Uridine diphosphate N-acetyl muramic acid (UDP NAM) is a critical intermediate in bacterial peptidoglycan (PG) biosynthesis. As the primary source of muramic acid that shapes the PG backbone, modifications installed at the UDP NAM intermediate can be used to selectively tag and manipulate this polymer via metabolic incorporation. However, synthetic and purification strategies to access large quantities of these PG building blocks, as well as their derivatives, are challenging. A robust chemoenzymatic synthesis was developed using an expanded NAM library to produce a variety of 2-N-functionalized UDP NAMs. In addition, a synthetic strategy to access bio-orthogonal 3-lactic acid NAM derivatives was developed. The chemoenzymatic UDP synthesis revealed that the bacterial cell wall recycling enzymes MurNAc/GlcNAc anomeric kinase (AmgK) and NAM α-1 phosphate uridylyl transferase (MurU) were permissive to permutations at the two and three positions of the sugar donor. We further explored the utility of these derivatives in the fluorescent labeling of both Gram (-) and Gram (+) PG in whole cells using a variety of bio-orthogonal chemistries including the tetrazine ligation. This report allows for rapid and scalable access to a variety of functionalized NAMs and UDP NAMs, which now can be used in tandem with other complementary bio-orthogonal labeling strategies to address fundamental questions surrounding PG's role in immunology and microbiology.
Bibliographical noteFunding Information:
We thank Dr. Jeffrey Caplan, the Director of Bioimaging, and the Delaware Biotechnology Institute for assistance with super-resolution microscopy. For financial support, this project was supported by the NIH U01 Common Fund program with grant number U01CA221230-01; Delaware COBRE program with a grant from the National Institute of General Medical Sciences, NIGMS P20GM104316-01A1. C.L.G. is a Pew Biomedical Scholar and Cottrell Scholar and thanks the Pew Foundation and the Research Corporation for Science Advancement. K.E.D. and M.R.J. thank the NIH for support through a CBI training grant, 5T32GM008550. For instrumentation support, the Delaware COBRE and INBRE programs supported this project with a grant from the National Institute of General Medical Sciences-NIGMS (5 P30 GM110758-02, P20GM104316-01A1, and P20 GM103446) from the National Institutes of Health.
© 2018 American Chemical Society.