Skip to main content
SpringerLink
Log in

Site-Specific Modification of Nucleobases in Oligonucleotides

  • Chapter
  • First Online:
Book cover Synthesis of Therapeutic Oligonucleotides

Abstract

Site-specific modification of oligonucleotides is a powerful approach for synthesis of oligonucleotides containing various derivatives of nucleobases, sugars, and phosphate backbones. By using this method, the structure of a modified moiety can be effectively screened to find artificial oligonucleotides having desired functions. In fact, many studies using a variety of site-specific modification methods have been reported to date. In this chapter, the site-specific modifications focusing on nucleobases within oligonucleotides are summarized. Moreover, as an experimental example of site-specific modification of an oligonucleotide, the preparation of N,N-disubstituted cytosine derivatives using reaction of 4-triazolylpyrimidin-2-one nucleobase with various secondary amines is demonstrated.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Chapter
USD 29.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 149.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Hardcover Book
USD 199.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Goodchild J (1990) Conjugations of oligonucleotides and modified oligonucleotides: a review of their synthesis and properties. Bioconjug Chem 1:165–187

    Article  CAS  PubMed  Google Scholar 

  2. Verma S, Eckstein F (1998) Modified oligonucleotides: synthesis and strategy for users. Annu Rev Biochem 67:99–134

    Article  CAS  PubMed  Google Scholar 

  3. Gramlich PME, Wirges CT, Manetto A, Carell T (2008) Postsynthetic DNA modification through the copper-catalyzed azide-alkyne cycloaddition reaction. Angew Chem Int Ed 47:8350–8358

    Article  CAS  Google Scholar 

  4. El-Sagheer AH, Brown T (2010) Click chemistry with DNA. Chem Soc Rev 39:1388–1405

    Article  CAS  PubMed  Google Scholar 

  5. Shaughnessy KH (2015) Palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides. Molecules 20:9419–9454

    Article  CAS  PubMed  Google Scholar 

  6. Sung WL (1981) Synthesis of 4-triazolopyrimidinone nucleotide and its application in synthesis of 5-methylcytosine-containing oligodeoxyribonucleotides. Nucleic Acids Res 9:6139–6151

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Sung WL (1982) Synthesis of 4-(1,2,4-triazol-1-yl)pyrimidin-2(1H)-one ribonucleotide and its application in synthesis of oligoribonucleotides. J Org Chem 47:3623–3628

    Article  CAS  Google Scholar 

  8. Webb TR, Matteucci MD (1986) Sequence-specific cross-linking of deoxyoligonucleotides via hybridization-triggered alkylation. J Am Chem Soc 108:2764–2765

    Article  CAS  Google Scholar 

  9. Webb TR, Matteucci MD (1986) Hybridization triggered cross-linking of deoxyoligonucleotides. Nucleic Acids Res 14:7661–7674

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Shigdel UK, Zhang J, He C (2008) Diazirine-based DNA photo-cross-linking probes for the study of protein-DNA interaction. Angew Chem Int Ed 47:90–93

    Article  CAS  Google Scholar 

  11. Semenyuk A, Darian E, Liu J, Majumdar A, Cuenoud B, Miller PS, MacKerell AD Jr, Seidman MM (2010) Targeting of an interrupted polypurine:polypyrimidine sequence in mammalian cells by a triplex-forming oligonucleotide containing a novel base analogue. Biochemistry 49:7867–7878

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hari Y, Akabane M, Hatanaka Y, Nakahara M, Obika S (2011) A 4-[(3R,4R)-dihydroxypyrrolidino]pyrimidin-2-one nucleobase for a CG base pair in triplex DNA. Chem Commun 47:4424–4426

    Article  CAS  Google Scholar 

  13. Hari Y, Akabane M, Obika S (2013) 2′,4′-BNA bearing a chiral guanidinopyrrolidine-containing nucleobase with potent ability to recognize the CG base pair in a parallel-motif DNA triplex. Chem Commun 49:7421–7423

    Article  CAS  Google Scholar 

  14. Fernandez-Forner D, Palom Y, Ikuta S, Pedroso E, Eritja R (1990) Synthesis and characterization of oligodeoxynucleotides containing the mutagenic base analogue 4-O-ethylthymine. Nucleic Acids Res 18:5729–5734

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Xu Y-Z, Zheng Q, Swann PF (1992) Synthesis of DNA containing modified bases by postsynthetic substitution. Synthesis of oligomers containing 4-substituted thymine: O 4-Alkylthymine, 5-methylcytosine, N 4-(dimethylamino)-5-methylcytosine, and 4-thiothymine. J Org Chem 57:3839–3845

    Article  CAS  Google Scholar 

  16. Xu Y-Z, Swann PF (1992) Chromatographic separation of oligodeoxynucleotides with identical length: application to purification of oligomers containing a modified base. Anal Biochem 204:185–189

    Article  CAS  PubMed  Google Scholar 

  17. Robles J, Grandas A, Pedroso E (2001) Synthesis of modified oligonucleotides containing 4-guanidino-2-pyrimidinone nucleobases. Tetrahedron 57:179–194

    Article  CAS  Google Scholar 

  18. Allerson CR, Chen SL, Verdine GL (1997) A chemical method for site-specific modification of RNA: the convertible nucleoside approach. J Am Chem Soc 119:7423–7433

    Article  CAS  Google Scholar 

  19. Komatsu Y, Kumagai I, Otsuka E (1999) Investigation of the recognition of an important uridine in an internal loop of a hairpin ribozyme prepared using post-synthetically modified oligonucleotides. Nucleic Acids Res 27:4314–4323

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. Aviñó A, García RG, Eritja R (2004) Synthesis of oligonucleotides containing 4-thiouridine using the convertible nucleoside approach and the 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl group. Nucleos Nucleot Nucleic Acids 23:1767–1777

    Article  CAS  Google Scholar 

  21. Schulhof JC, Molko D, Teoule R (1987) Facile removal of new base protecting groups useful in oligonucleotide synthesis. Tetrahedron Lett 28:51–54

    Article  CAS  Google Scholar 

  22. MacMillan AM, Verdine GL (1990) Synthesis of functionally tethered oligodeoxynucleotides by the convertible nucleoside approach. J Org Chem 55:5931–5933

    Article  CAS  Google Scholar 

  23. MacMillan AM, Verdine GL (1991) Engineering tethered DNA molecules by the convetible nucleoside approach. Tetrahedron 47:2603–2616

    Article  CAS  Google Scholar 

  24. MacMillan AM, Chen L, Verdine GL (1992) Synthesis of an oligonucleotide suicide substrate for DNA methyltransferases. J Org Chem 57:2989–2991

    Article  CAS  Google Scholar 

  25. Coleman RS, Siedlecki JM (1992) Synthesis of a 4-thio-2′-deoxyuridine-containing oligonucleotide. Development of the thiocarbonyl group as a linker element. J Am Chem Soc 114:9229–9230

    Article  CAS  Google Scholar 

  26. Coleman RS, Kesicki EA (1994) Synthesis and postsynthetic modification of oligonucleotides containing 4-thio-2′-deoxyuridine (ds4U). J Am Chem Soc 116:11636–11642

    Google Scholar 

  27. Rublack N, Nguyen H, Appel B, Springstubbe D, Strohbach D, Müller S (2011) Synthesis of specifically modified oligonucleotides for application in structural and functional analysis of RNA. J Nucleic Acids 2011:1–18

    Article  CAS  Google Scholar 

  28. Connolly BA, Newman PC (1989) Synthesis and properties of oligonucleotides containing 4-thiothymidine, 5-methyl-2-pyrimidinone-1-β-d-(2′-deoxyribose) and 2-thiothymidine. Nucleic Acids Res 17:4957–4974

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Draper DE (1984) Attachment of reporter groups to specific, selected cytidine residues in RNA using a bisulfite-catalyzed transamination reaction. Nucleic Acids Res 12:989–1002

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Miller PS, Cushman CD (1992) Selective modification of cytosines in oligodeoxyribonucleotides. Bioconjug Chem 3:74–79

    Article  CAS  PubMed  Google Scholar 

  31. Huang C-Y, Cushman CD, Miller PS (1993) Triplex formation by an oligonucleotide containing N 4-(3-acetamidopropyl)cytosine. J Org Chem 58:5048–5049

    Article  CAS  Google Scholar 

  32. Okamura H, Tanigushi Y, Sasaki S (2013) N-(Guanidinoethyl)-2′-deoxy-5-methylisocytidine exhibits selective recognition of a CG interrupting site for the formation of anti-parallel triplexes. Org Biomol Chem 11:3918–3924

    Article  CAS  PubMed  Google Scholar 

  33. Maier MA, Barber-Peoc’h I, Manoharan M (2002) Postsynthetic guanidinylation of primary amino groups in the minor and major grooves of oligonucleotides. Tetrahedron Lett 43:7613–7616

    Article  CAS  Google Scholar 

  34. Akabane-Nakata M, Obika S, Hari Y (2014) Synthesis of oligonucleotides containing N,N-disubstituted 3-deazacytosine nucleobases by post-elongation modification and their triplex-forming ability with double-stranded DNA. Org Biomol Chem 12:9011–9015

    Article  CAS  PubMed  Google Scholar 

  35. Kim SJ, Stone MP, Harris CM, Harris TM (1992) A postoligomerization synthesis of oligodeoxynucleotides containing polycyclic aromatic hydrocarbon adducts at the N6 position of deoxyadenosine. J Am Chem Soc 114:5480–5481

    Article  CAS  Google Scholar 

  36. Harris CM, Zhou L, Strand EA, Harris TM (1991) New strategy for the synthesis of oligodeoxynucleotides bearing adducts at exocyclic amino sites of purine nucleosides. J Am Chem Soc 113:4328–4329

    Article  CAS  Google Scholar 

  37. Wang H, Kozekov ID, Kozekova A, Tamura P, Marnett LJ, Harris TM, Rizzo CJ (2006) Site-specific synthesis of oligonucleotides containing malondialdehyde adducts of deoxyguanosine and deoxyadenosine via a post-synthetic modification strategy. Chem Res Toxicol 19:1467–1474

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Kierzek E, Kierzek R (2003) The synthesis of oligoribonucleotides containing N 6 -alkyladenosines and 2-methylthio-N 6 -alkyladenosines via post-synthetic modification of precursor oligomers. Nucleic Acids Res 31:4461–4471

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Gao H, Fathi R, Gaffney BL, Goswami B, Kung P-P, Rhee Y, Jin R, Jones RA (1992) 6-O-(Pentafluorophenyl)-2′-deoxyguanosine: a versatile synthon for nucleoside and oligonucleotide synthesis. J Org Chem 57:6954–6959

    Article  CAS  Google Scholar 

  40. Xu Y-Z, Zheng Q, Swann PF (1992) Synthesis and duplex stability of oligodeoxynucleotides containing 6-mercaptopurine. Tetrahedron Lett 33:5837–5840

    Article  CAS  Google Scholar 

  41. Xu Y-Z, Zheng Q, Swann PF (1992) Synthesis by post-synthetic substitution of oligomers containing guanine modified at the 6-position with S-, N-, O-derivatives. Tetrahedron 48:1729–1740

    Article  CAS  Google Scholar 

  42. Xu Y-Z (1996) Post-synthetic introduction of labile functionalities onto purine residues via 6-methylthiopurines in oligodeoxyribonucleotides. Tetrahedron 52:10737–10750

    Article  CAS  Google Scholar 

  43. Ferentz AE, Verdine GL (1991) Disulfide cross-linked oligonucleotides. J Am Chem Soc 113:4000–4002

    Article  CAS  Google Scholar 

  44. Erlanson DA, Chen L, Verdine GL (1993) DNA methylation through a locally unpaired intermediate. J Am Chem Soc 115:12583–12584

    Article  CAS  Google Scholar 

  45. Schmid N, Behr J-P (1995) Recognition of DNA sequences by strand replacement with polyamino-oligonucleotides. Tetrahedron Lett 36:1447–1450

    Article  CAS  Google Scholar 

  46. Ono A, Haginoya N, Kiyokawa M, Minakawa N, Matsuda A (1994) Nucleosides and nucleotides. 127. A novel and convenient post-synthetic modification method for the synthesis of oligodeoxyribonucleotides carrying amino linkers at the 5-position of 2′-deoxyuridine. Bioorg Med Chem Lett 4:361–366

    Article  CAS  Google Scholar 

  47. Haginoya N, Ono A, Nomura Y, Ueno Y, Matsuda A (1997) Nucleosides and nucleotides. 160. Oligodeoxyribonucleotides containing 5-(N-aminoalkyl)carbamoyl-2′-deoxyuridines by a new postsynthetic modification method and their thermal stability and nuclease-resistance properties. Bioconjug Chem 8:271–280

    Article  CAS  PubMed  Google Scholar 

  48. Ueno Y, Ogawa A, Nakagawa A, Matsuda A (1996) Nucleosides and nucleotides. 162. Facile synthesis of 5′,5′-linked oligodeoxyribonucleotides with the potential for triple-helix formation. Bioorg Med Chem Lett 6:2817–2822

    Article  CAS  Google Scholar 

  49. Nomura Y, Ueno Y, Matsuda A (1997) Site-specific introduction of functional groups into phosphodiester oligodeoxynucleotides and their thermal stability and nuclease-resistance properties. Nucleic Acids Res 25:2784–2791

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  50. Ono A, Dan A, Matsuda A (1993) Nucleosides and nucleotides. 121. Synthesis of oligonucleotides carrying linker groups at the 1′-position of sugar residues. Bioconjug Chem 4:499–508

    Article  CAS  PubMed  Google Scholar 

  51. Nomura Y, Haginoya N, Ueno Y, Matsuda A (1996) Nucleosides and nucleotides. 161. Incorporation of 5-(N-aminoalkyl)carbamoyl-2′-deoxycytidines into oligodeoxyribonucleotides by a convenient post-synthetic modification method. Bioorg Med Chem Lett 6:2811–2816

    Article  CAS  Google Scholar 

  52. Lietard J, Leumann CJ (2012) Synthesis, pairing, and cellular uptake properties of C(6′)-functionalized tricycle-DNA. J Org Chem 77:4566–4577

    Article  CAS  PubMed  Google Scholar 

  53. Hari Y, Osawa T, Obika S (2012) Synthesis and duplex-forming ability of oligonucleotides containing 4′-carboxythymidine analogs. Org Biomol Chem 10:9639–9649

    Article  CAS  PubMed  Google Scholar 

  54. Schlegel MK, Hütter J, Eriksson M, Lepenies B, Seeberger PH (2011) Defined presentation of carbohydrates on a duplex DNA scaffold. Chembiochem 12:2791–2800

    Article  CAS  PubMed  Google Scholar 

  55. Kahl JD, Greenberg MM (1999) Introducing structural diversity in oligonucleotides via photolabile, convertible C5-substituted nucleotides. J Am Chem Soc 121:597–604

    Article  CAS  Google Scholar 

  56. Beilstein AE, Grinstaff MW (2000) On-column derivatization of oligonucleotides with ferrocene. Chem Commun:509–510

    Google Scholar 

  57. Khan SI, Grinstaff MW (1999) Palladium(0)-catalyzed modification of oligonucleotides during automated solid-phase synthesis. J Am Chem Soc 121:4704–4705

    Article  CAS  Google Scholar 

  58. Rist M, Amann N, Wagenknecht H-A (2003) Preparation of 1-ethylnylpyrene-modified DNA via Sonogashira-type solid-phase couplings and characterization of the fluorescence properties for electron-transfer studies. Eur J Org Chem 2003:2498–2504

    Google Scholar 

  59. Mayer E, Valis L, Wagner C, Rist M, Amann N, Wagenknecht H-A (2004) 1-Ethylnylpyrene as a tunable and versatile molecular beacon for DNA. Chembiochem 5:865–868

    Article  CAS  PubMed  Google Scholar 

  60. Kottysch T, Ahlborn C, Brotzel F, Richert C (2004) Stabilizing or destabilizing oligodeoxynucleotide duplexes containing single 2′-deoxyuridine residues with 5-alkynyl substitutents. Chem Eur J 10:4017–4028

    Article  CAS  PubMed  Google Scholar 

  61. Baumhof P, Griesang N, Bächle M, Richert C (2006) Synthesis of oligonucleotides with 3′-terminal 5-(3-acylamidopropargyl)-3′-amino-2′,3′-dideoxyuridine residues and their reactivity in single-nucleotide steps of chemical replication. J Org Chem 71:1060–1067

    Article  CAS  PubMed  Google Scholar 

  62. Filichev V, Pedersen EB (2005) Stable and selective formation of Hoogsteen-type triplexes and duplexes using twisted intercalating nucleic acids (TINA) prepared via postsynthetic Sonogashira solid-phase coupling reactions. J Am Chem Soc 127:14849–14858

    Article  CAS  PubMed  Google Scholar 

  63. Beyer C, Wagenknecht (2010) In situ azide formation and “click” reaction of nile red with DNA as an alternative postsynthetic route. Chem Commun 46:2230–2231

    Article  CAS  Google Scholar 

  64. Wicke L, Engels JW (2012) Postsynthetic on column RNA labeling via Stille coupling. Bioconjug Chem 23:627–642

    Article  CAS  PubMed  Google Scholar 

  65. Cahová H, Jäschke A (2013) Nucleoside-based diarylethene photoswitches and their facile incorporation into photoswitchable DNA. Angew Chem Int Ed 52:3186–3190

    Article  CAS  Google Scholar 

  66. Lercher L, McGouran JF, Kessler BM, Schofield CJ, Davis BG (2013) DNA modification under mild conditions by Suzuki-Miyaura cross-coupling for the generation of functional probes. Angew Chem Int Ed 52:10553–10558

    Article  CAS  Google Scholar 

  67. Jeong HS, Hayashi G, Okamoto A (2015) Diazirine photocrosslinking recruits activated FTO demethylase complexes for specific N 6-methyladenosine recognition. ACS Chem Biol 10:1450–1455

    Article  CAS  PubMed  Google Scholar 

  68. Omumi A, Beach DG, Baker M, Gabryelski W, Manderville RA (2011) Postsynthetic guanine arylation of DNA by Suzuki-Miyaura cross-coupling. J Am Chem Soc 133:42–50

    Article  CAS  PubMed  Google Scholar 

  69. Minakawa N, Ono Y, Matsuda A (2003) A versatile modification of on-column oligodeoxynucleotides using a copper-catalyzed oxidative acetylenic coupling reaction. J Am Chem Soc 125:11545–11552

    Article  CAS  PubMed  Google Scholar 

  70. Nakahara M, Kuboyama T, Izawa A, Hari Y, Imanishi T, Obika S (2009) Synthesis and base-pairing properties of C-nucleotides having 1-substituted 1H-1,2,3-triazoles. Bioorg Med Chem Lett 19:3316–3319

    Article  CAS  PubMed  Google Scholar 

  71. Hari Y, Nakahara M, Pang J, Akabane M, Kuboyama T, Obika S (2011) Synthesis and triplex-forming ability of oligonucleotides bearing 1-substituted 1H-1,2,3-triazole nucleobases. Bioorg Med Chem 19:1162–1166

    Article  CAS  PubMed  Google Scholar 

  72. Hari Y, Nakahara M, Obika S (2013) Triplex-forming ability of oligonucleotides containing 1-aryl-1,2,3-triazole nucleobases linked via a two atom-length spacer. Bioorg Med Chem 21:5583–5588

    Article  CAS  PubMed  Google Scholar 

  73. Hari Y, Nakahara M, Ijitsu S, Obika S (2014) The ability of 1-aryltriazole-containing nucleobases to recognize a TA base pair in triplex DNA. Heterocycles 88:377–386

    Article  CAS  Google Scholar 

  74. Gutsmiedl K, Wirges CT, Ehmke V, Carell T (2009) Copper-free “click” modification of DNA via nitrile oxide-norbornene 1,3-dipolar cycloaddition. Org Lett 11:2405–2408

    Article  CAS  PubMed  Google Scholar 

  75. Schoch J, Wiessler M, Jäschke A (2010) Post-synthetic modification of DNA by inverse-electron-demand Diels-Alder reaction. J Am Chem Soc 132:8846–8847

    Article  CAS  PubMed  Google Scholar 

  76. Arndt S, Wagenknecht H-A (2014) “Photoclick” postsynthetic modification of DNA. Angew Chem Int Ed 53:14580–14582

    Article  CAS  Google Scholar 

  77. Borsenberger V, Howorka S (2009) Diene-modified nucleotides for the Diels-Alder-mediated functional tagging of DNA. Nucleic Acids Res 37:1477–1485

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  78. Dey S, Sheppard TL (2001) Ketone-DNA: a versatile postsynthetic DNA decoration platform. Org Lett 3:3983–3986

    Article  CAS  PubMed  Google Scholar 

  79. Raindlová V, Pohl R, Šanda M, Hocek M (2010) Direct polymerase synthesis of reactive aldehyde-functionalized DNA and its conjugation and staining with hydrazines. Angew Chem Int Ed 49:1064–1066

    Article  CAS  Google Scholar 

  80. Raindlová V, Pohl R, Hocek M (2012) Synthesis of aldehyde-linked nucleotides and DNA and their bioconjugations with lysine and peptides through reductive amination. Chem Eur J 18:4080–4087

    Article  CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Tokushima, Japan

    Yoshiyuki Hari

Authors
  1. Yoshiyuki Hari
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Yoshiyuki Hari .

Editor information

Editors and Affiliations

  1. Osaka University, Suita, Osaka, Japan

    Satoshi Obika

  2. Tokyo Institute of Technology, Yokohama, Kanagawa, Japan

    Mitsuo Sekine

Rights and permissions

Reprints and permissions

Copyright information

© 2018 Springer Nature Singapore Pte Ltd.

About this chapter

Check for updates. Verify currency and authenticity via CrossMark

Cite this chapter

Hari, Y. (2018). Site-Specific Modification of Nucleobases in Oligonucleotides. In: Obika, S., Sekine, M. (eds) Synthesis of Therapeutic Oligonucleotides. Springer, Singapore. https://doi.org/10.1007/978-981-13-1912-9_8

Download citation

Publish with us

Policies and ethics

Search

Navigation