Nucleoside phosphoramidite

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

• File:Nucl Amidite Synthesis1.png The common method involves treatment of a protected nucleoside bearing a single free hydroxy group with phosphorodiamidite under the catalytic action of a weak acid.{{cite journal|author1=Nielsen, J. |author2=Marugg, J. E. |author3=Taagaard, M. |author4=Van Boom, J. H. |author5=Dahl, O. |journal=Recl. Trav. Chim. Pays-Bas|year=1986|volume=105|issue=1|pages=33–34|doi=10.1002/recl.19861050106

|title=Polymer-supported synthesis of deoxyoligonucleotides using in situ prepared deoxynucleoside 2-cyanoethyl phosphoramidites}}{{cite journal|author1=Nielsen, J. |author2=Taagaard, M. |author3=Marugg, J. E. |author4=Van Boom, J. H. |author5=Dahl, O. |journal=Nucleic Acids Res.|year=1986|volume=14|issue=18|pages=7391–7403|doi=10.1093/nar/14.18.7391|title=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|pmc=311758 |pmid=3763407}} Although some bisamidites were reported as thermally unstable compounds,{{cite journal|author1=Nielsen, J. |author2=Marugg, J. E. |author3=Van Boom, J. H. |author4=Honnens, J. |author5=Taagaard, M. |author6=Dahl, O. |journal=J. Chem Res. Synopses|year=1986|issue=1|pages=26–27|title=Thermal instability of some alkyl phosphorodiamidites}} 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite, the amidite used to prepare commercial nucleoside phosphoramidites is relatively stable. It can be synthesized using a two-step, one-pot procedure and purified by vacuum distillation.{{cite journal|author1=Nielsen, J. |author2=Dahl, O. |journal=Nucleic Acids Res.|year=1987|volume=15|issue=8|page=3626|doi=10.1093/nar/15.8.3626|title=Improved synthesis of 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite (iPr2N)2POCH2CH2CN)|pmc=340760|pmid=3575107}} An excellent review outlines the use of the latter reagent in preparation of nucleosidic and non-nucleosidic phosphoramidites in great detail.{{cite journal|author=Beaucage, S. L.|journal=E-EROS Encyclopedia of Reagents for Organic Synthesis|year=2001|doi=10.1002/047084289X.rn00312|title=2-Cyanoethyl Tetraisopropylphosphorodiamidite|isbn=0471936235}}

• File:Nucl Amidite Synthesis2.pngIn the second method, the protected nucleoside is treated with the phosphorochloridite in the presence of an organic base, most commonly N-ethyl-N,N-diisopropylamine (Hunig's base).{{cite journal|author1=Sinha, N. D. |author2=Biernat, J. |author3=Koester, H. |journal=Tetrahedron Lett.|year=1983|volume=24|issue=52|pages=5843–5846|doi=10.1016/S0040-4039(00)94216-3

|title=β-Cyanoethyl N,N-dialkylamino/N-morpholinomonochloro phosphoamidites, new phosphitylating agents facilitating ease of deprotection and work-up of synthesized oligonucleotides}}

• File:Nucl Amidite Synthesis3.pngIn the third method,{{cite journal|author1=Marugg, J. E. |author2=Burik, A. |author3=Tromp, M. |author4=Van der Marel, G. A. |author5=Van Boom, J. H. |name-list-style=amp |journal=Tetrahedron Lett.|year=1986|volume=24|issue=20|pages=2271–22274|doi=10.1016/S0040-4039(00)84506-2 |title=A new and versatile approach to the preparation of valuable deoxynucleoside 3'-phosphite intermediates}} the protected nucleoside is first treated with chloro N,N,N',N'-tetraisopropyl phosphorodiamidite in the presence of an organic base, most commonly N-ethyl-N,N-diisopropylamine (Hunig's base) to form a protected nucleoside diamidite. The latter is treated with an alcohol respective to the desired phosphite protecting group, for instance, 2-cyanoethanol, in the presence of a weak acid.

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.

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 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.{{cite journal|author1=Guzaev, A. P. |author2=Manoharan, M. |journal=J. Am. Chem. Soc.|year=2001|volume=123|issue=5|pages=783–793|doi=10.1021/ja0016396

|title=2-Benzamidoethyl group - a novel type of phosphate protecting group for oligonucleotide synthesis|pmid=11456611 }}

• File:Nucl Amidite Nucleophiles.pngThe most important feature of phosphoramidites is their ability to undergo the phosphoramidite coupling reaction that is, to react with nucleophilic groups in the presence of an acidic azole catalyst, 1H-tetrazole, 2-ethylthiotetrazole,{{cite journal|doi=10.1080/15257779508014668

|date=Feb 1995|author1=Sproat, B. |author2=Colonna, F. |author3=Mullah, B. |author4=Tsou, D. |author5=Andrus, A. |author6=Hampel, A. |author7=Vinayak, R. |title=An efficient method for the isolation and purification of oligoribonucleotides|volume=14|issue=1&2|pages=255–273|issn=0261-3166|journal=Nucleosides & Nucleotides}} 2-benzylthiotetrazole,{{cite journal|doi=10.1002/1522-2675(20000906)83:9<2477::aid-hlca2477>3.0.co;2-9|date=Sep 2000|author1=Stutz, A. |author2=Hobartner, C. |author3=Pitsch, S. |title=Novel fluoride-labile nucleobase-protecting groups for the synthesis of 3'(2')-O-amino-acylated RNA sequences|volume=83|issue=9|pages=2477–2503|issn=0018-019X|journal=Helv. Chim. Acta}}{{cite journal|doi=10.1016/S0040-4039(01)02274-2

|date=Jan 2002|author1=Welz, R. |author2=Muller, S. |title=5-(Benzylmercapto)-1H-tetrazole as activator for 2'-O-TBDMS phosphoramidite building blocks in RNA synthesis|volume=43|issue=5|pages=795–797|issn=0040-4039|journal=Tetrahedron Letters}} 4,5-dicyanoimidazole,{{cite journal|doi=10.1093/nar/26.4.1046

|year=1998|author1=Vargeese, C. |author2=Carter, J. |author3=Yegge, J. |author4=Krivjansky, S. |author5=Settle, A. |author6=Kropp, E. |author7=Peterson, K. |author8=Pieken, W. |title=Efficient activation of nucleoside phosphoramidites with 4,5-dicyanoimidazole during oligonucleotide synthesis|volume=26|issue=4|pages=1046–1050|issn=0305-1048|journal=Nucleic Acids Res.|pmid=9461466|pmc=147346}} or a number of similar compounds. The reaction proceeds extremely rapidly. This very feature makes nucleoside phosphoramidites useful intermediates in oligonucleotide synthesis. Stereochemically, the phosphoramidite coupling leads to the epimerisation (forming of diastereomers) at the P(III) chiral center.

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.

• File:Nucl Amidite Oxidation.pngPhosphoramidites are readily oxidized with weak oxidating reagents, for instance, with aqueous iodine in the presence of weak bases or with hydrogen peroxide{{cite journal|author1=Gacs-Baitz, E. |author2=Sipos, F. |author3=Egyed, O. |author4=Sagi, G. |journal=Chirality|year=2009|volume=21|issue=7|pages=663–673|doi=10.1002/chir.20653

|title=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.|pmid=18937288 }} to form the respective phosphoramidates.

Similarly, phosphoramidites react with other chalcogens. When brought in contact with a solution of sulfur{{cite journal|author1=Nemer, M. J. |author2=Ogilvie, K. K. |journal=Tetrahedron Lett.|year=1980|volume=21|issue=43|pages=4153–4154|doi=10.1016/s0040-4039(00)93675-x

|title=Phosphoramidate analogs of diribonucleoside monophosphates.}} or a number of compounds collectively referred to as sulfurizing agents,{{cite journal|author1=Wilk, A. |author2=Uznanski, B. |author3=Stec, W. J. |journal=Nucleosides & Nucleotides|year=1991|volume=10|issue=1–3|pages=319–322|doi=10.1080/07328319108046469

|title=Assignment of absolute configuration at phosphorus in dithymidylyl(3',5')phosphormorpholidates and -phosphormorpholidothioates.}}{{cite journal|doi=10.1016/j.tetlet.2010.11.086|title=Reactivity of 3H-1,2,4-dithiazole-3-thiones and 3H-1,2-dithiole-3-thiones as sulfurizing agents for oligonucleotide synthesis |year=2011|author=Guzaev, A. P.|journal=Tetrahedron Letters|volume=52|issue=3 |pages=434–437}} phosphoramidites quantitatively form phosphorothioamidates. The reaction with selenium or selenium derivatives{{cite journal|author1=Holloway, G. A. |author2=Pavot, C. |author3=Scaringe, S. A. |author4=Lu, Y. |author5=Rauchfuss, T. B. |journal=ChemBioChem|year=2002|volume=3|issue=11|pages=1061–1065|doi=10.1002/1439-7633(20021104)3:11<1061::aid-cbic1061>3.0.co;2-9

|title=An organometallic route to oligonucleotides containing phosphoroselenoate.|pmid=12404630 |s2cid=18797616 }} produces phosphoroselenoamidates. In all reactions of this type, the configuration at the phosphorus atom is retained.

• File:Nucl Amidite Arbuzov.pngNucleoside phosphoramidites undergo Michaelis-Arbuzov reaction to form the respective phosphonamidates. One example describes the preparation of phosphonamidates in the presence of acrylonitrile.{{cite journal|doi=10.1021/op030035u

|title=Stereoselective Synthesis of Alkylphosphonates: A Facile Rearrangement of Cyanoethyl-Protected Nucleoside Phosphoramidites|year=2004|author1=Ravikumar, V. T. |author2=Kumar, R. K. |journal=Org. Process Res. Dev.|volume=8|issue=4|pages=603–608}} Reportedly, at room temperature the reaction is stereoselective with the retention of configuration at the phosphorus center. In contrast, when carried out at 55 °C, the reaction leads to racemized products.

• Similarly to phosphines and tertiary phosphites, phosphoramidites readily undergo Staudinger reaction.

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

The naturally occurring nucleotides (nucleoside-3'- or 5'-phosphates) and their phosphodiester analogs are insufficiently reactive to afford an expeditious 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 must 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{{cite web|url=https://products.appliedbiosystems.com/ab/en/US/adirect/ab?cmd=catNavigate2&catID=600634 |title=Beta-Cyanoethyl Phosphoramidites |publisher=Products.appliedbiosystems.com |access-date=2009-05-12}}{{cite web|url=http://www.biosearchtech.com |title=Biosearch Technologies |publisher=Biosearchtech.com |access-date=2009-05-12}}{{cite web|url=http://www.chemgenes.com |title=ChemGenes Corporation, a Biotechnology company |publisher=Chemgenes.com |access-date=2009-05-12}}{{cite web|author=M. Powell |url=http://www.glenresearch.com/Catalog/abi.html |title=Applied Biosystems Instruments |publisher=Glenresearch.com |date=2008-01-17 |access-date=2009-05-12}}{{cite web|url=http://www.thermo.com/com/cda/landingpage/0,,1383,00.html |title=Nucleic Acid Synthesis & Labeling |publisher=Thermo.com |date=2008-08-16 |access-date=2009-05-12 |url-status=dead |archive-url=https://web.archive.org/web/20090228191731/http://thermo.com/com/cda/landingpage/0,,1383,00.html |archive-date=February 28, 2009 }} and most common nucleoside phosphoramidite building blocks are briefly reviewed:

• The 5'-hydroxyl group is protected by an acid-labile DMT (4,4'-dimethoxytrityl) group.
• Thymine and uracil, nucleic bases of thymidine and uridine, respectively, do not have exocyclic amino groups and hence do not require any protection. In contrast, nucleic bases adenine, cytosine, and guanine bear the exocyclic amino groups, which are reactive with the activated phosphoramidites under the conditions of the coupling reaction. Although, at the expense of additional steps in the synthetic cycle, the oligonucleotide chain assembly may be carried out using phosphoramidites with unprotected amino groups,{{cite journal|author1=Gryaznov, S. M. |author2=Letsinger, R. L. |journal=J. Am. Chem. Soc.|year=1991|volume=113|issue=15|pages=5876–5877|doi=10.1021/ja00015a059|title=Synthesis of oligonucleotides via monomers with unprotected bases}} most often these are kept permanently protected over the entire length of the oligonucleotide chain assembly. The protection of the exocyclic amino groups must be orthogonal to that of the 5'-hydroxy group because the latter is removed at the end of each synthetic cycle. The simplest to implement and hence the most widely accepted is the strategy where the exocyclic amino groups bear a base-labile protection. Most often, two protection schemes are used.
• In the first, the standard and more robust scheme (Figure), Bz (benzoyl) protection is used for A, dA, C, dC, G, and dG are protected with isobutyryl group. More recently, Ac (acetyl) group is often used to protect C and dC as shown in Figure.{{cite journal|author1=Reddy, M. P. |author2=Hanna, N. B. |author3=Farooqui, F. |journal=Nucleosides & Nucleotides|year=1997|volume=16|pages=1589–1598|doi=10.1080/07328319708006236|title=Ultrafast Cleavage and Deprotection of Oligonucleotides Synthesis and Use of C^Ac Derivatives|issue=7–9 }}
• In the second, mild protection scheme, A and dA are protected with isobutyryl{{cite journal|doi=10.1016/S0040-4039(97)00568-6|title=Synthesis of oligonucleotides containing 3'-alkyl amines using N-isobutyryl protected deoxyadenosine phosphoramidite|year=1997|author=McMinn, D.|journal=Tetrahedron Lett.|volume=38|issue=18|page=3123}} or phenoxyacetyl groups (PAC).{{cite journal|author1=Schulhof, J. C. |author2=Molko, D. |author3=Teoule, R. |title=The final deprotection step in oligonucleotide synthesis is reduced to a mild and rapid ammonia treatment by using labile base-protecting groups|journal=Nucleic Acids Res.|year=1987|volume=15|pages=397–416|doi=10.1093/nar/15.2.397|pmid=3822812|issue=2|pmc=340442}} C and dC bear acetyl protection, and G and dG are protected with 4-isopropylphenoxyacetyl (i-Pr-PAC){{cite journal|doi=10.1016/S0960-894X(01)00161-5|title=Observation and elimination of N-acetylation of oligonucleotides prepared using fast-deprotecting phosphoramidites and ultra-mild deprotection|year=2001|author=Zhu, Q.|journal=Bioorg. Med. Chem. Lett.|volume=11|issue=9|pages=1105–7|pmid=11354354}} or dimethylformamidino (dmf){{cite journal|doi=10.1021/ja00268a052|title=Nucleotide chemistry. 16. Amidine protecting groups for oligonucleotide synthesis|year=1986|author=McBride, L. J.|journal=J. Am. Chem. Soc.|volume=108|page=2040|last2=Kierzek|first2=R.|last3=Beaucage|first3=S. L.|last4=Caruthers|first4=M. H.|issue=8}} groups. Mild protecting groups are removed more readily than the standard protecting groups. However, the phosphoramidites bearing these groups are less stable when stored in solution.
• The phosphite group is protected by a base-labile 2-cyanoethyl group.{{cite journal|author1=Sinha, N. D. |author2=Biernat, J. |author3=McManus, J. |author4=Koester, H. |title=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|journal=Nucleic Acids Res|year=1984|volume=12|pages=4539–4557|doi=10.1093/nar/12.11.4539|pmid=6547529|issue=11|pmc=318857}} Once a phosphoramidite has been coupled to the solid support-bound oligonucleotide and the phosphite moieties have been converted to the P(V) species, the presence of the phosphate protection is not mandatory for the successful conducting of further coupling reactions.{{cite journal|author1=Guzaev, A. P. |author2=Manoharan, M. |journal=J. Org. Chem.|year=2001|volume=66|issue=5|pages=1798–1804|doi=10.1021/jo001591e|title=Phosphoramidite Coupling to Oligonucleotides Bearing Unprotected Internucleosidic Phosphate Moieties|pmid=11262130}}

File:Rnaamidite.png

• In RNA synthesis, the 2'-hydroxy group is protected with TBDMS (t-butyldimethylsilyl) group.{{cite journal|author1=Ogilvie, K. K. |author2=Theriault, N. |author3=Sadana, K. L. |journal= J. Am. Chem. Soc.|year=1977|volume=99|issue=23|pages=7741–7743|doi=10.1021/ja00465a073|title=Synthesis of oligoribonucleotides |pmid=915168 }}{{cite journal|author1=Usman, N. |author2=Ogilvie, K. K. |author3=Jiang, M. Y. |author4=Cedergren, R. J. |journal= J. Am. Chem. Soc.|year=1987|volume=109|issue=25|pages=7845–7854|doi=10.1021/ja00259a037|title=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}}{{cite journal|author1=Usman, N. |author2=Pon, R. T. |author3=Ogilvie, K. K. |journal= Tetrahedron Lett.|year=1985|volume=26|issue=38|pages=4567–4570|doi=10.1016/S0040-4039(00)98753-7|title=Preparation of ribonucleoside 3'-O-phosphoramidites and their application to the automated solid phase synthesis of oligonucleotides }}{{cite journal|author1=Scaringe, S. A. |author2=Francklyn, C. |author3=Usman, N. |journal= Nucleic Acids Res.|year=1990|volume=18|issue=18|pages=5433–5441|doi=10.1093/nar/18.18.5433|title= Chemical synthesis of biologically active oligoribonucleotides using β-cyanoethyl protected ribonucleoside phosphoramidites|pmc=332221 |pmid=2216717}} or with TOM (tri-iso-propylsilyloxymethyl) group,{{cite journal|author1=Pitsch, S. |author2=Weiss, P. A. |author3=Wu, X. |author4=Ackermann, D. |author5=Honegger, T. |journal= Helv. Chim. Acta |year=1999|volume=82|issue=10|pages=1753–1761|doi=10.1002/(SICI)1522-2675(19991006)82:10<1753::AID-HLCA1753>3.0.CO;2-Y |title= Fast and reliable automated synthesis of RNA and partially 2'-O-protected precursors ("caged RNA") based on two novel, orthogonal 2'-O-protecting groups}}{{cite journal|author1=Pitsch, S. |author2=Weiss, P. A. |author3=Jenny, L. |author4=Stutz, A. |author5=Wu, X. |journal= Helv. Chim. Acta |year=2001|volume=84|issue=12|pages=3773–3795|doi=10.1002/1522-2675(20011219)84:12<3773::AID-HLCA3773>3.0.CO;2-E|title=Reliable chemical synthesis of oligoribonucleotides (RNA) with 2'-O-[(triisopropylsilyl)oxy]methyl(2'-O-tom)-protected phosphoramidites}} both being removable by treatment with fluoride ion.
• The phosphite moiety also bears a diisopropylamino (iPr₂N) group reactive under acidic conditions. On activation, the diisopropylamino group leaves, to be substituted by the 5'-hydroxy group of the support-bound oligonucleotide.

• DNA synthesis
• Nucleic acid analogues
• Oligonucleotide synthesis

• Comprehensive Natural Products Chemistry, Volume 7: DNA and Aspects of Molecular Biology. Kool, Eric T.; Editor. Neth. (1999), 733 pp. Publisher: (Elsevier, Amsterdam, Neth.)
• {{cite journal | author = Beaucage S. L., Iyer R. P. | year = 1992 | title = Advances in the synthesis of oligonucleotides by the phosphoramidite approach | url = https://zenodo.org/record/1259717| journal = Tetrahedron | volume = 48 | issue = 12| pages = 2223–2311 | doi=10.1016/s0040-4020(01)88752-4}}
• {{cite journal | author = Beaucage S. L., Iyer R. P. | year = 1993 | title = The functionalization of oligonucleotides via phosphoramidite derivatives | url = https://zenodo.org/record/1259711| journal = Tetrahedron | volume = 49 | issue = 10| pages = 1925–1963 | doi=10.1016/s0040-4020(01)86295-5}}
• {{cite journal | author = Beaucage S. L., Iyer R. P. | year = 1993 | title = The synthesis of modified oligonucleotides by the phosphoramidite approach and their applications | url = https://zenodo.org/record/1259715| journal = Tetrahedron | volume = 49 | issue = 28| pages = 6123–6194 | doi=10.1016/s0040-4020(01)87958-8}}
• Beaucage, S L. "Oligodeoxyribonucleotides synthesis. Phosphoramidite approach. Methods in Molecular Biology (Totowa, NJ, United States) (1993), 20 (Protocols for Oligonucleotides and Analogs), 33–61.
• {{cite journal | author = Reese C. B. | year = 2002 | title = The chemical synthesis of oligo- and poly-nucleotides: a personal commentary | journal = Tetrahedron | volume = 58 | issue = 44| pages = 8893–8920 | doi=10.1016/s0040-4020(02)01084-0}}
• Brown T., Brown D. J. S. 1991. In Oligonucleotides and Analogues. A Practical Approach, ed. F Eckstein, pp.  1 – 24. Oxford: IRL

{{organophosphorus}}

Category:Phosphorus-nitrogen compounds

pl:Amidofosforyny