Synthesis of 4′‐Methoxy 2′‐Deoxynucleoside Phosphoramidites for Incorporation into Oligonucleotides

Magdalena Petrová1, Ivan Rosenberg1

1 Department of Bioorganic and Medicinal Chemistry, The Institute of Organic Chemistry and Biochemistry of the Czech Academy of Sciences, Prague
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.38
DOI:  10.1002/cpnc.15
Online Posting Date:  September, 2016
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Abstract

This unit contains detailed synthetic protocols for the preparation of 4′‐methoxy 2′‐deoxynucleoside phosphoramidite monomers for A, G, C, T, and U. First, 3′‐silyl‐protected 2′‐deoxynucleosides (dNs) are converted in two steps to 4′,5′‐enol acetates as the key starting compounds. Next, 4′‐methoxy dNs are prepared by a one‐pot procedure comprising N‐iodosuccinimide‐promoted methoxylation, hydrolysis, and reduction of the formed intermediates. Finally, 3′‐phosphoramidites of 4′‐methoxy dNs are obtained by a routine three‐step procedure. Title phosphoramidite monomers are suitable for the synthesis of oligonucleotides on solid phase according to conventional amidite chemistry. 4′‐Methoxy substitution represents a simple modification of the sugar part of dNs, where β‐Derythro epimers preferentially adopt N‐type (C3′‐endo, RNA‐like) conformation. Moreover, it imparts superior chemical stability, nuclease resistance, and excellent hybridization properties to modified 4′‐methoxyoligodeoxynucleotides. The strong tendency toward RNA‐selective hybridization suggests its potential utilization in antisense and/or RNAi technologies. © 2016 by John Wiley & Sons, Inc.

Keywords: C3′‐endo conformation; 4′‐modified nucleosides; oligonucleotides; phosphoramidite; RNA mimics

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of 4′‐Methoxy 2′‐Deoxynucleoside Phosphoramidites 7a to 7e
  • Support Protocol 1: Synthesis of Protected 4′‐5′‐Enol Acetates 3a to 3e
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 4′‐Methoxy 2′‐Deoxynucleoside Phosphoramidites 7a to 7e

  Materials
  • 4′,5′‐enol acetates 3a‐e ( protocol 2Support Protocol)
  • Dry toluene
  • Dry argon gas (Ar)
  • Anhydrous methanol (MeOH)
  • N‐iodosuccinimide (NIS)
  • Acetonitrile (CH 3CN, HPLC grade)
  • Ammonium acetate (NH 4OAc)
  • Anhydrous dimethyl formamide (DMF)
  • 2 M triethylammonium bicarbonate (TEAB) buffer, pH 7.5 (appendix)
  • Sodium borohydride (NaBH 4)
  • Chloroform (CHCl 3), p.a. grade
  • Brine (saturated aqueous NaCl)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel (40 to 60 μm, Sigma‐Aldrich)
  • Ethanol (EtOH), p.a. grade
  • Acetone, p.a. grade
  • Toluene, p.a. grade
  • Ethyl acetate, p.a. grade
  • Luna C18 (15 µm; 100 Å Phenomenex, cat. no. 04K‐4273)
  • Dichloromethane (CH 2Cl 2), HPLC grade
  • Anhydrous pyridine
  • 4,4′‐Dimethoxytrityl chloride (DMTrCl)
  • Tetrabutylammonium fluoride trihydrate (TBAF⋅3H 2O)
  • Anhydrous tetrahydrofuran (THF; peroxide‐free)
  • Dowex 50WX2 resin, in triethylammonium (Et 3NH+) form (see recipe)
  • N,N‐diisopropylethylamine (DIPEA)
  • 2‐Cyanoethoxy‐N,N‐diisopropylaminochlorophosphine Sigma‐Aldrich, cat. no. 302309
  • Saturated aqueous sodium bicarbonate solution
  • 1 M triethylammonium acetate (TEAA) buffer, pH 7 ( appendix 2A)
  • Anhydrous benzene
  • 250‐ and 100‐mL round‐bottom flasks with ground‐glass stoppers
  • Rubber septa
  • Rotary evaporator
  • Magnetic stir bars
  • Magnetic stirring plate
  • Vacuum source (water aspirator/membrane pump; vacuum oil pump)
  • Analytical HPLC column: LUNA C18 (100 × 4.6 mm, 100Å, 5 µm, Phenomenex, cat. no. 00F‐4252‐E)
  • Waters LC‐MS Autopurification System comprising:
  • 515 HPLC pump
  • 3100 Mass detector
  • 600 controller
  • 2998 photodiode array (PDA)
  • Detector
  • Sample manager
  • 5 × 25‐cm precise glass column equipped with pistons (Omnifit EZ column, cat. no. 006EZ‐50‐25‐AF); for preparative chromatography in step 11
  • 3.5 × 25‐cm precise glass column equipped with pistons (Omnifit EZ column, cat. no. 006EZ‐35‐25‐AF); for preparative chromatography in step 31
  • 1‐liter, 500‐mL, and 250‐mL separatory funnels
  • UV lamp, 254 nm
  • Thin‐layer chromatography (TLC) plate (Silica gel 60 F254 aluminum sheets, Merck)
  • 4 × 20–cm sintered glass column
  • 1.6 × 20–cm sintered glass column
  • Lyophilizer
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and HPLC ( appendix 3E)

Support Protocol 1: Synthesis of Protected 4′‐5′‐Enol Acetates 3a to 3e

  Materials
  • 3′‐Otert‐butyldiphenylsilyl 2′‐deoxynucleosides 1 (see supporting information in Gao et al., )
  • 3′‐Otert‐butyldiphenylsilyl 2′‐deoxyadenosine 1a
  • 3′‐Otert‐butyldiphenylsilyl 2′‐deoxyguanosine 1b
  • 3′‐Otert‐butyldiphenylsilyl 2′‐deoxycytidine 1c
  • 3′‐Otert‐butyldiphenylsilyl thymidine 1d
  • 3′‐Otert‐butyldiphenylsilyl 2′‐deoxyuridine 1e
  • Anhydrous dimethyl formamide (DMF)
  • Dry argon gas (Ar)
  • Anhydrous dimethylsulfoxide (DMSO)
  • N‐(3‐dimethylaminopropyl)‐N′‐ethylcarbodiimide hydrochloride (EDC; Sigma‐Aldrich, cat. no. 03450)
  • Anhydrous pyridine
  • Trifluoroacetic acid (TFA)
  • Chloroform (CHCl 3), p.a. grade
  • Dry toluene
  • Acetone, p.a. grade
  • Diethyl ether
  • Brine (saturated aqueous NaCl solution)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Silica gel (40 to 60 μm, Sigma‐Aldrich)
  • Dichloromethane (CH 2Cl 2, HPLC grade)
  • Anhydrous acetonitrile (CH 3CN)
  • Potassium carbonate (K 2CO 3), anhydrous, finely ground
  • Acetic anhydride
  • Celite
  • Ethyl acetate, p.a. grade
  • Ethanol (EtOH), p.a. grade (TLC eluting solvent)
  • 500‐mL round‐bottom flasks with ground‐glass stoppers
  • Rubber septa
  • Magnetic stir bars
  • Magnetic stirring plate
  • 1000‐mL separatory funnels
  • Rotary evaporator
  • Vacuum source (water aspirator/membrane pump; vacuum oil pump)
  • UV lamp, 254 nm
  • Thin‐layer chromatography (TLC) plate (Silica gel 60 F254 aluminum sheets, Merck)
  • 4 × 20–cm sintered glass column
  • 2.5 × 20–cm sintered glass column
  • Analytical HPLC: LUNA C18 (100 × 4.6 mm, 5 µm, 100 Å Phenomenex, cat no. 00F‐4252‐E0)
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and HPLC ( appendix 3E)
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Figures

Videos

Literature Cited

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  Lee, R.C., Feinbaum, R.L., and Ambros, V. 1993. The C. elegans heterochronic gene lin‐4 encodes small RNAs with antisense complementarity to lin‐14. Cell 75:843‐854. doi: 10.1016/0092‐8674(93)90529‐Y.
  Liboska, R., Snášel, J., Barvík, I., Buděšínský, M., Pohl, R., Točík, Z., Páv, O., Rejman, D., Novák, P., and Rosenberg, I. 2011. 4′‐Alkoxy oligodeoxynucleotides: A novel class of RNA mimics. Org. Biomol. Chem. 9:8261‐8267. doi: 10.1039/c1ob06148h.
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  Petrová, M., Páv, O., Buděšínský, M., Zborníková, E., Novák, P., Rosenbergová, Š., Pačes, O., Liboska, R., Dvořáková, I., Šimák, O., and Rosenberg, I. 2015. Straightforward synthesis of purine 4′‐alkoxy‐2′‐deoxynucleosides: First report of mixed purine‐pyrimidine 4′‐alkoxyoligodeoxynucleotides as new RNA mimics. Org. Lett. 17:3426‐3429. doi: 10.1021/acs.orglett.5b01430.
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Key References
  Beaucage, S.C. 1993. Oligodeoxyribonuclotides synthesis. In Protocols for Oligonucleotides and Analogs: Synthesis and Properties: Methods in Molecular Biology, vol. 20 (S. Agrawal, ed.) Chapter 3, pp. 33‐61. Humana Press, Totawa, N.J.
  Wide coverage of the field of oligonucleotide chemistry is given in these two monographs in chapters of Methods in Molecular Biology edited by Sudhir Agrawal.
  Warren, W.J. and Vella, G. 1994. Analysis and purification of synthetic oligonucleotides by high‐performance liquid chromatography. In Protocols for Oligonucleotide Conjugates: Synthesis and Analytical Techniques: Methods in Molecular Biology, vol. 26 (S. Agrawal, ed.) Chapter 9, pp. 233‐264. Humana Press, Totowa, N.J.
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