Chemoenzymatic Synthesis of Bioorthogonal Peptidoglycan Derivatives: Tools to Remodel Bacterial Cell Wall
Authors: Jones Zachary 
Published: November 2023

The cell wall of bacteria is surrounded by a polymer known as peptidoglycan (PG). This material serves as a protective coat in Gram‐positive and Gram‐negative bacteria alike. The PG consists of alternating sugar monomers of N‐acetyl muramic acid (NAM) and N‐acetyl glucosamine (NAG) with layers linked by pentapeptide chains. In order to synthesize the PG, bacteria have a complex relay of enzymes that involve synthesizing NAG and NAM, attaching Uridine‐diphosphate (UDP) as a carrier molecule, adding the pentapeptide, and ultimately transferring the monomer across the cell membrane to be incorporated into the mature PG.

Glycan chemical synthesis, particularly of UDP sugar probes, has proven difficult to achieve with a reasonable yield. The objective of these experiments is to synthesize large‐scale quantities of UDP‐NAM derivatives and to utilize these chemical probes for incorporation into the PG of bacteria. By tracking the dynamics of PG, specifically the breakdown of these chemical probes, innate immune reactions and responses may be better understood.

The method we have developed utilizes the recycling enzymes anomeric NAM/NAG kinase (AmgK) and uridylyltransferase (MurU) in order to chemoenzymatically synthesize UDP‐NAM derivatives. Due to the relaxed substrate specificity of these enzymes, the NAM derivatives are able to be accepted into the active site so that the muramyl lactol will be converted to the charged UDP sugar donor.

In order to utilize AmgK and MurU, the enzymes were expressed and purified from Escherichia coli.1 The natural substrate NAM and its derivatives were then combined with AmgK and ATP to phosphorylate the anomeric carbon. The reaction was monitored by mass spectrometry and MurU was then added to facilitate the Uridine‐monophosphate addition.

Purification was carried out by high‐pressure liquid chromatography (HPLC) coupled to liquid chromatography mass spectrometry (LC/MS). A hydrophilic interaction liquid chromatography (HILIC) column was used along with a stationary phase of 92.5% Acetone/ H2O to 100% H2O as the mobile phase. Fractions were collected and the products were characterized by LC/MS and nuclear magnetic resonance (NMR) imaging.

Currently, large scale synthesis, purification, and characterization has been completed of the natural UDP‐NAM substrate, along with substrates which contain either an azide or alkyne at the 2‐N‐acetyl position. Additionally, the 2‐azido‐UDP‐NAM has successfully been incorporated into the PG of Lactobacillus acidophilus.

From these results, we can confirm that utilizing AmgK and MurU to create UDP‐NAM derivatives is viable, and thus we can expand the library of PG probes for cell wall remodeling. The next step is to attempt to label other biologically relevant bacteria, visualize the digestion of the labeled PG, and continue to explore the utility of these glycan probes.

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Acknowledgements:

Special thanks goes to Dr. Catherine Leimkuhler Grimes, Kristen DeMeester, Hai Liang, and the rest of the Grimes group for their support and collaboration on this project. Thanks to Mr. David Plastino for funding, as well as the Undergraduate Research Office at the University of Delaware. Thanks to Dr. PapaNii Asare‐Okai and COBRE for mass spectrometry facilities support.