Synthesis of vancomycin fluorescent probes that retain antimicrobial activity, identify Gram-positive bacteria, and detect Gram-negative outer membrane damage

Abstract

Antimicrobial resistance is an urgent threat to human health, and new antibacterial drugs are desperately needed, as are research tools to aid in their discovery and development. Vancomycin is a glycopeptide antibiotic that is widely used for the treatment of Gram-positive infections, such as life-threatening systemic diseases caused by methicillin-resistant Staphylococcus aureus (MRSA). Here we demonstrate that modification of vancomycin by introduction of an azide substituent provides a versatile intermediate that can undergo copper-catalysed azide−alkyne cycloaddition (CuAAC) reaction with various alkynes to readily prepare vancomycin fluorescent probes. We describe the facile synthesis of three probes that retain similar antibacterial profiles to the parent vancomycin antibiotic. We demonstrate the versatility of these probes for the detection and visualisation of Gram-positive bacteria by a range of methods, including plate reader quantification, flow cytometry analysis, high-resolution microscopy imaging, and single cell microfluidics analysis. In parallel, we demonstrate their utility in measuring outer-membrane permeabilisation of Gram-negative bacteria. The probes are useful tools that may facilitate detection of infections and development of new antibiotics.

Antibacterial Activity of Amphiphilic Janus Nanoparticles Enhanced by Polycationic Ligands

“…coli strain we used is highly sensitive to the staining conditions, particularly the incubation temperature and dye concentrations. This is consistent with previously reported observations. , Consequently, these experiments need careful optimization and execution of control experiments.…”

“…This is consistent with previously reported observations. 36,37 Consequently, these experiments need careful optimization and execution of control experiments.…”

A Far-Red Fluorescent Probe to VisualizeStaphylococcus aureusin Patient Samples

“…This MIC difference indicates that the attached fluorophore diminished vancomycin's affinity for its target D-alanyl-D-alanine, similar to other conjugates attached at the primary amine. 10,16 The lower toxicity of Van-JF669 compared to the parent antibiotic is an advantage since it allows for imaging live cells with limited impact on bacterial viability. We first evaluated the labeling efficiency of Van-JF669 with Staphylococcus epidermidis (S. epidermidis, ATCC 12228).…”

“…This kind of probe has been developed to image S. aureus by leveraging the binding of the glycopeptide vancomycin to D-alanyl-D-alanine moieties of lipid II (a precursor of peptidoglycan) and the mature peptidoglycan layer of bacteria. 10 Vancomycin labeled with the green fluorophore boron dipyrromethene (Van-BODIPY) 11 or with a near-infrared dye (Vanco-800CW) have been reported (Figure S1). 12 These probes, however, also exhibit drawbacks such as spectral overlap with host autofluorescence, which is particularly high in inflamed tissues (Van-BODIPY), 11,13 or an excitation wavelength that is not commonly available in fluorescence microscopes or flow cytometers (Vanco-800CW).…”

A Far-Red Fluorescent Probe to Visualize Gram-Positive Bacteria in Patient Samples

“…Van-JF 669 exhibited a minimum inhibitory concentration (MIC) of 5 mg L –1 (2.3 μM) against lab strain methicillin-resistant S. aureus (MRSA, ATCC 43300), lab strain methicillin-sensitive S. aureus (MSSA, ATCC 29213), and clinical isolate S. aureus (MSSA, PROSA28), which is significantly higher than vancomycin (MIC = 0.31 mg L –1 – 0.2 μM) in the same strains (Figure S7). This MIC difference indicates that the attached fluorophore diminished vancomycin’s affinity for its target d -ala- d -ala, similar to other conjugates attached at the primary amine. , The reduced toxicity suggests that our probe might operate differently from the original vancomycin, potentially offering an advantage by enabling live cell imaging without significantly affecting bacterial viability.…”