Synthesis Of A Reversible Fluorescent Probe With BODIPY As A Fluorophore And Real-time Monitoring Of GSH

Glutathione(GSH)is the most abundant non-protein biothiol in the cell,and plays an important role in maintaining the redox balance in the cell.The abnormal concentration causes abnormal reactions such as metabolism,stress and immunity.Therefore,finding a simple and convenient method for real-time quantitative detection of changes in GSH content in living cells is essential for understanding the pathological events associated with GSH.At present,there are two main methods for quantitative detection of GSH.cell lysate and GSH-S-transferase(GST)calculation method,and redox-sensitive fluorescent protein(roFPs)method.Although these two methods can quantify GSH,they are all insufficient.The former can not achieve real-time effective monitoring of GSH concentration,and the latter has the problem of the ratio of reduced glutathione to oxidized glutathione,which makes calculation There is a deviation in the value.Compared with the above two methods,the fluorescent probe method is more suitable for quantitative and real-time monitoring of GSH in vivo.Most of the fluorescent probes based on the Michael addition mechanism have been reported to be qualitative and incapable of quantitative and real-time monitoring.The performance is attributed to the problem of the difference between the amount of probes and GSH and the implementation strategy of real-time detection.The Kd value(dissociation equilibrium constant)plays a very important role in the reversible reaction,and its size is related to the affinity of the reversible reaction,while the affinity of the reversible reaction should be moderate,that is.the optimal Kd value is 3 mM.Therefore,the Michael addition receptor was constructed by modulating the group on the double bond in the Michael addition receptor,and the Kd value was used as an evaluation index.Based on this,this paper compares the five compounds synthesized and finds a combination of BODIPY,cyano,hydrogen and amide groups to construct the Michael Addition Receptor GP-2.which is easier to achieve Michael addition-removal reversibility.The reaction increases the sensitivity of the probe.The working curve of this probe has a high linear coefficient(R2=0.99),low detection limit and high accuracy,and its reaction with GSH is Kd=3.44 mM.The probe was used to quantify the GSH in HeLa cells(5.29 mM),which is very close to the previously reported GSH concentration in Hela cells,and proved to be fluorescent by comparison with the commonly used quantitative methods in molecular biology research.The accuracy and feasibility of the needle-quantitative GSH method provides a more convenient quantitative method for exploring GSH in living cells and or in vivo.