Nucleoside phosphoramidite

Protected 2'-deoxynucleoside phosphoramidites.

Nucleoside phosphoramidites are derivatives of natural or synthetic nucleosides. They are used to synthesize oligonucleotides, relatively short fragments of nucleic acid and their analogs. Nucleoside phosphoramidites were first introduced in 1981 by Beaucage and Caruthers.[1] In order to avoid undesired side reactions, reactive hydroxy and exocyclic amino groups present in natural or synthetic nucleosides are appropriately protected. As long as a nucleoside analog contains at least one hydroxy group, the use of the appropriate protecting strategy allows one to convert that to the respective phosphoramidite and to incorporate the latter into synthetic nucleic acids. In order to be incorporated in the middle of an oligonucleotide chain using phosphoramidite strategy, the nucleoside analog have to possess two hydroxy groups or, less often, a hydroxy group and another nucleophilic group (amino or mercapto). Examples include, but are not limited to, alternative nucleotides, LNA, morpholino, nucleosides modified at the 2'-position (OMe, protected NH2, F), nucleosides containing non-canonical bases (hypoxanthine and xanthine contained in natural nucleosides inosine and xanthosine, respectively, tricyclic bases such as G-clamp,[2] etc.) or bases derivatized with a fluorescent group or a linker arm.

Preparation of nucleoside phosphoramidites

There are three main methods for the preparation of nucleoside phosphoramidites.

Nucleoside phosphoramidites are purified by column chromatography on silica gel. To warrant the stability of the phosphoramidite moiety, it is advisable to equilibrate the column with an eluent containing 3 to 5% of triethylamine and maintain this concentration in the eluent throughout the entire course of the separation. The purity of a phosphoramidite may be assessed by 31P NMR spectroscopy. As the P(III) atom in a nucleoside phosphoramidite is chiral, it displays two peaks at about 149 ppm corresponding to the two diastereomers of the compound. The potentially present phosphite triester impurity displays peak at 138-140 ppm. H-phosphonate impurities display peaks at 8 and 10 ppm.

Chemical properties of phosphoramidite moiety

Nucleoside phosphoramidites are relatively stable compounds with a prolonged shelf-life when stored as powders under anhydrous conditions in the absence of air at temperatures below 4 °C. The amidites well withstand mild basic conditions. In contrast, in the presence of even mild acids, phosphoramidites perish almost instantaneously. The phosphoramidites are relatively stable to hydrolysis under neutral conditions. For instance, half-life of 2-cyanoethyl 5'-O-(4,4'-dimethoxytrityl)thymidine-3'-O-(N,N-diisopropylamino)phosphite in 95% aqueous acetonitrile at 25 °C is 200 h.[10]

When water is served as a nucleophile, the product is an H-phosphonate diester as shown in Scheme above. Due to the presence of residual water in solvents and reagents, the formation of the latter compound is the most common complication in the preparative use of phosphoramidites, particularly in oligonucleotide synthesis.

Similarly, phosphoramidites react with other chalcogens. When brought in contact with a solution of sulfur[15][16] or a number of compounds collectively referred to as sulfurizing agents,[17][18] phosphoramidites quantitatively form phosphorothioamidates. The reaction with selenium[15][16] or selenium derivatives[19] produces phosphoroselenoamidates. In all reactions of this type, the configuration at the phosphorus atom is retained.

(RO)2P-N(R1)2 + R2-N3 + H2O ---- (RO)2P(=O)-N(R1)2 + R2-NH2 + N2;

Protecting strategy

The naturally occurring nucleotides (nucleoside-3'- or 5'-phosphates) and their phosphodiester analogs are insufficiently reactive to afford an expedite synthetic preparation of oligonucleotides in high yields. The selectivity and the rate of the formation of internucleosidic linkages are dramatically improved by using 3'-O-(N,N-diisopropyl phosphoramidite) derivatives of nucleosides (nucleoside phosphoramidites) that serve as building blocks in phosphite triester methodology. To prevent undesired side reactions, all other functional groups present in nucleosides have to be rendered unreactive (protected) by attaching protecting groups. Upon the completion of the oligonucleotide chain assembly, all the protecting groups are removed to yield the desired oligonucleotides. Below, the protecting groups currently used in commercially available[21][22][23][24][25] and most common nucleoside phosphoramidite building blocks are briefly reviewed:

2'-O-Protected ribonucleoside phosphoramidites.

References

  1. Beaucage, S.L.; Caruthers M.H. (1981). "Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis". Tetrahedron Letters 22: 1859–1862. doi:10.1016/S0040-4039(01)90461-7.
  2. Lin, K.-Y., Matteucci, M. D. (1998). "A cytosine analog capable of clamp-like binding to a guanine in helical nucleic acids". J. Amer. Chem. Soc. 120 (33): 8531–8532. doi:10.1021/ja981286z.
  3. Nielsen, J.; Marugg, J. E.; Taagaard, M.; Van Boom, J. H.; Dahl, O. (1986). "Polymer-supported synthesis of deoxyoligonucleotides using in situ prepared deoxynucleoside 2-cyanoethyl phosphoramidites". Rec. Trav. Chim. Pays-Bas 105 (1): 33–34.
  4. Nielsen, J.; Taagaard, M.; Marugg, J. E.; Van Boom, J. H.; Dahl, O. (1986). "Application of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite for in situ preparation of deoxyribonucleoside phosphoramidites and their use in polymer-supported synthesis of oligodeoxyribonucleotides". Nucl. Acids Res. 14 (18): 7391–7403. doi:10.1093/nar/14.18.7391.
  5. Nielsen, J.; Marugg, J. E.; Van Boom, J. H.; Honnens, J.; Taagaard, M.; Dahl, O. (1986). "Thermal instability of some alkyl phosphorodiamidites". J. Chem Res. Synopses (1): 26–27.
  6. Nielsen, J.; Dahl, O. (1987). "Improved synthesis of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (iPr2N)2POCH2CH2CN)". Nucl. Acids Res. 15 (8): 3626. doi:10.1093/nar/15.8.3626.
  7. Beaucage, S. L. (2001). "2-Cyanoethyl Tetraisopropylphosphorodiamidite". e-EROS Encyclopedia of Reagents for Organic Synthesis. doi:10.1002/047084289X.rn00312.
  8. Sinha, N. D.; Biernat, J.; Koester, H. (1983). "β-Cyanoethyl N,N-dialkylamino/N-morpholinomonochloro phosphoamidites, new phosphitylating agents facilitating ease of deprotection and work-up of synthesized oligonucleotides". Tetrahedron Lett. 24 (52): 5843–5846. doi:10.1016/S0040-4039(00)94216-3.
  9. Marugg, J. E.; Burik, A.; Tromp, M.; Van der Marel, G. A.; Van Boom, J. H. (1986). "A new and versatile approach to the preparation of valuable deoxynucleoside 3'-phosphite intermediates". Tetrahedron Lett. 24 (20): 2271–22274. doi:10.1016/S0040-4039(00)84506-2.
  10. Guzaev, A. P.; Manoharan, M. (2001). "2-Benzamidoethyl group - a novel type of phosphate protecting group for oligonucleotide synthesis". J. Amer. Chem. Soc. 123 (5): 783–793. doi:10.1021/ja0016396.
  11. Sproat, B.; Colonna, F.; Mullah, B.; Tsou, D.; Andrus, A.; Hampel, A.; Vinayak, R. (Feb 1995). "An efficient method for the isolation and purification of oligoribonucleotides". Nucleosides & Nucleotides 14 (1&2): 255–273. doi:10.1080/15257779508014668. ISSN 0261-3166.
  12. Stutz, A.; Hobartner, C.; Pitsch, S. (Sep 2000). "Novel fluoride-labile nucleobase-protecting groups for the synthesis of 3'(2')-O-amino-acylated RNA sequences". Helv. Chim. Acta 83 (9): 2477–2503. doi:10.1002/1522-2675(20000906). ISSN 0018-019X.
  13. Welz, R.; Muller, S. (Jan 2002). "5-(Benzylmercapto)-1H-tetrazole as activator for 2'-O-TBDMS phosphoramidite building blocks in RNA synthesis". Tetrahedron Letters 43 (5): 795–797. doi:10.1016/S0040-4039(01)02274-2. ISSN 0040-4039.
  14. 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". Nucl. Acids Res. 26 (4): 1046–1050. doi:10.1093/nar/26.4.1046. ISSN 0305-1048. PMC 147346. PMID 9461466.
  15. 15.0 15.1 15.2 Gacs-Baitz, E.; Sipos, F.; Egyed, O.; Sagi, G. (2009). "Synthesis and structural study of variously oxidized diastereomeric 5'-dimethoxytrityl-thymidine-3'-O-[O-(2-cyanoethyl)-N,N-diisopropyl]-phosphoramidite derivatives. Comparison of the effects of the P=O, P=S, and P=Se functions on the NMR spectral and chromatographic properties.". Chirality 21 (7): 663–673. doi:10.1002/chir.20653.
  16. 16.0 16.1 Nemer, M. J.; Ogilvie, K. K. (1980). "Phosphoramidate analogs of diribonucleoside monophosphates.". Tetrahedron Lett. 21 (43): 4153–4154. doi:10.1016/s0040-4039(00)93675-x.
  17. Wilk, A.; Uznanski, B.; Stec, W. J. (1991). "Assignment of absolute configuration at phosphorus in dithymidylyl(3',5')phosphormorpholidates and -phosphormorpholidothioates.". Nucleosides & Nucleotides 10 (1-3): 319–322. doi:10.1080/07328319108046469.
  18. Guzaev, A. P. (2011). "Reactivity of 3H-1,2,4-dithiazole-3-thiones and 3H-1,2-dithiole-3-thiones as sulfurizing agents for oligonucleotide synthesis". Tetrahedron Letters 52: 434–437. doi:10.1016/j.tetlet.2010.11.086.
  19. Holloway, G. A.; Pavot, C.; Scaringe, S. A.; Lu, Y.; Rauchfuss, T. B. (2002). "An organometallic route to oligonucleotides containing phosphoroselenoate.". ChemBioChem 3 (11): 1061–1065. doi:10.1002/1439-7633(20021104)3:11<1061::aid-cbic1061>3.0.co;2-9.
  20. Ravikumar, V. T.; Kumar, R. K. (2004). "Stereoselective Synthesis of Alkylphosphonates: A Facile Rearrangement of Cyanoethyl-Protected Nucleoside Phosphoramidites". Org. Proc Res. Dev. 8 (4): 603–608. doi:10.1021/op030035u.
  21. "Beta-Cyanoethyl Phosphoramidites". Products.appliedbiosystems.com. Retrieved 2009-05-12.
  22. "Biosearch Technologies". Biosearchtech.com. Retrieved 2009-05-12.
  23. "ChemGenes Corporation, a Biotechnology company". Chemgenes.com. Retrieved 2009-05-12.
  24. M. Powell (2008-01-17). "Applied Biosystems Instruments". Glenresearch.com. Retrieved 2009-05-12.
  25. "Nucleic Acid Synthesis & Labeling". Thermo.com. 2008-08-16. Retrieved 2009-05-12.
  26. Gryaznov, S. M.; Letsinger, R. L. (1991). "Synthesis of oligonucleotides via monomers with unprotected bases". J. Amer. Chem. Soc. 113 (15): 5876–5877. doi:10.1021/ja00015a059.
  27. 27.0 27.1 Reddy, M. P.; Hanna, N. B.; Farooqui, F. (1997). "Ultrafast Cleavage and Deprotection of Oligonucleotides Synthesis and Use of CAc Derivatives". Nucleosides & Nucleotides 16: 1589–1598. doi:10.1080/07328319708006236.
  28. McMinn, D. (1997). "Synthesis of oligonucleotides containing 3'-alkyl amines using N-isobutyryl protected deoxyadenosine phosphoramidite". Tetrahedron Lett. 38: 3123. doi:10.1016/S0040-4039(97)00568-6.
  29. Schulhof, J. C.; Molko, D.; Teoule, R. (1987). "The final deprotection step in oligonucleotide synthesis is reduced to a mild and rapid ammonia treatment by using labile base-protecting groups". Nucleic Acids Res. 15 (2): 397–416. doi:10.1093/nar/15.2.397. PMC 340442. PMID 3822812.
  30. Zhu, Q. (2001). "Observation and elimination of N-acetylation of oligonucleotides prepared using fast-deprotecting phosphoramidites and ultra-mild deprotection". Bioorg. & Med. Chem. Lett. 11: 1105. doi:10.1016/S0960-894X(01)00161-5.
  31. McBride, L. J.; Kierzek, R.; Beaucage, S. L.; Caruthers, M. H. (1986). "Nucleotide chemistry. 16. Amidine protecting groups for oligonucleotide synthesis". J. Amer. Chem. Soc. 108: 2040. doi:10.1021/ja00268a052.
  32. Sinha, N. D.; Biernat, J.; McManus, J.; Koester, H. (1984). "Polymer support oligonucleotide synthesis. XVIII: use of β-cyanoethyl-N,N-dialkylamino-/N-morpholino phosphoramidite of deoxynucleosides for the synthesis of DNA fragments simplifying deprotection and isolation of the final product". Nucleic Acids Res 12 (11): 4539–4557. doi:10.1093/nar/12.11.4539. PMC 318857. PMID 6547529.
  33. Guzaev, A. P.; Manoharan, M. (2001). "Phosphoramidite Coupling to Oligonucleotides Bearing Unprotected Internucleosidic Phosphate Moieties". J. Org. Chem. 66 (5): 1798–1804. doi:10.1021/jo001591e. PMID 11262130.
  34. Ogilvie, K. K.; Theriault, N.; Sadana, K. L. (1977). "Synthesis of oligoribonucleotides". J. Amer. Chem. Soc. 99 (23): 7741–7743. doi:10.1021/ja00465a073.
  35. Usman, N.; Ogilvie, K. K.; Jiang, M. Y.; Cedergren, R. J. (1987). "The automated chemical synthesis of long oligoribuncleotides using 2'-O-silylated ribonucleoside 3'-O-phosphoramidites on a controlled-pore glass support: synthesis of a 43-nucleotide sequence similar to the 3'-half molecule of an Escherichia coli formylmethionine tRNA". J. Amer. Chem. Soc. 109 (25): 7845–7854. doi:10.1021/ja00259a037.
  36. Usman, N.; Pon, R. T.; Ogilvie, K. K. (1985). "Preparation of ribonucleoside 3'-O-phosphoramidites and their application to the automated solid phase synthesis of oligonucleotides". Tetrahedron Lett. 26 (38): 4567–4570. doi:10.1016/S0040-4039(00)98753-7.
  37. Scaringe, S. A.; Francklyn, C.; Usman, N. (1990). "Chemical synthesis of biologically active oligoribonucleotides using β-cyanoethyl protected ribonucleoside phosphoramidites". Nucl. Acids Res. 18 (18): 5433–5441. doi:10.1093/nar/18.18.5433.
  38. Pitsch, S.; Weiss, P. A.; Wu, X.; Ackermann, D.; Honegger, T. (1999). "Fast and reliable automated synthesis of RNA and partially 2'-O-protected precursors ("caged RNA") based on two novel, orthogonal 2'-O-protecting groups". Helv. Chim. Acta 82 (10): 1753–1761. doi:10.1002/(SICI)1522-2675(19991006)82:10<1753::AID-HLCA1753>3.0.CO;2-Y.
  39. Pitsch, S.; Weiss, P. A.; Jenny, L.; Stutz, A.; Wu, X. (2001). "Reliable chemical synthesis of oligoribonucleotides (RNA) with 2'-O-[(triisopropylsilyl)oxy]methyl(2'-O-tom)-protected phosphoramidites". Helv. Chim. Acta 84 (12): 3773–3795. doi:10.1002/1522-2675(20011219)84:12<3773::AID-HLCA3773>3.0.CO;2-E.

Further reading

See also