Solid‐Phase Oligonucleotide Labeling with DOTA

Lassi Jaakkola1, Alice Ylikoski1, Jari Hovinen1

1 PerkinElmer Life and Analytical Sciences, Turku, Finland
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.31
DOI:  10.1002/0471142700.nc0431s29
Online Posting Date:  June, 2007
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This unit describes a method to construct oligonucleotide conjugates labeled with 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA) on a solid support. A nucleosidic phosphoramidite that contains a protected DOTA ligand compatible with normal chain assembly is prepared first. As the chain assembly is completed, the oligonucleotide is deprotected and converted to the corresponding gadolinium(III) chelate, resulting in an oligonucleotide conjugate containing the chelate at the 5′‐terminus.

Keywords: solid support; oligonucleotides; DOTA; MRI; molecular imaging; phosphoramidites

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Table of Contents

  • Basic Protocol 1: Preparation of the Nucleosidic Phosphoramidite Tethered to DOTA
  • Basic Protocol 2: Synthesis of Oligonucleotide‐DOTA Conjugates
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Preparation of the Nucleosidic Phosphoramidite Tethered to DOTA

  • 1,4,7,10‐Tetraazacyclododecane‐1‐carboxymethylbenzyl ester (S.1; Heppeler et al., )
  • Dry acetonitrile, ≥99.5% pure, ≤0.005% H 2O (Merck)
  • Anhydrous potassium carbonate, p.a. (Merck)
  • Methyl bromoacetate, 99% pure (Acros Organics)
  • Silica gel 60, 0.063 to 0.200 nm (Merck)
  • Methanol (MeOH), ≥99.9% pure (Merck)
  • Dichloromethane (CH 2Cl 2), 99% pure (Lab‐Scan)
  • 10% palladium on activated carbon (Pd/C; Aldrich)
  • Hydrogen gas
  • Celite 521 (Aldrich)
  • 5′‐O‐(4,4′‐Dimethoxytrityl)‐2′‐deoxyuridine (S.4; Sigma)
  • N‐Trifluoroacetyl‐6‐aminohexan‐1‐ol (Sinha and Striepeke, )
  • Triphenylphosphine, 99% pure (Aldrich)
  • Dry tetrahydrofuran (THF), ≥99.5% pure, ≤0.0075% H 2O (Merck)
  • Diisopropyl azodicarboxylate (DIAD), 95% pure (Aldrich)
  • Diethyl ether, ≥99.7% pure (Merck)
  • Aqueous ammonia, p.a. 28% to 30% (Merck)
  • Sodium sulfate (Na 2SO 4), anhydrous, ≥99.0% pure (Merck)
  • N,N‐Diisopropylethylamine (DIPEA), ≥99.5% pure (Aldrich)
  • N,N‐Dimethylformamide (DMF), dry, 99.8% pure, ≤0.01% H 2O (Lab‐Scan)
  • O‐(7‐Azabenzotriazol‐1‐yl)‐N,N,N′,N′‐tetramethyluronium hexafluorophosphate (HATU; Applied Biosystems)
  • Sodium hydrogen carbonate (NaHCO 3) solutions, saturated and 5% (w/v)
  • 2‐Cyanoethyl‐N,N,N′,N′‐tetraisopropylphosphordiamidite, 97% pure (Aldrich)
  • 0.45 M 1H‐tetrazole in acetonitrile (Applied Biosystems)
  • Triethylamine (TEA), ≥99% pure (Merck)
  • Glass filters (3‐µm pore size)
  • Rotary evaporator equipped with an oil pump
  • Chromatography columns: 18 × 4 cm, 5 × 30 cm, 4 × 15 cm, and 4 × 2.5 cm
  • TLC plate: silica‐coated glass plate with fluorescent indicator (Merck silica gel 60 F 254)
  • Hydrogenation apparatus (Parr Instruments Company)
  • Ultrasonic bath
  • 254‐nm UV lamp
  • Reflux condenser
  • Additional reagents and equipment for TLC ( appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis of Oligonucleotide‐DOTA Conjugates

  • DOTA‐labeled phosphoramidite (S.8; see protocol 1)
  • Dry acetonitrile, ≥99.5%, ≤0.005% H 2O (Merck)
  • Deoxyribonucleoside phosphoramidites (e.g., Proligo)
  • 0.1 M NaOH
  • 1 M NH 4Cl
  • Aqueous ammonia, p.a. 28% to 30% (Merck)
  • Gadolinium(III) citrate: 0.1 M Gd(III)Cl 3/0.2 M citric acid
  • 20% polyacrylamide gel containing 7 M urea ( appendix 3B)
  • 1 M Na 2CO 3, pH 9.8
  • 1‐Butanol, ≥99.5% pure (Merck)
  • Sterile, nuclease‐free, deionized water
  • Automated DNA/RNA synthesizer (e.g., Applied Biosystems)
  • 0.2‐µmol DNA synthesis column (1000 Å CPG; Applied Biosystems)
  • 1‐mL syringes
  • 2‐mL vials
  • Speedvac evaporator (e.g., SPD121P, Savant)
  • NAP5 gel filtration columns (GE Healthcare)
  • Centrifuge (e.g., BR4i, JOUAN)
  • Additional reagents and equipment for automated DNA synthesis ( appendix 3C) and urea‐PAGE ( appendix 3B)
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Literature Cited

Literature Cited
   Aime, S., Botta, M., Fasano, M., and Terreno, E. 1999. Lanthanide(III) chelates for NMR biomedical applications. Chem. Soc. Rev. 27:19‐29.
   Anderson, C.J. and Welch, M.J. 1999. Radiometal‐labeled agents (non‐technetium) for diagnostic imaging. Chem. Rev. 99:2219‐2234.
   Butcher, W.W. and Westheimer, F.H. 1955. The lanthanum hydroxide gel promoted hydrolysis of phosphate esters. J. Am. Chem. Soc. 77:2420‐2424.
   Caravan, P., Ellison, J.J., McMurry, T.J., and Lauffer, R.B. 1999. Gadolinium(III) chelates as MRI contrast agents: Structure, dynamics, and applications. Chem. Rev. 99:2293‐2352.
   Heppeler, A., Froilevaux, S., Mäcke, H.R., Jermann, H.E., Béhé, M., Powell, P., and Hennig, M. 1999. Radiometal‐labeled macrocyclic chelator‐derivatised somatostatin analogue with superb tumor‐targeting properties and potential for receptor‐mediated tumor therapy. Chem. Eur. J. 5:1974‐1981.
   Hovinen, J. and Hakala, H. 2001. Versatile strategy for oligonucleotide derivatization. Introduction of lanthanide(III) chelates to oligonucleotides. Org. Lett. 3:2474‐2476.
   Hovinen, J. and Takalo, H. 2005. Oligonucleotide labeling reactants and their use. US Pat. 6,949,639.
   Jaakkola, L., Ylikoski, A., and Hovinen, J. 2006. Simple synthesis of a building block for solid phase labeling of oligonucleotides with 1,4,7,10‐tetraazacyclododecane‐1,4,7,10‐tetraacetic acid (DOTA). Bioconjug. Chem. 17:1105‐1107.
   Mitsunobu, O. 1981. The use of diethyl azodicarboxylate and triphenylphosphine in synthesis and transformation of natural products. Synthesis 1‐28.
   Paul‐Roth, C. and Raymond, K.N. 1995. Amide functional group contribution to the stability of gadolinium(III) complexes: DTPA derivatives. Inorg. Chem. 34:1408‐1412.
   Runge, V.M. 2000. Safety of approved MR contrast media for intravenous injection. J. Magn. Reson. Imaging 12:205‐213.
   Sinha, N.D. and Striepeke, S. 1991. Oligonucleotides with reporter groups attached to the 5′‐terminus. In Oligonucleotides and Analogues. A Practical Approach (F. Eckstein, ed.) p. 189. Oxford University Press, Oxford.
   Velikyan, I., Lendvai, G., Välilä, M., Roivainen, A., Yngve, U., Bergström, M., and Långström, B. 2004. Microwave‐accelerated 68Ga‐labelling of oligonucleotides. J. Labeled Comp. Radiopharm. 47:79‐89.
   Volkert, W.A. and Hoffman, T.J. 1999. Therapeutic radiopharmaceuticals. Chem. Rev. 99:2269‐2292.
   Woods, M., Kovacs, Z., and Sherry, A.D. 2002. Targeted complexes of lanthanide(III) ions as therapeutic and diagnostic pharmaceuticals. J. Supramol. Chem. 2:1‐15.
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