Chemoenzymatic Synthesis of Biotinylated Nucleotide Sugars as Substrates for Glycosyltransferases

摘要

The enzymatic oxidation of uridine 5'-diphospho-#alpha#-D-galactose (UDP-Gal) and uridine 5'-diphospho-N-acetyl-#alpha#-D-galactosamine (UDP-GalNAc) with galactose oxidase was combined with a chemical biotinylation step involving biotin-#edsilon#-amidocaproylhydrazino-#alpha#-D-galactose (UDP-6-biotinyl-Gal) and uridine 5'-diphospho-6-biotin-#edsilon#-amidocaproylhydrazino-N-acetyl-#alpha#-D-galactose (UDP-6-biotinyl-GalNAc) were synthesized on a 100-mg scale and characterized by mass spectrometry (fast atom bombardment and matrix-assisted laser desorption/ionization time of flight) and one/two dimensional NMR spectroscopy. It could be demonstrated for the first time, by use of UDP-6-biotinyl-Gal as a donor substrate, that the human recombinant galactosyltransferases #beta#3Gal-T5, #beta#4Gal-T1, and #beta#4Gal-T4 meidate biotinylation of the neoglycocon-jugate bovine serum albumin -p-aminophenyl N-acetyl-#beta#-D-glucosaminide (BSA-(GicNAc)_(17)) and ovalbumin. The detection of the biotin tag transferred by #beta#3Gal-T5 onto BSA-(GicNAc)_(17) with streptavidin-enzyme conjugates gave detection limits of 150 pmol of tagged GICNAc in a Western blot analysis and 1 pmol of tagged GicNAc in a microtiter plate assay. The dagree of Gal-biotin tag transfer onto agalactosylated hybrid N-glycans 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 substrate for human galactosyltransferases lies in the targeting of distinct acceptor structures, for example, under-galactosylated glycoconjgates, which are related to diseases, or in the quality glycoconjugates, which are related to diseases, or in the quality control of glycosylation of recombinant and native glycoproteins.