Design and Synthesis of Bis(Zn(II)–Dipicolylamine)-Based Fluorescent Artificial Chemosensors for Phosphorylated Proteins/Peptides

Abstract

Protein phosphorylation represents a ubiquitous mechanism for controlling diverse protein functions and plays central role in signal transduction cascades in living cells. In order to elucidate the complex network of phosphorylationbased signaling, it is desirable to develop versatile methods and molecular probes that can selectively recognize and detect the phosphoprotein of interest. We present herein the molecular recognition and fluorescence sensing of phosphorylated proteins/peptides by synthetic small molecules. The chemosensors are designed to utilize metal-ligand interaction as a main binding force and thus possess two Zn(II)-dipicolylamine(dpa)s as the binding sites for phosphate group(s) on the protein/peptide surface. We propose the two strategies for recognition of phosphorylated protein/peptide surface; (i) cooperative-binding strategy and (ii) cross-linking strategy. The binuclear anthracene-type chemosensors are designed to interact with a phosphate group on protein/peptide surface based on the cooperative-binding mode, in which the single phosphate group is recognized by the two Zn(II)-dpa sites of the chemosensor. The binding affinity of the chemosensors sensitively depends on the sequence of the phosphopeptide, and reaches to nearly 10 7 M À1 for a highly negatively charged peptide. In addition, the chemosensors increase their fluorescence upon binding to the phosphorylated proteins/peptides. Therefore, they can distinguish between phosphorylated and non-phosphorylated state of proteins/peptides by their fluorescence intensities. Detailed experiments clarify that the phosphate anion-assisted binding of the second Zn(II) to the binuclear chemosensors is crucial for the fluorescence increase upon binding to the phosphorylated derivatives. The other approach to recognize phosphorylated protein surface by synthetic small molecule is based on the cross-linking strategy. The chemosensors possess bipyridine as a spacer unit between two Zn(II)-dpa sites, which are arranged at appropriately distal position, thereby make possible the cross-linking interaction with two phosphate groups on protein surface. The chemosensors shows higher affinity for the bis-phosphorylated peptide than for the mono-phosphorylated one, indicating that the cross-linking interaction is effective for recognition of multisite phosphorylation domains of a protein. The results presented in this article are regarded as the first step toward detection of specific phosphorylation event of a protein in complicated biological systems using synthetic small molecules. The bioanalytical application of these chemosensors, such as the fluorescence detection of phosphatase catalyzed dephosphorylation and the selective staining of phosphoprotein in SDS-PAGE, are also discussed.Post-translational modification by phosphorylation represents a ubiquitous regulatory mechanism for controlling diverse protein functions.1 Especially, phosphorylation on serine, threonine, or tyrosine residues catalyzed by protein kinase forms the basis of cell signalin...

Metal complexes as “protein surface mimetics”

“…The bis(Zn(II)Dpa) receptors have been used as chemosensors by varying the bridging group between the two Zn(II) centres resulting in fluorescence changes on binding. 81 84 With doubly phosphorylate -helical peptides it was shown, by circular dichroism (CD), that with ) II -helical content of the peptide increases, and that that there is 10 -fold selectivity for doubly phosphorylated peptides over mono-phosphorylated peptides. 82,85 This approach has subsequently been used to develop an inhibitor (IC50 = M the phosphoprotein-protein interaction between the phosphorylated CTD peptide and the Pin1 WW domain.…”

New Method for Affinity Adsorbent Screening Using Gold Colloids

“…According to previous work, , metal ions play a very important role in the coordination interaction, so we investigated the interactions of linear polymers PAAm-Dpa(Zn 2+ ) and PAAm-Dpa (without Zn 2+ ions) with CG8-AuNPs. Static adsorption experiments on adsorbents A-AAm-Dpa(Zn 2+ ) and A-AAm-Dpa with CG-8 were also conducted.…”

Synthetic Multifunctional Nanoarchitecture in Lipid Bilayers: Ion Channels, Sensors, and Photosystems

“…116,117 The exceptional potential of the general optical transduction of reactions with rigid-rod pores for applications in domains such as drug discovery (enzyme inhibitor screening), diagnostics (multianalyte sensing), and so on invites to elaborate on diversity. 44,45 This may include variation in mode of detection (conductance), [117][118][119] 45 enzymes for inhibitor screening, [73][74][75][76] enzymes for signal generation, [43][44][45][73][74][75][76] reactive amplifiers, analytes, matrix, and so on. 44,45 The mainly academic nature of artificial photosynthesis in lipid bilayers, finally, invites to apply the lessons learned to advanced rigid-rod nanoarchitecture on conducting surfaces such as gold or ITO 102,103 with the hope to end up with photovoltaic devices.…”

“…Such optical transduction of enzyme activity with pores is of use to screen for inhibitors of enzymes that are attractive targets for drug discovery but otherwise difficult to address. [43][44][45][73][74][75][76] The combination of optical transduction by synthetic pores with signal generation by enzymes is of interest for sensing applications. [43][44][45] Sugar sensing by pore 41 in soft drinks was selected as illustrative example (Fig.…”