Synthesis and Properties of 7‐Substituted 7‐Deazapurine (Pyrrolo[2,3‐d]pyrimidine) 2′‐Deoxyribonucleosides

Frank Seela1, Xiaohua Peng1

1 Center for Nanotechnology (CeNTech), Münster
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.10
DOI:  10.1002/0471142700.nc0110s21
Online Posting Date:  July, 2005
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Abstract

7‐Substituted 7‐deazapurine (pyrrolo[2,3‐d]pyrimidine) 2′‐deoxyribonucleosides are synthesized by stereoselective nucleobase anion glycosylation. The introduction of a halogen at C7 is performed regioselectively either on the nucleobase or on the nucleoside. The pKa values of a series of 7‐deazapurine 2′‐deoxyribonucleosides are provided, and fluorescence properties are also discussed.

Keywords: pyrrolo[2,3‐d]pyrimidine; 7‐deazapurine; 2′‐deoxyribonucleoside; nucleobase anion glycosylation; stereoselectivity; regioselective halogenation; pKa values; fluorescence; quenching

     
 
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Table of Contents

  • Basic Protocol 1: Preparation of 7‐Substituted 7‐Deazapurine Nucleosides Related to 2′‐Deoxyadenosine and 2′‐Deoxyinosine
  • Basic Protocol 2: Preparation of 7‐IODO‐7‐Deazapurine Nucleosides Related to 2′‐Deoxyguanosine and 2′‐Deoxyxanthosine
  • Basic Protocol 3: Preparation of 7‐Bromo‐7‐Deaza‐2′‐Deoxyisoguanosine
  • Basic Protocol 4: Determination of pKa Values of Nucleosides
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Preparation of 7‐Substituted 7‐Deazapurine Nucleosides Related to 2′‐Deoxyadenosine and 2′‐Deoxyinosine

  Materials
  • Potassium hydroxide (KOH) powder (purity, ≥85%; Sigma)
  • Anhydrous acetonitrile (MeCN)
  • Tris[2‐(2‐methoxyethoxy)ethyl]amine (TDA‐1)
  • 4‐Chloro‐5‐iodo‐7H‐pyrrolo[2,3‐d]pyrimidine ( S.7; see Pudlo et al., )
  • 2‐Deoxy‐3,5‐di‐O‐(p‐toluoyl)‐α‐D‐erythro‐pentofuranosyl chloride ( S.8; see Hoffer, ; Rolland et al., )
  • Anhydrous dichloromethane (CH 2Cl 2)
  • Silica gel 60 (particle size, <0.063 mm; Merck)
  • Ethyl acetate (EtOAc)
  • Petroleum ether (b.p. range, 40° to 80°C)
  • Isopropanol (i‐PrOH)
  • Methanol (MeOH)
  • 1,4‐Dioxane
  • 25% (v/v) ammonium hydroxide (NH 4OH)
  • Palladium catalyst Pd(PPh 3) 4
  • Copper iodide (CuI)
  • Argon gas
  • Anhydrous dimethylformamide (DMF)
  • Triethylamine (Et 3N)
  • Cyclopentylacetylene
  • Sodium methoxide (NaOMe)
  • 2 M NaOH
  • 1 M HCl
  • 10‐, 50‐, and 100‐mL round‐bottom flasks
  • 3‐cm‐diameter Buchner funnel with filter paper circles
  • Rotary evaporator connected to a vacuum pump
  • 3 × 50– and 4 × 50–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)
  • Steel bomb (autoclave)
  • Reflux condenser
  • Heating mantle with controller
  • Additional reagents and equipment for flash chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 2: Preparation of 7‐IODO‐7‐Deazapurine Nucleosides Related to 2′‐Deoxyguanosine and 2′‐Deoxyxanthosine

  Materials
  • 7‐(2‐Deoxy‐β‐D‐erythro‐pentofuranosyl)‐2‐(formylamino)‐4‐methoxy‐7H‐pyrrolo[2,3‐d]pyrimidine ( S.12; see Seela and Driller, )
  • Anhydrous acetonitrile (MeCN)
  • Isobutyric anhydride
  • Triethylamine
  • Silica gel 60 (particle size, <0.063 mm; Merck)
  • Anhydrous dichloromethane (CH 2Cl 2)
  • Acetone
  • Cyclohexane
  • N,N‐Dimethylformamide (DMF)
  • N‐Iodosuccinimide
  • 5% (w/v) sodium bicarbonate (NaHCO 3)
  • Anhydrous sodium sulfate (Na 2SO 4)
  • 2 M NaOH
  • 1 M HCl
  • Methanol (MeOH)
  • Sodium methoxide (NaOMe)
  • 10% (v/v) acetic acid (AcOH) in H 2O
  • Sodium nitrite (NaNO 2)
  • Ethanol (EtOH)
  • Sodium iodide (NaI)
  • Chlorotrimethylsilane (Me 3SiCl)
  • 0.1 M NaH 2PO 4 buffer, pH 7.0 (see recipe)
  • 25‐, 50‐, and 250‐mL round‐bottom flasks
  • Rotary evaporator connected to a vacuum pump
  • 4 × 50–cm chromatography columns
  • 0.2‐mm‐thick silica gel 60 F 254 aluminum TLC plates (Merck)
  • 254‐nm UV lamp
  • 3‐cm‐diameter Buchner funnel with filter paper circles
  • High‐vacuum pump (final pressure, <1 mmHg)
  • Separatory funnel
  • 5‐cm‐diameter funnel with folded 10‐cm‐diameter Whatman no. 1 filter
  • Reflux condenser
  • Heating mantle with controller
  • Additional reagents and equipment for flash chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 3: Preparation of 7‐Bromo‐7‐Deaza‐2′‐Deoxyisoguanosine

  Materials
  • 2‐Amino‐4‐chloro‐7H‐pyrrolo[2,3‐d]pyrimidine ( S.17; see Seela et al., )
  • Anhydrous pyridine
  • Pivaloyl chloride
  • Potassium hydroxide (KOH) powder (purity, ≥85%; Sigma)
  • Anhydrous dichloromethane (CH 2Cl 2)
  • N‐Bromosuccinimide
  • Methanol (MeOH)
  • Anhydrous acetonitrile (MeCN)
  • Tris[2‐(2‐methoxyethoxy)ethyl]amine (TDA‐1)
  • 2‐Deoxy‐3,5‐di‐O‐(p‐toluoyl)‐α‐D‐erythro‐pentofuranosyl chloride ( S.8; see Hoffer, ; Rolland et al., )
  • Silica gel 60 (particle size, <0.063 mm; Merck)
  • Petroleum ether (b.p. range, 40° to 80°C)
  • 1,4‐Dioxane
  • 25% (v/v) ammonium hydroxide (NH 4OH)
  • 1:5 (v/v) acetic acid (AcOH)/H 2O
  • Sodium nitrite (NaNO 2)
  • Isopropanol (i‐PrOH)
  • 100‐ 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)
  • 5 × 50–cm chromatography columns
  • 0.2‐mm‐thick silica gel 60 F 254 aluminum TLC plates (Merck)
  • 254‐nm UV lamp
  • Steel bomb (autoclave)
  • 5 × 20–cm Serdolit AD‐4 column (resin particle size, 0.1 to 0.2 mm; Serva)
  • Additional reagents and equipment for flash chromatography ( appendix 3E) and thin‐layer chromatography (TLC; appendix 3D)

Basic Protocol 4: Determination of pKa Values of Nucleosides

  Materials
  • 4‐Amino‐5‐bromo‐7‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)‐3,7‐dihydro‐2H‐pyrrolo[2,3‐d]pyrimidin‐2‐one ( S.4c; see protocol 3)
  • Phosphate buffer solution, pH 4.5 (see recipe)
  • 1 M H 3PO 4
  • 3 M NaOH
  • 100‐mL volumetric flask
  • 100‐mL beaker
  • pH meter
  • UV‐Vis spectrophotometer
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Figures

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Literature Cited

Literature Cited
   Albert, A. and Serjeant, E.P. 1971. Determination of ionization constants by spectrometry. In The Determination of Ionization Constants, pp. 44‐64. Chapman & Hall, London.
   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.
   Hoffer, M. 1960. α‐Thymidin. Chem. Ber. 93:2777‐2781.
   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.
   LePecq, J.B. and Paoletti, C. 1967. A fluorescent complex between ethidium bromide and nucleic acids. Physical‐chemical characterization. J. Mol. Biol. 27:87‐106.
   Kasai, H., Ohashi, Z., Harada, F., Nishimura, S., Oppenheimer, N.J., Crain, P.F., Liehr, J.G., von Minden, D.L., and McCloskey, J.A. 1975. Structure of the modified nucleoside Q isolated from Escherichia coli transfer ribonucleic acid. 7‐(4,5‐cis‐Dihydroxy‐1‐cyclopenten‐3‐ylaminomethyl)‐7‐deazaguanosine. Biochemistry 14:4198‐4208.
   Kazimierczuk, Z., Cottam, H.B., Revankar, G.R., and Robins, R.K. 1984. Synthesis of 2′‐deoxytubercidin, 2′‐deoxyadenosine, and related 2′‐deoxynucleosides via a novel direct stereospecific sodium salt glycosylation procedure. J. Am. Chem. Soc. 106:6379‐6382.
   Kelley, S.O. and Barton, J.K. 1998. DNA‐mediated electron transfer from a modified base to ethidium: Pi‐stacking as modulator of reactivity. Chem. Biol. 5:413‐425.
   Kilpatrick, M.W. and Walker, R.T. 1982. The nucleotide‐sequence of the transfer‐RNA‐met(m) from the archaebacterium thermoplasma‐acidophilum. Zentralbl. Bakteriol. Mikrobiol. Hyg. 1 Abt. Orig. C 3:79‐89.
   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.
   Martin, J.C. (ed.) 1989. Nucleotide Analogues as Antiviral Agents. American Chemical Society, Washington, D.C.
   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.
   Prober, J.M., Trainor, G.L., Dam, R.J., Hobbs, F.W., Robertson, C.W., Zagursky, R.J., Cocuzza, A.J., Jensen, M.A., and Baumeister, K. 1987. A system for rapid DNA sequencing with fluorescent chain‐terminating dideoxynucleotides. Science 238:336‐341.
   Pudlo, J.S., Nassiri, M.R., Kern, E.R., Wotring, L.L., Drach, J.C., and Townsend, L.B. 1990. Synthesis, antiproliferative, and antiviral activity of certain 4‐substituted and 4,5‐disubstituted 7‐[(1,3‐dihydroxy‐2‐propoxy)methyl]pyrrolo[2,3‐d]pyrimidines. J. Med. Chem. 33:1984‐1992.
   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., Mittelbach, C., and Seela, F. 1999. 7‐Halogenated 7‐deaza‐2′‐deoxyinosines. Helv. Chim. Acta 82:12‐18.
   Revankar, G.R. and Robins, R.K. 1991. Pyrrolo[2,3‐d]pyrimidine (7‐deazapurine) nucleosides. In Chemistry of Nucleosides and Nucleotides (L.B. Townsend, ed.) pp. 200‐247. Plenum, New York.
   Rolland, V., Kotera, M., and Lhomme, J. 1997. Convenient preparation of 2‐deoxy‐3,5‐di‐O‐p‐toluoyl‐α‐D‐erythro‐pentofuranosyl chloride. Synth. Commun. 27:3505‐3511.
   Seela, F. and Becher, G. 2000. Synthesis, base pairing, and fluorescence properties of oligonucleotides containing 1H‐pyrazolo[3,4‐d]pyrimidin‐6‐amine (8‐aza‐7‐deazapurin‐2‐amine) as an analogue of purin‐2‐amine. Helv. Chim. Acta 83:928‐942.
   Seela, F. and Driller, H. 1989. 7‐Deaza‐2′‐deoxy‐O6‐methylguanosine: Selective N2‐formylation via a formamidine, phosphoramidite synthesis and properties of oligonucleotides. Nucleosides Nucleotides 8:1‐21.
   Seela, F. and Engelke, U. 1985. Ein Purin/7‐Desazapurin‐Dinucleosid‐Monophosphat mit 2‐Amino‐7H‐pyrrolo[2,3‐d]pyrimidin als fluoreszierender Base. Liebigs Ann. Chem. 1175‐1184.
   Seela, F. and Kehne, A. 1983. 2′‐Desoxytubercidin: Synthese eines 2′‐Desoxyadenosin‐Isosteren durch Phasentransferglycosylierung. Liebigs Ann. Chem. 876‐884.
   Seela, F. and Menkhoff, S. 1985. Synthese von 7‐Desaza‐2′‐desoxyinosin durch Phasentransferglycosylierung. Liebigs Ann. Chem. 1360‐1366.
   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 1203‐1210.
   Seela, F. and Shaikh, K. 2004. 7‐Halogenated 7‐deaza‐2′‐deoxyxanthine 2′‐deoxyribonucleosides. Helv. Chim. Acta 87:1325‐1332.
   Seela, F. and Steker, H. 1984. Synthese von 2′‐desoxyribofuranosiden des 7H‐Pyrrolo[2,3‐d]pyrimidins: Einfluβ des C‐2‐Substituenten auf Fluoreszenz. Liebigs Ann. Chem. 1719‐1730.
   Seela, F. and Thomas, H. 1994. Synthesis of certain 5‐substituted 2′‐deoxytubercidin derivatives. Helv. Chim. Acta 77:897‐903.
   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., Driller, H., and Liman, U. 1985. 7‐Desaza‐Isostere von 2′‐Desoxyxanthosin und 2′‐Desoxyspongosin: Synthese via Glycosylierung von 2,4‐Dichlor‐7H‐pyrrolo[2,3‐d]pyrimidin. Liebigs Ann. Chem. 312‐320.
   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., Chen, Y., Bindig, U., and Kazimierczuk, Z. 1994. Synthesis of 2′‐deoxyisoinosine and related 2′‐deoxyribonucleosides. Helv. Chim. Acta 77:194‐202.
   Seela, F., Chen, Y., and Sauer, M. 1998. Synthesis of 2′,3′‐didehydro‐2′,3′‐dideoxyisoinosine and oxidation of fluorescent 2‐hydroxypurine nucleosides by xanthine oxidase. Nucleosides Nucleotides 17:39‐52.
   Seela, F., Zulauf, M., Sauer, M., and Deimel, M. 2000. 7‐Substituted 7‐deaza‐2′‐deoxyadenosines and 8‐aza‐7‐deaza‐2′‐deoxyadenosines: fluorescence of DNA‐base analogues induced by the 7‐alkynyl side chain. Helv. Chim. Acta 83:910‐927.
   Seela, F., Chittepu, P., He, Y., He, J., and Xu, K. 2005. 6‐Azapyrimidine and 7‐deazapurine 2′‐deoxy‐2′‐fluoroarabinonucleosides: Synthesis, conformation and properties of oligonucleotides. Nucleosides Nucleotides Nucleic Acids In press.
   Simons, C. 2001. Nucleoside Mimetics: Their Chemistry and Biological Properties. Gordon and Breach Science Publishers, Amsterdam.
   Suhadolnik, R.J. 1970. Pyrrolopyrimidine nucleosides. In Nucleoside Antibiotics, pp. 298‐353. Wiley‐Interscience, New York.
   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.
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