The rise of antimicrobial resistance poses a critical challenge to global health, demanding the development of novel antibacterial drugs and research tools. Vancomycin, a glycopeptide antibiotic, is a crucial treatment for Gram-positive infections, including those caused by methicillin-resistant *Staphylococcus aureus* (MRSA). However, the emergence of vancomycin-resistant bacteria necessitates the development of new strategies. This research focuses on creating fluorescent vancomycin probes that retain their antimicrobial properties. These probes would be valuable tools to improve our understanding of bacterial interactions with antibiotics, aid in the development of new antibiotics, and contribute to novel diagnostic methods. Current fluorescent vancomycin analogues often suffer from significantly reduced antimicrobial activity compared to the parent compound. This reduction in activity is likely due to steric hindrance or electrostatic repulsion between the large, negatively charged fluorophores and the negatively charged bacterial cell wall. Therefore, the creation of fluorescent probes that maintain comparable potency is a crucial objective. The study aims to address this challenge by synthesizing new probes using a versatile intermediate and employing a method that allows facile incorporation of a range of fluorophores with different colours. The successful development of such probes would significantly benefit the fields of diagnostics and drug development, offering improved tools for visualizing bacterial infections and assessing the effectiveness of new antibiotics.

The use of fluorescent probes to visualize cellular structures and dynamics is essential for understanding the interactions between biomolecules in complex systems, including antibiotic-bacteria interactions. A widely used approach involves fluorescent dyes such as N-phenyl-1-naphthylamine (NPN) and SYTOX Green to assess membrane damage in Gram-negative bacteria. However, fluorescent probes derived from antibiotics offer potential advantages due to their mechanism-specific binding. The literature includes various examples of fluorescent vancomycin analogues conjugated with different fluorophores like BODIPY FL, fluorescein, rhodamine, and Alexa Fluor. However, many of these derivatives have shown significantly reduced antimicrobial activity compared to vancomycin. For example, fluorescein-linked vancomycin was considerably less potent than BODIPY-linked vancomycin against *B. subtilis*. This reduction in activity is likely due to the electrostatic repulsion between the negatively charged fluorescein and the negatively charged teichoic acids in the Gram-positive cell wall. The lack of consistency in antimicrobial activity among previously reported derivatives and the need for readily prepared, potent probes with diverse fluorophores motivated this research.

The researchers modified vancomycin at its C-terminal carboxy group, a site that doesn't appear to interfere with its binding to Lipid II or dimerization. This modification involved introducing an azide substituent, creating a versatile intermediate suitable for CuAAC reactions with various alkynes to generate fluorescent probes. Three azido-derivatized vancomycins (Van-C3-N3, Van-C8-N3, and Van-PEG3-N3) with different linkers were synthesized. The antimicrobial activity of these azido-vancomycin intermediates was assessed against various Gram-positive bacteria using broth microdilution. Next, these azides were conjugated with alkyne-derivatized fluorophores, 7-nitrobenzofurazan (NBD) (green) and 7-(dimethylamino)-coumarin-4-acetic acid (DMACA) (blue), using optimized CuAAC conditions. The optimized reaction conditions used a solvent mixture of DMF/t-BuOH/H2O and acetic acid to accelerate the reaction. The antimicrobial activity of the resulting fluorescent probes was assessed against various Gram-positive bacteria strains. The selectivity of these probes towards Gram-positive bacteria was evaluated using flow cytometry analysis, high-resolution microscopy imaging (SR-SIM and Airyscan confocal), and single-cell microfluidics analysis. The ability of the probes to detect outer-membrane permeabilization in Gram-negative bacteria was investigated using microscopy and flow cytometry after treatment with various membrane-active and inactive antibiotics. In single-cell microfluidics experiments, hundreds of individual *E. coli* cells immobilized within microfluidic channels were monitored over time for probe accumulation, with and without the addition of the outer membrane permeabilizer polymyxin B. A mathematical model was used to analyze the single-cell accumulation data to determine kinetic parameters of probe uptake.

The azido-vancomycin derivatives retained antimicrobial activity against Gram-positive bacteria, with Van-C8-N3 showing the highest potency. Fluorescent vancomycin probes, synthesized by CuAAC reactions, exhibited comparable or improved antimicrobial activity compared to vancomycin, demonstrating that the chosen fluorophores did not significantly impede their activity. Flow cytometry analysis demonstrated the selective labeling of *S. aureus* over *E. coli*, highlighting the probes' specificity for Gram-positive bacteria. Super-resolution microscopy confirmed the probes' binding to nascent peptidoglycan at the bacterial division septum. The probes successfully distinguished between Gram-positive strains with varying degrees of vancomycin resistance, with VRSA strains showing notably reduced staining. The probes effectively visualized membrane permeabilization in Gram-negative bacteria in *E. coli* mutants with impaired outer membranes (*lpxC* and DC2), and in wild-type *E. coli* under cold stress. Moreover, the fluorescent vancomycin probes facilitated the quantification of outer membrane damage caused by different membrane-active antibiotics using flow cytometry and plate reader assays. Single-cell microfluidics analysis coupled with mathematical modeling further confirmed the faster and greater accumulation of the vancomycin probe in the presence of polymyxin B, indicative of enhanced membrane permeability.

The study successfully addressed the research question by demonstrating the successful synthesis of fluorescent vancomycin probes that retain antimicrobial activity while specifically targeting Gram-positive bacteria. The improved activity compared to previous analogues emphasizes the importance of carefully selecting fluorophores with minimal electronic charges and steric hindrance. The ability of the probes to differentiate between Gram-positive bacteria with varying vancomycin resistance levels suggests their potential as diagnostic tools. The detection of outer membrane permeabilization in Gram-negative bacteria under specific conditions expands the utility of these probes to study the mechanisms of action of other antimicrobial compounds and the impact of bacterial mutations. The use of multiple techniques including flow cytometry, super-resolution microscopy, and single-cell microfluidics provided comprehensive data confirming the probes’ effectiveness and specificity. The developed methods for quantifying outer membrane damage can be applied in various scenarios, improving the understanding of antibiotic mechanisms and bacterial resistance.

This study successfully synthesized fluorescent vancomycin probes that maintain antimicrobial activity, selectively label Gram-positive bacteria, and detect Gram-negative outer membrane damage. These probes are valuable tools for studying bacterial interactions with antibiotics and have potential applications in diagnostics and drug discovery. Future research could focus on expanding the range of fluorophores used, testing the probes' efficacy against a wider range of bacterial species, and exploring their utility in in vivo models of infection.

The study primarily focused on a limited set of bacterial strains. Further investigation is required to determine the generalizability of the findings to a broader range of Gram-positive and Gram-negative bacteria. Although the probes showed significant success in visualizing membrane damage, more detailed mechanistic studies are needed to fully understand the interactions between the probes and bacterial membranes. Furthermore, the in vivo applications of these probes require further investigation.