The general objective of the proposed work is to demonstrate the ability of carbohydrates to mimic protein secondary structures such as beta-sheets and alpha-helices. The strategy taken to achieve this objective is to design a specific oligosaccharide and test its behavior (through spectroscopy and biochemical assays) in a biological context. A pre-requisite to this, however, is the ability to efficiently synthesize the target oligosaccharide. Significant effort, therefore is exerted in developing methodology for the synthesis of oligosaccharides. The final general objective is to identify molecules that may prove to be leads for novel molecular therapies for application in medicine. (i). Design and Synthesis of an alpha-(1-4)-linked trisaccharide that mimics the Bak BH3 domain. alpha-(1-4)-linked hexose oligosaccharides present functionality in a way similar to a peptidic alpha-helix. Specific peptide sequences can be mimicked by linking appropriate amino acid sidechain mimics to the carbohydrate scaffold. BH(Bcl-2 homology)3 domain peptides are alpha-helices that mediate protein-protein interaction with other Bcl-2 family proteins. BH3 domains have been shown to be sufficient for the induction of apoptosis in model systems. A trisaccharide mimic of the BH3 domain is proposed based on structural and alanine scan data. A novel one-pot glycosylation will be used in the synthesis of the trisaccharide. (ii). Synthesis of the Bcl-xL protein from an E. coli expression system. A recombinant form of human Bcl-xL(residues 1- 209)delta45-84 will be expressed in E. coli as described in the literature. (iii). Evaluation of Carbohydrate binding to Bcl-xL. An assay based on Bcl-xL fluorescence was used to collect alanine scan data for BH3 peptides. The same assay is proposed for measuring the affinity of the trisaccharide mimetic for Bcl-xL. NMR will also be used for determination of the carbohydrate conformation.
