3′-O-Caged 2′-Deoxynucleoside Triphosphates for Light-Mediated, Enzyme-Catalyzed, Template-Independent DNA Synthesis

Abstract

Synthesis, purification, and characterization of 3′-O-caged 2′-deoxyribonucleoside triphosphates (dNTPs), namely 3′-O-(2-nitrobenzyl)-2′-deoxy ribonucleoside triphosphates (NB-dNTPs) and 3′-O-(4,5-dimethoxy-2-nitrobenzyl)-2′-deoxy ribonucleoside triphosphates (DMNB-dNTPs), are discussed in detail. A total of eight 3′-O-caged dNTPs are synthesized with specific protocols depending on the nitrogenous base on the first carbon, i.e., adenine, guanine, thymine, and cytosine, as well as the photo-cleavable group, i.e, 2-nitrobenzyl and 4,5- dimethoxy-2-nitrobenzyl, to be attached in the 3′-O position. The purification of the synthesized compounds is done using ion-exchange and flash chromatography; this is followed by structural confirmation by nuclear magnetic resonance (NMR) and mass spectroscopy (MS). The efficiency of the designed compounds is tested by conducting and evaluating UV-cleaving experiments at 365 nm with proton NMR and LC-MS curves. Finally, the application of the 3′-O-cagged dNTPs in template-independent, enzyme-catalyzed, photo-mediated oligonucleotide synthesis is demonstrated. © 2017 by John Wiley & Sons, Inc.