| As an important part of thioredoxin（Trx）system,thioredoxin reductase（TrxR）ensures the redox balance in the biological system;whereby once the redox imbalance happens,it will lead to the occurrence of a variety of diseases,particularly cancer.Inhibition of overexpression of TrxR in cancer cells will lead to apoptosis of cancer cells,making TrxR become a very important target of antitumor drugs.It is of great significance to develop TrxR inhibitors.In addition,selenocysteine（Sec）is the key active site of TrxR.The content of Sec has a direct impact on the activity of TrxR,and is crucial to the development of physiology and pathology of related diseases.Therefore,it is extremely important to develop accurate visualization of Sec detection methods.To this end,TrxR inhibitors and Sec fluorescent probes were synthesized by 2,4-dinitrophenyl,and their properties were studied in this paper.It is known that 2,4-dinitrobenzene sulfonates are the fluorescence recognition group of Sec.However,to date,its inhibitory potential against TrxR has not been reported.In this paper,first of all,a series of2,4-dinitrophenyl TrxR inhibitors were designed and synthesized,and their TrxR inhibition performance and antitumor activity were studied.Then,in view of the problems of some Sec fluorescent probes with large self-interference of fluorescence caused by small Stokes shift,large light damage caused by short fluorescence emission wavelength,weak tissue penetration and low sensitivity,five novel small molecule Sec fluorescent probes were reasonably designed and synthesized based on the stronger nucleophilic properties of Sec than biothiols.Two isomeric Sec fluorescent probes were designed and synthesized based on phenomidazole derivatives.Probe A4 could solve the problem of small Stokes shift and large self-interference due to excited state intramolecular proton transfer（ESIPT）mechanism.Two long wavelength Sec fluorescent probes were designed and synthesized based on oxanthracene derivatives for the analysis of Sec in living cells due to intramolecular charge transfer（ICT）mechanism,which could solve the the problems of large light damage and shallow tissue penetration,etc.A ratiometric Sec fluorescent probe was designed and synthesized based on the connection between coumarin group（donor）and naphthalene dicarbonamide group（receptor）due to fluorescence resonance energy transfer（FRET）mechanism to solve the interference of instrument effect,probe molecular concentration and environmental factors,etc.The specific works of this paper are presented as follows:（1）Based on 2,4-dinitrobenzene group,a series of 2,4-dinitrobenzene sulfonamide,2,4-dinitrobenzene sulfonic acid ester and2,4-dinitrobenzene ether compounds（S1-S16）as TrxR inhibitors were synthesized.The inhibitory activity of the compound against TrxR was determined by TrxR assay kit.The inhibitory activity of the selective active compounds to A549,Hep G2 and Mc F-7 cells was studied and the structure-activity relationship was discussed.The IC50 of compound S2 to TrxR was 5.5±1.7μM,stronger than that of 2,4-dinitrochlorobenzene.Inhibitory activity of compounds S2,S5 and S13 to A549 cells,Hep G2cells and MCF-7 cells were stronger than the other compounds and the inhibitory activity of compound S2 was higher than that of 2,4-dinitrochlorobenzene to the three cancer cells.The structural-activity relationship indicated that the inhibitory activities against TrxR were found to be:2,4-dinitrobenzene sulfonyl esters>2,4-dinitrobenzene sulfonyl ethers>2,4-dinitrobenzene sulfonamides.This study proved all the aforementioned compounds could be used as TrxR inhibitors.Interestingly,this finding provided a theoretical background for the design,synthesis and screening of TrxR inhibitors.（2）Two isomerized Sec fluorescence probes A4 and B4 were developed,based on ESIPT and ICT mechanism,respectively.They could emit bright fluorescence after reaction with Sec to achieve quantitative detection of Sec and Sec identification imaging analysis in cells and zebrafishes.Their Stokes displacements were 125 nm and 82 nm,and the detection limit were 65.4 n M and 84.7 n M,respectively.A4 has a longer emission wavelength,a larger Stokes displacement and a smaller fluorescence self-interference than B4.（3）A Sec fluorescent probe YZ-A4 was developed based on benzoxanthene derivative,which reacted with Sec and emitted bright red fluorescence at 614 nm.It showed the advantages of fast response rate（3min）,good selectivity and high sensitivity.The quantitative detection range of the probe YZ-A4 to Sec was from 0 to 32μM,and the detection limit was as low as 11.2 n M.The fluorescent probe YZ-A4 could be used for Sec recognition imaging analysis in living cells and zebrafishes.（4）A Sec fluorescence probe YZ-M5 from xanthene derivative was reported.After reaction with Sec,the probe YZ-M5 emitted bright red fluorescence at 660 nm with a response time of 5 min.The quantitative detection range of the probe YZ-M5 to Sec was from 0 to 40μM,and the detection limit was as low as 18.5 n M.It showed the advantages of fast reaction rate,good selectivity and high sensitivity,etc.The fluorescent probe could be used for Sec recognition imaging analysis in living cells and living body（mice）.（5）Based on FRET and ICT mechanism,a FRET-ICT ratiometric fluorescent probe Q7 was designed and synthesized.It displayed two very bright blue and green fluorescence emission signal in the presence of Sec with self calibration,the distance between two emission peaks 68 nm,which could reduce or eliminate the interference of false signal caused by the mutual perturbation of light between two channels in imaging analysis.The detection limit was as low as 6.9 n M,which shows the better sensitivity.The results of imaging test in living cell and zebrafishes showed the high resolution imaging analysis of Sec by the conversion of blue and green fluorescence.This article is consisted of 84 figures,9 tables,184 references in total. |
