Synthesis and Characterization of the Spectroscopic and Imaging Utilities of Two Indole-Based Cyan Fluorescent Nucleoside Analogues

Indole is structurally similar to the nitrogenous bases of adenine and guanine. Therefore, it provides an ideal template for developing fluorescent nucleoside analogues that do not significantly perturb the native properties of the DNA or RNA system of interest, emit visible light with a spectrum that is sensitive to their local environments, and are bright enough for biological studies using fluorescence-based techniques. Herein, we demonstrate two such nucleoside analogues: 4-cyano-7-azaindole-2'-deoxyribonucleoside (4CN7AI-DNS) and 4-cyano-7-azaindole-2'-ribonucleoside (4CN7AI-NS). Compared to 2-aminopurine-2'-deoxyribonucleoside, the most commonly used fluorescent nucleoside analogue, and 4-cyanoindole-2'-deoxyribonucleoside, another recently developed indole-based fluorescent nucleoside analogue, the main advantage of 4CN7AI-DNS (and 4CN7AI-NS) is that its absorption spectrum is further red-shifted and its fluorescence spectrum, which is in the blue to green region of the visible spectrum, exhibits a larger and a more environmentally sensitive Stokes shift, hence enabling it to be useful not only in spectroscopic characterization of DNA (RNA) structures (e.g., G-quadruplex) and binding interactions, but also in DNA imaging applications.