Solid Phase Synthesis of Oligonucleotides

The fact that macroporous, highly cross-linked polystyrene does not swell makes this support particularly interesting for continuous-flow synthesis in columns. This support has also been successfully used as an alternative to CPG for the solid-phase synthesis of oligonucleotides [90,91]. Furthermore, because reagents do not need to penetrate into the polystyrene network, enzyme-mediated reactions should also proceed smoothly on macroporous polystyrene [85]. 

For the automated solid-phase synthesis of oligonucleotides, however, silica was found to be the support of choice [192-197]. Silica with large pore size (25-300 nm), so-called controlled pore glass (CPG), is generally used for this purpose. The main advantages of CPG, as compared with silica gel, are its more regular particle size and shape, and greater mechanical stability. 

Attachment of suitable linkers to the surface of silica can be achieved by transesterification with (3-aminopropyl)triethoxysilane, which leads to the support 2 (Figure 2.8) [198-200]. Alternatively, silica can be functionalized by reaction with alkyltri-chlorosilanes [201]. For the solid-phase synthesis of oligonucleotides, supports with a longer spacer, such as that in 3, have proven more convenient than 2 [202-206]. Supports 3, so-called LCAA-CPG (long chain alkylamine CPG [194,195]), are commercially available (typical loading 0.1 mmol/g) and are currently the most commonly used supports for the synthesis of oligonucleotides. For this purpose, protected nucleosides are converted into succinic acid monoesters, and then coupled to LCAA-CPG. CPG functionalized with a 3-mercaptopropyl linker has been used for the solid-phase synthesis of oligosaccharides [207]. 

Benzyl ethers bearing electron-donating groups can be cleaved by treatment with acids or oxidants. The rate of solvolysis increases with the number of electron-donating groups, and in the series benzyl < benzhydryl < trityl. For the solid-phase synthesis of oligonucleotides, the 5 -hydroxyl group is usually protected as 4,4/-dimethoxytrityl... 

Other known oligonucleotide analogs include oligomers in which the heterocyclic bases have been modified [155] or replaced by other types of compound [156], Solid-phase syntheses of hybrids of DNA with peptides [157-163], with carbohydrates [164,165], and with PNA (peptide nucleic acids, see Section 16.4.1.2 [166-168]) have also been reported, and several strategies have been developed that enable the preparation of oligonucleotides with a modified 5 - or 3 -terminus [169-174] or of cyclic oligonucleotides [122,175], Various techniques for the parallel solid-phase synthesis of oligonucleotides have been developed for the preparation of compound libraries [176-181],... 

Solid-Phase Synthesis of Oligonucleotide-Related Oligomers... 

A review on the 1,2,3-triazole formation via 1,3-dipolar cycloaddition of acetylenes with azides under mild conditions has been published <03H(60)1225>. The synthesis of a benzotriazole azo dye phosphoramidite and the subsequent use in solid phase synthesis of oligonucleotides has been reported <03TL1339>. The chemical reactivity of [l,2,3]triazolo[l,5-a]- and [l,5-c]-pyrimidinium salts has been published <03T4297>. A review on the use of benzotriazole as an ideal synthetic auxiliary has been disclosed <03CEJ4586>. 

The solid phase synthesis of oligonucleotides containing a site-specific modified psoralen derivative has been accomplished using reagent 197. The base... 

The phosphoramidate linkage in its aliphatic variant has been exploited by Gryaznov and Letsinger [252] for solid phase synthesis of oligonucleotide 3 -phosphates (Figure 19.9). After phosphate 2-cyanoethyl group removal by prior ammonolysis the phosphoramidate required milder conditions to break down (80% acetic acid, 4h at ambient temperature) that are tolerable for the purine... 

The solid-phase synthesis of oligonucleotides by the phosphoramidite approach requires the use of a solid support that is functionalized with an appropriate nucleoside. Solid supports are typically macroporous structures... 

Pon RT, Usman N, Damha MJ, Ogilvie KK. Prevention of guanine modification and chain cleavage during the solid-phase synthesis of oligonucleotides using phosphoramidite derivatives. Nucleic Acids Res 14 6453-6470, 1986. 

Usman N, Pon RT, Ogilvie KK. Preparation of ribonucleoside 3 -0-phos-phoramidites and their application to the automated solid-phase synthesis of oligonucleotides. Tetrahedron Lett 26 4567-4570, 1985. 

While a large number of improvements in procedures for solid phase synthesis of oligonucleotides have been described, the techniques have not altered fundamentally from those described in previous reports. Solid-phase synthesis using a continuous-flow phosphotriester method on a kieselguhr-polyamide support has been described. Other solid phases used as supports for synthesis include derivatized h.p.l.c.-grade silica gel, . derivatized cross-linked poly-... 

The 3 -isobut5Toylamino-thymidine derivative (117), on the other hand, reacts with 1 equiv of LR in the side chain to form the thioamide (118). Phosphite esters which are used as intermediates in solid-phase synthesis of oligonucleotides are converted into the corresponding phosphorothioates (119). Interestingly, LR can also be used as a potent catalyst for the s)uithesis of nucleosides. Tetramethylglycoluril (120) is readily converted to the monothiono- (121) or the dithionoglycolurU (122) depending on the applied molar amount of LR. ... 

The Alloc deprotection is also applicable to automated solid phase synthesis of oligonucleotides as well as linear and cyclic peptidesJ ... 

F. Morvan and J.-J. Vasseur, Inverse Solid Phase Synthesis of Oligonucleotides , in Collection Symposium Series, Institute of Organic Chemistry and Biochemistry Academy of Sciences of the Czech Republic, 2008, vol. 10, Chemistry of Nucleic Acid Components, p. 54. 

The solid-phase synthesis of oligonucleotides consists basically of the sequential repetition of reaction steps similar to those previously described, with the properly protected monomeric nucleotides (for reviews, see Kossel and Seliger, 1975 Amarnath and Broom, 1977). The essential steps are like those used in solid-phase peptide synthesis. 

Compared to solid-phase poljq)eptide synthesis, there has been far less activity in polymer-supported oligosaccharide synthesis. Unlike the solid-phase synthesis of oligonucleotides (Letsinger and Mahadevan, 1965), which closely followed the first reported solid-phase polypeptide... 

Wafers and Capsules. Another solution of compartmentalization of resin beads into permeable containers was reported by Beattie and Frost, who invented porous wafers that housed insoluble supports for the multiple solid-phase synthesis of oligonucleotides and peptides. The porous wafer was made from a Teflon ring covered on both sides by a porous Teflon membrane to form a cylindrical permeable container. The use of wafers was reduced to practice in a specialized column-based oligonucleotide synthesizer. ... 

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