Uridine 2′‐Carbamates: Facile Tools for Oligonucleotide 2′‐Functionalization

Vladimir A. Korshun1, Dmitry A. Stetsenko2, Michael J. Gait2

1 Shemyakin‐Ovchinnikov Institute of Bioorganic Chemistry, Moscow, 2 Medical Research Council, Laboratory of Molecular Biology, Cambridge
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.21
DOI:  10.1002/0471142700.nc0421s15
Online Posting Date:  February, 2004
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Abstract

A facile method for preparation of uridine 2′‐carbamate derivatives based on reaction of 3′,5′‐disilyl‐protected uridine with 1,1′‐carbonyldiimidazole followed by treatment with an aliphatic amine is presented. A phosphoramidite monomer suitable for automated oligonucleotide synthesis is obtained in a few steps. The compounds are useful for the introduction of various labels and modifications into an oligonucleotide chain. Although 2′‐carbamate modification is somewhat destabilizing for DNA‐DNA and DNA‐RNA duplexes, it is suitable for the direction of ligands into the minor groove of duplexes or at non‐base‐paired sites (e.g., loops and bulges) of oligonucleotides. Pyrene‐modified oligonucleotide 2′‐carbamates show a considerable increase in fluorescence intensity upon hybridization to a complementary RNA (but not DNA).

Keywords: nucleoside; uridine carbamate; oligonucleotide modification; duplex stability; pyrene; fluorescence

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of Uridine 2′‐Carbamate Phosphoramidites from Primary and Secondary Amines
  • Alternate Protocol 1: Preparation of Uridine 2′‐Carbamate Phosphoramidites from Primary Amines that Require Additional Side‐Chain Protection
  • Basic Protocol 2: Synthesis, Isolation, and Characterization of Oligonucleotides Containing Uridine 2′‐Carbamates
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of Uridine 2′‐Carbamate Phosphoramidites from Primary and Secondary Amines

  Materials
  • Uridine (S.1), 99% pure
  • Anhydrous pyridine, 99.8% pure (Aldrich)
  • Nitrogen (or argon) gas
  • Markiewicz reagent: 1,3‐dichloro‐1,1,3,3‐tetraisopropyldisiloxane, 96% pure (Lancaster)
  • Ethyl acetate (EtOAc), HPLC grade
  • 5% (w/v) sodium hydrogencarbonate (NaHCO 3)
  • Sodium sulfate (Na 2SO 4), anhydrous
  • Toluene, analytical grade
  • Silica gel: 0.040‐ to 0.063‐mm Macherey‐Nagel Kieselgel 60
  • Chloroform (CHCl 3), HPLC grade, ethanol free
  • Anhydrous dichloromethane (CH 2Cl 2), distilled from powdered CaH 2 (Fisher)
  • Hexane, HPLC grade
  • 1,1′‐Carbonyldiimidazole, 95% pure (Sigma)
  • Amine for S.3 conversion (select one):
    • Propargylamine (for S.4a)
    • N‐Methylpropargylamine (for S.4b)
    • 4‐Iodobenzylamine (Lancaster; for S.4c)
    • 1‐Pyrenemethylamine hydrochloride (Aldrich; for S.4d)
    • 2‐Aminomethyl‐15‐crown‐5 (for S.4e)
    • H‐Leu‐Phe‐NH 2 hydrochloride (for S.4f)
  • Triethylamine (TEA), ≥99% pure
  • Acetonitrile (CH 3CN), HPLC grade (optional; for S.4e and S.4f)
  • N,N‐Diisopropylethylamine (DIPEA), ≥99% pure (Aldrich; optional; for S.4f)
  • 5% (w/v) citric acid
  • Methanol (MeOH), analytical grade
  • Acetone, analytical grade
  • Dry tetrahydrofuran (THF), freshly distilled from LiAlH 4 (store over 4A molecular sieves under nitrogen)
  • Triethylamine trihydrofluoride
  • Absolute ethanol, analytical grade (optional; for S.5c and S.5d)
  • Diethyl ether, analytical grade (optional; for S.5c and S.5d)
  • 4,4′‐Dimethoxytrityl chloride (DMTr·Cl, 95% pure; Avocado Research Chemicals)
  • Diisopropylammonium tetrazolide
  • Bis(N,N‐diisopropylamino)‐2‐cyanoethoxyphosphine, 98% pure (Fluka)
  • 20% (w/v) sodium chloride (NaCl)
  • Rotary evaporator equipped with a water aspirator
  • 4 × 20–cm sintered glass chromatography column, porosity 3
  • Vacuum oil pump
  • TLC plate: silica‐coated aluminum plate with fluorescent indicator (Merck silica gel 60 F 254)
  • 254‐nm UV lamp
  • 30‐mL screw‐top Teflon bottle (Nalgene)
  • Additional reagents and equipment for thin‐layer chromatography (TLC, appendix 3D) and column chromatography ( appendix 3E)

Alternate Protocol 1: Preparation of Uridine 2′‐Carbamate Phosphoramidites from Primary Amines that Require Additional Side‐Chain Protection

  • 3′,5′‐O‐(Tetraisopropyldisiloxan‐1,3‐diyl)uridine (S.2; see protocol 1, step )
  • Amine for S.3 conversion (select one):
    • N‐(3‐Aminopropyl)‐1,3‐propanediamine, 98% pure (Aldrich; for S.4g)
    • Spermine, 99% pure (Aldrich; for S.4h)
    • 4,7,10‐Trioxa‐1,13‐tridecanediamine, 97% pure (Sigma; for S.4iS.4j)
    • 2‐(2‐Aminoethoxy)ethanol (for S.4k)
  • Reagent for amine protection (select one):
    • S‐Ethyl trifluorothioacetate, 97% pure (Aldrich; for S.4gS.4i)
    • Nα‐Fmoc‐Stert‐butylthio‐L‐cysteine pentafluorophenyl ester, 99% pure (Novabiochem; for S.4j)
    • Trimethylacetyl chloride, 99% pure (Aldrich; for S.4l)

Basic Protocol 2: Synthesis, Isolation, and Characterization of Oligonucleotides Containing Uridine 2′‐Carbamates

  Materials
  • Uridine 2′‐carbamate phosphoramidite(s) (S.7a‐j,l; see protocol 1 and protocol 2)
  • Acetonitrile, anhydrous
  • Phosphoramidites:
    • 2′‐Deoxyribonucleoside phosphoramidites (Transgenomics)
    • 2′‐O‐Methyl‐ribonucleoside phosphoramidites (2′‐OMe; Transgenomics)
    • 2′‐O‐[(Triisopropylsilyl)oxy]methyl‐ribonucleoside phosphoramidites (2′‐O‐TOM; Glen Research; also see units 2.9 & 3.8)
  • 30% ammonia
  • Nitrogen
  • Mobile phase A: 5% CH 3CN in 0.1 M triethylammonium acetate (TEAA), pH 7.0 (DMTr‐ON, HPLC)
  • Mobile phase B: 100% CH 3CN (DMTr‐ON, HPLC)
  • 1 to 400 mM sodium perchlorate in 20 mM Tris·Cl (pH 6.8; appendix 2A)/25% formamide (DMTr‐OFF, HPLC)
  • 15% (w/v) polyacrylamide gel ( appendix 3D) containing 2 M urea in 0.5× TBE electrophoresis buffer ( appendix 2A) (DMTr‐OFF, PAGE)
  • 0.5 M LiClO 4 (DMTr‐OFF, PAGE)
  • Matrix solution I: 40 mg/mL 2,6‐dihydroxyacetophenone in methanol
  • Matrix solution II: 80 mg/mL diammonium hydrogen citrate in water
  • Water aspirator
  • Screw‐capped tube (Sarstedt) or vial
  • Speedvac evaporator (Savant)
  • Spin‐X tube (Costar)
  • Reversed‐phase cartridges for DNA isolation (e.g., PolyPak, Glen Research; DMTr‐ON)
  • HPLC system (optional) with:
    • Column: 3.9 × 300–mm Phenomenex Bondclone 10 C18 column (DMTr‐ON) or 9 × 250–mm Dionex NucleoPac PA‐100 column (DMTr‐OFF)
    • Detector: 254 nm (DMTr‐ON) or 280 nm (DMTr‐OFF)
  • Lyophilizer
  • Microcon tube (Millipore) or NAP‐10 column (DMTr‐OFF, PAGE)
  • Additional reagents and equipment for automated solid‐phase oligonucleotide synthesis ( appendix 3C) and purification of oligonucleotides (units 10.1, 10.4, 10.5, 10.7 & 3.NaN)
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Figures

Videos

Literature Cited

Literature Cited
   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. Methods Enzymol. 154:287‐313.
   Dubey, I., Pratviel, G., and Meunier, B. 2000. Synthesis and DNA cleavage of 2′‐O‐amino‐linked metalloporphyrin‐oligonucleotide conjugates. J. Chem. Soc., Perkin Trans. 1:3088‐3095.
   Freier, S.M. and Altmann, K.‐H. 1997. The ups and downs of nucleic acid duplex stability: Structure‐stability studies on chemically‐modified DNA:RNA duplexes. Nucl. Acids Res. 25:4429‐4443.
   Korshun, V.A., Stetsenko, D.A., and Gait, M.J. 2002. Novel uridin‐2′‐yl carbamates: Synthesis, incorporation into oligodeoxyribonucleotides, and remarkable fluorescence properties of 2′‐pyren‐1‐ylmethylcarbamate. J. Chem. Soc., Perkin Trans. 1:1092‐1104.
   McGee, D.P.C., Sebesta D.P., O'Rourke, S.S., Martinez, R.L., Jung, M.E., and Pieken, W.A. 1996. Novel nucleosides via intramolecular functionalization of 2,2′‐anhydrouridine derivatives. Tetrahedron Lett. 37:1995‐1998.
   Prhavc, M., Lesnik, E.A., Mohan, V., and Manoharan, M. 2001. 2′‐O‐Carbamate‐containing oligonucleotides: Synthesis and properties. Tetrahedron Lett. 42:8777‐8780.
   Rannard, S.P. and Davis, N.P. 2000. The selective reaction of primary amines with carbonyl imidazole containing compounds: Selective amide and carbamate synthesis. Org. Lett. 2:2117‐2120.
   Seio, K., Wada, T., Sakamoto, K., Yokoyama, S., and Sekine, M. 1998. Chemical synthesis and properties of conformationally fixed diuridine monophosphates as building blocks of the RNA turn motif. J. Org. Chem. 63:1429‐1443.
   Silverman, S.K. and Cech, T.R. 1999. RNA tertiary folding monitored by fluorescence of covalently attached pyrene. Biochemistry 38:14224‐14237.
   Sproat, B.S. and Brown, D.M. 1985. A new linkage for solid phase synthesis of oligodeoxyribonucleotides. Nucl. Acids Res. 13:2979‐2987.
   Stolze, K., Holmes, S.C., Earnshow, D.J., Singh, M., Stetsenko, D.A., Williams, D., and Gait, M.J. 2001. Novel spermine‐amino acid conjugates and basic tripeptides enhance cleavage of the hairpin ribozyme at low magnesium ion concentration. Bioorg. Med. Chem. Lett. 11:3007‐3010.
   Wachter, A., Jablonski, J.‐A., and Ramachandran, K.L. 1986. A simple and efficient procedure for the synthesis of 5′‐aminoalkyl oligodeoxynucleotides. Nucl. Acids Res. 14:7985‐7994.
   Yamana, K., Zako, H., Asazuma, K., Iwase, R., Nakano, H., and Murakami, A. 2001. Fluorescence detection of specific RNA sequences using 2′‐pyrene‐modified oligoribonucleotides. Angew. Chem. Int. Ed. Engl. 40:1104‐1106.
   Zatsepin, T.S., Stetsenko, D.A., Arzumanov, A.A., Romanova, E.A., Gait, M.J., and Oretskaya, T.S. 2002. Synthesis of peptide‐oligonucleotide conjugates with single and multiple peptides attached to 2′‐aldehyde through thiazolidine, oxime and hydrazine linkages. Bioconjug. Chem. 13:822‐830.
   Zhang, L., Cui, Z., and Zhang, B. 2003. An efficient synthesis of 3′‐amino‐3′‐deoxyguanosine from guanosine. Helv. Chim. Acta 86:703‐710.
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