Figure 4.

Fmoc-trinucleotide approach for the creation of codon-based amino acid substitutions in a non-saturating mode. The starting sphere represents a DMTr-protected oligonucleotide growing in a solid support. Synthesis proceeds in common 3′ to 5′ direction. In the first step, the DMTr group is removed by acid treatment (H⁺). A part of the growing oligo reacts with a diluted solution of a single Fmoc-trinucleotide phosphoramidite (XXX-O-Fmoc), or a mixture, without performing capping, oxidation and detritylation. The remaining chains are saturated with three sequential ordinary couplings (W-O-DMTr) to assemble the wild-type codon to be partially replaced. Removal of both protecting groups by sequential acid and base (B⁻) treatments prepares the oligo for a new cycle of mutagenesis.