Department of Chemistry, Konstanz Research School Chemical Biology, University of Konstanz, Universitätsstr. 10, 78457 Konstanz, Germany.
Org Biomol Chem. 2013 Dec 28;11(48):8298-305. doi: 10.1039/c3ob41751d. Epub 2013 Oct 30.
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.
三磷酸腺苷（ATP）类似物用两种适合进行Förster 共振能量转移（FRET）的染料标记，有可能成为持续研究消耗 ATP 的酶的酶活性的有用工具。在这里，我们提出了一种合成策略，可以直接获得这些 ATP 类似物。早期的研究表明，在 O2'-和γ-位置修饰 ATP 是设计这些探针的一个非常有前途的起点。我们合成了用附着在这些位置的五种不同染料组合修饰的探针，并研究了它们在非切割状态以及酶水解后的荧光特性。所有呈现的探针在切割后都极大地改变了它们的荧光特性。它们包括比率型 FRET 探针和暗猝灭类似物。对于典型的体外应用，磺化聚甲川染料 Sulfo-Cy3 和 Sulfo-Cy5 的组合似乎最有前途，因为它们在水性缓冲液中的溶解度很好，并且在切割后荧光特性有很大的变化。对于这种染料组合，我们还合成了分别在γ-和 C2-或 O3'-位置修饰的类似物，因为这些附着点也被某些消耗 ATP 的酶很好地接受。这些类似物的荧光特性变化相当大。总的来说，我们提出了新的基于 ATP 的 FRET 探针，它们有可能能够监测消耗 ATP 的酶的酶活性。
