Allylmalonamide as a bivalent linker: synthesis of biantennary GM3-saccharide--keyhole limpet hemocyanin glycoconjugate and the immune response in mice.
A biantennary GM3-saccharide (sialyllactoside) derivative (4) was constructed using allylmalonic acid as a bivalent linker, both carboxylic acids of which were condensed with 3-aminopropyl lactoside (2) prior to enzymatic sialylation with a fusion enzyme. While ozonolysis of its allyl group generated a saccharide having a terminal aldehyde (6), we were unable to couple 6 directly to protein by reductive amination. However, extension of the spacer by means of introducing a maleimide group to 6 through its aldehyde group to give 7 enabled the latter to be successfully coupled to thiolated proteins. 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 obtained by immunizing mice with the biantennary sialyllactoside-KLH conjugate (13) together with MPL adjuvant were analyzed by ELISA. Using several structurally related saccharide-HSA conjugates as screening antigens, it was concluded that anti-sialyllactoside antibodies, both IgG and IgM, were effectively raised. This was further supported by competitive inhibition experiments using lactoside (1), sialyllactoside (8) and biantennary sialyllactoside (4) as inhibitors.
Zou
W
W
Institute for Biological Sciences, National Research Council of Canada, Ottawa. Wei.Zou@nrc.ca
Abraham
M
M
Gilbert
M
M
Wakarchuk
W W
WW
Jennings
H J
HJ
D000276:Adjuvants, Immunologic / Q000737:chemistry*; D000498:Allyl Compounds / Q000737:chemistry*; D000818:Animals; D000917:Antibody Formation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D005679:G(M3) Ganglioside / Q000737:chemistry*; D006001:Glycoconjugates / Q000138:chemical synthesis* / Q000276:immunology; D006433:Hemocyanins / Q000737:chemistry*; D009682:Magnetic Resonance Spectroscopy; D008314:Malonates / Q000737:chemistry*; D013058:Mass Spectrometry; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data
D000276:Adjuvants, Immunologic; D000498:Allyl Compounds; D003432:Cross-Linking Reagents; D005679:G(M3) Ganglioside; D006001:Glycoconjugates; D008314:Malonates; C409212:allylmalonic acid; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin