Facile Synthesis and Metabolic Incorporation of m-DAP Bioisosteres Into Cell Walls of Live Bacteria

Abstract

Bacterial cell walls contain peptidoglycan (PG), a scaffold that provides proper rigidity to resist lysis from internal osmotic pressure and a barrier to protect cells against external stressors. It consists of repeating sugar units with a linkage to a stem peptide that becomes highly crosslinked by cell wall transpeptidases (TP). Because it is an essential component of the bacterial cell, the PG biosynthetic machinery is often the target of antibiotics. For this reason, cellular probes that advance our understanding of PG biosynthesis and its maintenance can be powerful tools to reveal novel drug targets. While synthetic PG fragments containing L-Lysine in the 3rd position on the stem peptide are easier to access, those with meso-diaminopimelic acid (m-DAP) pose a severe synthetic challenge. Herein, we describe a solid phase synthetic scheme based on the widely available Fmoc-protected L-Cysteine building block to assemble meso-cystine (m-CYT), which mimics key structural features of m-DAP. To demonstrate proper mimicry of m-DAP, cell wall probes were synthesized with m-CYT in place of m-DAP and evaluated for their metabolic processing in live bacterial cells. We found that m-CYT-based cell wall probes were properly processed by TPs in various bacterial species that endogenously contain m-DAP in their PG. We anticipate that this strategy, which is based on the use of inexpensive and commercially available building blocks, can be widely adopted to provide greater accessibility of PG mimics for m-DAP containing organisms.