Profluorescent Fluoroquinolone-Nitroxides for Investigating Antibiotic–Bacterial Interactions

Abstract

Fluorescent probes are widely used for imaging and measuring dynamic processes in living cells. Fluorescent antibiotics are valuable tools for examining antibiotic–bacterial interactions, antimicrobial resistance and elucidating antibiotic modes of action. Profluorescent nitroxides are ‘switch on’ fluorescent probes used to visualize and monitor intracellular free radical and redox processes in biological systems. Here, we have combined the inherent fluorescent and antimicrobial properties of the fluoroquinolone core structure with the fluorescence suppression capabilities of a nitroxide to produce the first example of a profluorescent fluoroquinolone-nitroxide probe. Fluoroquinolone-nitroxide (FN) 14 exhibited significant suppression of fluorescence (>36-fold), which could be restored via radical trapping (fluoroquinolone-methoxyamine 17) or reduction to the corresponding hydroxylamine 20. Importantly, FN 14 was able to enter both Gram-positive and Gram-negative bacterial cells, emitted a measurable fluorescence signal upon cell entry (switch on), and retained antibacterial activity. In conclusion, profluorescent nitroxide antibiotics offer a new powerful tool for visualizing antibiotic–bacterial interactions and researching intracellular chemical processes.

Nitroxide functionalized antibiotics are promising eradication agents against Staphylococcus aureus biofilms

“…We rationalized that by localising the antibiotic directly at the site of nitroxide-mediated dispersal, it could more effectively eradicate dispersed cells than in combination treatment. To test this hypothesis, we utilized several ciprofloxacin-nitroxide hybrids that we have previously generated (Figure 2) (25, 27). Cipro-PROXYL, cipro-TEMPO, and cipro-TMIO (Figure 2) were first screened in MIC assays for activity against planktonic S. aureus ATCC 29213 (Table 1).…”

“…Chemical structures of CTEMPO, ciprofloxacin, cipro-PROXYL,(25) cipro-TEMPO,(25) cipro-TEMPO,(25) fluoro-TEMPO,(27) and fluoro-TEMPOMe (27). …”

“…To investigate the impact of nitroxide-antibiotic hybrids on established S. aureus biofilms in more detail, we utilized the recently reported profluorescent fluoroquinolone-nitroxide hybrid fluoro-TEMPO (27). Fluoro-TEMPO was found to be most active against S. aureus and emit bright fluorescence upon cell entry (27), making it a valuable switch-on fluorescent probe for studying S. aureus biofilm eradication by nitroxide-antibiotic hybrids.…”

“…To investigate the impact of nitroxide-antibiotic hybrids on established S. aureus biofilms in more detail, we utilized the recently reported profluorescent fluoroquinolone-nitroxide hybrid fluoro-TEMPO (27). Fluoro-TEMPO was found to be most active against S. aureus and emit bright fluorescence upon cell entry (27), making it a valuable switch-on fluorescent probe for studying S. aureus biofilm eradication by nitroxide-antibiotic hybrids. We first established the MBEC value for fluoro-TEMPO (MBEC ≤720 µM; Table 2) and found it to be at least 5-times more potent than ciprofloxacin treatment of S. aureus ATCC 29213 biofilms, further supporting the increased potency of tethered nitroxide-antibiotic hybrids.…”

“…The antibiotic ciprofloxacin (Sigma, Australia) was prepared in aqueous hydrochloric acid (0.1 M) at a concentration of 60.35 mM (stock solution). Cipro-PROXYL, cipro-TEMPO, cipro-TMIO, fluoro-TEMPO, and fluoro-TEMPOMe were synthesized in-house utilizing previously established procedures (25, 27), and stock solutions were prepared in DMSO (8 mM). All stock solutions were stored in the absence of light at −20 °C.…”