Synthesis of N2‐Substituted Deoxyguanosine Nucleosides from 2‐Fluoro‐6‐O‐(Trimethylsilylethyl)‐2′‐Deoxyinosine

Thomas M. Harris1, Constance M. Harris1

1 Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 1.3
DOI:  10.1002/0471142700.nc0103s00
Online Posting Date:  May, 2001
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Abstract

Syntheses of N2‐substituted nucleosides have been studied for many years, primarily with ribonucleosides. However, the primary route to these compounds requires acidic conditions that are too vigorous for the acid‐labile deoxyribonucleosides. The current strategy takes advantage of methods for low‐temperature, nonaqueous diazotization of ribosides in organic solvents using t‐butyl nitrate as the diazotizing agent and HF/pyridine as the fluoride source for the preaparation of a 2‐fluoro‐2?‐deoxyinosine derivative that can be used to synthesize N2‐substituted deoxyguanosine.

     
 
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Table of Contents

  • Basic Protocol 1: Synthesis of 2‐Fluoro‐6‐O‐(Trimethylsilylethyl)‐2′‐Deoxyinosine using 2‐N‐3′,5′‐O‐Triacetyl‐2′‐Deoxyguanosine
  • Alternate Protocol 1: Synthesis of 2‐Fluoro‐6‐O‐(Trimethylsilylethyl)‐2′‐Deoxyinosine Using 3′,5′‐O‐Diacetyl‐2′‐Deoxyguanosine
  • Basic Protocol 2: General Guidelines for Synthesis of N2‐Substituted Nucleosides
  • Alternate Protocol 2: Synthesis of 2‐N‐(3‐Aminopropyl)‐2′‐Deoxyguanosine
  • Alternate Protocol 3: Synthesis of 2‐N‐[2(S)‐1‐Hydroxybut‐3‐en‐2‐yl]‐2′‐Deoxyguanosine
  • Support Protocol 1: Setting Up a Nitrogen Atmosphere
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of 2‐Fluoro‐6‐O‐(Trimethylsilylethyl)‐2′‐Deoxyinosine using 2‐N‐3′,5′‐O‐Triacetyl‐2′‐Deoxyguanosine

  Materials
  • 2′‐Deoxyguanosine (dG) monohydrate
  • Pyridine, anhydrous (Aldrich; packed under nitrogen in a Sure/Seal bottle)
  • Acetic anhydride, freshly distilled
  • Triethylamine (d 0.726), distilled from calcium hydride
  • 4‐Dimethylaminopyridine (DMAP)
  • Dry nitrogen (N 2) or argon (Ar)
  • Methanol, anhydrous
  • Methylene chloride (CH 2Cl 2), anhydrous
  • Anisaldehyde/sulfuric acid spray (see recipe)
  • Acetonitrile
  • Dioxane, anhydrous, distilled from sodium metal before use
  • Triphenylphosphine
  • 2‐Trimethylsilylethanol
  • Diethyl azodicarboxylate (DEAD; from a fresh, unopened bottle)
  • 0.35 M sodium methoxide in methanol (see recipe)
  • Aqueous acetic acid: 6.2 mL glacial acetic acid in 30 mL water
  • Sodium sulfate (Na 2SO 4), anhydrous
  • 63‐ to 200‐mesh silica gel
  • Sand
  • Dry ice/acetonitrile cooling bath (−35° to −40°C)
  • 70% HF/pyridine solution (Aldrich)
  • t‐Butyl nitrite
  • Potassium carbonate (K 2CO 3)
  • Ethyl acetate
  • 2‐liter round‐bottom flask
  • Rotary evaporator equipped with a condenser cooled with chilled water or a dry ice condenser
  • Reflux condenser with 24/40 joint and gas inlet adapter
  • Temperature‐controlled oil bath (up to ∼115°C)
  • 0.25‐mm silica gel 60 F‐254 glass thin‐layer chromatography (TLC) plates
  • UV light source
  • Vacuum system (oil pump) capable of creating <1 mmHg pressure, with manifold and cold trap
  • Filter paper (Whatman no. 1, 7‐cm diameter)
  • Buchner funnel
  • 500‐mL and l‐liter Erlenmeyer flasks
  • 1‐liter, three‐neck flask with 24/40 joints (oven dried) and rubber septa
  • 10‐mL glass syringes (oven dried)
  • 1‐liter, single‐neck flask with 24/40 joint
  • Water aspirator
  • 500‐mL separatory funnels
  • Heavy‐walled glass column (5‐cm i.d. × 40‐cm length)
  • Abderhalden apparatus (drying pistol; 78°C)
  • 50‐mL polypropylene conical tubes with rubber septa
  • 23‐G syringe needles
  • 20‐mL plastic syringes with 3‐in. (7.6‐cm) 20‐G needles
  • 1‐mL glass syringe (oven dried)
  • Additional reagents and equipment for performing reactions under nitrogen (see protocol 6)

Alternate Protocol 1: Synthesis of 2‐Fluoro‐6‐O‐(Trimethylsilylethyl)‐2′‐Deoxyinosine Using 3′,5′‐O‐Diacetyl‐2′‐Deoxyguanosine

  • Concentrated ammonium hydroxide (NH 4OH)
  • 1‐liter, three‐neck, round‐bottom flask with 24/40 joints

Basic Protocol 2: General Guidelines for Synthesis of N2‐Substituted Nucleosides

  Materials
  • 2‐Fluoro‐6‐O‐(trimethylsilylethyl)‐2′‐deoxyinosine ( S.4; see protocol 1 or protocol 2)
  • Amine of choice
  • N,N‐Diisopropylethylamine (DIEA), anhydrous
  • Dimethylsulfoxide (DMSO), anhydrous, vacuum distilled from calcium hydride
  • 0.1 M aqueous acetic acid or 1 M tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF)
  • Methanol, anhydrous
  • 0.1 M sodium bicarbonate (NaHCO 3)
  • Vial or test tube with secure cap
  • Temperature‐controlled oil bath or heating block at 45° to 60°C
  • Rotary evaporator with water aspirator and oil pump
  • Additional reagents and equipment for thin‐layer chromatography (TLC; see protocol 1), or high‐performance liquid chromatography (HPLC)

Alternate Protocol 2: Synthesis of 2‐N‐(3‐Aminopropyl)‐2′‐Deoxyguanosine

  • 1,3‐Diaminopropane
  • Acetonitrile
  • Concentrated NH 4OH
  • Silica gel
  • Sand
  • Tetrahydrofuran (THF), anhydrous
  • 0.1 M ammonium formate in water, pH 6.4
  • 12 × 75–mm glass test tube or conical vial with magnetic stir bar
  • 2.5 × 20–cm column
  • 100‐mL round‐bottom flask
  • 10 × 250–mm C18 reversed‐phase HPLC column (e.g., YMC‐ODS‐AQ column; YMC)

Alternate Protocol 3: Synthesis of 2‐N‐[2(S)‐1‐Hydroxybut‐3‐en‐2‐yl]‐2′‐Deoxyguanosine

  • 2(S)‐Amino‐3‐butenol
  • 0.1 M ammonium formate in water, pH 6.4
  • 12 × 75–mm glass test tube or conical vial with a magnetic stir bar
  • Lyophilizer
  • 10 × 250–mm C18 reversed‐phase HPLC column (e.g., YMC‐ODS‐AQ column; YMC)
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Figures

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Literature Cited

Literature Cited
   Acedo, M., Fabrega, C., Avino, A., Goodman, M., Fagan, P., Wemmer, D., and Eritja, R. 1994. A simple method for N‐15 labelling of exocyclic amino groups in synthetic oligodeoxynucleotides. Nucl. Acids Res. 22:2982‐2989.
   Adib, A., Potier, P.F., Doronina, S., Huc, I., and Behr, J.‐P. 1997. A high‐yield synthesis of deoxy‐2‐fluoroinosine and its incorporation into oligonucleotides. Tetrahedron Lett. 38:2989‐2992.
   Aldrich Chemical. 1983. Technical Bulletin AL‐134: Handling Air‐Sensitive Reagents. Aldrich Chemical Co., Milwaukee, Wis.
   Allerson, C.R., Chen, S.L., and Verdine, G.L. 1997. A chemical method for site‐specific modification of RNA: The convertible nucleoside approach. J. Am. Chem. Soc. 119:7423‐7433.
   Bergstrom, D.E. and Gerry, N.P. 1994. Precision sequence‐specific cleavage of a nucleic acid by a minor‐groove‐directed metal‐binding ligand linked through N‐2 of deoxyguanosine. J. Am. Chem. Soc.116:12067‐12068.
   DeCorte, B.L., Tsarouhtsis, D., Kuchimanchi, S., Cooper, M.D., Horton, P., Harris, C.M., and Harris, T.M. 1996. Improved strategies for postoligomerization synthesis of oligodeoxynucleotides bearing structurally defined adducts at the N2 position of deoxyguanosine. Chem. Res. Toxicol.9:630‐637.
   Diaz, A.R., Eritja, R., and Garcia, R.G. 1997. Synthesis of oligodeoxynucleotides containing 2‐substituted guanine derivatives using 2‐fluoro‐2′‐deoxyinosine as common nucleoside precursor. Nucleosides Nucleotides 16:2035‐2051.
   Edwards, C., Boche, G., Steinbrecher, T., and Scheer, S. 1997. Synthesis of 2‐substituted 2′‐deoxyguanosines and 6‐O‐allylguanines via activation of C‐2 by a trifluoromethanesulfonate group. J. Chem. Soc. Perkin Trans 1 1887‐1893.
   Eritja, R., Acedo, M., Avino, A., and Fabrega, C. 1995. Preparation of oligonucleotides containing non‐natural base analogues. Nucleosides Nucleotides 14:821‐824.
   Erlanson, D.A., Chen, L., and Verdine, G.L. 1993. DNA methylation through a locally unpaired intermediate. J. Am. Chem. Soc. 115:12583‐12584.
   Gaffney, B.L. and Jones, R.A. 1982. A new strategy for the protection of deoxyguanosine during oligonucleotide synthesis. Tetrahedron Lett. 23:2257‐2260.
   Gerster, J.F. and Robins, R.K. 1965. Purine nucleosides. X. The synthesis of certain naturally occurring 2‐substituted amino‐9‐β‐D‐ribofuranosylpurin‐6(1H)‐ones (N2‐substituted guanosines). J. Am. Chem. Soc. 87:3752‐3759.
   Gerster, J.F. and Robins, R.K. 1966. Purine nucleosides. XIII. The synthesis of 2‐fluoro‐ and 2‐chloroinosine and certain derived purine nucleosides. J. Org. Chem. 31:3258‐3262.
   Harris, C.M., Zhou, L., Strand, E.A., and Harris, T.M. 1991. A new strategy for synthesis of oligonucleotides bearing adducts at exocyclic amino sites of purine nucleosides. J. Am. Chem. Soc. 113:4328‐4329.
   Himmelsbach, F., Schulz, B.S., Trichtinger, T., Charubala, R., and Pfleiderer, W. 1984. The p‐nitrophenylethyl (NPE) group. A versatile new blocking group for phosphate and aglycone protection in nucleosides and nucleotides. Tetrahedron 40:59‐72.
   Lee, H., Hinz, M., Stezowski, J.J., and Harvey, R.G. 1990. Syntheses of polycyclic aromatic hydrocarbon‐nucleoside and oligonucleotide adducts specifically alkylated on the amino functions of deoxyguanosine and deoxyadenosine. Tetrahedron Lett. 31:6773‐6776.
   Lee, H., Luna, E., Hinz, M., Stezowski, J.J., Kiselyov, A.S., and Harvey, R.G. 1995. Synthesis of oligonucleotide adducts of the bay region diol epoxide metabolites of carcinogenic polycyclic aromatic hydrocarbons. J. Org. Chem. 60:5604‐5613.
   Matsuda, A., Shinozaki, M., Suzuki, M., Watanabe, K., and Miyasaka, T. 1986. A convenient method for the selective acylation of guanine nucleosides. Synthesis 385‐386.
   Mitsunobu, O. 1981. The use of diethyl azodicarboxylate and triphenylphosphine in synthesis and transformation of natural products. Synthesis1‐28.
   Montgomery, J.A. and Hewson, K. 1960. Synthesis of potential anticancer agents. XX. 2‐Fluoropurines. J. Am. Chem. Soc. 82:463‐468.
   Ramasamy, K., Zounes, M., Gonzalez, C., Freier, S.M., Lesnik, E.A., Cummins, L.L., Griffey, R.H., Monia, B.P., and Cook, P.D. 1994. Remarkable enhancement of binding affinity of heterocycle‐modified DNA to DNA and RNA. Synthesis, characterization and biophysical evaluation of N2‐imidazolylpropylguanine and N2‐imidazolylpropyl‐2‐aminoadenine modified oligonucleotides. Tetrahedron Lett. 35:215‐218.
   Robins, M.J. and Uznanski, B. 1981. Nucleic acid related compounds. 34. Non‐aqueous diazotization with tert‐butyl nitrite. Introduction of fluorine, chlorine, and bromine at C‐2 of purine nucleosides. Can. J. Chem. 59:2608‐2611.
   Sangaiah, R., Gold, A., Ball, L.M., Matthews, D.L., and Toney, G.E. 1992. Synthesis and resolution of putative diastereomeric N2‐deoxyguanosine and N6‐deoxyadenosine adducts of biologically active cyclopentaPAH. Tetrahedron Lett. 33:5487‐5490.
   Schmid, N. and Behr, J.‐P. 1995. Recognition of DNA sequences by strand replacement with polyamino‐oligonucleotides. Tetrahedron Lett. 36:1447‐1450.
   Steinbrecher, T., Wameling, C., Oesch, F., and Seidel, A. 1993. Activation of the C2 position of purine by the trifluoromethanesulfonate group: Synthesis of N2‐alkylated deoxyguanosines. Angew. Chem. Int. Ed. Engl. 32:404‐406.
   Tsarouhtsis, D., Kuchimanchi, S., DeCorte, B.L., Harris, C.M., and Harris, T.M. 1995. Synthesis of oligonucleotides containing interchain cross‐links of bifunctional pyrroles. J. Am. Chem. Soc. 117:11013‐11014.
   Wang, G. and Bergstrom, D.E. 1993. Synthesis of oligonucleotides containing N2‐[2‐(imidazol‐4‐ylacetamido)ethyl]‐2′‐deoxyguanosine. Tetrahedron Lett. 34:6725‐6728.
   Wolfe, S.A. and Verdine, G.L. 1993. Ratcheting torsional stress in duplex DNA. J. Am. Chem. Soc. 115:12585‐12586.
   Woo, J., Sigurdsson, S.T., and Hopkins, P.B. 1993. DNA interstrand cross‐linking reactions of pyrrole‐derived, bifunctional electrophiles: Evidence for a common target site in DNA. J. Am. Chem. Soc. 115:3407‐3415.
   Zajc, B., Lakshman, M.K., Sayer, J.M., and Jerina, D.M. 1992. Epoxide and diol epoxide adducts of polycyclic aromatic hydrocarbons at the exocyclic amino group of deoxyguanosine. Tetrahedron Lett. 33:3409‐3412.
Key References
   Gerster and Robins, , . See above.
  These papers describe the synthesis and some of the reactions of 2‐fluoro‐6‐O‐benzyl‐inosine.
   Tsarouhits et al., ; DeCorte et al., . See above.
  Synthesis of 2‐fluoro‐6‐O‐(trimethylsilylethyl)‐2′‐deoxyinosine and the related phosphoramidite; oligonucleotide synthesis and substitution reactions.
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