Turn-On Fluorescence Sensing of Nucleoside Polyphosphates Using a Xanthene-Based Zn(II) Complex Chemosensor

Abstract

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).

Ternary complex formation and competition quench fluorescence of ZnAF family zinc sensors

“…The published conditional Zn(II) binding constants of ZnAF-1 and ZnAF-2F were determined by competitive fluorescence titrations with NTA. 9,17 We additionally confirmed them by analogous competitive titrations, but using EGTA as a 15 competitor, 18 in the 100 mM Hepes at pH 7.4.…”

Mitochondria Regulate Neutrophil Activation by Generating ATP for Autocrine Purinergic Signaling

“…Although this method was highly sensitive, it had major drawbacks including the need for an excitation wavelength in the ultraviolet range, which resulted in significant phototoxicity (23). The small molecular probe we used in the current study was developed in the laboratory of Dr. Hamachi (11,24). This ATP probe features a membrane anchor that has allowed us to attach the probe to the cell surface of living PMNs and to assess ATP concentration changes at the cell surface for prolonged periods of time.…”

Mitochondria Are Gate-keepers of T Cell Function by Producing the ATP That Drives Purinergic Signaling

“…7E). To assess concomitant intracellular ATP production, we used an intracellular ATP probe, 1-2Zn(II) that was also developed in the laboratory of Prof. Hamachi and is structurally identical to 2-2Zn(II), except that 1-2Zn(II) lacks the membrane anchor residue and can be taken up by cells (30). Using this probe, we found that mitochondrial firing in response to cell stimulation resulted in rapid bursts of intracellular ATP release, apparently by the release of ATP from activated mitochondria (supplemental Movie S8).…”