Chemoenzymatic Synthesis of Biotinylated Nucleotide Sugars as Substrates for Glycosyltransferases

The enzymatic oxidation of uridine 5′‐diphospho‐α‐ D ‐galactose (UDP‐Gal) and uridine 5′‐diphospho‐ N ‐acetyl‐α‐ D ‐galactosamine (UDP‐GalNAc) with galactose oxidase was combined with a chemical biotinylation step involving biotin‐ε‐amidocaproylhydrazide in a one‐pot synthesis. The novel nucleotide sugar derivatives uridine 5′‐diphospho‐6‐biotin‐ε‐amidocaproylhydrazino‐α‐ D ‐galactose (UDP‐6‐biotinyl‐Gal) and uridine 5′‐diphospho‐6‐biotin‐ε‐amidocaproylhydrazino‐ N ‐acetyl‐α‐ D ‐galactosamine (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 β3Gal‐T5, β4Gal‐T1, and β4Gal‐T4 mediate biotinylation of the neoglycoconjugate bovine serum albumin–p‐aminophenyl N ‐acetyl‐β‐ D ‐glucosaminide (BSA–(GlcNAc) 17 ) and ovalbumin. The detection of the biotin tag transferred by β3Gal‐T5 onto BSA–(GlcNAc) 17 with streptavidin–enzyme conjugates gave detection limits of 150 pmol of tagged GlcNAc in a Western blot analysis and 1 pmol of tagged GlcNAc in a microtiter plate assay. The degree 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 β3Gal‐T5, β4Gal‐T4, or β4Gal‐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 glycoconjugates, which are related to diseases, or in the quality control of glycosylation of recombinant and native glycoproteins.