Design And Synthesis Of Novel H₂S₂/Thiol Two-Photon Probes And Investigation Of Their Spectral Properties

The technology of fluorescence imaging has been widely used in biotechnology, ion detection, medical clinical diagnosis, environmental science, materials science and other fields because of its easy operation, fast response, high sensitivity, easy to operate and reproduce, simple instrument, destructive biological samples etc. Apart from that, researchers can design and synthesize probes according to their own requirements, so the fluorescent probes and fluorescence imaging techniques are loved by the majority of scientists. Lot of biology studies have shown that the biological function of the hydrogen sulfide is actually regulated by H2Sn (n> 1). Compared with OPM, two-photon imaging techniques which were excited by two photons have many advantages, such as low biological samples injury, deep penetration, high-resolution imaging and three-dimensional imaging. These advantages are well suited for living tissue imaging. However, the H2Sn situ imaging in vivo has not been implemented, so to build a new type of two-photon fluorescence probe to detect the H2Sn in vivo biological sample is of great importance. On the other hand, thiol is the constitution of the essential part of many biological proteins and small molecules in cells which plays a very important role in cellular antioxidant, especially in cellular redox reaction, carbon dioxide fixation reaction, and methyl transfer reaction, the reaction of coenzyme. Therefore, in clinical medicine and biochemistry fields, the design of a fluorescent probe to detect the concentration of thiol has a very important significance. The main works of this paper are as follows:Firstly, a turn-on two-photon fluorescent probe GCTPOC-H2S2 was designed which was based on GCTPOC and 2-fluoro-5-nitro-benzoic acid methyl ester. GCTPOC had an adjustable two-photon cross-section, which was this effectively regulated by the mechanism of ICT. The structure of 2-fluoro-5-nitro-benzoic acid moiety was a typical fluorescent quencher. Baced on the mechanism of ICT and the typical fluorescent quenching function, probe GCTPOC-H2S2 had high sensitivity and selectivity of H2Sn, which was very suitable for H2Sn situ imaging in live mice.Secondly, we designed and synthesized a two-photon thiol probe BTh- thiol which was based on the benzothiazole derivative. The probe had a good selectivity to in neutral buffered solution of cysteine (CyS) and homocysteine (Hcy). The probe BTh-thiol could react with Cys/Hcy and generated thioether through the reaction of conjugate addition and then through cyclization ammonia to form 3-benzothiazol-7-hydroxy coumarin. Although two kinds of thiol Cys/Hcy had the similar structure, homocysteine (Hcy) had another methylene compared with cysteine (Cys). So there were differences in the kinetics of ring-forming process, we could distinguish Cys from Hcy by researching the differences in dynamics. Because of the structure of glutathione (GSH), GSH could generated thioether through the reaction of conjugate addition only when reacted with BTh-thiol, which could not release hydroxy, so the fluorescence did not change significantly, which demonstrated that the probe could be used to detect three thiols (Cys/Hcy/GSH) by the changes of fluorescence intensity and duration of reactions.