Design And Synthesis Of Functional Peptides And Their Applications

Due to the inherent biocompatibility, biodegradability, specific protein recognition ability, easy to modify and purify at laboratory, peptides have been extensively used in the field of drug research and development. More importantly, during the last decade, researchers have demonstrated that some peptides can self-assembly into various well-ordered nanostructures which are attractive to many applications (e.g., drug delivery, biosensor, catalysis, nanodevice and so on). Their ability to form such structures is due to structural complementarities coupled with hydrogen bonding, electrostatic interaction, hydrophobic affinity and π-stacking (aromatic amino acids). Based on the above considerations, we designed and synthesized a series of peptides, and studied their applications in the field of antitumor, anti-neurodegerative disease, catalysis and biosensior.(1) Ferrocenoyl RGD conjugates Fc-RGD and Fc-Am-RGD (Am:6-aminohexanoic acid) were synthesized, and the antitumor activities in vitro were investigated. The cell uptake of the conjugates by B16murine melanoma cells was measured using HPLC-electrochemical method. The antitumor activities of the conjugates were also evaluated by MTT and flow cytometric measurements. The experimental results revealed that Fc-RGD and Fc-Am-RGD exhibit more effective antitumor activities than their parent compounds RGD and Fc-COOH. Moreover, it is found that Fc-Am-RGD yields the lowest IC50values of5.2±1.4μmol·L-1toward B16cells. The HPLC-electrochemical studies confirmed the insertion of flexible alkyl spacer Am between Fc and RGD significantly increases the cell uptake toward B16cells and consequently improves the antitumor activity. Our results suggest that Fc-RGD and Fc-Am-RGD conjugates are potential candidates for cancer treatment.(2) The conjugates of ferrocene and Gly-Pro-Arg (GPR) tripeptide, GPR-Fca and Fc-GPR (Fc:ferrocene; Fca:ferrocene amino acid) were synthesized by HOBt/HBTU protocol in solution. These ferrocene GPR conjugates were employed to inhibit Aβ(1-42) fibrillogenesis and to disaggregate preformed A(3fibrils. The inhibitory properties of ferrocene GPR conjugates on Aβ(1-42) fibrillogenesis were evaluated by thioflavin T fluorescence assay, and confirmed by AFM analysis. The interaction between the ferrocene GPR conjugates and Aβ(1-42) was monitored by electrochemical means. Our results showed that both GPR and GPR-Fca can significantly inhibit the fibril formation of Aβ(1-42), and cause disaggregation of the preformed fibrils. As expected, GPR-Fca shows stronger inhibitory effect on Aβ(1-42) fibrillogenesis than that of its parent peptide GPR. In contrast, Fc-GPR shows no inhibitory effect on fibrillogenesis of Aβ(1-42). Furthermore, GPR-Fca demonstrates significantly protection against Aβ-induced cytotoxicity and exhibits high resistance to proteolysis and good lipophilicity.(3) The aggregation and fibrillation of α-syn have been implicated as a causative factor in Parkinson’s disease (PD). α-syn can binds to copper and the aggregation of α-syn is stimulated in the presence of copper attracted great attention. Particularly, recently studies have shown that the toxicity of α-syn aggregates significantly increased in the presence of copper, which make researchers to believe that α-syn-Cu2+aggregates play key role in PD. In this study,N-methylated peptide (VAQKTmV) was synthesized and employed as an inhibitor to prevent the aggregation of a-syn-Cu2+. The inhibitory properties of VAQKTmV on a-syn-Cu2+fibrillation were evaluated by ThT fluorescence assay, and confirmed by AFM analysis. Our results demonstrated that VAQKTmV can not only strongly inhibit the aggregation of α-syn-Cu2+, but also significantly prevent the α-syn-Cu2+-induced cytotoxicity toward SH-SY5Y cells.(4) In this study, circular dichroism confirmed that natively unstructured α-syn in aqueous solution was transformed to its α-helical conformation upon addition of trifluoroethanol. Electrochemical and UV-visible spectroscopic experiments reveal that both Cu+and Cu2+are stabilized, with the former being stabilized by about two orders of magnitude. Compared to unstructured α-syn, α-helical α-syn stabilizes Cu+by more than three orders of magnitude. Through the measurements of H2O2and hydroxyl radicals (OH·) in solutions containing different forms of Cu2+(free and complexed by unstructured or α-helical α-syn), we demonstrate that the significantly enhanced Cu+binding affinity helps inhibit the production of highly toxic reactive oxygen species, especially the hydroxyl radicals. Our study provides strong evidence that, as a possible means to prevent neuronal cell damage, conversion of the natively unstructured a-syn to its a-helical conformation in vivo could significantly attenuate the copper-modulated ROS production.(5) Self-assembly peptides, due to their inherent biocompatibility, molecular-recognition capability and functional flexibility, have been recognized as very useful building block for creating nanostructures. In this study, aniline-GGAAKLVFF peptide (AFP) was prepared by solid-phase synthesis. The AFP peptide can readily self-assemble into amyloid-like fibrils in a simple way, then the negatively charged Pt NPs were directly assemble onto the positively charged surface of the fibrils via electrostatic interaction. The Pt-AFP fibrils exhibited excellent electrocatalytic activities toward oxygen reduction, which is of special interest for polymer electrolyte fuel cells, batteries and other electrode applications.(6) Polyaniline has been of great interest to many researchers due to its excellent electrical properties, is a unique type of conducting polymer. In this study, aniline was conjugated to the N-terminal of amyloid-like peptide GGAAKLVFF by solid-phase synthesis. The aniline-GGAA-KLVFF peptide not only can readily self-assemble into fibrils in a simple way, but also forms polyaniline by the extruding aniline of peptide fibrils in the present of H2O2and HRP. The polyaniline nanowire retains polyaniline full electrochemical activity and was successfully used as signal amplification to fabricate a sensitive nucleic acid detecter.