Herein we report a convenient approach for the preparation of fully protected trinucleotide synthons to be used for the synthesis of gene libraries. The trinucleotide synthons bear β-cyanoethyl groups at the phosphate residues, and thus can be used in standard oligonucleotide synthesis without additional steps for deprotection and work-up.

The use of pre-formed trinucleotides, representing codons of the 20 canonical amino acids, for oligonucleotide-directed mutagenesis offers the advantage of controlled randomization and generation of “smart libraries”. We here present a method for the preparation of fully protected trinucleotides on solid phase. The key issue of our strategy is the linkage of the starting nucleoside to the solid support via a traceless disulfide linker. Upon trinucleotide assembly, the disulfide bridge is cleaved under reducing conditions, and the fully protected trinucleotide is released with a terminal 3′-OH group.