Imaging of antitubercular dimeric boronic acids at the mycobacterial cell surface by click-probe capture

Dimeric boronic acids kill Mycobacterium tuberculosis (Mtb) by targeting mycobacterial specific extracellular glycans, removing the requirement for a therapeutic agent to permeate the complex cell envelope. Here we report the successful development and use of new ‘clickable’ boronic acid probes as a powerful method to enable the direct detection and visualisation of this unique class of cell-surface targeting antitubercular agents.

All reactions were performed using oven dried. Thin Layer Chromatography (TLC) was carried out using Merck aluminium backed sheets coated with 60 F254 silica gel. Visualisation of the silica plates was achieved using a UV Lamp (l = 254 nm), and/or potassium permanganate (1.5 g KMnO4, 10 g KCO3 and 1.25 mL 10% NaOH in 200 mL water) or Alizarin Red S (1 mM alizarin red S in acetone). 1 Flash chromatography was carried out using Merck silica gel 60, 35-75 μm as the stationary phase (Sigma Aldrich) unless otherwise stated. Mobile phases are reported in solvent ratios.
Proton ( 1 H) and carbon ( 13 H) NMR spectra were obtained at 298 K. 1 H NMR and 13 C DEPT NMR were recorded on a Bruker DPX-500 instrument. NMR were fully assigned using COSY, HSQC and HMBC. Mass spectra were recorded on a Bruker HCT Ultra spectrometer using electrospray ionisation (ESI).

Determination of minimum inhibitory concentrations
The minimum inhibitory concentrations (MIC) of all compounds were determined using the resazurin reduction microplate assay (REMA) as described previously. triazol-4-yl)methyl]amine (100 µM) and sodium ascorbate (2.5 mM) in PBST were also prepared.

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Control cells resuspended in PBST with no dye were also prepared. Cells for microscopy were resuspended in 7H9 media containing 4 drops/mL of NucBlue live DAPI stain, incubated at room temperature in the dark with shaking for 20 mins, centrifuged (2,200 g, 5 mins, 4 °C), washed once (PBST) and resuspended in PBST. Cells for flow cytometry were resuspended in PBST for flow cytometry analysis.

Confocal microscopy
The labelled mycobacteria were spotted onto glass slides that had been prepared with 7H10 media (Middlebrook) containing 1.5% agarose and allowed to air-dry. Cover slips were placed over the sample which was fixed with an adhesive. Microscopy was performed on an LSM 880 confocal microscope