4-(2-Aminooxyethoxy)-2-(ethylureido)quinoline−Oligonucleotide Conjugates: Synthesis, Binding Interactions, and Derivatization with Peptides
Identifieur interne : 001A94 ( Istex/Corpus ); précédent : 001A93; suivant : 001A954-(2-Aminooxyethoxy)-2-(ethylureido)quinoline−Oligonucleotide Conjugates: Synthesis, Binding Interactions, and Derivatization with Peptides
Auteurs : Tomoko Hamma ; Paul S. MillerSource :
- Bioconjugate Chemistry [ 1043-1802 ] ; 2003.
Abstract
Oligo-2‘-O-methylribonucleotides conjugated with 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) and 4-ethoxy-2-(ethylureido)quinoline (EOQ) were prepared by reaction of the AOQ or EOQ phosphoramidite with the protected oligonucleotide on a controlled pore glass support. Deprotection with ethylenediamine enabled successful isolation and purification of the highly reactive AOQ-conjugated oligomer. Polyacrylamide gel electrophoresis mobility shift experiments showed that the dissociation constants of complexes formed between an AOQ- or EOQ-conjugated 8-mer and complementary RNA or 2‘-O-methyl-RNA targets (9- and 10-mers) were in the low nM concentration range at 37 °C, whereas no binding was observed for the corresponding nonconjugated oligomer, even at a concentration of 500 nM. Fluorescence studies suggested that this enhanced affinity is most likely due to the ability of the quinoline ring of the AOQ or EOQ group to stack on the last base pair formed between the oligomer and target, thus stabilizing the duplex. The binding affinity of a 2‘-O-methyl RNA 15-mer, which contained an alternating methylphosphonate/phosphodiester backbone, for a 59-nucleotide stem-loop HIV TAR RNA target, increased 2.3 times as a consequence of conjugation with EOQ. The aminooxy group of AOQ-conjugated oligomers is a highly reactive nucleophile, which reacts readily with aldehydes and ketones to form stable oxime derivatives. This feature was used to couple an AOQ−oligomer with leupeptin, a tripeptide that contains a C-terminus aldehyde group. A simple method was developed to introduce a ketone functionality into peptides that contain a cysteine residue by reacting the peptide with bromoacetone. The resulting keto-peptide was then coupled to the AOQ−oligomer. This procedure was used to prepare oligonucleotide conjugates of a tetrapeptide, RGDC, and a derivative of HIV tat peptide having a C-terminus cysteine. The combination of the unique reactivity of the aminooxy group and enhanced binding affinity conferred by its quinoline ring suggests that AOQ may serve as a useful platform for the preparation of novel oligonucleotide conjugates.
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DOI: 10.1021/bc025638+
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<record><TEI wicri:istexFullTextTei="biblStruct"><teiHeader><fileDesc><titleStmt><title>4-(2-Aminooxyethoxy)-2-(ethylureido)quinoline−Oligonucleotide Conjugates: Synthesis, Binding Interactions, and Derivatization with Peptides</title><author><name sortKey="Hamma, Tomoko" sort="Hamma, Tomoko" uniqKey="Hamma T" first="Tomoko" last="Hamma">Tomoko Hamma</name><affiliation><mods:affiliation>Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns HopkinsUniversity, 615 North Wolfe Street, Baltimore, Maryland 21205</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: Division of Basic Science, Fred HutchinsonCancer Research Center, 1100 Fairview Avenue North Mail StopA3-015, Seattle, WA 98109.</mods:affiliation></affiliation></author><author><name sortKey="Miller, Paul S" sort="Miller, Paul S" uniqKey="Miller P" first="Paul S." last="Miller">Paul S. Miller</name><affiliation><mods:affiliation>Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns HopkinsUniversity, 615 North Wolfe Street, Baltimore, Maryland 21205</mods:affiliation></affiliation><affiliation><mods:affiliation> Corresponding author. Phone: 410-955-3489. Fax: 410-955-2926. E-mail: pmiller@jhsph.edu.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:D22D50DF700312506B9A74AAE89CFC7812D22C6D</idno><date when="2003" year="2003">2003</date><idno type="doi">10.1021/bc025638+</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-P7LNH6TZ-J/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">001A94</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">001A94</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">4-(2-Aminooxyethoxy)-2-(ethylureido)quinoline−Oligonucleotide
Conjugates: Synthesis, Binding Interactions, and Derivatization
with Peptides</title><author><name sortKey="Hamma, Tomoko" sort="Hamma, Tomoko" uniqKey="Hamma T" first="Tomoko" last="Hamma">Tomoko Hamma</name><affiliation><mods:affiliation>Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns HopkinsUniversity, 615 North Wolfe Street, Baltimore, Maryland 21205</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: Division of Basic Science, Fred HutchinsonCancer Research Center, 1100 Fairview Avenue North Mail StopA3-015, Seattle, WA 98109.</mods:affiliation></affiliation></author><author><name sortKey="Miller, Paul S" sort="Miller, Paul S" uniqKey="Miller P" first="Paul S." last="Miller">Paul S. Miller</name><affiliation><mods:affiliation>Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns HopkinsUniversity, 615 North Wolfe Street, Baltimore, Maryland 21205</mods:affiliation></affiliation><affiliation><mods:affiliation> Corresponding author. Phone: 410-955-3489. Fax: 410-955-2926. E-mail: pmiller@jhsph.edu.</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">2003</date><date type="published">2003</date><biblScope unit="vol">14</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="320">320</biblScope><biblScope unit="page" to="330">330</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">Oligo-2‘-O-methylribonucleotides conjugated with 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) and 4-ethoxy-2-(ethylureido)quinoline (EOQ) were prepared by reaction of the AOQ or EOQ phosphoramidite with the protected oligonucleotide on a controlled pore glass support. Deprotection with ethylenediamine enabled successful isolation and purification of the highly reactive AOQ-conjugated oligomer. Polyacrylamide gel electrophoresis mobility shift experiments showed that the dissociation constants of complexes formed between an AOQ- or EOQ-conjugated 8-mer and complementary RNA or 2‘-O-methyl-RNA targets (9- and 10-mers) were in the low nM concentration range at 37 °C, whereas no binding was observed for the corresponding nonconjugated oligomer, even at a concentration of 500 nM. Fluorescence studies suggested that this enhanced affinity is most likely due to the ability of the quinoline ring of the AOQ or EOQ group to stack on the last base pair formed between the oligomer and target, thus stabilizing the duplex. The binding affinity of a 2‘-O-methyl RNA 15-mer, which contained an alternating methylphosphonate/phosphodiester backbone, for a 59-nucleotide stem-loop HIV TAR RNA target, increased 2.3 times as a consequence of conjugation with EOQ. The aminooxy group of AOQ-conjugated oligomers is a highly reactive nucleophile, which reacts readily with aldehydes and ketones to form stable oxime derivatives. This feature was used to couple an AOQ−oligomer with leupeptin, a tripeptide that contains a C-terminus aldehyde group. A simple method was developed to introduce a ketone functionality into peptides that contain a cysteine residue by reacting the peptide with bromoacetone. The resulting keto-peptide was then coupled to the AOQ−oligomer. This procedure was used to prepare oligonucleotide conjugates of a tetrapeptide, RGDC, and a derivative of HIV tat peptide having a C-terminus cysteine. The combination of the unique reactivity of the aminooxy group and enhanced binding affinity conferred by its quinoline ring suggests that AOQ may serve as a useful platform for the preparation of novel oligonucleotide conjugates.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>oligonucleotides</json:string><json:string>chem</json:string><json:string>oligonucleotide</json:string><json:string>aminooxy</json:string><json:string>acetonitrile</json:string><json:string>peptide</json:string><json:string>oligomer</json:string><json:string>room temperature</json:string><json:string>spectrometry</json:string><json:string>hplc</json:string><json:string>mmol</json:string><json:string>quinoline</json:string><json:string>aminooxy group</json:string><json:string>bioconjugate</json:string><json:string>phosphoramidite</json:string><json:string>oligomers</json:string><json:string>bioconjugate chem</json:string><json:string>duplex</json:string><json:string>mass spectrometry</json:string><json:string>aldehyde</json:string><json:string>binding affinity</json:string><json:string>oxime</json:string><json:string>chloroform</json:string><json:string>reactive</json:string><json:string>phase hplc</json:string><json:string>adduct</json:string><json:string>calcd</json:string><json:string>quinoline ring</json:string><json:string>cytosine</json:string><json:string>ketone</json:string><json:string>aqueous acetonitrile</json:string><json:string>antisense</json:string><json:string>methanol</json:string><json:string>linear gradient</json:string><json:string>conjugation</json:string><json:string>derivative</json:string><json:string>carbonate</json:string><json:string>hamma</json:string><json:string>reaction mixture</json:string><json:string>bromoacetone</json:string><json:string>biochemistry</json:string><json:string>nucleic acid</json:string><json:string>lett</json:string><json:string>nucleic</json:string><json:string>column chromatography</json:string><json:string>molecular weight</json:string><json:string>potassium carbonate</json:string><json:string>cysteine residue</json:string><json:string>apparent dissociation constant</json:string><json:string>antisense oligonucleotides</json:string><json:string>tetrahedron lett</json:string><json:string>ethyl acetate</json:string><json:string>acetic acid</json:string><json:string>emission maximum</json:string><json:string>sodium phosphate</json:string><json:string>sodium chloride</json:string><json:string>michael adduct</json:string><json:string>silica</json:string><json:string>monomethoxytrityl chloride</json:string><json:string>binding mode</json:string><json:string>mass spectrum</json:string><json:string>dimethyloxime derivative</json:string><json:string>retention time</json:string><json:string>centrifuge tube</json:string><json:string>base pair</json:string><json:string>chemoselective ligation</json:string><json:string>ammonium hydroxide</json:string><json:string>double bond</json:string><json:string>last base pair</json:string><json:string>deae sephadex column</json:string></teeft></keywords><author><json:item><name>HAMMA Tomoko</name><affiliations><json:string>Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns HopkinsUniversity, 615 North Wolfe Street, Baltimore, Maryland 21205</json:string><json:string>Current address: Division of Basic Science, Fred HutchinsonCancer Research Center, 1100 Fairview Avenue North Mail StopA3-015, Seattle, WA 98109.</json:string></affiliations></json:item><json:item><name>MILLER Paul S.</name><affiliations><json:string>Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns HopkinsUniversity, 615 North Wolfe Street, Baltimore, Maryland 21205</json:string><json:string>Corresponding author. Phone: 410-955-3489. Fax: 410-955-2926. E-mail: pmiller@jhsph.edu.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-P7LNH6TZ-J</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>Oligo-2‘-O-methylribonucleotides conjugated with 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) and 4-ethoxy-2-(ethylureido)quinoline (EOQ) were prepared by reaction of the AOQ or EOQ phosphoramidite with the protected oligonucleotide on a controlled pore glass support. Deprotection with ethylenediamine enabled successful isolation and purification of the highly reactive AOQ-conjugated oligomer. Polyacrylamide gel electrophoresis mobility shift experiments showed that the dissociation constants of complexes formed between an AOQ- or EOQ-conjugated 8-mer and complementary RNA or 2‘-O-methyl-RNA targets (9- and 10-mers) were in the low nM concentration range at 37 °C, whereas no binding was observed for the corresponding nonconjugated oligomer, even at a concentration of 500 nM. Fluorescence studies suggested that this enhanced affinity is most likely due to the ability of the quinoline ring of the AOQ or EOQ group to stack on the last base pair formed between the oligomer and target, thus stabilizing the duplex. The binding affinity of a 2‘-O-methyl RNA 15-mer, which contained an alternating methylphosphonate/phosphodiester backbone, for a 59-nucleotide stem-loop HIV TAR RNA target, increased 2.3 times as a consequence of conjugation with EOQ. The aminooxy group of AOQ-conjugated oligomers is a highly reactive nucleophile, which reacts readily with aldehydes and ketones to form stable oxime derivatives. This feature was used to couple an AOQ−oligomer with leupeptin, a tripeptide that contains a C-terminus aldehyde group. A simple method was developed to introduce a ketone functionality into peptides that contain a cysteine residue by reacting the peptide with bromoacetone. The resulting keto-peptide was then coupled to the AOQ−oligomer. This procedure was used to prepare oligonucleotide conjugates of a tetrapeptide, RGDC, and a derivative of HIV tat peptide having a C-terminus cysteine. The combination of the unique reactivity of the aminooxy group and enhanced binding affinity conferred by its quinoline ring suggests that AOQ may serve as a useful platform for the preparation of novel oligonucleotide conjugates.</abstract><qualityIndicators><score>10</score><pdfWordCount>8135</pdfWordCount><pdfCharCount>52786</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>11</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>740</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>302</abstractWordCount><abstractCharCount>2154</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>4-(2-Aminooxyethoxy)-2-(ethylureido)quinoline−Oligonucleotide Conjugates: Synthesis, Binding Interactions, and Derivatization with Peptides</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>14</volume><issue>2</issue><pages><first>320</first><last>330</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-P7LNH6TZ-J</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></inist></categories><publicationDate>2003</publicationDate><copyrightDate>2003</copyrightDate><doi><json:string>10.1021/bc025638+</json:string></doi><id>D22D50DF700312506B9A74AAE89CFC7812D22C6D</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-P7LNH6TZ-J/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-P7LNH6TZ-J/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-P7LNH6TZ-J/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-P7LNH6TZ-J/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">4-(2-Aminooxyethoxy)-2-(ethylureido)quinoline−Oligonucleotide
Conjugates: Synthesis, Binding Interactions, and Derivatization
with Peptides</title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2003 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2003</date><date type="Copyright" when="2003">2003</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">4-(2-Aminooxyethoxy)-2-(ethylureido)quinoline−Oligonucleotide
Conjugates: Synthesis, Binding Interactions, and Derivatization
with Peptides</title><author xml:id="author-0000"><persName><surname>Hamma</surname><forename type="first">Tomoko</forename></persName><affiliation><orgName type="laboratory">Department of Biochemistry and Molecular Biology</orgName><orgName type="department">Bloomberg School of Public Health</orgName><orgName type="institution">Johns Hopkins University</orgName><address><addrLine>615 North Wolfe Street</addrLine><addrLine>Baltimore</addrLine><addrLine>Maryland 21205</addrLine></address></affiliation><note place="foot" n="bc0256381AF2"><ref>†</ref><p>
Current address: Division of Basic Science, Fred Hutchinson
Cancer Research Center, 1100 Fairview Avenue North Mail Stop
A3-015, Seattle, WA 98109.</p></note></author><author xml:id="author-0001" role="corresp"><persName><surname>Miller</surname><forename type="first">Paul S.</forename></persName><affiliation><orgName type="laboratory">Department of Biochemistry and Molecular Biology</orgName><orgName type="department">Bloomberg School of Public Health</orgName><orgName type="institution">Johns Hopkins University</orgName><address><addrLine>615 North Wolfe Street</addrLine><addrLine>Baltimore</addrLine><addrLine>Maryland 21205</addrLine></address></affiliation><affiliation role="corresp"> Corresponding author. Phone: 410-955-3489. Fax: 410-955-2926. E-mail: pmiller@jhsph.edu.</affiliation></author><idno type="istex">D22D50DF700312506B9A74AAE89CFC7812D22C6D</idno><idno type="ark">ark:/67375/TPS-P7LNH6TZ-J</idno><idno type="DOI">10.1021/bc025638+</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">2003</date><date type="published">2003</date><biblScope unit="vol">14</biblScope><biblScope unit="issue">2</biblScope><biblScope unit="page" from="320">320</biblScope><biblScope unit="page" to="330">330</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>Oligo-2‘-<hi rend="italic">O</hi>-methylribonucleotides conjugated with 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ)
and 4-ethoxy-2-(ethylureido)quinoline (EOQ) were prepared by reaction of the AOQ or EOQ
phosphoramidite with the protected oligonucleotide on a controlled pore glass support. Deprotection
with ethylenediamine enabled successful isolation and purification of the highly reactive AOQ-conjugated oligomer. Polyacrylamide gel electrophoresis mobility shift experiments showed that the
dissociation constants of complexes formed between an AOQ- or EOQ-conjugated 8-mer and
complementary RNA or 2‘-<hi rend="italic">O</hi>-methyl-RNA targets (9- and 10-mers) were in the low nM concentration
range at 37 °C, whereas no binding was observed for the corresponding nonconjugated oligomer, even
at a concentration of 500 nM. Fluorescence studies suggested that this enhanced affinity is most likely
due to the ability of the quinoline ring of the AOQ or EOQ group to stack on the last base pair formed
between the oligomer and target, thus stabilizing the duplex. The binding affinity of a 2‘-<hi rend="italic">O</hi>-methyl
RNA 15-mer, which contained an alternating methylphosphonate/phosphodiester backbone, for a 59-nucleotide stem-loop HIV TAR RNA target, increased 2.3 times as a consequence of conjugation with
EOQ. The aminooxy group of AOQ-conjugated oligomers is a highly reactive nucleophile, which reacts
readily with aldehydes and ketones to form stable oxime derivatives. This feature was used to couple
an AOQ−oligomer with leupeptin, a tripeptide that contains a C-terminus aldehyde group. A simple
method was developed to introduce a ketone functionality into peptides that contain a cysteine residue
by reacting the peptide with bromoacetone. The resulting keto-peptide was then coupled to the AOQ−oligomer. This procedure was used to prepare oligonucleotide conjugates of a tetrapeptide, RGDC,
and a derivative of HIV tat peptide having a C-terminus cysteine. The combination of the unique
reactivity of the aminooxy group and enhanced binding affinity conferred by its quinoline ring suggests
that AOQ may serve as a useful platform for the preparation of novel oligonucleotide conjugates.
</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/bc025638+</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>4-(2-Aminooxyethoxy)-2-(ethylureido)quinoline−Oligonucleotide
Conjugates: Synthesis, Binding Interactions, and Derivatization
with Peptides</article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Hamma</surname><given-names>Tomoko</given-names></name><xref rid="bc0256381AF2"><sup>†</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Miller</surname><given-names>Paul S.</given-names></name><xref rid="bc0256381AF1">*</xref></contrib><aff>Department of Biochemistry and Molecular Biology, Bloomberg School of Public Health, Johns Hopkins
University, 615 North Wolfe Street, Baltimore, Maryland 21205 </aff></contrib-group><author-notes><fn id="bc0256381AF2"><label>†</label><p>
Current address: Division of Basic Science, Fred Hutchinson
Cancer Research Center, 1100 Fairview Avenue North Mail Stop
A3-015, Seattle, WA 98109.</p></fn><corresp id="bc0256381AF1">
Corresponding author. Phone: 410-955-3489. Fax: 410-955-2926. E-mail: pmiller@jhsph.edu.</corresp></author-notes><pub-date pub-type="epub"><day>6</day><month>02</month><year>2003</year></pub-date><pub-date pub-type="ppub"><day>19</day><month>03</month><year>2003</year></pub-date><volume>14</volume><issue>2</issue><fpage>320</fpage><lpage>330</lpage><history><date date-type="received"><day>13</day><month>11</month><year>2002</year></date><date date-type="rev-recd"><day>09</day><month>01</month><year>2003</year></date><date date-type="asap"><day>6</day><month>02</month><year>2003</year></date><date date-type="issue-pub"><day>19</day><month>03</month><year>2003</year></date></history><permissions><copyright-statement>Copyright © 2003 American Chemical Society</copyright-statement><copyright-year>2003</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>Oligo-2‘-<italic toggle="yes">O</italic>-methylribonucleotides conjugated with 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ)
and 4-ethoxy-2-(ethylureido)quinoline (EOQ) were prepared by reaction of the AOQ or EOQ
phosphoramidite with the protected oligonucleotide on a controlled pore glass support. Deprotection
with ethylenediamine enabled successful isolation and purification of the highly reactive AOQ-conjugated oligomer. Polyacrylamide gel electrophoresis mobility shift experiments showed that the
dissociation constants of complexes formed between an AOQ- or EOQ-conjugated 8-mer and
complementary RNA or 2‘-<italic toggle="yes">O</italic>-methyl-RNA targets (9- and 10-mers) were in the low nM concentration
range at 37 °C, whereas no binding was observed for the corresponding nonconjugated oligomer, even
at a concentration of 500 nM. Fluorescence studies suggested that this enhanced affinity is most likely
due to the ability of the quinoline ring of the AOQ or EOQ group to stack on the last base pair formed
between the oligomer and target, thus stabilizing the duplex. The binding affinity of a 2‘-<italic toggle="yes">O</italic>-methyl
RNA 15-mer, which contained an alternating methylphosphonate/phosphodiester backbone, for a 59-nucleotide stem-loop HIV TAR RNA target, increased 2.3 times as a consequence of conjugation with
EOQ. The aminooxy group of AOQ-conjugated oligomers is a highly reactive nucleophile, which reacts
readily with aldehydes and ketones to form stable oxime derivatives. This feature was used to couple
an AOQ−oligomer with leupeptin, a tripeptide that contains a C-terminus aldehyde group. A simple
method was developed to introduce a ketone functionality into peptides that contain a cysteine residue
by reacting the peptide with bromoacetone. The resulting keto-peptide was then coupled to the AOQ−oligomer. This procedure was used to prepare oligonucleotide conjugates of a tetrapeptide, RGDC,
and a derivative of HIV tat peptide having a C-terminus cysteine. The combination of the unique
reactivity of the aminooxy group and enhanced binding affinity conferred by its quinoline ring suggests
that AOQ may serve as a useful platform for the preparation of novel oligonucleotide conjugates.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bc025638+</meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec id="d7e121"><title>Introduction</title><p>Recent advances in chemical syntheses of modified
oligonucleotides have enabled broad applications of antisense oligonucleotides as research tools in molecular
biology; as diagnostic agents; and as potential therapeutics (see recent reviews in refs <italic toggle="yes">1</italic> and<italic toggle="yes"> 2</italic>). The efficacy of
antisense oligonucleotides relies on stable and specific
hybridization with their RNA targets and on effective
internalization by cells. A considerable amount of research has been devoted to synthesizing oligonucleotides
that possess unique sugar, phosphate, or base moieties
or with chemically reactive functional groups in an effort
to enhance the binding affinities of the oligonucleotides
for their targets. Furthermore, efforts have also been
made to conjugate antisense oligonucleotides to small
ligands, peptides, or proteins to facilitate their uptake
by cells.
</p><p>Chemically reactive functional groups such as psoralen
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00003" ref-type="bibr"></xref>, <xref rid="bc0256381b00004" ref-type="bibr"></xref></named-content></italic>), dimethylanthraquinone (<italic toggle="yes"><xref rid="bc0256381b00005" ref-type="bibr"></xref></italic>), mitomycin (<italic toggle="yes"><xref rid="bc0256381b00006" ref-type="bibr"></xref></italic>), and
2-amino-6-vinylpurine (<italic toggle="yes"><xref rid="bc0256381b00007" ref-type="bibr"></xref></italic>) have been conjugated to oligonucleotides to achieve oligomer-target cross-linking.
However, these agents require photoactivation, reduction,
or acidic conditions to trigger the cross-linking reaction.
In contrast, groups such as aromatic 2-chloroethylamine
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00008" ref-type="bibr"></xref>, <xref rid="bc0256381b00009" ref-type="bibr"></xref></named-content></italic>), cyclopropapyrroloindole (<italic toggle="yes"><xref rid="bc0256381b00010" ref-type="bibr"></xref></italic>), transplatin (<italic toggle="yes"><xref rid="bc0256381b00011" ref-type="bibr"></xref></italic>), and
5-methyl-N<sup>4</sup>, N<sup>4</sup>-ethanocytosine (<italic toggle="yes"><xref rid="bc0256381b00012" ref-type="bibr"></xref></italic>), when incorporated
into oligonucleotides, can carry out alkylation when the
oligonucleotide hybridizes to its target. Efficient covalent
bond formation between an oligonucleotide and its target
has been shown to correlate with the antisense efficacy
in the cell (<italic toggle="yes">6</italic>,<italic toggle="yes"> 11</italic>,<italic toggle="yes"> 13</italic>).
</p><p>The aminooxy group reacts readily with a variety of
electrophiles. The high nucleophilicity of this group is
attributed to the unshared pair of electrons on the oxygen
atom adjacent to the amino nucleophilic center, inducing
the so-called alpha effect (<italic toggle="yes"><xref rid="bc0256381b00014" ref-type="bibr"></xref></italic>). The high reactivity of the
aminooxy group toward aldehydes and ketones (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00015" ref-type="bibr"></xref>−<xref rid="bc0256381b00016" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0256381b00017" ref-type="bibr"></xref></named-content></italic>)
has been utilized for chemoselective conjugation of peptides (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00018" ref-type="bibr"></xref>, <xref rid="bc0256381b00019" ref-type="bibr"></xref></named-content></italic>), proteins (<italic toggle="yes"><xref rid="bc0256381b00020" ref-type="bibr"></xref></italic>), carbohydrates (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00021" ref-type="bibr"></xref>−<xref rid="bc0256381b00022" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0256381b00023" ref-type="bibr"></xref></named-content></italic>), and
oligonucleotides (<italic toggle="yes">15</italic>,<italic toggle="yes"> 23−26</italic>), resulting in formation of a
highly stable oxime linkage (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00015" ref-type="bibr"></xref>, <xref rid="bc0256381b00027" ref-type="bibr"></xref></named-content></italic>). Alkoxyamines have
also been shown to react with the 5−6 double bonds of
cytosine and uracil to form covalent Michael adducts (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00028" ref-type="bibr"></xref>−<xref rid="bc0256381b00029" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0256381b00030" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0256381b00031" ref-type="bibr"></xref></named-content></italic>). It thus appeared possible that an aminooxy group,
when appended to an oligonucleotide, might increase the
binding affinity between the oligomer and its target as a
result of covalent Michael adduct formation with a
cytosine residue in the target.
</p><p>To explore these possibilities, we have designed an
antisense oligonucleotide bearing a 5‘-4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) group. The interactions between this AOQ-derivatized oligonucleotide
and complementary RNA and 2‘-<italic toggle="yes">O</italic>-methyl-RNA targets
were characterized by gel mobility shift assays and
steady state fluorescence measurements. In addition, a
novel, simple method was developed to conjugate the
AOQ-derivatized oligonucleotide with small peptides. The
increased binding of the AOQ-derivatized oligonucleotide
to its target and its successful conjugation with biologically active peptides suggests that the AOQ group may
be a useful modification for enhancing the efficacy of
antisense oligonucleotides.
</p></sec><sec id="d7e236"><title>Experimental Procedures</title><p><bold>Materials.</bold> 2-Amino-4-hydroxyquinoline hydrate (97%,Lancaster), 2-cyanoethyl diisopropyl chlorophosphoramidite (Chemgenes Corp.), bromoethane (98%,Acros Organics), and bromoacetone (1-bromo-2-propanone) (95%,
Chem Service Inc.) were commercial products. Protected
2‘-<italic toggle="yes">O</italic>-methylribonucleoside-3‘-<italic toggle="yes">O</italic>-(β-cyanoethyl-<italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>-diisopropyl)phosphoramidites, ribonucleoside-3‘-<italic toggle="yes">O</italic>-(β-cyanoethyl-<italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>-diisopropyl)-phosphoramidites, controlled pore
glass supports, 4,5-dicyanoimidazole, 5-ethylthio-1<italic toggle="yes">H</italic>-tetrazole, and oligonucleotide synthesis reagents were
purchased from Glen Research, Inc. Protected 2‘-<italic toggle="yes">O</italic>-methylribonucleoside-3‘-<italic toggle="yes">O</italic>-(<italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>-diisopropyl)methylphosphonamidites were obtained from Chemgenes Corp.
Ethyl acetate, dichloromethane, diisopropylethylamine,
and triethylamine were stored over calcium hydride,
whereas pyridine, chloroform, and <italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>-dimethylformamide were stored over Linde-type 4 Å molecular sieves.
Silica gel thin-layer chromatography (TLC) was carried
out using Whatman aluminum-backed sheets that contained fluorescent indicator. Column chromatography
was performed on Davisil-grade 634-type 60 Å silica gel,
100−200 mesh, obtained from Fisher Scientific. DEAE
Sephadex A-25 columns were purchased from Sigma.
Snake venom phosphodiesterase (SVP) from <italic toggle="yes">Crotalus
durissus</italic> was purchased from Boehringer Mannheim, and
calf intestinal alkaline phosphatase (CIP) and T4 polynucleotide kinase (PNK) were obtained from New England Biolabs. Synthetic leupeptin, H-Leu-Leu-Arg-H
and synthetic tetrapeptide, H-Arg-Gly-Asp-Cys-OH, were
obtained from Alexis Biochemicals and American Peptide
Company, respectively. The derivative of HIV Tat peptide
H-Gly-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-Pro-Pro-Gln-Cys-OH was synthesized and purified by the Protein/Peptide Sequencing Core Facility at Johns Hopkins
University.
</p><p><bold>General Methods.</bold> <sup>1</sup>H NMR spectra were acquired on
a Bruker AMX 300 MHz spectrometer. Samples were
prepared in dimethyl sulfoxide, and the chemical shifts
were reported in ppm relative to the internal residual
solvent peaks (DMSO: 2.5 ppm). MALDI-TOF mass
spectrometry was carried out at the Johns Hopkins
University Mass Spectrometry Facility and ESI mass
spectrometry by the Scripps Research Institute. Reversed-phase (RP) HPLC was carried out on a Microsorb C-18
column (0.46 × 15 cm, C18, 10 μm) purchased from
Varian Analytical using a flow rate of 1.0 mL/min. Strong
anion exchange (SAX) HPLC was carried out on a
Dynamax II column (0.46 × 25 cm) purchased from
Rainin Instruments Co. using a flow rate of 0.6 mL/min.
For the oligonucleotides, the columns were monitored at
260 and 290 nm for analytical runs and preparative runs,
respectively. Peptides were analyzed and purified on the
Microsorb C-18 column, monitored at 215 nm. Polyacrylamide gel electrophoresis was performed on 20 × 20 ×
0.75 cm gels containing either 8% or 20% acrylamide in
1x TBE. Urea, 7 M, was added for denaturing gels. The
gels were run in 1× TBE buffer, which contained 89 mM
Tris, 89 mM boric acid, and 0.2 mM ethylendiaminetetraacetate (EDTA) at pH 8.0.
</p><p><bold>Oligonucleotides</bold><italic toggle="yes">.</italic> The sequences of the oligonucleotides are shown in Figure <xref rid="bc0256381f00001"></xref>. They were synthesized on
an Applied Biosystems Model 392 DNA/RNA synthesizer
using commercially available phosphoramidites and phosphonamidites as described previously (<italic toggle="yes"><xref rid="bc0256381b00032" ref-type="bibr"></xref></italic>). The oligonucleotides were removed from the support and deprotected as described previously (<italic toggle="yes"><xref rid="bc0256381b00032" ref-type="bibr"></xref></italic>). Twenty <italic toggle="yes">A</italic><sub>260</sub> units of
each crude oligomer, 1676, 1843, 1855, 1856, 1885, 1886,
1887, or 2016 were purified by SAX HPLC using a 30
min linear gradient of 0−0.4 M ammonium sulfate. RNA
oligomer 1960 was purified on 20% polyacrylamide gels
run under denaturing conditions (<italic toggle="yes"><xref rid="bc0256381b00032" ref-type="bibr"></xref></italic>). The oligomers were
desalted using SEP PAK C-18 cartridges. The compositions of the oligomers were assessed by enzymatic digestion using SVP and CIP (<italic toggle="yes"><xref rid="bc0256381b00033" ref-type="bibr"></xref></italic>), and/or their molecular
weights were determined by MALDI-TOF mass spectrometry. The extinction coefficients of the oligonucleotides were determined as described previously (<italic toggle="yes"><xref rid="bc0256381b00033" ref-type="bibr"></xref></italic>):
1676, 1.3 × 10<sup>5</sup>; 1843, AOQ-1843, EOQ-1843, 6.23 × 10<sup>4</sup>;
1855, 1856, 1885, 1886, 1887, 7.67 × 10<sup>4</sup>; 1960, 1.0 ×
10<sup>5</sup>; and 2016, 5.2 × 10<sup>4</sup>. HIV TAR RNA was prepared
by in vitro transcription of a plasmid carrying a TAR
RNA template (<italic toggle="yes"><xref rid="bc0256381b00032" ref-type="bibr"></xref></italic>). The 73 nucleotide run off transcript
contained the 59 nucleotide TAR RNA sequence (underlined) 5‘GAAUACUCAAGCU<underline>GGGUCUCUCUGGUUAGAUUAGAUCUGAGCCUGGGAGCUCUCUGGCUAACUAGGGAACCC</underline>G.
<fig id="bc0256381f00001" position="float" orientation="portrait"><label>1</label><caption><p>Sequences of antisense oligonucleotides and their targets.<bold>X</bold> is AOQ, EOQ, or no modification. The symbols mr and r
indicate 2‘-<italic toggle="yes">O</italic>-methylribonucleoside or ribonucleotide respectively; p is a phosphodiester linkage, and <underline>p</underline> a methylphosphonate linkage.</p></caption><graphic xlink:href="bc0256381f00001.tif" position="float" orientation="portrait"></graphic></fig></p><p><bold>2-(2-Hydroxyethylureido)-4-hydroxyquinoline (2)</bold><italic toggle="yes">.</italic>
2-Amino-4-hydroxyquinoline hydrate (<bold>1</bold>) (640 mg, 4 mmol,
see Scheme 1) was coevaporated with anhydrous pyridine
and dissolved in 40 mL of dry pyridine. To this stirred
solution was added 10 equiv of 4-nitrophenylchloroformate (8.1 g, 40 mmol) dissolved in 20 mL dry chloroform
(20 mL), and the reaction was allowed to stir for 60 min
at room temperature. A thick orange suspension was
obtained that was treated with 41 equiv of ethanolamine
(10 mL, 164 mmol) and further stirred for 2 h at room
temperature. The reaction was complete as determined
by silica gel TLC [chloroform/methanol (4:1, v/v)], with
conversion of the starting material (<italic toggle="yes">R<sub>f</sub></italic> = 0.25) to a faster-moving product (<italic toggle="yes">R<sub>f</sub></italic> = 0.34). The suspension was filtered
through a 60 mL coarse sintered glass funnel, and the
residue was washed briefly with 20% methanol in chloroform. The filtrate was concentrated under vacuum on a
rotary evaporator to a syrup that was dissolved in a
minimum amount of chloroform and applied to a column
packed with a Davisil-grade 634-type 60 Å silica gel. The
column was eluted using a gradient from 10% to 20%
methanol in chloroform. Fractions containing the desired
product were combined and concentrated to yield <bold>2</bold> as a
brown syrup (1.2 g or 4 mmol, quantitative yield). <sup>1</sup>H
NMR (<italic toggle="yes">d<sub>6</sub></italic>- DMSO) δ (ppm): 4.82 (s; 1H, ureido-N<underline>H</underline>), 4.64
(s; 1H, ureido-N<underline>H</underline>), 7.98−7.24 (m; 4H, quinoline H5−8),
7.00 (t; 1H, quinoline H3), 5.44 (s; 1H, quinoline −OH),
3.91−3.87 (t; 1H, −NHCH<sub>2</sub>CH<sub>2</sub>O<underline>H</underline>), 3.54−3−3.48 (t; 2H,
−NHC<underline>H<sub>2</sub></underline>C<underline>H<sub>2</sub></underline>OH), 3.20−3.16 (t; 2H, −NHC<underline>H<sub>2</sub></underline>C<underline>H<sub>2</sub></underline>OH).
<fig id="bc0256381h00001" position="float" orientation="portrait"><label></label><graphic xlink:href="bc0256381h00001.tif" position="float" orientation="portrait"></graphic></fig></p><p><bold>2-(2-</bold><bold><italic toggle="yes">O</italic></bold><bold>-Monomethoxytritylethylureido)-4-hydroxyquinoline (3)</bold>. A solution of compound <bold>2</bold> (1.2 g, 4 mmol)
in 40 mL of dry pyridine was treated with 5 equiv of
monomethoxytrityl chloride (6.2 g, 20 mmol) for 4 h at
room temperature. The clear orange solution was cooled
in an ice bath, diluted with 10 mL of methanol and 2
mL of triethylamine, and then concentrated under vacuum
on a rotary evaporator. The product, <bold>3</bold>, which was
purified by silica gel column chromatography using a
gradient of 0−10% methanol in chloroform, was obtained
as a brown syrup (1.4 g or 2.69 mmol, 67%). <italic toggle="yes">R<sub>f</sub></italic> : 0.54
[chloroform/methanol (9:1, v/v)].
</p><p><bold>2-(2-</bold><bold><italic toggle="yes">O</italic></bold><bold>-Monomethoxyltritylethylureido)-4-(2-phthalimidooxyethoxy)quinoli</bold><bold>ne (4)</bold><italic toggle="yes">.</italic> Compound <bold>3</bold> (1.4
g, 2.7 mmol) and 3 equiv of 2-phthalimidooxyethyl
bromide (2.2 g, 8.1 mmol) were dissolved in dry <italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>-dimethylformamide (10.8 mL), and the solution was
treated with 3 equiv of dry powdered potassium carbonate (1.1 g, 8.1 mmol) overnight at room temperature. The
potassium carbonate was removed by filtration, and the
filtrate was concentrated under vacuum. The product, <bold>4</bold>,
which was purified by silica gel column chromatography
using a gradient of 0−5% methanol in chloroform, was
obtained as a yellow syrup (1.4 g or 1.8 mmol, 67%). <italic toggle="yes">R<sub>f</sub></italic>:
0.28 [methanol:chloroform (1:19, v/v)].
</p><p><bold>2-(2-Hydroxyethylureido)-4-(2-phthalimidooxyethoxy)quinoline (5)</bold><italic toggle="yes">. </italic>Compound <bold>4</bold> (1.4 g, 1.8 mmol)
was dissolved in 80% aqueous acetic acid (46 mL), and
the solution was incubated at 37 °C for 45 min. The acetic
acid was removed by coevaporation with 95% ethanol,
and the product, <bold>5</bold>, was precipitated by addition of
absolute ethanol. The precipitate was collected on a fine
porosity sintered glass funnel and dried under vacuum
in a desiccator overnight to yield a light yellow powder
(483 mg or 0.90 mmol, 50%). <italic toggle="yes">R<sub>f</sub></italic>: 0.1 [methanol:chloroform
(1:4 v/v)]. The overall yield starting from <bold>1</bold> was 22%. The
molecular weight of <bold>5</bold> was confirmed by ESI mass
spectrometry (MH<sup>+</sup>: calcd 437.4, found 437). <sup>1</sup>H NMR
(<italic toggle="yes">d<sub>6</sub></italic>- DMSO) δ (ppm): 9.60 (s; 1H, ureido-N<underline>H</underline>), 9.53 (s;
1H, ureido-N<underline>H</underline>), 7.83 (s; 4H, phthalimide), 7.78−7.14 (m;
4H, quinoline H5−8), 6.77 (s; 1H, quinoline H3), 4.86−4.83 (t; 1H, −NHCH<sub>2</sub>CH<sub>2</sub>O<underline>H</underline>), 4.67−4.45 (t; 4H,
−OC<underline>H<sub>2</sub></underline>C<underline>H<sub>2</sub></underline>O−), 3.58−3−3.30 (m; 4H, −NHC<underline>H<sub>2</sub></underline>C<underline>H<sub>2</sub></underline>OH).
</p><p><bold>2-[(2-Ureido)-4-(2-phthalimidooxyethoxy)quinoline]-ethyl-1-</bold><bold><italic toggle="yes">O</italic></bold><bold>-</bold><bold>β</bold><bold>-cyanoethyl-</bold><bold><italic toggle="yes">N</italic></bold>,<bold><italic toggle="yes">N</italic></bold><bold>-diisopropylphosphoramidite (6)</bold><italic toggle="yes">.</italic> Compound <bold>5</bold> (15 mg, 0.03 mmol) was
dissolved in dry dichloromethane (600 μL). Diisopropylethylamine (104.5 μL, 0.60 mmol) and 2-cyanoethyl
diisopropyl chlorophosphoramidite (41.7 μL, 0.19 mmol)
were added via syringe to the stirred solution, and the
reaction was allowed to proceed at room temperature for
1 h. The product <bold>6</bold>, whose <italic toggle="yes">R<sub>f</sub></italic> was 0.21 on silica gel TLC
[ethyl acetate:triethylamine, (95.5:0.5, v/v)], was purified
by silica gel column chromatography using ethyl acetate/triethylamine (95.5:0.5, v/v). Fractions containing <bold>6</bold> were
combined and evaporated under vacuum to yield a clear
light yellow syrup, which was dissolved in a minimum
volume of dry dichloromethane (∼300 μL), and the
solution was added dropwise with stirring to hexane at
room temperature. The resulting precipitate was collected
by centrifugation and dried under vacuum to yield a
white solid (10.9 mg or 0.017 mmol, 57%). <sup>1</sup>H NMR (<italic toggle="yes">d<sub>6</sub></italic>-DMSO) δ (ppm): 9.70 (s; 1H, ureido-N<underline>H</underline>), 9.58 (s; 1H,
ureido-N<underline>H</underline>), 7.83 (s; 4H, phthalimide), 7.79−7.15 (m, 4H;
quinoline H5−8), 6.73 (s; 1H, quinoline H3), 4.68−4.46
(m, 4H, −OC<underline>H<sub>2</sub></underline>C<underline>H<sub>2</sub></underline>O−), 4.03−3.40 (m; −NHC<underline>H<sub>2</sub></underline>C<underline>H<sub>2</sub></underline>O−), 2.91−2.73 (t; 4H, cyanoethyl), 1.23−1.12 (m;
24H, diisopropyl), 1.09−1.05 (m; 14H, diisopropyl).
The MALDI-TOF mass spectrum was consistent for
C<sub>25</sub>H<sub>24</sub>N<sub>5</sub>O<sub>8</sub>P (<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>: calc. 553.5, found 554.4), indicating
that <bold>6</bold> had converted to its H-phosphonate derivative
during analysis.
</p><p><bold>2-(2-Hydroxyethylureido)-4-ethoxyquinoline (7)</bold>.
Compound <bold>3</bold> (1.52 g, 2.9 mmol) and bromoethane (873
μL, 12 mmol) were dissolved in 11.7 mL of dry dimethylformamide, and the solution was treated with dry,
powdered potassium carbonate (1.6 g, 12 mmol) for 14 h
at room temperature to give a new product whose <italic toggle="yes">R<sub>f</sub></italic> was
0.75 [methanol: chloroform, (1:19 v/v)] on silica gel TLC.
The potassium carbonate was removed by filtration and
washed with several portions of dichloromethane. The
filtrate was evaporated under vacuum, and the residue
was treated with 58 mL of 80% aqueous acetic acid at
37 °C for 90 min. The acetic acid was removed by
coevaporation with 95% ethanol, and the resulting compound <bold>7 </bold>was purified by silica gel column chromatography using 0−5% methanol in chloroform. Fractions
containing <bold>7</bold> were combined and concentrated under
vacuum to give an oil, <italic toggle="yes">R</italic><sub>f</sub> = 0.18 [methanol: chloroform,
(1:19 v/v)]. The oil was dissolved in a minimum volume
of methanol, and heated water (50 °C) was added
dropwise with stirring until the solution became turbid.
This solution was heated until everything dissolved. The
solution was then cooled slowly to 4 °C. The resulting
solid was collected by filtration, washed several times
with ice-cold water, and dried under vacuum. The product
was obtained as a white powder (18 mg or 0.67 mmol,
17%), <italic toggle="yes">R<sub>f</sub></italic>: 0.32 [ethyl acetate:triethylamine, (99.5:0.5,
v/v)]. MALDI-TOF mass spectrometry (<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>: calcd 275.3,
found 276.1).
</p><p><bold>2-[(2-Ureido)-4-ethoxyquinoline]-ethyl-1-</bold><bold><italic toggle="yes">O</italic></bold><bold>-</bold><bold>β</bold><bold>-cyanoethy-</bold><bold><italic toggle="yes">N</italic></bold>,<bold><italic toggle="yes">N</italic></bold><bold>-diisopropyl-phosphoramidite (8)</bold><italic toggle="yes">. </italic>The
synthesis of phosphoramidite <bold>8</bold> was identical to that of
phosphoramidite <bold>6</bold> described above with the following
modifications. After phosphitylation, the crude phosphoramidite was applied to a silica gel column using a
mixture of petroleum ether and ethyl acetate (2:1 v/v),
and the column was eluted with 0.5% triethylamine in
ethyl acetate. The purified phosphoramidite was not
precipitated in hexane. Rather, the oily product was
transferred to an autosampler vial, evaporated under
vacuum for 30 min, and the resulting oily residue was
used immediately for conjugation to the oligonucleotide
as described below.
</p><p><bold>Preparation of AOQ- and EOQ-Conjugated Oligonucleotides. </bold>AOQ-1843, AOQ-2106, EOQ-1843, and
EOQ-1676 were synthesized as follows. The oligo 2‘-<italic toggle="yes">O</italic>-methylribonucleotides were synthesized as described
above, and the 5‘-DMT group was removed on the
synthesizer. The oligonucleotide−CPG (10 mg) was mixed
with AOQ phosphoramidite <bold>6</bold> in a 1.5 mL centrifuge tube
or EOQ phosphoramidite <bold>8</bold> in a 4 mL autosampler vial.
A 0.4 M solution of tetrazole in anhydrous acetonitrile
was added, and the final concentration of the phosphoramidite was adjusted to 0.2 M by addition of anhydrous
acetonitrile. The reaction mixture was incubated for 10
min at room temperature. The CPG was transferred to
a synthesis cartridge and washed with 10 mL of acetonitrile, after which the oxidation and capping reactions
were performed on the DNA synthesizer.
</p><p>AOQ-1843 and AOQ-2106 were deprotected and purified as follows. The oligonucleotide−CPG, 10 mg, was
treated with 30 μL of a solution containing ethylenediamine, acetonitrile, 95% ethanol, and water (20:9:9:2 v/v)
for 6 h at room temperature. The reaction mixture was
cooled in an ice−water bath and neutralized by addition
of 120 μL of ice-cold 2 N hydrochloric acid. The deprotected oligomer was purified immediately by C-18 reversed phase HPLC using a 20 mL linear gradient of
0−50% acetonitrile in 50 mM sodium phosphate, pH 5.8.
The oligomer was then desalted using a C-18 SEP PAK
cartridge. The 50% aqueous acetonitrile solution containing the oligomer (9.9 <italic toggle="yes">A</italic><sub>260</sub> units AOQ-1843; 0.34 <italic toggle="yes">A</italic><sub>260</sub> units
of AOQ-2106) was evaporated to dryness, and the dry
residue was stored at −20 °C. The oligomers were
analyzed by MALDI-TOF mass spectrometry: AOQ-1843
(<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>: calcd + K<sup>+</sup> 3001.8, found 3006.1); AOQ-2106 (<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>:
calcd 1966.8, found 1967.1).
</p><p>The EOQ-derivatized oligonucleotides were each deprotected and purified as follows. The oligonucleotide−CPG
was treated with 400 μL of concentrated ammonium
hydroxide at 65 °C for 5 h. After evaporation of solvents,
the EOQ−oligonucleotide was purified by C-18 reversed-phase HPLC using a 20 mL linear gradient of 2−50%
acetonitrile in 50 mM sodium phosphate, pH 7.8. Each
purified EOQ−oligonucleotide was desalted on a C-18
SEP PAK cartridge and stored as a solution in 50%
aqueous acetonitrile at 4 °C. The oligonucleotides were
analyzed by MALDI-TOF mass spectrometry: EOQ-1843
(<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>: calcd 2934.1, found 2935); EOQ-1676 (<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>: calcd
+ K<sup>+</sup> 5283.8, found 5279.0).
</p><p><bold>Gel Mobility Shift Experiments.</bold> Electrophoretic gel
mobility shift assays were carried out as described
previously (<italic toggle="yes"><xref rid="bc0256381b00032" ref-type="bibr"></xref></italic>). Oligonucleotide solutions with concentrations ranging from 0.5 nM to 100 μM were prepared in
binding buffer that contained 100 mM sodium chloride,
10 mM Tris, pH 7.5, and 0.5 mM EDTA in diethylpyrocarbonate treated water. Solutions containing 0.2 nM of
5‘-[<sup>32</sup>P]-end-labeled 1855, 1856, 1885, 1886, 1887, 1960,
or TAR RNA (specific activity, 1000 Ci/mmol) were
prepared in the same buffer. Equal volumes of oligomer
and target solution (total volume, 10 μL) were mixed in
0.5 mL centrifuge tubes, and 1 μL of autoclaved 80%
glycerol was added. Duplexes formed between 10-mer
targets 1855, 1856, 1885, 1886, 1887, 1960, and 8-mer
oligomers 1843, AOQ-1843, EOQ-1843, were incubated
at 37 °C for 1 h. Duplexes formed between TAR RNA and
15-mer oligomers 1676 or EOQ-1676 were incubated for
24 h at 37 °C. The solutions were loaded onto a polyacrylamide gel using a 20 μL syringe. Complexes formed
with the short targets 1855, 1856, 1885, 1856, 1857, or
1960 were separated on a 20% nondenaturing acrylamide
gel run at 500−600 V for 1.5 h at the same temperature
at which the sample was incubated. In the case of the
TAR RNA target, electrophoresis was carried out in a
similar manner on an 8% nondenaturing polyacrylamide
gel at 200−300 V for 2 h. The wet gels were visualized
by phosphorimaging and quantitated using the phosphorimager software package. The apparent dissociation
constants were determined as the half-maximal point on
a plot of percent complex versus the log of oligomer
concentration. Averages were obtained from at least three
separate experiments.
</p><p><bold>AOQ-1843</bold><bold>−</bold><bold>Leupeptin Conjugate.</bold> Leupeptin (1.25
μmol) was mixed with 5 <italic toggle="yes">A</italic><sub>260</sub> units (0.8 μmol) of AOQ-1843 in 54 μL of 50% aqueous acetonitrile solution, and
the solution was incubated for 60 min at room temperature. The AOQ-1843−leupeptin conjugate was purified
by C-18 reversed-phase HPLC using a 20 mL linear
gradient of 2−50% acetonitrile in 50 mM sodium phosphate, pH 5.8. The conjugate, which was obtained in
quantitative yield, was desalted on a SEP PAK C-18
cartridge and analyzed by MALDI-TOF mass spectrometry (<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>: calcd 3373.4, found 3372.4).
</p><p><bold>AOQ-2106</bold><bold>−</bold><bold>RGDC or </bold><bold>−</bold><bold>Tat Conjugate.</bold> A 0.2 M
solution of bromoacetone in 50 μL of 50% aqueous
acetonitrile was added to either RGDC peptide (0.5 mg,
1.0 μmol) or Tat peptide (0.5 mg, 0.3 μmol) in a 1.5 mL
centrifuge tube and the solution incubated for 1 h at room
temperature. The reaction was shielded from light by
covering the tube with aluminum foil during the incubation period. The resulting keto-peptide was purified by
C-18 reversed phase HPLC using a 20 mL linear gradient
of 2−50% acetonitrile in 0.1% trifluoroacetic acid. The
fraction containing the keto-peptide was evaporated to
dryness and redissolved in 50% aqueous acetonitrile. The
keto-peptides were analyzed by MALDI-TOF mass spectrometry: keto-RDGC (<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>: calcd 506, found 506); keto-tat (<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>: calcd 1879, found 1880).
</p><p>Keto-RDGC (19.5 nmol) was reacted with 0.1 <italic toggle="yes">A</italic><sub>260</sub> unit
(1.95 nmol) of AOQ-2106 in 53 μL of 50% aqueous
acetonitrile 1 h at room temperature. The resulting AOQ-2106−RDGC conjugate was purified by C-18 reversed-phase HPLC using a 20 mL linear gradient of 2−50%
acetonitrile in 0.1% trifluoroacetic acid. Two peaks were
observed that corresponded to the two oxime isomers. The
conjugate was analyzed by MALDI-TOF mass spectrometry (<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>, calcd 2455, found 2457 for peaks 1 and 2, see
Figure <xref rid="bc0256381f00006"></xref>).
</p><p>Keto−tat peptide (2.5 nmol) and 1.0 <italic toggle="yes">A</italic><sub>260</sub> unit of AOQ-2106 (19.5 nmol) were incubated in 25 μL of 50% aqueous
acetonitrile for 1 h at room temperature. The resulting
AOQ-2106−tat conjugate was separated from the unreacted AOQ-2106 by ion exchange chromatography. The
reaction mixture was diluted with 75 μL of 50% aqueous
acetonitrile, and the solution was applied to a small (0.6
× 1.2 cm) DEAE Sephadex A-25 column which had been
preequilibrated with 50% aqueous acetonitrile. The column was washed with 50% aqueous acetonitrile, and the
AOQ-2106−tat conjugate (1 nmol) was collected. The
conjugate was analyzed by MALDI-TOF mass spectrometry: (<italic toggle="yes">m</italic>/<italic toggle="yes">z</italic>, calcd 3828, found 3829).
</p></sec><sec id="d7e868"><title>Results and Discussion</title><p><bold>AOQ- and EOQ-Conjugated Oligonucleotides.</bold> The
structures of the 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) and 4-ethoxy-2-(ethylureido)quinoline
(EOQ) groups are shown in Figure <xref rid="bc0256381f00001"></xref>. EOQ lacks an
aminooxy group and thus is a nonreactive analogue of
AOQ. When bound to its target, the quinoline ring of an
AOQ- or EOQ-conjugated oligonucleotide can potentially
stack on the last base pair formed between the oligomer
and the target as shown in Figure <xref rid="bc0256381f00002"></xref>A. In this binding
mode, the aminooxy group of the AOQ−oligomer could
form a Michael adduct by reacting with the 5,6 double
bond of a cytosine residue in the target as shown
schematically in Figure <xref rid="bc0256381f00002"></xref>B. Although such adduct formation would be expected to be transient, it could decrease
the dissociation rate of the duplex, and consequently
enhance the binding affinity of the AOQ−oligomer. In
addition, the highly reactive aminooxy group can serve
as a site for conjugation with other molecules that bear
an aldehyde or ketone function.
<fig id="bc0256381f00002" position="float" orientation="portrait"><label>2</label><caption><p>Binding model. (A) The oval indicates either the AOQ or the EOQ group. (B) Possible stacking interaction between quinoline ring and neighboring bases. Left, target strand; right, antisense oligonucleotide strand.</p></caption><graphic xlink:href="bc0256381f00002.tif" position="float" orientation="portrait"></graphic></fig></p><p>To test these possibilities, AOQ or EOQ was conjugated
via a phosphodiester linkage to the 5‘-ends of oligo-2‘-<italic toggle="yes">O</italic>-methylribonucleotides as shown in Figure <xref rid="bc0256381f00001"></xref>. The sequences of these oligomers, with the exception of oligomer
2106, are complementary to the apical stem loop of HIV
TAR RNA. Oligomer 1843 is complementary to nucleotides 31−38 of TAR RNA. Oligomer 1676, which contains alternating methylphosphonate/phosphodiester internucleotide linkages, is complementary to nucleotides
21−38 of TAR RNA.
</p><p>AOQ− and EOQ−phosphoramidites were prepared as
shown in Scheme 1. 2-Amino-4-hydroxyquinoline, <bold>1</bold>, was
converted to its <italic toggle="yes">p</italic>-nitrophenylcarbamate derivative, <bold>1a</bold>,
by reaction with <italic toggle="yes">p</italic>-nitrophenylchloroformate. Compound
<bold>1a</bold> was not isolated but was converted to the hydroxyethylureido derivative, <bold>2</bold>, by treatment with ethanolamine. The primary hydroxyl group of <bold>2</bold> was selectively
protected by reaction with monomethoxytrityl chloride
to give <bold>3</bold>. The 4-hydroxy group of <bold>3</bold> was then alkylated
with 2-phthalimidooxyethyl bromide in the presence of
potassium carbonate to give the protected AOQ derivative
<bold>4</bold>. Treatment of <bold>4</bold> with 80% acetic acid removed the
monomethoxytrityl group to produce compound <bold>5</bold>. The
corresponding EOQ derivative <bold>7</bold> was prepared by alkylation of <bold>3</bold> with bromoethane in the presence of potassium
carbonate followed by removal of the monomethoxytrityl
group. AOQ and EOQ derivatives <bold>5</bold> and <bold>7</bold> were each
converted to their phosphoramidite derivatives, <bold>6</bold> and <bold>8</bold>,
respectively, by reaction with 2-cyanoethyl chlorodiisopropylphosphoramidite. The phosphoramidites were purified by silica gel column chromatography. The AOQ
phosphoramidite, <bold>6</bold>, was precipitated from hexane and
was obtained as a white powder. In contrast the EOQ
phosphoramidite, <bold>8</bold>, failed to precipitate and was obtained as an oil.
</p><p>Both phosphoramidites were very reactive and under
some conditions appeared to be susceptible to conversion
to their H-phosphonate derivatives. For example, mass
spectrometric analysis of AOQ phosphoramidite <bold>6</bold> gave
a mass consistent with that of the H-phosphonate of AOQ
(<bold>6b</bold>). This species most likely arose from intramolecular
displacement of the phosphorus <italic toggle="yes">N</italic>,<italic toggle="yes">N</italic>-diisopropylamino
group by the nitrogen of the ureido linkage to produce a
five-member cyclic phosphoramidite (<bold>6a</bold>). The cyclic
phosphoramidite would be expected to be very reactive
and readily react with traces of water in the solvent to
give the H-phosphonate derivative. Consistent with this
explanation is the observation that acetonitrile solutions
of the AOQ or EOQ phosphoramidite showed conversion
to a slower migrating product when monitored by silica
gel TLC. The formation of this new compound correlated
with a decrease in coupling efficiency to the oligonucleotides, indicating the formation of an inactive species.
</p><p>The AOQ- and EOQ-conjugated oligonucleotides were
prepared by first synthesizing the oligomers on controlled
pore glass supports and removing the 5‘-terminal dimethoxytrityl group. The supports were then transferred to
an eppendorf tube or a glass autosampler vial that
contained the dry phosphoramidite. A solution of tetrazole in dry acetonitrile was then added via a syringe and
the coupling reaction was allowed to proceed for 10 min.
The support was then returned to the DNA synthesis
column, and capping and oxidation were carried out on
the DNA synthesizer.
</p><p>We initially tried deprotecting the AOQ-conjugated
oligonucleotide by standard treatment with concentrated
ammonium hydroxide solution at 65 °C for 5 h. However,
a multitude of peaks were observed when the reaction
mixture was analyzed by reversed phase HPLC (data not
shown). Many of these peaks were detected at both 260
and 320 nm, indicating the presence of the AOQ group.
However, among the AOQ containing peaks, only one
possessed a reactive aminooxy group, as assessed by its
ability to react with acetone to form the dimethyloxime
derivative. Thus, it appeared that most of the AOQ-containing oligomers represented species where the aminooxy group had reacted with some component in the
deprotection reaction solution. This observation is in
agreement with that of Salo et al., who reported that
attempts to purify aminooxy containing oligomers were
unsuccessful when concentrated ammonium hydroxide
was used for deprotection (<italic toggle="yes"><xref rid="bc0256381b00016" ref-type="bibr"></xref></italic>). To circumvent this
problem, an alternate deprotection method was developed
which involved treatment of the oligonucleotide with
ethylenediamine (EDA) at room temperature (<italic toggle="yes"><xref rid="bc0256381b00034" ref-type="bibr"></xref></italic>). To
preclude generation of side products resulting from
transamination of C by EDA during deprotection, N<sup>4</sup>-acetyl-C or N<sup>4</sup>-isobutryl-C protected phosphoramidite and
methylphosphonamidite, respectively, were used to synthesize the oligonucleotides (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00034" ref-type="bibr"></xref>, <xref rid="bc0256381b00035" ref-type="bibr"></xref></named-content></italic>). In separate experiments, we confirmed that no transamination occurred
when acetyl- or isobutryl-protected C was treated with
ethylenediamine under these conditions.
</p><p>The AOQ−oligo was cleaved from the support, and all
the protecting groups, including the phthalimide group,
were completely removed after treatment with ethylenediamine for 6 h at room temperature. For example, as
shown in Figure <xref rid="bc0256381f00003"></xref>B for AOQ-1843, essentially a single
peak was observed when the reaction mixture was
analyzed by C-18 reversed phase HPLC. This peak, which
adsorbed at both 260 and 320 nm, had a longer retention
time than that of nonconjugated oligomer, 1843, which
did not absorb at 320 nm. As shown in Figure <xref rid="bc0256381f00003"></xref>D, AOQ-1843 reacted readily with acetone to give the dimethyloxime derivative, whose retention time was greater than
that of AOQ-1843. The deprotected AOQ−oligo was
immediately purified by C-18 reversed-phase HPLC.
Storage of the purified AOQ−oligo in aqueous acetonitrile
over time resulted in the generation of additional compounds as observed by C-18 reversed phase HPLC (data
not shown). These compounds most likely result from
reaction of the aminooxy group with minute amounts of
aldehydes and/or ketones present in the solution as
previously observed by others (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00015" ref-type="bibr"></xref>, <xref rid="bc0256381b00017" ref-type="bibr"></xref></named-content></italic>). To prevent these
unwanted reactions, the solution containing the AOQ−oligo after desalting was immediately evaporated to
dryness and stored at −20 °C. Under these conditions,
the dry AOQ−oligo proved to be stable, and the aminooxy
function remained active.
<fig id="bc0256381f00003" position="float" orientation="portrait"><label>3</label><caption><p>Reverse-phase HPLC profile of (A) unmodified oligomer 1843, (B) AOQ-1843, (C) EOQ-1843, (D) dimethyloxime derivative of AOQ-1843, (E) leupeptin conjugated AOQ-1843. A 20 mL linear gradient of 2−50% acetonitrile (1 mL/min) in 50 mM sodium phosphate buffer (pH 5.8) was used.</p></caption><graphic xlink:href="bc0256381f00003.tif" position="float" orientation="portrait"></graphic></fig></p><p>In contrast to the AOQ−oligomers, the EOQ−oligonucleotides were successfully deprotected by treatment
with concentrated ammonium hydroxide at 65 °C. As
shown in Figure <xref rid="bc0256381f00003"></xref>C, EOQ-1843 gave a single peak whose
retention time on C-18 reversed phase HPLC was approximately 6 min longer than that of unconjugated 1843.
As was the case for the AOQ−oligomers, the EOQ−oligonucleotides absorbed at both 260 and 320 nm.
MALDI-TOF mass spectrometric analyses of both the
AOQ- and EOQ oligomers gave molecular weights consistent with the structures of the derivatized oligomers.
</p><p><bold>Interactions of AOQ- and EOQ-Conjugated Oligonucleotides with </bold><bold>Complementary Oligonucleotide
Targets.</bold> The interactions of AOQ-1843 and EOQ-1843
with oligo-2‘-<italic toggle="yes">O</italic>-methylribonucleotides 1855, 1856, 1885,
1886, and 1887 and with oligoribonucleotide 1960 were
studied by gel electrophoretic mobility shift assays at 37
°C. The sequences of the target oligonucleotides are
shown Figure <xref rid="bc0256381f00001"></xref>. AOQ- and EOQ-1843 are each complementary to nucleotides 2−9 of the target oligomers.
Hybridization of AOQ- or EOQ-1843 to targets 1855,
1856, 1885, 1886, or 1960 gives a duplex with a 3‘-overhanging end, whereas hybridization to 1887 produces
a duplex that lacks a 3‘-overhanging base. Most of the
binding studies were carried out using the oligo-2‘-<italic toggle="yes">O</italic>-methylribonucleotide targets because these oligomers are
easier to synthesize and handle than are RNA targets.
Duplexes formed between AOQ- or EOQ-1843 and either
the 2‘-<italic toggle="yes">O</italic>-methylribonucleotide or RNA target are predicted to be in the A-type conformation, and thus the
binding affinities of the oligomers for either target were
expected to be the same.
</p><p>As shown in Table <xref rid="bc0256381t00001"></xref>, conjugation of oligomer 1843 with
either AOQ or EOQ dramatically increased its binding
affinity to all the targets studied at 37 °C. Thus, the
dissociation constants, <italic toggle="yes">K</italic><sub>d</sub>, of AOQ- or EOQ-1843 to these
targets are in the low nanomolar range, whereas no
binding was observed between 1843 and any of the
targets, even at a concentration of 500 nM, which was
the highest concentration of oligomer tested. As expected,
the <italic toggle="yes">K</italic><sub>d</sub> values of duplexes formed with 2‘-<italic toggle="yes">O</italic>-methylribonucleotide target 1856 or RNA target 1960, which have
identical sequences, are almost identical. This result
confirms that the 2‘-<italic toggle="yes">O</italic>-methylribonucleotide targets are
good models for the RNA target in these binding studies.
<table-wrap id="bc0256381t00001" position="float" orientation="portrait"><label>1</label><caption><p>Apparent Dissociation Constant of Duplexes Formed between AOQ- or EOQ-1843 and Various Targets</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 colname="3"></oasis:entry><oasis:entry namest="4" nameend="6"><italic toggle="yes">K</italic><sub>d</sub> (nM)<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry namest="1" nameend="1"></oasis:entry><oasis:entry namest="2" nameend="2">target</oasis:entry><oasis:entry namest="3" nameend="3"></oasis:entry><oasis:entry namest="4" nameend="4">1843</oasis:entry><oasis:entry namest="5" nameend="5">EOQ-1843</oasis:entry><oasis:entry namest="6" nameend="6">AOQ-1843
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">2‘-<italic toggle="yes">O</italic>-Me
</oasis:entry><oasis:entry colname="2">1855
</oasis:entry><oasis:entry colname="3">mrACUGGGAGCU
</oasis:entry><oasis:entry colname="4">>500<italic toggle="yes"><sup>b</sup></italic><sup></sup></oasis:entry><oasis:entry colname="5">4.6 ± 2.5
</oasis:entry><oasis:entry colname="6">9.2 ± 1.0
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">1856
</oasis:entry><oasis:entry colname="3">mrACUGGGAGCC
</oasis:entry><oasis:entry colname="4">>500
</oasis:entry><oasis:entry colname="5">5.8 ± 1.7
</oasis:entry><oasis:entry colname="6">8.2 ± 2.4
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">1885
</oasis:entry><oasis:entry colname="3">mrACUGGGAGCG
</oasis:entry><oasis:entry colname="4">>500
</oasis:entry><oasis:entry colname="5">1.9 ± 0.4
</oasis:entry><oasis:entry colname="6">5.5 ± 4.1
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">1886
</oasis:entry><oasis:entry colname="3">mrACUGGGAGCA
</oasis:entry><oasis:entry colname="4">>500
</oasis:entry><oasis:entry colname="5">1.6 ± 0.4
</oasis:entry><oasis:entry colname="6">5.2 ± 0.9
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1"></oasis:entry><oasis:entry colname="2">1887
</oasis:entry><oasis:entry colname="3">mrACUGGGAGC
</oasis:entry><oasis:entry colname="4">>500
</oasis:entry><oasis:entry colname="5">15.3 ± 0.1
</oasis:entry><oasis:entry colname="6">19.6 ± 3.5
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">RNA
</oasis:entry><oasis:entry colname="2">1960
</oasis:entry><oasis:entry colname="3">rACUGGGAGCC
</oasis:entry><oasis:entry colname="4">>500
</oasis:entry><oasis:entry colname="5">5.7 ± 1.2
</oasis:entry><oasis:entry colname="6">7.1 ± 1.6</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Apparent dissociation constants were determined at 37 °C in
a buffer containing 100 mM sodium chloride, 10 mM Tris pH 7.5,
0.5 mM EDTA. <italic toggle="yes"><sup>b</sup></italic><sup></sup>The highest concentration used for the measurements.</p></table-wrap-foot></table-wrap></p><p>Targets 1855, 1856, 1885, 1886, and 1960 each have a
3‘-overhanging base adjacent to the oligomer binding site.
Removal of this base to give target 1887 results in
approximately 3−10 times increase in the <italic toggle="yes">K</italic><sub>d</sub> of the
duplex. This suggests that to some extent binding
involves the 3‘-base residue. Saito et al. have demonstrated that 2-amino-1,8-naphthyridine can bind to guanine via formation of specific hydrogen bonds (<italic toggle="yes"><xref rid="bc0256381b00036" ref-type="bibr"></xref></italic>). The
ureidoquinoline moiety of AOQ or EOQ is structurally
similar to 2-amino-1,8-naphthyridine and thus could
potentially form similar hydrogen bonds with the 3‘-C
residue of targets 1856 or 1960, as illustrated in Figure
<xref rid="bc0256381f00004"></xref>. However, as shown in Table <xref rid="bc0256381t00001"></xref>, the <italic toggle="yes">K</italic><sub>d</sub>s of the duplexes
formed with 1855, 1856, 1885, or 1886, each of which has
a different 3‘-base residue, are very similar, and thus it
is unlikely that such hydrogen bonding interactions are
the source of enhanced binding exhibited by AOQ- or
EOQ-1843.
<fig id="bc0256381f00004" position="float" orientation="portrait"><label>4</label><caption><p>Potential hydrogen bond formation between the AOQ or EOQ group and a cytosine in the target strand.</p></caption><graphic xlink:href="bc0256381f00004.tif" position="float" orientation="portrait"></graphic></fig></p><p>Both AOQ- and EOQ-1843 showed increased binding
affinities compared to that of 1843, suggesting that the
common aromatic quinoline ring of AOQ and EOQ
contributes significantly to the binding interaction. Steady-state fluorescence experiments at room temperature were
used to further investigate the binding mode. Because
there were no significant differences in the <italic toggle="yes">K</italic><sub>d</sub>s of AOQ-
or EOQ-1843, EOQ-1843 was used in these studies. The
quinoline ring of the EOQ group absorbs UV light at 310
and 322 nm and fluoresces at 351 nm when excited at
314 nm. This fluorescence was used to study the interaction of the quinoline fluorophore with neighboring
bases in the duplexes. Strong quenching and a concomitant red shift of the emission maximum have been
observed upon the intercalative binding of polycyclic
aromatic hydrocarbon-derivatized oligonucleotides with
complementary targets (<italic toggle="yes">37</italic>,<italic toggle="yes"> 38</italic>, and references therein).
On the other hand, moderate quenching and very little
or no red shift of the emission maximum has been
interpreted as pi-stacking of the fluorophore to the
exterior of the last base pair of the duplex (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00038" ref-type="bibr"></xref>−<xref rid="bc0256381b00039" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0256381b00040" ref-type="bibr"></xref></named-content></italic>).
</p><p>As shown in Figure <xref rid="bc0256381f00005"></xref>, the emission maximum of EOQ-1843 was clearly observed at 351 nm when excited at 314
nm, whereas 1843 did not show such emission. Duplexes
formed between EOQ-1843 and targets 1855, 1856, 1885,
1886, or 1887 all showed a moderate decrease in fluorescence intensity and no shift in the emission maximum.
As expected, there was no change in fluorescence intensity when equal amounts of EOQ-1843 and 1843 were
mixed because these two oligomers do not form a duplex.
These results are consistent with stacking of the quinoline ring on the end of the duplex rather than full
intercalation between the last two base pairs of the
duplex. In this binding mode, further stabilization could
occur if the quinoline ring interacts with a 3‘-overhanging
base as shown in Figure <xref rid="bc0256381f00002"></xref>A. The observation that 1855
and 1856, which have a pyrimidine overhang, have
slightly higher <italic toggle="yes">K</italic><sub>d</sub>s than 1885 and 1886, which have a
purine overhang, is consistent with this model. Thus, the
quinoline ring may stack more effectively with the purine
overhang leading to a lower <italic toggle="yes">K</italic><sub>d</sub>. In essence, the AOQ and
EOQ groups may serve as “caps” to prevent fraying of
terminal bases on the duplexes (<italic toggle="yes">39</italic>,<italic toggle="yes"> 41−43</italic>).
<fig id="bc0256381f00005" position="float" orientation="portrait"><label>5</label><caption><p>Emission spectra of EOQ-1843 with its complementary target. Emission spectra were measured in a buffer containing 100 mM sodium chloride, 10 mM Tris pH 7.5, and 0.5 mM ethylenediaminetetraacetate at room temperature. 1843 only (+), EOQ-1843 only (○), EOQ-1843:1843 duplex (·), and EOQ-1843:1855 duplex (□).</p></caption><graphic xlink:href="bc0256381f00005.tif" position="float" orientation="portrait"></graphic></fig></p><p>The <italic toggle="yes">K</italic><sub>d</sub>s of duplexes formed by EOQ-1843 are 1.2−3.2
times lower than those of duplexes formed by AOQ-1843.
This result suggests that Michael adduct formation by
AOQ-1843 either does not take place, or if it does, that
such formation does not contribute to the overall stability
of the duplex. Deuterium exchange experiments were
carried out to directly investigate the possible formation
of Michael adducts. Equal amounts of AOQ-1843 and
RNA target 1960 were incubated at room temperature
overnight in buffer prepared with deuterated water. RNA
1960, which contains two cytosines in proximity to the
AOQ binding site, was then digested with a combination
of snake venom phosphodiesterase and calf intestinal
phosphatase, and the resulting cytidine was isolated by
C-18 reversed phase HPLC. The molecular weight of the
cytidine was then determined by EIS mass spectrometry.
If a Michael adduct formed, deuterium from the buffer
would be incorporated at the 5 position of cytidine,
resulting in a 1 mass unit increase in the molecular
weight. No mass increase was observed for the recovered
cytidine, suggesting that the aminooxy group of AOQ-1843 does not participate to any significant extent in
Michael adduct formation with the C residues of target
RNA. A control experiment using 1-(2-aminooxyethyl)
cytosine, which undergoes intramolecular Michael adduct
formation (<italic toggle="yes"><xref rid="bc0256381b00044" ref-type="bibr"></xref></italic>), showed that adduct formation could be
detected by both NMR and mass spectrometry (data not
shown). The inability of AOQ-1843 to form a Michael
adduct may be due to unfavorable positioning of the
aminooxy group when AOQ-1843 is bound to its target.
</p><p>AOQ- or EOQ-1843 shows at least a 2 orders of
magnitude increased binding affinity for the short, linear
RNA target, 1960, compared to the nonconjugated oligomer, 1843. To determine if an EOQ-conjugated oligomer
could also bind to a longer, structured RNA target, we
studied the interaction between EOQ-1676 and HIV TAR
RNA. This RNA target, whose sequence and secondary
structure are shown in Figure <xref rid="bc0256381f00001"></xref>, consists of two stems,
20 base pairs and 4 base pairs in length, joined by a 3
base bulge and capped with a 6 base loop. We have shown
previously that 1676, which contains a nuclease resistant
alternating methylphosphonate/phosphodiester backbone, binds with high affinity to this target at 37 °C (<italic toggle="yes"><xref rid="bc0256381b00032" ref-type="bibr"></xref></italic>).
The interactions of 1676 and EOQ-1676 with TAR RNA
were analyzed by gel mobility shift experiments. As
shown in Table <xref rid="bc0256381t00002"></xref>, EOQ-conjugated 1676 forms a duplex
with TAR RNA whose apparent <italic toggle="yes">K</italic><sub>d</sub> is approximately 2.3
times less than that of the duplex formed by nonconjugated 1676. These results demonstrate that conjugation
with EOQ can also enhance the binding affinity of
oligonucleotides for targets with extensive secondary
structure.
<table-wrap id="bc0256381t00002" position="float" orientation="portrait"><label>2</label><caption><p>Apparent Dissociation Constants of TAR RNA Duplexes</p></caption><oasis:table colsep="2" rowsep="2"><oasis:tgroup cols="2"><oasis:colspec colnum="1" colname="1"></oasis:colspec><oasis:colspec colnum="2" colname="2"></oasis:colspec><oasis:tbody><oasis:row><oasis:entry namest="1" nameend="1">oligomer</oasis:entry><oasis:entry namest="2" nameend="2"><italic toggle="yes">K</italic><sub>d</sub> (nM)<italic toggle="yes"><sup>a</sup></italic><sup></sup></oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">1676
</oasis:entry><oasis:entry colname="2">101 ± 19
</oasis:entry></oasis:row><oasis:row><oasis:entry colname="1">EOQ-1676
</oasis:entry><oasis:entry colname="2">43 ± 7.4</oasis:entry></oasis:row></oasis:tbody></oasis:tgroup></oasis:table><table-wrap-foot><p><italic toggle="yes"><sup>a</sup></italic><sup></sup> Apparent dissociation constants were determined at 37 °C in
a buffer containing 100 mM sodium chloride, 10 mM Tris pH 7.5,
0.5 mM EDTA.</p></table-wrap-foot></table-wrap></p><p><bold>Conjugation of AOQ-Derivatized Oligonucleotide
with Peptides.</bold> The aminooxy group reacts readily with
aldehydes and ketones (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00015" ref-type="bibr"></xref>−<xref rid="bc0256381b00016" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0256381b00017" ref-type="bibr"></xref></named-content></italic>), and the resulting oxime
linkage is very stable under physiological conditions (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00015" ref-type="bibr"></xref>,
<xref rid="bc0256381b00027" ref-type="bibr"></xref></named-content></italic>). This chemistry has been widely utilized for chemoselective ligation of a variety of compounds. In particular,
Forget et al. have recently reported the successful
preparation of peptide−oligonucleotide conjugates in
which aldehyde containing peptides are conjugated with
aminooxy-derivatized oligonucleotides or vice versa (<italic toggle="yes"><xref rid="bc0256381b00015" ref-type="bibr"></xref></italic>).
</p><p>We have explored oxime formation to conjugate AOQ−oligonucleotides to small peptides. The AOQ group may
be advantageous for this purpose because as shown
above, AOQ-derivatized oligonucleotides have increased
binding affinity for their RNA targets. Thus, the AOQ
group could provide a duplex-stabilizing platform for
chemoselective ligation of AOQ−oligonucleotides to a
variety of compounds.
</p><p>Initially, we conjugated commercially available, synthetic leupeptin to AOQ-1843. This tripeptide has a
unique aldehyde group at its C-terminus that can react
selectively with the aminooxy group of AOQ-1843 to form
an oxime. As shown in the chromatogram in Figure <xref rid="bc0256381f00002"></xref>E,
the conjugation reaction was complete within 60 min at
room temperature when carried out in 50% aqueous
acetonitrile. The peptide−oligo conjugate was purified by
C-18 reversed phase HPLC and its structure was confirmed by MALDI-TOF mass spectrometry analysis.
</p><p>Conjugations between an oligonucleotide aminooxy
group and a peptide aldehyde group are very efficient.
Introduction of aldehyde groups into peptides can be
accomplished by periodate oxidation of an N-terminus
serine or threonine (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00027" ref-type="bibr"></xref>, <xref rid="bc0256381b00045" ref-type="bibr"></xref></named-content></italic>), or by use of specific linker
resins during solid-phase peptide synthesis (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00046" ref-type="bibr"></xref>, <xref rid="bc0256381b00047" ref-type="bibr"></xref></named-content></italic>). An
alternative approach would be to introduce into the
peptide postsynthetically, a reactive functional group that
could be conjugated with an aminooxy group of the
oligonucleotide. To this end we developed a method to
introduce a ketone functional group into a peptide by
alkylating a cysteine residue in the peptide with bromoacetone, as illustrated in Scheme 2. The ketone functionality could be introduced anywhere in a peptide that
already contains a cysteine residue or the peptide could
be synthesized with a cysteine residue at its N terminus.
The carbonyl group of the resulting keto-peptide then
serves as a reactive site for conjugation with the aminooxy group of the oligonucleotide. This procedure is in
some ways analogous to the recently reported procedure
in which ketone modified DNA is conjugated with aminooxy-derivatized compounds (<italic toggle="yes"><xref rid="bc0256381b00048" ref-type="bibr"></xref></italic>).
<fig id="bc0256381h00002" position="float" orientation="portrait"><label></label><graphic xlink:href="bc0256381h00002.tif" position="float" orientation="portrait"></graphic></fig></p><p>We tested this procedure on a commercially available
tetrapeptide, RGDC, and on a cysteine-modified 14 amino
acid peptide derived from the transduction domain of HIV
Tat protein. The RGD peptide motif has been used for
cell targeting and gene delivery (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00049" ref-type="bibr"></xref>, <xref rid="bc0256381b00050" ref-type="bibr"></xref></named-content></italic>), and the Tat
peptide has been shown to promote the delivery of
macromolecules into cells (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00051" ref-type="bibr"></xref>−<xref rid="bc0256381b00052" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bc0256381b00053" ref-type="bibr"></xref></named-content></italic>).
</p><p>The C-terminus cysteine residue of each peptide was
first reacted with a 10−33-fold excess of bromoacetone
to give the keto-peptide. The reaction was complete
within 1 h as assayed by C-18 reversed phase HPLC.
Three major peaks were observed in the chromatogram.
The first peak was the unreacted bromoacetone, the
second peak was the desired keto-peptide, and the third
peak was the disulfide-linked peptide dimer. The keto-peptide was obtained in 50% yield for each peptide after
purification by HPLC.
</p><p>The keto-peptides were then reacted with the short
oligo-2‘-<italic toggle="yes">O</italic>-methylribonucleotide, AOQ-2106. As shown by
the chromatograms in Figure <xref rid="bc0256381f00006"></xref>, the conjugation reaction
between keto-RGD and AOQ-2106 was complete within
1 h at room temperature. The peptide−oligonucleotide
conjugate appeared as two peaks in the chromatogram.
Subsequent characterization of each peak by MALDI-TOF mass spectrometry showed that they had identical
molecular weights. Thus these two peaks are attributed
to the two isomeric oximes shown in Scheme 2.
<fig id="bc0256381f00006" position="float" orientation="portrait"><label>6</label><caption><p>Reversed-phase (RP) HPLC profile of the reaction of keto-RGDC peptide and AOQ-2106 in 50% acetonitrile/water at room temperature. A 20 min linear gradient of 2−50% acetonitrile in 50 mM sodium phosphate (pH 5.8) was used. (A)<italic toggle="yes">T</italic> = 0 h, (B) <italic toggle="yes">T</italic> = 1 h.</p></caption><graphic xlink:href="bc0256381f00006.tif" position="float" orientation="portrait"></graphic></fig></p><p>Keto-derivatized Tat peptide was reacted with AOQ-2016 in 50% acetonitrile/water at room temperature.
Reactions were run using a 5-fold excess of keto-peptide
to oligonucleotide or with a 8-fold excess of oligonucleotide
to keto-peptide. Examination of the reaction mixtures by
MALDI-TOF mass spectrometry showed the presence of
a compound whose molecular weight was consistent with
the composition of the AOQ-2016-Tat conjugate. In
addition to the conjugate, AOQ-2106 or keto-Tat peptide
was also detected in the mass spectrum, depending on
whether the reaction was run using an excess of oligonucleotide or an excess of keto-peptide.
</p><p>Although we were able to detect the oligonucleotide−peptide conjugate by mass spectrometry, we were unable
to detect the conjugate by C-18 reversed phase HPLC. A
variety of solvent systems and columns were tested
including those previously reported to be effective in
eluting oligonucleotide−Tat conjugates (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bc0256381b00054" ref-type="bibr"></xref>, <xref rid="bc0256381b00055" ref-type="bibr"></xref></named-content></italic>). This
apparent irreversible absorption to the C-18 column
appears to be a property of the conjugate because when
unmodified 2106 and Tat peptide were mixed, both
compounds could be detected by C-18 reversed phase
HPLC.
</p><p>We found that the Tat-2106 conjugate could be purified
by DEAE Sephadex chromatography. The keto-Tat peptide has a net positive charge of +9, whereas AOQ-2106
has net negative charge of −4. Thus, the AOQ-2106−Tat conjugate would be expected to have a net positive
charge of +5. We exploited this charge difference to purify
the conjugate. Mass spectrometry showed that all of the
keto-Tat could be converted to conjugate by carrying out
the reaction in the presence of excess AOQ-2106. The
excess AOQ-2106 was removed by simply passing the
reaction mixture through a DEAE Sephadex column. The
negatively charged AOQ-2106 was retained on the column, while the AOQ-2106−Tat conjugate, which is
positively charged, passed through the column. The
molecular weight of the purified oligo−Tat conjugate was
confirmed by MALDI-TOF mass spectrometry analysis.
Using this procedure, pure AOQ-2106−Tat conjugate was
obtained at 39% yield.
</p></sec><sec id="d7e1402"><title>Conclusions</title><p>AOQ- and EOQ-conjugated oligo-2‘-<italic toggle="yes">O</italic>-methylribonucleotides show enhanced binding affinities for complementary RNA targets. This most likely results from favorable
stacking interactions between the quinoline ring and the
terminal base pair of the duplex formed by the oligomer
and the target. Although we could find no evidence that
the aminooxy group of AOQ reacts with 5,6 double bond
of cytosines in the target RNA, this functional group does
provide a site for efficient conjugation of AOQ−oligonucleotides to peptides or other moieties that bear an
aldehyde or ketone group. The combination of the unique
reactivity of the aminooxy group and enhanced binding
affinity conferred by its quinoline ring suggest that AOQ
may serve as a useful platform for the preparation of
novel oligonucleotide conjugates.
</p></sec></body><back><ack><title>Acknowledgments</title><p>The authors thank Dr. Eric Hildebrand for assistance
with the fluorescence experiments, Dr. Anne Noronha for
help obtaining the mass spectra, and Dr. David Noll for
helpful comments and suggestions. This research was
supported by a grant from the National Institutes of
Health (GM57140).
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Phone: 410-955-3489. Fax: 410-955-2926. E-mail: pmiller@jhsph.edu.</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">2003-02-06</dateCreated><dateIssued encoding="w3cdtf">2003-03-19</dateIssued><copyrightDate encoding="w3cdtf">2003</copyrightDate></originInfo><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>Oligo-2‘-O-methylribonucleotides conjugated with 4-(2-aminooxyethoxy)-2-(ethylureido)quinoline (AOQ) and 4-ethoxy-2-(ethylureido)quinoline (EOQ) were prepared by reaction of the AOQ or EOQ phosphoramidite with the protected oligonucleotide on a controlled pore glass support. Deprotection with ethylenediamine enabled successful isolation and purification of the highly reactive AOQ-conjugated oligomer. Polyacrylamide gel electrophoresis mobility shift experiments showed that the dissociation constants of complexes formed between an AOQ- or EOQ-conjugated 8-mer and complementary RNA or 2‘-O-methyl-RNA targets (9- and 10-mers) were in the low nM concentration range at 37 °C, whereas no binding was observed for the corresponding nonconjugated oligomer, even at a concentration of 500 nM. Fluorescence studies suggested that this enhanced affinity is most likely due to the ability of the quinoline ring of the AOQ or EOQ group to stack on the last base pair formed between the oligomer and target, thus stabilizing the duplex. The binding affinity of a 2‘-O-methyl RNA 15-mer, which contained an alternating methylphosphonate/phosphodiester backbone, for a 59-nucleotide stem-loop HIV TAR RNA target, increased 2.3 times as a consequence of conjugation with EOQ. The aminooxy group of AOQ-conjugated oligomers is a highly reactive nucleophile, which reacts readily with aldehydes and ketones to form stable oxime derivatives. This feature was used to couple an AOQ−oligomer with leupeptin, a tripeptide that contains a C-terminus aldehyde group. A simple method was developed to introduce a ketone functionality into peptides that contain a cysteine residue by reacting the peptide with bromoacetone. The resulting keto-peptide was then coupled to the AOQ−oligomer. This procedure was used to prepare oligonucleotide conjugates of a tetrapeptide, RGDC, and a derivative of HIV tat peptide having a C-terminus cysteine. The combination of the unique reactivity of the aminooxy group and enhanced binding affinity conferred by its quinoline ring suggests that AOQ may serve as a useful platform for the preparation of novel oligonucleotide conjugates.</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>2003</date><detail type="volume"><caption>vol.</caption><number>14</number></detail><detail type="issue"><caption>no.</caption><number>2</number></detail><extent unit="pages"><start>320</start><end>330</end></extent></part></relatedItem><relatedItem type="references" ID="bc0256381b00001" displayLabel="bibbc0256381b00001"><titleInfo><title>Nucleic acid therapeutics: state of the art and future prospects</title></titleInfo><titleInfo contentType="CDATA"><title>Nucleic acid therapeutics: state of the art and future prospects</title></titleInfo><name type="personal"><namePart type="family">GEWIRTZ</namePart><namePart type="given">A. 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