Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015;34(3):180-98.
doi: 10.1080/15257770.2014.978011.

Practical synthesis of cytidine-5-carboxamide-modified nucleotide reagents

John C Rohloff  1 , Catherine Fowler, Brian Ream, Jeffrey D Carter, Greg Wardle, Tim Fitzwater
Affiliations

Practical synthesis of cytidine-5-carboxamide-modified nucleotide reagents

John C Rohloff et al. Nucleosides Nucleotides Nucleic Acids. 2015.

Abstract

Chemically-modified derivatives of cytidine, bearing a 5-(N-substituted-carboxamide) functional group, are new reagents for use in aptamer discovery via the SELEX process (Systematic Evolution of Ligands by EXponential enrichment). Herein, we disclose a practical synthesis of 5-(N-benzylcarboxamide)-2'-deoxycytidine, and the corresponding 5-(N-1-naphthylmethylcarboxamide)- and 5-(N-3-phenylpropylcarboxamide)-2'-deoxycytidine analogs, as both the suitably-protected 3'-O-cyanoethylphosphoramidite reagents (CEP; gram scale) and the 5'-O-triphosphate reagents (TPP; milligram-scale). The key step in the syntheses is a mild, palladium(0)-catalyzed carboxyamidation of an unprotected 5-iodo-cytidine. Use of the CEP reagents for solid-phase oligonucleotide synthesis was demonstrated and incorporation of the TPP reagents by KOD polymerase in a primer extension assay confirmed the utility of these reagents for SELEX. Finally, the carboxyamidation reaction was also used to prepare the nuclease-resistant sugar-variants: 5-(N-benzylcarboxamide)-2'-O-methyl-cytidine and 5-(N-3-phenylpropylcarboxamide)-2'-deoxy-2'-fluoro-cytidine.

Keywords: Aptamer; SELEX; cytidine-5-carboxamide; modified nucleotide.

PubMed Disclaimer

Figures

None
Chemical structures of representative 2′-deoxyuridine-5-carboxamides.
Figure 2
Figure 2. General synthetic process for uridine-5-carboxamides via Matsuda's “activated ester” intermediate 1.
Figure 3
Figure 3. Synthesis scheme for 5-(N-substituted-carboxamide)-2′-deoxycytidine CEP reagents by carboxyamidation of 5-iodo-2′-deoxycytidine.
Figure 4
Figure 4. Synthesis scheme for 5-(N-substituted-carboxamide)-2′-deoxycytidine-5′-O-triphosphate reagents.
Figure 5
Figure 5. Synthesis scheme for 2′-substituted-cytidine nucleosides by carboxyamidation of a 5-iodocytidine.
Figure 6
Figure 6. Polyacrylamide gel image of Primer Extension Assay with dNTP's, as described in Material and Methods. Lane 1: dAdGdT (negative control); Lane 2: dAdGdTdC (control); Lane 3: dAdGdT + 10a; Lane 4: dAdGdT + 10b; Lane 5: dAdGdT + 10c; Lane 6: 20/200 DNA Ladder.
See this image and copyright information in PMC

References

    1. Gold L. Ayers D. Bertino J. Bock C. Bock A. Brody E.N. Carter J. Dalby A. Eaton B. Fitzwater T. Aptamer-based proteomic technology for biomarker discovery. PLoS ONE. 2010;5(12):e15004. - PMC - PubMed
    1. Hollenstein M. Synthesis of deoxynucleoside triphosphates that include proline, Urea, or sulfonamide groups and their polymerase incorporation into DNA. Chem., A Eur. J. 2012;18:13320–13330. - PubMed
    1. Imaizumi Y. Kasahara Y. Fujita H. Kitadume S. Ozaki H. Endoh T. Kuwahara M. Sugimoto N. Efficacy of base-modification on target binding of small molecule DNA aptamers. J. Am. Chem. Soc. 2013;135(25):9412–9419. - PubMed
    1. Davies D.R. Gelinas A.D. Zhang C. Rohloff J.C. Carter J.D. O’Connell D. Waugh S.M. Wolk S.M. Mayfield W.S. Burgin A.B. Edwards T.E. Stewart L.J. Gold L. Janjic N. Jarvis T.C. Unique motifs and hydrophobic interactions shape the binding of modified DNA ligands to protein targets. PNAS. 2012;190(49):19971–19976. - PMC - PubMed
    1. Lee K.Y. Kang H. Ryu S.H. Lee D.S. Lee J.H. Kim S. Bioimaging of nucleolin aptamer-containing 5-(N-benzylcarboxamide)-2′-deoxyuridine more capable of specific binding to targets in cancer cells. J. Biomed. Biotechnol. 2010 article ID 168306, 9 pages. - PMC - PubMed

MeSH terms