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

Abstract

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.

Solid-phase synthesis and structural characterisation of phosphoroselenolate-modified DNA: a backbone analogue which does not impose conformational bias and facilitates SAD X-ray crystallography

“…Furthermore, subsequent quenching of the reaction following addition of water resulted in detritylation in the absence of a strong organic base but if such a base was used during work-up, further decyanoethylation of the phosphoroselenolate was evident. 63 In order to ameliorate issues associated with b-elimination of the cyanoethyl function, methyl-protection of the phosphate was adopted. This provides greater resilience towards phosphate deprotection in the presence of stronger organic bases such as triethylamine and has recently been utilised for the preparation of di-and trinucleotide phosphoramidites.…”

“…This provides greater resilience towards phosphate deprotection in the presence of stronger organic bases such as triethylamine and has recently been utilised for the preparation of di-and trinucleotide phosphoramidites. [63][64][65] The methyl-protected 3 0 -H-phosphonates (3a-e) were prepared from the corresponding phosphoramidites under modied literature conditions. 66 Following extractive work-up, the materials were pure by 31 P NMR.…”

Synthesis of fully protected trinucleotide building blocks on a disulphide-linked soluble support

“…1). 4,11,12 Traditionally, trinucleotide synthons have been prepared in solution, paying special attention to the pair of orthogonal protecting groups for the 5 0 -and 3 0 -OH functions, when synthesizing a dinucleotide that subsequently can be extended in 5 0 -or 3 0 -direction. 1,[13][14][15][16][17][18][19] Synthesis in solution requires isolation of products aer each synthesis step, which can become rather tedious.…”

“…1,[13][14][15][16][17][18][19] Synthesis in solution requires isolation of products aer each synthesis step, which can become rather tedious. Therefore, we 11,20 and others 19 have developed strategies for trinucleotide synthesis on solid support as an attractive alternative to protocols in solution.…”

“…With regard to trinucleotide synthesis, an oxalyl anchor 19 or a disulphide linker have been described. 11,21 In recent years, protocols for the synthesis of oligonucleotides on soluble supports have emerged, 12,[21][22][23][24] and those have also been used with particular attention to the preparation of fully protected trinucleotides. 12,21 The general strategy involves iterative cycles of reaction steps in solution and precipitation for isolation/purication of reaction products.…”