Design,Synthesis And Application Of Novel Selective Probes For Thiol Group

Proteins play an essential role in the regulation of physiological and pathological processes,especially many active proteins with catalytic activity have become the target for drug development.At present,although a large number of proteins related to various diseases have been identified as drug targets,as many as 85-90% of human proteome are still defined as "undruggable" target.This could be accounted for by lack of effective chemical compounds that can mediate the protein functions.Activity-based protein profiling(ABPP)has been a powerful method for exploring the function of "undruggable" proteins by modifying the active sites of proteins,but this approach relies on a variety of novel electrophiles.Therefore,this paper aims to develop novel electrophilic reagents for ABPP technology to study the cancer mechanism and drug development.Thiol is an important functional group of sulfur-containing amino acids and proteins,which plays an important role in biological system,such as cysteine(Cys),homocysteine(Hcy)and glutathione(GSH).Proteins containing thiols have become the target of many anticancer drugs such as afatinib,osibetinib.Due to the strong nucleophilic properties,cysteine is the main binding site.At present,electrophilic probes targeting cysteine are mainly based on the Michael addition reaction of α,β-unsaturated amide with cysteine.Although many probes have been developed for cysteine modification,such as iodoacetamide,a large number of cysteine in proteins are still not accessible.Therefore,it is highly desirable to develop new electrophilic warhead for cysteine modification,so as to facilitate the exploration of the mechanism of disease and drug developments.In this paper,we developed two novel two-photon probes TP-1/2 for selective detection of thiol groups.Both probes have shown good selectivity to thiols under both in vitro and in situ conditions.TP-1 reacts very quickly with thiol group,the reaction completes within a few minutes and gives little fluorescence.The detection limit of TP-1 for thiol group is at as low as nmol level,and the products are stable.In addition,this probe has two-photon absorption and fluorescence "turn-on" effect based on PET.Compared with ordinary one-photon fluorescence probes,these probes show low fluorescence background,strong tissue penetration and haves been successfully used in-situ tissue imaging.In addition,we have developed a series of new electrophilic probes based on isonitrile.By studying the stability of these probes,we found that they were stable.Moreover,our results showed this kind of the probe was a broad spectrum electrophilic probe,which could modify cysteine,lysine,serine,glutamic acid and other nucleophilic amino acids.Based on ABPP,Hep G-2 living cells were labeled with this kind of probe,and the active sites of important proteins were identified.Therefore,two types of probes were developed in this paper,which were TP-1/2probe for quantification of thiols and living cell imaging,and broad-spectrum isonitrile electrophilic probe for labeling various kinds of nucleophilic amino acids.Our research enriched the molecular probe library that can be used for ABPP technology,which proved new tool probe for the study of "undruggable" proteins,and development of potential drug targets and lead compounds.