Solid-Phase Enzymatic Synthesis of a Sialyl Lewis X Tetrasaccharide on a Sepharose Matrix

Thiopyridyl Sepharoses with different linker arm lengths were prepared from epoxy Sepharose 6B by reaction first with 1,8-diamino-3,6-dioxaoctane and then with, sucessively, diethoxy-3-cyclobutene-1,2-dione (squaric acid diethyl ester) and 1,8-diamino-3,6-dioxaoctane in several cycles, followed by reaction of the obtained amino Sepharoses with, successively, thiobutyrolactone and 2,2'-dithiopyridine. The thiopyridyl Sepharoses were reacted with the glucosamine derivative 2-(3'-mercaptobutyrylamido)ethyl 2-acetamido-2-deoxy-beta-D-glucopyranoside, giving GlcNAc Sepharoses with different linker lengths. Enzymatic galactosylation of these with beta-(1-4)-galactosyltransferase and UDP-galactose gave yields varying between 70 and 98%, and there was a clear correlation between linker length and yield. A GlcNAc Sepharose with a long linker was then used in a solid-phase synthesis of a sialyl Le x tetrasaccharide. The three required enzymes (galactosyl-, sialyl, and fucosyltransferase) and nucleotide sugars were reacted consecutively with the GlcNAc Sepharose, giving, after cleavage from Sepharose with DTT, the free sialyl Le x tetrasaccharide derivative in a 57% total yield after purification.