Synthesis and fluorescence characteristics of ATP-based FRET probes

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HARDT, Normann, Stephan M. HACKER, Andreas MARX, 2013. Synthesis and fluorescence characteristics of ATP-based FRET probes. In: Organic & Biomolecular Chemistry. 11(48), pp. 8298-8305. ISSN 1477-0520. eISSN 1477-0539. Available under: doi: 10.1039/C3OB41751D

@article{Hardt2013-12-28Synth-25795, title={Synthesis and fluorescence characteristics of ATP-based FRET probes}, year={2013}, doi={10.1039/C3OB41751D}, number={48}, volume={11}, issn={1477-0520}, journal={Organic & Biomolecular Chemistry}, pages={8298--8305}, author={Hardt, Normann and Hacker, Stephan M. and Marx, Andreas} }

Organic & Biomolecular Chemistry ; 11 (2013), 48. - S. 8298-8305 Hacker, Stephan M. Synthesis and fluorescence characteristics of ATP-based FRET probes Hardt, Normann Adenosine triphosphate (ATP) analogues labelled with two dyes suitable for undergoing Förster Resonance Energy Transfer (FRET) have the potential to be valuable tools to continuously study the enzymatic activity of ATP consuming enzymes. Here, we present a synthesis strategy that allows obtaining these ATP analogues in a straight-forward manner. Earlier studies indicate that modifying ATP at the O2′- and the γ-position is a very promising starting point for the design of these probes. We synthesized probes modified with five different combinations of dyes attached to these positions and investigated their fluorescence characteristics in the non-cleaved state as well as after enzymatic hydrolysis. All presented probes largely change their fluorescence characteristics upon cleavage. They include ratiometric FRET probes as well as dark quenched analogues. For typical in vitro applications a combination of the sulfonated polymethine dyes Sulfo-Cy3 and Sulfo-Cy5 seems to be most promising due to their excellent solubility in aqueous buffer and a large change of fluorescence characteristics upon cleavage. For this combination of dyes we also synthesized analogues modified at the γ- and the C2- or the O3′-position, respectively, as these attachment sites are also well accepted by certain ATP consuming enzymes. These analogues show comparably large changes in fluorescence characteristics. Overall, we present new ATP-based FRET probes that have the potential to enable monitoring the enzymatic activity of ATP consuming enzymes. 2013-12-28 Marx, Andreas terms-of-use Marx, Andreas eng 2014-01-10T10:08:09Z Hacker, Stephan M. Hardt, Normann 2014-01-10T10:08:09Z

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