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Effect of the peptide moiety of Lipid II on bacterial transglycosylase.

The rise of antibiotic-resistant bacteria, such as VRE (vancomycin resistant Enterococcus), MRSA (methicillin resistant Staphylococcus aureus), MDR-TB, and XDR-TB (multidrug resistant and extensively drug-resistant tuberculosis) has stimulated the development of new antibiotics and new strategies to tackle the problem of antibiotic resistance. In bacterial cell-wall biosynthesis, the bifunctional enzyme on the surface, such as E. coli PBP1b, possesses two catalytic domains for transpeptidase and transglycosylase activity. The transpeptidase responsible for the cross-linking of peptidoglycans has long been a target for antibiotic discovery and development. 2] In contrast, no antibiotics, except moenomycin used in animal feeds, have been developed to target the transglycosylase (TGase), which catalyzes the polymerization of Lipid II to form a peptidoglycan (Figure 1). Because TGase is essential for bacteria and does not have a eukaryotic counterpart, it is thought to be an attractive target for antibiotic discovery and development. Lipid II (1) consists of a disaccharide, a pyrophosphate, an undecaprenol lipid tail, and an oligopeptide (d-lactyl-lalanyl-g-d-glutamyl-meso-diaminopimelyl (or l-lysyl-dalanyl-d-alanine) moieties (Figure 1). Notably, the first GlcNAc linked to lactic acid to form a ubiquitous component in cell walls is also called MurNAc (N-acetylmuramic acid), which is only found in bacteria. Currently, little information is available regarding the interaction of Lipid II with TGase, presumably because of difficulties in the synthesis and modification of Lipid II (1). In this report, we systematically study how the various Lipid II constituents interact with TGases. Although the peptide moiety in Lipid II is for the transpeptidation process, the last cross-linking step between neighboring peptidoglycan chains catalyzed by transpeptidase, the function of the peptide moiety in Lipid II towards TGase is still unclear. We have recently reported a method for the synthesis of Lipid II and derivatives, and the X-ray crystal structure of MRSA TGase in complex with a Lipid II analogue to elucidate the mechanism of Lipid II polymerization. Herein, we describe the preparation of Lipid II analogues with varying peptide moieties for evaluation as TGase substrates, as an effort toward development of new antibiotics. Five analogues 1–5 were designed (Figure 2). Compounds 1–3 contain a peptide chain (R) prepared by sequential removal of the two amino acids from the end of the peptide stem; 4 has no peptide chain, but a methyl group instead; and 5 was prepared from 1 by adding the fluorophore, 6-[N-(7-nitrobenz-2-oxa-1,3-diazol4-yl)amino]hexanoyl (NBD-X), to the e-NH2 group of the lysine residue. Our synthetic strategy for 1–3 is depicted in Scheme 1. The lactyl group was installed at the C3 hydroxy group of 6 to obtain 7 in 72 % yield. The anomeric thiol group in 7 was removed and the product subjected to phosphorylation to give 8 as a single diastereomer. Deprotection of the C4 OTBS group in 8, followed by ester hydrolysis gave the acid intermediate, which was individually coupled with peptides and subsequently debenzylated to give 12. Finally, conjugation of 12 with an undecaprenyl phosphate (C55P), and global deprotection gave the corresponding 1–3 in 31–48% yields over two steps. Compound 4 was directly prepared from 6 Figure 1. Formula of Lipid II (1) for bacterial transglycosylase (TGase) and transpeptidase (TPase).