Efficient coupling of glycopeptides to proteins with a heterobifunctional reagent

A heterobifunctional linking reagent containing a masked aldehydo group and acyl hydrazide was synthesized for coupling of glycopeptides and other amino-containing compounds to proteins. After conversion to acyl azide, the reagent reacts with the amino group of a glycopeptide, and the modified glycopeptide is deacetalized with a weak acid to unmask the aldehydo group, which is then conjugated to bovine serum albumin (BSA) by reductive alkylation with pyridine-borane. The overall reaction scheme proceeds under relatively mild conditions. When the protein amino group was in a large excess (greater than 6-fold) of the aldehyde reagent, the efficiency of conjugation was as high as 88% even at submicromole levels. As a test case for application of this reagent, 6-aminohexyl beta-D-galactopyranoside (Gal-AH) was attached to the linking reagent and conjugated to BSA at various aldehyde-to-protein molar ratios ranging from 25 to 200. The level of O-galactosyl residue incorporated into BSA by this reagent far exceeded that observed in a similar reductive alkylation involving S-galactoside reagents [Lee, R. T., & Lee, Y. C. (1980) Biochemistry 19, 156-163]. By use of the present conjugating procedure, as many as 112 mol of Gal-AH residues were incorporated per mole of BSA, which represents near total modification of the amino groups. Some binding characteristics of the new BSA derivatives were studied in the mammalian hepatic galactose/N-acetylgalactosamine specific lectin system along with other types of BSA derivatives (containing S-galactosyl residues). In general, the behavior of the new derivatives was similar to that of other types. For instance, the affinity increased exponentially at low sugar substitution levels (up to 30 mol of galactosyl residues/mol of BSA), and the slope of exponential increase and affinity at a given sugar substitution level was similar to those of other types.