Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates

Paul S. Miller, M. Parameswara Reddy, Akira Murakami, Kathleen Blake, Shwu Bin Lin, Cheryl H. Agris

Research output: Contribution to journalArticle

Abstract

Oligodeoxyribonucleoside methylphosphonates of defined sequence of the type d-Np(Np)nN, where n is 6-13, are readily prepared on insoluble polystyrene supports by use of protected 5′-(dimethoxytrityl)deoxyribonucleoside 3′-(methylphosphonic imidazolides) as synthetic intermediates. The imidazolides are prepared in situ by reaction of protected 5′-(dimethoxytrityl)deoxyribonucleoside with methylphosphonic bis(imidazolide) and can be stored in the reaction solution for up to 2 weeks at 4 °C with no loss in activity. The condensation reaction is accelerated by the presence of tetrazole, which appears to act as an acid catalyst. The half-life for dimer formation on the polystyrene support is 5 min, and the reaction is 95% complete after 60 min. Although similar kinetics are observed when controlled pore glass is used as the support, the extent of the reaction does not go beyond 78%, even after prolonged incubation. In order to simplify purification and sequence analysis of the oligomer, the 5′-terminal nucleoside unit is linked via a phosphodiester bond. This linkage may be introduced by either an o-chlorophenyl phosphotriester method or a cyanoethyl phosphoramidite method. The latter procedure simplifies the deprotection step, since the cyanoethyl group is readily cleaved by ethylenediamine, which also removes the base protecting groups and cleaves the oligomer from the support. The singly charged oligomers are easily purified by affinity chromatography on DEAE-cellulose. The chain lengths of the oligomers were confirmed after 5'-end labeling with polynucleotide kinase by partial hydrolysis of the methylphosphonate linkages with 1 M aqueous piperidine followed by polyacrylamide gel electrophoresis of the hydrolysate. The positions of purine and pyrimidine bases were confirmed by treatment of 5′-end labeled oligomers with acid and hydrazine, respectively. These experiments show that Oligodeoxyribonucleoside methylphosphonates can be prepared and characterized by procedures analogous to those used to prepare oligodeoxyribonucleotides.

Original languageEnglish (US)
Pages (from-to)5092-5097
Number of pages6
JournalBiochemistry®
Volume25
Issue number18
StatePublished - 1986

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Solid-Phase Synthesis Techniques
Oligomers
Deoxyribonucleosides
ethylenediamine
hydrazine
Polystyrenes
Polynucleotide 5'-Hydroxyl-Kinase
DEAE-Cellulose
Acids
Oligodeoxyribonucleotides
Affinity chromatography
Affinity Chromatography
Nucleosides
Condensation reactions
Glass
Sequence Analysis
Half-Life
Polyacrylamide Gel Electrophoresis
Electrophoresis
Hydrolysis

ASJC Scopus subject areas

  • Biochemistry

Cite this

Miller, P. S., Parameswara Reddy, M., Murakami, A., Blake, K., Lin, S. B., & Agris, C. H. (1986). Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates. Biochemistry®, 25(18), 5092-5097.

Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates. / Miller, Paul S.; Parameswara Reddy, M.; Murakami, Akira; Blake, Kathleen; Lin, Shwu Bin; Agris, Cheryl H.

In: Biochemistry®, Vol. 25, No. 18, 1986, p. 5092-5097.

Research output: Contribution to journalArticle

Miller, PS, Parameswara Reddy, M, Murakami, A, Blake, K, Lin, SB & Agris, CH 1986, 'Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates', Biochemistry®, vol. 25, no. 18, pp. 5092-5097.
Miller PS, Parameswara Reddy M, Murakami A, Blake K, Lin SB, Agris CH. Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates. Biochemistry®. 1986;25(18):5092-5097.
Miller, Paul S. ; Parameswara Reddy, M. ; Murakami, Akira ; Blake, Kathleen ; Lin, Shwu Bin ; Agris, Cheryl H. / Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates. In: Biochemistry®. 1986 ; Vol. 25, No. 18. pp. 5092-5097.
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abstract = "Oligodeoxyribonucleoside methylphosphonates of defined sequence of the type d-Np(Np)nN, where n is 6-13, are readily prepared on insoluble polystyrene supports by use of protected 5′-(dimethoxytrityl)deoxyribonucleoside 3′-(methylphosphonic imidazolides) as synthetic intermediates. The imidazolides are prepared in situ by reaction of protected 5′-(dimethoxytrityl)deoxyribonucleoside with methylphosphonic bis(imidazolide) and can be stored in the reaction solution for up to 2 weeks at 4 °C with no loss in activity. The condensation reaction is accelerated by the presence of tetrazole, which appears to act as an acid catalyst. The half-life for dimer formation on the polystyrene support is 5 min, and the reaction is 95{\%} complete after 60 min. Although similar kinetics are observed when controlled pore glass is used as the support, the extent of the reaction does not go beyond 78{\%}, even after prolonged incubation. In order to simplify purification and sequence analysis of the oligomer, the 5′-terminal nucleoside unit is linked via a phosphodiester bond. This linkage may be introduced by either an o-chlorophenyl phosphotriester method or a cyanoethyl phosphoramidite method. The latter procedure simplifies the deprotection step, since the cyanoethyl group is readily cleaved by ethylenediamine, which also removes the base protecting groups and cleaves the oligomer from the support. The singly charged oligomers are easily purified by affinity chromatography on DEAE-cellulose. The chain lengths of the oligomers were confirmed after 5'-end labeling with polynucleotide kinase by partial hydrolysis of the methylphosphonate linkages with 1 M aqueous piperidine followed by polyacrylamide gel electrophoresis of the hydrolysate. The positions of purine and pyrimidine bases were confirmed by treatment of 5′-end labeled oligomers with acid and hydrazine, respectively. These experiments show that Oligodeoxyribonucleoside methylphosphonates can be prepared and characterized by procedures analogous to those used to prepare oligodeoxyribonucleotides.",
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