Application Of Several Reactive Fluorescence Probes For Phosphate Ion Detection For Detection, Synthesis And Biomedical Applications

It still remains a huge challenge to determine the intracellular concentration of some biological important species for conundrum of these species including low concentration, high reactivity and short lifetimes. Recently, the detecting technology of fluorescent sensors have been popular for its many advantages, comparing with other biological detecting technologies, such as good sensitivity, excellent selectivity, rapid response, and non-invasive detection. As one kind of the fluorescent sensors, reaction-based fluorescent sensor is becoming a new favorite originated in it’s distinguished characteristics of exclusive detection and high selectivity.In this thesis, we focused on the detection of phosphate ion(Pi), which is an important downstream metabolic product of nucleotides, playing pivotal roles in energy transduction and storage in biological systems. We have designed and synthesized several reaction-based fluorescent probes for phosphate ion detection, also, their photophysical and biological properties have been studied. It was found that these fluorescent probes exhibit excellent selectivity and good spectrum signal responding to Pi. Further more, some of these fluorescent probes have been applied successfully in the study of biological imaging for phosphate ion detection. Using 1,8-naphthyridine-based boron complexes, coumarin and cyanine as fluorophores respectively, we have designed and synthesized several high selective probes for Pi detection, including 1, 2, SN1, SN2 and SN3.1. Two 1,8-naphthyridine-based N,O-chelated boron complexes(1 and 2) were designed as novel phosphate ion(Pi) probes, based on a methoxy oxalyl group as reaction site. Dramatic color changes from colorless to light yellow were observed by addition of Pi to solutions of 1 and 2 in DMSO/HEPES buffer(0.02 M, pH 7.4)(V/V = 8:2). Fluorescence quenching was observed in solutions of 1 and 2 upon addition of Pi. The invivo imaging results demonstrated that 1 and 2 were able to permeate into HeLa cells and detect Pi via a strong fluorescence intensity change.2. Using 7-amino-4-methyl-2H-chromen-2-one or 7-amino ethyl-4-methyl-2H-chromen-2-one as fluorophore, and using methyl oxalyl chloride as reaction group, we synthesized two reaction-based fluorescent probes SN1 and SN2. These two novel probes displayed an instant turn-on fluorescence response specific towards Pi without interference from ATP and PPi, which were characterized by 1H NMR titration and MS. Probe SN1 was successfully used to image endogenous and exogenous Pi in living cells and to trace Pi released from ATP by apyrase in C. Elegans.3. Using IR-780 cyanine as fluorophore and methyl oxalyl chloride as reaction group, we synthesized a reaction-based fluorescent probe SN3. This Pi probe SN3 displayed enhancement in fluorescence and colorimetric response towards Pi with a distinct color change from green to yellow in aqueous media. SN3 was successfully used to image endogenous and exogenous Pi in living cells.