New Benzimidazole-Based pH-Sensitive Fluorescent Probes

Abstract

This article is devoted to the synthesis and investigation of a family of new benzimidazole compounds with a propylsulfonate moiety, synthesized by condensation of salicylic aldehyde or its 5-substituted derivatives with 3-(2,3-dimethylbenzimidazol-1-ium-1-yl)propane-1-sulfonate. The structure of the obtained dyes was confirmed using NMR, FT-IR, and HRMS. Absorption and photoluminescence properties were studied in phosphate buffers over a wide pH range, and changes in the absorption and fluorescence spectra of DMSO solutions upon titration with DIPEA and HCl were also studied. It was found that all the target compounds possess pH-sensitive optical properties and can be used as fluorescent probes, while methoxycarbonyl-substituted derivative 3c demonstrated the most prominent optical and fluorescent response starting from pH ~ 4.5. The toxicity of the compounds was studied using whole-cell bioluminescent bacterial sensors. The effect on the biomass and metabolic activity of strains Staphylococcus aureus ATCC 6538-P FDA 209-P and Escherichia CDC F-50 bacterial biofilms was also investigated. In the final stage of the study, bioimaging experiments were carried out using the selected most promising dye 3c and biofilms. It was demonstrated that the dye can be excited by light with wavelengths of 458 nm or 750 nm in multiphoton mode. Importantly, when biofilms are incubated in the dye solution for 3 h, only the extracellular matrix is stained. However, if the staining time is increased to 24 h, dye penetration into bacterial cells is observed, resulting in a second photoluminescence maximum during sample analysis. It is important to note that when biofilms are incubated in a dye solution for 3 h, only the extracellular matrix is stained, while with longer staining, penetration of the dye into bacterial cells is observed, and a second photoluminescence maximum appears during sample analysis. The results obtained demonstrate a high potential of using benzimidazole-based compounds as pH-sensitive fluorescent probes operating in a biologically relevant pH range, which can be used for imaging of bacterial biofilms.

Keywords: benzimidazole; bioimaging; fluorescence; molecular docking; pH; sulfonic acids; whole-cell lux-biosensors.

Scheme 1

Synthesis of benzimidazolium derivatives 3a–c and numbering of atoms for NMR spectra correlations.

Figure 1

Absorption and photoluminescence spectra (λ_ex = 440 nm) of compounds 3a (a,d), 3b (b,e) and 3c (c,f) (100 μM) in pH buffers.

Scheme 2

Graphic representation of fluorescence intensity versus pH. The darker the shade of orange, the more intense the fluorescence.

Figure 2

The dependence curve of the fluorescence intensity at the maximum on the pH and pKa value for compounds 3a (a), 3b (b), and 3c (c).

Figure 3

Absorbance (a,c) and photoluminescence (λ_ex = 440 nm) (b,d) spectra changes in DMSO solution of compound 3c (100 μM) during the addition of HCl (a,b) and DIPEA (c,d).

Scheme 3

Structural transformations and fluorescent properties changes in benzimidazolium derivatives 3a–c upon the addition of acids or bases.

Figure 4

Toxicity of compounds 3a–c at concentrations of 10⁻⁸–10⁻⁴ M using the V. aquamarinus strain VKPM B-11245. The dotted lines in the diagram indicate the boundaries of the zones of acceptable toxicity, toxicity, and high toxicity. * Differences from the control are statistically significant, t-test; p < 0.05.

Figure 5

Changes in the toxicity of compounds 3a (a), 3b (b), 3c (c), depending on concentrations (10⁻⁸–10⁻⁴ M). The dotted line in the diagram indicates the boundary between weak and moderate toxicity. * Differences from the control are statistically significant, t-test; p < 0.05.

Figure 6

The effect of compounds 3a–c on the biomass, metabolic activity, and viable cell count of bacterial biofilms of E. coli CDC F-50 and S. aureus ATCC 6538-P FDA 209-P strains, depending on concentration (10⁻⁸–10⁻⁴ M). * Differences from control are statistically significant, t-test; p < 0.05.

Figure 7

The image of E. coli CDC-F50 biofilm treated by a solution of dye 3c (10⁻⁴ M, excitation in multiphoton mode with λ = 725 nm) during 3 h, obtained as a result of the bioimaging experiment.

Figure 8

Lambda-stack (left) and snap (right) images of E. coli CDC-F50 biofilm treated by a solution of dye 3c (10⁻⁴ M, excitation in multiphoton mode with λ = 725 nm) during 24 h obtained as a result of the bioimaging experiment.

Figure 9

Lambda-stack (left) and snap (right) images of S. aureus ATCC 6538-P FDA 209-P biofilm treated by solution of dye 3c (10⁻⁴ M, excitation in multiphoton mode with λ = 725 nm) during 24 h obtained as a result of the bioimaging experiment.