Fluorescent N2,N3-epsilon-adenine nucleoside and nucleotide probes: synthesis, spectroscopic properties, and biochemical evaluation.

N1,N(6)-ethenoadenine, epsilon-A, nucleos(t)ides have been previously applied as fluorescent probes in numerous biochemical systems. However, these epsilon-A analogues lack the H-bonding capability of adenine. To improve the fluorescence characteristics while preserving the H-bonding pattern required for molecular recognition, we designed a novel probe: N(2),N3-etheno-adenosine, (N(2),N3-epsilon-A). Here, we describe four novel syntheses of the target epsilon-nucleoside and related analogues. These methods are short, facile, and provide the product regiospecifically. In addition, we report the absorption and emission spectra of N(2),N3-epsilon-A and the dependence of the spectral features on the pH and polarity of the medium. Specifically, maximum emission of N(2),N3-epsilon-A in water is observed at 420 nm (phi=0.03, excitation at 290 nm). The biochemical relevance of the new probe was evaluated with respect to the P2Y(1) receptor and NTPDases 1 and 2. N(2),N3-epsilon-ATP was found to be almost equipotent with ATP at the P2Y(1) receptor and was hydrolyzed by NTPDases 1 and 2 at about 80 % of the rate of ATP. Furthermore, protein binding does not seem to shift the fluorescence of N(2),N3-epsilon-ATP. Based on the fluorescence and full recognition by ATP-binding proteins, we propose N(2),N3-epsilon-ATP and related nucleo(s)tides as unique probes for the investigation of adenine nucleo(s)tide-binding proteins as well as for other biochemical applications.