We have developed a robust solid-phase protocol which allowed the synthesis of chimeric oligonucleotides modified with phosphodiester and O-methylphosphonate linkages as well as their P-S and P-N variants. The novel O-methylphosphonate-derived modifications were obtained by oxidation, sulfurization, and amidation of the O-methyl-(H)-phosphinate internucleotide linkage introduced into the oligonucleotide chain by H-phosphonate chemistry using nucleoside-O-methyl-(H)-phosphinates as monomers. The H-phosphonate coupling followed by oxidation after each cycle enabled us to successfully combine H-phosphonate and phosphoramidite chemistries to synthesize diversely modified oligonucleotide strands.

Institute of Organic Chemistry and Biochemistry AS CR v v i Flemingovo nám 2 166 10 Prague 6 Czech Republic

Zobrazit více v PubMed

Šípová H., Špringer T., Rejman D., Šimák O., Petrová M., Novák P., Rosenbergová Š., Páv O., Liboska R., Barvík I., et al. 5′-O-Methylphosphonate nucleic acids—new modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes. Nucleic Acids Res. 2014;42:5378–5389. doi: 10.1093/nar/gku125. PubMed DOI PMC

Kostov O., Páv O., Buděšínský M., Liboska R., Šimák O., Petrová M., Novák P., Rosenberg I. 4-Toluenesulfonyloxymethyl-(H)-phosphinate: A reagent for the introduction of O- and S-Methyl-(H)-phosphinate Moieties. Org. Lett. 2016;18:2704–2707. PubMed

Kostov O., Páv O., Rosenberg I. Nucleoside-O-methyl-(H)-phosphinates: Novel monomers for the synthesis of methylphosphonate oligonucleotides using H-phosphonate chemistry. Curr. Protoc. Nucleic Acid Chem. 2017;70:4.76.1–4.76.22. PubMed

Garegg P.J., Lindh I., Regberg T., Stawinski J., Strömberg R., Henrichson C. Nucleoside H-phosphonates. III. Chemical synthesis of oligodeoxyribonucleotides by the hydrogenphosphonate approach. Tetrahedron Lett. 1986;27:4051–4054. doi: 10.1016/S0040-4039(00)84908-4. DOI

Froehler B.C., Ng P.G., Matteucci M.D. Synthesis of DNA via deoxynudeoside H-phosphonate intermediates. Nucleic Acids Res. 1986;14:5399–5407. doi: 10.1093/nar/14.13.5399. PubMed DOI PMC

Liu C., Cozens C., Jaziri F., Rozenski J., Maréchal A., Dumbre S., Pezo V., Marlière P., Pinheiro V.B., Groaz E., et al. Phosphonomethyl oligonucleotides as backbone-modified artificial genetic polymers. J. Am. Chem. Soc. 2018;140:6690–6699. doi: 10.1021/jacs.8b03447. PubMed DOI

Garegg P.J., Regberg T., Stawinski J., Stromberg R. Nucleoside phosphonates: Part 7. Studies on the oxidation of nucleoside phosphonate esters. J. Chem. Soc. Perkin Trans. 1. 1987:1269–1273. doi: 10.1039/P19870001269. DOI

Cullis P.M., Lee M. The mechanism of iodine-water oxidation of H-phosphonate diesters. J. Chem. Soc. Chem. Commun. 1992:1207–1208. doi: 10.1039/C39920001207. DOI

Wallin R., Kalek M., Bartoszewicz A., Thelin M., Stawinski J. On the sulfurization of H-phosphonate diesters and phosphite triesters using elemental sulfur. PhosphorusSulfurSilicon Relat. Elem. 2009;184:908–916. doi: 10.1080/10426500802715619. DOI

Mohe N.U., Padiya K.J., Salunkhe M.M. An efficient oxidizing reagent for the synthesis of mixed backbone oligonucleotides via the H-phosphonate approach. Bioorg. Med. Chem. 2003;11:1419–1431. doi: 10.1016/S0968-0896(02)00615-6. PubMed DOI

Nilsson J., Stawinski J. Oxidative coupling of H-phosphonate and H-phosphonothioate diesters. Iodine as a reagent and a catalyst. Collect. Symp. Ser. 2002;5:87–92.

Nilsson J., Stawinski J. Controlling stereochemistry during oxidative coupling. Preparation of Rp or Sp phosphoramidates from one P-chiral precursor. Chem. Commun. 2004:2566–2567. doi: 10.1039/b411451e. PubMed DOI

Stawinski J., Stromberg R. Di- and oligonucleotide synthesis using H-phosphonate chemistry. Methods Mol. Biol. (CliftonN.J.) 2005;288:81–100. doi: 10.1002/chin.200541254. PubMed DOI

Maier M.A., Guzaev A.P., Manoharan M. Synthesis of chimeric oligonucleotides containing phosphodiester, phosphorothioate, and phosphoramidate linkages. Org. Lett. 2000;2:1819–1822. doi: 10.1021/ol005842h. PubMed DOI

Bartoszewicz A., Kalek M., Stawinski J. The Case for the intermediacy of monomeric metaphosphate analogues during oxidation of H-phosphonothioate, H-phosphonodithioate, and H-phosphonoselenoate monoesters: Mechanistic and synthetic studies. J. Org. Chem. 2008;73:5029–5038. doi: 10.1021/jo8006072. PubMed DOI

Le Corre S.S., Berchel M., Couthon-Gourvès H., Haelters J.-P., Jaffrès P.-A. Atherton–Todd reaction: Mechanism, scope and applications. Beilstein J. Org. Chem. 2014;10:1166–1196. doi: 10.3762/bjoc.10.117. PubMed DOI PMC

Letsinger R.L., Singman C.N., Histand G., Salunkhe M. Cationic oligonucleotides. J. Am. Chem. Soc. 1988;110:4470–4471. doi: 10.1021/ja00221a089. DOI

Jung P.M., Histand G., Letsinger R.L. Hybridization of alternating cationic/anionic oligonucleotides to RNA segments. Nucleosides Nucleotides. 1994;13:1597–1605. doi: 10.1080/15257779408012174. DOI

Paul S., Roy S., Monfregola L., Shang S., Shoemaker R., Caruthers M.H. Oxidative substitution of boranephosphonate diesters as a route to post-synthetically modified DNA. J. Am. Chem. Soc. 2015;137:3253–3264. doi: 10.1021/ja511145h. PubMed DOI

Brill W.K.D. Thioalkylation of nucleoside-H-phosphonates and its application to solid phase synthesis of oligonucleotides. Tetrahedron Lett. 1995;36:703–706. doi: 10.1016/0040-4039(94)02380-T. DOI

Reese C.B., Yan H. Solution phase synthesis of ISIS 2922 (Vitravene) by the modified H-phosphonate approach. J. Chem. Soc. Perkin Trans. 1. 2002:2619–2633. doi: 10.1039/b208802a. DOI

Alefelder S., Patel B.K., Eckstein F. Incorporation of terminal phosphorothioates into oligonucleotides. Nucleic Acids Res. 1998;26:4983–4988. doi: 10.1093/nar/26.21.4983. PubMed DOI PMC

Andrus A., Efcavitch J.W., McBride L.J., Giusti B. Novel activating and capping reagents for improved hydrogen-phosphonate DNA synthesis. Tetrahedron Lett. 1988;29:861–864. doi: 10.1016/S0040-4039(00)82467-3. DOI

Ugi I., Jacob P., Landgraf B., Ruppf C., Lemmen P., Verfürth U. Phosphite oxidation and the preparation of five-membered cyclic phosphorylating reagents via the phosphites. Nucleosides Nucleotides. 1988;7:605–608. doi: 10.1080/07328318808056294. DOI

Reynolds M.A., Hogrefe R.I., Jaeger J.A., Schwartz D.A., Riley T.A., Marvin W.B., Daily W.J., Vaghefi M.M., Beck T.A., Knowles S.K., et al. Synthesis and thermodynamics of oligonucleotides containing chirally pure R P methylphosphonate linkages. Nucleic Acids Res. 1996;24:4584–4591. doi: 10.1093/nar/24.22.4584. PubMed DOI PMC

Dellinger D.J., Sheehan D.M., Christensen N.K., Lindberg J.G., Caruthers M.H. Solid-phase chemical synthesis of phosphonoacetate and thiophosphonoacetate oligodeoxynucleotides. J. Am. Chem. Soc. 2003;125:940–950. doi: 10.1021/ja027983f. PubMed DOI

Glen Research Unicap Phosphoramidite, An Alternative to Acetic Anhydride Capping. [(accessed on 9 October 2018)]; Available online: http://www.glenresearch.com/GlenReports/GR17-13.html.

Vlaho D., Damha M.J. Synthesis of chimeric oligonucleotides having modified internucleotide linkages via an automated H-phosphonate/phosphoramidite approach. Curr. Protoc. Nucleic Acid Chem. 2018;73:e53. doi: 10.1002/cpnc.53. PubMed DOI