Design,synthesis And Application Of Fluorescent Probes Based On BODIPY Fluorophore For Detection Of Metabolizing Enzymes Activities

Metabolizing enzymes are a kind of protein which responsible for the metabolism and detoxification of various xenobiotics and endogenous substrates,thereby play key roles in maintaining endogenous physiological balance of body.The dysfunction of enzymes has been proved to be closely related to many diseases,so it is of great importance to develop the practical tool for real-time and sensitive evaluating metabolizing enzymes activities.Compared with traditional testing methods,fluorescent probe-based assays have drawn increasing attention for their ability of visualization real activities of enzymes,as well as many other inherent advantages including excellent sensitivity,ultrahigh imaging resolution and in situ sensing character.Metabolizing enzymes is a large family with wide variety,and part of them have been identified containing multiple isoforms,while fluorescent probes with isoform-specificity for relevant metabolizing enzymes were rare reported at present,and the employing of probes as tools for researches on enzymes-associated physiological function expecting to be further explored.In this paper,difluoroboron dipyrromethene(BODIPY)was employed as a basic scaffold to construct fluorescent substrates based on its desirable spectroscopic properties and feasibility in structural modification.On the basis of substrate preference of target enzymes and the relationship between structure and spectroscopic properties of BODIPY fluorophore,we designed and developed highly specific fluorescent probes to monitor the activity of carboxylesterase 1(CES1),glutathione S-transferase(GSTs),uridine 5’-diphospho-glucuronosyltransferase 1A1(UGT1A1),and further explored the application of relevant probes in the biological system.(1)As a key member of body’s natural defense system,CES1 plays crucial roles in detoxification and defense processes.In this part,a series of fluorescent candidates for CES1 were rationally designed based on the metabolism characteristics of CES1(CES1 preferentially hydrolyzes ester substrates with a large carboxyl group and a small alcohol group)and the relationship between spectroscopic properties of BODIPY and electronic properties of the fluorophore’s meso substituents.Following molecular docking-based virtual screening combined with reaction phenotyping-based experimental screening,a long-wavelength(595 nm)and highly specific fluorescent probe(MMB)for CES1 was developed.MMB exhibited ultra-sensitivity and strong anti-interference ability,as well as showed high affinity with CES1(Km=2.1 μM),which held great promise for applications in detection of CES1 activity in complex biological samples and high throughput screening of CES1 inhibitors.Moreover,MMB showed well cell membrane permeable and poor cytotoxicity,and has been successfully applied to monitoring the real activities of CES1 in situ in various biological systems including living cells,tissue slices,organs,and zebrafish.(2)As a crucial biochemical barrier for body’s defense,GSTs play key roles in detoxification of various xenobiotics.In this part,a highly specific fluorescent probe(BDCN)for GSTs was constructed by employing Photoinduced Electron Transfer mechanism on the basis of the relationship between structure and spectroscopic properties of BODIPY and the fact that GSTs catalyze glutathione to attack the electrophilic centers of compounds.BDCN exhibited high sensitivity and strong anti-jamming capability,which has been successfully applied to monitoring the real activities of GSTs in a range of biological systems including living cells,organs microsomes and tumor tissue slices.Furthermore,using BDCN,the fact that the increase of GSTs expression would contribute to the drug resistance of cancer cells towards cisplatin to a large extent was revealed,which would further reduce the effectiveness of chemotherapy of cisplatin,all these results were of great reference value to the prescription for cancer treatments,as well as provide a rapid and effective tool for personalized use of cisplatin in clinic.Refer to the structure character of BDCN,a two-photon probe BNPA for GSTs was developed by retaining 3,4-dinitrobenzene unit as recognition site and introducing the phenylethynyl group into 2 position of BODIPY core to increase the two-photon absorption cross section of fluorophore.BNPA exhibited high selectivity and prominent sensitivity,importantly,could be applied to sensing the endogenous GSTs activities in living cells and deep tissues by two-photon imaging with minimum autofluorescence background interference.Moreover,using BNPA,the down regulation of GSTs activities during a-Naphthylisothiocyanate(ANIT)induced liver injury was revealed,which would provide vital guidance for study of GSTs-associated liver diseases.(3)UGT1A1,one of most important phase Ⅱ metabolizing enzymes,plays a predominant role in detoxification of various endogenous toxic substrates(such as bilirubin)and maintaining metabolic balance of body.A series of potential fluorescent substrates for UGT1A1 were designed and constructed based on the relationship between structure and spectroscopic properties of BODIPY and the fact that UGT1A1 mediates glucuronidation of hydroxyl.An Off-On fluorescent probe(BUHE)for UGT1A1 was developed by further modification of the superior substrate to improve subtype selectivity on the basis of the larger catalytic cavity of UGT1A1.BUHE exhibited prominent specificity and good anti-interference ability,as well as showed high affinity with UGT1A1(Km=0.59 μM).Moreover,the enzymatic reaction exhibited typical Michaelis-Menten kinetics.BUHE has been used as a practical tool for evaluating and high throughput screening of UGT1A1 inhibitors.Meanwhile,both inhibition curve and IC50 value of Neobavaisoflavone in UGT1A1 were similar to that in human liver microsomes(HLMs),suggesting that BUHE could be applied to screening of UGT1A1 inhibitors by employing HLMs as enzyme source replacing high-cost recombinant UGT1A1.Furthermore,BUHE exhibited well cell membrane permeable and poor cytotoxicity,which has also been successfully applied to in situ monitoring UGT1A1 activities in complicated biological systems including living cells,tissue slices and organs.