Synthesis and evaluation of potential inhibitors of chondroitin AC lyase from Flavobacterium heparinum

Chondroitin AC lyase from Flavobacterium heparinum degrades chondroitin sulfate glycosamino-glycans via an elimination mechanism, resulting in disaccharides or oligosaccharides with Delta4,5-unsaturated uronic acid residues at their nonreducing end. The syntheses and testing of two potential inhibitors of this lyase are described. Methyl O-(2-acetamido-2-deoxy-beta-D-galactopyranosyl)(1–>4)-alpha-L-threo-hex -4-enopyranoside, 1, has the trigonal geometry at C5 of the uronic acid moiety expected at the transition state, yet retains the ``leaving group{''} sugar moiety. Surprisingly, compound 1 showed no inhibition of the enzyme. The novel 5-nitro sugar, phenyl (5S)-5-nitro-beta-D-xylopyranoside, 2, is a monosaccharide nitro analogue of the natural substrate, with C5 being a carbon acid of pK(a) 8.8. The rate of reprotonation of the anion generated at this center is sufficiently low that the anion of 2 can be used directly in initial steady-state velocity measurements without significant interference from the conjugate carbon acid. The anion of compound 2 was found to be a competitive inhibitor with a K-i value of 5 mM, whereas the conjugate acid had a K-i value of 35 mM.