The Development Of Benzimidazole-based Ratiometric Fluorescent Probes And Their Applications

Ratiometric fluorescent probes allow the measurement of emission intensities at two different wavelengths, which can overcome the drawbacks of intensity-based measurements due to a built-in correction for environmental effects (i.e. canceling artifacts due to instrumental efficiency and light intensity) and increase the selectivity, sensitivity and dynamic ranges of the method. The design mechanisms for ratiometric fluorescent probes are based on fluorescence resonance energy transfer (FRET), excited-state intramolecular proton transfer (ESIPT), intramolecular charge transfer (ICT) and so on.2-(2-hydroxyphenyl) benzimidazole (HPBI) could undergo an ESIPT process upon light excitation and displayed fluorescence emission at 360 and 454 nm. If the hydroxyl group of HPBI was modified, ESIPT process of HPBI would be switched off and its spectral characteristics should change. Based on the consideration, three ratiometric fluorescent probes for F-, alkaline phosphatase (ALP) and P2O74- were designed based on modulation of ESIPT process via modification of the functional hydroxyl group of HPBI in the thesis. In addition, we developed 2-(4-aldehydephenyl)benzimidazole (4) as a novel ratiometric fluorescent probe for HSO3-. Compound 4 could undergo an intramolecular charge transfer (ICT) from electron rich benzimidazole moiety to electron deficient aldehyde group upon photoexcitation. Thus,4 could emit two emission bands. If the aldehyde group on 4 interacted with HSO3-, the electron deficient capability of compound 4 decreased and the ICT process would be switched off, which resulted fluorescent spectral characteristics of the system changed. Based on the mechanism,4 displayed a ratiometric response to HSO3-. The thesis composes of five chapters as follows:In chapter 1, several mechanisms for designing ratiometric fluorescent probes were introduced, and research content of the thesis was presented.In chapter 2, a ratiometric fluorescent probe for fluoride ion employing ESIPT process. The probe 1 was developed based on modulation of ESIPT process of HPBI through the hydroxyl group protection/deprotection reaction. Because the ESIPT process was switched off, the probe 1 showed only fluorescence emission maximum at 360 nm. Upon treatment with fluoride in aqueous DMF solution, the TBS protective group of probe 1 was removed readily and ESIPT of the probe was switched on. Accordingly, it was observed that the fluorescence emission at 360 nm showed "turn-off", while the fluorescence emission at 454 nm showed "turn on". The proposed probe showed excellent selectivity toward fluoride ion.In chapter 3, an ESIPT-based approach to ratiometric fluorescent detection of ALP. The probe 2 was readily prepared by the reaction of HPBI with phosphorus oxychloride (POCl3) and then hydrolyzed to give the desired product. In the absence of ALP, free 2 exhibited one typical emission peak at 363 nm. However, addition of ALP to the solution of 2 induced formation of a new emission peak at 430 nm because 2 was hydrolyze into HPBI by ALP and ESIPT of the probe was switched on. Moreover, with an increasing amount of ALP, the emission band of probe 2 at 360nm gradually decreased with the concomitant growth of emission band at 430 nm. The fluorescent intensity ratio at 430 and 363 nm (I430/I363) increased linearly with the activity of ALP up to 0.050 U mL-1 with a detection limit of 0.0013 UmL-1.In chapter 4, a new P2O74--selective ratiometric synchronous fluorescent probe was designed and synthesized. The probe 3 was prepared by the reaction of HPBI with Zn2+ Because its hydroxyl group was bound, the ESIPT process of HPBI was switched off and probe 3 showed fluorescence emission maximum at 418 nm. Upon introducing P2O74- to a solution of probe 3, P2O74- could compete effectively Zn2+ with HPBI, which resulted in the decomposition of 3 into HPBI and restoration of the ESIPT process. However, the emission wavelength of 3 (418 nm) was close to the maximum emission wavelength (454 nm) of HPBI and their fluorescence spectra were prone to overlap. Therefore, synchronous fluorescence technique was applied in this experiment. HPBI showed another fluorescence emission at 364 nm and its stockes shift (△λ) was very close to that of 3. Therefore, fluorescence emission spectra of 3 could distinguish from the emission band of HPBI at 364 nm by synchronous fluorescence technique with a△λof 40 nm. Addition of P2O74- to a solution of 3 induced a decrease of the emission band at 418 nm and an increase of a new fluorescence peak at 364 nm simultaneously. Based on above mechanism, a ratiometric synchronous fluorescent probe for P2O74- was developed. The proposed probe showed excellent selectivity toward P2O74-over other common anions.In chapter 5, a novel ratiometric fluorescent probe for HSO3- was developed based on ICT process. The probe 4 showed two fluorescence emission bands centered at 368 nm and 498 nm, respectively. Upon addition of HSO3-, the aldehyde of 4 reacted with bisulfite and produced an aldehyde-bisulfite adduct, which resulted in a decrease of the emission band at 498 nm and an increase of a fluorescence peak at 368 nm due to switching off ICT process of probe 4. The fluorescent intensity ratio at 368 and 498 nm (I368/I498) increased linearly with HSO3- concentration in the range 2×10-6-2.0×10-4mol L-1. The proposed method showed high sensitivity and excellent selectivity toward HSO3-.