Microwave‐Assisted Phosphitylation of DNA and RNA Nucleosides and Their Analogs

Tim Efthymiou1, Ramanarayanan Krishnamurthy1

1 Department of Chemistry, The Scripps Research Institute, La Jolla, California
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 2.19
DOI:  10.1002/0471142700.nc0219s60
Online Posting Date:  March, 2015
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Abstract

Microwave‐assisted chemical phosphitylation of novel nucleoside analogs containing a ribulose sugar unit was successful with yields ranging from 50% to 79% using 2‐cyanoethyl‐N,N‐diisopropyl chlorophosphoramidite as the phosphitylating reagent. The resultant phosphoramidite products remained intact, with no signs of degradation over extended reaction times (up to 60 min) at an elevated temperature (65°C). When the same microwave‐mediated phosphitylating protocols were applied to canonical DNA and RNA nucleoside monomers as substrates, using either 2‐cyanoethyl‐N,N,‐diisopropyl chlorophosphoramidite or 2‐cyanoethyl‐N,N,N′,N′‐tetraisopropyl phosphane with an activator, 40% to 90% yields of DNA and RNA phosphoramidites were obtained within 10 to 15 min. These results demonstrate that microwave‐assisted phosphitylation is an efficient alternative to standard phosphitylating conditions that can be expanded and refined to include a variety of substrates. © 2015 by John Wiley & Sons, Inc.

Keywords: microwave; phosphoramidite; nucleoside; ribulose; phosphitylation

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of β‐L‐Pentulofuranosyl Phosphoramidite Building Blocks
  • Basic Protocol 2: Microwave‐Assisted Phosphitylation of Canonical DNA and RNA Nucleosides Using 2‐Cyanoethyl‐N,N‐Diisopropyl Chlorophosphoramidite
  • Alternate Protocol 1: Microwave‐Assisted Phosphitylation of Canonical DNA and RNA Nucleosides Through Activation of 2‐Cyanoethyl‐N,N,N′,N′‐Tetraisopropyl Phosphane
  • Support Protocol 1: Preparation of Pyridinium Hydrochloride Activating Reagent
  • Commentary
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of β‐L‐Pentulofuranosyl Phosphoramidite Building Blocks

  Materials
  • Nucleoside substrates:
    • 1′‐O‐(4,4′‐Dimethoxytrityl)‐4′‐O‐triisopropylsilyloxymethyl‐β‐L‐ribulofuranosyl thymidine (1a; Stoop et al., )
    • N6‐Benzoyl‐1′‐O‐dimethoxytrityl‐4′‐O‐triisopropylsilyloxymethyl‐β‐L‐ribulofuranosyl‐adenosine (1b; Stoop et al.,  )
    • N4‐Benzoyl‐1′‐O‐dimethoxytrityl‐4′‐O‐triisopropylsilyloxymethyl‐β‐L‐ribulofuranosyl‐cytidine (1c; Efthymiou,  )
    • N2‐Acetyl‐O6‐diphenylcarbamoyl‐1′‐O‐dimethoxytrityl‐4′‐O‐triisopropylsilyloxymethyl‐β‐L‐ribulofuranosyl‐guanosine (1d; Efthymiou, )
  • Anhydrous toluene (Fisher)
  • Argon gas (Praxair)
  • Nitrogen gas (Praxair)
  • Anhydrous dichloromethane (CH 2Cl 2; EMD Millipore)
  • Anhydrous N,N‐diisopropyl‐N‐ethylamine ((iPr) 2NEt, Hunig's base; Aldrich)
  • 2‐Cyanoethyl‐N,N,‐diisopropyl chlorophosphoramidite (ChemGenes)
  • 4‐Dimethylaminopyridine (DMAP; Acros)
  • Ethyl acetate (EtOAc; Fisher)
  • Hexanes (Fisher)
  • Triethylamine (TEA; Aldrich)
  • Saturated sodium bicarbonate (NaHCO 3; Fisher)
  • Sodium sulfate (Na 2SO 4; Fisher)
  • Deuterated chloroform (CDCl 3; Oakwood)
  • Saturated sodium chloride (NaCl; Fisher)
  • Silica gel (60 Å, 230‐400 mesh; Silicycle)
  • 2‐5 mL microwave reaction tubes (Biotage), oven‐dried with a magnetic stir bar
  • Metal tube caps with 20‐mm PTFE (blue)/silicon (white) septa (Biotage)
  • 100‐ and 250‐mL round‐bottom flasks, oven‐dried
  • Rotary evaporator (Buchi) equipped with a vacuum pump (Welch)
  • High‐vacuum oil pump (Welch)
  • Red rubber septa: 14/20, 24/40 (Aldrich)
  • Balloons
  • 1‐ and 3‐mL plastic syringes (Air‐tite)
  • Needles: 18 awg, 20 awg, 4‐in. 20 awg
  • Microwave tube crimper and decapper (Biotage)
  • Glove box/bag (Fisher)
  • Microwave synthesizer (Initiator Classic, Biotage)
  • Fluorescent (F 254) silica TLC plates (EMD or equivalent)
  • UV lamp (Spectroline)
  • 2‐mL glass screw‐cap sample vials
  • Vacuum adapter: 24/40 (ChemGlass)
  • 250‐mL separatory funnels
  • 250‐mL Erlenmeyer flasks
  • 3 × 30− and 2 × 20−cm glass chromatography columns
  • 16 × 150−mm test tubes
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Microwave‐Assisted Phosphitylation of Canonical DNA and RNA Nucleosides Using 2‐Cyanoethyl‐N,N‐Diisopropyl Chlorophosphoramidite

  Materials
  • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyadenosine (3a; ChemGenes)
  • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyguanosine (3b; ChemGenes)
  • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)guanosine (3c; ChemGenes)
  • 20‐G needles
  • 10‐mL round‐bottom flasks
  • Additional reagents and equipment for microwave‐assisted phosphitylation (see protocol 1)

Alternate Protocol 1: Microwave‐Assisted Phosphitylation of Canonical DNA and RNA Nucleosides Through Activation of 2‐Cyanoethyl‐N,N,N′,N′‐Tetraisopropyl Phosphane

  Additional Materials (also see protocol 1)
  • Nucleoside substrates:
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐deoxythymidine (5a; ChemGenes)
    • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)uridine (5c; ChemGenes)
    • N4‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxycytidine (5b; ChemGenes)
    • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)adenosine (5d; ChemGenes)
    • N4‐Acetyl‐5′‐(4,4′‐dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)cytidine (5e; ChemGenes)
    • N6‐Benzoyl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyadenosine (3a; ChemGenes)
    • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐deoxyguanosine (3b; ChemGenes)
    • N2‐Isobutyryl‐5′‐O‐(4,4′‐dimethoxytrityl)‐2′‐O‐(tert‐butyldimethylsilyl)guanosine (3c; ChemGenes)
  • Activator: 5‐ethylthio‐1H‐tetrazole (Glen Research) or anhydrous pyridinium hydrochloride (see protocol 4Support Protocol for drying procedure)
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropyl phosphane (ChemGenes)
  • 10‐mL round‐bottom flasks

Support Protocol 1: Preparation of Pyridinium Hydrochloride Activating Reagent

  Materials
  • Silicon oil (Acros)
  • Pyridinium hydrochloride (Acros)
  • Benzene (EMD Millipore)
  • Argon gas (Praxair)
  • Ring rod apparatus, including stand, clamps, clamp holders, and flask neck clips (24/40)
  • 200‐mL round‐bottom flask (24/40 neck)
  • Fume hood with running water tap and drain
  • Magnetic stirring hot plate (IKA)
  • 500‐mL crystallization dish
  • Dean‐Stark apparatus (24/40 neck; ChemGlass)
  • Tygon tubing
  • Red rubber septum: 24/40
  • Balloon (Aldrich)
  • Rotary evaporator (Buchi) equipped with a vacuum pump (Welch)
  • Needle: 18 awg
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Figures

Videos

Literature Cited

Literature Cited
  Alvarado‐Urbina, G., Sathe, G.M., Liu, W.‐C., Gillen, M.F., Duck, P.D., Bender, R., and Ogilvie, K.K. 1981. Automated synthesis of gene fragments. Science. 214:270‐274.
  Amigues, E.J., Hardacre, C., Keane, G., Migaud, M.E., Norman, S.E., and Pitner, W.R. 2009. Green Chem. 11:1391‐1396.
  Beaucage, S. 2003. 2‐Cyanoethyl‐tetraisopropylphosphoramidite. e‐EROS Encyclopedia of Reagents for Organic Synthesis, John Wiley & Sons, DOI: 10.1002/047084289X.rn00312.
  Caruthers, M.H., Barone, A.D., Beaucage, S.L., Dodds, D.R., Fisher, E.F., McBride, L.J., Matteucci, M., Stabinsky, Z., and Tang, J.‐Y. 1987. Chemical Synthesis of Deoxyoligonucleotides by the Phosphoramidite Method. Method. Enzymol. 154:287‐313.
  Catana, D.‐A., Maturano, M., Payrastre, C., Lavedan, P., Tarrat, N., and Escudier, J.‐M. 2011. Synthesis of phostone‐constrained nucleic acid (P‐CAN) dinucleotides through intramolecular Arbuzov's reaction. Eur. J. Org. Chem. 6857‐6863.
  Efthymiou, T. 2014. Post‐doctoral Report. Krishnamurthy group, TSRI.
  Grünfeld, P. and Richert, C. 2006. Effect of microwave irradiation on phoshporamidite couplings on controlled pore glass. Nucleosides, Nucleotides, Nucleic Acids 25:815‐821.
  Hardcare, C., Huang, H., James, S.L., Migaud, M.E., Norman, S.E., and Pitner, W.R. 2011. Chem. Commun. 47:5846‐5848.
  Heller, S.T., Fu, T., and Sarpong, R. 2012. Dual Bronsted acid/nucleophilic activation of carbonylimidazole derivatives. Org. Lett. 14:1970‐1973.
  McBride, L.J. and Caruthers, M.H. 1983. An investigation of several deoxynucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides. Tetrahedron Lett. 24:245‐248.
  Meher, G. and Krishnamurthy, R. 2011. An expedient synthesis of L‐ribulose and derivatives. Carbohyd. Res. 346:703‐707.
  Meher, G., Efthymiou, T., Stoop, M., and Krishnamurthy, R. 2014. Microwave‐assisted preparation of nucleoside‐phosphoramidites. Chem. Comm. 50:7463‐7465.
  Meng, M., Ahlborn, C., Bauer, M., Plietzsch, O., Soomro, S.A., Singh, A., Muller, T., Wenzel, W., Brase, S., and Richert, C. 2009. Two base pair duplexes suffice to build a novel material. Chem. Bio. Chem. 10:1335‐1339.
  Ogilvie, K.K., Usman, N., Nicoghosian, K., and Cedergren, R.J. 1988. Total chemical synthesis of a 77‐nucleotide‐long RNA sequence having methionine‐acceptance activity. Proc. Natl. Acad. Sci. U.S.A. 85:5764‐5768.
  Ohtsuka, E., Ikehara, M., and Soll, D. 1982. Recent developments in the chemical synthesis of polynucleotides. Nucleic Acid Res. 10:6553‐6570.
  Scaringe, S.A., Francklyn, C., and Usman, N. 1990. Chemical synthesis of biologically active oligoribonucleotides using β‐cyanoethyl protected ribonucleoside phosphoramidites. Nucleic Acids Res. 18:5433‐5441.
  Sinden, R.R. 1994. DNA Structure and Function. Academic Press, San Diego.
  Stoop, M., Meher, G., Karri, P., and Krishnamurthy, R. 2013. Chemical etiology of nucleic acid structure: The pentulofuranosyl oligonucleotide systems: The (1′→3′)‐β‐L‐ribulo, (4′→3′)‐α‐L‐xylulo, and (1′→3′)‐α‐L‐xylulo nucleic acids. Chem. Eur. J. 19:15336‐15345.
  Usman, N., Ogilvie, K.K., Jiang, M.‐Y., and Cedergren, R.J. 1987. Automated chemical synthesis of long oligoribonucleotides using 2′‐O‐silylated ribonucleoside 3′‐O‐phosphoramidites on a controlled‐pore glass support: Synthesis of a 43‐nucleotide sequence similar to the 3′‐half molecule of an Escherichia coli formylmethionine tRNA. J. Am. Chem. Soc. 109:7845‐7854.
  Weixlbaumer, A., Murphy, FV 4th, Dziergowska, A., Malkiewicz, A., Vendeix, F.A.P., Agris, P.F., and Ramakrishnan, V. 2007. Mechanism for expanding the decoding capacity of transfer RNAs by modification of uridines. Nat. Struct. Mol. Biol. 14:498‐502.
Internet Resources
  Behlke, M.A. and Devor, E.J. 2005. Chemical Synthesis of Oligonucleotides. Integrated DNA Technologies. http://www.crchudequebec.ulaval.ca/ronyweb/DOWNLOADS/Chemical_Synthesis_of_Oligonucleotides.pdf.
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