Designing Fluorescent Probes For Detecting Mitochondrial Superoxide Radical Anion And Viscosity

As the primary reactive oxygen species(ROS),superoxide radical anion(O₂^·-)could be transformed into other ROS by enzymatic or non-enzymatic ways.It is mainly produced in mitochondria and features with high selectivity,short half-life period and low concentration.It functions as a signal molecule to mediate diverse cellular physiological responses.However,abnormal level of O₂^·-could damage oxidatively lipids,DNA and protein,resulting in oxidative stress,which is closely related to development of various diseases such as cancer,stroke and atherosclerosis.Therefore,it is of concern to construct powerful fluorescent probes for visualizing fluctuation of O₂^·-in mitochondria.Visocisty,as a vital micro-environment parameter,has an essential effect on transportation,signaling and interaction among reactive species.Normal viscosity is the prerequisite for mitochondrial function.And the aberrant increase of mitochondrial viscosity would reduce the activity of electron transport chain,promote the release of cytochrome C,and cause diseases such as malignant tumor and atherosclerosis.It is known that cellular oxidative stress induces an increased viscosity,while changes in viscosity affect the diffusion of redox signaling molecules.The construction of multifunctional fluorescent probes for simultaneously detecting redox signaling molecules and cellular microenvironment parameters will help us clarify the relationship between them.However,to our knowledge,there are no fluorescent probes for simultaneous detection of viscosity and O₂^·-.Accordingly,two fluorescent probes were designed and synthesized in this paper:(1)Ratiometric fluorescent probes are highly demanded because they are based on the quantitativet analysis by the ratio of dual-wavelength intensity,which effectively avoids data distortion caused by background interference.We constructed a hybrid fluorescent probe JCOU-OS by bridging the julolidine coumarin and pyridinium modified with diphenyl phosphinate.After reacting with O₂^·-,the push-pull electron effect of the system changes,which triggers a ratiometric fluorescence response(resulting in a blue-shift in fluorescence emission).The probe has advantages of superior selectivity,high sensitivity,large emission wavelength difference and mitochondrial targeting ability.It was successfully applied to image mitochondrial O₂^·-in human breast cancer cell MCF-7 and track dynamic changes of O₂^·-in cancer cells treated by the anticancer drug cisplatin.(2)We bridged diethylaminobenzene with diphenylphosphinate-modified pyridinium moiety by an olefinic bond to firstly construct the probe V-OS for visualizing simultaneously mitochondrial viscosity and O₂^·-in a two-channel mode.V-OS induces a turn-on response either at 625 nm to viscosity which blocks intramolecular rotation,or at 530 nm to O₂^·-via nucleophilic attack of O₂^·-followed by1,6-elimination.The probe was successfully applyed to map the burst of O₂^·-and the increase of viscosity during cellular ferroptosis process and cerebral ischemia reperfusion injury of living mice.(3)Killing cancer cells by promoting generation of ROS has surfaced as an important anticancer strategy.However,it remains a challenge to design ROS-generating(prooxidative)anticancer agents with the unique ability to generate ROS in cancer cells efficiently and selectively.A previous investigation by our group reveals that by virtue of its Michael aceptor units,a curcumin mono-carbonyl analog A1 is a stronger ROS generator and ROS-dependent killer against cancer cells than the parent curcumin.To further improving its selectivity,we designed a class of pre-Michael molecules based on a strategy which features with the protection of the carbonyl group in A1 by a cyanoester group,and the grafting of the responsive moieties to redox characteristic of cancer cells.We speculate that pre-Michael aceptors could be activated in cancer cells to release in situ the active Michael acceptors,which generate ROS and killing cancer cells selectively.Due to the constraint of time,we only completed the synthesis of a carboxylesterase-activated pre-Michael receptor molecule A1-OAc.The evaluation on its biological activity and the synthesis of other related molecules are currently underway.