Design,Synthesis And Application Of Dual Key And Lock-based Fluorescent Probes

Fluorescent sensing technology has become excellent tools for applications in various fields owing to the advantages of good selectivity,high sensitivity and temporal-spatial resolution,as well as real-time detection of cellular physiological or pathological.Nowadays,a variety of small-molecule fluorescent probes have been developed to detect important markers related to diseases in living organisms.However,a pathological process usually needs two or more indicators to be determined together.It is still a challenge to detect markers in specific diseased tissues or organelles with single response probes.Dual key and lock response fluorescent probes contain two responsive sites to allow for dual stimuli.Therefore,the single molecule dual key and lock response probe could overcome shortcomings of the single response probe and improve the accuracy of detection.In addition,dual key and lock response fluorescent probes for real-time imaging of multiple biomarkers could study the relationship between biomarkers and distinguish them in the pathological pathway of organisms.Hence,taking peroxynitrite(ONOO-)and membrane tension,hydrogen peroxide(H2O2)and viscosity,hydrogen sulfide(H2S)and viscosity as markers,multiple kinds of fluorescent probes with dual key and lock response were designed and synthesized,which could accurately detect several pathological processes and reveal the relationship between related pathological processes.Combined with multiple physiological pathological processes such as pyroptosis,inflammatory and ferroptosis,the application of dual key and lock response probes for early diagnosis of disease was studied by cell and in vivo imaging in many aspects.(1)The dual key and lock response fluorescent probe 2-1 was constructed by coumarin derivatives as a fluorophore,which was modified with alkyl of liposolubility and sulfonic acid group of water-solubility to endow it with amphiphilic ability.2-1 could be embedded into the phospholipid bilayer of cell membrane through hydrophobic action.The fluorophore was covalency connected with pyridine propane sulfonic acid group by single bond,which gave it rotation characteristics to achieve the ability to detect cell membrane tension.Meanwhile,2-1 could be used to identify ONOO-through oxidation reaction to realize the strategy of dual key and lock response.Experimental results showed that the ratio signal(I417 nm/I602 nm)of 2-1 could be changed after reacting with ONOO(response time 5 min),and could target and aggregate the cell membrane to detect the changes of cell membrane tension during pyroptosis induced by palmitic acid(PA).In the OGD/R cell experiment,pyroptosis could be significantly alleviated by adding cell pyroptosis inhibitor GM-1.The dual-response performance of 2-1 is expected to realize the detection of signal pathway in the process of pyroptosis by inflammation induced.(2)Quinoline derivatives as a fluorophore and borate ester group as the H2O2 recognition site,a dual key and lock response fluorescence probe 3-1 was designed and synthesized.The ratio of fluorescence signal of 3-1 changed after reacting with H2O2(response time 40 min)at high viscosity,which had large Stokes shift(174 nm).The probe showed that N-(2-(cyclohexyloxy)-4-nitrophenyl)methanesulfonamide(NS-398)could be used as antiinflammation drugs,which was proved by the experiment of the cell inflammation induced by lipopolysaccharide and interferon-y.Meanwhile,3-1 can distinguish oxidative stress to realize the accurate diagnosis of cellular inflammation.In addition,formaldehyde(FA)extracted from aldehyde tanned fur of leather products induced inflammation in cells and in vivo.The experiment showed that 3-1 could realize the imaging of FA induced neuroinflammation in cells and in vivo.Besides,FA in fur products have an effect on the health of human body,thus the fur industry needs to strictly control the FA content in fur products in strict accordance with national.(3)A near-infrared dual key and lock response fluorescent probe 4-1 was constructed by isophorone derivative as a fluorophore,which could detect H2O2 in complex inflammatory biological systems.The probe with the viscosity sensitivity a boronate group was easily cleaved by H2O2 which then release fluorophore through 1,6-elimination and generate H2S from COS catalysed by carbon anhydrase(CA).The dual response to H2O2 and viscosity in vitro,4-1 has obvious the enhancement of fluorescence signal and has the excellent detection limit(0.37?M).Meanwhile,4-1 exhibited good biocompatibility and enabled fluorescent imaging towards endogenous/exogenous H2O2 under compatible viscosity.In addition,the experiment of cellular inflammatory induced by LPS showed that 41 could not only realize the accurate diagnosis of inflammation,but also effectively alleviate the inflammatory damage caused by the release of H2S and the removal of H2O2.(4)Quinoline-malononitrile derivatives as a fluorophore,azide benzene as the identify unit of H2S,a dual key and lock response fluorescent probe 5-1 was constructed.The fluorophore connected with azide group by covalent bond through a thiocarbamate(H2S precursor).5-1 with NIR luminescence was endowed with strong tissue penetration,minor cell and tissue damage,and low auto fluorescence interference.Changes in viscosity altered the degree of rotation between thiophene and the isophorone derivative.Therefore,5-1 was capable to accurate understanding of ferroptosis progression through the ability of viscosity and H2S dual response.Meanwhile,5-1 could release H2S in the process of detecting H2S to achieve the stability of H2S content.This strategy could eliminate the damage of 5-1 to redox homeostasis of cell in the detection process and avoid the aggravation of ferroptosis of cells,which resulted in the detection results more accurate.The results showed that a ferroptosis model by erastin did not change levels of Fe2+,malondialdehyde(MDA)and glutathione(GSH),which demonstrated that the degree of cell ferroptosis was not affected by the introduction of 5-1.In contrast,ferroptosis progression was significantly promoted when acetazolamide was used to inhibit the release of H2S by 5-1.These results indicated that the dual key and lock response fluorescent 5-1 could achieve the high-fidelity in situ imaging analysis of the degree of ferroptosis.