Fluorescent N²,N3‐ε‐Adenine Nucleoside and Nucleotide Probes: Synthesis, Spectroscopic Properties, and Biochemical Evaluation

Abstract

N1,N 6 -ethenoadenine, ε-A, nucleos(t)ides have been previously applied as fluorescent probes in numerous biochemical systems. However, these ε-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-ε-A). Here, we describe four novel syntheses of the target ε-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-ε-A and the dependence of the spectral features on the pH and polarity of the medium. Specifically, maximum emission of N 2 ,N3-ε-A in water is observed at 420 nm (ϕ=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-ε-ATP was found to be almost equipotent with ATP at the P2Y 1 receptor and was hydrolyzed by NTPDases 1 and 2at about 80% of the rate of ATP. Furthermore, protein binding does not seem to shift the fluorescence of N 2 ,N3-ε-ATP. Based on the fluorescence and full recognition by ATP-binding proteins, we propose N 2 ,N3-ε-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.

Evidence for the Existence of a Specific G Protein‐Coupled Receptor Activated by Guanosine

“…14 For the synthesis of thio derivatives 8 – 11 , compound 4 was reacted with the appropriate thiols in the presence of potassium carbonate, and after evaporation, the crude mixture was treated with methanolic ammonia to give the desired 6‐thio derivatives. Synthesis of compound 8 was already described, but our procedure afforded an improved yield 15. Access to compound 11 was reported with a different procedure, starting from a nucleoside in which the 2‐amino group had been previously protected 16.…”

“…(2 R ,3 R ,4 S ,5 R )‐2‐[2‐Amino‐6‐(methylthio)‐9 H ‐purin‐9‐yl]‐5‐(hydroxymethyl)tetrahydrofuran‐3,4‐diol (8). Sodium methanethiolate (198 mg, 2.82 mmol) was added to a solution of 4 (200 mg, 0.47 mmol) in dry DMF (7 mL), and the mixture was stirred at RT for 1 h. MeOH (7 mL) was then added, and the mixture was stirred for another 2 h. After removing the volatiles, the residue was separated by flash column chromatography, eluting with CHCl 3 /MeOH (98:2) to give 8 as a white powder 15. Yield 38 %; mp: 125–126 °C; ESI‐MS: positive mode m / z : 314.0 ([ M +H] + ), 336.0 ([ M +Na] + ), 648.9 ([2 M +Na] + ), negative mode m / z : 347.5 ([ M +Cl] − ), 624.2 ([2 M ‐H] − ); Anal (C 11 H 15 N 5 O 4 S) C, H, N.…”

Development of selective agonists and antagonists of P2Y receptors

“…8-Aza and 1-deaza modifications are generally tolerated at P2Y 1 and P2Y 12 receptors [ 22 , 34 , 87 ], but other modifications, such as N1, N6-etheno, result in inactivity at P2Y receptors [ 32 ]. A fluorescent derivative of ATP 19 containing a tricyclic nucleobase is strikingly potent in activation of the P2Y 1 receptor [ 33 ].…”

8-(p-CF3-cinnamyl)-modified purine nucleosides as promising fluorescent probes

“…19 Previously, we have extended the adenine chromophore at C2,N3-positions to give N 2 ,N3-etheno-adenosine, 6, as a novel probe exhibiting improved fluorescence characteristics (l em 420 nm; f 0.03). 20 The limitations of fluorescent dyes used in current hybridization-based technologies have encouraged us to develop novel intrinsically fluorescent adenine and guanine nucleosides that will be later incorporated into oligonucleotides. These probes include a minimal extension at the C8-position of adenosine (A) or guanosine (G), rather than attachment of large hydrophobic dyes to the nucleobase.…”