Design And Synthesis Of Indole-substituted Flavonol Functional Molecules Combined With Cys Fluorescent Probe And CO Photo-releasing And Its Application

Cysteine（Cys）is a natural amino acid containing a sulfhydryl group and plays an indispensable role in regulating physiological processes of physiological metabolism and cell homeostasis.Its abnormal concentration in the body is closely related to the pathogenesis of cardiovascular and cerebrovascular diseases,neurodegenerative diseases,cancer and other major diseases.A trace amount of carbon monoxide（CO）is an important gas signaling molecule in the body and plays an important regulatory role in many physiological and pathological processes.Photo-induced carbon monoxide releasing molecules（Photo CORMs）can realize the spatiotemporally controllable release of CO.Therefore,fluorescent probe that organically combine Cys detection and Photo CORM is of great significance for understanding the biological functions of Cys and CO in organisms.In this paper,we designed,synthesized and characterized a cysteine fluorescent probe of flavonols with indole-substituted flavonols and MICA（2-（1-methyl-1H-indol-3-yl）-4-oxo-4H-chromen-3-yl-acrylate）as the precursor of photoCORM,and extended the A ring on the basis of MICA,designed,synthesized and characterized MICB（2-（1-methyl-1H-indol-3-yl）-4-oxo-4H-benzo[g]chromen-3-yl acrylate）.The responsiveness of MICA and MICB to Cys in vivo and in vitro was comprehensively evaluated.The fluorophores HMIA（3-hydroxy-2-（1-methyl-1H-indol-3-yl）-4H-chromen-4-one）and HMIB（3-hydroxy-2-（1-methyl-1H-indol-3-yl）-4H-benzo[g]chromen-4-one）under O₂ light release performance of CO was studied.The results of the study are as follows:1.Probes MICA and MICB are fast,highly selective（vs.Hcy/GSH）,and highly sensitive（DL:92 n M;86.5 n M,3σ/k）,in a wide linear range（0-2.4 equiv.and 0.4-2 equiv.）for sensing of endogenous and exogenous Cys(λ_em:464 nm,λ_ex:400 nm andλ_em:507 nm,λ_ex:430 nm).2.Probes MICA and MICB can detect and image endogenous and exogenous Cys in He La cells and zebrafish.The probes,fluorophores,and their photolysis product all have good biocompatibility,membrane permeability and low toxicity to He La cells and zebrafish.3.Compared with 3-hydroxyflavonol（3-FL）,the absorption peak of HMIA whose B ring is substituted by indole is red-shifted by 52 nm.On the basis of B ring being substituted by indole,the A ring is expanded to naphthalene ring.The red-shift of the HMIB absorption peak is as high as 88 nm（vs.HMIA red-shifted by 88 nm）.4.HMIA and HMIB generated by the sensing reaction of precursors MICA and MICB with Cys are used as photoCORM,which can provide precise controllable quantitative linear CO in aerobic environment by adjusting the light intensity,light duration,and photoCORM dose.In biological environment,the location of HMIA and HMIB and the CO release process can be tracked in real time without adding any other fluorophores or CO probes to the body.This work offers promising applications for the detection and imaging of exogenous and endogenous Cys,as well as spatiotemporally controllable CO release in living systems.