Nucleoside phosphoramidite

Nucleoside phosphoramidites are derivatives of natural or synthetic nucleosides. They are used to synthesize oligonucleotides, relatively short fragments of nucleic acid and their analogs. Nucleoside phosphoramidites were first introduced in 1981 by Beaucage and Caruthers. In order to avoid undesired side reactions, reactive hydroxy and exocyclic amino groups present in natural or synthetic nucleosides are appropriately protected. As long as a nucleoside analog contains at least one hydroxy group, the use of the appropriate protecting strategy allows one to convert that to the respective phosphoramidite and to incorporate the latter into synthetic nucleic acids. In order to be incorporated in the middle of an oligonucleotide chain using phosphoramidite strategy, the nucleoside analog have to possess two hydroxy groups or, less often, a hydroxy group and another nucleophilic group (amino or mercapto). Examples include, but are not limited to, alternative nucleotides, LNA, morpholino, nucleosides modified at the 2'-position (OMe, protected NH₂, F), nucleosides containing non-canonical bases (hypoxanthine and xanthine contained in natural nucleosides inosine and xanthosine, respectively, tricyclic bases such as G-clamp, etc.) or bases derivatized with a fluorescent group or a linker arm.

There are three main methods for the preparation of nucleoside phosphoramidites.

Nucleoside phosphoramidites are purified by column chromatography on silica gel. To warrant the stability of the phosphoramidite moiety, it is advisable to equilibrate the column with an eluent containing 3 to 5% of triethylamine and maintain this concentration in the eluent throughout the entire course of the separation. The purity of a phosphoramidite may be assessed by ³¹P NMR spectroscopy. As the P(III) atom in a nucleoside phosphoramidite is chiral, it displays two peaks at about 149 ppm corresponding to the two diastereomers of the compound. The potentially present phosphite triester impurity displays peak at 138-140 ppm. H-phosphonate impurities display peaks at 8 and 10 ppm.

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