Phosphoramidite approach

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. ... 

These groups, along with a number of other trialkylsilylethyl derivatives, were examined for protection of phosphorothioates. Only the phenyl-substituted silyl derivative was useful, because simple trialkylsilyl derivatives were prone to acid-catalyzed thiono-thiolo rearrangement. Other trialkylsilylethyl derivatives also suffer from inherent instability upon storage,but the trimethylsilylethyl group has been used successfully in the synthesis of the very sensitive agrocin 84 and for intemucleotide phosphate protection with the phosphoramidite approach. 

Another convenient phosphitylating reagent for use in an oligodeoxyribonucleotide synthesis based on the phosphoramidite approach is bis(tetrazolyl)morpholinophosphine. This is generally prepared in situ from morpholinophosphordichloridite containing di-isopropylethylamine and two equivalents of tetrazole in dichloromethane/pyridine at... 

Approaches to glycosyl phosphates based on P(m)-intermediates 4.1 Phosphoramidite approach in the synthesis of glycosyl phosphates... 

Phosphoramidite approach for the instalment of oc-anomeric phosphate in lipid A synthesis... 

The preparation of twin ribozymes included both chemical and enzymatic procedures. Basically, in our laboratory RNA strands longer than 60 nucleotides are transcribed from synthetic DNA templates using T7 RNA-polymerase. By using 5-benzylmercapto-lH-tetrazole [18a,b] as activator in the phosphoramidite approach, however, we also succeeded in synthesizing a chemical version of HP-... 

Beaucage SL, Iyer RP, Advances in the synthesis of oligonucleotides by the phosphoramidite approach, Tetrahedron, 48 2223-2311, 1992. 

The Synthesis of Modified Oligonucleotides by the Phosphoramidite Approach and Their Applications, Beaucage, S. L. Iyer, R. P Tetrahedron 1993, 49, 6123-6194. 

In 1981, Beaucage and Caruthers4 extended the domain of phosphorus(lll) chemistry with the development of the phosphoramidite approach to oligonucleotide synthesis (see reviews in section 1.9.5.) which, in its later variants, is unsurpassed in its speed and efficiency in both solid and solution phase. An early synthesis of thymidylic acid derivative 1.7 illustrates the phosphoramidite approach [Scheme 7.1]. The sequence begins with the conversion of the 3 -hy-droxyl of nucleoside 1.1 to the phosphoramidite 13 by reaction with chloro-... 

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... 

Limbach PA, Crain PF, McCloskey JA. Summary the modified nucleosides of RNA. Nucleic Acids Res. 1994 22 2183-2196. Rozenski J, Crain PF, McCloskey JA. The RNA modification database 1999 update. Nucleic Acids Res. 1999 27 196-197. Beaucage SL, Iyer RP. The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications. Tetrahedron 1993 49 6123-6194. 

Bis(hydroxymethyl)phosphonic acid esters that incorporated thymine were employed as a backbone to prepare short oligonucleotide chains. This chain was prepared by condensation of the bis(4,4 -dimethoxytrityl) protected phosphonic acid and iV or N -(2-hydroxyethyl)thymine in the presence of l-(2-mesitylenesul-fonyl)-3-nitro-l,2,4-triazole or by an Appel reaction with or N -(2-aminoethyl)thymine (89a-h). Selective removal of one DMT-group and phos-phitylation yielded the building blocks for solid supported synthesis of the short oligomers by the phosphoramidite approach. Holy has reported the synthesis of 8-amino and 8-substituted amino derivatives of acyclic purine nucleotide analogues. The 8-amino, 8-methylamino- and 8-dimethylamino-adenine and -guanine analogues of iV-(2-phosphonomethoxyethyl) and (S)-iV-(3-hydroxy-2-phosphono-methoxy-propyl) derivatives of purines (90a-i), were prepared by... 

Oligonucleotides containing the isonucleoside (59), that has an extended phosphodiester linkage were prepared by the phosphoramidite approach. Such homo-oligonucleotides adopt an A-form conformation as demonstrated by... 

This group was developed for 5 -hydroxyl protection in oligonucleotide synthesis. It is stable to the conditions for nucleotide coupling using the phosphoramidite approach. It is not stable to acid or to 12/pyridine/THF, conditions used for phosphite oxidation. It has been used to prepare a 20-mer. ... 

Prepared for use in the phosphoramidite approach, the amidite reagent, (CF3)2CHCH20P(N/Pr)2 is stable to distillation unlike the cyanoethyl version which tends to decompose. It is cleaved rapidly with ammonia from the internucleotidic bonds. ... 

The 5 -phosphoramidite of the novel bicyclic nucleoside (14), restricted to an S-type conformation, has been synthesised in eight steps via double inversion of configuration at the C4 position of protected l-(3 -deoxy-p-D-psicofuranosyl)uracyl and introduction of an azide moiety. The secondary amino group at C4 permitted the subsequent incorporation of (14) into oligonucleotides via a 5 -3 directed variation of the standard phosphoramidite approach for synthesis of oligonucleotides. Thermal denaturation studies showed rather large decreases in duplex stabilities toward complementary DNA and RNA. ... 

Phosphorus protection. The protecting group is stable to acid and is removed by fragmentation using aqueous NH4OH. It is introduced by the phosphoramidite approach and is useful for internucleotidic bonds in oligodeoxynucleotide synthesis. 

Figure 1 Oligonucleotide synthesis on solid support via the phosphoramidite approach.

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... 

Kim S-G, Eida K, Takaku H. Use of the hexafluoro-2-butyl protecting group in the synthesis of DNA fragments via the phosphoramidite approach on solid-support. Bioorg Med Chem Lett 5 1663-1666, 1995. 

Ravikumar VT, Cheruvallath ZS, Cole DL. 4-Cyano-2-butenyl group A new type of protecting group in oligonucleotide synthesis via phosphoramidite approach. Tetrahedron Lett 37 6643-6646, 1996. 

Iwai S, Ohtsuka E. 5 -Levulinyl and 2 -tetrahydrofuranyl protection for the synthesis of oligoribonucleotides by the phosphoramidite approach. Nucleic Acids Res 16 9443-9456, 1988. 

Sakatsume O, Yamaguchi T, Ishikawa M, Hirao I, Miura K, Takaku H. Solid-phase synthesis of oligoribonucleotides by the phosphoramidite approach using 2 -0-l-(2-chloroethoxy)ethyl protection. Tetrahedron 47 8717-8728, 1991. 

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