Chemoenzymatic synthesis of biotinylated nucleotide sugars as substrates for glycosyltransferases

The enzymatic oxidation of uridine 5'-diphospho-alpha -D-galactose (UDP-Gal ) and uridine 5'-diphospho-N-acetyl-alpha -D-galactosamine (UDP-GalNAc) wit h galactose oxidase was combined with a chemical biotinylation step involvi ng biotin-epsilon -amidocaproylhydrazide in a one-pot synthesis. The novel nucleotide sugar derivatives uridine 5'-diphospho-6-biotin-epsilon -amidoca proylhydrazino-alpha -D-galactose (UDP-6-biotinyl-Gal) and uridine 5'-dipho spho-6-biotin-epsilon -amidocaproythydrazino-N-acetyl-alpha -D-galactosomin e (UDP-6-biotinyl-GalNAc) were synthesized on a 100-mg scale and characteri zed by mass spectrometry (fast atom bombardment and matrix-assisted laser d esorption/ionization time of flight) and one/two dimensional NMR spectrosco py. It could be demonstrated for the first time, by use of UDP-6-biotinyl-G al as a donor substrate, that the human recombinant galactosyltransferases beta 3Gal-T5, beta 4Gal-T1, and beta 4Gal-T4 mediate biotinylation of the n eoglycoconjugate bovine serum albumin-beta -aminophenyl N-acetyl-beta -D-gl ucosaminide (BSA - (GlcNAc)(17)) and ovalbumin. The detection of the biotin tag transferred by beta 3Gal-T5 onto BSA-(GlcNAc)(17) with streptavidin - enzyme conjugates gave detection limits of 150 pmol of togged GlcNAc in a W estern blot analysis and I pmol of togged GlcNAc in a microtiter plate assa y. The degree of Gal-biotin tag transfer onto agalactosylated hybrid N-glyc ans present at the single glycosylation site of ovalbumin was dependent on the Gal-T used (either beta 3Gal-T5, beta 4Gal-T4, or beta 4Gal-T1), which indicates that the acceptor specificity may direct the transfer of the Gal- biotin tag. The potential of this biotinylated UDP-Gal as a novel donor sub strate for human galactosyltransferases lies in the targeting of distinct a cceptor structures, for example, under-galactosylated glycoconjugates, whic h are related to diseases, or In the quality control of glycosylation of re combinant and native glycoproteins.