Fluorescent N super(2),N3-[epsiv]-Adenine Nucleoside and Nucleotide Probes: Synthesis, Spectroscopic Properties, and Biochemical Evaluation

N1,N super(6)-ethenoadenine, [epsiv]-A, nucleos(t)ides have been previously applied as fluorescent probes in numerous biochemical systems. However, these [epsiv]-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 super(2),N3-etheno-adenosine, (N super(2),N3-[epsiv]-A). Here, we describe four novel syntheses of the target [epsiv]-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 super(2),N3-[epsiv]-A and the dependence of the spectral features on the pH and polarity of the medium. Specifically, maximum emission of N super(2),N3-[epsiv]-A in water is observed at 420 nm ([phgr]=0.03, excitation at 290 nm). The biochemical relevance of the new probe was evaluated with respect to the P2Y sub(1) receptor and NTPDases 1 and 2. N super(2),N3-[epsiv]-ATP was found to be almost equipotent with ATP at the P2Y sub(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 super(2),N3- [epsiv]-ATP. Based on the fluorescence and full recognition by ATP-binding proteins, we propose N super(2),N3-[epsiv]-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.