A supramolecular ON-OFF-ON fluorescence assay for selective recognition of GTP

With the objective of developing small molecule based receptors for nucleosides and nucleotides, interactions of a cyclic donor-acceptor conjugate 1 with adenosine, AMP, ADP, CTP, UTP, ITP, ATP, and GTP have been investigated by absorption, steady-state, and time-resolved fluorescence, cyclic voltammetry (CV), NMR, and fluorescence indicator displacement techniques. Titration of 1 with the fluorescent indicator, 8-hydroxy-1,3,6-pyrene trisulfonate (HPTS), resulted in nearly complete fluorescence quenching of HPTS, along with 25% hypochromicity in its absorption spectrum. Benesi-Hildebrand analysis gave a 1:1 stoichiometry for the complex between the receptor 1 and HPTS with an association constant (Kass) of 4.66 × 104 M-1 in buffer. The driving force for such a complexation was evaluated to be the synergistic effects of π-stacking and electrostatic interactions inside the cavity as confirmed by the effect of ionic strength, temperature, and the negative results obtained with the model compound 2. Titration of the nonfluorescent complex [1·HPTS] with various nucleosides and nucleotides resulted in revival of fluorescence of the indicator, HPTS. It was observed that GTP induces maximum displacement of HPTS from the complex [1·HPTS] with an overall fluorescence enhancement of ca. 150-fold. The addition of adenosine, AMP, ADP, CTP, and UTP showed negligible changes, whereas ca. 45- and 50-fold enhancement was observed with ATP and ITP, respectively. The competitive displacement of the indicator by various analytes is found to be in the order GTP (buffer) ≈ GTP (biofluid) ≫ ITP ≈ ATP > UTP > CTP ≈ ADP ≈ AMP ≈ Ade. By virtue of having a better π-electron cloud, GTP undergoes effective electronic, π-stacking, and electrostatic interactions inside the cavity and forms a stable complex with the receptor 1. The uniqueness of this assay is that it differentiates GTP from ATP and other nucleotides and signals the event through a visual turn on" fluorescence mechanism in buffer as well as in biological fluids. Copyright © 2006 American Chemical Society."