Designing Fluorescent Probes For Detecting Hydrogen Peroxide And Developing Tyrosinaseactivated Anticancer Theranostic Prodrug

Hydrogen peroxide（H₂O₂）,the most stable active oxygen species（ROS）,functions as either an important participant in maintaining intracellular redox homeostasis,or a signaling molecule in regulating physiological process including cellular growth,proliferation,and migration.However,its excessive production can promote oxidative stress and further accelerate the development of various disease.Developing fluorescent probes for detecting levels of intracellular H₂O₂ has great significance to clarify its role in various physiological and pathological process.Boronates have been widely used as response groups to construct H₂O₂ fluorescent probes,which can be finally transformed into phenols via nucleophilic attack of H₂O₂,Bayer-Villiger oxidation-like rearrangement and hydrolysis.Even with many advantages,such as low cytotoxicity and high sensitivity,Boronate-based probes show poor selectivity over other ROS,especially peroxynitrite,thereby limiting their further application.Therefore,it remains highly needed for developing boronate-based probes with better selectivity.Excited-state intramolecular proton transfer（ESIPT）-based dyes has attracted much attention for their large Stokes shift,double emission and environmental sensitivity.2-（2’-Hydroxy-3’-methoxyphenyl）benzothiazole（HMBT）is a common ESIPT dye that can be easily synthesized and modificated.In this thesis,two H₂O₂fluorescent probes with good selectivity over other ROS including peroxynitrite were developed by elongating conjugated links of HMBT via carbon-carbon double bonds.The main research contents are summarized as follows:（1）The ratio fluorescent probe,quantitative signal of which is obtained from ratio of two wavelengths,can avoid the data distortion caused by background interference.In this case,boronate-modified pyridylacrylonitrile was conjugated to HMBT scaffold,generating a ratio probe THMP.The emission of probe was blueshifting from red to green when H₂O₂ was added.The probe can not only target mitochondria by virtue of its pyridinium unit but also effectively distinguish peroxynitrite（ONOO^-）from H₂O₂via oxidative cleavage of its carbon-carbon double bond by ONOO^-,which results in quenching of the green fluorescent.The probe was successfully applied to indicate the concentration of mitochondrial H₂O₂ and its increase during ferroptosis of Hep G2,highlighting that it is a powerful tool for detecting mitochondrial H₂O₂.（2）Near infrared fluorescence imaging has attracted much attention due to its deep tissue penetration and low light damage.Herein,a near-infrared fluorophore THMT-OH characterized by cooperation of ESIPT and intramolecular charge transfer（ICT）processes was constructed,and further developed for a turn-on dectection of H₂O₂ by introducing an electron withdrawing group 2-dicyanomethylene-3-cyano-4,5,5-trimethyl-2,5-dihydrofuran and boronate into HMBT.The probe THMT features that a)quick response to H₂O₂（20 min）;b)near infrared emission;c)easy preparation from cheap materials;d)good selectivity towards H₂O₂ over ONOO^-.The probe was successfully applied for monitoring the production of exogenous and endogenous H₂O₂in Hep G2 cells.It is urgent to develop effective anti-melanoma drugs because melanoma is a highly aggressive malignant skin cancer with high incidence rate,metastasis and mortality.The theranostic prodrugs can track the release,localization and activity of drugs in real time by emitting fluorescence signals while providing chemotherapy,which provide valuable information on mode of action of drugs.We previously designed a catechol-type diphenylbutadiene,which could construct a catalytic two electron redox cycle with overexpressed tyrosinase（TYR）and NAD（P）H:quinone oxidoreductase-1（NQO1）in melanoma cells to facilitate selective generation of H₂O₂and death of melanoma cells（Free Radic.Biol.Med.2019,130,489）.Considering the oxidative and metabolic instability of catechols,we further designed TYR-activated anticancer theranostic prodrugs.（3）Herein,we engineered a TYR-activated anticancer theranostic prodrug T-PDHH by using m-hydroxybenzyl alcohol to mask the 4-hydroxyphenol in a catechol-type diphenylhexatriene（DHH）.We reasoned that with the aid of TYR,the-OH of m-hydroxybenzyl alcohol can be oxidatively converted into o-quinone,triggering 1,6-elimination to release free DHH in melanoma cells,which generates H₂O₂ according to the above catalytic redox cycle to selectively killed melanoma cells.In addition,DHH shows strong blue fluorescence emission at 475 nm under 370 nm excitation（free radical.Biol.Med.2019,134,406）,allowing real-time visualization of its release,localization and cytotoxicity.T-PDHH was synthesized by 6 step reactions in total yield of 8%with metal-catalyzed coupling as the key step.Limited by time,exploring the activation and activity mechanisms of T-PDHH is still in progress.