The Research On Design, Synthesis And Performance Of New Fluorescent Probes For Metal Ions

Heavy metal ion pollution poses severe risks in human health andthe environment. Metal ion’s alterations in its cellular homeostasis areconnected to serious diseases. Fluorescent probe has become a rapidlyexpanding area of analytical chemistry, because it offers the advantagesof simple sample preparation, noninvasive detection, high selectivity andsensitivity. The interest in developing fluorescent probes for the detectionof transition metal ions in complex aqueous solutions has been stimulatedby their important applications including environmental monitoring andwastewater treatment.We report herein a new class of metal fluorescent probes based onmetal-dependent peptidase hydrolysis. The probe contains the basicspecific metal-dependent peptide bond hydrolysis sequence. The substratewas labeled with a fluorophore at the N-terminal and a quencher at theC-terminal Lys side chain. In the presence of analyte, the substrate wascarries out catalytic reactions by analyte, such as hydrolytic cleavage of the substrate at the cleavage site. Upon cleavage, the fluorophore wasreleased and far away from quencher, leading to its increasedfluorescence. In the present study, because of introducing amino acids asbinding site of probe, it increased probe’s water-solubility. Moreover, theprobes displayed high selectivity and sensitivity responses to target metalions in buffered aqueous solution. Besides, we designed and synthesizedZn²⁺fluorescent probe based on PET, and studied the property of theprobe. The main studies as follows:Firstly, the Ni²⁺fluorescent probe based on metal-dependentpeptidase hydrolysis has been designed and synthesized. The probecontains the basic specific Ni²⁺-dependent peptide bond hydrolysissequence （Gly-Ala-Ser-Arg-His-Trp-Lys-Phe-Lys）. The substrate waslabeled with a fluorophore at the N-terminal and a quencher at theC-terminal Lys side chain. Initially, the MOCAc（（7-methoxycoumarin-4-yl）acetyl-） emission was quenched by the nearbyquencher. In the presence of Ni²⁺, the substrate was irreversibly cleaved atthe cleavage site between Ala and Ser, leading to a20-fold increase influorescence intensity. The probe combines the high selectivity of apeptidase with high sensitivity and selectivity in aqueous media.Secondly, a new fluorescent probe for Cu²⁺ion was designed on thebasis of the sequence-specific cleavage of the peptide bond by thepeptidase. In the fluorescent probe system, the substrate （Ser-Asp-Lys-Ser-His-Thr-Lys） was labeled with a6-FAM fluorophore（6-carboxyfluorescein） at the N-terminal and a Dabcyl quencher4-（4′-dimethylaminophenylazo）benzoic acid at the ε-N of C-terminal Lys.In the presence of Cu²⁺, the substrate strand is cleaved at the cleavage sitebetween Asp and Lys, and the release of the cleaved fragment results insignificant fluorescence increase. The design was aided by the FRETstudy that showed a “turn-on” responding for Cu²⁺in aqueous media.Under optimum conditions, the novel probe described here had a linearresponse range for Cu²⁺with a detection limit of10nM.Thirdly, we designed and synthesized a Zn²⁺fluorescent probe basedon PET. Primary analytic experiment showed that it weakly responds toZn²⁺ion.In summary, we report here a new application for metal-dependentpeptidase as a unique class of probes for metal ions. Given probes’desirable high water solubility, high sensitivity and selectivity, ourmolecular engineering design may prove useful in the future developmentof other peptidase-based probes for toxicological and environmentalmonitoring.