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.
Sharon
Einat
E
Department of Chemistry, Bar-Ilan University, Ramat-Gan 52900, Israel.
Lévesque
Sébastien A
SA
Munkonda
Mercedes N
MN
Sévigny
Jean
J
Ecke
Denise
D
Reiser
Georg
G
Fischer
Bilha
B
D000225:Adenine / Q000031:analogs & derivatives*; D000241:Adenosine / Q000031:analogs & derivatives*; D000249:Adenosine Monophosphate / Q000031:analogs & derivatives*; D000251:Adenosine Triphosphatases / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives*; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry; D001081:Apyrase / Q000037:antagonists & inhibitors / Q000737:chemistry; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D003500:Cyclization; D004791:Enzyme Inhibitors / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D015394:Molecular Structure; D058910:Purinergic P2 Receptor Agonists; D051381:Rats; D058472:Receptors, Purinergic P2Y1; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D015703:Antigens, CD; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; C546382:P2RY1 protein, human; D058910:Purinergic P2 Receptor Agonists; D058472:Receptors, Purinergic P2Y1; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C027201:ectoATPase; D001081:Apyrase; C067869:CD39 antigen; D000225:Adenine; D000241:Adenosine; D002118:Calcium