Synthesis of a 4‐Selenothymidine Phosphoramidite and Incorporation into Oligonucleotides

Jia Sheng1, Zhen Huang1

1 Georgia State University, Atlanta, Georgia
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.19
DOI:  10.1002/0471142700.nc0119s32
Online Posting Date:  March, 2008
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The detailed synthetic protocol for a 4‐selenothymidine phosphoramidite and its use to prepare modified oligonucleotides is described here. The Se‐phosphoramidite synthesis was achieved by developing a useful protection and deprotection system for the selenium functionality. The coupling reaction of the Se‐phosphoramidite during solid‐phase oligonucleotide synthesis is quantitative, and the oligonucleotides containing the Se‐modification are stable. Based on crystal structure analysis, the selenium‐modified oligonucleotides retain base‐pairing like their native counterparts, and the derivatized DNA structure is virtually identical to the native structure. This achievement will present a novel opportunity for structural studies of nucleic acids and their protein complexes, because selenium can resolve the phase problem in macromolecular X‐ray crystallography. In addition, this atom‐specific replacement of oxygen with selenium will provide a useful tool for investigating biochemical and biophysical properties of nucleic acids and their protein complexes. Curr. Protoc. Nucleic Acid Chem. 32:1.19.1‐1.19.13. © 2008 by John Wiley & Sons, Inc.

Keywords: nucleic acid; selenium; derivatization; structure determination; X‐ray crystallography

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

Table of Contents

  • Introduction
  • Basic Protocol 1: Preparation of the 4‐Selenothymidine Phosphoramidite
  • Support Protocol 1: Synthesis of DI(2‐Cyanoethyl) Diselenide
  • Basic Protocol 2: Synthesis, Purification, and Characterization of Oligonucleotides Containing 4‐Selenothymidine
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of the 4‐Selenothymidine Phosphoramidite

  Materials
  • 5′‐O‐Dimethoxytrityl‐thymidine (5′‐O‐DMTr‐thymidine, S.1, ChemGenes, 99.5% pure)
  • Acetonitrile (CH 3CN, Fluka, anhydrous, purity >99%)
  • Argon
  • 1‐(Trimethylsilyl)imidazole (TMS‐Im, Aldrich, purity >99%)
  • 1,2,4‐Triazole (Aldrich, 98% pure)
  • Phosphorus oxychloride (POCl 3, Fluka, purity >98%)
  • Triethylamine (Et 3N, Aldrich, anhydrous, 99% pure)
  • Methanol (MeOH)
  • Methylene chloride (CH 2Cl 2, Fluka, purity >99.5%)
  • Ethyl acetate (EtOAc)
  • NaCl, aqueous, saturated
  • MgSO 4 (anhydrous)
  • Hexane
  • Tetrahydrofuran (THF, Fluka, purity >99%)
  • 1.0 M triethylamine trihydrofluoride (Et 3N·3HF, Aldrich) in THF
  • Sodium borohydride (NaBH 4, Aldrich, 98% pure)
  • Ethanol (absolute)
  • Di(2‐cyanoethyl) diselenide ((NCCH 2CH 2Se) 2, d = 1.8 g/mL, see protocol 2)
  • Silica gel (porosity, 60 Å; particle size, 40 to 63 µm; 230 × 400 mesh)
  • N,N‐Diisopropylethylamine (DIPEA, Aldrich, 99% pure)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite (ChemGenes Corporation)
  • NaHCO 3, saturated, aqueous
  • Petroleum ether
  • 25‐, 50‐, and 100‐mL round‐bottom flasks
  • Separatory funnels
  • Vacuum oil pump
  • Syringe packed with cotton for filtration
  • Rotary evaporator
  • 22 × 457–mm chromatography columns
  • Al 2O 3 column (neutral, 20 × 4 cm)
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)
NOTE: All the reactions in this protocol are carried out at room temperature unless otherwise specified.NOTE: Drying steps use a vacuum oil pump for high vacuum and a rotary evaporator for reduced pressure.

Support Protocol 1: Synthesis of DI(2‐Cyanoethyl) Diselenide

  Materials
  • Selenium metal
  • Sodium borohydride (NaBH 4, Aldrich, 98% pure)
  • Dioxane
  • Ethanol (EtOH, absolute)
  • Argon
  • 3‐Bromopropionitrile (Aldrich, 99%)
  • 10% (v/v) acetic acid
  • Ethyl acetate (EtOAc)
  • NaCl, aqueous, saturated
  • MgSO 4, anhydrous
  • Silica gel (porosity, 60 Å; particle size, 40 to 63 µm; 230 × 400 mesh)
  • Methylene chloride (CH 2Cl 2, Fluka, purity >99.5%)
  • Hexane
  • 250‐mL flask
  • Separatory funnel
  • Rotary evaporator
  • Chromatography column
  • Additional reagents and equipment for column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis, Purification, and Characterization of Oligonucleotides Containing 4‐Selenothymidine

  Materials
  • 4‐Selenothymidine phosphoramidite (see protocol 1)
  • Acetonitrile (CH 3CN), anhydrous
  • Regular ultra‐mild phosphoramidites: Pac‐dA‐CE, iPr‐Pac‐dG‐CE, Ac‐dC‐CE, dT‐CE (Glen Research; abbreviations: Ac, acetyl; CE, cyanoethyl; iPr, isopropyl; Pac, phenoxyacetyl)
  • 50 mM K 2CO 3 in methanol
  • 20 mM triethylammonium acetate (TEAA) buffer, pH 7.0
  • Acetonitrile (CH 3CN), HPLC grade
  • 30% (v/v) trichloroacetic acid (TCA), aqueous
  • Argon
  • 3‐Hydroxypicolinic acid (3‐HPA)
  • Diammonium citrate
  • NaCl
  • NaH 2PO 4
  • Na 2HPO 4
  • EDTA
  • MgCl 2
  • Nucleic Acid Mini‐Screen Kit (Hampton Research, http://www.hamptonresearch.com)
  • ABI392 DNA/RNA synthesizer (also see appendix 3C)
  • Screw‐cap tubes or vials
  • 13‐mm syringe filter with 0.2‐µm nylon membrane (Life Sciences)
  • HPLC system (optional) with detector at 260 and/or 369 nm
  • RP‐HPLC column: 21.2 × 250–mm Zorbax RX‐C8 (Agilent Technology) or 21 × 250–mm XB‐C18 (Welch Materials; http://www.instrument.com)
  • Lyophilizer
  • UV spectrophotometer
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C), and MALDI‐TOF mass spectrometry (unit 10.1), and determination of UV melting curves (unit 7.3)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Brandt, G., Carrasco, N., and Huang, Z. 2006. Efficient substrate cleavage catalyzed by hammerhead ribozymes derivatized with selenium for X‐ray crystallography. Biochemistry 45: 8972‐8977.
   Buzin, Y., Carrasco, N., and Huang, Z. 2004. Synthesis of selenium‐derivatized cytidine and oligonucleotides for X‐ray crystallography using MAD. Org. Lett. 6: 1099‐1102.
   Carrasco, N. and Huang, Z. 2004. Enzymatic synthesis of phosphoroselenoate DNA using thymidine 5′‐(a‐P‐seleno)triphosphate and DNA polymerase for X‐ray crystallography via MAD. J. Am. Chem. Soc. 126: 448‐449.
   Carrasco, N., Ginsburg, D., Du, W., and Huang, Z. 2001. Synthesis of selenium‐derivatized nucleosides and oligonucleotides for X‐ray crystallography. Nucleosides Nucleotides Nucleic Acids 20: 1723‐1734.
   Carrasco, N., Buzin, Y., Tyson, E., Halpert, E., and Huang, Z. 2004. Selenium derivatization and crystallization of DNA and RNA oligonucleotides for X‐ray crystallography using multiple anomalous dispersion. Nucleic Acids Res. 32: 1638‐1646.
   Carrasco, N., Caton‐Williams, J., Brandt, G., Wang, S., and Huang, Z. 2006. Efficient enzymatic synthesis of phosphoroselenoate RNA by using adenosine 5′‐(alpha‐P‐seleno)triphosphate. Angew. Chem. Int. Ed. Engl. 45: 94‐97.
   Chen, C.S. and Stadtman, T.C. 1980. Selenium‐containing tRNAs from Clostridium sticklandii: Cochromatography of one species with L‐prolyl‐tRNA. Proc. Natl. Acad. Sci. U.S.A. 77: 1403‐1407.
   Du, Q., Carrasco, N., Teplova, M., Wilds, C.J., Egli, M., and Huang, Z. 2002. Internal derivatization of oligonucleotides with selenium for X‐ray crystallography using MAD. J. Am. Chem. Soc. 124: 24‐25.
   Egli, M., Pallan, P.S., Pattanayek, R., Wilds, C.J., Lubini, P., Minasov, G., Dobler, M., Leumann, C.J., and Eschenmoser, A. 2006. Crystal structure of homo‐DNA and nature's choice of pentose over hexose in the genetic system. J. Am. Chem. Soc. 128: 10847‐10856.
   Jiang, J., Sheng, J., Carrasco, N., and Huang, Z. 2007. Selenium derivatization of nucleic acids for crystallography. Nucleic Acids Res. 35: 477‐485.
   Lezius, A.G. and Scheit, K.H. 1967. Enzymatic synthesis of DNA with 4‐thio‐thymidine triphosphate as substitute for dTTP. Eur. J. Biochem. 3: 85‐94.
   Logan, G., Igunbor, C., Chen, G.X., Davis, H., Simon, A., Salon, J., and Huang, Z. 2006. A novel and simple strategy for incorporation, protection, and deprotection of selenium functionality. Synlett 10: 1554‐1558.
   Mautner, H.G. 1956. The synthesis and properties of some selenopyridines and selenopyrimidines. J. Am. Chem. Soc. 78: 5292‐5294.
   Moroder, H., Kreutz, C., Lang, K., Serganov, A., and Micura, R. 2006. Synthesis, oxidation behavior, crystallization and structure of 2′‐methylseleno guanosine containing RNAs. J. Am. Chem. Soc. 128: 9909‐9918.
   Salon, J., Chen, G., Portilla, Y., Germann, M.W., and Huang, Z. 2005. Synthesis of a 2′‐Se‐uridine phosphoramidite and its incorporation into oligonucleotides for structural study. Org. Lett. 7: 5645‐5648.
   Salon, J., Sheng, J., Jiang, J., Chen, G., Caton‐Williams, J., and Huang, Z. 2007. Oxygen replacement with selenium at the thymidine 4‐position for the Se base pairing and crystal structure studies. J. Am. Chem. Soc. 129: 4862‐4863.
   Serganov, A., Yuan, Y.R., Pikovskaya, O., Polonskaia, A., Malinina, L., Phan, A.T., Hobartner, C., Micura, R., Breaker, R.R., and Patel, D.J. 2004. Structural basis for discriminative regulation of gene expression by adenine‐ and guanine‐sensing mRNAs. Chem. Biol.11: 1729‐1741.
   Serganov, A., Keiper, S., Malinina, L., Tereshko, V., Skripkin, E., Hobartner, C., Polonskaia, A., Phan, A.T., Wombacher, R., Micura, R., Dauter, Z., Jaschke, A., and Patel, D.J. 2005. Structural basis for Diels‐Alder ribozyme‐catalyzed carbon‐carbon bond formation. Nat. Struct. Mol. Biol. 12: 218‐224.
   Sheng, J., Jiang, J., Salon, J., and Huang, Z. 2007. Synthesis of a 2′‐Se‐thymidine phosphoramidite and its incorporation into oligonucleotides for crystal structure study. Org. Lett. 9: 749‐752.
   Sintim, H.O. and Kool, E.T. 2006. Enhanced base pairing and replication efficiency of thiothymidines, expanded‐size variants of thymidine. J. Am. Chem. Soc. 128: 396‐397.
   Sismour, A.M. and Benner, S.A. 2005. The use of thymidine analogs to improve the replication of an extra DNA base pair: A synthetic biological system. Nucleic Acids Res. 33: 5640‐5646.
   Teplova, M., Wilds, C.J., Wawrzak, Z., Tereshko, V., Du, Q., Carrasco, N., Huang, Z., and Egli, M. 2002. Covalent incorporation of selenium into oligonucleotides for X‐ray crystal structure determination via MAD: Proof of principle. Multiwavelength anomalous dispersion. Biochimie 84: 849‐858.
   Wilds, C.J., Pattanayek, R., Pan, C., Wawrzak, Z., and Egli, M. 2002. Selenium‐assisted nucleic acid crystallography: Use of DNA phosphoroselenoates for MAD phasing. J. Am. Chem. Soc. 124: 14910‐14916.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library