Synthesis of ganglioside analogues as biological probes for enzymes that modify sialic acids

Abstract

Ganglioside GM3, a glycosphingolipid found in the outer membrane of all mammalian cells, is expressed at much higher concentrations than normal in cancer cells. It has been reported that GM3 can inhibit signal transduction via the EGF receptor kinase, ultimately down regulating cell growth. In contrast to this, de-N-acetyl GM3, another ganglioside that has been found in a number of cancer cell lines, is reported to be a potent promoter of cell growth. Evidence suggests that GM3 and de-N-acetyl GM3 are inter-converted by enzymes that remove and replace the N-acetyl group on sialic acid. In this thesis, the syntheses of a number of simplified analogues of gangliosides are described, including some in which an inter-glycosidic oxygen atom is replaced by sulfur. Synthesis of O-linked ganglioside analogues was achieved by both a totally chemical route and also by a chemo-enzymatic route, involving the use of a recombinant construct of trypanosoma cruzi trans-sialidase for glycosylation with sialic acid. A novel strategy for the synthesis of ganglioside analogues that contain a sialic acid α(2-->3) galactose thioglycosidic linkage, is also described. These compounds were evaluated as substrates and inhibitors for Clostridium perfringens neuraminidase and Trypanosoma cruzi trans-sialidase. Some of the ganglioside analogues described herein were used to develop biochemical assays for detecting the putatative de-N-acetylase and N-acetyltransferase activities that would be required for the inter-conversion of gangliosides GM3 and de-N-acetyl GM3.