Synthesis of 5-Hydroxymethylcytidine- and 5-Hydroxymethyl­uridine-Modified RNA

Abstract

We report on the syntheses of 5-hydroxymethyl-uridine [5hm(rU)] and -cytidine [5hm(rC)] phosphoramidites and their incorporation into RNA by solid-phase synthesis. Deprotection of the oligonucleotides is accomplished in a straightforward manner using standard conditions, confirming the appropriateness of the acetyl protection used for the pseudobenzylic alcohol moieties. The approach provides robust access to 5hm(rC/U)-modified RNAs that await applications in pull-down experiments to identify potential modification enzymes. They will also serve as synthetic probes for the development of high-throughput-sequencing methods in native RNAs.

1 Introduction

2 Protection Strategies Reported for the Synthesis of 5hm(dC)-Modified DNA

3 Synthesis of 5-Hydroxymethylpyrimidine-Modified RNA

3.1 Synthesis of 5hm(rC) Phosphoramidite

3.2 Synthesis of 5hm(rU) Phosphoramidite

3.3 Synthesis of 5hm(rC)- and 5hm(rU)-Modified RNA

4 Conclusions

Key words

nucleosides - oligonucleotides - modification - methylation - epigenetics

• References

• 1 Shen L, Song CX, He C, Zhang Y. Annu. Rev. Biochem. 2014; 83: 585
• 2 Koh KP, Rao A. Curr. Opin. Cell Biol. 2013; 25: 152
• 3 Breiling A, Lyko F. Epigenetics Chromatin 2015; 8: 24
• 4 Goll MG, Bestor TH. Annu. Rev. Biochem. 2005; 74: 481
• 5 Kumar S, Cheng X, Klimasauskas S, Mi S, Posfai J, Roberts RJ, Wilson GG. Nucleic Acids Res. 1994; 22: 1
• 6 Zheng G, Fu Y, He C. Chem. Rev. 2014; 114: 4602
• 7 Münzel M, Globisch D, Carell T. Angew. Chem. Int. Ed. 2011; 50: 6460
• 8 Shukla A, Sehgal M, Singh TR. Gene 2015; 564: 109
• 9 Motorin Y, Lyko F, Helm M. Nucleic Acids Res. 2010; 38: 1415
• 10 Amort T, Soulière MF, Wille A, Jia X.-Y, Fiegl H, Wörle H, Micura R, Lusser A. RNA Biol. 2013; 10: 1003
• 11 Edelheit S, Schwartz S, Mumbach MR, Wurtzel O, Sorek R. PLoS Genetics 2013; 9: e1003602
• 12 Squires JE, Patel HR, Nousch M, Sibbritt T, Humphreys DT, Parker BJ, Suter CM, Preiss T. Nucleic Acids Res. 2012; 40: 5023
• 13 Wang X, He C. Mol. Cell 2014; 56: 5
• 14 Liu N, Pan T. Transl. Res. 2015; 165: 28
• 15 Fu L, Guerrero CR, Zhong N, Amato NJ, Liu Y, Liu S, Cai Q, Ji D, Jin SG, Niedernhofer LJ, Pfeifer GP, Xu GL, Wang Y. J. Am. Chem. Soc. 2014; 136: 11582
• 16 Höbartner C. Angew. Chem. Int. Ed. 2011; 50: 4268
• 17 Raiber EA, Beraldi D, Ficz G, Burgess HE, Branco MR, Murat P, Oxley D, Booth MJ, Reik W, Balasubramanian S. Genome Biol. 2012; 13: R69
• 18 Booth MJ, Branco MR, Ficz G, Oxley D, Krueger F, Reik W, Balasubramanian S. Science 2012; 336: 934
• 19 Booth MJ, Marsico G, Bachman M, Beraldi D, Balasubramanian S. Nat. Chem. 2014; 6: 435
• 20 Dominissini D, Moshitch-Moshkovitz S, Schwartz S, Salmon-Divon M, Ungar L, Osenberg S, Cesarkas K, Jacob-Hirsch J, Amariglio N, Kupiec M, Sorek R, Rechavi G. Nature 2012; 485: 201
• 21 Sowers LC, Beardsley GP. J. Org. Chem. 1993; 58: 1664
• 22 Shakya N, Srivastav NC, Bhavanam S, Tse C, Desroches N, Agrawal B, Kunimoto DY, Kumar R. Bioorg. Med. Chem. 2012; 20: 4088
• 23 Tardy-Planechaud S, Fujimoto J, Lin SS, Sowers LC. Nucleic Acids Res. 1997; 25: 553
• 24 Hansen AS, Thalhammer A, El-Sagheer AH, Brown T, Schofield CJ. Bioorg. Med. Chem. Lett. 2011; 21: 1181
• 25 de Kort M, de Visser PC, Kurzeck J, Meeuwenoord NJ, van der Marel GA, Rüger W, van Boom JH. Eur. J. Org. Chem. 2001; 2075
• 26 Dai Q, Song C.-X, Pan T, He C. J. Org. Chem. 2011; 76: 4182
• 27 Münzel M, Globisch D, Trindler C, Carell T. Org. Lett. 2010; 12: 5671
• 28 Schröder AS, Steinbacher J, Steigenberger B, Gnerlich FA, Schiesser S, Pfaffeneder T, Carell T. Angew. Chem. Int. Ed. 2014; 53: 315
• 29 Johnsson RA, Bogojeski JJ, Damha MJ. Bioorg. Med. Chem. Lett. 2014; 24: 2146
• 30 Reddy MP, Hanna NB, Farooqui F. Tetrahedron Lett. 1994; 35: 4311
• 31 Hunziker J. Bioorg. Med. Chem. Lett. 1999; 9: 201
• 32 Tona R, Bertolini R, Hunziker J. Org. Lett. 2000; 2: 1693
• 33 Abdel-Rahman AA.-H, El Ashry ES. Synlett 2002; 2043
• 34 Chung R, Anderson KS. Tetrahedron Lett. 2006; 47: 8361
• 35 Huber SM, van Delft P, Mendil L, Bachman M, Smollett K, Werner F, Miska EA, Balasubramanian S. ChemBioChem 2015; 16: 752
• 36 Abdel Rahman AA.-H, Wada T, Saigo K. Tetrahedron Lett. 2001; 42: 1061
• 37 Höbartner C, Micura R. J. Am. Chem. Soc. 2004; 126: 1141
• 38 Fauster K, Hartl M, Santner T, Aigner M, Kreutz C, Bister K, Ennifar E, Micura R. ACS Chem. Biol. 2012; 7: 581
• 39 Košutić M, Jud L, Da Veiga C, Frener M, Fauster K, Kreutz C, Ennifar E, Micura R. J. Am. Chem. Soc. 2014; 136: 6656
• 40 Hakimelahi GH, Proba ZA, Ogilvie KK. Tetrahedron Lett. 1981; 22: 4775
• 41 Pitsch S, Weiss PA, Jenny L, Stutz A, Wu X. Helv. Chim. Acta 2001; 84: 3773
• 42 Neuner S, Santner T, Kreutz C, Micura R. Chem. Eur. J. 2015; 21: 11634
• 43 Micura R. Angew. Chem. Int. Ed. 2002; 41: 2265
• 44 Höbartner C, Kreutz C, Flecker E, Ottenschlaeger E, Pils W, Grubmayr K, Micura R. Monatsh. Chem. 2003; 134: 851