The Design And Synthesis Of Fluorescent Probes For The Selective Detection Of Reducing Substances And Its Application In Bioimaging

Thiols, which are components of many proteins and simple molecules, play an important role in the cellular antioxidant defense system. Alternations in the level of cellular thiols have been linked to a number of diseases, such as leucocyte loss, psoriasis, liver damage, cancer, and AIDS. Thus, the quantitative detection of intracellular thiols is of great importance for investigating cell functions. In recent years, more and more studies focus on detecting intracellular thiols and the detection methods have been developed. Among these methods, using fluorescent probes has many advantages such as high sensitivity, high selectivity, easy to operate, and the availability of a wide range of indicator dyes. What is more attractive is that fluorescent probes can penetrate into cells without any damage, and combine with thiols to form strongly fluorescent substance. With the help of confocal microscopic imaging, visualizing and real-time detecting of cellular thiols can be achieved.At present, much attention has been paid to develop fluorescence probes for biological thiols. However, these fluorescence probes cannot react with thiols fast enough to make real-time detection under physiological conditions. Therefore, we are interested in developing a highly sensitive and selective fluorescence probe that exhibits a fast thiol-quantification reaction. Based on the changes in spectrum characters of the fluorescent probes reacting with thiols, we have achieved the detection of them. We have carried out two aspects of investigation:First, a novel fluorescent probe was prepared bearing two maleimide groups attached directly to the acridine derivative for fast nonprotein thiol detection based on thiol-maleimide bioconjugate reaction. The probe was weak-fluorescent, upon selective reaction with non-protein thiols with a ratio of 1:2 (Probe/ Glutathione(GSH)), strong fluorescence generates (λex/λem = 380/440 nm). During the reaction of dimaleimide fluorogens with bulkier protein thiols, very little dithiolated adduct is formed, and formation of monothiolated adduct predominates. However, when the dimaleimide fluorogens reacts with nonprotein thiols, the dithiolated adduct appears to be the sole contributor to the observed fluorescence. So the probe is highly selective to nonprotein thiols. The reaction went to complete in less than 1 min after mixing in simulated physiological condition. The probe features excellent immunity to interferences, including protein thiols, reactive oxygen species (ROS), metal ions and biological antioxidants. There was a good linearity between fluorescence intensity and GSH concentrations in the range of 1.0×10^-8-1.5×10^-6 M. The detection limit of 7.7 nM was achieved. The distance between the two maleimide moiety is a very important parameter for the rate of thiol-maleimide bioconjugate reaction. So we synthesized ortho- and meta- substituted dimaleimide derivatives of acridine via conventional routes, and the experiment showed the ortho-substituted probe indeed has a faster rate than the meta-substituted one.Second, this paper presented the design and synthesis of a novel rhodamine 110-based fluorescenct probe for detecting small peptides including di-Cys.