Synthesis of galactofuranose-based acceptor substrates for the study of galactan biosynthesis.

摘要

Polysaccharides play important roles in all organisms, from humans to bacteria. Their significance is highlighted by their prevalence: carbohydrate polymers are the most abundant organic materials on Earth. Glycan biosynthesis occurs without a template, and is regulated by the delicate balance of enzymes and building blocks. The molecular details of polysaccharide assembly remain poorly understood. A rule of glycobiology is that one enzyme is responsible for one type of glycosidic linkage, and yet a number of examples exist of one enzyme functioning to produce polymers containing two types of linkages. One example is the glycosyltransferase GlfT2. GlfT2 is responsible for the assembly of an important component of the mycobacterial cell wall known as galactan. The galactan is a linear polymer of galactofuranose sugars connected via alternating beta-(1,5) and beta-(1,6) bonds. The mechanistic details of GlfT2 activity are unknown, in part because of the challenge associated with obtaining appropriate substrates. Here, I describe the production of a large collection of chemical tools that allowed investigations into GlfT2 activity and mechanism.;The putative natural substrate for GlfT2 is a tetrasaccharide glycolipid. To explore the substrate requirements for GlfT2, I developed a synthetic route using ruthenium-catalyzed cross-metathesis as the penultimate transformation. This strategy allowed independent variation of glycone and aglycone to generate glycolipid acceptors. With the appropriate synthetic tools, we determined that a single galactofuranose residue with a lipid of sufficient length is the minimal substrate for GlfT2 polymerization activity. We showed that GlfT2 is processive and bifunctional, and achieves this activity with a single active site.;Furthermore, my synthetic strategy allowed exploration of the importance of the lipid functionality of the substrates. Data generated by varying acceptor lipid length suggests that GlfT2 controls galactan length using a bivalent tethering mechanism, and a minimum lipid length is required for processive polymerization.;Finally, I have synthesized trisaccharide substrates containing natural alternating linkages, as well as substrates with repeating, non-alternating glycosidic linkages. In concert with computation and crystallography, we shall use these tools to probe the importance of regiochemistry to GlfT2 activity and to explore the impact of alternating regiochemsitry on galactan structure and conformation.