Allylmalonamide as a bivalent linker: Synthesis of biantennary GM(3)-saccharide-Keyhole limpet hemocyanin glycoconjugate and the immune response in mice
W. Zou et al., Allylmalonamide as a bivalent linker: Synthesis of biantennary GM(3)-saccharide-Keyhole limpet hemocyanin glycoconjugate and the immune response in mice, GLYCOCON J, 16(9), 1999, pp. 507-515
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.