N-Acetylneuraminic acid (NeuAc) is a sialic acid which constitutes terminal positions in glycoproteins and glycolipids. It is part of the antigenic determinant of many forms of cancer (S. Hakomori, Chem. Phys. Lipids. 42, 209 (1986)) and hence a cancer "vaccine" could be made from an appropriate multivalent macromolecular form of NeuAc. To explore the possibility of inducing anti-NeuAc antibodies, we have synthesized polyvalent conjugates of $\alpha$NeuAc or NeuAc$\alpha$2-3Gal$\beta$1-4Glc (sialyl2-3lactose) on protein carriers. This was accomplished by reductive amination with 2-oxoethyl $\alpha$-NeuAc or with the concealed aldehyde of the reducing sugar (glucose) onto the lysine residues of the proteins Bovine Serum Albumin (BSA) and Tetanus Toxoid (TT). A new procedure involving a Michael-type addition employed the $\epsilon$-amino groups of the lysine residues in a 1,4 nucleophilic addition to the acrylamide functional group in two derivatives of NeuAc; the N-acryloylaminoethylthiopropyl glycoside, and the N-acryloylsialyl2-3lactosylamine. This technique does not require the use of reagents and was performed under mild basic conditions. Polyacrylamide copolymers were also synthesized by radical co-polymerization of acrylamide with NeuAc monomers, providing a polymer (Mw $\approx$ 100 kDa) with pendant NeuAc. Reactive monomers giving different spacer arms between the sugar and the polymer backbone were developed, including the allyl glycoside, a more reactive N-acryloylaminoethylthiopropyl glycoside and several lactose derivatives. The polymers were found to have a better shelf life than the proteins, and had the advantage of sharing only the NeuAc moiety. Some of the NeuAc/protein conjugates were used to immunize rabbits, and the antibodies formed were screened with polyacrylamide copolymers with pendant NeuAc. The polymer with spacer was found to react better with the antibodies in immunoprecipitation and enzyme-linked immunosorbent assay (ELISA). Inhibition of ELISA experiments were done using synthesized derivatives of NeuAc to determine the binding specificity of the antibodies. It was found that the antibodies recognized the glycerol side chain, acid function, spacer, and to a lesser extent, the acetamido function of NeuAc. The same methodology was used to map the binding site of the lectin from Triticum vulgaris (called Wheat germ agglutinin, WGA) which binds NeuAc. Nuclear magnetic resonance (nmr) studies were undertaken to determine the acidity constants (pK$\sb{\rm a}$) and conformation of several NeuAc derivatives. Also studied was the lactone of NeuAc$\alpha$2-3Gal$\beta$1-4Glc which is believed to be responsible for the immunogenicity of certain cancer cells (T. Dohi, G. Nores and S. Hakomori, Cancer Res. 48, 5680 (1988)). This work was complemented by molecular modelling using MM2. A model was proposed for the conformation of the lactone which is consistent with the n.m.r. evidence.
| Identifer | oai:union.ndltd.org:uottawa.ca/oai:ruor.uottawa.ca:10393/5987 |
| Date | January 1990 |
| Creators | Laferrière, Craig A. |
| Publisher | University of Ottawa (Canada) |
| Source Sets | Université d’Ottawa |
| Detected Language | English |
| Type | Thesis |
| Format | 198 p. |
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