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.


Experimental Procedures Preparative Methods
Reactions requiring anhydrous conditions were carried out in dry solvents stored over molecular sieves (THF, DCM, toluene, pyridine, DMF from Sigma Aldrich) which were used as received. These moisture-and air-sensitive reactions were conducted under nitrogen using Schlenk-technique.

Analytical Methods
NMR spectra were acquired on devices from the company Bruker (AV 300, AV 600). All 13 C-NMR-spectra were recorded with 1 H-broadband decoupling. Calibration of the chemical shift was conducted using the solvent residual signals. Numbering of the denoted compounds arises from the IUPAC nomenclature. Mass spectra were recorded with an Agilent 1260 liquid chromatography coupled accurate mass time-of-flight 6230 detector. Standard analysis of reaction time courses were conducted with an Agilent 1260 infinity liquid chromatography coupled quadrupole mass spectrometer 6120 detector.

UV-VIS absorption and fluorescence spectra
The concentration of DMSO stock solution of control probe 10 (10 mM) and nitroreductase probe 12 (10 mM) were diluted to 20 μM in the according buffer. The UV-Visible spectra were recorded using a Tecan Spark™ 10M Multimode Microplate Reader, wavelength interval: 5.0 nm. Fluorescence spectroscopic studies were also performed at the excitation wavelength of 355 nm, wavelength interval: 5.0 nm.

Determination of luminescent lifetimes
The luminescence emission decays of the compounds (20 μM in Tris-HCl buffer, pH 7.4) were recorded with an Edinburgh photonics FLS980 with the excitation wavelength set to 355 nm and the emission monitored at 550 nm. The luminescence emission was fitted to a single exponential decay as the most suitable fit according to R 2 and χ 2 to obtain the lifetime constants. Data is representative of a single experiment.

Determination of luminescence quantum yield
The total quantum yield of the complex was determined in aq. PBS pH 7.4 with an integrating sphere using an Edinburgh photonics FLS980. The quantum yield was calculated using the direct excitation method, consecutively exciting a reference sample solution containing aq. PBS and a probe containing sample solution set to an optical density of around 0.1 at the excitation wavelength. All emissions were corrected by the wavelength sensitivity of the spectrometer. The total quantum yield was then calculated according to the equation Φtot = (Es-Er)/(Sr-Ss), where Es and Er correspond to the integrated emission peaks of the sample and the reference, and Ss and Sr correspond to the integrated scatter peaks of the sample and the reference, respectively. [1] Data is representative of a single experiment.
NTR activity in bacterial lysates. The six bacterial strains were cultured for 12 h in Luria-Bertani (LB) medium at 37 °C, then harvested and washed twice with Tris buffer (pH 7.4). The washed cells were resuspended in Tris buffer with an OD600 of 10.0. The cell suspension was then aliquoted and lysed by sonication. The lysates were then treated with 20 μM of nitroreductase probe 12 for 2 h with or without dicoumarin (0.1 mM) and fluorescence of the reaction solutions was measured using a Tecan Spark™ 10M Multimode Microplate Reader. (λex/λem = 355/550 nm) NTR activity in live bacteria. The three bacterial strains were cultured for 12 h in Luria-Bertani (LB) medium at 37 °C, and then harvested and washed twice with Tris buffer (pH 7.4). The washed cells were resuspended in Tris buffer with an OD600 of 2.0. The cell suspension was then aliquoted and treated with 20 μM of nitroreductase probe 12 for 4 h and 12 h with or without dicoumarin (0.1 mM). Fluorescence of the bacterial solutions was measured using a Tecan Spark™ 10M Multimode Microplate Reader (λex/λem = 355/550 nm).
Uptake of probe 12 and reference 10 in live bacteria. E. coli, K. pneumoniae, A. baumannii and E. cloacae cells were cultured for 12 h in Luria-Bertani (LB) medium at 37 °C, and then harvested and washed twice with Tris buffer (pH 7.4). The washed cells were resuspended in Tris buffer with an OD600 of 2.0. The cell suspension was then aliquoted and treated with 20 μM of probe 12 and reference 10 for 12 h. The cells were then washed twice with 1× PBS buffer. Fluorescence of the bacterial solutions was measured (λex/λem = 355/550 nm).
Fluorescence lifetime imaging of live E. coli. E.coli (ATCC25922) cells cultured overnight in LB medium were harvested and washed twice with PBS buffer solution. The washed cells were resuspended in 50 mM Tris-HCl (pH 7.4) buffer with an OD600 of 0.5 -0.7. Then 500 μL aliquots were treated with 20 μM of probe 12. After incubation at 37 °C for 4 h, a drop of the suspension was added into an 8well chamber followed by covering with agarose pads. Fluorescence lifetime images were acquired by an ISS Q2 confocal laser scanning system coupled to a Nikon TE2000 microscope with the 60×/1.2 NA WI objective lens. The excitation wavelength of the probe 12 was 375 nm (5000 Hz repetition rate), fluorescence emission and lifetime signals were collected through a 641/75 nm long-pass edge filter.

(4-Amino-2-bromophenyl)methanol
A solution of methyl 4-amino-2-bromobenzoate (2, 500 mg, 2.17 mmol) in THF (7 ml) was added dropwise to a solution of lithiumtetrahydroaluminate (3.26 ml, 6.52 mmol, 2 M in THF) in THF (7 ml) at 0°C. The reaction was stirred for 3 h while cooling. Water was added carefully, then 2 M NaOH was added and the mixture was stirred until a fine precipitate had formed. The mixture was filtered over celite, ethyl acetate was added and the org. layer was separated. The aq. phase was extracted with ethyl acetate and the combined org. layers were washed with brine, dried over MgSO4 and filtered. The crude was purified by flash chromatography (SiO2, cHex/EA 3:1 -1:1) to provide 299 mg (67%) (4-amino-2-bromophenyl)methanol as colorless crystalline solid.

Caged antenna (8)
Boc-protected antenna precursor (349 mg, 0.63 mmol) was dissolved in DCM/TFA (10 ml, 1:1) and the solution was stirred for 30 min. The solvents were evaporated and the residue was suspended in water/acetonitrile and lyophilized to obtain 183 mg (64%) of the title compound (8) as pale yellow powder which was directly used for the next step without further purification.

Detection Limit
The detection limit was calculated according to the formula 3σ/k, where σ is the standard deviation of the background noise of 20 µM probe collected at 12 time points and k is the slope of the linear fit obtained from enzyme concentration vs probe emission intensity at 550 nm, 20 µM probe (inset Fig. 1A). With σ = 2.357 and k = 1617 ml/µg, 3σ/k = 4.4 ng/ml. Figure S10. Fluorescence emission spectra of probe 12 (20 µM) with NTR (1 μg/mL) and NADH (50 µM) and with added NTR inhibitor dicoumarin (12.5, 25, 50 μM). Purple: probe in pH 7.4 Tris buffer (control). All measurements were carried out after mixing for 2 h at 37 °C. λex = 355nm. Figure S11. Quantification of emission intensity of probe 12 at 550 nm incubated with NTR and dicoumarin as shown in Figure 9.