Phosphoramidites synthesis

The chemical synthesis of RNA has the distinct advantage of allowing for site-selective incorporation of modified nucleosides into the oligonucleotide chain. The early phosphodi-ester method developed by Khorana (30) was followed by the H-phosphonate and phosphoramidite approaches (31). Although the H-phosphonate approach has distinct advantages over phosphoramidite synthesis, it is not as widely used. Therefore, the focus of this section will be on the more commonly employed... 

A new method for the removal of the 5 -0-DMT group during the synthesis of ODNs on a microarray has been studied" which uses all the steps involved in phosphoramidite synthesis, but uses a photogenerated acid (PGA) rather than trichloroacetic acid to deprotect. The photolabile 3 -0- [2-(2-nitrophenyl)propoxy]carbonyl -protected 5 -phosphoramidites (5) have been prepared for the 5 ->3 light-directed synthesis of DNA on microarrays." Developments for the synthesis of high-density DNA probe arrays employ nucleoside monomers protected with 5 -(a-methyl-6-nitropiperonyloxycarbonyl) (MeN-POC) with proximity photolithography, which is currently capable of printing 10 m probe features at a density of 10 probes/cm. "... 

Kondo, H, Ichikawa, Y, Wong, C-H, (i-Sialyl phosphite and phosphoramidite synthesis and application to the chemoenzymatic synthesis of CMP-sialic acid and sialyl oligosaccharides, J. Am. Chem. Soc., 114, 8748-8750, 1992. 

Most of the linkers described for the synthesis of 3 -aminoalkyl ohgonucleotides employ the carbamate (urethane) linkage to the polymer. A 2,2 -dithiodiethanol-derivatised silica gel support (Section 19.3.2.3) has been transformed by Asseline and Thuong [287] into the N-ro-hydroxyhexylcarbamate (Figure 19.12) via the consecutive treatments with l,T-carbonyldiimidazole and 6-amino-l-hexanol followed by the phosphoramidite synthesis of a DNA undecamer. Deprotection by 0.1 M DTT in concentrated aqueous ammonia unmasked the [3-mercaptoethyl carbamate, which fragments under basic conditions with the elimination of thiirane to furnish 3 -aminohexyl oligodeoxyribonucleotide. The structure of the latter has been confirmed by the reaction with acridine-9-isothiocyanate. 

In the phosphite triester synthesis (phosphoramidite synthesis), an efficient coupling reaction takes place between the 3 -hydroxyl group of the chain and 5 -DMTr-(A -acy-lated)-deoxyribonucleoside 3 -0-(MA -diisopropylamino)phosphite. 

As regards the modification of intemucleosidic links, bis(deoxynucleosidyl)-trimethylsilyl phosphites 190 can be converted to phosphorofluoridates 191 by the use of S02C1F,230 and dibenzoyltetrasulfide has been advocated as a rapid sulfur transfer agent in the synthesis of nucleoside phosphorothioates, being used to prepare a phosphorothioate after each cycle of a phosphoramidite synthesis.231 Oxidation of intemucleosidic H-phosphonothioates (2 -deoxy series) with iodine in aqueous acetonitrile in the presence of trimethylamine gives... 

Eritja, R., Smirnov, V, and Caruthers, M. H (1990) 0-aryl phosphoramidites Synthesis, reactivity and evaluation of their use for solid-phase synthesis of oligonucleotides Tetrahedron 46,721-730. 

Some of the concepts most attractive for the simplification of oligonucleotide synthesis and, thus, also for reducing cost and effort of potential large-scale preparations, come from a combination of two of the three operations essential for conventional chain-elongation. Although the combination of protection and activation principles plays a minor role in phosphate-diester and phosphate-triester chemistry, this is the basis of success of the phosphoramidite synthesis (23,24). The relative stability of nucleoside phosphoramidites at room temperature and their fast and efficient activation by tetrazole (23) (see also 25 for mechanistic studies) are essential to today s most utilized process of internucleotide bond formation. 

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