Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents
Identifieur interne : 002196 ( Istex/Corpus ); précédent : 002195; suivant : 002197Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents
Auteurs : Richard T. Pon ; Shuyuan Yu ; Yogesh S. SanghviSource :
- Bioconjugate Chemistry [ 1043-1802 ] ; 1999.
Abstract
Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT), N-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior to other additives and high nucleoside loadings (up to 60 μmol/g) and rapid coupling reactions (≤10 min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3‘-succinyl or 3‘-hydroquinone-O,O‘-diacetic acid (HQDA or Q-Linker) carboxyl groups through a primary ester linkage. Alternatively, supports derivatized with succinic acid or the Q-Linker were attached directly to the 3‘-OH group of nucleosides through a secondary ester linkage. Uronium reagents (HATU or HBTU) gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP) reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.
Url:
DOI: 10.1021/bc990063a
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents</title><author><name sortKey="Pon, Richard T" sort="Pon, Richard T" uniqKey="Pon R" first="Richard T." last="Pon">Richard T. Pon</name><affiliation><mods:affiliation>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Calgary.</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. Phone: (403)220-4277. Fax: (403) 283-4907. E-mail: rtpon@ucalgary.ca.</mods:affiliation></affiliation></author><author><name sortKey="Yu, Shuyuan" sort="Yu, Shuyuan" uniqKey="Yu S" first="Shuyuan" last="Yu">Shuyuan Yu</name><affiliation><mods:affiliation>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Calgary.</mods:affiliation></affiliation></author><author><name sortKey="Sanghvi, Yogesh S" sort="Sanghvi, Yogesh S" uniqKey="Sanghvi Y" first="Yogesh S." last="Sanghvi">Yogesh S. Sanghvi</name><affiliation><mods:affiliation>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</mods:affiliation></affiliation><affiliation><mods:affiliation> Isis Pharmaceuticals.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:AF27C373BD6F54EEFC99A285270A4577271529AD</idno><date when="1999" year="1999">1999</date><idno type="doi">10.1021/bc990063a</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-64GCR0WK-7/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002196</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002196</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">Rapid Esterification of Nucleosides to Solid-Phase Supports for
Oligonucleotide Synthesis Using Uronium and Phosphonium
Coupling Reagents</title><author><name sortKey="Pon, Richard T" sort="Pon, Richard T" uniqKey="Pon R" first="Richard T." last="Pon">Richard T. Pon</name><affiliation><mods:affiliation>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Calgary.</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. Phone: (403)220-4277. Fax: (403) 283-4907. E-mail: rtpon@ucalgary.ca.</mods:affiliation></affiliation></author><author><name sortKey="Yu, Shuyuan" sort="Yu, Shuyuan" uniqKey="Yu S" first="Shuyuan" last="Yu">Shuyuan Yu</name><affiliation><mods:affiliation>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Calgary.</mods:affiliation></affiliation></author><author><name sortKey="Sanghvi, Yogesh S" sort="Sanghvi, Yogesh S" uniqKey="Sanghvi Y" first="Yogesh S." last="Sanghvi">Yogesh S. Sanghvi</name><affiliation><mods:affiliation>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</mods:affiliation></affiliation><affiliation><mods:affiliation> Isis Pharmaceuticals.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Bioconjugate Chemistry</title><title level="j" type="abbrev">Bioconjugate Chem.</title><idno type="ISSN">1043-1802</idno><idno type="eISSN">1520-4812</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">1999</date><date type="published">1999</date><biblScope unit="vol">10</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="1051">1051</biblScope><biblScope unit="page" to="1057">1057</biblScope></imprint><idno type="ISSN">1043-1802</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">1043-1802</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT), N-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior to other additives and high nucleoside loadings (up to 60 μmol/g) and rapid coupling reactions (≤10 min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3‘-succinyl or 3‘-hydroquinone-O,O‘-diacetic acid (HQDA or Q-Linker) carboxyl groups through a primary ester linkage. Alternatively, supports derivatized with succinic acid or the Q-Linker were attached directly to the 3‘-OH group of nucleosides through a secondary ester linkage. Uronium reagents (HATU or HBTU) gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP) reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>nucleoside</json:string><json:string>ester</json:string><json:string>dmap</json:string><json:string>reagent</json:string><json:string>hbtu</json:string><json:string>oligonucleotide</json:string><json:string>chem</json:string><json:string>oligonucleotide synthesis</json:string><json:string>tetrahedron</json:string><json:string>esterification</json:string><json:string>linker</json:string><json:string>uronium</json:string><json:string>hobt</json:string><json:string>tetrahedron lett</json:string><json:string>lett</json:string><json:string>hatu</json:string><json:string>mmol</json:string><json:string>phosphonium</json:string><json:string>pybrop</json:string><json:string>carboxyl</json:string><json:string>hexafluorophosphate</json:string><json:string>hydroxyl</json:string><json:string>nucleic acid</json:string><json:string>brop</json:string><json:string>derivatized</json:string><json:string>synthesizer</json:string><json:string>oligonucleotides</json:string><json:string>bioconjugate</json:string><json:string>acetonitrile</json:string><json:string>bioconjugate chem</json:string><json:string>peptide</json:string><json:string>succinylated</json:string><json:string>pybop</json:string><json:string>trityl</json:string><json:string>equiv</json:string><json:string>primary ester linkage</json:string><json:string>nucleoside loading</json:string><json:string>nucleic</json:string><json:string>secondary ester linkage</json:string><json:string>ester linkage</json:string><json:string>nucleoside nucleotide</json:string><json:string>amino</json:string><json:string>trityl analysis</json:string><json:string>amino acid</json:string><json:string>rapid esterification</json:string><json:string>amide</json:string><json:string>linkage</json:string><json:string>bromophosphonium reagent</json:string><json:string>free group</json:string><json:string>carboxyl derivatized</json:string><json:string>room temperature</json:string><json:string>hbtu mmol</json:string><json:string>phosphonium salt</json:string><json:string>succinylated support</json:string><json:string>best result</json:string><json:string>final product</json:string></teeft></keywords><author><json:item><name>PON Richard T.</name><affiliations><json:string>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</json:string><json:string>University of Calgary.</json:string><json:string>To whom correspondence should be addressed. Phone: (403)220-4277. Fax: (403) 283-4907. E-mail: rtpon@ucalgary.ca.</json:string></affiliations></json:item><json:item><name>YU Shuyuan</name><affiliations><json:string>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</json:string><json:string>University of Calgary.</json:string></affiliations></json:item><json:item><name>SANGHVI Yogesh S.</name><affiliations><json:string>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</json:string><json:string>Isis Pharmaceuticals.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-64GCR0WK-7</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT), N-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior to other additives and high nucleoside loadings (up to 60 μmol/g) and rapid coupling reactions (≤10 min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3‘-succinyl or 3‘-hydroquinone-O,O‘-diacetic acid (HQDA or Q-Linker) carboxyl groups through a primary ester linkage. Alternatively, supports derivatized with succinic acid or the Q-Linker were attached directly to the 3‘-OH group of nucleosides through a secondary ester linkage. Uronium reagents (HATU or HBTU) gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP) reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.</abstract><qualityIndicators><refBibsNative>true</refBibsNative><abstractWordCount>161</abstractWordCount><abstractCharCount>1180</abstractCharCount><keywordCount>0</keywordCount><score>8.795</score><pdfWordCount>4863</pdfWordCount><pdfCharCount>33096</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>7</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>695</pdfWordsPerPage><pdfText>true</pdfText></qualityIndicators><title>Rapid Esterification of Nucleosides to Solid-Phase Supports for Oligonucleotide Synthesis Using Uronium and Phosphonium Coupling Reagents</title><genre><json:string>research-article</json:string></genre><host><title>Bioconjugate Chemistry</title><language><json:string>unknown</json:string></language><issn><json:string>1043-1802</json:string></issn><eissn><json:string>1520-4812</json:string></eissn><volume>10</volume><issue>6</issue><pages><first>1051</first><last>1057</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-64GCR0WK-7</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - chemistry, organic</json:string><json:string>2 - chemistry, multidisciplinary</json:string><json:string>2 - biochemistry & molecular biology</json:string><json:string>2 - biochemical research methods</json:string></wos><scienceMetrix><json:string>1 - natural sciences</json:string><json:string>2 - chemistry</json:string><json:string>3 - organic chemistry</json:string></scienceMetrix><scopus><json:string>1 - Physical Sciences</json:string><json:string>2 - Chemistry</json:string><json:string>3 - Organic Chemistry</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Pharmacology, Toxicology and Pharmaceutics</json:string><json:string>3 - Pharmaceutical Science</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Pharmacology, Toxicology and Pharmaceutics</json:string><json:string>3 - Pharmacology</json:string><json:string>1 - Physical Sciences</json:string><json:string>2 - Engineering</json:string><json:string>3 - Biomedical Engineering</json:string><json:string>1 - Physical Sciences</json:string><json:string>2 - Chemical Engineering</json:string><json:string>3 - Bioengineering</json:string><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biotechnology</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string><json:string>4 - biotechnologie</json:string></inist></categories><publicationDate>1999</publicationDate><copyrightDate>1999</copyrightDate><doi><json:string>10.1021/bc990063a</json:string></doi><id>AF27C373BD6F54EEFC99A285270A4577271529AD</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-64GCR0WK-7/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-64GCR0WK-7/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-64GCR0WK-7/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-64GCR0WK-7/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">Rapid Esterification of Nucleosides to Solid-Phase Supports for
Oligonucleotide Synthesis Using Uronium and Phosphonium
Coupling Reagents</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 1999 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">1999</date><date type="Copyright" when="1999">1999</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">Rapid Esterification of Nucleosides to Solid-Phase Supports for
Oligonucleotide Synthesis Using Uronium and Phosphonium
Coupling Reagents</title><author xml:id="author-0000" role="corresp"><persName><surname>Pon</surname><forename type="first">Richard T.</forename></persName><note place="foot" n="bc990063aAF2"><ref>†</ref><p>
University of Calgary.</p></note><affiliation role="corresp"> To whom correspondence should be addressed. Phone: (403) 220-4277. Fax: (403) 283-4907. E-mail: rtpon@ucalgary.ca.</affiliation></author><author xml:id="author-0001"><persName><surname>Yu</surname><forename type="first">Shuyuan</forename></persName><affiliation><orgName type="laboratory">Department of Molecular Biology and Biochemistry</orgName><orgName type="institution">University of Calgary</orgName><orgName type="institution">3350 Hospital Drive N.W.</orgName><address><addrLine>Calgary</addrLine><addrLine>Alberta</addrLine><addrLine>Canada T2N 4N1</addrLine><addrLine>and Isis Pharmaceuticals</addrLine><addrLine>Development Chemistry Department</addrLine><addrLine>2280 Faraday Avenue</addrLine><addrLine>Carlsbad</addrLine><addrLine>California 92008</addrLine></address></affiliation><note place="foot" n="bc990063aAF2"><ref>†</ref><p>
University of Calgary.</p></note></author><author xml:id="author-0002"><persName><surname>Sanghvi</surname><forename type="first">Yogesh S.</forename></persName><affiliation><orgName type="laboratory">Department of Molecular Biology and Biochemistry</orgName><orgName type="institution">University of Calgary</orgName><orgName type="institution">3350 Hospital Drive N.W.</orgName><address><addrLine>Calgary</addrLine><addrLine>Alberta</addrLine><addrLine>Canada T2N 4N1</addrLine><addrLine>and Isis Pharmaceuticals</addrLine><addrLine>Development Chemistry Department</addrLine><addrLine>2280 Faraday Avenue</addrLine><addrLine>Carlsbad</addrLine><addrLine>California 92008</addrLine></address></affiliation><note place="foot" n="bc990063aAF3"><ref>‡</ref><p>
Isis Pharmaceuticals.</p></note></author><idno type="istex">AF27C373BD6F54EEFC99A285270A4577271529AD</idno><idno type="ark">ark:/67375/TPS-64GCR0WK-7</idno><idno type="DOI">10.1021/bc990063a</idno></analytic><monogr><title level="j" type="main">Bioconjugate Chemistry</title><title level="j" type="abbrev">Bioconjugate Chem.</title><idno type="acspubs">bc</idno><idno type="coden">bcches</idno><idno type="pISSN">1043-1802</idno><idno type="eISSN">1520-4812</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">1999</date><date type="published">1999</date><biblScope unit="vol">10</biblScope><biblScope unit="issue">6</biblScope><biblScope unit="page" from="1051">1051</biblScope><biblScope unit="page" to="1057">1057</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.41" when="2020-04-06"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract><p>Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents
and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT),
<hi rend="italic">N</hi>-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior
to other additives and high nucleoside loadings (up to 60 μmol/g) and rapid coupling reactions (≤10
min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3‘-succinyl or 3‘-hydroquinone-<hi rend="italic">O,O‘</hi>-diacetic acid (HQDA or <hi rend="italic">Q-Linker</hi>) carboxyl groups through a <hi rend="italic">primary </hi>ester linkage.
Alternatively, supports derivatized with succinic acid or the <hi rend="italic">Q-Linker</hi> were attached directly to the
3‘-OH group of nucleosides through a <hi rend="italic">secondary </hi>ester linkage. Uronium reagents (HATU or HBTU)
gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP)
reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster
than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make
this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2020-04-06" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bc</journal-id><journal-id journal-id-type="coden">bcches</journal-id><journal-title-group><journal-title>Bioconjugate Chemistry</journal-title><abbrev-journal-title>Bioconjugate Chem.</abbrev-journal-title></journal-title-group><issn pub-type="ppub">1043-1802</issn><issn pub-type="epub">1520-4812</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/bc</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bc990063a</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>Rapid Esterification of Nucleosides to Solid-Phase Supports for
Oligonucleotide Synthesis Using Uronium and Phosphonium
Coupling Reagents</article-title></title-group><contrib-group><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Pon</surname><given-names>Richard T.</given-names></name><xref rid="bc990063aAF1">*</xref><xref rid="bc990063aAF2"><sup>†</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Yu</surname><given-names>Shuyuan</given-names></name><xref rid="bc990063aAF2"><sup>†</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Sanghvi</surname><given-names>Yogesh S.</given-names></name><xref rid="bc990063aAF3"><sup>‡</sup></xref></contrib><aff>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,
Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,
2280 Faraday Avenue, Carlsbad, California 92008</aff></contrib-group><author-notes><corresp id="bc990063aAF1">
To whom correspondence should be addressed. Phone: (403)
220-4277. Fax: (403) 283-4907. E-mail: rtpon@ucalgary.ca.</corresp><fn id="bc990063aAF2"><label>†</label><p>
University of Calgary.</p></fn><fn id="bc990063aAF3"><label>‡</label><p>
Isis Pharmaceuticals.</p></fn></author-notes><pub-date pub-type="epub"><day>10</day><month>09</month><year>1999</year></pub-date><pub-date pub-type="ppub"><day>15</day><month>11</month><year>1999</year></pub-date><volume>10</volume><issue>6</issue><fpage>1051</fpage><lpage>1057</lpage><history><date date-type="received"><day>24</day><month>05</month><year>1999</year></date><date date-type="rev-recd"><day>04</day><month>08</month><year>1999</year></date><date date-type="asap"><day>10</day><month>09</month><year>1999</year></date><date date-type="issue-pub"><day>15</day><month>11</month><year>1999</year></date></history><permissions><copyright-statement>Copyright © 1999 American Chemical Society</copyright-statement><copyright-year>1999</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents
and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT),
<italic toggle="yes">N</italic>-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior
to other additives and high nucleoside loadings (up to 60 μmol/g) and rapid coupling reactions (≤10
min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3‘-succinyl or 3‘-hydroquinone-<italic toggle="yes">O,O‘</italic>-diacetic acid (HQDA or <italic toggle="yes">Q-Linker</italic>) carboxyl groups through a <italic toggle="yes">primary </italic>ester linkage.
Alternatively, supports derivatized with succinic acid or the <italic toggle="yes">Q-Linker</italic> were attached directly to the
3‘-OH group of nucleosides through a <italic toggle="yes">secondary </italic>ester linkage. Uronium reagents (HATU or HBTU)
gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP)
reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster
than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make
this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bc990063a</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="d7e151"><title>Introduction</title><p>We have introduced a new dicarboxylic acid linker arm
(<italic toggle="yes"><xref rid="bc990063ab00001" ref-type="bibr"></xref></italic>), hydroquinone-<italic toggle="yes">O,O‘</italic>-diacetic acid (HQDA<xref rid="bc990063ab00001" ref-type="bibr"></xref> or <italic toggle="yes">Q-Linker</italic>), which allows oligodeoxynucleotides to be cleaved from
the support approximately 30 times faster than a traditional succinic acid linker arm (i.e., 2 min vs 60 min).
We have also developed very fast (≤60 s) and efficient
(only 0.05 mmol of nucleoside required/g of support)
coupling procedures for attaching the first nucleoside to
amino derivatized solid-phase supports (<italic toggle="yes"><xref rid="bc990063ab00002" ref-type="bibr"></xref></italic>). Recently, we
have used these improvements to recycle and reuse
hydroxyl derivatized solid-phase supports multiple times,
without having to remove the support from the synthesis
column (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00003" ref-type="bibr"></xref>, <xref rid="bc990063ab00004" ref-type="bibr"></xref></named-content></italic>).
</p><p>The support recycling required <italic toggle="yes">ester</italic> attachments,
which were more easily cleaved than the usual<italic toggle="yes"> amide</italic>
linkages. Coupling through ester linkages is also important when cellulose (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00005" ref-type="bibr"></xref>, <xref rid="bc990063ab00006" ref-type="bibr"></xref></named-content></italic>), soluble poly(ethylene glycol)
(<italic toggle="yes"><xref rid="bc990063ab00007" ref-type="bibr"></xref></italic>), poly(ethylene glycol)-polystyrene (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00008" ref-type="bibr"></xref>, <xref rid="bc990063ab00009" ref-type="bibr"></xref></named-content></italic>), oxime (<italic toggle="yes"><xref rid="bc990063ab00010" ref-type="bibr"></xref></italic>),
photolabile (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00011" ref-type="bibr"></xref>−<xref rid="bc990063ab00012" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00013" ref-type="bibr"></xref></named-content></italic>), or other hydroxyl (<italic toggle="yes"><xref rid="bc990063ab00014" ref-type="bibr"></xref></italic>) supports are
used. There has also been interest in ester linkages for
the synthesis of alcohol peptides (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00015" ref-type="bibr"></xref>−<xref rid="bc990063ab00016" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00017" ref-type="bibr"></xref></named-content></italic>), depsipeptides
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00018" ref-type="bibr"></xref>, <xref rid="bc990063ab00019" ref-type="bibr"></xref></named-content></italic>), oligonucleotide−peptides (<italic toggle="yes"><xref rid="bc990063ab00020" ref-type="bibr"></xref></italic>), and combinatorial
libraries (<italic toggle="yes"><xref rid="bc990063ab00021" ref-type="bibr"></xref></italic>). Unfortunately, the reduced nucleophilicity
of hydroxyl groups relative to amino groups makes
coupling through ester linkages more difficult, and so the
coupling reactions are longer and often produce lower
support loadings. This problem becomes even more
pronounced when coupling must be made through a
secondary alcohol (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00022" ref-type="bibr"></xref>, <xref rid="bc990063ab00023" ref-type="bibr"></xref></named-content></italic>) such as the 3‘-OH group in
nucleosides. Therefore, we wanted to find coupling conditions which would form the required ester linkages faster
and more efficiently than the carbodiimide coupling
reagents previously used (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00022" ref-type="bibr"></xref>−<xref rid="bc990063ab00023" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00024" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00025" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00026" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00027" ref-type="bibr"></xref></named-content></italic>).
</p><p>We investigated a number of commonly used uronium
and phosphonium coupling reagents. These reagents
have become very popular for peptide (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00028" ref-type="bibr"></xref>, <xref rid="bc990063ab00029" ref-type="bibr"></xref></named-content></italic>) and PNA
synthesis (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00030" ref-type="bibr"></xref>−<xref rid="bc990063ab00031" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00032" ref-type="bibr"></xref></named-content></italic>) because they are stable, nonhygroscopic, and easy to handle. Similar amide linkage formation in oligonucleotide synthesis has also been performed
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00033" ref-type="bibr"></xref>−<xref rid="bc990063ab00034" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00035" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00036" ref-type="bibr"></xref></named-content></italic>). Recently, PyBOP<sup>1</sup> and photolabile protecting
groups have allowed functionalization of oligonucleotides
while still immobilized on the support (<italic toggle="yes"><xref rid="bc990063ab00037" ref-type="bibr"></xref></italic>). Various
phosphonium salt reagents have also been used to
activate phosphate (<italic toggle="yes"><xref rid="bc990063ab00038" ref-type="bibr"></xref></italic>), phosphonate (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00039" ref-type="bibr"></xref>, <xref rid="bc990063ab00040" ref-type="bibr"></xref></named-content></italic>) and phosphorothioate/dithioate compounds (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00041" ref-type="bibr"></xref>, <xref rid="bc990063ab00042" ref-type="bibr"></xref></named-content></italic>). However,
ester bond formation using these reagents has not been
as common. BOP and PyBOP have been used to prepare
primary, secondary, <italic toggle="yes">tert</italic>-butyl, or cyclic esters of amino
acids (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00043" ref-type="bibr"></xref>−<xref rid="bc990063ab00044" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00045" ref-type="bibr"></xref></named-content></italic>), and 2,2,2-trihaloethyl esters have been
converted into other esters by phosphonium salts generated in situ (<italic toggle="yes"><xref rid="bc990063ab00046" ref-type="bibr"></xref></italic>). Occasionally, HATU (<italic toggle="yes"><xref rid="bc990063ab00047" ref-type="bibr"></xref></italic>), HBTU (<italic toggle="yes"><xref rid="bc990063ab00048" ref-type="bibr"></xref></italic>),
and TBTU (<italic toggle="yes"><xref rid="bc990063ab00049" ref-type="bibr"></xref></italic>) have also been used to attach amino acids
to hydroxyl resins for peptide or PNA synthesis.
</p><p>In this report, we describe conditions for efficient and
rapid coupling of nucleosides, via succinyl or hydroquinone-<italic toggle="yes">O,O‘</italic>-diacetyl ester linkages, to solid-phase supports using a number of commonly available uronium and
phosphonium coupling reagents in acetonitrile solution.
Conditions for coupling through both primary (Scheme
<xref rid="bc990063ah00001"></xref>) and secondary ester linkages (Scheme <xref rid="bc990063ah00002"></xref>) have been
developed using 1 equiv of DMAP as a coupling additive.
These esterification reactions are much faster (1−60 min)
than previous ester coupling reactions (<italic toggle="yes">22, 23, 43−49</italic>),
yield excellent nucleoside loadings (up to ∼60 μmol/g),
and can be readily implemented on automated DNA
synthesizers.
<fig id="bc990063ah00001" position="float" fig-type="scheme" orientation="portrait"><label>1</label><graphic xlink:href="bc990063ah00001.gif" position="float" orientation="portrait"></graphic></fig><fig id="bc990063ah00002" position="float" fig-type="scheme" orientation="portrait"><label>2</label><graphic xlink:href="bc990063ah00002.gif" position="float" orientation="portrait"></graphic></fig></p></sec><sec id="d7e324"><title>Materials and Methods</title><p><bold>Materials and General Methods.</bold> LCAA-CPG (120−200 mesh, 500 Å, 90−120 μmol/g NH<sub>2</sub> groups) was
obtained from CPG Inc. (Lincoln Park, NJ). Hydroxyl-CPG (<italic toggle="yes"><xref rid="bc990063ab00004" ref-type="bibr"></xref></italic>) and succinyl-CPG (<italic toggle="yes"><xref rid="bc990063ab00022" ref-type="bibr"></xref></italic>) were prepared as previously described. HBTU and HOBT were from Quantum
Biotechnologies (Montreal, PQ, Canada), HATU and
HOAT were from Perseptive Biosystems (Framingham,
MA), BOP and HBPyU were from Sigma Chemical (St.
Louis, MO), PyBOP, PyBrOP, TNTU, and TPTU were
from Chem-Impex Intl. (Wood Dale, IL), BrOP and
HBPipU were from Fluka Chemie (Buchs, Switzerland),
and TOTU was from Aldrich Chemicals (Milwaukee, WI).
</p><p><bold>Caution.</bold> Precautions must be taken to avoid skin
exposure to any coupling reagent since allergic sensitivity
may develop in users.
</p><p>Nucleoside-3‘-<italic toggle="yes">O</italic>-succinates were purchased from Sigma
Chemical and nucleoside-3‘-<italic toggle="yes">O</italic>-hydroquinone-<italic toggle="yes">O,O‘</italic>-diacetic
acid hemiesters were prepared as previously described
(<italic toggle="yes"><xref rid="bc990063ab00001" ref-type="bibr"></xref></italic>) and used as a mixture of both hemi- and diesters.
DIEA was distilled and stored over molecular sieves and
acetonitrile was freshly distilled from CaH<sub>2</sub>. Quantitative
analysis of dimethoxytrityl colors was performed using
5% dichloroacetic acid/1,2-dichloroethane (±5% accuracy).
Synthesis, cleavage, and deprotection of trial oligonucleotides (data not shown) proceeded without modification.
</p><p><bold>Primary Esterification.</bold> OH-CPG (0.5 g), nucleoside
<bold>2</bold> or <bold>3</bold> (0.1 mmol), HBTU (0.1 mmol), and coupling
additive (DMAP, HOBT, HOAT, or NMI, 0.1 mmol) were
combined in a septum-sealed 10 mL vial. DIEA (3 mmol,
520 μL) and acetonitrile (2.48 mL) were added, via
syringe, and the reaction shaken at room temperature.
Aliquots (∼10−20 mg) were removed at various intervals,
washed with CH<sub>2</sub>Cl<sub>2,</sub> MeOH, and CH<sub>2</sub>Cl<sub>2</sub>, dried, and the
progress of the reaction was determined by trityl analysis
(<italic toggle="yes"><xref rid="bc990063ab00027" ref-type="bibr"></xref></italic>). The CPG was filtered off and washed with CH<sub>2</sub>Cl<sub>2</sub>,
MeOH, and CH<sub>2</sub>Cl<sub>2</sub>. The results for nucleosides <bold>2a</bold><bold>−</bold><bold>d</bold>,
using HBTU/HOBT and coupling times of either 10, 30,
or 60 min were, for <bold>2a</bold>, 7, 14, and 20; for <bold>2b</bold>, 6, 12, and
16; for <bold>2c</bold>, 10, 20, and 22; and for <bold>2d</bold>, 13, 28, and 40 μmol/g. Other coupling results are shown in Figures <xref rid="bc990063af00001"></xref> and <xref rid="bc990063af00002"></xref>.
</p><p>Automated primary esterification reactions (see Table
<xref rid="bc990063at00001"></xref>) were performed on a PE Biosystems 394 DNA synthesizer as previously described for primary amide
coupling reactions (<italic toggle="yes"><xref rid="bc990063ab00002" ref-type="bibr"></xref></italic>). However, OH-CPG was used
instead of LCAA-CPG, equal nucleoside and coupling
reagents concentrations (0.05−2 M as described in the
discussion), a 5 min capping step was included to acetylate unreacted hydroxyl groups on the support, and in
the case of nucleoside <bold>2d</bold>, 1:1 acetonitrile/CH<sub>2</sub>Cl<sub>2</sub> was
required for solubility.
<table-wrap id="bc990063at00001" position="float" orientation="portrait"><label>1</label><caption><p>Automated Coupling of Nucleoside-3‘-<italic toggle="yes">O</italic>-succinates to OH-CPG through Primary
Ester Linkages Using Different Coupling Reagents and
DMAP.</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="3"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">reagent</oasis:entry><oasis:entry namest="2" nameend="2">nucleoside<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry><oasis:entry namest="3" nameend="3">nucleoside loading (μmol/g)<italic toggle="yes"><sup>c</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">BOP
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">25
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">BrOP
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">57
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HATU
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">30
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HATU
</oasis:entry><oasis:entry colname="2">2b
</oasis:entry><oasis:entry colname="3">31
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HATU
</oasis:entry><oasis:entry colname="2">2c
</oasis:entry><oasis:entry colname="3">33
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HATU
</oasis:entry><oasis:entry colname="2">2d<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="3">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HBTU
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">23
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HBTU
</oasis:entry><oasis:entry colname="2">2b
</oasis:entry><oasis:entry colname="3">18
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HBTU
</oasis:entry><oasis:entry colname="2">2c
</oasis:entry><oasis:entry colname="3">19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HBTU
</oasis:entry><oasis:entry colname="2">2d<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="3">11
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HBPipU
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">21
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HBPyU
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">20
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PyBOP
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">20
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PyBrOP
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">47
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">TNTU
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">2
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">TOTU
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">26
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">TPTU
</oasis:entry><oasis:entry colname="2">2a
</oasis:entry><oasis:entry colname="3">2</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Nucleosides <bold>2a</bold><bold>−</bold><bold>c</bold>, coupling reagents, and coupling additives
were used at a concentration of 0.05 M in acetonitrile with a wait
step of 600 s during the coupling reaction.<italic toggle="yes"><sup>b</sup></italic><sup></sup> Nucleoside <bold>2d</bold> required
1:1 acetonitrile/dichloromethane for solubility.<italic toggle="yes"><sup>c</sup></italic><sup></sup> Loading determinations by trityl assay are ±5%.</p></table-wrap-foot></table-wrap></p><p><bold>Preparation of HQDA-CPG 7. </bold>LCAA-CPG (1.0 g),
HQDA (0.4 mmol, 90 mg), DMAP (0.38 mmol, 46 mg),
and HBTU (0.38 mmol, 144 mg) were combined in a glass
screw capped vial or flask and then DIEA (0.8 mmol, 140
μL) and acetonitrile (10 mL) were added via syringe. The
reaction was shaken at room temperature (2 h). The
support was filtered off and washed with acetonitrile,
MeOH, and finally CH<sub>2</sub>Cl<sub>2</sub>. After drying, the amount of
HQDA on the support (∼40 μmol/g) was determined by
derivatizing a small aliquot with <italic toggle="yes">N</italic>-monomethoxytrityl-6-amino-1-hexanol (<italic toggle="yes"><xref rid="bc990063ab00001" ref-type="bibr"></xref></italic>) followed by trityl analysis.
</p><p><bold>Secondary Esterification.</bold> 5‘-Dimethoxytritylthymidine <bold>1d</bold> (0.1 mmol, 54 mg), HATU or HBTU (0.1 mmol),
DMAP (12 mg), and carboxyl-CPG <bold>6</bold> or <bold>7</bold> (0.25 g) were
combined in a 4 mL glass vial. Acetonitrile (1 mL) was
added and the mixture shaken at room temperature.
Aliquots (∼10−20 mg) were removed at various intervals,
washed with CH<sub>2</sub>Cl<sub>2,</sub> MeOH, and CH<sub>2</sub>Cl<sub>2</sub>, dried, and the
progress of the reaction was determined by trityl analysis. The loadings obtained on succinylated support <bold>6</bold> after
coupling times of 5, 15, 30, and 60 min were HATU/DMAP, 34, 47, 53, and 63; HBTU/DMAP, 45, 51, 61, and
59 μmol/g. The loading obtained on the <italic toggle="yes">Q-Linker</italic> support
<bold>7</bold> after 2 h was 43 μmol/g.
</p><p>The above reaction was also performed with succinylated support <bold>6</bold> using one-half the amount of nucleoside,
and HATU/DMAP or HBTU/DMAP. The loadings obtained after coupling times of 5, 15, 30, and 60 min were,
for HATU/DMAP, 17, 21, 22, and 23 and, for HBTU/DMAP, 15, 17, 18, and 19 μmol/g.
</p><p>Automated secondary esterification reactions were
performed as above, except a 0.05 M solution of <bold>1d</bold> and
DIEA was used as the nucleoside reagent and carboxyl-CPG supports <bold>6</bold> and <bold>7</bold> were used instead of OH-CPG (see
Table <xref rid="bc990063at00002"></xref>).
<table-wrap id="bc990063at00002" position="float" orientation="portrait"><label>2</label><caption><p>Automated Coupling of 5‘-Dimethoxytritylthymidine<bold>1d</bold> to Carboxyl Derivatized
CPG through Secondary Ester Linkages.</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="6"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:colspec colnum="3" colname="3"></oasis:colspec><oasis:colspec colnum="4" colname="4"></oasis:colspec><oasis:colspec colnum="5" colname="5"></oasis:colspec><oasis:colspec colnum="6" colname="6"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2"></oasis:entry><oasis:entry namest="3" nameend="6">coupling time and nucleoside
loading obtained (μmol/g)<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry namest="1" nameend="1">coupling reagent<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry namest="2" nameend="2">support<italic toggle="yes"><sup>c</sup></italic><sup></sup></oasis:entry><oasis:entry namest="3" nameend="3">60 s</oasis:entry><oasis:entry namest="4" nameend="4">150 s</oasis:entry><oasis:entry namest="5" nameend="5">300 s</oasis:entry><oasis:entry namest="6" nameend="6">600 s
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">BrOP/DMAP
</oasis:entry><oasis:entry colname="2">6
</oasis:entry><oasis:entry colname="3">17
</oasis:entry><oasis:entry colname="4">30
</oasis:entry><oasis:entry colname="5">37
</oasis:entry><oasis:entry colname="6">44
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PyBrOP/DMAP
</oasis:entry><oasis:entry colname="2">6
</oasis:entry><oasis:entry colname="3">26
</oasis:entry><oasis:entry colname="4">34
</oasis:entry><oasis:entry colname="5">39
</oasis:entry><oasis:entry colname="6">43
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HATU/DMAP
</oasis:entry><oasis:entry colname="2">6
</oasis:entry><oasis:entry colname="3">10
</oasis:entry><oasis:entry colname="4">14
</oasis:entry><oasis:entry colname="5">20
</oasis:entry><oasis:entry colname="6">28
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HBTU/DMAP
</oasis:entry><oasis:entry colname="2">6
</oasis:entry><oasis:entry colname="3">9
</oasis:entry><oasis:entry colname="4">12
</oasis:entry><oasis:entry colname="5">15
</oasis:entry><oasis:entry colname="6">24
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">BrOP/DMAP
</oasis:entry><oasis:entry colname="2">7
</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6">13
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HATU/DMAP
</oasis:entry><oasis:entry colname="2">7
</oasis:entry><oasis:entry colname="3">18
</oasis:entry><oasis:entry colname="4">20
</oasis:entry><oasis:entry colname="5">21
</oasis:entry><oasis:entry colname="6">25
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">HBTU/DMAP
</oasis:entry><oasis:entry colname="2">7
</oasis:entry><oasis:entry colname="3">10
</oasis:entry><oasis:entry colname="4">13
</oasis:entry><oasis:entry colname="5">19
</oasis:entry><oasis:entry colname="6">22
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">PyBrOP/DMAP
</oasis:entry><oasis:entry colname="2">7
</oasis:entry><oasis:entry colname="3"></oasis:entry><oasis:entry colname="4"></oasis:entry><oasis:entry colname="5"></oasis:entry><oasis:entry colname="6">9
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">TOTU/DMAP
</oasis:entry><oasis:entry colname="2">7
</oasis:entry><oasis:entry colname="3">17
</oasis:entry><oasis:entry colname="4">19
</oasis:entry><oasis:entry colname="5">19
</oasis:entry><oasis:entry colname="6">20</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Loading determinations by trityl assay are ±5%.<italic toggle="yes"><sup>b</sup></italic><sup></sup> 0.05 M <bold>1d</bold>
and DIEA solution was used with 0.05 M coupling reagent/DMAP
solution.<italic toggle="yes"><sup>c</sup></italic><sup></sup> The carboxyl loadings of the succinylated <bold>6</bold> and <italic toggle="yes">Q-Linker</italic> <bold>7</bold> supports were respectively, 90 and 40 μmol/g.</p></table-wrap-foot></table-wrap></p></sec><sec id="d7e886"><title>Results and Discussion</title><p><bold>Coupling through Primary Ester Linkages.</bold> The
hydroxyl-derivatized solid-phase support (OH-CPG) was
prepared from long-chain alkylamine controlled pore
glass (LCAA-CPG), succinic anhydride, and 6-aminohexan-1-ol as previously described (<italic toggle="yes"><xref rid="bc990063ab00004" ref-type="bibr"></xref></italic>). Although, our
initial experiments with this support, <italic toggle="yes">Q-Linker</italic> nucleosides <bold>3a</bold><bold>−</bold><bold>d, </bold>and an HBTU/HOBT coupling reagent were
satisfactory, we found that this method did not work well
with the 3‘-O-succinylated nucleosides <bold>2a</bold><bold>−</bold><bold>d</bold>. For example, the <italic toggle="yes">Q-Linker</italic> nucleosides gave loadings of 30−50
μmol/g while the 3‘-O-succinylated nucleosides produced
only 6−13 μmol/g during a 10 min long coupling reaction.
</p><p>Then we used DMAP as an additive, instead of HOBT,
to improve coupling performance. A catalytic amount of
DMAP is commonly used with carbodiimide coupling
reagents (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00022" ref-type="bibr"></xref>−<xref rid="bc990063ab00023" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00024" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00025" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00026" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00027" ref-type="bibr"></xref></named-content></italic>), and recently, we found that DMAP
greatly increases the speed and efficiency of amide
formation with uronium or phosphonium coupling reagents (<italic toggle="yes"><xref rid="bc990063ab00002" ref-type="bibr"></xref></italic>). However, there have only been a few other
reports of DMAP used with uronium (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00047" ref-type="bibr"></xref>−<xref rid="bc990063ab00048" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00049" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00050" ref-type="bibr"></xref></named-content></italic>) or phosphonium (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00046" ref-type="bibr"></xref>, <xref rid="bc990063ab00051" ref-type="bibr"></xref></named-content></italic>) coupling reagents. This is because
DMAP is known to cause racemization during long amino
acid couplings (<italic toggle="yes"><xref rid="bc990063ab00052" ref-type="bibr"></xref></italic>) as well as partial loss of base-sensitive
Fmoc protecting groups (<italic toggle="yes"><xref rid="bc990063ab00045" ref-type="bibr"></xref></italic>). However, in oligonucleotide
synthesis, these concerns are not an issue. Instead, it is
important that the reagents not cause base modifications,
such as those previously reported with sulfonic acid
coupling reagents (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00053" ref-type="bibr"></xref>, <xref rid="bc990063ab00054" ref-type="bibr"></xref></named-content></italic>) or acetic anhydride/DMAP
capping reagents (<italic toggle="yes"><xref rid="bc990063ab00055" ref-type="bibr"></xref></italic>). Although, only the first nucleoside
and not subsequent bases are exposed to the uronium/phosphonium salt and DMAP coupling conditions, we
looked for base modifications by treating nucleoside
samples (<bold>1c</bold><bold>−</bold><bold>d</bold> or <bold>3c</bold><bold>−</bold><bold>d)</bold> with an excess (2 equiv) of
coupling reagent (HBTU, BrOP, or PyBrOP) and DMAP
in either the presence or absence of phenoxyacetic acid
(2 equiv). Phosphorothioate oligonucleotide 20-mers made
from supports derivatized with the HBTU/DMAP reagent
were also analyzed by CE, AX-HPLC, and MS (<italic toggle="yes"><xref rid="bc990063ab00003" ref-type="bibr"></xref></italic>). In both
cases, no unexpected products were detected (results not
shown). It is also noteworthy that no base modifications
have been reported in any previous applications of
uronium or phosphonium coupling reagents in oligonucleotide synthesis (<italic toggle="yes">33−35, 38, 40−42</italic>) or PNA synthesis (<italic toggle="yes">30−32, 47</italic>).
</p><p>To evaluate the effect of DMAP as an additive, identical coupling reactions using the succinylated nucleosides<bold>
2a </bold>and <bold>2d</bold> were setup with HBTU solutions (0.033 M)
containing either HOBT or DMAP and samples were
removed for trityl analysis at various times. In both
cases, the DMAP coupling reactions were much better
than the HOBT reactions (Figure <xref rid="bc990063af00001"></xref>a). The nucleoside
loadings obtained with DMAP were 1.5−2 times greater
than with HOBT and these loadings were obtained in
only 10 min instead of 60 min.
<fig id="bc990063af00001" position="float" orientation="portrait"><label>1</label><caption><p>Comparison of HOBT (1 equiv) and DMAP (1 equiv) as additives in the coupling of nucleoside-3‘-carboxylates to OH-CPG through primary ester linkages. 0.2 mmol/g of nucleoside and coupling reagent were used at a concentration of 0.033 M. (a) Nucleosides<bold>2a</bold> and <bold>2d</bold> with a 3‘-succinyl linker arm. (b)
Nucleoside <bold>3d</bold> with a 3‘-HQDA linker arm.</p></caption><graphic xlink:href="bc990063af00001.gif" position="float" orientation="portrait"></graphic></fig></p><p>A similar experiment with the <italic toggle="yes">Q-Linker</italic> nucleoside <bold>3d</bold>
was also performed (Figure <xref rid="bc990063af00001"></xref>b). In this case, the <italic toggle="yes">Q-Linker</italic>
nucleoside reacted much faster than the succinylated
nucleosides using either HOBT or DMAP. However, the
DMAP reaction was still much better. For example, after
only a 1 min coupling, the respective loadings for the
HOBT and DMAP reactions were 12 and 45 μmol/g.
</p><p>The rapid couplings observed were ideal for implementation on an automated DNA synthesizer. Automation
allows different coupling reagents and additives to be
easily compared under identical conditions and allows
inexpensive underivatized supports to be used for oligonucleotide synthesis (<italic toggle="yes"><xref rid="bc990063ab00002" ref-type="bibr"></xref></italic>). Therefore, an automated PE
Biosystems 394 DNA synthesizer was programmed to fill
a synthesis column with a mixture of nucleoside and
coupling reagent solutions (4.0 s), wait a defined time
for coupling (60−600 s), acetylate unreacted hydroxyl
groups (300 s), and then continue with oligonucleotide
synthesis. The nucleoside loading was determined by
measurement of the trityl color released during detritylation (<italic toggle="yes"><xref rid="bc990063ab00027" ref-type="bibr"></xref></italic>).
</p><p>In the first set of automated coupling experiments, the
effect of different additives on the esterification of the
<italic toggle="yes">Q-Linker</italic> nucleoside <bold>3d</bold> to OH-CPG using HBTU was
investigated. Five identical sets of experiments were
performed using either 0.1 M HBTU, HBTU/DMAP (1:1), HBTU/NMI (1:1) HBTU/HOBT/DIEA (1:1:1), or HBTU/HOAT/DIEA (1:1:1) and coupling times of up to 10 min.
The results (Figure <xref rid="bc990063af00002"></xref>) showed that HBTU alone could
produce some esterification (22 μmol/g) to the <italic toggle="yes">Q-Linker</italic>
after 10 min. However, inclusion of 1 equiv of either NMI,
HOAT, or HOBT with the HBTU almost doubled the
nucleoside loading (37−42 μmol/g) within the same
coupling time. However, a remarkable increase in the
speed and amount of esterification was obtained when
DMAP was used. Approximately the same nucleoside
loading (37 μmol/g) was obtained using DMAP for 1 min,
as produced in 10 min with either NMI, HOAT, or HOBT.
Coupling for 10 min with DMAP produced more than
three times the loading (65 μmol/g) as HBTU alone and
∼1.5 times the loading of the other three additives.
<fig id="bc990063af00002" position="float" orientation="portrait"><label>2</label><caption><p>Comparison of different coupling additives in the coupling of 0.1 M nucleoside-3‘-<italic toggle="yes">O</italic>-HQDA <bold>3d</bold> through a primary
ester linkage to OH-CPG by HBTU.</p></caption><graphic xlink:href="bc990063af00002.gif" position="float" orientation="portrait"></graphic></fig></p><p>In a second set of experiments, the coupling efficiency
of the four <italic toggle="yes">Q-Linker</italic> nucleosides <bold>3a</bold><bold>−</bold><bold>d</bold> with either 0.075
M HBTU/DMAP or 0.2 M HBTU/HOBT was compared
(10 min coupling in each case). The average loading for
the four nucleosides using 0.075 M HBTU/DMAP was 66
μmol/g, while the average loading using 0.2 M HBTU/HOBT was only 46 μmol/g. Therefore, even with more
than twice the reagent concentration, the HBTU/HOBT
coupling reagent was less satisfactory than HBTU/DMAP.
</p><p>Coupling experiments were also performed with 0.075
M solutions of <italic toggle="yes">Q-Linker</italic> nucleoside <bold>3d</bold>, DMAP, and either
HATU, HBTU, BrOP, or PyBrOP. These experiments
showed that the uronium reagents were slightly better
(69−71 μmol/g) than the bromophosphonium reagents
(53−56 μmol/g) when used with a <italic toggle="yes">Q-Linker</italic> nucleoside.
</p><p>Finally, the automated primary esterification experiments were performed using OH-CPG, and 0.05 M
solutions of 3‘-succinylated nucleosides <bold>2a</bold><bold>−</bold><bold>d</bold>, DMAP,
and 11 coupling reagents (Table <xref rid="bc990063at00001"></xref>). When the results for
the 3‘-succinylated nucleoside <bold>2a</bold> were compared, the best
coupling reagents were the bromophosphonium reagents
BrOP and PyBrOP. These two reagents produced significantly higher nucleoside loadings (47−57 μmol/g) than
the other coupling reagents, and the order of esterification efficiency was BrOP > PyBrOP > HATU > TOTU
> BOP > HBTU > HBPipU ≈ HBPyU ≈ PyBOP ≫
TNTU ≈ TPTU. The only unsatisfactory results (2 μmol/g) were obtained using the last two reagents. Similar
results were also obtained with nucleosides <bold>2b</bold>,<bold>c</bold> and
HATU/DMAP or HBTU/DMAP. Poorer results were
obtained with succinylated thymidine nucleoside <bold>2d</bold>,
possibly because a 1:1 mixture of acetonitrile/dichloromethane instead of acetonitrile was required for solubility.
</p><p><bold>Coupling through Secondary Ester Linkages.</bold> We
also investigated a scheme, which did not require prior
synthesis of a separate nucleoside-3‘-O-carboxylate (i.e.,
<bold>2 </bold>or<bold> 3</bold>) for each base. In this strategy, the dicarboxylic
acid linker was first added to the support to produce
carboxylic acid derivatized supports <bold>6</bold> and <bold>7</bold> (Scheme <xref rid="bc990063ah00002"></xref>).
The carboxyl supports were then coupled to a nucleoside's
3‘-OH position via a secondary ester linkage. This
strategy has three important advantages. First, the cost
of nucleosides with free 3‘-OH groups is less than the cost
of nucleosides with 3‘-carboxyl groups. Second, nucleosides, which may be rare or very expensive, do not need
to be converted into 3‘-carboxylic acid derivatives before
being attached to the support. Finally, the strategy uses
readily available amino-derivatized supports, instead of
hydroxyl supports, as the starting materials. However,
esterification to a secondary hydroxyl group is more
difficult, and long coupling times (24 h) are required if
carbodiimide coupling reagents (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00022" ref-type="bibr"></xref>, <xref rid="bc990063ab00023" ref-type="bibr"></xref></named-content></italic>) are used. Therefore, we were very interested to know if our faster
coupling conditions could be used to improve this approach.
</p><p>Succinylated-CPG <bold>6</bold> (90 μmol/g) was prepared from
LCAA-CPG and succinic anhydride, (<italic toggle="yes"><xref rid="bc990063ab00022" ref-type="bibr"></xref></italic>) and HQDA-CPG
<bold>7</bold> (40−45 μmol/g) was prepared by coupling HQDA to
LCAA-CPG using HBTU/DMAP. Although, the carboxyl
loading of support <bold>7</bold> was not as high as support <bold>6</bold>, the
loading was still satisfactory for most purposes.
</p><p>Nucleoside coupling was performed by shaking succinylated-CPG support <bold>6</bold> with either HATU/DMAP or
HBTU/DMAP and either 0.2 or 0.4 mmol of nucleoside
<bold>1d</bold> (0.05 and 0.1 M respectively) per gram of support.
Unreacted carboxyl groups on the support were not
blocked since carboxyl sites do not interfere with subsequent phosphoramidite synthesis (<italic toggle="yes"><xref rid="bc990063ab00056" ref-type="bibr"></xref></italic>). These initial
experiments showed that nucleoside loadings within the
range most commonly used (∼20−50 μmol/g) could be
obtained within 15−30 min. A similar experiment, with
HQDA-CPG <bold>7</bold> resulted in all the carboxyl groups (∼40
μmol/g) being derivatized with nucleoside <bold>1d</bold>. Thus, both
the HATU/DMAP and HBTU/DMAP reagents were
significantly faster than the previously used carbodiimide
reagents (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00022" ref-type="bibr"></xref>, <xref rid="bc990063ab00023" ref-type="bibr"></xref></named-content></italic>).
</p><p>Next, we adapted this coupling procedure to run on
our automated DNA synthesizer. We examined coupling
of 0.05 M <bold>1d</bold> to supports <bold>6</bold> or <bold>7</bold> using either BrOP,
PyBrOP, HATU, HBTU or TOTU, and DMAP. Loadings
were determined after coupling times of either 60, 150,
300, or 600 s <bold>(</bold>Table <xref rid="bc990063at00002"></xref>). Nucleoside loadings within the
ideal range ∼30−40 μmol/g were obtained on succinylated support <bold>6</bold> using either BrOP/DMAP or PyBrOP/DMAP and coupling times of 150−600 s. The nucleoside
loadings obtained on the <italic toggle="yes">Q-Linker</italic> support <bold>7</bold> were lower
(20−25 μmol/g), probably due to the lower (∼40 μmol/g)
carboxylic acid loading of the support, but still useful.
Also, the uronium coupling reagents were more satisfactory for coupling to the <italic toggle="yes">Q-Linker</italic> than the bromophosphonium reagents. Finally, it should be noted that the
thymidine compound <bold>1d</bold> with a free 3‘-OH group was
much more soluble than the thymidine compound <bold>2d</bold>
with a 3‘-succinyl group and so there were no solubility
problems with any of the four 3‘-OH nucleosides <bold>1a</bold><bold>−</bold><bold>d</bold>.
</p><p>The above method is well-suited for automation on
high-throughput oligonucleotide synthesizers (<italic toggle="yes"><xref rid="bc990063ab00057" ref-type="bibr"></xref></italic>) because the reagents are inexpensive, easy to handle, stable
in solution, and fast enough to allow nucleoside attachment in about the same time period as a typical phosphoramidite coupling cycle. In particular, the cost savings
obtained by using less expensive 3‘-OH nucleosides and
carboxyl derivatized supports is a significant improvement to our previous proposal to use nucleoside-3‘-carboxylates and amino supports (<italic toggle="yes"><xref rid="bc990063ab00002" ref-type="bibr"></xref></italic>).
</p><p>We believe that using automated on-line nucleoside
attachment through amide or ester linkages is superior
to adding the first base as a phosphoramidite derivative,
as proposed for other “universal” supports (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc990063ab00058" ref-type="bibr"></xref>−<xref rid="bc990063ab00059" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc990063ab00060" ref-type="bibr"></xref></named-content></italic>). This
is because our approach eliminates the need for either
additional reagents or deprotection time to 3‘-dephosphorylate the final product. There is also no possibility
of 3‘-phosphorylated impurities contaminating the final
product if 3‘-dephosphorylation is incomplete. Indeed,
when the linker arm is the <italic toggle="yes">Q-Linker</italic>, the products can
be very quickly removed (2 min) from the support with
the desired 3‘-OH terminus. Although, base deprotection
must still be performed by conventional means, this can
be done away from the synthesizer and the solid-phase
support. This allows equipment to be reused sooner,
allows easy separation of the final product from the
support, and reduces any contamination from the support
(i.e., dissolved silica).
</p><p>In conclusion, we have found that uronium or phosphonium salt coupling reagents used with 1 equiv of
DMAP are very efficient coupling reagents for both ester
and amide couplings. The coupling reactions are very fast
and both primary and secondary hydroxyl groups can
form ester linkages to supports in 10 min or less.
Esterification was obtained with a number of different
reagents; however, the best reagents differed with the
linker arm used. With the <italic toggle="yes">Q-Linker</italic>, the best results were
obtained with uronium coupling reagents, while the best
results with the succinyl linker were obtained with
bromophosphonium reagents. Primary ester linkages,
used to couple nucleoside-3‘-carboxylates to OH-CPG, are
best when short coupling times, high nucleoside loadings,
and only the four common bases are required, i.e., the
large-scale synthesis of antisense oligonucleotides (<italic toggle="yes"><xref rid="bc990063ab00003" ref-type="bibr"></xref></italic>). On
the other hand, secondary ester couplings, used to attach
nucleosides with free 3‘-OH groups to carboxyl derivatized supports, are more suitable when the cost of the
nucleosides is a concern, i.e., high volume, small-scale
synthesis or use of rare or modified nucleosides.
</p><p>Although our work has only used these conditions for
coupling nucleosides to solid-phase supports, ester formation is of general synthetic interest. We hope the utility
of the above coupling conditions will encourage others
to use these reagents in the synthesis of other bioconjugates or combinatorial products.
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Phone: (403)220-4277. Fax: (403) 283-4907. E-mail: rtpon@ucalgary.ca.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">YU</namePart><namePart type="given">Shuyuan</namePart><affiliation>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</affiliation><affiliation> University of Calgary.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><name type="personal"><namePart type="family">SANGHVI</namePart><namePart type="given">Yogesh S.</namePart><affiliation>Department of Molecular Biology and Biochemistry, University of Calgary, 3350 Hospital Drive N.W.,Calgary, Alberta, Canada T2N 4N1, and Isis Pharmaceuticals, Development Chemistry Department,2280 Faraday Avenue, Carlsbad, California 92008</affiliation><affiliation> Isis Pharmaceuticals.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">1999-09-10</dateCreated><dateIssued encoding="w3cdtf">1999-11-15</dateIssued><copyrightDate encoding="w3cdtf">1999</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>Nucleosides can be esterified to solid-phase supports using uronium or phosphonium coupling reagents and a coupling additive, such as 1-hydroxybenzotriazole (HOBT), 7-aza-1-hydroxybenzotriazole (HOAT), N-methylimidazole (NMI), or 4-(dimethylamino)pyridine (DMAP). However, DMAP was far superior to other additives and high nucleoside loadings (up to 60 μmol/g) and rapid coupling reactions (≤10 min) were possible. Hydroxyl-derivatized CPG was attached to nucleosides with 3‘-succinyl or 3‘-hydroquinone-O,O‘-diacetic acid (HQDA or Q-Linker) carboxyl groups through a primary ester linkage. Alternatively, supports derivatized with succinic acid or the Q-Linker were attached directly to the 3‘-OH group of nucleosides through a secondary ester linkage. Uronium reagents (HATU or HBTU) gave the best results with the HQDA linker arm, while the bromophosphonium (BrOP or PyBrOP) reagents were best with the succinyl linker arm. In all cases, the coupling reactions were much faster than previous methods using carbodiimide coupling reagents. The ease and speed of the reaction make this support derivatization procedure suitable for automated in situ couplings on DNA synthesizers.</abstract><relatedItem type="host"><titleInfo><title>Bioconjugate Chemistry</title></titleInfo><titleInfo type="abbreviated"><title>Bioconjugate Chem.</title></titleInfo><genre type="journal" authority="ISTEX" authorityURI="https://publication-type.data.istex.fr" valueURI="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</genre><identifier type="ISSN">1043-1802</identifier><identifier type="eISSN">1520-4812</identifier><identifier type="acspubs">bc</identifier><identifier type="coden">BCCHES</identifier><identifier type="uri">pubs.acs.org/bc</identifier><part><date>1999</date><detail type="volume"><caption>vol.</caption><number>10</number></detail><detail type="issue"><caption>no.</caption><number>6</number></detail><extent unit="pages"><start>1051</start><end>1057</end></extent></part></relatedItem><relatedItem type="references" ID="bc990063ab00001" displayLabel="bibbc990063ab00001"><titleInfo><title>Hydroquinone-O,O‘-diacetic acid (‘Q-linker') as a replacement for succinyl and oxalyl linker arms in solid-phase oligonucleotide synthesis</title></titleInfo><titleInfo contentType="CDATA"><title>Hydroquinone-O,O‘-diacetic acid (‘Q-linker') as a replacement for succinyl and oxalyl linker arms in solid-phase oligonucleotide synthesis</title></titleInfo><name type="personal"><namePart type="family">PON</namePart><namePart type="given">R. 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S.</namePart></name><name type="personal"><namePart type="family">MUTHINI</namePart><namePart type="given">S.</namePart></name><name type="personal"><namePart type="family">VIERRA</namePart><namePart type="given">M.</namePart></name><name type="personal"><namePart type="family">ACOSTA</namePart><namePart type="given">L.</namePart></name><name type="personal"><namePart type="family">SMITH</namePart><namePart type="given">T. 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