Metabolic labelling of the carbohydrate core in bacterial peptidoglycan and its applications

Bacterial cells are surrounded by a polymer known as peptidoglycan (PG), which protects the cell from changes in osmotic pressure and small molecule insults. A component of this material, N-acetyl-muramic acid (NAM), serves as a core structural element for innate immune recognition of PG fragments. We report the synthesis of modifiable NAM carbohydrate derivatives and the installation of these building blocks into the backbone of Gram-positive and Gram-negative bacterial PG utilizing metabolic cell wall recycling and biosynthetic machineries. Whole cells are labelled via click chemistry and visualized using super-resolution microscopy, revealing higher resolution PG structural details and allowing the cell wall biosynthesis, as well as its destruction in immune cells, to be tracked. This study will assist in the future identification of mechanisms that the immune system uses to recognize bacteria, glean information about fundamental cell wall architecture and aid in the design of novel antibiotics.

coli MurQ-KU cells pre-treated with 1 for 45 min were then used to invade J774 cells for 1 h. Cells were fixed and treated with Az488 (green) and click conditions. SIM microscopy shows J774 macrophages without any 488 fluorescence. Cellular DNA was S15 labeled with DAPI (blue) (scale bars, 10 m). Images are representative of a minimum of 3 fields viewed per replicate with at least 2 technical replicates and the experiment was conducted in at least 3 biological replicates. b, All cells treated the same as described in

Materials.
All reagents were purchased from Sigma Aldrich, Fisher Scientific, Alfa Aesar or Invitrogen and used without further purification, unless otherwise noted. NMR solvents were purchased from Cambridge Isotope Laboratories, Inc.

Synthetic Procedures.
General Procedures. Unless otherwise noted, all reactions were performed in flame or oven dried flasks equipped with rubber septa, positive pressure of nitrogen, and magnetic stirring. All solvents were anhydrous and transferred via stainless steel syringe or cannula. Reactions were monitored by electrospray ionization liquid chromatography mass spectrometry (ESI LC-MS) and thin layer chromatography (TLC) in which glass plates coated with silica gel (250 m, Silica Gel HL, Sorbent Technologies) were used and visualized with shortwave 254 nm UV light or developed upon heating with panisaldehyde or KMnO 4 . Flash chromatography was carried out on silica gel (60 Å, 40-63 m, Sorbent Technologies). Analytical and semi-preparative HPLC was performed on an Agilent Series 1100 using a Phenomenex ® Luna 5 m C18 column (250 x 10.00 nm). Preparative HPLC purification was performed on a Waters 2767 Sample Manager with HPLC and SQD2 MS using a Sunfire ® Prep C18 OBD 5m 19x100mm or 4.6x50mm columns.
Instrumentation. All NMR spectra were recorded on Bruker AV 400 MHz and AV III 600 MHz spectrometers. Proton chemical shifts were recorded in parts per million (ppm) on the  scale, downfield from tetramethylsilane and referenced from an internal standard S38 of residual protium in the NMR solvents (CHCl 3 :  7.26, D 2 HCOD 3.30). Data for 13 C NMR were reported in ppm downfield from tetramethylsilane and referenced based on the chemical shift from the carbon resonances of the solvent (CDCl 3 :  77.16, CD 3 OD 49.0). NMR data were reported as follows: chemical shift, multiplicity (s = singlet d = doublet, t = triplet, q = quartet, m = multiplet, br = broad), coupling constant in Hz, integration, and assignment based on two dimensional COSY, HSQC and HMBC experiments. Low-resolution mass spectra (LRMS) were obtained on a Shimadzu LCMS 2020 instrument using electrospray ionization (ESI), while high-resolution mass spectra (HR-MS), ESI mode, were obtained on a Thermo Q-Exactive Orbitrap at the Mass Spectroscopy Facility at the Department of Chemistry, University of Delaware. IR spectra were obtained on an IR100 with an ATR probe.  N,N'sulfuryldiimidazole (5.00 g, 25.2 mmol, 1.0 eq) was then suspended in 50 mL of anhydrous dichloromethane at 0C under N 2 . Methyl trifluoromethanesulfonate (2.56 mL, 22.7 mmol, 0.9 eq) was added dropwise over 15 minutes at 0C. The reaction stirred at 0C for 2 hours. The solvent was decanted off and 3-(imidazole-1-sulfonyl)-1-methyl-3H-imidazol-1-ium triflate was isolated as a white solid was washed three times each with 50 mL of cold dichloromethane and dried under high vacuum for 10 minutes and immediately used in the next reaction.
3-(Imidazole-1-sulfonyl)-1-methyl-3H-imidazol-1-ium triflate (9.31 g, 25.2 mmol, 1.0 eq) was dissolved in 30 mL of deionized H 2 O followed by 30 mL of ethyl acetate at 0C. This solution stirred at 0C for 30 minutes. NaN 3 (1.97 g, 30.24 mmol, 1.2 eq) was added slowly and the reaction mixture stirred at 0C for 1 hour. The phases were separated and the organic layer was collected, dried over Na 2 SO 4 , and filtered. The filtrate containing the imidazole-1-sulfonyl azide was used directly in the diazotransfer reaction without further purification.
Preparation of (2S,3R,4R,5S,6R)-6-(acetoxymethyl)-3-azidotetrahydro-2H-pyran-2,4,5-triyl triacetate (4). To the imidazole-1-sulfonyl azide ethylacetate solution (40 mL, S40 25.2 mmol) was added sequentially D glucosamine HCl (6.52 g, 30.2 mmol, 1.2 eq), 82 mL of anhydrous methanol, K 2 CO 3 (6.27 g, 45.4 mmol, 1.8 eq), and anhydrous CuSO 4 (0.0483g, 0.302 mmol, 0.012 eq) at room temperature under N 2 . The reaction continued to stir at room temperature for 16 hours. The reaction was filtered over celite and washed with 20 mL methanol. The solvent was then evaporated under reduced pressure and dried on the high vacuum overnight to yield a light yellow foam. To the light yellow foam was added 52 mL of anhydrous pyridine at 0C under N 2 . To this mixture was added a solution of Ac 2 O (17 mL, 176 mmol, 7.0 eq) and DMAP (0.2709 g, 2.22 mmol, 0.088 eq) dropwise at 0C. The reaction warmed slowly to room temperature and continued to stir for 20 h. Product formation was confirmed by TLC (3:2 hexanes : ethyl acetate r f : 0.5) with PAA staining. The reaction mixture was diluted with 100 mL of deionized water. The water layer was extracted three times with ethyl acetate (200 mL total). The organic layers were combined and washed three times with 1N HCl. The organic layer was dried over Na 2 SO 4 , filtered, and condensed. The brown oily residue was purified by flash chromatography 3:2 hexanes : ethyl acetate to yield tan foam (5.88g, 63% over 4 steps). 1  (2R,3S,4R,5R)-2-(acetoxymethyl)-5-azido-6-hydroxytetrahydro-2H-pyran-3,4-diyl diacetate (5). 4 (4.25 g, 11.4 mmol, 1.0 eq) and hydrazine acetate (1.26 g, 13.7 mmol, 1.2 eq) was dissolved in 11.7 mL of anhydrous N,N dimethylformamide under N 2 . Reaction warmed to 50C and continued to stir under N 2 for 20 minutes. TLC 10% EtOAc/DCM confirmed product formation and disappearance of starting material. Reaction was cooled to room temperature, diluted with 12 mL of dichloromethane. The organic layer was washed with deionized water, saturated NaHCO 3 and brine. The organic layer was dried over Na 2 SO 4 , filtered and condensed to yield a yellow oil. The crude product was purified with flash chromatography with a gradient of 100% DCM to 10% EtOAC/DCM to 20% EtOAc/DCM. The purified product was isolated as a colorless oil (3.40 g, 90%). 1  Anhydrous DMF (233 L, 3.00 mmol, 0.2 eq) was added and the reaction stirred for 35 minutes. (COCl) 2 (2M, 9.01 mL) was added dropwise and the reaction stirred at room temperature for 1.5 hours. The reaction was filtered, washed with DCM and the solvent was evaporated under reduced pressure without heat. The yellow oil was then coevaporated twice with benzene and dried under high vacuum for 20 minutes. In a separate reaction flask, Ag 2 CO 3 (41.4 g, 0.150 mol, 10 eq), AgOTf (0.088 g, 3.45 mmol, 0.23 eq), and anhydrous BnOH (7.77 mL, 75.1 mmol, 5.0 eq) were suspended in 400 mL of anhydrous dichloromethane under N 2 with 4Å molecular sieves. The mixture was cooled to 0C and stirred for 15 minutes. At the same time, the yellow oily intermediate was dissolved in 170 mL of anhydrous dichloromethane under N 2 with 4Å molecular sieves and stirred at room temperature for 15 minutes. The solution containing the intermediate was added dropwise to the reaction flask. The reaction slowly warmed to room temperature and continued to stir for 15 hours. The reaction mixture was filtered over celite. Product formation was confirmed by LC/MS and TLC (30% EtOAc/Hex). The organic layer was washed three times with deionozed water, dried over Na 2 SO 4 , filtered and condensed. The resulting residue was purified with flash chromatography 0% to 16% EtOAc in hexanes. Purified product was isolated as a colorless oil (5.39 g, 85%). 1  The reaction was put under vacuum and heated to 60C for 1.5 hours. PhCH(OMe) 2 (4.12 mL, 27.5 mmol, 3.0 eq) was added to the reaction at 60C and continued to stir under vacuum for 1.5 hours. TLC (100% DCM) confirmed that the reaction was complete. Once the vacuum was removed and the flask cooled to room temperature, the reaction was quenched with 15 mL of saturated NaHCO 3 and stirred for 20 minutes. The reaction was diluted with DCM and extracted three times. The organic layers were combined and washed three times with 1N HCl. The organic layer was dried over Na 2 SO 4 , filtered, and condensed to yield a yellow oil. The product was purified with column chromatography (0 to 5% EtOAc in hexanes to 50% EtOAc in hexanes). The clean product was isolated as a colorless oil (quantitative). 1 2S,4aR,7R,8R,8aS)
To 10 (0.089 g, 0.35 mmol, 1.0 eq) and Na 2 CO 3 (0.250 g, 2.36 mmol, 6.7 eq) was added 8 mL of anhydrous MeOH under N 2 . 2,5-dioxopyrrolidin-1-yl 2-azidoacetate (0.186 g, 0.94 mmol, 2.7 eq) was added in two additions every 30 minutes. Reaction was monitored by TLC (25% MeOH/EtOAc) and LC/MS ESI neg [M-1] -= 333. Once complete, the reaction was filtered and evaporated under reduced pressure without heat. The off-white solid was purified on the Waters preparative HPLC/MS. Crude oil was dissolved in DI H 2 O 0.1% formic acid (50mg/mL) and purified on the Waters preparative HPLC/MS with the method as follows: flow rate 20 mL/min, 0.1% formic acid in millipure H 2 O as eluent A and 0.1% formic acid in HPLC grade acetonitrile as eluent B.