Synthesis of 4‐Cyanoindole Nucleosides, 4‐Cyanoindole‐2ʹ‐Deoxyribonucleoside‐5ʹ‐Triphosphate (4CIN‐TP), and Enzymatic Incorporation of 4CIN‐TP into DNA

Abstract

4‐Cyanoindole‐2ʹ‐deoxyribonucleoside (4CIN) is a fluorescent isomorphic nucleoside analogue with superior spectroscopic properties in terms of Stokes shift and quantum yield in comparison to the widely utilized isomorphic nucleoside analogue, 2‐aminopurine‐2ʹ‐deoxyribonucleoside (2APN). Notably, when inserted into single‐ or double‐stranded DNA, 4CIN experiences substantially less in‐strand fluorescence quenching compared to 2APN. Given the utility of these properties for a spectrum of research applications involving oligonucleotides and oligonucleotide‐protein interactions (e.g., enzymatic processes, DNA hybridization, DNA damage), we envision that additional reagents based on 4‐cyanoindole nucleosides may be widely utilized. This protocol expands on the previously published synthesis of 4CIN to include synthetic routes to both 4‐cyanoindole‐ribonucleoside (4CINr) and 4‐cyanoindole‐2ʹ‐deoxyribonucleoside‐5ʹ‐triphosphate (4CIN‐TP), as well as a method for the enzymatic incorporation of 4CIN‐TP into DNA by a polymerase. These methods are anticipated to further enable the utilization of 4CIN in diverse applications involving DNA and RNA oligonucleotides. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of 4‐cyanoindole‐2ʹ‐deoxyribonucleoside (4CIN) and 4CIN phosphoramidite 4 Basic Protocol 2: Synthesis of 4‐cyanoindole‐ribonucleoside (4CINr) Basic Protocol 3: Synthesis of 4‐cyanoindole‐2ʹ‐deoxyribonucleoside‐5ʹ‐triphosphate (4CIN‐TP) Basic Protocol 4: Steady state incorporation kinetics of 2AP‐TP and 4CIN‐TP by a DNA polymerase

Synthesis and photophysical characterization of fluorescent indole nucleoside analogues

“…were dissolved in anhydrous DMF (15 mL) and stirred under N 2 for 18 hours. The reaction was quenched with the addition of saturated bicarbonate solution (5 mL), extracted into dichloromethane (DCM, 50 mL), washed with 3 ′ ,5 ′ -Di-O-tert-butyldimethylsilyl-6-cyanopurine-2 ′ -deoxyribonucleoside (9). Nucleoside 9 was synthesized through modication to a known procedure.…”

“…[5][6][7] Recently, 4-cyanoindole (4CI) was developed into the corresponding uorescent nucleoside 4-cyanoindole-2 ′ -deoxyribonucleoside (4CIN), which is superior to 2APN in all the aforementioned photophysical properties. 8 4CI has been incorporated into a ribonucleoside (4CINr) and a 2 ′ -deoxyribonucleoside-2 ′ -triphosphate (4CINTP), 9 and 4CIN has served as a useful probe for DNA 8,10 and RNA applications. 11 To further optimize 4CIN and interrogate the scope of structural modications that can contribute to favorable photophysical properties, a library of uorescent nucleosides combining the structural features of 4CIN and 2APN were synthesized and their photophysical properties characterized (Fig.…”

Covalent labeling of nucleic acids

“…7B) was synthesized and successfully incorporated into DNA by DNA polymerase I Klenow exo À fragment. 113 Similar to DNA polymerases, RNA polymerases were also exploited to introduce fluorescently labeled nucleotides into nascent RNA. Thieno [3,4-d]pyrimidine represents an isomorphic GTP analog termed thGTP (Fig.…”

Blue Fluorescence of Cyano-tryptophan Predicts Local Electrostatics and Hydrogen Bonding in Biomolecules

“…The incorporation of cyanoindole (CI) into DNA as a fluorescent nucleoside has recently been reported. 51,63 To this effect, we have extended our field− frequency studies on 4-cyanoindole (1) and 5-cyanoindole (2). Similar to 3 and 4, cyanoindoles also show robust yet separate linear correlations for non-H-bonding and aqueous H-bonding environments (Figure 4).…”