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 phosphorylation-based 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 -1 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.
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