Phosphoramidite method oligonucleotide synthesis

Oligonucleotide Synthesis (phosphoramidite method - Lessinger) Review Tetrahedron 1992, 48,2223... 

The disadvantage of this method is that the dichloridites and monochloridites are sensitive to water and thus could not be used readily in automated oligonucleotide synthesis. This problem was overcome by Beaucage and Caruthers, who developed the phosphoramidite approach. In this method, derivatives of the form R 0P(NR2)2 react with one equivalent of an alcohol (catalyzed by species such as l//-tetrazole) to form diesters, R OP(OR")NR2, which usually are stable, easily handled solids. These phosphoroamidites are easily converted to phosphite triesters by reaction with a second alcohol (catalyzed by l//-tetrazole). Here, again, oxidation of the phosphite triester with aqueous iodine affords the phosphate triester. Over the years, numerous protective groups and amines have been examined for use in this approach. Much of the work has been reviewed. ... 

Modified oligonucleotides, synthesis by phosphoramidite method and application 93T6123. 

A selective method which involves the selective pivaloyloxymethyl protection of the N1 of pseudouridine followed by methylation at N3 was developed to prepare the 5-benzhydryloxybis(trimethylsilyloxy)silyl, bis(2-acetoxy-ethoxy)me-thyl- protected phosphoramidite derivative (139) of the nucleoside 3-methylpseu-douridine. The methylated pseudouridine phosphoramidite was successfully used in oligonucleotide synthesis for the NMR study of helix 69 of E. coli 23S rRNA. 2-Thiouridines incorporating 2 -modified nucleoside phosphoramidites... 

An additional reason for the inferior performance of the H-phosphonate method compared to the phosphoramidite lies in its mechanism, which makes the synthesis very sensitive to pre-activation of the H-phosphonate monoesters with the conventional coupling agents, which must be avoided [138], However, the main appeal of the H-phosphonate approach lies in the ease it offers for the synthesis of backbone analogues modified at the phosphorus atom phosphorothioates [139], phos-photriesters [126] and especially phosphoramidates [126, 140, 141]. Now it is rarely used for routine oligonucleotide synthesis but very often for the preparation of variously modified analogues. Further developments in the H-phosphonate method have been made in the last decade by Polish [142] and Japanese researchers [143]. 

Figure 7. Synthesis cycle in standard oligonucleotide synthesis using the phosphoramidite method. A 5 -OH-group is released by acid cleavage of the DMT-group B Substitution of the N(iPr)2-group by the 5 -OHgroup is catalyzed by l//-tetrazole C Unreacted 5 -OH-functional groups are capped by catalyzed acetylation D Cyanoethyl-phosphite is oxidized by iodine E Oligomer is cleaved from spacer by concentrated ammonium hydroxide solution and kept at 55 °C for 8 h for complete deprotection.

Crosslinking of DNA and proteins is a versatile method to study protein-DNA interactions. Among others, phosphoramidites of brominated or iodinated 2 -deoxycytidines [325], 2 -desoxuridines [326,327] and recently also of 2 -deoxyadenosines 132 have been used [328]. Nucleophilic replacement of the bromine by an amino group during deprotection does not occur when reaction conditions of concentrated ammonia solution for 24 h at room temperature are used after oligonucleotide synthesis. 

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