Staphylococcus aureus Penicillin-Binding Protein 2 Can Use Depsi-Lipid II Derived from Vancomycin-Resistant Strains for Cell Wall Synthesis

Abstract

Vancomycin-resistant Staphylococcus aureus (S. aureus) (VRSA) uses depsipeptide-containing modified cell-wall precursors for the biosynthesis of peptidoglycan. Transglycosylase is responsible for the polymerization of the peptidoglycan, and the penicillin-binding protein 2 (PBP2) plays a major role in the polymerization among several transglycosylases of wild-type S. aureus. However, it is unclear whether VRSA processes the depsipeptide-containing peptidoglycan precursor by using PBP2. Here, we describe the total synthesis of depsi-lipid I, a cell-wall precursor of VRSA. By using this chemistry, we prepared a depsi-lipid II analogue as substrate for a cell-free transglycosylation system. The reconstituted system revealed that the PBP2 of S. aureus is able to process a depsi-lipid II intermediate as efficiently as its normal substrate. Moreover, the system was successfully used to demonstrate the difference in the mode of action of the two antibiotics moenomycin and vancomycin.

Keywords: antibiotics; enzymes; lipids; peptides; vancomycin.

Figure 1

Structure of bacterial cell wall building blocks, wild-type lipid II (1) and depsi-lipid II (2), and their mode of interaction with vancomycin.

Figure 2

Late-stage cell wall synthesis of S. aureus. In vancomycin-resistant S. aureus. The d-Ala-d-Ala-terminal of the pentapeptide moiety is replaced with d-Ala-d-Lac. M=MurNAc; G=N-acetylglucosamine (GlcNAc); MurG=N-acetylglucosamyl transferase; FemA, FemB, and FemX: peptidyltransferases.

Figure 3

Retrosynthetic analysis for depsi-lipid I and its analogue. Bn=benzyl, Teoc=2-(trimethylsilyl)ethoxycarbonyl, TMSE=2-(trimethylsilyl)ethyl, Ac=acyl.

Figure 4

Synthesis of the phosphomuramyl peptide 8. Reagents and conditions: a) l-alanine-2-(trimethylsilyl)ethyl ester, diisopropylethylamine (DIPEA), 1-hydroxybenzotirazole (HOBt), benzotriazolyl-1-oxy-tripyrrolidinophosphonium hexafluorophosphate (PyBOP), THF/CH₂Cl₂, RT, 59 %; b) 10 % Pd/C, H₂ gas, MeOH/EtOAc, RT, 83 %; c) 1 H-tetrazole, dibenzyl-N,N-diisopropylphosphoramidite, −40 to −10 °C, then meta-chloroperbenzoic acid (m-CPBA), −40 °C, CH₂Cl₂, 56 %; d) TBAF, THF, 0 °C to RT, 92 %.

Figure 5

Synthesis of the depsi-lipid I and its C35 analogue. Reagents and conditions: a) compound 9, O-(7-azabenzotriazol-1-yl)-tetramethyluroium hexafluorophosphate (HATU), DIPEA, DMF, 0 °C, 1 h, 81 %; b) 10 % Pd/C, H₂ gas, MeOH, RT, 30 min, 94 %; c) 80 % AcOH(aq), RT, 4 d, 91 %; d) pyridine, N,N′-carbonyldiimidazole (CDI), THF/DMF (4:1), RT, 3 h, then MeOH; undecaprenylphosphate ammonium salt 5 or 6, 1 H-tetrazole, THF/DMF (4:1), RT, 2 d, (78 %); f) TBAF, THF/DMF, 0 °C to RT, 2 d, 23 % (depsi-lipid I, 3) or 34 % (depsi-lipid I analogue 4).

Figure 6

Inhibitory effect of vancomycin and moenomycin on the PBP2 reaction. ○ and ▵=lipid II, • and ▴=depsi-lipid II. PG: peptidoglycan.