An Activatable Lanthanide Luminescent Probe for Time‐Gated Detection of Nitroreductase in Live Bacteria

Abstract

Herein we report the development of a turn‐on lanthanide luminescent probe for time‐gated detection of nitroreductases (NTRs) in live bacteria. The probe is activated through NTR‐induced formation of the sensitizing carbostyril antenna and resulting energy transfer to the lanthanide center. This novel NTR‐responsive trigger is virtually non‐fluorescent in its inactivated form and features a large signal increase upon activation. We show that the probe is capable of selectively sensing NTR in lysates as well as in live bacteria of the ESKAPE family which are clinically highly relevant multiresistant pathogens responsible for the majority of hospital infections. The results suggest that our probe could be used to develop diagnostic tools for bacterial infections.

9-Nitrobenzo[b]quinolizinium as a fluorogenic probe for the detection of nitroreductase in vitro and in Escherichia coli

“…Thus, a continuous increase of the fluorescence intensity was observed with increasing pH and temperature up to pH 7 and 37 °C, whereas at larger values the fluorescence intensity started to decrease. The effect of the temperature on the reaction of 1b with nitroreductase is in good agreement with the reported activities of the enzyme towards nitroarene derivatives [9][10][11]15 but the optimal pH value lies usually slightly higher at pH 7.4. [9][10][11]15 The lower fluorescence intensity at higher pH values is presumably caused by a slow degradation of the substrate by nucleophilic addition of the hydroxide ion to the benzo[b]quinolizinium at C6 position and subsequent ring opening reaction, as usually observed for this class of compounds.…”

“…The effect of the temperature on the reaction of 1b with nitroreductase is in good agreement with the reported activities of the enzyme towards nitroarene derivatives [9][10][11]15 but the optimal pH value lies usually slightly higher at pH 7.4. [9][10][11]15 The lower fluorescence intensity at higher pH values is presumably caused by a slow degradation of the substrate by nucleophilic addition of the hydroxide ion to the benzo[b]quinolizinium at C6 position and subsequent ring opening reaction, as usually observed for this class of compounds. 20 Furthermore, excitation spectra indicated that the new long-wavelength absorption maxima at 447 nm and 480 nm correspond to the newly formed emitting species (Figure 2, Figure S4A).…”

“…In summary, it was demonstrated that the nitrobenzoquinolizinium 1b has the potential to act as a probe for the fluorimetric detection of nitroreductase with a comparatively good sensing performance. 5,[9][10][11] The fluorogenic reaction in the enzyme binding site proceeds on formation of an "over-reduced" intermediate 4 that is eventually transformed to the fluorescent 9-aminobenzo[b]quinolizinium (1c) upon release from the binding site. These results shall constitute the starting point of the development of a novel class of watersoluble, nitroreductase-targeting fluorescent probes based on the benzo[b]quinolizinium fluorophore, which can be utilized for the fluorimetric detection of the hypoxia status of cells.…”

Activated molecular probes for enzyme recognition and detection

“…In recent years, various enzyme-sensitive probes have been reported 117 . These probes use enzymes to specifically recognize the performance of a reaction substrate and have various design mechanisms for enzyme detection, including in-situ self-assembly, intramolecular cyclization, FRET, ICT, and group self-elimination.…”

Small Molecule Fluorescent Probes for Sensing and Bioimaging of Nitroreductase

“…Brennecke et al . published their NTR responsive lanthanide probe 21 [59] . The nitro group on this probe was reduced to its corresponding amine due to reducing action of NTR, thereby releasing iminoquinone methide.…”