A method was developed for stepwise synthesis of oligonucleotides of denned sequence using 2′(3′)-O-dihydrocinnamoyl-nucleoside 5′-diphosphates as substrates for polynucleotide phosphorylase [EC 2.7.7.8]. Polynucleotide phosphorylase from Thermus thermophilus catalyzed the transfer of one 2′(3′)-O-dihydrocinnamoyl-adenylate residue from 2′(3′)-blocked ADP to the 3′-terminus of the primer trinucleoside diphosphate, ApApA. The product was 2′(3′)-substituted triadenylyladenosine. The blocking group, dihydrocinnamoyl, could be removed completely from the product without destruction of the phosphodiester bond using α-chymotrypsin [EC 3.4.21.1] at neutral pH.