Novel enzymatic synthesis of spacer-linked P(k) trisaccharide targeting for neutralization of Shiga toxin.

A novel alkyl spacer-conjugated derivative of P(k) trisaccharide (P(k)), one of the active receptors of Shiga toxins (Stxs; Stx1 and Stx2) produced by pathogenic Escherichia coli (STEC), was designed and synthesized by a combination of cellulase-mediated condensation from Trichoderma reesei and α1,4-galactosyltransferase (LgtC) from Neisseria gonorrhoeae. The specific activity of N. gonorrhoeae LgtC was 66U/mg, which was 13-fold higher than that from N. meningitidis expressed in E. coli. 5-trifluoroacetamidopentyl-β-P(k) (TFAP-P(k)) was synthesized (yield of 86%, based on the amount of TFAP-lactose added) and its binding to Stx1a-B and Stx2a-B was evaluated. The dissociation constants (KDs) of Stx1a-B and Stx2a-B to the spacer-linked P(k), immobilized on a CM5 sensor chip, were 6.8×10(-6) M (kon=4.1×10(1)M(-1)S(-1), koff=2.8×10(-4)S(-1)) and 2.2×10(-5)M (kon=3.9×10(2)M(-1)S(-1), koff=8.6×10(-3)S(-1)), respectively. This result suggests that the monovalent P(k)-derivative, conjugated to a pentylamino group, represents a promising Stx-neutralizing agent. This cellulase-mediated condensation using cellulase and glycosyltransferase is a valuable tool for the synthesis of spacer-linked oligosaccharide.