Synthesis of a 2‐Selenothymidine Phosphoramidite and Its Incorporation into Oligodeoxyribonucleotides

Wen Zhang1, Zhen Huang1

1 Georgia State University, Atlanta, Georgia
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.23
DOI:  10.1002/0471142700.nc0123s42
Online Posting Date:  September, 2010
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

The detailed synthetic protocol for a 2‐selenothymidine phosphoramidite and its use in preparing Se‐derivatized oligonucleotides are described here. The Se‐modified phosphoramidite synthesis was achieved by activating a 2‐thiothymidine derivative, followed by introduction of selenium functionality. The coupling reaction yield of the 2‐selenothymidine phosphoramidite during solid‐phase synthesis is high (>95%), and the oligonucleotides containing the 2‐selenothymidine derivatization are stable. Curr. Protoc. Nucleic Acid Chem. 42:1.23.1‐1.23.13. © 2010 by John Wiley & Sons, Inc.

Keywords: nucleic acid; selenium; derivatization; base pairing; 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 2‐Selenothymidine Phosphoramidite
  • Support Protocol 1: Synthesis of Iodopropionitrile
  • Basic Protocol 2: Synthesis, Purification, and Characterization of Oligonucleotides Containing 2‐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 2‐Selenothymidine Phosphoramidite

  Materials
  • 4,4′‐Dimethoxytrityl chloride
  • 2‐Thiothymidine ( S.1; ChemGenes, 99.5% pure)
  • 4‐Dimethylaminopyridine (DMAP, Aldrich, purity >99%)
  • Pyridine (Aldrich, anhydrous, purity >99%)
  • Argon
  • Ethyl acetate (EtOAc)
  • Methylene chloride (dichloromethane, CH 2Cl 2; Fluka, purity >99.5%)
  • Methanol (MeOH)
  • MgSO 4 (anhydrous)
  • Silica gel (porosity, 60 Å; particle size, 40 to 63 µm; 230 × 400 mesh)
  • N, N‐Dimethylformamide (DMF; Aldrich, anhydrous, 99% pure)
  • Iodomethane (CH 3I; Aldrich, 99% pure)
  • 1,8‐Diazabicyclo[5.4.0]undec‐7‐ene (DBU; Aldrich, 98% pure)
  • Chloroform (CHCl 3)
  • Selenium (Se; Fluka, 95% pure)
  • Sodium borohydride (NaBH 4; Aldrich, 98% pure)
  • Ethanol (absolute)
  • NaCl, aqueous, saturated
  • Iodopropionitrile (ICH 2CH 2CN; protocol 2)
  • N,N‐Diisopropylethylamine (DIPEA; Aldrich, 99% pure)
  • 2‐Cyanoethyl N,N‐diisopropylchlorophosphoramidite (ChemGenes Corporation)
  • Pentane
  • 25‐, 50‐, and 100‐mL round‐bottom flasks
  • Vacuum oil pump
  • 1‐ and 5‐mL syringes
  • Rubber septum
  • Rotary evaporator
  • Separatory funnels
  • 22 × 457–mm silica gel chromatography columns
  • Stir bar
  • 100‐mL beakers
  • Additional reagents and equipment for performing thin‐layer chromatography ( appendix 3D) and column chromatography ( appendix 3E)

Support Protocol 1: Synthesis of Iodopropionitrile

  Materials
  • Potassium iodide (Aldrich, 95%)
  • Acetone
  • Argon
  • 3‐Bromopropionitrile (Aldrich, 99%)
  • Ethyl acetate (EtOAc)
  • MgSO 4, anhydrous
  • 100‐mL round‐bottom flasks
  • Stir bar
  • Condenser
  • Heating plate
  • Separatory funnel
  • Rotary evaporator

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

  Materials
  • 2‐Selenothymidine phosphoramidite ( S.6; protocol 1)
  • Acetonitrile (CH 3CN), anhydrous
  • 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 M K 2CO 3 in methanol
  • 2 M 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
  • ABI3400 DNA/RNA synthesizer
  • Screw‐cap tubes or vials
  • 13‐mm syringe filter with 0.2‐µm nylon membrane (Life Sciences)
  • RP‐HPLC column: 21.2 × 250–mm Zorbax RX‐C8 (Agilent Technology) or 21 × 250–mm XB‐C18 (Welch Materials; http://www.instrument.com)
  • HPLC system with detector at 260 nm
  • Lyophilizer
  • Microcentrifuge tubes
  • Microcentrifuge
  • UV spectrophotometer
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C), 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
   Basu, A.K., Loechler, E.L., Leadon, S.A., and Essigmann, J.M. 1989. Genetic effects of thymine glycol: Site‐specific mutagenesis and molecular modeling studies. Proc. Natl. Acad. Sci. U.S.A. 86:7677‐7781.
   Carrasco, N. and Huang, Z. 2004. Enzymatic synthesis of phosphoroselenoate DNA using thymidine 5′‐(α‐P‐seleno)triphosphate and DNA polymerase for X‐ray crystallography via MAD. J. Am. Chem. Soc. 126:448‐449.
   Cate, J.H., Gooding, A.R., Podell, E., Zhou, K., Golden, B.L., Kundrot, C.E., Cech, T.R., and Doudna, J.A. 1996. Crystal structure of group I ribozyme domain: Principles of RNA packing. Science 273:1678‐1685.
   Caton‐Williams, J. and Huang, Z. 2008. Synthesis and DNA‐polymerase incorporation of colored 4‐selenothymidine triphosphate for polymerase recognition and DNA visualization. Angew. Chem. Int. Ed. Engl. 47:1723‐1725.
   Drew, H.R., Wing, R.M., Takano, T., Broka, C., Tanaka, S., Itakura, K., and Dickerson, R.E. 1981. Structure of a B‐DNA dodecamer: Conformation and dynamics. Proc. Natl. Acad. Sci. U.S.A. 78:2179‐2183.
   Eoff, R.L., Irimia, A., Egli, M., and Guengerich, F.P. 2007. Sulfolobus solfataricus DNA polymerase Dpo4 is partially inhibited by “Wobble” pairing between O6‐methylguanine and cytosine, but accurate bypass is preferred. J. Biol. Chem. 282:1456‐1467.
   Hassan, A.E.A., Sheng, J., Zhang, W., and Huang, Z. 2010. High fidelity of base pairing by 2‐selenothymidine in DNA. J. Am. Chem. Soc. 132:2120‐2121.
   Herschlag, D. 1991. Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: More isn't always better. Proc. Natl. Acad. Sci. U.S.A. 88:6921‐6925.
   Jiang, J., Sheng, J., Carrasco, N., and Huang, Z. 2007. Selenium derivatization of nucleic acids for crystallography. Nucleic Acids Res. 35:477‐485.
   Montange, R.K. and Batey, R.T. 2006. Structure of the S‐adenosylmethionine riboswitch regulatory mRNA element. Nature 441:1172‐1175.
   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.
   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.
   Sheng, J., Salon, J., Gan, J.‐H., and Huang, Z. 2010. Synthesis and crystal structure study of 2′‐Se‐adenosine‐derivatized DNA. Sci. China, Ser. B: Chem. 53:78.
   Shiue, C.Y. and Chu, S.H. 1975. A facile synthesis of l‐β‐D‐arabinofuranosyl‐2‐seleno and −4‐selenouracil and related compounds. J. Org. Chem. 40:2971.
   Spratt, T.E. and Levy, D.E. 1997. Structure of the hydrogen bonding complex of O6‐methylguanine with cytosine and thymine during DNA replication. Nucleic Acids Res. 25:3354‐3361.
   Sussman, J.L. and Kim, S. 1976. Three‐dimensional structure of a transfer RNA in two crystal forms. Science 192:853‐858.
   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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library