Fluorescent ε-ATP analogues for probing physicochemical properties of proteins. Synthesis, biochemical evaluation, and sensitivity to properties of the medium

Sharon, Einat; Zündorf, Gregor; Lévesque, Sébastien A.; Beaudoin, Adrien R.; Reiser, Georg; Fischer, Bilha

doi:10.1016/j.bmc.2004.09.011

Cited by 6 publications

(4 citation statements)

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“…8-Aza and 1-deaza modifications are generally tolerated. A fluorescent adenine-modified derivative of ATP (5) behaved as a potent P2Y 1 receptor agonist (Sharon et al, 2004). The 2-position of the adenine ring can accommodate a wide variety of substituents, with resultant activation of both P2Y 1 and P2Y 12 receptors.…”

Section: A Chemical Structure Of Agonist and Antagonist Ligandsmentioning

confidence: 99%

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

et al. 2006

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Section: A Chemical Structure Of Agonist and Antagonist Ligandsmentioning

confidence: 99%

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

et al. 2006

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“…One of the earliest fluorescent base analogs, etheno‐adenosine, has been used occasionally since the early 1970s as a probe of nucleic acid interactions (79–83). Its absorption and fluorescence properties appear favorable to act as a fluorescence probe for DNA conformation and interactions, as well as an excitation acceptor from the normal DNA bases ( λ abs ∼310 nm, λ em ∼ 410 nm), but it cannot form base‐pairing hydrogen bonds and tends to locate itself outside the normal stack of bases in B‐DNA.…”

Section: Introductionmentioning

confidence: 99%

Sequence, Structure and Energy Transfer in DNA†

Nordlund¹

2007

Photochemistry and Photobiology

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Excitation energy transfer in DNA has similarities to charge transfer, but the transport is of an excited state, not of mass or charge. Use of the fluorescent, modified adenine base 2-aminopurine (2AP) as an energy trap in short (3- to 20-base) single- and double-stranded DNA oligomers is reviewed. Variation of 2AP's neighboring sequence shows (1) relatively efficient transfer from adenine compared to that from cytosine and thymine, (2) efficient transfer from guanine, but only when 2AP is at the 3' end, (3) approximate equality of efficiencies for 3' to 5' and 5' to 3' directional transfer in adenine tracks. The overall, average transfer distance at room temperature is about four adenine bases or less before de-excitation. The transfer fluorescence excitation spectral shape is similar to that of the absorption spectrum of the neighboring normal bases, confirming that initial excitation of the normal bases, followed by emission from 2AP (i.e. energy transfer), is occurring. Transfer apparently may take place both along one strand and cross-strand, depending on the oligomer sequence. Efficiency increases when the temperature is decreased, rising above 50% (overall efficiency) in decamers of adenine below -60 degrees C (frozen media). Modeling of the efficiencies of transfer from the nearest several adenine neighbors of 2AP in these oligomers suggests that the nearest two neighbors transfer with near 100% efficiency. As bases in B DNA, as well as in single-stranded DNA, are separated by less than 5 A (less than the size of a base), standard Förster transfer theory should not apply. Indeed, while both theory and experiment show efficiency decreasing with donor-acceptor distance, the experimental dependence clearly disagrees with Förster 1/r6 dependence. It is not yet clear what the best theoretical approach is, but any calculation must deal accurately with the excited states of bases, including strong base-base interactions and structural fluctuations, and should reflect the increase of efficiency with temperature decrease and the relative insensitivity to strandedness (single, double). Attempts to use DNA as a molecular "fiber optic" face three primary challenges. First, reasonable efficiency over more than a base or two occurs only in adenine stretches at temperatures well below freezing. Second, transfer in these adenine tracks is efficient in both directions. Third, absorption of UV light occurs randomly, making excitation at a specific site on this "fiber optic" a challenge.

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“…Whereas the well‐established fluorescent probe N1,N 6 ‐etheno‐ATP (ε‐ATP) needs further modifications to gain an affinity for the binding site of P2Y 1 ‐R,43 analogue 18 a was found to be almost equipotent with ATP. Namely, the introduction of the 2,3‐aminoetheno group to create a fluorophore has no negative effect on the potency of ATP at the P2Y 1 receptor.…”

Section: Resultsmentioning

confidence: 99%

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

et al. 2006

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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.

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Fluorescent ε-ATP analogues for probing physicochemical properties of proteins. Synthesis, biochemical evaluation, and sensitivity to properties of the medium

Cited by 6 publications

References 75 publications

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

Sequence, Structure and Energy Transfer in DNA†

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

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Fluorescent ε-ATP analogues for probing physicochemical properties of proteins. Synthesis, biochemical evaluation, and sensitivity to properties of the medium

Cited by 6 publications

References 75 publications

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy

Sequence, Structure and Energy Transfer in DNA†

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

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Product

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