Base‐Modified Oligodeoxyribonucleotides: Using Pyrrolo[2,3‐d]pyrimidines to Replace Purines

Frank Seela1, Xiaohua Peng1

1 Center for Nanotechnology (CeNTech), Münster
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.25
DOI:  10.1002/0471142700.nc0425s20
Online Posting Date:  April, 2005
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Abstract

Phosphoramidites are first synthesized from suitably protected 7‐deazapurine 2′‐deoxyribonuclosides that have halogen or alkynyl subsituents at the 7 position. Oligonucleotides containing 7‐deazapurines (pyrrolo[2,3‐d]pyrimidines) in place of the canonical nucleobases are then prepared by solid‐phase synthesis. These oligonucleotides are subsequently allowed to hybridize, and the stabilities of the resulting duplexes are determined. Comparison of Tm values indicates that the presence of 7‐halo or 7‐alkynyl substituents increases duplex stability relative to duplexes involving oligonucleotides that contain the corresponding parent purines.

Keywords: pyrrolo[2,3‐d]pyrimidines; 7‐deazapurines; nucleobase analogs; halogen substituents; alkynyl groups; nucleobase protection; phosphoramidites; solid‐phase synthesis; oligonucleotides; mass spectra; duplex formation; base pair stability; Tm values; thermodynamic data

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of the Phosphoramidite of 7‐Deaza‐7‐Iodo‐2′‐Deoxyadenosine
  • Basic Protocol 2: Synthesis of the Phosphoramidite Of 7‐Deaza‐7‐Iodo‐2′‐Deoxyguanosine
  • Basic Protocol 3: Synthesis of the Phosphoramidite of 7‐Deaza‐2′‐Deoxyisoguanosine
  • Basic Protocol 4: Synthesis and Purification of Oligonucleotides Containing 7‐Deaza‐2′‐Deoxyribonucleosides
  • Basic Protocol 5: Determination of Transition (Melting) Temperature and Thermodynamic Data
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of the Phosphoramidite of 7‐Deaza‐7‐Iodo‐2′‐Deoxyadenosine

  Materials
  • 4‐Amino‐7‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)‐5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidine (systematic numbering; S.9d; see unit 1.10), also known as 7‐deaza‐7‐iodo‐2′‐deoxyadenosine (purine numbering)
  • Methanol (MeOH)
  • N,N‐Dimethylformamide dimethylacetal
  • Silica gel 60 for flash chromatography (particle size, <0.063 mm; Merck)
  • Anhydrous dichloromethane (CH 2Cl 2)
  • Anhydrous pyridine
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • 5% (w/v) sodium bicarbonate (NaHCO 3)
  • Brine (i.e., saturated aqueous NaCl)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Anhydrous N,N‐diisopropylethylamine (DIPEA; 99.5% pure)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite
  • Anhydrous triethylamine (Et 3N)
  • Acetone
  • 10‐, 25‐, 50‐, and 100‐mL round‐bottom flasks
  • Rotary evaporator connected to a vacuum pump
  • 4 × 70–cm chromatography columns
  • 0.2‐mm‐thick silica gel 60 F 254 aluminum TLC plates (Merck)
  • 254‐nm UV lamp
  • High‐vacuum pump (final pressure, <1 mmHg)
  • 100‐mL separatory funnels
  • 5‐cm‐diameter funnel with folded 10‐cm‐diameter Whatman no. 1 filter
  • Argon source
  • Additional reagents and equipment for flash chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 2: Synthesis of the Phosphoramidite Of 7‐Deaza‐7‐Iodo‐2′‐Deoxyguanosine

  Materials
  • 4‐Amino‐7‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)‐5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidin‐2‐one (systematic numbering; S.12d; see unit 1.10), also known as 7‐deaza‐7‐iodo‐2′‐deoxyguanosine (purine numbering)
  • Anhydrous pyridine
  • Chlorotrimethylsilane (Me 3SiCl)
  • Isobutyric anhydride
  • 25% (v/v) ammonium hydroxide (NH 4OH)
  • 4,4′‐Dimethoxytrityl chloride (DMTr‐Cl)
  • 5% (w/v) sodium bicarbonate (NaHCO 3)
  • Anhydrous dichloromethane (CH 2Cl 2)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Silica gel 60 for flash chromatography (particle size, <0.063 mm; Merck)
  • Acetone
  • Methanol (MeOH)
  • Anhydrous N,N‐diisopropylethylamine (DIPEA)
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite
  • Anhydrous triethylamine (Et 3N)
  • Ethyl acetate (EtOAc)
  • 10‐, 25‐, 50‐, and 250‐mL round‐bottom flasks
  • Rotary evaporator connected to a vacuum pump
  • 3‐cm‐diameter Buchner funnel with filter paper circles
  • High‐vacuum pump (final pressure, <1 mmHg)
  • 100‐mL separatory funnels
  • 5‐cm‐diameter funnel with folded 10‐cm‐diameter Whatman no. 1 filter
  • 4 × 70–cm chromatography columns
  • 0.2‐mm‐thick silica gel 60 F 254 aluminum TLC plates (Merck)
  • 254‐nm UV lamp
  • Argon source
  • Additional reagents and equipment for flash chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 3: Synthesis of the Phosphoramidite of 7‐Deaza‐2′‐Deoxyisoguanosine

  Materials
  • 4‐Amino‐7‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)‐1,7‐dihydro‐2H‐pyrrolo[2,3‐d]pyrimidin‐2‐one (systematic numbering; S.15a; see Seela and Wei, ), also known as 7‐deaza‐2′‐deoxyisoguanosine (purine numbering)
  • Anhydrous pyridine
  • Diphenylcarbamoyl chloride
  • Anhydrous N,N‐diisopropylethylamine (DIPEA)
  • Crushed ice
  • 5% (w/v) sodium bicarbonate (NaHCO 3)
  • Anhydrous dichloromethane (CH 2Cl 2)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • Silica gel 60 for flash chromatography (particle size, <0.063 mm; Merck)
  • Methanol (MeOH)
  • Chlorotrimethylsilane (Me 3SiCl)
  • Isobutyryl chloride
  • 25% (v/v) ammonium hydroxide (NH 4OH)
  • 4,4′‐Dimethoxtrityl chloride (DMTr‐Cl)
  • Acetone
  • 2‐Cyanoethyl‐N,N‐diisopropylchlorophosphoramidite
  • Brine (i.e., saturated aqueous NaCl)
  • Anhydrous triethylamine (Et 3N)
  • 10‐, 50‐, 100‐, and 250‐mL round‐bottom flasks
  • 50‐ and 100‐mL separatory funnels
  • 5‐cm‐diameter funnel with folded 10‐cm‐diameter Whatman no. 1 filter
  • Rotary evaporator connected to a vacuum pump
  • 4 × 70–cm chromatography columns
  • 0.2‐mm‐thick silica gel 60 F 254 aluminum TLC plates (Merck)
  • 254‐nm UV lamp
  • High‐vacuum pump (final pressure, <1 mmHg)
  • Argon source
  • Additional reagents and equipment for flash chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 4: Synthesis and Purification of Oligonucleotides Containing 7‐Deaza‐2′‐Deoxyribonucleosides

  Materials
  • Phosphoramidite solutions (0.1 M in MeCN): 2′‐deoxyadenylate, 2′‐deoxythymidylate, 5‐methyl‐2′‐deoxyisocytidylate, and 7‐deaza‐2′‐deoxyisoguanylate ( S.6a; see protocol 3)
  • 25% (v/v) aqueous ammonium hydroxide (NH 4OH)
  • Mobile phase A: acetonitrile (MeCN)
  • Mobile phase B: 95:5 (v/v) 0.1 M TEAA buffer (pH 7.0)/acetonitrile (see recipe)
  • 2.5% (v/v) dichloroacetic acid (Cl 2CHCOOH) in anhydrous dichloromethane (CH 2Cl 2)
  • Anhydrous triethylamine (Et 3N)
  • Methanol (MeOH)
  • 0.1 M Tris·Cl, pH 8.9 (see recipe)
  • 36.2 U/mL snake venom phosphodiesterase I (SVPD; EC 3.1.4.1; Crotalus adamanteus; Amersham)
  • 1 U/µL alkaline phosphatase (EC 3.1.3.1; calf intestine; Roche Diagnostics)
  • ABI 392‐08 automated DNA synthesizer (Applied Biosystems)
  • SpeedVac evaporator (Savant)
  • HPLC apparatus, including:
    • 4 × 125–mm and 4 × 250–mm RP‐18 LiChrospher columns (Merck)
    • 655A‐12 HPLC pump (Merck/Hitachi)
    • 655A variable‐wavelength monitor (Merck/Hitachi)
    • L‐5000 controller unit (Merck/Hitachi)
    • D‐2000 chromatogram peak integrator (Merck/Hitachi)
  • 5‐mL polypropylene tubes
  • 1‐mL quartz cuvette (1‐cm path length)
  • UV‐Vis spectrophotometer
  • Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C) using phosphoramidite chemistry (unit 3.3), reversed‐phase high‐performance liquid chromatography (RP‐HPLC; unit 10.5), and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS; unit 10.1)
NOTE: Phosphoramidites should be dried under high vacuum (<1 mmHg) for 20 to 24 hr at 40°C before being applied to the DNA synthesizer.
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Figures

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Literature Cited

   Balow, G., Mohan, V., Lesnik, E.A., Johnston, J.F., Monia, B.P., and Acevedo, O.L. 1998. Biophysical and antisense properties of oligodeoxynucleotides containing 7‐propynyl‐ , 7‐iodo‐ and 7‐cyano‐7‐deaza‐2‐amino‐2′‐deoxyadenosines. Nucl. Acids Res. 26:3350‐3357.
   Barr, P.J., Thayer, R.M., Laybourn, P., Najarian, R.C., Seela, F., and Tolan, D.R. 1986. 7‐Deaza‐2′‐deoxyguanosine‐5′‐triphosphate: Enhanced resolution in M13 dideoxy sequencing. Biotechniques 4:428‐432.
   Buhr, C.A., Wagner, R.W., Grant, D., and Froehler, B.C. 1996. Oligodeoxynucleotides containing C‐7 propyne analogs of 7‐deaza‐2′‐deoxyguanosine and 7‐deaza‐2′‐deoxyadenosine. Nucl. Acids Res. 24:2974‐2980.
   Latimer, L.J. and Lee, J.S. 1991. Ethidium‐bromide does not fluoresce when intercalated adjacent to 7‐deazaguanine in duplex DNA. J. Biol. Chem. 266:13849‐13851.
   Li, H., Peng, X., and Seela, F. 2004. Fluorescence quenching of parallel‐stranded DNA bound ethidium bromide: The effect of 7‐deaza‐2′‐deoxyisoguanosine and 7‐halogenated derivatives. Bioorg. Med. Chem. Lett. 14:6031–6034.
   McDowell, J.A. and Turner, D.H. 1996. Investigation of the structural basis for thermodynamic stabilities of tandem GU mismatches: Solution structure of (rGAG GU CUC)2 by two‐dimensional NMR and simulated annealing. Biochemistry 35:14077‐14089.
   Mizusawa, S., Nishimura, S., and Seela, F. 1986. Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy‐7‐deazaguanosine triphosphate in place of dGTP. Nucl. Acids Res. 14:1319‐1324.
   Okamoto, A., Tanaka, K., and Saito, I. 2002. 2‐Amino‐7‐deazaadenine forms stable base pairs with cytosine and thymine. Bioorg. Med. Chem. Lett. 12:97‐99.
   Ramzaeva, N. and Seela, F. 1995. 7‐Substituted 7‐deaza‐2′‐deoxyguanosines: Regioselective halogenation of pyrrolo[2,3‐d]pyrimidine nucleosides. Helv. Chim. Acta 78:1083‐1090.
   Ramzaeva, N. and Seela, F. 1996. Duplex stability of 7‐deazapurine DNA: Oligonucleotides containing 7‐bromo or 7‐iodo‐7‐deazaguanine. Helv. Chim. Acta 79:1549‐1558.
   Ramzaeva, N., Mittelbach, C., and Seela, F. 1997. 7‐Deazaguanine DNA: Oligonucleotides with hydrophobic or cationic side chains. Helv. Chim. Acta 80:1809‐1822.
   Seela, F. and Chen, Y. 1997. Methylated DNA: The influence of 7‐deaza‐7‐methylguanine on the structure and stability of oligonucleotides. Helv. Chim. Acta 80:1073‐1086.
   Seela, F. and Driller, H. 1989. Alternating d(G‐C)3 and d(C‐G)3 hexanucleotides containing 7‐deaza‐2′‐deoxyguanosine or 8‐aza‐7‐deaza‐2′‐deoxyguanosine in place of dG. Nucl. Acids Res. 17:901‐910.
   Seela, F. and Kaiser, K. 1986. Phosphoramidites of base‐modified 2′‐deoxyinosine isosteres and solid‐phase synthesis of d(GCI*CGC) oligomers containing an ambiguous base. Nucl. Acids Res. 14:1825‐1844.
   Seela, F. and Kehne, A. 1983. 2′‐Desoxytubercidin: Synthese eines 2′‐desoxyadenosin‐isosteren durch phasentransferglycosylierung. Liebigs Ann. Chem. 876‐884.
   Seela, F. and Kehne, A. 1985. 2′‐Desoxytubercidin: Synthese des O‐3′‐phosphoramidites und kondensation zu 2′‐desoxytubercidylyl(3′→5′)‐2′‐desoxytubercidin. Tetrahedron 41:5387‐5392.
   Seela, F. and Mersmann, K. 1993. 7‐Deazaguanosine: Synthesis of an oligoribonucleotide building block and disaggregation of the UGGGGU G4 structure by the modified base. Helv. Chim. Acta 76:1435‐1449.
   Seela, F. and Peng, X. 2004. Regioselective syntheses of 7‐halogenated 7‐deazapurine nucleosides related to 2‐amino‐7‐deaza‐2′‐deoxyadenosine and 7‐deaza‐2′‐deoxyisoguanosine. Synthesis 8:1203‐1210.
   Seela, F. and Shaikh, K.I. 2005. Oligonucleotides containing 7‐propynyl‐7‐deazaguanine: Synthesis and base pair stability. Tetrahedron In press.
   Seela, F. and Thomas, H. 1994. Synthesis of certain 5‐substituted 2′‐deoxytubercidin derivatives. Helv. Chim. Acta 77:897‐903.
   Seela, F. and Thomas, H. 1995. Duplex stabilization of DNA: Oligonucleotides containing 7‐substituted 7‐deazaadenines. Helv. Chim. Acta 78:94‐108.
   Seela, F. and Wei, C. 1997. 7‐Deazaisoguanine quartets: Self‐assembled oligonucleotides lacking the Hoogsteen motif. Chem. Commun. 1869‐1870.
   Seela, F. and Wei, C. 1999. The base‐pairing properties of 7‐deaza‐2′‐deoxyisoguanosine and 2′‐deoxyisoguanosine in oligonucleotide duplexes with parallel and antiparallel chain orientation. Helv. Chim. Acta 82:726‐745.
   Seela, F. and Zulauf, M. 1996. Palladium‐catalyzed cross coupling of 7‐iodo‐2′‐deoxytubercidin with terminal alkynes. Synthesis 726‐730.
   Seela, F. and Zulauf, M. 1998. 7‐Deazaadenine‐DNA: Bulky 7‐iodo substituents or hydrophobic 7‐hexynyl chains are well accommodated in the major groove of oligonucleotide duplexes. Chem. Eur. J. 4:1781‐1790.
   Seela, F. and Zulauf, M. 1999. Oligonucleotides containing 7‐deazaadenines: The influence of the 7‐substituent chain length and charge on the duplex stability. Helv. Chim. Acta 82:1878‐1898.
   Seela, F., Steker, H., Driller, H., and Bindig, U. 1987. 2‐Amino‐2′‐desoxytubercidin und verwandte pyrrolo[2,3‐d]pyrimidinyl‐2′‐desoxyribofuranoside. Liebigs Ann. Chem. 15‐19.
   Seela, F., Westermann, B., and Bindig, U. 1988. Liquid‐liquid and solid‐liquid phase‐transfer glycosylation of pyrrolo[2,3‐d]pyrimidines: Stereospecific synthesis of 2‐deoxy‐β‐D‐ ribofuranosides related to 2′‐deoxy‐7‐carbaguanosine. J. Chem. Soc. Perkin Trans. I:697‐702.
   Seela, F., Berg, H., and Rosemeyer, H. 1989. Bending of oligonucleotides containing an isosteric nucleobase: 7‐Deaza‐2′‐deoxyadenosine replacing dA within d(A)6 tracts. Biochemistry 28:6193‐6198.
   Seela, F., Wiglenda, T., Rosemeyer, H., Eickmeier, H., and Reuter, H. 2002. 7‐Deaza‐2′‐ deoxyxanthosine dihydrate forms water‐filled nanotubes with C‐H⋅⋅⋅O hydrogen bonds. Angew. Chem. Int. Ed. Engl. 41:603‐605.
   Seela, F., Shaikh, K., and Wiglenda, T. 2003. Synthesis and properties of halogenated 7‐deaza‐2′‐ deoxyxanthosine and protected derivatives for oligonucleotides synthesis. Nucleosides Nucleotides Nucl. Acids 22:1239‐1241.
   Seela, F., Chittepu, P., He, Y., He, J., and Xu, K. 2005a. 6‐Azapyrimidine and 7‐deazapurine 2′‐deoxy‐2′‐fluoroarabinonucleosides: Synthesis, conformation and properties of oligonucleotides. Nucleosides Nucleotides Nucleic Acids In press.
   Seela, F., Peng, X., and Ming, X. 2005b. 7‐Deazapurine‐2,6‐diamine and 7‐deazaguanine: Synthesis and property of 7‐substituted nucleosides and oligonucleotides. Nucleosides Nucleotides Nucleic Acids In press.
   Ti, G.S., Gaffney, B.L., and Jones, R.A. 1982. Transient protection: Efficient one‐flask syntheses of protected deoxynucleosides. J. Am. Chem. Soc. 104:1316‐1319.
   Winkeler, H.D. and Seela, F. 1983. Synthesis of 2‐amino‐7‐(2′‐deoxy‐β‐D‐erythro‐pentofuranosyl)‐3,7‐dihydro‐4H‐pyrrolo[2,3‐d]pyrimidin‐4‐one, a new isostere of 2′‐deoxyguanosine. J. Org. Chem. 48:3119‐3122.
Internet Resources
   http://www.seela.net
  Research web site of Dr. Frank Seela, author of this unit.
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