Design,Synthesis And Application Of Peptidase-Activated Fluorescent Probes

Peptidases,as one of polypeptide hydrolase for response cell reaction,are widely existed in animals,plants and microorganisms with important roles in nutrition intake,signal transduction,cell differentiation,maturation and apoptosis.The occurrence,development and metastasis of malignant tumors are often accompanied by the over-expression of peptidases.In gut microbes,the activity of peptidases is also different.During recent few years,fluorescence imaging has attracted more attention for exploring the physiology and pathology function of peptidases in organism owing to the advantages of high sensitivity,strong specificity,good biocompatibility and non-invasive.Based to the large stokes shift and near-infrared wavelength emission properties fluorophore of(E)-2-(3-(4-aminostyryl)-5,5-dimethylcyclohex-2-en-1-ylidene)malononitrile ADM,this dissertation established three fluorescent probes for enzyme function assessment,disease diagnosis,microorganism angling and inhibitor screening.According to the metabolic characteristic of dipeptidyl peptidase Ⅳ(DPP Ⅳ),a fluorescent probe GP-DM was synthesized for accurately and dynamically monitoring DPP Ⅳ activity in vitro and in vivo.GP-DM exhibited higher metabolic activity than other human serine hydrolases and strong anti-interference ability to a complex biological matrix,which was fully characterized in a series of phenotyping reactions and inhibition assays.GP-DM can be used to evaluate DPP Ⅳ activity in a variety of biological samples(plasma,tissue particles)and tumor cells,as well as performing real-time imaging of tumor-bearing nude mice and zebrafish.And,it is revealed that DPP Ⅳ is closely associated with the migration and proliferation of cancer cells,while its inhibition may be a promising cancer therapeutic approach.A fluorescent probe ADMG has been designed and developed for the sensing of bacterialγ-GT owing to γ-glutamyl-transferase(y-GT)in the important role to maintain intracellular redox homeostasis.Using the probe on mouse intestinal imaging,it was discovered that y-GT over-expression in intestinal bacteria mainly distributed in the duodenum section.With the guidance of the sensing of y-GT by using ADMG,it is found that the high fluorescence intensity intestinal bacteria strains were isolated from duodenum.After 16S DNA sequencing identification,these bacteria strains were K.pneumoniae CAV1042,K.pneumoniae XJRML-1,and E.faecalis,respectively.This result provided a new idea for rapid recognition of intestinal bacteria according to the over-expression functional enzyme.A fluorescent probe ADMP was constructed for detection pyroglutamate aminopeptidase 1(PGP-1)by using L-pyroglutamic acid as recognition group.Four PGP-1 inhibitors were obtained from 74 kinds of natural products guided by ADMP through high-throughput screening,namely menadione,alantolactone,constunolide and carnosic acid,respectively.Then,using ADMP for angling gut microbes from human feces,one of bacteria and fungi were distinguished with over-expression of PGP-1.After the gene sequencing identification,the bacteria is K.pneumonia 1557 and the fungi is Trichosporon asteroids.Finally,the assays of fluorescence imaging and flow cytometry were proved the reliability of detect endogenous PGP-1 and the practicability of inhibitor screening by ADMP,which further shown that fluorescence imaging can effectively improve the efficiency of microbial recognition.