Efficient coupling of glycopeptides to proteins with a heterobifunctional reagent

Abstract

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.

Analysis of the molecular interaction of glycosylated proteins with rabbit liver asialoglycoprotein receptors using surface plasmon resonance spectroscopy

“…Since the chance of multivalent binding with liver ASGPRs is expected to be higher for highly galactosylated BSA, more energy would be required for dissociation. In addition, clustering effect of liver ASGPRs has been observed in the Triton-solubilized form as well as on the hepatocyte surface [16][17][18][19]. These findings would support the present results.…”

Neoglycoconjugates from synthetic tetra- and hexasaccharides that mimic the terminus of the O-PS of Vibrio cholerae O:1, serotype Inaba

“…The reaction was set up so that at the onset of the reaction the molar 25/BSA ratio was 100:1. With the 59 lysine groups in BSA 22, 23 (molecular mass 66,430 Da) we deemed this excess to be sufficient to achieve the required higher carbohydrate-onto-protein loading, while maintaining a reasonable reaction rate. When the molar carbohydrate/BSA ratio of ∼6:1 had been reached (3 h, as shown by SELDI-TOF MS), a portion of the reaction mixture was withdrawn and processed.…”

Eukaryotic glycosylation: whim of nature or multipurpose tool?

“…logical testing [269][270][271][272]. Coupling of purified glycans from natural sources is thus no longer the only approach to recruit complex oligosaccharides as tools for receptor search [273][274][275][276], whose concepts and successes will be presented in the section on glycan recognition. When no further processing will reduce the size of the Man 5 GlcNAc 2 chain, it is committed to the generation of high-mannose-type glycans, which are common products of yeasts but are also present in animal glycoproteins such as ovalbumin.…”