Gram-Scale Chemical Synthesis of Base-Modified Ribonucleoside-5'-O-Triphosphates

This unit delineates a simple, reliable, straight-forward, general, and efficient chemical method for the synthesis of modified nucleoside-5'-O-triphosphates such as 5-methylcytidine-5'-O-triphosphate (5-Me-CTP), pseudouridine-5'-O-triphosphate (pseudo-UTP), and N¹ -methylpseudouridine-5'-O-triphosphate (N¹ -methylpseudo-UTP), starting from the corresponding nucleoside. The reaction utilizes an improved protection-free "one-pot, three-step" Ludwig synthetic strategy that involves the monophosphorylation of the nucleoside with phosphorous oxychloride followed by reaction with tributylammonium pyrophosphate and subsequent hydrolysis of the resulting cyclic intermediate to furnish the corresponding ribonucleoside triphosphate (NTP) in moderate yields. It is noteworthy that the reaction affords high purity (>99.5%) NTPs after DEAE Sepharose column purification. © 2016 by John Wiley & Sons, Inc.