Chemical Synthesis of RNA with Base‐Labile 2′‐O‐(Pivaloyloxymethyl)‐Protected Ribonucleoside Phosphoramidites

Thomas Lavergne1, Michaël Janin1, Christelle Dupouy1, Jean‐Jacques Vasseur1, Françoise Debart1

1 IBMM, University of Montpellier, Montpellier, France
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 3.19
DOI:  10.1002/0471142700.nc0319s43
Online Posting Date:  December, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The efficiency of chemical RNA synthesis has been radically improved by the use of pivaloyloxymethyl (PivOM) groups as 2′‐protection, containing an acetal spacer that minimizes the steric effect of the ester group on the neighboring amidite during the coupling. However, the major benefit of the base‐labile PivOM groups is their simple removal upon standard basic conditions applied to deprotect the RNA and release it from solid supports. Combined with standard acyl groups for nucleobases, cyanoethyl groups for phosphates, and base‐cleavable linkers, PivOM groups make RNA deprotection as simple as DNA deprotection. Thus, no additional deprotection step with tedious desalting workup procedures is required, and RNA synthesis becomes as convenient and efficient as DNA synthesis. Curr. Protoc. Nucleic Acid Chem. 43:3.19.1‐3.19.27. © 2010 by John Wiley & Sons, Inc.

Keywords: RNA; pivaloyloxymethyl; PivOM; amidites; ammonia deprotection

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of the 2′‐O‐PivOm‐Ribonucleosides
  • Support Protocol 1: Preparation of Iodomethyl Pivalate
  • Alternate Protocol 1: Preparation of 5′‐O‐DMTr‐2′‐O‐PivOm‐Uridine Ribonucleoside Using 3′,5′‐O‐Tips Uridine
  • Alternate Protocol 2: Synthesis of the N4‐Ac‐5′‐O‐DMTr‐2′‐O‐PivOm‐Cytidine
  • Alternate Protocol 3: Synthesis of the N6‐PAC‐5′‐O‐DMTr‐2′‐O‐PivOM‐Adenosine
  • Alternate Protocol 4: Synthesis of the N2‐iPrPAC‐5′‐O‐DMTr‐2′‐O‐PivOm‐Guanosine
  • Basic Protocol 2: Synthesis of 2′‐O‐PivOm‐Protected Phosphoramidites
  • Basic Protocol 3: Assembly of 2′‐O‐PivOm‐Protected Phosphoramidites on DNA Synthesizer
  • Basic Protocol 4: Deprotection and Cleavage of RNA Sequences Assembled from 2′‐O‐PivOm‐Protected Phosphoramidites
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Synthesis of the 2′‐O‐PivOm‐Ribonucleosides

  Materials
  • n‐Dibutyltin oxide (DBTO, Aldrich, purum)
  • Tetrabutylammonium bromide (TBAB, Aldrich, purissimum)
  • 5′‐O‐Dimethoxytritylated, N‐acylated ribonucleosides:
    • 5′‐O‐DMTr‐uridine (S.1a,Chemgenes, cat. no. PM‐2112)
    • N4‐Ac‐5′‐O‐DMTr‐cytidine (S.1b, Chemgenes, cat. no. PM‐2114)
    • N6‐PAC‐5′‐O‐DMTr‐adenosine (S.1c, Chemgenes, cat. no. PM‐2203)
    • N2iPrPAC‐5′‐O‐DMTr‐guanosine (S.1d, Chemgenes, cat. no. PM‐2209)
  • Phosphorus pentoxide (P 2O 5)
  • Acetonitrile (CH 3CN, H 2O < 50 ppm)
  • Chloromethylpivalate (PivOM‐Cl, Aldrich, 97%)
  • Dichloromethane (CH 2Cl 2, Aldrich, purissimum)
  • 94:6 (v/v) dichloromethane/methanol (for U)
  • Celite
  • 100:1 (v/v) dichloromethane/triethylamine
  • Silica gel (Macherey Nagel, http://www.macherey‐nagel.de; 40 to 63 µm)
  • Sand
  • Acetone (Aldrich, ReagentPlus)
  • 1,2‐dichloroethane (DCE; Aldrich, H 2O < 0.005%)
  • 92:8 (v/v) dichloromethane/methanol (for C)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Saturated aqueous sodium chloride (NaCl)
  • Sodium sulfate (Na 2SO 4, anhydrous)
  • Iodomethylpivalate (PivOM‐I; protocol 2)
  • Methanol (MeOH)
  • 95:5 (v/v) dichloromethane/methanol (for A and G)
  • Saturated aqueous sodium thiosulfate (Na 2S 2O 3)
  • Argon source
  • Desiccator
  • 1‐L and 500‐mL round‐bottom flasks equipped with a reflux condenser and a stir bar
  • Balloon
  • Rubber septum
  • Syringe
  • 25‐mL measuring cylinder
  • 75° and 80°C oil baths
  • TLC sheets (Macherey‐Nagel, silica gel 60 F254 aluminum sheets)
  • 254‐nm UV lamp
  • 10‐cm‐diameter fritted glass filter
  • Rotary evaporator equipped with a vacuum pump
  • 6‐cm‐diameter chromatography column (60‐cm length)
  • 1‐L separatory funnel
  • 1‐L Erlenmeyer flask
  • Additional reagents and equipment for thin‐layer chromatography ( appendix 3D)

Support Protocol 1: Preparation of Iodomethyl Pivalate

  • Calcium chloride (CaCl 2)
  • Sodium iodide (NaI, Aldrich, purissimum)
  • 18‐crown‐6 (Acros Organics, 99%)
  • Sodium sulfate (Na 2SO 4, anhydrous)
  • 100‐mL round bottom flask equipped with a guard
  • Syringe or measuring cylinder
  • 5‐cm fritted glass filter
  • 250‐mL separatory funnel

Alternate Protocol 1: Preparation of 5′‐O‐DMTr‐2′‐O‐PivOm‐Uridine Ribonucleoside Using 3′,5′‐O‐Tips Uridine

  • 3′,5′‐O‐(tetraisopropyldisiloxane‐1,3‐diyl)‐uridine (3′,5′‐O‐TIPS‐uridine; see unit 2.10)
  • Dimethylsulfoxide (DMSO), dry
  • Acetic anhydride (Ac 2O)
  • Acetic acid (AcOH)
  • Potassium carbonate (K 2CO 3)
  • Diethyl ether (Et 2O; Aldrich)
  • Cyclohexane (Carlo Erba, http://www.carloerbareagenti.com/; >99%)
  • Methanol (MeOH)
  • Sulfuryl chloride (1 M solution in CH 2Cl 2)
  • Deuterated chloroform (Eurisotop, 99.8% D)
  • Sodium pivalate (Aldrich, 97%)
  • 15‐crown‐5 (Acros Organics, 99%)
  • Ethyl acetate (AcOEt; Carlo Erba, http://www.carloerbareagenti.com/; >99%)
  • Tetrahydrofuran (THF; Carlo Erba, http://www.carloerbareagenti.com/; >99.5%)
  • Triethylamine trihydrofluoride (Et 3N 3HF; Aldrich, 98%)
  • 2 M triethylamine acetate (TEAAc), pH 7 (Fluka, purissimum, for HPLC)
  • Pyridine, dry (Carlo Erba, 99.5%, http://www.carloerbareagenti.com/; redistilled over CaCl 2)
  • 4,4′‐dimethoxytrityl chloride (Chemgenes, 97%)
  • 80:20 dichloromethane/cyclohexane containing 1% triethylamine
  • 1‐L, 500 mL, and 250 mL round‐bottom flasks
  • 4‐cm‐diameter chromatography column (50‐cm length)

Alternate Protocol 2: Synthesis of the N4‐Ac‐5′‐O‐DMTr‐2′‐O‐PivOm‐Cytidine

  • N4‐Ac‐3′,5′‐O‐TIPS‐ cytidine (unit 2.10)
  • 85:15 (v/v) CH 2Cl 2/acetone
  • 70:30 (v/v) CH 2Cl 2/acetone
  • 50:50 (v/v) cyclohexane/ethyl acetate (AcOEt)
  • 70:30 (v/v) ethyl acetate (AcOEt)

Alternate Protocol 3: Synthesis of the N6‐PAC‐5′‐O‐DMTr‐2′‐O‐PivOM‐Adenosine

  • N6‐PAC‐3′,5′‐O‐TIPS‐ adenosine (unit 2.10)
  • 70:30 and 50:50 (v/v) cyclohexane/ethyl acetate (AcOEt)
  • 60:40 (v/v) cyclohexane/AcOEt
  • 90:10 v/v CH 2Cl 2/MeOH
  • 60:40 (v/v) CH 2Cl 2/AcOEt

Alternate Protocol 4: Synthesis of the N2‐iPrPAC‐5′‐O‐DMTr‐2′‐O‐PivOm‐Guanosine

  • N2iPrPAC‐3′,5′‐O‐TIPS‐ guanosine (unit 2.10)
  • 50:50 (v/v) cyclohexane/ethyl acetate (AcOEt)
  • 40:60 (v/v) cyclohexane/AcOEt
  • Ethyl acetate (AcOEt)
  • 95:5 (v/v) AcOEt/MeOH

Basic Protocol 2: Synthesis of 2′‐O‐PivOm‐Protected Phosphoramidites

  Materials
  • Argon source
  • Dichloromethane (CH 2Cl 2)
  • Aluminum oxide 90 active neutral
  • Phosphorus pentoxide (P 2O 5)
  • 5′‐O‐Dimethoxytrityl‐2′‐O‐pivaloyloxymethyl N‐acyl ribonucleosides ( protocol 1 or Alternate Protocols protocol 31 protocol 64):
    • 5′‐O‐DMTr‐2′‐O‐PivOM‐uridine
    • N4‐Ac‐5′‐O‐DMTr‐2′‐O‐PivOM‐cytidine
    • N6‐PAC‐5′‐O‐DMTr‐2′‐O‐PivOM‐adenosine
    • N2iPrPAC‐5′‐O‐DMTr‐2′‐O‐PivOM‐guanosine
  • N‐ethyl‐N,N‐diisopropylamine (Aldrich)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite (Chemgenes, cat. no. RN‐1505)
  • 90:10 (v/v) dichloromethane/acetone (for monitoring of the reaction)
  • Pyridine
  • Ethyl acetate (AcOEt; Carlo Erba, http://www.carloerbareagenti.com/; >99%)
  • Saturated aqueous sodium bicarbonate (NaHCO 3)
  • Saturated aqueous sodium chloride (NaCl)
  • Silica gel (Macherey Nagel, http://www.macherey‐nagel.de; 40 to 63 µm)
  • Sand
  • Acetonitrile (CH 3CN, H 2O < 50 ppm)
  • Dioxane (dried through aluminum oxide column)
  • 50‐mL pressure‐equalizing dropping funnels
  • 50‐mL and 10‐mL round‐bottom flasks equipped with stir bars
  • Rubber septum
  • Balloon
  • Syringe
  • TLC sheets (Macherey‐Nagel, silica gel 60 F254 aluminum sheets)
  • 254‐nm UV lamp
  • 1‐L separatory funnels
  • 500‐mL Erlenmeyer flask
  • Rotary evaporator with vacuum pump
  • Disposable filter device (Whatman, 0.45‐µm nylon membrane)
  • Additional reagents and equipment for thin‐layer chromatography ( appendix 3D)

Basic Protocol 3: Assembly of 2′‐O‐PivOm‐Protected Phosphoramidites on DNA Synthesizer

  Materials
  • 0.1 M PivOM‐amidite solution in CH 3CN ( protocol 7)
  • Phosphorus pentoxide (P 2O 5)
  • 0.1 M 5‐benzylthio‐1H‐tetrazole (5‐benzylmercaptotetrazole or BMT) in CH 3CN (Biosolve, Chemgenes)
  • Argon gas
  • Controlled‐pore glass (CPG) with attached nucleoside (30 to 50 µmol/g; Glen Research, Chemgenes)
  • Capping solutions A and B (see Table 3.19.1, Biosolve, Chemgenes)
  • Oxidizing solution (see Table 3.19.1, Biosolve, Chemgenes)
  • Deblocking solution (see Table 3.19.1, Biosolve, Chemgenes)
  • Extra‐dry acetonitrile (Biosolve, H 2O < 0.003%)
  • Desiccator
  • Vials and bottles for attachment of the phosphoramidites and reagents to the synthesizer
  • Automated DNA synthesizer (ABI 381A or 394, Applied Biosystems)
  • Empty 1‐µmol synthesis column fitted with filters (Glen Research)
  • Additional reagents and equipment for automated DNA synthesis (see appendix 3C)

Basic Protocol 4: Deprotection and Cleavage of RNA Sequences Assembled from 2′‐O‐PivOm‐Protected Phosphoramidites

  Materials
  • Argon source
  • Dry CH 3CN
  • 1,8‐diazabicyclo‐[5.4.0]undec‐7‐ene (DBU) (Aldrich)
  • PivOM‐protected oligoribonucleotides attached to solid support ( protocol 8)
  • 28% aqueous ammonia
  • Isopropylamine (purissimum, >99.5%, Aldrich)
  • 5‐mL and 100‐mL round‐bottom flasks
  • Stir bar
  • Rubber septum
  • Balloon
  • 1‐mL and 2‐mL plastic syringes with Luer fittings
  • 4‐mL screw‐cap glass vials
  • 2‐mL microcentrifuge tubes
  • Rotary evaporator (or SpeedVac evaporator)
  • Pasteur pipets with cotton filters
  • Additional reagents and equipment for oligoribonucleotide purification by HPLC (unit 10.5)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Cieslak, J., Grajkowski, A., Kauffman, J.S., Duff, R.J., and Beaucage, S.L. 2008. The 4‐(N‐dichloroacetyl‐N‐methylamino)benzyloxymethyl group for 2′‐hydroxyl protection of ribonucleosides in the solid‐phase synthesis of oligoribonucleotides. J. Org. Chem. 73:2774‐2783.
   Fire, A., Xu, S., Montgomery, M.K., Kostos, S.A., Driver, S.E., and Mello, C. 1998. Potent and specific genetic interference by double‐stranded RNA in Caenorhabditis elegans. Nature 391:806‐811.
   Kempe, T., Chow, F., Sundquist, W.I., Nardi, T.J., Paulson, B., and Peterson, S.M. 1982. Selective 2′‐benzoylation at the cis 2′,3′‐diols of protected ribonucleosides: New solid phase synthesis of RNA and DNA‐RNA mixtures. Nucleic Acids Res. 10:6695‐6714.
   Lackey, J.G., Sabatino, D., and Damha, M.J. 2007. Solid‐phase synthesis and on‐column deprotection of RNA from 2′‐ (and 3′‐)O‐levulinated (Lv) ribonucleoside monomers. Org. Lett. 9:789‐792.
   Lackey, J.G., Mitra, D., Somoza, M.M., Cerrina, F., and Damha, M.J. 2009. Acetal Levulinyl Ester (ALE) groups for 2′‐Hydroxyl protection of ribonucleosides in the synthesis of oligoribonucleotides on glass and microarrays. J. Am. Chem. Soc. 131:8496‐8502.
   Lavergne, T., Bertrand, J.‐R., Vasseur, J.‐J., and Debart, F. 2008. A base‐labile group for 2′‐OH protection of ribonucleosides: A major challenge for RNA synthesis. Chem. Eur. J. 14:9135‐9138.
   Ohgi, T., Masutomi, Y., Ishiyama, K., Kitagawa, H., Shiba, Y., and Yano, J. 2005. A new RNA synthetic method with a 2′‐O‐(2‐Cyanothoxymethyl) protecting group. Org. Lett. 7:3477‐3480.
   Pitsch, S., Weiss, P.A., Jenny, L., Stutz, A., and Wu, J.‐C. 2001. Reliable chemical synthesis of oligoribonucleotides (RNA) with 2′‐O‐[(Triisopropylsilyl)oxy]methyl(2′‐O‐tom)‐protected phosphoramidites. Helv. Chim. Acta 84:3773‐3795.
   Saneyoshi, H., Ando, K., Seio, K., and Sekine, M. 2007. Chemical synthesis of RNA via 2′‐O‐cyanoethylated intermediates. Tetrahedron 63:11195‐11203.
   Scaringe, S.A., Wincott, F.E., and Caruthers, M.H. 1998. Novel RNA synthesis method using 5′‐O‐silyl‐2′‐O‐orthoester protecting groups. J. Am. Chem. Soc. 120:11820‐11821.
   Semenyuk, A., Földesi, A., Johansson, T., Estmer‐Nilsson, C., Blomgren, P., Brännvall, M., Kirsebom, L.A., and Kwiatkowski, M. 2006. Synthesis of RNA using 2′‐O‐DTM protection. J. Am. Chem. Soc. 128:12356‐12357.
   Umemoto, T. and Wada, T. 2004. Oligoribonucleotide synthesis by the use of 1‐(2‐cyanoethoxy)ethyl (CEE) as a 2′‐hydroxy protecting group. Tetrahedron Lett. 45:9529‐9531.
   Usman, N., Pon, R.T., and Ogilvie, K.K. 1985. Preparation of ribonucleoside 3′‐O‐phosphoramidites and their application to the automated solid phase synthesis of oligonucleotides. Tetrahedron Lett. 26:4567‐4570.
   Zhou, C., Honcharenko, D., and Chattopadhyaya, J. 2007. 2‐(4‐Tolylsulfonyl)ethoxymethyl (TEM)‐ a new 2′‐OH protecting group for solid‐supported RNA synthesis. Org. Biomol. Chem. 5:333‐343.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library