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. 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. To be incorporated in the middle  of an oligonucleotide chain using phosphoramidite strategy, the nucleoside analog must 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 NH, 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. Preparation. 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 P 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. Chemical properties of phosphoramidite moiety. Nucleoside phosphoramidites are relatively stable compounds with a prolonged shelf-life when stored as powders under anhydrous conditions in the absence of air at temperatures below 4 °C. The amidites withstand mild basic conditions. In contrast, in the presence of even mild acids, phosphoramidites perish almost instantaneously. The phosphoramidites are relatively stable to hydrolysis under neutral conditions. For instance, half-life of 2-cyanoethyl 5'-"O"-(4,4'-dimethoxytrityl)thymidine-3'-"O"-(N,N-diisopropylamino)phosphite in 95% aqueous acetonitrile at 25 °C is 200 h. When water is served as a nucleophile, the product is an H-phosphonate diester as  shown in Scheme above. Due to the presence of residual water in solvents and reagents, the formation of the latter compound is the most common complication in the preparative use of phosphoramidites, particularly in oligonucleotide synthesis. Similarly, phosphoramidites react with other chalcogens. When brought in contact with a solution of sulfur or a number of compounds collectively referred to as sulfurizing agents, phosphoramidites quantitatively form phosphorothioamidates. The reaction with selenium or selenium derivatives produces phosphoroselenoamidates. In all reactions of this type, the configuration at the phosphorus atom is retained. (RO)P-N(R) + R-N + HO ---- (RO)P(=O)-N(R) + R-NH  + N; Protecting strategy. The naturally occurring nucleotides (nucleoside-3'- or 5'-phosphates) and their phosphodiester analogs are insufficiently reactive to afford an expeditious synthetic preparation of oligonucleotides in high yields. The selectivity and the rate of the formation of internucleosidic linkages are dramatically improved by using 3'-"O"-("N","N"-diisopropyl phosphoramidite) derivatives of nucleosides (nucleoside phosphoramidites) that serve as building blocks in phosphite triester methodology. To prevent undesired side reactions, all other functional groups present in nucleosides must be rendered unreactive (protected) by attaching protecting groups. Upon the completion of the oligonucleotide chain assembly, all the protecting groups are removed to yield the desired  oligonucleotides. Below, the protecting groups currently used in commercially available and most common nucleoside phosphoramidite building blocks are briefly reviewed: