11] Coupling of aldobionic acids to proteins using water-soluble carbodiimide

Abstract

Synthetic carbohydrate–protein conjugates have proved to be valuable tools in immunochemical investigations. This chapter describes a method for the preparation of such conjugates. It involves the oxidation of an oligosaccharide to an aldonic acid derivative, which is then coupled to free amino groups in the protein using a water-soluble carbodiimide as the activating agent. The oligosaccharide aldonic acid can be prepared in essentially quantitative yield by oxidation of the oligosaccharide with alkaline iodine solution or with bromide–lead carbonate solution. The oligosaccharide aldonic acid reacts with the carbodiimide to form an o-acylisourea derivative, which reacts further with the free amino functions of the protein to form amide linkages. When bovine serum albumin (BSA) was used as the carrier protein, about 35 of the 60 amino groups present in the protein were substituted when the protein/carbohydrate ratio was 1: 500. By this procedure, several di- and trisaccharide aldonic acids have been linked to different proteins such as BSA , ovalbumin, and concanavalin A.