Design,syntheses And Applications Of Enzyme-activated Fluorescent Probes Based On O-heterocyclic Quinolinium Fluorophore For Bioanalysis

As the indispensable components of organisms,bio-enzymes are a class of natural catalysts with high efficiency and specificity for catalyzing biochemical reactions and regulating physiology activities,and play crucial roles in maintaining the body’s overall functions.Serine hydrolases(SHs)are one of the largest and most diverse enzyme classes in the mammal body.There are more than 200 SHs in the human body,that not only participate in various physiological processes,but also are found to be related to many diseases,such as obesity,inflammation,cancer,liver injury,diabetes,bacterial infection and Alzheimer’s disease(AD).Therefore,the development of effective methods for accurate,real-time and visual monitoring of the distribution and activity of SHs in vivo is significant for both exploring the physiological functions of SHs in organisms and improving diagnosis or therapy of diseases.Organic fluorophore-based probes have many advantages,including easy regulation of molecular chemical structure,high sensitivity and biosecurity,by which the non-invasive,non-radioactive and real-time fluorescence imaging of SHs in vivo can be performed.Due to their great application prospects in biochemical and clinical researches,organic small-molecule fluorescent probes have attracted much attention from researchers.In this work,two kinds of fluorophores with intramolecular charge transfer(ICT)effect were designed and synthesized based on chemical structure regulation strategy and results of theoretical calculation and molecular docking simulations.To meet the requirements of fluorescent visualization of SHs,four enzyme-activated fluorescent probes were developed by modifying corresponding recognition groups on the above fluorophores for sensing carboxylesterase-2(CES2)and butyrylcholinesterase(BCh E)in vivo.Fluorescent properties,sensing mechanisms,biocompatibility,analytical performance and application potential of the developed probes were investigated and evaluated.The detailed research works are listed as follows:(1)A fluorophore PFQ was designed and synthesized by regulating the electronwithdrawing group and the electron-donating group at opposite sides of the molecule.The intramolecular charge transfer(ICT)was attributed as the photoluminescence mechanism of PFQ.PFQ exhibits a red maximum emission wavelength(655 nm),good photostability and a wide application range of environmental p H.A large Stokes shift(135 nm)facilitates the suppression of spectral interference induced by the bandwidth and the scattering of excitation light during the analyses of biosamples.A sensitive CES2-activatable fluorescent probe PFQ-E was developed by modifying PFQ with benzoyl group as the recognition group.PFQ-E exhibits a weak fluorescence signal.After the cleavage of the benzoyl group by CES2-catalyzed hydrolysis reaction,the fluorophore PFQ will be generated and result in a fluorescence "turn-on" response.PFQ-E exhibits high sensitivity,good selectivity and strong affinity towards CES2,and displays good photostability and biocompatibility,which make it suitable for bioanalysis and bioimaging.The spatial distribution and activity variation of endogenous CES2 in tissues as well as living cells were monitored by PFQ-E,and the down-regulation of CES2 activity in acetaminophen-induced liver injury(AILI).The production and remediation pathways of AILI and its underlying molecular mechanism were revealed.(2)A highly sensitive fluorescent probe PFQ-B was also developed by linking cyclopropanecarbonyl group as the recognition group to PFQ.The fluorescence signal of PFQ-B is very weak,while BCh E can catalyze the hydrolysis of PFQ-B to release the fluorophore PFQ,resulting in a bright red fluorescence signal.PFQ-B exhibits excellent performance,including good water solubility,high sensitivity,good photostability,good selectivity and a wide application range of p H,and can be employed for the quantitative detection of BCh E by fluorometry and colorimetry.Results of cell viability and hemolysis experiments demonstrated the low cytotoxicity and good biocompatibility of PFQ-B.PFQ-B can also be utilized to visualize endogenous BCh E in living cells and tissues.Results of the cell substructure localization experiments revealed the good cell-penetrating ability and the non-nucleus distribution of PFQ-B.PFQ-B was applied to imaging BCh E in brain tissues of AD mouse models and normal mice.Results demonstrated the excellent performance of PFQ-B for discriminating AD tissues.(3)A near-infrared(NIR)fluorophore CHQ was designed and synthesized by introducing dihydroxanthene group to PFQ.The rigid ring chemical structure of dihydroxanthene group will inhibit the rotation of single bonds,enhance the planarity,and improve the overall conjugation degree of the CHQ molecule.Both the excitation and the emission maxima of CHQ are in the NIR region(715 nm and 775 nm,respectively),which improves tissue penetration depth,and eliminates autofluorescence and photodamage of biosamples during analyses.A novel NIR fluorescent probe CHQ-E was developed for the rapid and sensitive detection of CES2 by linking a recognition group(benzoyl)to CHQ.The benzoyl group can be easily cleaved from CHQ-E by CES2,resulting in the "turn-on" fluorescence.CHQ-E displayed superior analytical performance for sensing endogenous CES2 in living Hep G2 and HCT-116 cells,including high sensitivity,good photostability and biosafety.Results of tomographic scanning experiment using hepatic tissue of mouse revealed the three-dimensional imaging capability of CHQ-E.CHQ-E was applied to monitoring in vivo CES2 activity in drug-induced hepatotoxicity and remediation models,demonstrating its broad potential for the assessment of liver health status.(4)By respectively employing CHQ and cyclopropanecarbonyl group as the fluorophore and the recognition group,a sensitive NIR fluorescent probe CHQ-B was also developed to investigate the expression of BCh E in tissues of diabetic mouse models.The fluorescence signal of CHQ-B is very weak due to the inhibition of ICT process induced by the cyclopropanecarbonyl group.After the cyclopropanecarbonyl group is specifically cleaved from CHQ-B by the BCh E-catalyzed hydrolysis reaction,and the consequent product CHQ will cause 30-fold fluorescence "turn-on" response.The probe CHQ-B exhibits excellent photostability,good water solubility,and highly sensitive and selective responses towards BCh E,and is suitable for quantitative detection of BCh E by fluorescent and colorimetric modes.Additionally,the good biocompatibility of CHQ-B facilitates its application in fluorescent imaging of BCh E in vivo.Experimental results of fluorescence imaging diabetic mouse models confirm the up-regulation of BCh E expression induced by diabetes.In summary,the chemical structure regulation strategy as well as the recognition group modification strategy were proved to be effective for the development of organic small-molecule fluorescent probes with enzyme-activated response.All the organic small-molecule fluorescent probes developed in this work are adequate for both quantitative detection of the activity and visual imaging of the distribution of corresponding SHs in living cells or tissues,and are easy to be applied to related research processes of enzymatic functions or the production and remediation pathways of enzyme-related diseases and their underlying molecular mechanism.The strategies of chemical structure regulation and modification of fluorophore molecules proposed in this work can provide effective reference for similar research works in the future.The probes developed in this work provide novel tools for clinical diagnosis and therapy.