Synthesis of lactic acid functionalized N-acetylmuramic acid derivatives and progress toward pathogen cell wall labeling

Abstract

Bacterial peptidoglycan (PG), a thick mesh-like polymer of alternating N-acetylmuramic acid (NAM) and N-acetylglucosamine (NAG) crosslinked by a peptide chain, not only protects bacteria from their environments, but also breaks down under various circumstances into fragments that can trigger a response from the innate immune system. One such fragment, muramyl dipeptide (MDP), is a synthetically-derived fragment of PG that is known as the minimal component for activation of Nod2, a cytosolic innate immune receptor. Probes of PG have been a sought after in innate immune research, and previous innovations by Grimes and coworkers have made a bioorthogonally-reactive probe that includes this base structure of MDP by modifications at the 2N-acetyl position and allows metabolic incorporation through a Pseudomonas putida NAM recycling pathway. However, several pathogenic species including Mycobacterium tuberculosis modify PG directly at this position. Therefore, modified tools that functionalize at another position of MDP may be necessary to label these bacteria. ☐ The following thesis outlines an approach to circumventing this issue using a novel probe that is a NAM derivative with an azide group appended to the methyl group of the lactic acid moiety, which is an unprecedented modification in the chemical literature. The probe's incorporation into bacterial PG in whole cells does not match its predecessor, instead, it accumulates in a region of hypothesized PG de novo biosynthesis in dividing cells. It is proposed that the modification results in the accumulation of Lipid I, a membrane-bound precursor to immature PG, by preventing the addition of a NAG residue to form Lipid II via steric hindrance.