Synthesis of Trinucleotide Building Blocks in Solution and on Solid Phase

Abstract

We have developed two methods, in solution and on solid phase, that give easy access to trinucleotide phosphoramidites capable of undergoing coupling reactions by the solid‐phase phosphoramidite approach. The solution protocol is characterized by application of 5′‐O‐dimethoxytrityl (DMT) and 3′‐O‐tert‐butyldimethylsilyl (TBDMS) as a pair of orthogonal protecting groups and 2‐cyanoethyl (CE) for protection of the phosphate. Starting with suitably functionalized monomers, synthesis proceeds in the 3′‐ to 5′‐direction, delivering the fully protected trinucleotide. The 3′‐O‐protecting group is cleaved followed by phosphitylation of the free 3′‐OH group. The solid‐phase protocol is based on standard phosphoramidite chemistry in conjunction with a dithiomethyl linkage connecting the 3′‐starting nucleoside to the polymer. The disulfide bridge can be cleaved under neutral conditions for release of the trinucleotide from the support preserving all other protecting groups. © 2018 by John Wiley & Sons, Inc.

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

“…The absorption of the DMT-cation was measured at 498 nm, and the corresponding DMT concentration was determined by comparison to a standard curve. For the detailed procedure see Suchsland et al [10] and Supplementary Materials S1.…”

“…The synthesis of 3′-O-thiomethyl-modified nucleosides (in Scheme 1(3a-d)) is described in detail in Suchsland et al [10]. See Supplementary Materials S2 for 1 H and 13 C NMR spectra.…”

“…Trinucleotide assembly on solid support can proceed by standard phosphoramidite chemistry and, thus, is a valuable strategy for the preparation of fully protected trinucleotides, until the major hurdles of supported oligonucleotide assembly on soluble polymers have been overcome. Our initial protocol for solid phase trinucleotide assembly has been described recently [10]. We here wish to report our studies regarding the choice of a suitable polymer, efficient conjugation chemistry for linking the starting nucleoside to the support, and detachment of the fully protected trinucleotide.…”

Liquid‐Phase Oligonucleotide Synthesis: Past, Present, and Future Predictions

“…Both SPOS and LPOS utilize a support that is inert during the ON synthesis cycles. Although di‐ and trinucleotide synthons have been possible to construct in solution phase without applying any soluble polymer (Arunachalam, Wichert, Appel, & Müller, ; Jabgunde, Molina, Virta, & Lönnberg, ; Kungurtsev, Lönnberg, & Virta, ; Current Protocols article: Suchsland, Appel, & Müller, ), the use of a soluble support is indispensable for the synthesis of longer ONs, as it facilitates an easier purification of the elongated chain from undesired excess soluble reagents. During ON synthesis, successful product separation is accomplished because of the macromolecular properties of the soluble support.…”

“…In addition, the utilization of the blockmer approach over monomeric building‐block addition is expected to furnish full‐length ON that is devoid of n‐1 truncated products, making the purification task easier. For example, convergent LPOS dinucleotide and trinucleotide synthesis was successfully carried out using the phosphoramidite strategy (Chen et al., ; Current Protocols article: Suchsland et al., ) or H ‐phosphonate chemistry (Reese & Yan, ).…”