Department of Chemistry and Biochemistry, San Diego State University , San Diego, California 92182, United States.
J Am Chem Soc. 2017 Feb 1;139(4):1372-1375. doi: 10.1021/jacs.6b10410. Epub 2017 Jan 20.
Most fluorescent nucleoside analogues are quenched when base stacked and some maintain their brightness, but there has been little progress toward developing nucleoside analogues that markedly increase their fluorescence upon duplex formation. Here, we report on the design and synthesis of a new tricyclic cytidine analogue, 8-diethylamino-tC (8-DEA-tC), that responds to DNA duplex formation with up to a 20-fold increase in fluorescent quantum yield as compared with the free nucleoside, depending on neighboring bases. This turn-on response to duplex formation is the greatest of any reported nucleoside analogue that can participate in Watson-Crick base pairing. Measurements of the quantum yield of 8-DEA-tC mispaired with adenosine and, separately, opposite an abasic site show that there is almost no fluorescence increase without the formation of correct Watson-Crick hydrogen bonds. Kinetic isotope effects from the use of deuterated buffer show that the duplex protects 8-DEA-tC against quenching by excited state proton transfer. These results, supported by DFT calculations, suggest a rationale for the observed photophysical properties that is dependent on duplex integrity and the electronic structure of the analogue.
大多数荧光核苷类似物在碱基堆积时会被猝灭，而有些则保持其亮度，但在开发能够在形成双链体时显著增加其荧光的核苷类似物方面几乎没有进展。在这里，我们报告了一种新的三环胞苷类似物 8-二乙氨基-tC（8-DEA-tC）的设计和合成，与游离核苷相比，8-DEA-tC 在形成 DNA 双链体时的荧光量子产率增加了多达 20 倍，这取决于相邻碱基。这种对双链体形成的开环响应是任何已报道的可参与 Watson-Crick 碱基配对的核苷类似物中最大的。对与腺苷错配和单独与无碱基位点相对的 8-DEA-tC 的量子产率的测量表明，在没有形成正确的 Watson-Crick 氢键的情况下，几乎没有荧光增加。使用氘化缓冲液的动力学同位素效应表明，双链体保护 8-DEA-tC 免受激发态质子转移猝灭。这些结果，得到了 DFT 计算的支持，为观察到的光物理性质提供了一个依赖于双链体完整性和类似物电子结构的理由。
