Solid‐Phase Synthesis of DNA and RNA 5′‐O‐Triphosphates Using cycloSal Chemistry

Ivo Sarac1, Chris Meier1

1 Department of Chemistry, University of Hamburg, Hamburg
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.67
DOI:  10.1002/0471142700.nc0467s64
Online Posting Date:  March, 2016
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A chemical method for the synthesis of short strands of DNA and RNA 5′‐O‐triphosphates is described that makes use of the conventional coupling and oxidizing reagents that are readily available on standard DNA/RNA synthesizers. After automated solid‐phase synthesis of oligonucleotides and 5′‐O‐detritylation, a novel cycloSal‐phosphoramidite, 5‐chloro‐saligenyl‐N,N‐diisopropylphosphoramidite, was coupled to the 5′‐hydroxyl group and the product oxidized. The resulting support‐bonded 5′‐cycloSal‐oligonucleotide was reacted with pyrophosphate to yield 5′‐O‐triphosphorylated DNA/RNA oligonucleotides after cleavage from the support in high purity and excellent yields. The reaction sequence was adapted to be used completely on a standard automated oligonucleotide synthesizer. © 2016 by John Wiley & Sons, Inc.

Keywords: automated solid‐phase synthesis; 5′‐O‐triphosphate; cycloSal; modified DNA and RNA synthesis; modified oligonucleotides

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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 5‐Chlorosaligenyl‐N,N‐Diisopropylphosphoramidite
  • Support Protocol 1: Preparation of Bis(Tetra‐n‐Butylammonium) Dihydrogen Pyrophosphate
  • Basic Protocol 2: Synthesis of DNA and RNA 5′‐O‐Triphosphates
  • Reagents and Solutions
  • Commentary
  • Figures
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Basic Protocol 1: Synthesis of 5‐Chlorosaligenyl‐N,N‐Diisopropylphosphoramidite

  • Lithium aluminum hydride (LiAlH 4)
  • Diethyl ether, anhydrous (Acros, cat. no. 10417372)
  • 5‐Chlorosalicylic acid (Sigma‐Aldrich, cat. no. C70908)
  • Distilled water
  • 10% sulfuric acid
  • Sodium sulfate
  • Chloroform
  • Phosphorus trichloride (distilled)
  • Pyridine, anhydrous (Acros, cat. no. 10700693)
  • Diisopropylamine, anhydrous (DIPA; see recipe)
  • n‐Hexane, HPLC grade (VWR Chemicals, cat. no. BDH24575)
  • Silica gel (Macherey‐Nagel, cat. no. 815381.25)
  • Pressure‐equalizing dropping funnel
  • Stirrer and stirring bar
  • Condenser
  • Separator funnel
  • Rotary evaporator
  • Büchner funnel
  • Glass column (3 cm diameter)
  • Schlenk filtration device
  • Kugelrohr distillation apparatus

Support Protocol 1: Preparation of Bis(Tetra‐n‐Butylammonium) Dihydrogen Pyrophosphate

  • Disodium dihydrogen pyrophosphate (Sigma‐Aldrich, ≥99%)
  • RNAse free water, sterile
  • 10% hydrochloric acid (aqueous solution)
  • DOWEX 50WX8, hydrogen form (50 to 100 mesh; Sigma‐Aldrich, cat. no. 217492)
  • 40% tetra‐n‐butylammonium hydroxide in water (Sigma‐Aldrich, cat. no. 86854)
  • N,N‐Dimethylformamide, anhydrous (Acros, cat. no. 348431000)
  • 4 Å molecular sieves
  • Glass column (3 cm diameter)

Basic Protocol 2: Synthesis of DNA and RNA 5′‐O‐Triphosphates

  • Long‐chain alkylamine controlled‐pore glass (LCAA‐CPG), 1000 Å, succinyl linker, standard base protection
  • DNA standard base protected 3′‐O‐phosphoramidites (Glen Research)
  • RNA standard base protected 3′‐O‐phosphoramidites (Sigma‐Aldrich)
  • Acetonitrile (≤30 ppm water content, Sigma‐Aldrich)
  • TCA Deblock (3% trichloroacetic acid in dichloromethane, Sigma‐Aldrich)
  • 5‐Benzylthio‐1H‐tetrazole (BTT) activator (0.3 M in acetonitrile; Link, cat. no. 3160/3162)
  • 0.02 M iodine in THF/pyridine/water (89.6:0.4:10) oxidizer solution (Link, cat. no. 4132)
  • Cap A (10% acetic anhydride in tetrahydrofuran/pyridine, Sigma‐Aldrich, cat. no. 555339)
  • Cap B (10% N‐methylimidazole in tetrahydrofuran, Sigma‐Aldrich, cat. no. L350000‐HH)
  • 100 mM 5‐chlorosaligenyl‐N,N‐diisopropylphosphoramidite 4 (in acetonitrile; protocol 1)
  • Argon gas
  • 0.5 M bis(tetra‐n‐butylammonium) dihydrogen pyrophosphate in N,N‐dimethylformamide (see protocol 2Support Protocol)
  • N,N‐Dimethylformamide, anhydrous (Acros, cat. no. 348431000)
  • Concentrated ammonium hydroxide/40% methylamine solution in water (AMA; 1:1, v/v, mixture of 28% to 30% ammonium hydroxide solution in water [Sigma‐Aldrich, cat. no. 221228] and 40% methylamine solution in water [Sigma‐Aldrich, cat. no. 426466])
  • 1 M tetra‐n‐butylammonium fluoride in tetrahydrofuran (TBAF; Sigma‐Aldrich, cat. no. 216143)
  • Ammonium acetate (Sigma‐Aldrich, cat. no. A1542)
  • Ethanol, absolute (Sigma‐Aldrich, cat. no. 32205)
  • Acetonitrile, HPLC grade (VWR Chemicals, cat. no. 83639.320)
  • 3‐Hydroxypicolinic acid (Bruker Daltonics)
  • Ammonium citrate dibasic (Merck Millipore, cat. no. 1011540500)
  • 1.5‐mL sterile plastic centrifuge tubes
  • 1‐mL plastic syringes
  • Disposable syringe filter (Chromafil RC‐20/15 MS, Macherey‐Nagel)
  • Sterile glass pipet
  • Thermomixer
  • Speed‐vac concentrator
  • Reversed phase (RP)‐HPLC
NOTE: In general, standard long‐chain alkylamine controlled‐pore glass (LCAA‐CPG; 1000 Å, succinyl linker, standard base protection) is used as a solid support. We have not observed any difference in quality when using polystyrene‐based Custom Primer Support (GE Healthcare).
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Literature Cited

Literature Cited
  Allam, R., Pawar, R.D., Kulkarni, O.P., Hornung, V., Hartmann, G., Segerer, S., Akira, S., Endres, S., and Anders, H.‐J. 2008. Viral 5′‐triphosphate RNA and non‐CpG DNA aggravate autoimmunity and lupus nephritis via distinct TLR‐independent immune responses. Eur. J. Immunol. 38:3487‐3498. doi: 10.1002/eji.200838604
  Beaucage, S.L. and Caruthers, M.H. 2000. Synthetic strategies and parameters involved in the synthesis of oligodeoxyribonucleotides according to the phosphoramidite method. Curr. Protoc. Nucl. Acid Chem. 3.3.1‐3.3.20. doi: 10.1002/0471142700.nc0303s00
  De Clercq, E. 2009. The history of antiretrovirals: Key discoveries over the past 25 years. Rev. Med. Virol. 19:287‐299. doi: 10.1002/rmv.624
  Ekland, E., Szostak, J., and Bartel, D. 1995. Structurally complex and highly active RNA ligases derived from random RNA sequences. Science 269:364‐370. doi: 10.1126/science.7618102
  Goldeck, M., Tuschl, T., Hartmann, G., and Ludwig, J. 2014. Efficient solid‐phase synthesis of pppRNA by using product‐specific labeling. Angew. Chem. Int. Ed. 53:4694‐4698. doi: 10.1002/anie.201400672
  Haugner III, J.C. and Seelig, B. 2013. Universal labeling of 5′‐Triphosphate RNAs by artificial RNA ligase enzyme with broad substrate specificity. Chem. Commun. 49:7322‐7324. doi: 10.1039/c3cc44454F
  Hogrefe, R.I., McCaffrey, A.P., Borozdina, L.U., McCampbell, E.S., and Vaghefi, M.M. 1993. Effect of excess water on the desilylation of oligoribonucleotides using tetrabutylammonium fluoride. Nucl. Acids Res. 21:4739‐4741. doi: 10.1093/nar/21.20.4739
  Hornung, V., Ellegast, J., Kim, S., Brzózka, K., Jung, A., Kato, H., Poeck, H., Akira, S., Conzelmann, K.‐K., Schlee, M., Endres, S., and Hartmann, G. 2006. 5′‐Triphosphate RNA is the ligand for RIG‐I. Science 314:994‐997. doi: 10.1126/science.1132505
  Jemielity, J., Kowalska, J., Rydzik, A.M., and Darzynkiewicz, E. 2010. Synthetic mRNA cap analogs with a modified triphosphate bridge – synthesis, applications and prospects. New J. Chem. 34:829‐844. doi: 10.1039/c0nj00041h
  Peyrane, F., Selisko, B., Decroly, E., Vasseur, J.‐J., Benarroch, D., Canard, B., and Alvarez, K. 2007. High‐yield production of short GpppA‐ and 7MeGpppA‐capped RNAs and HPLC‐monitoring of methyltransfer reactions at the guanine‐N7 and adenosine‐2′O positions. Nucleic Acids Res. 35:e26. doi: 10.1093/nar/gkl1119
  Poeck, H., Besch, R., Maihoefer, C., Renn, M., Tormo, D., Morskaya, S.S., Kirschnek, S., Gaffal, E., Landsberg, J., Hellmuth, J., Schmidt, A., Anz, D., Bscheider, M., Schwerd, T., Berking, C., Bourquin, C., Kalinke, U., Kremmer, E., Kato, H., Akira, S., Meyers, R., Hacker, G., Neuenhahn, M., Busch, D., Rothenfusser, J., Prinz, M., Hornung, V., Endres, S., Tuting, T., and Hartmann, G. 2008. 5′‐Triphosphate‐siRNA: Turning gene silencing and Rig‐I activation against melanoma. Nat. Med. 14:1256‐1263. doi: 10.1038/nm.1887
  Sarac, I. and Meier, C. 2015. Efficient automated solid‐phase synthesis of DNA and RNA 5′‐triphosphates. Chem. Eur. J. 21:16421‐16426. doi: 10.1002/chem.201502844
  Strömberg, R. and Stawinski, J. 2004. Synthesis of oligodeoxyribo‐ and oligoribonucleotides according to the H‐phosphonate method. Curr. Protoc. Nucl. Acid Chem. 19:3.4.1‐3.4.15. doi: 10.1002/0471142700.nc0304s19
  Thillier, Y., Decroly, E., Morvan, F., Canard, B., Vasseur, J.‐J., and Debart, F. 2012. Synthesis of 5′ cap‐0 and cap‐1 RNAs using solid‐phase chemistry coupled with enzymatic methylation by human (guanine‐N7)‐methyl transferase. RNA 18:856‐868. doi: 10.1261/rna.030932.111
  Zlatev, I., Lavergne, T., Debart, F., Vasseur, J.‐J., Manoharan, M., and Morvan, F. 2010. Efficient solid‐phase chemical synthesis of 5′‐triphosphates of DNA, RNA, and their analogues. Org. Letters 12:2190‐2193. doi: 10.1021/ol1004214
Key References
  Warnecke, S. and Meier, C. 2009. Synthesis of nucleoside di‐ and triphosphates and dinucleoside polyphosphates with cycloSal‐nucleotides. J. Org. Chem. 74:3024‐3030.
  Basic mechanism and explanation of the cycloSal method.
  Wolf, S., Zismann, T., Lunau, N., and Meier C. 2009. Reliable synthesis of various nucleoside diphosphate glycopyranoses. Chem. Eur. J. 15:7656‐7664.
  Synthesis of NDP‐glycopyranoses using the cycloSal method.
  Tonn, V.C. and Meier, C. 2011. Solid‐phase synthesis of (poly)phosphorylated nucleosides and conjugates. Chem. Eur. J. 17:9832‐9842.
  First solid‐phase‐based approach using the cycloSal method to synthesize phosphate‐bridged bioconjugates.
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