Synthesis And Application Of Thiazolothiazole Derivatives With Photo-Responsive Oxidase Activity

Photo-responsive nanozymes have been extensively investigated for applications in various fields,including biosensing,antibacterial and photodynamic therapy due to their characteristics of non-invasiveness,high spatial and temporal resolution,greenness,safety,and economy.How to construct highly catalytically active photoresponsive nanozyme is a hot research topic in this field.Thiazolothiazole is a novel type of fluorophore with a unique fused-bicyclic aromatic framework and exhibits a large molar extinction coefficient and generates reactive oxygen species（ROS）under light conditions.These features favour its applications in photoactive oxygen catalysis.However,to date,no work has been directed at classifying the light-driven oxidase-like activity of thiazolothiazole derivatives.Based on this,eight kinds of thiazolothiazole derivatives with different structural substitutions through the reaction of Menschutkin and Zincke were designed and synthesized after which the photooxidase activity and structure-activity relationship of these compounds combined with molecular simulation technology.The composite nanozyme system with higher activity was formed via supramolecular self-assembly to assembly thiazolothiazole derivatives with macrocyclic molecule Curcubit[8]uril（CB[8]）.On the basis of obtaining highly active oxidase,these designed thiazolothiazole derivatives were assessed for their ability to be used in glutathione（GSH）colorimetric sensor analysis method system as well as photosterilization system.The results obtained in this study provide important reference and basis for the research and development of photoinduced oxidase of thiazolothiazole derivatives.The detailed description is listed as follows:1.The synthesis of thiazolothiazole derivatives and their activity of lightdriven mimetic enzymesEight kinds of thiazolothiazole derivatives with different structural substitutions were synthesized by Menschutkin and Zincke reactions,and their photo-oxidase activity was determined by 3,3’,5,5’-tetramethylbenzidine（TMB）oxidation reaction.The redox potential,highest occupied molecular orbital（HOMO）and lowest unoccupied molecular orbital（LUMO）energy levels of eight compounds in an air atmosphere were obtained by combining cyclic voltammetry properties and molecular simulation techniques.Then,the effects of different substituents on redox potential and HOMO and LUMO energy levels of eight compounds were compared.Studies have shown that increasing the conjugation structure and the electron cloud density of thiazolothiazole derivatives can effectively reduce the electron excitation energy and further promote the photo-induced electron transfer efficiency,resulting in effectively improving the photo-oxidase activity.2.The mechanism of light-driven oxidase-like activity of thiazolothiazole derivatives and its application colorimetric sensing of glutathione（GSH）Based on the above synthesized thiazolothiazole derivatives,N,N’dichlorophenzyled dipyridinium thiazolo[5,4-d]thiazole difluoride(TTz-Cl²⁺)with moderate oxidase activity was selected as the representative compound.The mechanism for these compounds as a photo-oxidase was explored by radical scavenging experiments,electron spin resonance spectroscopy,cyclic voltammetry performance and comparison of oxidase activity in air and nitrogen atmospheres.As a result of illumination by visible light,TTz-Cl²⁺can absorb photons which results in the generation of excited TTz-Cl²⁺.Afterwards,the excited TTz-Cl²⁺obtains an electron from the HAc-NaAc buffer to form an extremely stable radical cation(TTzCl^·+).Finally,when the TTz-Cl^·+ radical cation returns to the parent TTz-Cl²⁺,the dissolved oxygen is converted into the relevant ROS(O₂^·-,·OH)for oxidation of TMB.Given that the oxidation of TMB by TTz-Cl²⁺photo-oxidase activity and the reduction of TMBox by GSH,TTz-Cl²⁺was applied as a functional colorimetric probe for a highly sensitive analysis and detection of GSH in cell lysate,realizing the rapid and sensitive determination of GSH.3.The self-assembly of thiazolothiazole-cucurbituril and its photobacterial applicationIn view of the research results of 1 and the need to further enhance the activity of photooxidase,TTz-Cl²⁺ was applied as a model compound to explore the noncovalent self-assembly properties with macrocyclic molecule CB[8]and its effect on photo-oxidase activity by combining with Job Plot curve,MALDI-TOF-MS,nuclear magnetic titration,electron transmission microscope,photocurrent experiment,electron spin resonance experiments and steady-state kinetic experiments.Studies have shown that 1:1 head-to-tail complex was formed between TTz-Cl²⁺and CB[8]through hydrophobic and ion-dipole interactions.The enzymatic catalytic efficiency of TTz-Cl²⁺increased from 3.58 S-1·M-1 to 131 S-1·M-1（i.e.about 36.59 fold）,which is mainly attributed to the fact that the self-assembled complex(CB[8]-TTz-Cl²⁺)has stronger radical generating ability.In addition,the antibacterial experiments in vitro suggested that the antibacterial effect of CB[8]-TTz-Cl²⁺against Staphylococcus aureus was 100%at low concentration（15 μM）and short time irradiation（5 min）,and is expected to be applied in photo-induced sterilization.