Cell-Permeable Fluorogenic Probes for Identification and Imaging Nitroreductases in Live Bacterial Cells

Area of Science:

• Chemical Biology
• Molecular Imaging
• Biotechnology

Background:

• Enzyme-activated fluorogenic probes are crucial for monitoring biological processes.
• Developing probes that penetrate bacterial cell envelopes is essential for intracellular analysis.
• Bacterial nitroreductase (NTR) is a key target for studying bacterial physiology and drug resistance.

Purpose of the Study:

• To design, synthesize, and evaluate novel cyanine 5-based fluorogenic probes for bacterial nitroreductase (NTR) detection.
• To assess the cell permeability and selectivity of the developed probes in both Gram-positive and Gram-negative bacteria.
• To demonstrate the utility of the probes for real-time intracellular imaging of NTR in live bacterial cells.

Main Methods:

• Synthesis of two series of cyanine 5-based fluorogenic probes.
• Enzymatic assays to evaluate probe activation by bacterial nitroreductase (NTR).
• Confocal microscopy for live bacterial cell imaging and assessment of cell permeability.

Main Results:

• Probe 3 exhibited a 10-fold fluorescence enhancement upon catalytic reduction by NTR.
• The probe is cell-permeable across both Gram-positive and Gram-negative bacterial envelopes.
• Probe 3 demonstrated high selectivity for NTR, enabling background signal minimization.
• Real-time intracellular imaging of NTR in live bacteria was successfully achieved.

Conclusions:

• Developed cyanine 5-based fluorogenic probes are effective tools for detecting bacterial nitroreductase (NTR).
• Probe 3 offers excellent cell permeability, selectivity, and fluorescence response for live bacterial imaging.
• These probes advance the capability for real-time intracellular visualization of bacterial targets.