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. 2018 Aug 1;140(30):9458-9465.
doi: 10.1021/jacs.8b03304. Epub 2018 Jul 23.

Synthesis of Functionalized N-Acetyl Muramic Acids To Probe Bacterial Cell Wall Recycling and Biosynthesis

Kristen E DeMeester, Hai Liang, Matthew R Jensen, Zachary S Jones, Elizabeth A D'Ambrosio, Samuel L Scinto, Junhui Zhou, Catherine L Grimes

Synthesis of Functionalized N-Acetyl Muramic Acids To Probe Bacterial Cell Wall Recycling and Biosynthesis

Kristen E DeMeester et al. J Am Chem Soc. .

Abstract

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.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1. E. coli QKU cells fluorescently modified with the lactic acid NAM derivative 14 and WGA co-staining
DIC and maximum intensity projection 2D images from super-resolution SIM Z-stacks of E. coli QKU cells treated with 14, fosfomycin, IPTG for 45 min and clicked with Alk488 (green) and then stained with tetramethylrhodamine WGA 561 (red) of whole cell (white arrow, top) and dividing cell (white arrow, bottom) (scale bars, 10 μm). Images are representative of a minimum of three fields viewed per replicate with at least two technical replicates and the experiment was conducted in three biological replicates.
Figure 2
Figure 2. E. coli QKU cells modified with 8 for tetrazine-TCO ligation
DIC and 2D images from super-resolution SIM Maximum Intensity Projection Images Z-stacks of E. coli QKU cells treated with 8 and IPTG for 20 min and clicked with TCO-TAMRA (yellow) (scale bars, 2 μm). Images are representative of a minimum of three fields viewed per replicate with at least two technical replicates and the experiment was conducted in at least three biological replicates.
Figure 3
Figure 3. L. acidophilus labeling with azido UDP NAM 3b and WGA costaining
DIC and 2D Maximum Intensity Projection images from super-resolution SIM Z-stacks L. acidophilus cells treated with 3b, clicked with Alk488 (green) and treated with WGA-561 (red) (scale bars, 10 μm). Images are representative of a minimum of three fields viewed per replicate with at least two technical replicates and the experiment was conducted in at three biological replicates.
Scheme 1
Scheme 1
Peptidoglycan Recycling and Biosynthetic Pathways
Scheme 2
Scheme 2
Library of 2-N NAM and UDP NAM probes
Scheme 3
Scheme 3
Synthesis of lactic acid N3 functionalized NAM a. Ac2O, DMAP, pyridine (85%), b. PhSH, SnCl4, DCM, reflux (54%), c. NaOMe, MeOH, r.t. (quant), d. PhCH(OMe)2, TsOH, DMF, 70°C (72%), e. NaH, 15, DMF (57%), f. K2CO3, MeI, DMF, r.t. (80%), g. DDQ, DCM/H2O (quant), h. MsCl, pyridine/DCM (80%), i. 1. NaN3, DMF, 70°C; 2. IRA H+, H2O, 95°C; 3. TCCA (12% over 3 steps).
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