Research Summary, Rob White and Coworkers
In our Bioorganic Research Program, chemical principles and techniques are combined with microbiological and biochemical methods to solve mechanistic problems at the interface of chemistry with biological disciplines. We are interested in the three areas outlined below. There is considerable synergy between projects. For example, carbanion chemistry is a common theme for both synthetic and mass spectral studies, and the isotopically labelled compounds utilized in metabolic studies also provide key substrates for mass spectral investigations.
1. Amino Acid Catabolism in Anaerobic Bacteria
The anaerobes Fusobacterium nucleatum and Fusobacterium varium are normal constituents of the human gastrointestinal tract. F. nucleatum has been implicated in periodontal disease and there is considerable interest in the in vivo effects of butyrate, the main metabolite product excreted by fusobacteria. While amino acids serve as important energy sources, there is considerable ambiguity in assigning the pathways employed, a challenging task given the recognized genetic heterogeneity within fusobacteria. Our metabolic studies concentrate on substrate-induced changes in the exometabolome, the use of isotopes to define substrate-product relationships, and the identification of specific enzyme-catalyzed steps within a pathway. Our identification of fundamental metabolism in anaerobes is necessary to define the complicated bacteria-host interactions.
2. Synthesis of Potential Enzyme Inhibitors / Antibiotics
The peptidoglycan component of the bacterial cell wall is formed from sugars and amino acids via a multistep biosynthetic pathway. The clinically successful β-lactam (penicillins and cephalosporins) and glycopeptide (vancomycin) antibiotics act by inhibiting the final cross-linking step, a metabolic process that has no counterpart in mammalian metabolism. As a result, these antibiotics normally display minimal side-effects. In response to the widespread problem of antibiotic resistance in pathogenic bacteria, other steps in peptidoglycan biosynthesis are attractive potential targets for novel inhibitors based on substrate and transition-state structures. Methodology developed in our laboratory is being utilized to synthesize novel amino acids that may influence several enzyme-catalyzed steps in peptidoglycan biosynthesis and thereby exhibit antibacterial properties.
3. Chemistry of Gas-Phase Ions Using Tandem Mass Spectrometry (MS/MS)
Negative-ion mass spectrometry is important for the analysis of samples containing drugs, herbicides and by-products of wastewater disinfection. While negative ions are now generated easily by electrospray ionization (ESI), collision-induced dissociation (CID) reactions of negative ions are both less studied and less understood than those of positive ions. Early negative-ion, high-energy MS/MS studies focussed on a limited range of structures, while recent studies are mostly on peptides. Our initial work, however, shows that a rich chemistry is associated with the fragmentations of carboxylate anions and that subtle structural differences can open up different fragmentation pathways. Using chemical synthesis, we are constructing several series of anions that differ in a rational systematic fashion to define the relationship between structure and anion reactivity. Our mechanistic interpretations of the low-energy CID spectra of these anions are leading to an increased understanding of their chemical properties in the gas phase. For example, the predominant fragmentation reaction of the β-alanine anion has been identified as a McLafferty-type rearrangement.
