Development Of Accurate SA-β-gal Probes To Visually Diagnose Aging

In this dissertation,a series of studies have been carried out around the accurate diagnosis of aging.Aging is a physiological phenomenon that the function of organism gradually declines with time.It is often accompanied by a variety of age-related diseases,which seriously threatens the health and life of human beings.The development of rapid,convenient and high-throughput methods for aging diagnosis is crucial to the research of aging and the development of anti-aging drugs.Studies have shown that the accumulation of senescent cells is the main cause of aging,and the detection of senescent cells is an important way to diagnose aging.Therfore,the development of tools for accurately detect senescence is of great significance in the research of diagnosis and treatment of aging diseases.Senescence associated β-galactosidase(SA-β-gal)is a characteristic biomarker of senescent cells and has been used as the "golden standard" for senescent cells detection.In recent years,a large number of β-galactosidase((3-gal)fluorescent probes have been developed for tumor diagnosis.However,there are only a few SA-β-gal fluorescent probes for aging diagnosis and still lack of excellent near-infrared species-specific SA-β-gal probes.It should be noted that the catalytic pocket of β-gal from different species are different.SA-β-gal fluorescent probes with no differential response to β-gal from other species(such as bacteria)may cause false positive detection.Therefore,it is very important to design and synthesize novel near-infrared and species-specific SA-(3-gal probes to realize accurate visualization of aging.The first part of this dissertation is about the design and synthesize of new near-infrared SA-β-gal fluorescent probe and its application in accurate visualization of aging.We designed near-infrared fluorescent probe KSL0401 by conjugating the coumarin-dicyanoisophorone hybrid fluorophore KSL0401-OH with an enzyme-activatable functional group(β-D-galactose residue).We tested the spectral properties of KSL0401 to study its response to β-gal,and conducted HPLC and HRMS to verify its sensing mechanism.The results indicated that probe KSL0401 can be correctly identified by β-gal and emit remarkable near-infrared fluorescence signal at 706 nm along with an stokes shift of 218 nm,which effectively reduces the signal-to-noise ratio and realizes the detection of β-gal based on the change of fluorescence signal.At the same time,the results of anti-interference experiment,fluorescence titration experiment and time kinetics experiment indicated that probe KSL0401 has high sensitivity and rapid response to β-gal.We established a variety of chemically induced senescent cells,and then incubated with KSL0401 for the confocal imaging.The results proved that probe KSL0401 can accurately distinguish between senescent cells and young cells,and is a potential diagnostic tool in the research of accurate visualization of agingThe second part of this dissertation is about the design and synthesize of first-generation species-specific S A-β-gal fluorescent probes and its application in accurate diagnosis of aging.We synthesized a kind of species-specific probes that can selectively identify human SA-β-gal and realize more accurate detection of aging in response to the differences of β-gal from different species.We selected the 2-(2’-hydroxyphenyl)benzothiazole(HBT)scaffold as the fluorophore due to its exibility in structural modication.KSL01-KSL12 were designed and synthesized by adjusting the size of electron-withdrawing aldehyde group on the 3’-position or 5’-position to realize the specific detection of β-gal from different species.E.coli β-gal is universally used for the in vitro study of β-gal fluorescent probes.However,we found that the domain of human β-gal was very different from E.coli β-gal,while it was very similar to that of A.oryzae β-gal,indicating that A.oryzae β-gal is a more suitable human β-gal model than E.coli β-gal.According to the spectral properties of the probes after incubated with E.coli β-gal or A.oryzae β-gal,we found KSL08-KSL12 are species-specific SA-β-gal fluorescent probes Representative probe KSL04 and KSL11 showed excellent optical properties,and were taken for further study.Confocal imaging was conducted after probes were incubated with LacZ-transfected cells which is overexpressed E,coli β-gal and LY3177833-induced senescent cells which is overexpressed SA-β-gal,the results indicated that KSL11 can specifically response to human SA-β-gal but not to E.coli β-gal.And KSL11 can avoid the interference of E.coli β-gal in simulated E.coli infected kidney section of mouse.It proved that KSL11 can realize more accurate diagnosis of aging.In addition,we incubated MRC5 cells of different passage numbers with the probe KSL04,and then conducted the confocal imaging.The results indicated that KSL04 can detect the senescence degree of MRC5 cells from different passage numbers via the change of fluorescence intensity.When senescent MRC5 cells of passage 29 were incubated with anti-aging drug Rapamycin,the fluorescence intensity of the treatment group was significantly decreased compared with the control.It indicated that KSL04 can visually evaluate the anti-aging effect of Rapamycin at cellular level.Subsequently,we conducted confocal imaging of the main organs and tissue sections from mice with different age after incubated with our probes.The results showed that the accumulation amount of SA-β-gal in the kidney is the largest and the amount of SA-β-gal increases significantly with age,indicating that kidney is one of the organs with the most severe agingTherefore,we have developed several fluorescent probes based on SA-β-gal which is the classic biomarker of senescence,and reported species-specific SA-β-gal fluorescent probes for the first time.These probes can be used for the accurately diagnosis and tracing of aging,and can evaluate the efficacy of anti-aging drugs.This makes it is possible to establish a cell model for accurate and rapid evaluation of the efficacy of anti-aging drugs.The species-specific fluorescent probes we reported can avoid the false positive brought by bacteria and provide more scientific diagnostic tools for the research of aging and the development of anti-aging drugs.