Ratiometric fluorescence sensing is a useful technique for the precise and quantitative analysis of biological events occurring under complex conditions, such as those inside cells. We report herein the design of new ratiometric chemosensors for nucleoside polyphosphates such as ATP that are based on binding-induced modulation of fluorescence resonance energy transfer (FRET) coupled with a turn-on fluorescence-sensing mechanism. We designed these new FRET-based ratiometric chemosensors by utilizing spectral overlap changes to modulate the FRET efficiency. Introduction of coumarin fluorophores as the FRET donors into a binuclear zinc complex as the FRET acceptor provided the ratiometric chemosensors. These chemosensors exhibited a clear dual-mission signal change upon binding with strong affinity (K(app) ≈ 10(6)-10(7) M(-1)) to nucleoside polyphosphates in aqueous solution, whereas no detectable emission change was observed with monophosphates and phosphodiester species or various other anions. These chemosensors were used for real-time fluorescence monitoring of enzyme reactions such as saccharide synthesis by glycosyltransferase and phosphorylation by protein kinase, both of which involve nucleoside polyphosphates as substrates. The utility of ratiometric sensing by chemosensors was further demonstrated in a fluorescence-imaging study of the nucleoside polyphosphates inside living cells, wherein we ratiometrically visualized the stimulus-responsive concentration change of ATP, an indicator of the cellular energy level.

ATP and its derivatives (nucleoside polyphosphates (NPPs)) are implicated in many biological events, so their rapid and convenient detection is important. In particular, live cell detection of NPPs at specific local regions of cells could greatly contribute understanding of the complicated roles of NPPs. We report herein the design of two new fluorescent chemosensors that detect the dynamics of NPPs in specific regions of living cells. To achieve imaging of NPPs on plasma membrane surfaces (2-2Zn(II)), a lipid anchor was introduced into xanthene-based Zn(II) complex 1-2Zn(II), which was previously developed as a turn-on type fluorescent chemosensor for NPPs. Meanwhile, for subcellular imaging of ATP in mitochondria, we designed rhodamine-type Zn(II) complex 3-2Zn(II), which possesses a cationic pyronin ring instead of xanthene. Detailed spectroscopic studies revealed that 2-2Zn(II) and 3-2Zn(II) can sense NPPs with a several-fold increase of their fluorescence intensities through a sensing mechanism similar to 1-2Zn(II), involving binding-induced recovery of the conjugated form of the xanthene or pyronin ring. In live cell imaging, 2-2Zn(II) containing a lipid anchor selectively localized on the plasma membrane surface and detected the extracellular release of NPPs during cell necrosis induced by streptolysin O. On the other hand, rhodamine-type complex 3-2Zn(II) spontaneously localized at mitochondria inside cells, and sensed the local increase of ATP concentration during apoptosis. Multicolor images were obtained through simultaneous use of 2-2Zn(II) and 3-2Zn(II), allowing detection of the dynamics of ATP in different cellular compartments at the same time.

Small-molecule ligands that control the spatial location of proteins in living cells would be valuable tools for regulating biological systems. However, the creation of such molecules remains almost unexplored because of the lack of a design methodology. Here we introduce a conceptually new type of synthetic ligands, self-localizing ligands (SLLs), which spontaneously localize to specific subcellular regions in mammalian cells. We show that SLLs bind their target proteins and relocate (tether) them rapidly from the cytoplasm to their targeting sites, thus serving as synthetic protein translocators. SLL-induced protein translocation enables us to manipulate diverse synthetic/endogenous signaling pathways. The method is also applicable to reversible protein translocation and allows control of multiple proteins at different times and locations in the same cell. These results demonstrate the usefulness of SLLs in the spatial (and temporal) control of intracellular protein distribution and biological processes, opening a new direction in the design of small-molecule tools or drugs for cell regulation.

We report a general strategy to create small-molecule fluorescent probes for the nucleus in living cells. Our strategy is based on the attachment of the DNA-binding Hoechst compound to a fluorophore of interest. Using this approach, simple fluorescein, BODIPY, and rhodamine dyes were readily converted to novel turn-on fluorescent nucleus-imaging probes.