2D Strategy for the Construction of an Enzyme-Activated NIR Fluorophore Suitable for the Visual Sensing and Profiling of Homologous Nitroreductases from Various Bacterial Species

Nitroreductases (NTRs) mediate the reduction of nitroaromatic compounds to the corresponding nitrite, hydroxylamine, or amino derivatives. The activity of NTRs in bacteria facilitates the metabolic activation and antibacterial activity of 5-nitroimidazoles. Therefore, NTR activity correlates with the drug susceptibility and resistance of pathogenic bacteria. As such, it is important to develop a rapid and visual assay for the real-time sensing of bacterial NTRs for the evaluation and development of antibiotics. Herein, an activatable near-infrared fluorescent probe (HC–NO2) derived from a hemicyanine fluorophore was designed and developed based on two evaluation factors, including the calculated partition coefficient (Clog P) and fluorescence wavelength. Using HC–NO2 as the special substrate of NTRs, NTR activity can be assayed efficiently, and then, bacteria can be imaged based on the detection of NTRs. More importantly, a sensitive in-gel assay using HC–NO2 has been developed to selectively identify NTRs and sensitively determine NTR activity. Using the in-gel assay, NTRs from various bacterial species have been profiled visually from the “fluorescence fingerprints”, which facilitates the rapid identification of NTRs from bacterial lysates. Thus, various homologous NTRs were identified from three metronidazole-susceptible bacterial species as well as seven unsusceptible species, which were confirmed by the whole-genome sequence. As such, the evaluation of NTRs from different bacterial species should help improve the rational usage of 5-nitroimidazole drugs as antibiotics.


S5
Compound 1: 3-Nitrophenol (2 g, 14 mmol) and HMTA (2.42 g, 17 mmol) were dissolved in TFA (10 mL) and the mixture was refluxed for 12 hours. After consumption the starting material, the reaction mixture was concentrated in vacuo and purified on silica gel (hexane/AcOEt: 10/1) to afford a white solid (440 mg, 19%). 1  Compound 2: To a solution of DMF (22.4 mL, 290 mmol) in CHCl 3 (150 mL) at 0 °C PBr 3 was lowly added slowly dropwise (24.8 mL, 261 mmol). After 1 hour, cyclohexanone (10 mL, 96.8 mmol) was added and the mixture was stirred overnight at 25 °C. Then, the red solution was poured onto ice and pH adjusted to 7 using solid NaHCO 3 . The organic layer was separated and the aqueous layer was extracted with DCM (150 mL×3). The organic layer was combined, and dried over Na 2 SO 4 , filtered and concentrated in vacuo to provide compound 2 as yellow oil (12.4 g). Without any purification, the yellow oil was direct used for the next step. General procedure for compound 3: To a solution of nitro-salicylaldehyde (167 mg, 1 mmol) in DMF (10 mL), was added compound 2 (277 mg, 1.2 mmol) and Cs 2 CO 3 (978mg, 3 mmol), which was stirred for 2 hours at room temperature. Then, the mixture was diluted in deionized water (50 mL) and extracted twice with AcOEt (50 mL×3); the organic layer was dried over Na 2 SO 4 , filtered and concentrated in vacuo.

Determination of the quantum yield (QY)
The fluorescence quantum yields for the compounds were measured using an
Using the same procedure as above, the fluorescence response of HC-NO 2 (10 μM)

Investigation about inhibitory effects of compounds on NTR
Pre-incubation was conducted for the PB solution containing NTR, NADH, and compounds to be tested with different concentrations for 3 min at 37 °C. Then, the fluorescent probe HC-NO 2 (10 μM) was added into the solution to start the enzymatic reaction. The following procedures were the same as mentioned above. Compared with the fluorescence intensity of the control group, the fluorescence intensities of the tested groups were applied to determine the inhibitory rate using GraphPad Prism 6.0. A bacterial suspension (2 μL) was dropped onto a glass slide, which was then subjected to confocal laser scanning microscopy to obtain the fluorescence images (λ ex = 633 nm, λ em = 690 -750 nm).

Fluorescence imaging of bacterial cells by HC-NO
For above mentioned bacterial suspensions, flow cytometric analysis was performed using a Biosciences Accuri C6 flow cytometer (λ ex = 640 nm, λ em > 670 nm).

Fluorescence imaging of bacterial colonies by HC-NO 2 on agar plates
Various bacteria were cultured in a liquid medium to afford a stock bacterial suspension (OD 600 0.8). After dilution (10 5 ×), the bacterial strains were coated on to agar plates as culture medium followed by cultivation at 37 °C for 24 h. For anaerobic bacteria, the cultivation was conducted in an anaerobic box at 37 °C for 48 h. The bacterial colonies were observed by naked eye. HC-NO 2 solution was dropped onto the bacterial colonies carefully followed by 1h incubation at 37 °C. For inhibitory experiments (menadione, 50 μM), the inhibitor was added at the same time. After the co-incubation, the agar plates were then imaged using a fluorescence scanner (λ ex = 635 nm, λ em = 720 ± 20 nm).

Native-PAGE electrophoresis for bacterial lysates and NTR proteins
Three anaerobic bacteria (B. fragilis, B. thetaiotaomicron, and B.            Figure S18. Sensitivity investigation for NTR assay on native PAGE-gel stained using silver. Table S2. Inhibitory effect of metronidazole on various bacteria. Figure S19. Total protein profiling of various bacteria species on native gel stained using Coomassie brilliant blue.