Nitroreductase-triggered activation of a novel caged fluorescent probe obtained from methylene blue

Abstract

A near-infrared fluorescent probe based on methylene blue (p-NBMB) was developed for the detection of nitroreductase. Conjugating methylene blue with a p-nitrobenzyl moiety enables it to be activated by nitroreductase-catalyzed 1,6-elimination, resulting in the release of an active methylene blue fluorophore.

Fig. 1

Heterogeneity of p-NBMB in addition of NTR (1 unit) with NAD(P)H (1 mM). Thin film fluorescence emission spectra were obtained at 541 nm excitation.

Fig. 2

Fluorescence spectra of p-NBMB (1 μM, 10 μM, 100 μM) with NTR (1 unit) in the presence of NAD(P)H (1 mM) and the negative control without NTR. Spectra were acquired in 10 mM PBS, pH 7.4 with the excitation at 580 nm.

Fig. 3

Fluorescence response of p-NBMB to nitroreductase. (a) IVIS fluorescence images of p-NBMB (10 μM, 100 μM) with nitroreductase (1 and 2 unit/ml, 1 mM NAD(P)H) and the control without nitroreductase. (b) Quantitation of fluorescence from each group. Data shown as mean ± SD (n = 3). Spectra were acquired after incubation at 37 °C overnight in 10 mM PBS (pH 7.4).

Fig. 4

IVIS fluorescence images of p-NBMB (1 – 50 μM) incubated with intact E. coli K12 strain XL1-Blue (10⁹ CFU/mL) at 37 °C for 4 hours.

Fig. 5

The best binding pose of p-NBMB (orange carbons) to NTR. FMN is shown as cyan carbons. The substrate binding pocket of NTR (PDB 1F5V) was generated using Fred v3.0.0 of OpenEye software suite. The enzyme is represented as cartoon and colored by secondary structure (prepared with Maestro).

Scheme 1

Mechanism of switching on the fluorescence probe, p-NBMB, upon reduction of a p-nitrobenzyl moiety after nitroreductase-mediated activation.