Enzymic glycosylation
seems to be an attractive possibility for modification of glycoproteins'. In a recent project, we have been engaged in sialylation of glycoproteins in order to increase their plasmatic lifetime*. In N-glycoproteins, oligosaccharide and polypeptide components are linked by the residue, 2-acetamido-l-N-(L-aspart-4-oyl)-2-deoxy-P-D-glucopyranosyIamine (6). Therefore, we selected the glycosylasparagine derivative 6 as a model compound in studies of enzymic glycosylation, and we report herein the enzymic synthesis of the sialyl glycopeptide 8 from the well-known3 derivative 6 via the galactosylated intermediate 7 on a semipreparative scale (50 pmol). Two enzymic steps were involved, i.e., D-galactosylation catalyzed by immobilized N-acetyllactosamine synthase (EC 2.4.1.90), and sialylation catalyzed by soluble CMP-N-acetylneuraminate:
/3-D-galactopyranosyl-(l-+4)-Z-acetamido-2-deoxy-P_D-glucopyranose cu-(2-+6)-sialyltransferase (EC 2.4.99.1). The preparation of several sialyloligosaccharides by enzymic synthesis has already been reported by different groups 4,5, but this is the first report of an enzyme-catalyzed synthesis of a glycopeptide. The structure of compounds 7 and 8 are not known to be present in any glycoproteins, but the glycosylasparagine derivatives 6, 7, and 8 have been isolated from the urine of patients suffering from aspartylglucosaminuria6-s.
Compound 6 was synthesized by published procedures with minor modifications. Thus, silver azide, which was formerly used9, was advantageously replaced by tetrabutylammonium azide for the conversion of chloride 1 into azide 2 (70% yield). The 'H-n.m.r. spectrum of 6 was identical with the one reported by Vliegenthart et aZ.'O for compound 6 isolated from natural sources. However, we observed, for the H-l resonance of the /3-D-GlcpNAc residue, a broad signal instead of the expected doublet. This anomaly was previously reported by Brisson and Carver" for N,N'-*To whom correspondence should be addressed. +Recipient of a fellowship supported by the CNRS and Elf-Aquitaine.