Allylmalonamide as a bivalent linker: Synthesis of biantennary GM(3)-saccharide-Keyhole limpet hemocyanin glycoconjugate and the immune response in mice

A biantennary GM(3)-saccharide (sialyllactoside) derivative (4) was constru cted using allylmalonic acid as a bivalent linker, both carboxylic acids of which were condensed with 3-aminopropyl lactoside (2) prior to enzymatic s ialylation with a fusion enzyme. While ozonolysis of its allyl group genera ted a saccharide having a terminal aldehyde (6), we were unable to couple 6 directly to protein by reductive amination. However, extension of the spac er by means of introducing a maleimide group to 6 through its aldehyde grou p to give 7 enabled the latter to be successfully coupled to thiolated prot eins. The average ratios of saccharide to protein were observed to be 35 in KLH conjugate (13) and 9-12 in HSA conjugates (14 and 15). The antisera ob tained by immunizing mice with the biantennary sialyllactoside-KLH conjugat e (13) together with MPL adjuvant were analyzed by ELISA. Using several str ucturally related saccharide-HSA conjugates as screening antigens, it was c oncluded that anti-sialyllactoside antibodies, both IgG and IgM, were effec tively raised. This was further supported by competitive inhibition experim ents using lactoside (1), sialyllactoside (8) and biantennary sialyllactosi de (4) as inhibitors.