Study On The Fluorescence Detection Of Cysteine And Its Sulfinated Modified Products

Biothiols are important reducing substances in cells and the main components of most protein macromolecules,and they play a vital role in biological physiological activities.Cysteine(Cys),as one of the most important small-molecule biological thiols,is not only the precursor of glutathione(GSH),acetyl-Co A and taurine,but also involved in multiple cell and life activities,such as biocatalysis,post-translational modification,and detoxification of exogenous substances.Cys plays a key role in maintaining the physiological functions of cells.Elevated plasma homocysteine(Hcy)levels are risk factors for Alzheimer's disease,folic acid and cobalamin(vitamin B12)deficiency,and cardiovascular disease.Protein oxidative modification usually refers to the structural and conformational changes caused by the oxidation reaction of biological macromolecules induced by reactive oxygen species(ROS)in cells.As an important molecular switch,protein oxidative modification can quickly and effectively regulate the process of signal transduction.Protein sulfinated modification(RSO₂H),as a form of peroxidative modification of the cysteine sulfhydryl group of protein,affects various signal transduction of cells and various physiological processes of the organism,and is closely related to various oxidative stress diseases and cancers.Therefore,realizing the timely,rapid and specific detection of cysteine/homocysteine and protein cysteine sulfhydryl modification will help to better study the exact mechanism of oxidative modification,which is of great significance for the early diagnosis and treatment of diseases.At present,many scholars and experts have developed many fluorescent probes for the detection of small molecule biological thiols.However,high-specificity,real-time and rapid monitoring of changes in the content of small-molecule biothiols in cells is still one of our research hotspots.So far,there are only scattered reports on the selective detection of protein cysteine sulfhydrylation in cells,and there are still few studies on in situ imaging of protein cysteine sulfhydryl modification.A chemical probe that can specifically react with the sulfhydryl modification of protein cysteine is still an important problem.In order to better solve the above problems,we can conduct more intuitive,sensitive and real-time monitoring of the sulfonation modification of small molecules of thiol cysteine/homocysteine and protein cysteine,which are reducing substances in cells.Two fluorescent probes were designed and synthesized,and the application of these two fluorescent probes was discussed.Specifically:(1)A new type of fluorescent probe PNC-SH with 5-nitro-2-furoate as a small molecule thiol recognition group was designed and synthesized for intracellular cysteine/homocysteine fluorescence imaging with rapid,high sensitivity and high specificity at the same time of acid.In PBS buffer solution(10 m M,p H=7.4),the PNC-SH probe can react quickly with small molecule thiol cysteine/homocysteine within 5 minutes,the fluorescence intensity is significantly enhanced,and Cys and Hcy were quantitatively detected,and the detection limits were as low as0.17?M and 0.20?M,respectively.In addition,we successfully used the PNC-SH probe to perform fluorescence imaging of the small molecule thiol Cys/Hcy in human normal liver cells and human liver cancer cells.We also successfully detected and imaged Cys/Hcy in zebrafish using the probe PNC-SH.(2)We designed and synthesized a tetrazine compound tag TNO that can specifically label protein cysteine sulfhydrylation sites,and generate fluorescent probes in situ based on the tetrazine bioorthogonal elimination reaction,the fluorescence of the alkenyl ether probe BDNE is turned on in situ.Therefore,it is expected to realize real-time in situ fluorescence imaging of intracellular protein cysteine sulfhydrylation,providing a new tool for the study of intracellular protein sulfination with high selectivity,speed and efficiency.