Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates
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 imi...
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Veröffentlicht in: | Biochemistry (Easton) 1986-09, Vol.25 (18), p.5092-5097 |
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creator | MILLER, P. S REDDY, M. P MURAKAMI, A BLAKE, K. R LIN, S.-B AGRIS, C. H |
description | 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 stores in the reaction solution for up to 2 weeks at 4 degrees 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. |
doi_str_mv | 10.1021/bi00366a017 |
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S ; REDDY, M. P ; MURAKAMI, A ; BLAKE, K. R ; LIN, S.-B ; AGRIS, C. H</creator><creatorcontrib>MILLER, P. S ; REDDY, M. P ; MURAKAMI, A ; BLAKE, K. R ; LIN, S.-B ; AGRIS, C. H</creatorcontrib><description>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 stores in the reaction solution for up to 2 weeks at 4 degrees 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.</description><identifier>ISSN: 0006-2960</identifier><identifier>EISSN: 1520-4995</identifier><identifier>DOI: 10.1021/bi00366a017</identifier><identifier>PMID: 3768335</identifier><language>eng</language><publisher>Washington, DC: American Chemical Society</publisher><subject>Base Sequence ; Biological and medical sciences ; Biotechnology ; Carbohydrates. Nucleosides and nucleotides ; Chemistry ; Exact sciences and technology ; Fundamental and applied biological sciences. Psychology ; Genetic engineering ; Genetic technics ; Indicators and Reagents ; Kinetics ; Methods. Procedures. Technologies ; Nucleosides, nucleotides and oligonucleotides ; Oligodeoxyribonucleotides - chemical synthesis ; Organic chemistry ; Organophosphorus Compounds - chemical synthesis ; Preparations and properties ; Structure-Activity Relationship ; Synthetic digonucleotides and genes. 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H</creatorcontrib><title>Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates</title><title>Biochemistry (Easton)</title><addtitle>Biochemistry</addtitle><description>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 stores in the reaction solution for up to 2 weeks at 4 degrees 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.</description><subject>Base Sequence</subject><subject>Biological and medical sciences</subject><subject>Biotechnology</subject><subject>Carbohydrates. Nucleosides and nucleotides</subject><subject>Chemistry</subject><subject>Exact sciences and technology</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Genetic engineering</subject><subject>Genetic technics</subject><subject>Indicators and Reagents</subject><subject>Kinetics</subject><subject>Methods. Procedures. Technologies</subject><subject>Nucleosides, nucleotides and oligonucleotides</subject><subject>Oligodeoxyribonucleotides - chemical synthesis</subject><subject>Organic chemistry</subject><subject>Organophosphorus Compounds - chemical synthesis</subject><subject>Preparations and properties</subject><subject>Structure-Activity Relationship</subject><subject>Synthetic digonucleotides and genes. 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H</creator><general>American Chemical Society</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope></search><sort><creationdate>19860909</creationdate><title>Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates</title><author>MILLER, P. S ; REDDY, M. P ; MURAKAMI, A ; BLAKE, K. R ; LIN, S.-B ; AGRIS, C. H</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-p235t-5289271f45f356e1df14020a00406a70c3a6ef27703434cf91561bcfdbec39dc3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1986</creationdate><topic>Base Sequence</topic><topic>Biological and medical sciences</topic><topic>Biotechnology</topic><topic>Carbohydrates. Nucleosides and nucleotides</topic><topic>Chemistry</topic><topic>Exact sciences and technology</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Genetic engineering</topic><topic>Genetic technics</topic><topic>Indicators and Reagents</topic><topic>Kinetics</topic><topic>Methods. Procedures. Technologies</topic><topic>Nucleosides, nucleotides and oligonucleotides</topic><topic>Oligodeoxyribonucleotides - chemical synthesis</topic><topic>Organic chemistry</topic><topic>Organophosphorus Compounds - chemical synthesis</topic><topic>Preparations and properties</topic><topic>Structure-Activity Relationship</topic><topic>Synthetic digonucleotides and genes. Sequencing</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>MILLER, P. S</creatorcontrib><creatorcontrib>REDDY, M. P</creatorcontrib><creatorcontrib>MURAKAMI, A</creatorcontrib><creatorcontrib>BLAKE, K. R</creatorcontrib><creatorcontrib>LIN, S.-B</creatorcontrib><creatorcontrib>AGRIS, C. H</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>MEDLINE - Academic</collection><jtitle>Biochemistry (Easton)</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>MILLER, P. S</au><au>REDDY, M. P</au><au>MURAKAMI, A</au><au>BLAKE, K. R</au><au>LIN, S.-B</au><au>AGRIS, C. H</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates</atitle><jtitle>Biochemistry (Easton)</jtitle><addtitle>Biochemistry</addtitle><date>1986-09-09</date><risdate>1986</risdate><volume>25</volume><issue>18</issue><spage>5092</spage><epage>5097</epage><pages>5092-5097</pages><issn>0006-2960</issn><eissn>1520-4995</eissn><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 stores in the reaction solution for up to 2 weeks at 4 degrees 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.</abstract><cop>Washington, DC</cop><pub>American Chemical Society</pub><pmid>3768335</pmid><doi>10.1021/bi00366a017</doi><tpages>6</tpages></addata></record> |
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subjects | Base Sequence Biological and medical sciences Biotechnology Carbohydrates. Nucleosides and nucleotides Chemistry Exact sciences and technology Fundamental and applied biological sciences. Psychology Genetic engineering Genetic technics Indicators and Reagents Kinetics Methods. Procedures. Technologies Nucleosides, nucleotides and oligonucleotides Oligodeoxyribonucleotides - chemical synthesis Organic chemistry Organophosphorus Compounds - chemical synthesis Preparations and properties Structure-Activity Relationship Synthetic digonucleotides and genes. Sequencing |
title | Solid-phase syntheses of oligodeoxyribonucleoside methylphosphonates |
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