Fluorescence sensing with small molecular chemosensors is a versatile technique for elucidation of function of various biological substances. We now report a new fluorescent chemosensor for nucleoside polyphosphates such as ATP using metal-anion coordination chemistry. The chemosensor 1-2Zn(II) is comprised of the two sites of 2,2'-dipicolylamine (Dpa)-Zn(II) as the binding motifs and xanthene as a fluorescent sensing unit for nucleoside polyphosphates. The chemosensor 1-2Zn(II) selectively senses nucleoside polyphosphates with a large fluorescence enhancement (F/F(o) > 15) and strong binding affinity (K(app) approximately = 1 x 10(6) M(-1)), whereas no detectable fluorescence change was induced by monophosphate species and various other anions. The 'turn-on,' fluorescence of 1-2Zn(II) is based on a new mechanism, which involves the binding-induced recovery of the conjugated form of the xanthene ring from its nonfluorescent deconjugated state which was formed by an unprecedented nucleophilic attack of zinc-bound water. The selective and highly sensitive ability of 1-2Zn(II) to detect nucleoside polyphosphates enables its bioanalytical applications in fluorescence visualization of ATP particulate stores in living cells, demonstrating the potential utility of 1-2Zn(II).

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.

This study has successfully demonstrated that the cooperative action of artificial receptors with semi-wet supramolecular hydrogels may produce a unique and efficient molecular recognition device not only for the simple sensing of phosphate derivatives, but also for discriminating among phosphate derivatives. We directly observed by confocal laser scanning microscopy that fluorescent artificial receptors can dynamically change the location between the aqueous cavity and the hydrophobic fibers upon guest-binding under semi-wet conditions provided by the supramolecular hydrogel. On the basis of such a guest-dependent dynamic redistribution of the receptor molecules, a sophisticated means for molecular recognition of phosphate derivatives can be rationally designed in the hydrogel matrix. That is, the elaborate utilization of the hydrophobic fibrous domains, as well as the water-rich hydrophilic cavities, enables us to establish three distinct signal transduction modes for phosphate sensing: the use of (i) a photoinduced electron transfer type of chemosensor, (ii) an environmentally sensitive probe, and (iii) an artificial receptor displaying a fluorescence resonance energy transfer type of fluorescent signal change. Thus, one can selectively sense and discriminate the various phosphate derivatives, such as phosphate, phospho-tyrosine, phenyl phosphate, and adenosine triphosphate, using a fluorescence wavelength shift and a seesaw type of ratiometric fluorescence change, as well as a simple fluorescence intensity change. It is also shown that an array of the miniaturized hydrogel is promising for the rapid and high-throughput sensing of these phosphate derivatives.

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