Benzimidazole–Piperazine–Coumarin/Cucurbit[7]uril Supramolecular Photoinduced Electron Transfer Fluorochromes for Detection of Carnosol by Stimuli-Responsive Dye Displacement and pKa Tuning

A new fluorescent dye (4PBZC) comprising coumarin (C), piperazine (P), and benzimidazole (BZ) was designed, prepared, and complexed to cucurbit[7]uril (CB7) to detect carnosol (CAR), an anti-breast cancer drug, in sub-nanomolar concentrations utilizing the supramolecular indicator displacement assay strategy, the CB7-assisted pKa shift, and the CB7-retarded photoinduced electron transfer process. The host–guest complexation was confirmed by UV–visible absorption, fluorescence, and 1H NMR spectroscopy, which established the binding of 4PBZC to CB7. CB7 preferentially binds the indicator dye (4PBZC) via the protonated BZ residue compared to the neutral BZ one, demonstrated by a higher binding constant of the complex in its di-protonated form, which led to an increase in the pKa of the BZ moiety by ca. 3.0 units after the addition of CB7. In aqueous solution (pH 6), switching the emission signals between 4PBZH+C/CB7 (ON state) and 4PBZC (OFF state) was achieved by displacement of the protonated dye from the cavity of CB7 by the CAR analyte. An efficient sensor was obtained for the sensitive detection of CAR in aqueous solution with a low detection limit of 0.148 ng/mL (0.45 nM) and a linear range from 20 to 627 ng/mL.

The formation of host−guest inclusion complexes between 4PBZC and 4PBZH + C and CB7 was confirmed by UV−visible and NMR titrations at different pH (pD) values. The appearance of isosbestic points at 300 nm ( Figure S2a) and the fitting analysis confirmed the 2:1 binding stoichiometry for the 4PBZH + C/CB7 complex, see Experimental Section. The di-protonated dye enters the CB7 cavity through its BZ and coumarin rings ( Figure S4). Inclusion was also confirmed because two proton resonances, H-1, and H-2, shifted upfield by approximately 0.4−0.9 ppm with the addition of 1 equivalent of CB7, while other proton resonances H-8, H-9, H-3, and H-4 slightly shifted to higher ppm upon addition of one equivalent of CB7 whereas H-6, H-7 and the methyl protons exhibited insignificant shifts. Furthermore, consequently to the addition of 2.0 molar equivqlent of CB7, two resonance proton H-6, H7 and the methyl proton shifted upfield, indicating the encalpsulation of the coumarin unit ( Figure S4). S3 Figure S2. Variations in the UV−visible absorption spectra of 5 μM of 4PBZH + C at pH 3 (a) and 4PBZC at pH 9 (b) with the addition of up to 300 μM of CB7. Figure S3. Binding curve resulted from the measurements the UV−visible absorption spectra of 4PBZH + C (5 µM) with different concentrations of CB7 at pH 3 in aqueous solution at 298 K with the nonlinear fit to a 2:1 host−guest binding model (solid line and Experimental Section). The change in optical density (ΔOD) is the difference between the absorbance in the absence and presence of CB7. S4 Figure S4. 1 H NMR spectra of 1 mM 4PBZH + C with CB7 (0−4 molar equivalents) in D2O of pD 3 (400 MHz) with CB7 peaks indicated.
The extracted 2:1 host−guest stability constants ( Figure S3) based on the UV−visible absorption data was 5.5 × 10 6 M −2 ( Figure S2a) at approximately pH 3 (pD 3). In contrast, very low binding affinity of the mono-protonated form with CB7 was noticed from the corresponding binding UV-titration data at pH 9 ( Figure S2b). The NMR titration data ( Figure S4), however, allowed us to extract a 1:1 host−guest stability constant ( Figure S5) of 2.4 × 10 4 M −1 at approximately pH (pD) of 3 by monitoring the shifts in the NMR peak position of H-1 proton.
Although the 1 H NMR results clearly indicated 2:1 binding stoichiometry ( Figure S4) in agreement with the optical data, the data in Figure S5 were fitted according to a 1:1 binding model, due to the fact that 1 H NMR shifts of the BZ due to complexation with CB7 is independent on the binding of the fluorophore (C), albeit both have experienced complexation induced down-field shifts. The spectra in Figure S6 reveal a significant fluorescence enhancement upon the addition of HCl in the absence or presence of CB7 with less amounts of acid needed in the latter case. The di-S6 protonated and mono-protonated forms emit a fluorescence signal in water at 410 nm and 460 nm, respectively, in the absence ( Figure S6a) and the presence of CB7 ( Figure S6b). The changes which give the plot in Figure 5 agree with the host-induced pKa shift in Figure 2, that, both the free or CB7-complexed BZ unit are either protonated at pH< 4 or neutral at pH> 6. Addition of CB7 ( Figure S7) slightly enhanced the emission from the mono-protonated form, possibly because of some restriction imposed by the cavity of the host to their free rotation (confinement effects). 1 Figure S7. Variations in the fluorescence spectra of 5 μM of 4PBZH + C (a) and 4PBZC (b) with the addition of up to 300 μM of CB7, at pH 3 and pH 9, respectively.
A blend of 19.6 mmol of 2-chrolobenzimidazole amounting to 3.0 g and 3.7 g N-tertbutoxycarbonyl piperazine, which equivalent to 20 mmol, were added to 40 mL of 1-butanol and

Preparation of 2-(piperazin-1-yl)-1H-benzo[d]imidazole
Scheme S2.   In the titration test, the complete centralization of the 4PBZC was kept consistent and that of the host, while contender/competitor were slowly expanded at steady pH. The pH of known volume of H2O was first acclimated to bring down pH, somewhere in the range of 2 and 3, to  The intramolecular interaction between CB7 and 4PBZC and 4PBZC/CB7 and carnosol was quantified by the affinity constant known as the association equilibrium constant or binding constant (K). This is associated with the binding/unbinding interaction of receptor/guest (4PBZC) and ligand/host (CB7), as describe below: For an equilibrium reaction, the forward rate of reaction (kf) should be equal to the backward rate of reaction (kb) provided the equation of reaction is balanced.
Subtracting equation (7) from (5), we obtain: The titration by using a UV-Visible spectrophotometer of 4PBZC with CB7 was also conducted to obtain the binding constant (K′) between two CB7 macrocycles and one 4PBZC The absorbance of the free 4PBZC (Y4PBZC) is: