Using chemical synthesis to analyze polymerization and inhibition mechanisms of peptidoglycan glycosyltransferases

Peptidoglycan glycosyltransferases (PGTs) are essential enzymes for bacterial survival and have tremendous potential as antibiotic targets. This potential has not yet been realized for two main reasons: (1) mechanistic studies of these enzymes have been hampered by a lack of available substrates to monitor the enzymatic activity; (2) the only known inhibitor that targets glycosyltransferases is moenomycin A, which has excellent antibiotic activity, but poor pharmacokinetic properties. The polyisoprenoid lipid phosphoglycerate moiety is mainly responsible for the poor pharmacokinetic properties, however its role in enzymatic inhibition and antibiotic activity remains ambiguous. This thesis presents studies aimed at confronting the above issues by two principal routes: (1) syntheses of Lipid IV, a different substrate from Lipid II, in order to explore detailed mechanistic and structural information of PGTs; (2) use of the degradation/reconstruction method to synthesize moenomycin analogs with modifications on the lipid phosphoglycerate moiety to understand the structural requirements for enzyme inhibition and antibiotic activity.;First, the first total synthesis of heptaprenyl-Lipid IV was accomplished. The challenging β-(1,4) linkage between two N-acetyl glucosamine derivatives was constructed regio- and stereo-selectively using sulfoxide glycosylation method. The different reactivity of equatorial hydroxyls in the glycosyl donor and acceptor was observed and utilized for chemoselective glycosylations, which allowed a highly convergent synthesis of the tetrasaccharide backbone of Lipid IV. The Tin(II) pyrophosphate coupling method developed for the synthesis of Lipid I was employed successfully to achieve the pyrophosphate bond in Lipid IV. Then, an efficient SDS-PAGE method was developed to separate the polyglycan chains generated from the glycosyltransfer reaction. With both Lipid II and Lipid IV substrates, the method permitted us to analyze the product distribution pattern and evaluate the polymerization process in details. Lastly, the degradation and reconstruction of moenomycin A was illustrated. An efficient synthesis of a chiral 2-O-alkyl glycerol was completed and utilized in the synthesis of chiral 2-O-moenocinyl glycerate. Moenomycin analogs with single change in the lipid and phosphoric acid segments were obtained enlisting the degradation/reconstruction method. Comparison of in vitro and in vivo performances of the analogs assists the investigation of the function of the lipid phosphoglycerate in enzyme inhibition and antibiotic activity.