Developing Hydroxylphenylpolyenylpyridinium-based Two-photon Fluorescent Probes For Detecting NQO1 And TrxR

Imbalance of intracellular redox homeostasis plays a pivotal role in the development of various diseases,such as cancer,neurodegenerative and cardiovascular diseases.Oxidoreductases are the important components to maintain this balance.Probing their function and level is of great significance for diagnosis and treatment of diseases in pathlolgy and physiology.Two-photon fluorescence imaging technology has become a powerful tool for detecting and imaging target bio-analytes in complicated biological systems due to its excellent sensitivity,non-destructive rapid analysis,high spatio-temporal resolution and deep tissue penetration.Herein we developed the novel enzyme-activated two-photon fluorescent probes based on the hydroxylphenylpolyenylpyridinium chromophores conjugated with the response groups.The main research contents are summarized as follows:(1)NAD(P)H:quinone oxidoreductase 1(NQO1)plays a critical role in the detoxification of quinone and is recognized as a potential therapeutic target.Spatio-temporally tracking and visualizing NQO1 is thus highly desirable for early diagnosis of the diseases associated with NQO1 and monitoring the related efficacy of therapy.Herein we designed a class of push-pull hydroxylphenylpolyenylpyridinium fluorophores differentiated by length of the conjugated links for imaging of NQO1 via an intramolecular charge transfer process,based on their coupling with trimethyl lock quinone.After careful screening we finally identified the novel probe(QBMP)as a potent ratiometric two-photon fluorescent probe for detection of mitochondrial NQO1 with superior properties,including excellent selectivity,rapid response time(4 min),large Stokes shift(162 nm),ultralow detection limit(0.9 nM)and deep tissue penetration(225 ?m).More importantly,this probe was successfully applied for distinguishing different NQO1-expressing cancer cells and normal cells,revealing decreased NQO1 activity in a cellular Parkinson's disease model and screening NQO1-inducers as neuroprotective agents as exemplified by dietary [6]-shogaol and its analogs.(2)The thioredoxin system,consists of thioredoxin reductase(TrxR),thioredoxin(Trx)and NADPH,and plays an important role in maintaining intracellular redox homeostasis and regulating redox signal events.Among which,TrxR is the only known enzyme to catalyze the NADPH-dependent reduction of Trx,and has surfaced as a promising target for developing anticancer agents.We used hydroxylphenylpolyenylpyridinium as the fluorophore,1,2-dithiolane as the reaction site and carbonate as the linker to develop the TrxR probe,DSMP.It manifests a plethora of excellent properties including large Stokes shift,quick response,excellent two-photon property,mitochondria-targeting,ratiometric two-photon imaging of mitochondrial TrxR.