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). While synthetic PG fragments containing L-Lysine in the 3 rd 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 widely available building blocks 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. Additionally, we have used hybrid Quantum Mechanical/Molecular Mechanical (QM/MM) and Molecular Dynamics (MD) simulations to explore the influence of m-DAP analogs on the PG crosslinking. The results showed that crosslinking mechanism of transpeptidases occurred through a concerted process. 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.

Metabolic Processing of Selenium-based Bioisostere of meso-diaminopimelic Acid in Live Bacteria

“…We had previously shown that the carboxylic acid in the sidechain of the acyl-acceptor was critical for recognition and crosslinking in M. smegmatis . 27 To confirm those results in the context of the seleno-based probes, we synthesized two additional stem peptide analogs ( TriLys and TriSeLys ), in which the acyl-acceptor amino group is retained. Consistently, cellular fluorescence labeling decreased considerably for both probes ( Fig.…”

“…Initially, it should be pointed out that starting points for MM and QM/MM simulations were obtained from our previous study. 27 In our previous QM/MM mechanistic studies involving Ldt systems, 48 we described that the catalytic mechanism starts with the formation of the ionic-pair, by a proton transfer from Cys346 to His328. Therefore, we started our calculation from product state of Ldt inhibition reaction with ionic-pair of Cys/His catalytic residues.…”

“…1A ). 27 While m -CYT was well tolerated as a surrogate for m -DAP in live bacteria, as demonstrated by its metabolic incorporation into the PG, we recognized that it may be important to retain the same number of atoms in the sidechain, which m -CYT does not. Moreover, the reduction of the disulfide bond in m -CYT may prove to be a challenge in certain conditions such as assays that require reducing agents and in intracellular compartments of mammalian cells.…”

Structure Activity Relationship of the Stem Peptide in Sortase A mediated Ligation from Staphylococcus aureus

“…Our group, and others, previously showed that synthetic acyl-acceptor strands modified with an N-terminal fluorophore are well tolerated by cell wall crosslinking enzymes (PBPs and Ldts). [37][38][39][40][41][42][43] We envisioned that a similar tolerability would enable the structure-activity relationship of srtA. As wildtype S. aureus displays pentaglycine cross-bridge (G5) appended to its 3 rd position lysine sidechain, we first sought to determine the importance of the glycine cross-bridge length.…”

Non-invasive Fluorescence Imaging of Gut Commensal Bacteria in Live Mice

“…Instead, we envisioned that administration of a synthetic stem peptide analog conjugated to a NIR fluorophore would circumvent this challenge (Figure 4A). We [41][42][43][44] , and others [45][46][47] , have shown that treatment of bacterial cells with stem peptide analogs also result in their incorporation the PG by PG-related transpeptidases (Figure 4B). The principal advantage of stem peptide analogs is that our laboratory previously showed that the N-terminus of a tetra-or penta-stem peptide analog demonstrates tolerability to large chemical modifications.…”