Synthesis and Evaluation of Photoactivatable D-Amino Acid Probes of Bacterial Peptidoglycan Biosynthesis

Ph. D. Thesis.The bacterial cell wall contains peptidoglycan (PG), a mesh-like polymer of glycan strands crosslinked by short peptide chains. PG is essential for bacterial cell viability and as such PG biosynthesis is an important antibiotic target. Despite considerable study, there remain many PG associated enzymes with unknown function, identification of which will allow us to develop the antibiotics of the future. This project is focused on the development of D-amino acid based photoaffinity probes, for the covalent capture of PG associated proteins. Following the evaluation of a number of strategies, a photoactivatable fluorescent D-amino acid probe was successfully synthesised, methoxy coumarin trifluoromethyl diazirine D-alanine or MCTDA.Addionally it can be shown by LCMS that MCTDA is accepted as a substrate by the bacterial transpeptidase enzyme LdtD and that, through the action of LdtD, MCTDA can be successfully incorporated into both isolated muropeptide and whole PG of E. coli. Evaluation of photoactivated capture of PG associated proteins by incorporated MCTDA is ongoing in the laboratory (Figure 1). (please see electronic version of thesis for figure1.) Figure 1: Synthesis of photoactivatable amino acid probe MCTDA and incorporation into PG and muropeptide (E. coli) by LdtD