-3-l-Fucα1-4-d-Glcβ1-3-d-GalNAcα1-]. The O-antigen gene cluster of E. coli O157 contains the genes responsible for the assembly of this repeating unit and includes wbdN.

11-Phenoxyundecyl phosphate as a 2-acetamido-2-deoxy-α-d-glucopyranosyl phosphate acceptor in O-antigen repeating unit assembly of Salmonella arizonae O:59.

A synthesis of 11-phenoxyundecyl phosphate and its biochemical transformation (using GlcNAc-P transferase from Salmonella arizonae O:59 membranes catalysing transfer of GlcNc-phosphate from UDP-GlcNAc on lipid-phosphate) into P(1)-11-phenoxyundecyl, P(2)-2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate are described.

Chemical synthesis of dolichyl phosphates, their analogues and derivatives and application of these compounds in biochemical assays.

Several methods for simple and efficient chemical synthesis of dolichyl phosphates and their analogues and derivatives are briefly summarized with a special emphasis on chemical modification of phosphoryl group and preparation of dolichyl phosphates labelled at the omega-end and at the gamma-isoprene unit of the isoprene chain by fluorescent groups, 2-aminopyridine and 1-aminonaphtalene residues. Additionally, data on biochemical assays with application of the compounds mentioned above are presented.