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Synthesis of 1,5‐Anhydrohexitol Building Blocks for Oligonucleotide Synthesis

Irene M. Lagoja1,  Arnaud Marchand1,  ArthurVan Aerschot1,  Piet Herdewijn1

1Rega Institute for Medical Research, Leuven, Belgium

Publication Name: 
Current Protocols in Nucleic Acid Chemistry
Unit Number: 
UNIT 1.9
DOI: 
10.1002/0471142700.nc0109s14
Online Posting Date: 
November, 2003
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Abstract

This unit describes in detail, the optimized preparations of 1,5-anhydrohexitol and the 1,5-anhydrohexitol building blocks for oligonucleotide synthesis (hG, hA, hC, hT).

Keywords: hexitol nucleic acid (HNA); 1,5-anhydrohexitol building blocks; hA; hG; hC; hT

     
 
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Table of Contents

  • Unit Introduction
  • Basic Protocol 1: Preparation of 1,5-Anhydro-4,6-O-Benzylidine-3-Deoxy-d-Glucitol
  • Basic Protocol 2: Synthesis of 1¢,5¢-Anhydro-2¢,3¢-Dideoxy-2¢-(N2-Isobutyrylguanin-9-yl)-6¢-O-Monomethoxytrityl-d-Arabinohexitol
  • Basic Protocol 3: Synthesis of 1¢,5¢-Anhydro-6¢-Monomethoxytrityl-2¢,3¢-Dideoxy-2¢-(N6-Benzoyladenin-9-yl)-d-Arabinohexitol
  • Basic Protocol 4: Synthesis of 1¢,5¢-Anhydro-6¢-O-Monomethoxytrityl-2¢,3¢-Dideoxy-2¢-(Thymin-1-yl)-d-Arabinohexitol
  • Basic Protocol 5: Synthesis of 1¢,5¢-Anhydro-2¢-(N4-Benzoylcytosin-1-yl)-2¢,3¢-Dideoxy-6¢-Monomethyoxytrityl-d-Arabinohexitol
  • Commentary
  • Bibliography
  • Figures
     
 
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Materials

Basic Protocol 1: Preparation of 1,5-Anhydro-4,6-O-Benzylidine-3-Deoxy-d-Glucitol

 Materials
  • 2,3,4,6-Tetra-O-acetyl--d-bromoglucose
  • Diethyl ether: reflux overnight on sodium (Na, FeCl2, Et2O) and distill
  • Azoisobutyronitrile [2,2¢-azobis(2-methylpropionitrile); AIBN]
  • Tri-n-butyltin hydride
  • Precoated silica gel TLC plates (Alugram Sil G/UV254)
  • Dichloromethane
  • Anisaldehyde/sulfuric acid spray (unit 1.3)
  • Potassium fluoride dihydrate
  • Sodium sulfate
  • Silica gel (0.060 to 0.200 nm)
  • Methanol
  • 0.1 N sodium methoxide, freshly prepared from sodium and dry methanol
  • Acetic acid (glacial not necessary)
  • Toluene
  • Zinc chloride (dry fresh before use)
  • Benzaldehyde
  • Ethyl acetate
  • n-Hexane
  • Dibutyltinoxide
  • Benzene
  • Dioxane (reflux overnight on lithium aluminum hydride and distill)
  • p-Toluenesulfonyl chloride (for S.3a) or p-toluoyl chloride (for S.3b)
  • Delite Celite
  • 4-(Dimethylamino)pyridine (DMAP)
  • Dry ice/isopropanol
  • Thiophosgene
  • 2,4-Dichlorophenol
  • 1 M potassium dihydrogenphosphate solution, pH 5
  • Nitrogen gas
  • Oil bath and magnetic stirrer
  • Rotary evaporator equipped with a vacuum pump and cooling trap
  • 5 × 35–, 6 × 50–, and 5 × 20–cm chromatography columns
  • 2 × 18–cm test tubes
  • 1-L round-bottom flask with rubber stopper
  • Dropping funnel
  • Glass funnel
  • Dean-Stark condenser
  • UV lamp, 254 nm
  • Additional reagents and equipment for thin-layer chromatography (TLC; appendix 3D) and column chromatography (appendix 3E)

NOTE: The 1H and 13C NMR spectra given as examples were determined with a JEOL FX 90Q spectrometer or 400 MHz Bruker AMX with tetramethylsilane as internal standard. Electron-impact mass spectra (EIMS) and chemical-ionization mass spectra (CIMS) were obtained using a KRATOS Concept 1H mass spectrometer. Abbreviations: s, singlet; d, doublet; dd, double doublet; t, triplet; br s, broad signal; m, multiplet; ddd, double doublet of doublet; and dm, double multiplet.

Basic Protocol 2: Synthesis of 1¢,5¢-Anhydro-2¢,3¢-Dideoxy-2¢-(N2-Isobutyrylguanin-9-yl)-6¢-O-Monomethoxytrityl-d-Arabinohexitol

 Materials
  • 6-Chloro-9H-purine-2-amine
  • 1,5-Anhydro-4,6-O-benzylidene-3-deoxy-d-glucitol (S.5; see Basic Protocol 1)
  • Triphenylphosphine
  • Dioxane (reflux overnight on lithium aluminum hydride and distill)
  • Nitrogen gas
  • Diisopropyl azodicarboxylate (DIAD)
  • n-Hexane
  • Ethyl acetate
  • 10% (v/v) HCl
  • Dichloromethane (store over phosphorous pentoxide and distill before use)
  • Phenolpthalein solution
  • 4 N sodium hydroxide
  • Phosphorous pentoxide
  • Pyridine (reflux overnight over potassium hydroxide distill before use)
  • Bis(trimethylsilyl)acetamide (BSA)
  • Isobutyric anhydride
  • 25% (v/v) ammonia
  • Diethyl ether
  • Dimethylformamide (DMF; remove water by distillation with benzene followed by distillation under vacuum)
  • 4-Monomethoxytrityl chloride (MMTr·Cl)
  • Methanol
  • Saturated sodium bicarbonate solution
  • Sodium sulfate
  • Toluene
  • Dropping funnel
  • Rotary evaporator equipped with a vacuum pump and cooling trap
  • Oil bath and magnetic stirrer
  • 5 × 35– and 4 × 25–cm chromatography columns
  • Additional reagents and equipment for TLC and column chromatography (see Basic Protocol 1 and Critical Parameters)

NOTE: The 1H NMR and 13C NMR spectra were determined with a 200 MHz Varian Gemini spectrometer with tetramethylsilane as internal standard. Abbreviations: s, singlet; d, doublet; dd, double doublet; t, triplet; br s, broad signal; m, multiplet; ddd, double doublet of doublet; dm, double multiplet. Liquid secondary-ion (LSIMS) mass spectra were obtained using a KRATOS Concept 1H mass spectrometer.

Basic Protocol 3: Synthesis of 1¢,5¢-Anhydro-6¢-Monomethoxytrityl-2¢,3¢-Dideoxy-2¢-(N6-Benzoyladenin-9-yl)-d-Arabinohexitol

 Materials
  • Adenine
  • Lithium hydride
  • 12-Crown-4
  • Dimethylformamide (DMF; remove water by distillation with benzene followed by distillation under vacuum)
  • Nitrogen gas
  • 1,5-Anhydro-4,6-O-benzylidene-3-deoxy-2-O-(p-tolylsulfonyl)-d-ribohexitol (S.4a; see Basic Protocol 1)
  • n-Hexane
  • Ethyl acetate
  • Dichloromethane
  • Saturated sodium bicarbonate solution
  • Sodium sulfate
  • Methanol
  • Pyridine (reflux over potassium hydroxide overnight and distill)
  • Benzoyl chloride
  • 25% (v/v) ammonia
  • Toluene
  • 80% (v/v) acetic acid
  • Diethyl ether: reflux overnight on sodium (Na, FeCl2, Et2O) and distill
  • Pyridine (reflux over potassium hydroxide overnight and distill)
  • 4-Monomethoxytrityl chloride (MMTr·Cl)
  • Oil bath and magnetic stirrer
  • Rotary evaporator equipped with a vacuum pump and cooling trap
  • 3 × 30–, 5 × 40–, and 4 × 30–cm chromatography columns
  • Additional reagents and equipment for TLC and column chromatography (see Basic Protocol 1 and Critical Parameters)

NOTE: The 1H NMR and 13C NMR spectra were determined with a 200 MHz Varian Gemini spectrometer with tetramethylsilane as internal standard. Abbreviations: s, singlet; d, doublet; dd, double doublet; t, triplet; br s, broad signal; m, multiplet; ddd, double doublet of doublet; dm, double multiplet. Liquid secondary-ion (LSIMS) mass spectra were obtained using a KRATOS Concept 1H mass spectrometer.

Basic Protocol 4: Synthesis of 1¢,5¢-Anhydro-6¢-O-Monomethoxytrityl-2¢,3¢-Dideoxy-2¢-(Thymin-1-yl)-d-Arabinohexitol

 Materials
  • N3-Benzoylthymine
  • 1,5-Anhydro-4,6-O-benzylidene-3-deoxy-d-glucitol (S.5; see Basic Protocol 1)
  • Triphenylphosphine
  • Tetrahydrofuran (THF; reflux overnight on lithium aluminum hydride and distill)
  • Nitrogen gas
  • Diethyl azodicarboxylate (DEAD)
  • n-Hexane
  • Ethyl acetate
  • Saturated ammonia in methanol
  • Dichloromethane
  • Toluene
  • 80% (v/v) acetic acid
  • Methanol
  • Pyridine (reflux over potassium hydroxide overnight and distill)
  • Monomethoxytrityl chloride (MMTr·Cl)
  • Saturated sodium bicarbonate solution
  • Sodium sulfate
  • Oil bath and magnetic stirrer
  • Dropping funnel
  • Rotary evaporator equipped with a vacuum pump and cooling trap
  • 3 × 35– and 4 × 35–cm chromatography columns
  • Additional reagents and equipment for TLC and column chromatography (see Basic Protocol 1 and Critical Parameters)

NOTE: The 1H NMR and 13C NMR spectra were determined with a 200-MHz Varian Gemini spectrometer with tetramethylsilane as internal standard. Liquid secondary-ion (LSIMS) mass spectra were obtained using a KRATOS Concept 1H mass spectrometer. Abbreviations: s, singlet; d, doublet; dd, double doublet; t, triplet; br s, broad signal; m, multiplet; ddd, double doublet of doublet; dm, double multiplet.

Basic Protocol 5: Synthesis of 1¢,5¢-Anhydro-2¢-(N4-Benzoylcytosin-1-yl)-2¢,3¢-Dideoxy-6¢-Monomethyoxytrityl-d-Arabinohexitol

 Materials
  • N4-Benzoyluracil
  • 1,5-Anhydro-4,6-O-benzylidene-3-deoxy-d-glucitol (S.5; see Basic Protocol 1)
  • Triphenylphosphine
  • Dioxane (reflux overnight on lithium aluminum hydride and distill)
  • Nitrogen gas
  • Diethyl azodicarboxylate (DEAD)
  • Saturated ammonia in methanol
  • Dichloromethane
  • Methanol
  • Toluene
  • Triazole
  • Pyridine (reflux overnight over potassium hydroxide and distill)
  • Phosphoroxy chloride
  • 25% ammonia
  • Sodium sulfate
  • Benzoyl chloride
  • Saturated sodium bicarbonate solution
  • Trifluoroacetic acid (TFA)
  • Diethyl ether: reflux overnight on sodium (Na, FeCl2, Et2O) and distill
  • Monomethoxytrityl chloride (MMTr·Cl)
  • Dropping funnel
  • Rotary evaporator equipped with a vacuum pump and cooling trap
  • 4 × 30– and 3 × 35–cm chromatography columns
  • Oil bath and magnetic stirrer
  • Drying tube
  • Additional reagents and equipment for TLC and column chromatography (see Basic Protocol 1 and Critical Parameters)

NOTE: The 1H NMR and 13C NMR spectra were determined with a 200 MHz Varian Gemini spectrometer with tetramethylsilane as an internal standard. Abbreviations: s, singlet; d, doublet; dd, double doublet; t, triplet; br s, broad signal; m, multiplet; ddd, double doublet of doublet; dm, double multiplet. Liquid secondary-ion (LSIMS) mass spectra were obtained using a KRATOS Concept 1H mass spectrometer.

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Figures

  • Figure 1.9.1
    Preparation of 1,5-anhydro-4,6-O-benzylidine-3-deoxy-d-glucitol (S.5). Abbreviations: AIBN, azoisobutyronitrile; Bu2SnO, dibutyltinoxide; Bu3SnH, tributyltinhydride; DMAP, 4-(dimethylamino)pyridine; NaOMe, sodium methoxide; PhCHO, benzaldehyde; TolCl, toluoyl chloride; TsCl, tosyl chloride.

    View Image
  • Figure 1.9.2
    Preparation of protected hG (S.9). Abbreviations: BSA, bis(trimethylsilyl)acetamide; DIAD, diisopropyl azodicarboxylate; DMF, dimethylformamide; MMTr·Cl, 4-monomethoxytrityl chloride; Ph3P, triphenylphosphine.

    View Image
  • Figure 1.9.3
    Preparation of protected hA (S.13). Abbreviations: BzCl, benzoyl chloride; DMF, dimethylformamide; HOAc, acetic acid; MMTr·Cl, monomethoxytrityl chloride.

    View Image
  • Figure 1.9.4
    Preparation of protected hT (S.16). Abbreviations: DEAD, diethyl azodicarboxylate; THF, tetrahydrofuran; Ph3P, triphenylphosphine; HOAc, acetic acid; MMTr·Cl, monomethoxytrityl chloride.

    View Image
  • Figure 1.9.5
    Preparation of protected hC (S.20). Abbreviations: BzCl, benzoylchloride; DEAD, diethyl azodicarboxylate; MMTr·Cl, 4-monomethoxy chloride; POCl3, phosphoroxy chloride; TFA, trifluoroacetic acid.

    View Image

Literature Cited

 Literature Cited
    Atkins, D., Miller, M., De Bouvere, B., Van Aerschot, A., and Herdewijn, P. 2000. Evaluation of the cellular uptake of hexitol nucleic acids in HeLa cells. Pharmazie 55:615-617.
    De Bouvere, P., Kerremans, L., Rozenski, J., Janssen, G., Van Aerschot, A., Claes, P., Busson, R., and Herdewijn, P. 1997. Improved synthesis of anhydrohexitol building blocks for oligonucleotide synthesis. Liebigs Ann./Recueil 1453-1461.
    De Winter, H., Lescrinier, E., Van Aerschot, A., and Herdewijn, P. 1998. Molecular dynamics simulation to investigate differences in minor groove hydration of HNA/RNA hybrids as compared to HNA/DNA complexes. J. Am. Chem. Soc. 120:5381-5394.
    Hendrix, C., Verheggen, I., Rosemeyer, H., Seela, F., Van Aerschot, A., and Herdewijn, P. 1997a. 1¢,5¢-Anhydrohexitol oligonucleotides: Synthesis, base pairing and recognition by regular oligodeoxyribonucleotides and oligoribonucleotides. Chem. Eur. J. 3:110-119.
    Hendrix, C., Rosemeyer, H., De Bouvere, B., Van Aerschot, A., Seela, F., and Herdewijn, P. 1997b. 1¢,5¢-Anhydrohexitol oligonucleotides: Hybridization and strand displacement with oligoribonucleotides, interaction with RNase H and HIV reverse transcriptase. Chem. Eur.J. 3:1513-1520.
    Kocienski, P. and Pant, C. 1982. A convenient preparation of some 2,3,4,6-tetraacetyl-1,5-anhydro-d-hexitols. Carbohydr. Res. 110:330–332.
    Kozlov, I.A., Politis, P.K., Pitsch, S., Herdewijn, P., and Orgel, L.E. 1999a. A highly enantio-selective hexitol nucleic acid template for nonenzymatic oligoguanylate synthesis. J. Am. Chem. Soc. 121:1108-1109.
    Kozlov, I.A., Politis, P.K., Van Aerschot, A., Busson, R., Herdewijn, P., and Orgel, L.E. 1999b. Nonenzymatic synthesis of RNA and DNA oligomers on hexitol nucleic acid templates: The importance of the A structure. J. Am. Chem. Soc. 121:2613-2656.
    Lavrik, I.N., Avdeeva, O.N., Dontsova, O.A., Froeyen, M., and Herdewijn, P.A. 2001. Translational properties of mHNA, a messenger RNA containing anhydrohexitol nucleotides. Biochemistry 40:11777-11784.
    Lescrinier, E., Esnouf, R.M., Schraml, J., Busson, R., and Herdewijn, P. 2000a. Solution structure of a hexitol nucleic acid duplex with four consecutive T.T base pairs. Helv. Chim. Acta 83:1291-1310.
    Lescrinier, E., Esnouf, R.M., Schraml, J., Busson, R., Heus, H.A., Hilbers, C.W., and Herdewijn, P. 2000b. Solution structure of a HNA-RNA hybrid. Chem. & Biol. 7:719-731.
    Vastmans, K., Pochet, S., Peys, A., Kerremans, L., Van Aerschot, A., Hendrix, C., Marlire, P., and Herdewijn, P. 2000. Enzymatic incorporation in DNA of 1,5-anhydrohexitol nucleotides. Biochemistry 39:12757-12765.
    Verheggen, I., Van Aerschot, A., Toppet, S. Snoeck, R., Janssen, G., Balzarini, J., De Clercq, E., and Herdewijn, P. 1993. Synthesis and antiherpes virus activity of 1,5-anhydrohexitol nucleosides. J. Med. Chem. 36:2033-2039.
    Verheggen, I., Van Aerschot, A., Van Meervelt, L., Rozenski, J., Wiebe, L., Snoek, R., Andrei, G., Balzarini, J., Claes, P., De Clercq, E., and Herdewijn, P. 1995. Synthesis, biological evaluation, and structure analysis of a series of new 1,5-anhydrohexitol nucleosides J. Med. Chem. 38:826-35.
     
 
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