This unit describes postsynthetic modification of oligonucleotides (ONs) containing 2'-deoxy-5-trifluoromethyluridine and 2'-deoxy-5-trifluoromethylcytidine. In ONs, the trifluoromethyl group at the C5 position of pyrimidine bases is converted into a variety of carboxylic acid equivalents using alkaline and amine solutions. In addition, treating fully protected and controlled pore glass (CPG)-attached ONs with methylamine and sodium hydroxide aqueous solution results in deprotection of all protecting groups (except the 4,4'-dimethoxytrityl group), cleavage from CPG, and simultaneous conversion of the trifluoromethyl group to afford the corresponding ONs containing 5-substituted pyrimidine bases. © 2019 by John Wiley & Sons, Inc.