Various Coupling Agents in the Phosphoramidite Method for Oligonucleotide Synthesis

  • Masaki Tsukamoto
  • Yoshihiro Hayakawa


This review selects some representative coupling agents used for internucleotide bond formation reactions in the phosphoramidite method, which is now the most widely employed method for the chemical synthesis of oligodeoxyribonucleotides and oligoribonucleotides, and it describes their utility, efficiency, and drawbacks. Moreover, the mechanism of the coupling of the nucleoside phosphoramidite and nucleoside promoted by the coupling agent is discussed in some cases. The selected coupling agents are 1H-tetrazole, 5-ethylthio-1H-tetrazole (ETT), 5-benzylthio-1H-tetrazole (BTT), 5-[3,5-bis(trifluoromethyl)phenyl]-1H-tetrazole (Activator 42), 4,5-dicyanoimidazole (DCI), certain carboxylic acids, and various acid/azole complexes such as benzimidazolium triflate (BIT) and saccharin 1-methylimidazole (SMI).


Synthesis of oligonucleotides Phosphoramidite method 1H-Tetrazole Acid/azole complexes DNA oligomer RNA oligomer 


  1. 1.
    Beaucage SL, Iyer RP (1992) Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 48:2223–2311Google Scholar
  2. 2.
    Reese CB (2002) The chemical synthesis of oligo- and poly-nucleotides: a personal commentary. Tetrahedron 58:8893–8920Google Scholar
  3. 3.
    (2006) Chapter 4 synthesis of oligonucleotides. In: Blackburn GM, Gait MJ, Loakes D, Williams DM (eds) Nucleic acids in chemistry and biology, 3rd edn. The Royal Society of Chemistry, Cambridge, pp 143–166Google Scholar
  4. 4.
    Khorana HG (1968) Nucleic acid synthesis. Pure Appl Chem 17:349–381Google Scholar
  5. 5.
    Khorana HG (1968) Synthesis in the study of nucleic acids. Fourth Jubilee Lect Biochem J 109:709–725Google Scholar
  6. 6.
    Letsinger RL, Mahadevan V (1965) Oligonucleotide synthesis on a polymer support. J Am Chem Soc 87:3526–3527Google Scholar
  7. 7.
    Letsinger RL, Ogilvie KK (1967) Convenient method for stepwise synthesis of oligothymidylate derivatives in large-scale quantities. J Am Chem Soc 89:4801–4803Google Scholar
  8. 8.
    Reese CB (1978) The chemical synthesis of oligo- and poly-nucleotides by the phosphotriester approach. Tetrahedron 34:3143–3179Google Scholar
  9. 9.
    Letsinger RL, Finnan JL, Heavner GA, Lunsford WB (1975) Nucleotide chemistry. XX. Phosphite coupling procedure for generating internucleotide links. J Am Chem Soc 97:3278–3279Google Scholar
  10. 10.
    Letsinger RL, Lunsford WB (1976) Synthesis of thymidine oligonucleotides by phosphite triester intermediates. J Am Chem Soc 98:3655–3661Google Scholar
  11. 11.
    Beaucage SL, Caruthers MH (1981) Deoxynucleoside phosphoramidites–A new class of key intermediates for deoxypolynucleotide synthesis. Tetrahedron Lett 22:1859–1862Google Scholar
  12. 12.
    Caruthers MH (1985) Gene synthesis machines: DNA chemistry and its uses. Science 230:281–285Google Scholar
  13. 13.
    Caruthers MH (1991) Chemical synthesis of DNA and DNA analogs. Acc Chem Res 24:278–284Google Scholar
  14. 14.
    Beaucage SL, Caruthers MH (2001) Synthetic strategies and parameters involved in the synthesis of oligodeoxyribonucleotides according to the phosphoramidite method. In: Beaucage SL, Bergstrom DE, Glick GD, Jones RA (eds) Current protocols in nucleic acid chemistry. Wiley, New York, pp 3.3.1–3.3.20Google Scholar
  15. 15.
    Caruthers MH (2013) Chemical synthesis of DNA, RNA, and their analogues. Chem Int 35:8–11Google Scholar
  16. 16.
    Corby NS, Kenner GW, Todd AR (1952) 704. Nucleotides. Part XVI. Ribonucleoside-5′ phosphites. A new method for the preparation of mixed secondary phosphites. J Chem Soc:3669–3675Google Scholar
  17. 17.
    Hall RH, Todd A, Webb RF (1957) 644. Nucleotides. Part XLI. Mixed anhydrides as intermediates in the synthesis of dinucleoside phosphates. J Chem Soc:3291–3296Google Scholar
  18. 18.
    Froehler BC, Ng PG, Matteucci MD (1986) Synthesis of DNA via deoxynudeoside H-phosphonate intermediates. Nucleic Acids Res 14:5399–5407Google Scholar
  19. 19.
    Strömberg R, Stawinski J (2001) Synthesis of oligodeoxyribo- and oligoribonucleotides according to the H-phosphonate method. In: Beaucage SL, Bergstrom DE, Glick GD, Jones RA (eds) Current protocols in nucleic acid chemistry. Wiley, New York, pp 3.4.1–3.4.15Google Scholar
  20. 20.
    Sanghvi YS (2000) Large-scale oligonucleotide synthesis. Org Proc Res Dev 4:168–169Google Scholar
  21. 21.
    Dorsett Y, Tuschl T (2004) siRNAs: applications in functional genomics and potential as therapeutics. Nat Rev Drug Discov 3:318–329Google Scholar
  22. 22.
    (2006) Chapter 5 nucleic acids in biotechnology. In: Blackburn GM, Gait MJ, Loakes D, Williams DM (eds) Nucleic acids in chemistry and biology, 3rd edn. The Royal Society of Chemistry, Cambridge, pp 167–208Google Scholar
  23. 23.
    Keefe AD, Pai S, Ellington A (2010) Aptamers as therapeutics. Nat Rev Drug Discov 9:537–550Google Scholar
  24. 24.
    Hughes RA, Miklos AE, Ellington AD (2011) Gene synthesis: methods and applications. Methods Enzymol 498:277–309Google Scholar
  25. 25.
    Kosuri S, Church GM (2014) Large-scale de novo DNA synthesis: technologies and applications. Nat Meth 11:499–507Google Scholar
  26. 26.
    Dickey DD, Giangrande PH (2016) Oligonucleotide aptamers: a next-generation technology for the capture and detection of circulating tumor cells. Methods 97:94–103Google Scholar
  27. 27.
    Hyodo M, Hayakawa Y (2004) An improved method for synthesizing cyclic bis(3′–5′)diguanylic acid (c-di-GMP). Bull Chem Soc Jpn 77:2089–2093Google Scholar
  28. 28.
    Hyodo M, Sato Y, Hayakawa Y (2006) Synthesis of cyclic bis(3′-5′)diguanylic acid (c-di-GMP) analogs. Tetrahedron 62:3089–3094Google Scholar
  29. 29.
    Hyodo M, Hayakawa Y (2008) Synthesis, chemical properties and biological activities of cyclic bis(3′–5′)diguanylic acid (c-di-GMP) and its analogues. In: Modified nucleosides. Wiley-VCH, pp 343–363Google Scholar
  30. 30.
    Schwede F, Genieser H-G, Rentsch A (2017) The chemistry of the noncanonical cyclic dinucleotide 2′3′-cGAMP and its analogs. In: Seifert R (ed) Non-canonical cyclic nucleotides. Springer, Cham, pp 359–384Google Scholar
  31. 31.
    Hayakawa Y, Uchiyama M, Noyori R (1986) Nonaqueous oxidation of nucleoside phosphites to the phosphates. Tetrahedron Lett 27:4191–4194Google Scholar
  32. 32.
    Wincott FE (2001) Strategies for oligoribonucleotide synthesis according to the phosphoramidite method. In: Beaucage SL, Bergstrom DE, Glick GD, Jones RA (eds) Current protocols in nucleic acid chemistry. Wiley, New York, pp 3.5.1–3.5.12Google Scholar
  33. 33.
    Bellon L (2001) Oligoribonucleotides with 2′-O-(tert-butyldimethylsilyl) groups. In: Beaucage SL, Bergstrom DE, Glick GD, Jones RA (eds) Current protocols in nucleic acid chemistry. Wiley, New York, pp 3.6.1–3.6.13Google Scholar
  34. 34.
    Hayakawa Y (2001) Toward an ideal synthesis of oligonucleotides: development of a novel phosphoramidite method with high capability. Bull Chem Soc Jpn 74:1547–1565Google Scholar
  35. 35.
    Tsukamoto M, Hayakawa Y (2005) Strategies useful for the chemical synthesis of oligonucleotides and related compounds. In: Atta-Ur-Rahman, Hayakawa Y (eds) Frontiers in organic chemistry, vol 1. Bentham, Hilversum, pp 3–40Google Scholar
  36. 36.
  37. 37.
    Höbartner C, Wachowius F (2010) Chemical synthesis of modified RNA. In: Mayer G (ed) The chemical biology of nucleic acids. Wiley, Chichester, pp 1–37Google Scholar
  38. 38.
    Wei X (2013) Coupling activators for the oligonucleotide synthesis via phosphoramidite approach. Tetrahedron 69:3615–3637Google Scholar
  39. 39.
    Ohkubo A, Seio K, Sekine M (2006) DNA synthesis without base protection using the phosphoramidite approach. In: Beaucage SL, Bergstrom DE, Herdewijn P, Matsuda A (eds) Current protocols in nucleic acid chemistry. Wiley, Hoboken, pp 3.15.1–3.15.22Google Scholar
  40. 40.
    Hayakawa Y, Kawai R, Kataoka M (2001) Nucleotide synthesis via methods without nucleoside-base protection. Eur J Pharm Sci 13:5–16Google Scholar
  41. 41.
    Benson FR (1947) The chemistry of the tetrazoles. Chem Rev 41:1–61Google Scholar
  42. 42.
  43. 43.
    Wang Z, Olsen P, Ravikumar VT (2007) A novel universal linker for efficient synthesis of phosphorothioate oligonucleotides. Nucleosides Nucleotides Nucleic Acids 26:259–269Google Scholar
  44. 44.
    LeProust EM, Peck BJ, Spirin K, McCuen HB, Moore B, Namsaraev E, Caruthers MH (2010) Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process. Nucleic Acids Res 38:2522–2540Google Scholar
  45. 45.
    Dellinger DJ, Monfregola L, Caruthers M, Roy M (2015) US Patent 0,315,227 A1Google Scholar
  46. 46.
    Vargeese C, Carter J, Yegge J, Krivjansky S, Settle A, Kropp E, Peterson K, Pieken W (1998) Efficient activation of nucleoside phosphoramidites with 4,5-dicyanoimidazole during oligonucleotide synthesis. Nucleic Acids Res 26:1046–1050Google Scholar
  47. 47.
    Scaringe SA, Francklyn C, Usman N (1990) Chemical synthesis of biologically active oligoribonucleotides using β-cyanoethyl protected ribonucleoside phosphoramidites. Nucleic Acids Res 18:5433–5441Google Scholar
  48. 48.
    Wincott F, DiRenzo A, Shaffer C, Grimm S, Tracz D, Workman C, Sweedler D, Gonzalez C, Scaringe S, Usman N (1995) Synthesis, deprotection, analysis and purification of RNA and ribosomes. Nucleic Acids Res 23:2677–2684Google Scholar
  49. 49.
    Dahl BH, Nielsen J, Dahl O (1987) Mechanistic studies on the phosphoramidite coupling reaction in oligonucleotide synthesis. I. Evidence for nudeophilic catalysis by tetrazole and rate variations with the phosphorus substituents. Nucleic Acids Res 15:1729–1743Google Scholar
  50. 50.
    Berner S, Mūhlegger K, Seliger H (1989) Studies on the role of tetrazole in the activation of phosphoramidites. Nucleic Acids Res 17:853–864Google Scholar
  51. 51.
    McBride LJ, Caruthers MH (1983) An investigation of several deoxynucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides. Tetrahedron Lett 24:245–248Google Scholar
  52. 52.
    Pon RT, Damha MJ, Ogilvie KK (1985) Modification of guanine bases by nucleoside phosphoramidite reagents during the solid phase synthesis of oligonucleotides. Nucleic Acids Res 13:6447–6465Google Scholar
  53. 53.
    Lieber E, Enkoji T (1961) Synthesis and properties of 5-(substituted) mercaptotetrazoles. J Org Chem 26:4472–4479Google Scholar
  54. 54.
    LeBlanc BW, Jursic BS (1998) Preparation of 5-alkylthio and 5-arylthiotetrazoles from thiocyanates using phase transfer catalysis. Synth Commun 28:3591–3599Google Scholar
  55. 55.
    Wright P, Lloyd D, Rapp W, Andrus A (1993) Large scale synthesis of oligonucleotides via phosphoramidite nucleosides and a high-loaded polystyrene support. Tetrahedron Lett 34:3373–3376Google Scholar
  56. 56.
    Scaringe SA, Wincott FE, Caruthers MH (1998) Novel RNA synthesis method using 5′-O-silyl-2′-O-orthoester protecting groups. J Am Chem Soc 120:11820–11821Google Scholar
  57. 57.
    Ohgi T, Masutomi Y, Ishiyama K, Kitagawa H, Shiba Y, Yano J (2005) A new RNA synthetic method with a 2′-O-(2-cyanoethoxymethyl) protecting group. Org Lett 7:3477–3480Google Scholar
  58. 58.
    Semenyuk A, Földesi A, Johansson T, Estmer-Nilsson C, Blomgren P, Brännvall M, Kirsebom LA, Kwiatkowski M (2006) Synthesis of RNA using 2′-O-DTM protection. J Am Chem Soc 128:12356–12357Google Scholar
  59. 59.
    Zhou C, Honcharenko D, Chattopadhyaya J (2007) 2-(4-Tolylsulfonyl)ethoxymethyl (TEM)-a new 2′-OH protecting group for solid-supported RNA synthesis. Org Biomol Chem 5:333–343Google Scholar
  60. 60.
    Lackey JG, Sabatino D, Damha MJ (2007) Solid-phase synthesis and on-column deprotection of RNA from 2′- (and 3′-) O-levulinated (Lv) ribonucleoside monomers. Org Lett 9:789–792Google Scholar
  61. 61.
    Krotz AH, Klopchin PG, Walker KL, Srivatsa GS, Cole DL, Ravikumar VT (1997) On the formation of longmers in phosphorothioate oligodeoxyribonucleotide synthesis. Tetrahedron Lett 38:3875–3878Google Scholar
  62. 62.
    Welz R, Müller S (2002) 5-(Benzylmercapto)-1H-tetrazole as activator for 2′-O-TBDMS phosphoramidite building blocks in RNA synthesis. Tetrahedron Lett 43:795–797Google Scholar
  63. 63.
    Wu X, Pitsch S (1998) Synthesis and pairing properties of oligoribonucleotide analogues containing a metal-binding site attached to β-D-allofuranosyl cytosine. Nucleic Acids Res 26:4315–4323Google Scholar
  64. 64.
    Saneyoshi H, Ando K, Seio K, Sekine M (2007) Chemical synthesis of RNA via 2′-O-cyanoethylated intermediates. Tetrahedron 63:11195–11203Google Scholar
  65. 65.
    Shiba Y, Masuda H, Watanabe N, Ego T, Takagaki K, Ishiyama K, Ohgi T, Yano J (2007) Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2′-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate. Nucleic Acids Res 35:3287–3296Google Scholar
  66. 66.
    Lavergne T, Bertrand JR, Vasseur JJ, Debart F (2008) A base-labile group for 2′-OH protection of ribonucleosides: a major challenge for RNA synthesis. Chem Eur J 14:9135–9138Google Scholar
  67. 67.
    Cieślak J, Grajkowski A, Kauffman JS, Duff RJ, Beaucage SL (2008) The 4-(N-dichloroacetyl-N-methylamino)benzyloxymethyl group for 2′-hydroxyl protection of ribonucleosides in the solid-phase synthesis of oligoribonucleotides. J Org Chem 73:2774–2783Google Scholar
  68. 68.
    Gaglione M, Potenza N, Di Fabio G, Romanucci V, Mosca N, Russo A, Novellino E, Cosconati S, Messere A (2013) Tuning RNA interference by enhancing siRNA/PAZ recognition. ACS Med Chem Lett 4:75–78Google Scholar
  69. 69.
    Reddy KS (2008) US Patent 7,339,052 B2Google Scholar
  70. 70.
    Wolter A, Leuck M (2006) US Patent 0,247,431 A1Google Scholar
  71. 71.
    Utagawa E, Ohkubo A, Sekine M, Seio K (2007) Synthesis of branched oligonucleotides with three different Sequences using an oxidatively removable tritylthio group. J Org Chem 72:8259–8266Google Scholar
  72. 72.
    Leszczynska G, Pieta J, Wozniak K, Malkiewicz A (2014) Site-selected incorporation of 5-carboxymethylaminomethyl(-2-thio)uridine into RNA sequences by phosphoramidite chemistry. Org Biomol Chem 12:1052–1056Google Scholar
  73. 73.
    Woodward DW (1950) US Patent 2,534,331Google Scholar
  74. 74.
    Persson T, Kutzke U, Busch S, Held R, Hartmann RK (2001) Chemical synthesis and biological investigation of a 77-mer oligoribonucleotide with a sequence corresponding to E. coli tRNAAsp. Bioorg Med Chem 9:51–56Google Scholar
  75. 75.
    Lackey JG, Mitra D, Somoza MM, Cerrina F, Damha MJ (2009) Acetal levulinyl ester (ALE) groups for 2′-hydroxyl protection of ribonucleosides in the synthesis of oligoribonucleotides on glass and microarrays. J Am Chem Soc 131:8496–8502Google Scholar
  76. 76.
    Reddy MP, Farooqui F (1996) US Patent 5,574,146Google Scholar
  77. 77.
    Tsukamoto M, Nurminen EJ, Iwase T, Kataoka M, Hayakawa Y (2004) Internucleotide-linkage formation via the phosphoramidite method using a carboxylic acid as a promoter. Nucleic Acids Symp Ser 48:25–26Google Scholar
  78. 78.
    Hayakawa Y, Iwase T, Nurminen EJ, Tsukamoto M, Kataoka M (2005) Carboxylic acids as promoters for internucleotide-bond formation via condensation of a nucleoside phosphoramidite and a nucleoside: relationship between the acidity and the activity of the promoter. Tetrahedron 61:2203–2209Google Scholar
  79. 79.
    Brill WKD, Nielsen J, Caruthers MH (1991) Synthesis of deoxydinucleoside phosphorodithioates. J Am Chem Soc 113:3972–3980Google Scholar
  80. 80.
    Hayakawa Y, Kataoka M, Noyori R (1996) Benzimidazolium triflate as an efficient promoter for nucleotide synthesis via the phosphoramidite method. J Org Chem 61:7996–7997Google Scholar
  81. 81.
    Hayakawa Y, Kawai R, Hirata A, Sugimoto J-i, Kataoka M, Sakakura A, Hirose M, Noyori R (2001) Acid/azole complexes as highly effective promoters in the synthesis of DNA and RNA oligomers via the phosphoramidite method. J Am Chem Soc 123:8165–8176Google Scholar
  82. 82.
    Nurminen E, Lönnberg H (2004) Mechanisms of the substitution reactions of phosphoramidites and their congeners. J Phys Org Chem 17:1–17Google Scholar
  83. 83.
    Sinha ND, Zedalis WE, Miranda GK (2003) WO Patent 004,512 A1Google Scholar
  84. 84.
    Sinha ND, Foster P, Kuchimanchi SN, Miranda G, Shaikh S, Michaud D (2007) Highly effective non-explosive activators based on saccharin for the synthesis of oligonucleotides and phosphoramidites. Nucleosides Nucleotides Nucleic Acids 26:1615–1618Google Scholar
  85. 85.
    Russell MA, Laws AP, Atherton JH, Page MI (2008) The mechanism of the phosphoramidite synthesis of polynucleotides. Org Biomol Chem 6:3270–3275Google Scholar

Copyright information

© Springer Nature Singapore Pte Ltd. 2018

Authors and Affiliations

  1. 1.Graduate School of Information ScienceNagoya UniversityNagoyaJapan
  2. 2.Department of Applied Chemistry, Faculty of EngineeringAichi Institute of TechnologyToyotaJapan

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