A new and convenient approach for the preparation of β-cyanoethyl protected trinucleotide phosphoramidites

Abstract

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.

Synthesis of Trinucleotide Building Blocks in Solution and on Solid Phase

“…Likewise, protecting groups at the 3′‐OH position had to be removed under rather harsh conditions, thus causing damage to other functionalities of the trinucleotide (Lyttle et al., ; Virnekas et al., ). Among the different 3′‐ O ‐protecting groups, 2‐azidomethylbenzoyl and TBDMS stand out as the best performing functionalities (Janczyk et al., ; Yagodkin et al., ). The 2‐azidomethylbenzoyl group can be selectively removed with triphenylphosphine/H 2 O, inducing reduction of the azide to a primary amine followed by spontaneous intramolecular cyclization to release isoindoline‐1‐one and the free 3′‐OH group.…”

“…Therein, it is important to use a mild agent, such as 3HF/triethylamine, because the harder fluoride in tert‐ butylammonium fluoride (TBAF) would also cleave the phosphotriester bond. Both strategies, 3′‐ O ‐2‐azidomethylbenzoyl and 3′‐ O ‐TBDMS protection, have been successfully used for preparation of trinuleotide synthons by phosphotriester chemistry or by the phosphoramidite approach (Janczyk et al., ; Yagodkin et al., ). Herein, the protocol using the 3′‐ O ‐TBDMS strategy is described.…”

“…This is achieved only with 2-cyanoethyl-N,N,N ,Ntetraisopropylphosphorodiamidite in combination with 5-benzylmercaptotetrazole as phosphitylation agent (Fig. 5) as described in Janczyk, Appel, Springstubbe, Fritz, & Muller, 2012. We describe a protocol that considers the more labile nature of the 2-cyanoethyl group but can be applied to trimers with the more robust methyl protecting group on the phosphate moieties as well.…”

Solid Phase Assembly of Fully Protected Trinucleotide Building Blocks for Codon-Based Gene Synthesis

“…Furthermore, the generation of stop codons cannot be prevented, which leads to abortion fragments instead of the full-length enzymes [5]. A possibility to overcome these problems is the use of trinucleotide building blocks (Figure 1), which represent codons of the canonical amino acids and thus permit the controlled synthesis of a fully or partially randomized gene library without stop codons or codon bias [6]. There are several but not easy ways to synthesize such trinucleotide building blocks, either in solution [7] or, as a more recent addition to the field, on soluble support [8,9].…”

Generation of highly diverse peptide library by linear-double-stranded DNA based AND gate genetic circuit in mammalian cells

“…When the polypeptide length reaches 20 amino acids, about 47% of the sequences contain pre-stop codon. One potential solution to avoid stop codon is to synthesize PCR primer using trimer phosphoramidites (39,40).…”