In recent years glycobiology has emerged as an important area in which chemists and biochemists continue to make key contributions. Glycomolecules include simple sugars, oligo- and polysaccharides, glycoproteins and lipids. The enzymes responsible for creation of glycosidic bonds to various sugars are called glycosyltransferases, a broad group of enzymes for which mechanisms and inhibitors are only now starting to be known and identified. Glycosyltransferase inhibitors are important tools for better understanding the enzyme mechanism(s) and provide opportunities for manipulating the biology that arises due to glycosyltransferase activity.

In our mechanistic work (see Project Two) we have shown that sialyltransferases (one class of glycosyltransferase) catalyze a reaction in which the transition state develops a positive charge on the flattened transferring sugar residue and places the leaving group in a position above the plane of the flattened sugar. We have devised synthetic routes to inhibitor molecules that are designed to mimic the transition state structure, so called “transition state analog inhibitors”. We have made both geometric and charge transition state analogs and are now turning towards new hybrid compounds.

Our synthetic strategy involves as the key step cyclopropanation of suitably functionalized alkenes, in either inter- or intra- molecular reactions to afford cyclopropanes suitably functionalized for transformation into our target compounds.

The mechanisms of sialyltransferase enzymes are only now being revealed via the application of kinetic isotope effect (KIE) experiments. The KIE arises when a heavy isotope labeled molecule reacts at a different rate than a light labeled substrate, for example, deuterium (2H) labeled substrates can react at a different rate than protium (1H) labeled ones. In this work, the observation of a KIE provides information on the structure of the enzyme-bound transition state both in terms of charges that develop and geometry, and as an additional benefit, one can deduce information about the kinetic mechanism of the enzyme.

As a prerequisite to making KIE measurements, we synthesize isotope labeled substrates for the enzyme using a combination of chemical and multi-enzyme synthetic methods that allows us to avoid the use of protecting groups during the synthesis. In this way we have synthesized substrates with 2H, 3H, 14C, 13C, and 18O isotope labels at strategic positions in the molecule. As part of our work, we cloned and overexpressed a number of the enzymes required for this synthesis, and we have cloned and expressed human sialyltransferase in insect cells to facilitate our work.

Our studies into sialyltransferases have revealed that the reaction mechanism involves formation of a flattened oxocarbenium ion at the sugar residue, with little if any nucleophilic participation by the other substrate. The leaving group cytidine monophosphate (CMP) is relatively distant from the glycosidic carbon to which it was formerly attached. In recent work, we have used heavy atom 18O isotope effects to probe interactions between the enzyme and the phosphate oxygens of the CMP leaving group.

Neuronal nicotinic acetylcholine receptors (nAChr's) are ion channels which transmit an electrical signal upon binding of acetylcholine in the synaptic cleft. This class of receptor derives its name from the observation that it can be activated by treatment with nicotine. It represents an important target for the development of treatments for addictions, and is also thought to be important in cognition, memory and as a target for treatment of dementia associated with Alzheimer's.

In our collaboration with Dr. Roger Papke, we seek to determine what structural features on a given agonist or antagonist are responsible for the efficacy, potency, and/or receptor subtype specificity. One of the challenges in this project are that high resolution three dimensional structures of intact receptors are not available. However, the combination of synthesis, molecular biology and electrophysiology is a powerful way to allow us to characterize structure/function relationships between the receptor and its ligands.