Nucleoside‐O‐Methyl‐(H)‐Phosphinates: Novel Monomers for the Synthesis of Methylphosphonate Oligonucleotides Using H‐Phosphonate Chemistry

Ondřej Kostov1, Ondřej Páv1, Ivan Rosenberg1

1 Department of Bioorganic and Medicinal Chemistry, Institute of Organic Chemistry and Biochemistry of the CAS, Prague
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.76
DOI:  10.1002/cpnc.35
Online Posting Date:  September, 2017
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This unit comprises the straightforward synthesis of protected 2′‐deoxyribonucleoside‐O‐methyl‐(H)‐phosphinates in both 3′‐ and 5′‐series. These compounds represent a new class of monomers compatible with the solid‐phase synthesis of oligonucleotides using H‐phosphonate chemistry and are suitable for the preparation of both 3′‐ and 5′‐O‐methylphosphonate oligonucleotides. The synthesis of 4‐toluenesulfonyloxymethyl‐(H)‐phosphinic acid as a new reagent for the preparation of O‐methyl‐(H)‐phosphinic acid derivatives is described. © 2017 by John Wiley & Sons, Inc.

Keywords: RNase H; methylphosphonate; modified oligonucleotide; nucleoside‐(H)‐phosphinates; H‐phosphonate chemistry

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Table of Contents

  • Introduction
  • Basic Protocol 1: 2′‐Deoxyribonucleoside‐O‐Methyl‐(H)‐Phosphinates 8a–d and 9a–d
  • Basic Protocol 2: Synthesis of Short Phosphonate Oligonucleotide via H‐Phosphonate Chemistry
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: 2′‐Deoxyribonucleoside‐O‐Methyl‐(H)‐Phosphinates 8a–d and 9a–d

  • Phosphinic acid (puriss. p.a., 49.5% to 50.5% aqueous solution)
  • Dry argon (Ar atmosphere can be introduced through a rubber septum)
  • Trifluoroacetic acid (TFA)
  • Triethyl orthoacetate (Sigma‐Aldrich, cat. no. 75580)
  • Chloroform (CHCl 3) p.a. grade
  • Saturated aqueous sodium bicarbonate solution (sat. aq. NaHCO 3)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Paraformaldehyde
  • Triethylamine (Et 3N, TEA)
  • Ethyl acetate, p.a. grade, anhydrous (TLC eluting solvent)
  • 1‐Methylimidazole (MeIm; Sigma‐Aldrich, cat. no. M50834)
  • Silica gel (40 to 60 μm, Sigma‐Aldrich).
  • 4‐Toluenesulfonyl chloride (tosyl chloride; TsCl)
  • Toluene, p.a. grade (TLC eluting solvent)
  • Dichloromethane (DCM), anhydrous
  • Absolute ethanol
  • Chlorotrimethylsilane (TMSCl; Sigma‐Aldrich, cat. no. 92360)
  • Ethanol, p.a. grade (TLC eluting solvent)
  • Acetonitrile (CH 3CN), HPLC grade
  • Acetone, p.a. grade (TLC eluting solvent)
  • HPLC‐grade H 2O
  • Dowex® 50WX2 (Na+ form; see recipe)
  • Diethyl ether
  • Phosphorus pentoxide
  • 3′‐O‐Dimethoxytrityl‐2′‐deoxynucleotides 6a–d (Rejman et al., )
  • 5′‐O‐Dimethoxytrityl‐2′‐deoxynucleotides 7a–d (Rejman et al., )
  • Ammonium acetate (NH 4OAc)
  • Dimethylformamide (DMF; anhydrous)
  • 60% sodium hydride in mineral oil (NaH)
  • 2 M triethylammonium bicarbonate (TEAB) buffer, pH 7.5 ( appendix 2A)
  • Acetic acid
  • 0.1 M triethylammonium bromide (TEAB)
  • Round‐bottom flasks with glass stoppers: 250‐mL, 500‐mL, 1000‐mL
  • Rotary evaporator
  • Vacuum source (water aspirator/membrane pump; vacuum oil pump)
  • Laboratory balance
  • Magnetic stir bars
  • Magnetic stirrer
  • Separatory funnels: 100‐mL, 1000‐mL
  • Sintered glass filter; S3
  • Vacuum distillation apparatus
  • Addition funnel
  • Thin‐layer chromatography (TLC) plate (Silica gel 60 F254 aluminum sheets, Merck; also see appendix 3D)
  • 6 × 20‐cm and 8 × 20–cm sintered glass chromatography columns
  • UV detector
  • Gas balloon or flow bubbler
  • UV lamp, 254 nm
  • Analytical HPLC: LUNA C18 (100 × 4.6 mm, 3 µm, Phenomenex); linear gradient of acetonitrile in 0.1 M triethylammonium acetate, unless stated otherwise.
  • Waters LC‐MS Autopurification System comprising 515 HPLC pump, 3100 Mass detector, 600 controller, 2998 photodiode array (PDA) detector and sample manager
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and HPLC ( appendix 3E)

Basic Protocol 2: Synthesis of Short Phosphonate Oligonucleotide via H‐Phosphonate Chemistry

  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Sigma‐Aldrich, cat. no. 139009)
  • HPLC‐grade H 2O
  • Carbon dioxide gas
  • Monomers 8a–d or 9a–d ( protocol 1)
  • Chloroform
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Anhydrous dioxane (peroxide‐free)
  • Dry argon gas
  • LCAA‐dT‐5′‐CPG (Glen Research, cat. no. 20‐0302‐10)
  • Acetonitrile, anhydrous
  • Pyridine, anhydrous
  • 3‐Å molecular sieves
  • 2‐chloro‐5,5‐dimethyl‐1,3,2‐dioxaphosphorinane 2‐oxide (CDDO; Sigma‐Aldrich)
  • Carbon tetrachloride (CCl 4)
  • Methanol
  • Triethylamine (Et 3N, TEA)
  • 1‐Methylimidazole (MeIm; Sigma‐Aldrich, cat. no. M50834)
  • Benzenethiol (Sigma‐Aldrich)
  • Dimethylformamide (DMF)
  • Gaseous ammonia (NH 3)
  • Sodium acetate
  • NaCl
  • 0.1 M aqueous triethylammonium hydrogen carbonate buffer (pH 8)
  • CentriVap evaporator
  • 100‐mL separatory funnel
  • DNAPac PA100 column (9 × 250 mm, Dionex)
  • Luna C18(2) column (5 μm; 10 × 50 mm; Phenomenex)
  • Additional reagents and equipment for oligonucleotide synthesis ( appendix 3C; Ellington & Pollard, ), thin‐layer chromatography (TLC; appendix 3D), and HPLC ( appendix 3E)
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Literature Cited

Literature Cited
  Crooke, S. T., & Geary, R. S. (2013). Clinical pharmacological properties of mipomersen (Kynamro), a second generation antisense inhibitor of apolipoprotein B. British Journal of Clinical Pharmacology, 76, 269–276. doi: 10.1111/j.1365‐2125.2012.04469.x.
  Dean, N. M., & Bennett, C. F. (2003). Antisense oligonucleotide‐based therapeutics for cancer. Oncogene, 22, 9087–9096. doi: 10.1038/sj.onc.1207231.
  Ellington, A., & Pollard, J. D. (2001). Introduction to the synthesis and purification of oligonucleotides. Current Protocols in Nucleic Acid Chemistry, 00, A.3C.1–A.3C.22. doi: 10.1002/0471142700.nca03cs00.
  Kole, R., Krainer, A. R., & Altman, S. (2012). RNA therapeutics: Beyond RNA interference and antisense oligonucleotides. Nature Reviews Drug Discovery, 11, 125–140. doi: 10.1038/nrd3625.
  Kostov, O., Páv, O., Buděšínský, M., Liboska, R., Šimák, O., Petrová, M., … Rosenberg, I. (2016). 4‐Toluenesulfonyloxymethyl‐(H)‐phosphinate: A reagent for the introduction of O‐ and S‐methyl‐(H)‐phosphinate moieties. Organic Letters, 18, 2704–2707. doi: 10.1021/acs.orglett.6b01167.
  Marwick, C. (1998). First “antisense” drug will treat CMV retinitis. Journal of the American Medical Association, 280, 871. doi: 10.1001/jama.280.10.871‐JMN0909‐6‐1.
  Minshull, J., & Hunt, T. (1986). The use of single‐stranded DNA and RNase H to promote quantitative 'hybrid arrest of translation' of mRNA/DNA hybrids in reticulocyte lysate cell‐free translations. Nucleic Acids Research, 14, 6433–6451. doi: 10.1093/nar/14.16.6433.
  Prakash, T. P., Siwkowski, A., Allerson, C. R., Migawa, M. T., Lee, S., Gaus, H. J., … Bhat, B. (2010). Antisense oligonucleotides containing conformationally constrained 2′,4′‐(N‐Methoxy)aminomethylene and 2′,4′‐aminooxymethylene and 2′‐O,4′‐C‐aminomethylene bridged nucleoside analogues show improved potency in animal models. Journal of Medicinal Chemistry, 53, 1636–1650. doi: 10.1021/jm9013295.
  Rejman, D., Masojídkovaá, M., & Rosenberg, I. (2004). Nucleosidyl‐O‐methylphosphonates: A pool of monomers for modified oligonucleotides. Nucleosides, Nucleotides Nucleic & Acids, 23, 1683−1705. doi: 10.1081/NCN‐200033912.
  Šípová, H., Špringer, T., Rejman, D., Šimák, O., Petrova, M., Novak, P., … Homola, J. (2014). 5′‐O‐Methylphosphonate nucleic acids—New modified DNAs that increase the Escherichia coli RNase H cleavage rate of hybrid duplexes. Nucleic Acids Research, 42, 5378−5389. doi: 10.1093/nar/gku125.
  Southwell, A. L., Skotte, N. H., Bennett, C. F., & Hayden, M. R. (2012). Antisense oligonucleotide therapeutics for inherited neurodegenerative diseases. Trends in Molecular Medicine, 18, 634–643. doi: 10.1016/j.molmed.2012.09.001.
  Wheeler, T. M., Leger, A. J., Pandey, S. K., MacLeod, A. R., Nakamori, M., Cheng, S. H., … Thornton, C. A. (2012). Targeting nuclear RNA for in vivo correction of myotonic dystrophy. Nature, 488, 111–115. doi: 10.1038/nature11362.
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