Abstract

This unit describes two methods for preparing oligonucleotides containing an O⁶ ?2??deoxyguanosine?alkyl?O⁶ ?2??deoxyguanosine interstrand cross?link by a solid?phase synthesis approach. Depending on the desired orientation of the cross?link in the DNA duplex, either a bis? or a mono?phosphoramidite synthesis strategy can be employed. Both procedures require the preparation of a protected 2??deoxyguanosine?containing dimer where the two nucleosides are attached at the O⁶ ?atoms by an alkyl linker. This linker is introduced as a protected diol using two successive Mitsunobu reactions to produce a cross?linked amidite that is incorporated into an oligonucleotide via solid?phase synthesis. The use of a protected diol lends versatility to this method, as cross?links of variable chain length may be prepared. The bis?phosphoramidite approach is a direct method to preparing the cross?linked duplex, whereas the mono?phosphoramidite strategy requires additional manipulation of the solid support to prepare cross?linked oligonucleotides. Once all synthetic steps are completed, these oligonucleotides can then be removed from the solid support and deprotected, and then purified via ion?exchange HPLC to produce sufficient quantities of substrates that can be used in DNA repair studies. Curr. Protoc. Nucleic Acid Chem. 44:5.9.1?5.9.19. © 2011 by John Wiley & Sons, Inc.

Keywords: interstrand cross?link; phosphoramidite; oligonucleotide synthesis; chemical synthesis; solid?phase synthesis; DNA repair

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Introduction
Basic Protocol 1: Protection of the Diol with a tert‐Butyldiphenyl Silyl Group
Basic Protocol 2: Synthesis of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine‐3′‐O‐bis‐Phosphoramidites
Basic Protocol 3: Synthesis of 1‐{O6‐[3′‐O‐tert‐Butyldimethysilyl‐5′‐O‐Dimethoxytrityl‐N2‐Phenoxyacetyl‐2′‐Deoxyguanidyl]}‐7‐{O6‐[5′‐O‐Dimethoxytrityl‐N2‐Phenoxyacetyl‐2′‐Deoxyguanidyl‐3′‐O‐(β‐2‐Cyanoethyl‐N,N′‐Diisopropyl)Phosphoramidite]}‐Heptane
Basic Protocol 4: Solid‐Phase Synthesis and Deprotection of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine Oligonucleotides
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Protection of the Diol with a tert‐Butyldiphenyl Silyl Group

Materials

Argon gas
1,4‐butanediol or 1,7‐heptanediol (Aldrich Chemical)
Imidazole (Aldrich Chemical)
Anhydrous tetrahydrofuran (THF; Aldrich Chemical)
Tert ‐butyldiphenylchlorosilane (TBDPS‐Cl; Aldrich Chemical)
Dichloromethane (DCM, reagent grade; Caledon Laboratory Chemicals)
5% (w/v) Aqueous sodium bicarbonate (NaHCO₃ )
Magnesium sulfate (MgSO₄ )
Hexanes (reagent grade; Caledon Laboratory Chemicals)
Ethyl acetate (EtOAc, reagent grade)

50‐mL round‐bottom flask with a rubber septum
Magnetic stir plate and stir bar
25‐mL glass syringe
TLC plate, EMD silica gel 60 F₂₅₄
Rotary evaporator and chemically resistant dry vacuum pump
500‐mL separatory funnel
Glass funnel and filter paper
Silica gel (60 Å, 230 to 400 mesh)
3 × 50–cm and 5 × 50–cm chromatography column
Vacuum manifold
Vacuum pump

Additional reagents and equipment for thin‐layer chromatography ( appendix 3D ) and column chromatography ( appendix 3E )

Basic Protocol 2: Synthesis of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine‐3′‐O‐bis‐Phosphoramidites

Materials

3′‐O ‐alloxycarbonyl‐5′‐O ‐dimethoxytrityl‐N² ‐phenoxyacetyl‐2′‐deoxyguanosine (see McManus et al., )
Anhydrous 1,4‐dioxane
1‐O ‐tert ‐Butyldiphenylsilylbutanol ( S.1 ; protocol 1 )
Triphenylphosphine (Aldrich Chemical)
Argon gas
Diisopropylazodicarboxylate (Aldrich Chemical)
Dichloromethane (DCM; reagent grade)
5% (w/v) Aqueous sodium bicarbonate (NaHCO₃ )
Magnesium sulfate (MgSO₄ )
Silica gel (60 Å, 230 to 400 mesh)
Hexanes (reagent grade)
Ethyl acetate (EtOAc; reagent grade)
Anhydrous tetrahydrofuran (THF)
Tetrabutylammonium fluoride (1 M in THF)
Sodium sulfate (Na₂ SO₄ )
Palladium (0) tetrakistriphenylphosphine (Aldrich Chemical)
Butylamine (Aldrich Chemical)
Formic acid (Aldrich Chemical)
Diisopropylethylamine (Aldrich Chemical)
N ,N ‐diisopropylamino cyanoethyl phosphonamidic chloride (ChemGenes)

10‐, 50, 100, and 250‐mL round‐bottom flasks with a rubber septum
Magnetic stir plate and stir bar
1‐, 2‐, 10‐, and 25‐mL glass syringes
Rotary evaporator and chemically resistant dry vacuum pump
100‐ and 250‐mL Separatory funnel
Glass funnel and filter paper
2 × 50–cm and 10 × 70–cm flash chromatography columns
Vacuum manifold
Vacuum pump

Additional reagents and equipment for column chromatography ( appendix 3E )

Basic Protocol 3: Synthesis of 1‐{O6‐[3′‐O‐tert‐Butyldimethysilyl‐5′‐O‐Dimethoxytrityl‐N2‐Phenoxyacetyl‐2′‐Deoxyguanidyl]}‐7‐{O6‐[5′‐O‐Dimethoxytrityl‐N2‐Phenoxyacetyl‐2′‐Deoxyguanidyl‐3′‐O‐(β‐2‐Cyanoethyl‐N,N′‐Diisopropyl)Phosphoramidite]}‐Heptane

Materials

3′‐O ‐alloxycarbonyl‐5′‐O ‐dimethoxytrityl‐N² ‐phenoxyacetyl‐2′‐deoxyguanosine (see McManus et al., )
Anhydrous 1,4‐dioxane (Aldrich Chemical)
1‐O‐tert ‐Butyldiphenylsilylheptanol ( S.2 ; protocol 1 )
Triphenylphosphine (Aldrich Chemical)
Argon gas
Dichloromethane (DCM; reagent grade)
5% (w/v) Aqueous sodium bicarbonate (NaHCO₃ )
Magnesium sulfate (MgSO₄ )
Silica gel (60 Å, 230 to 400 mesh)
Anhydrous tetrahydrofuran (THF)
Tetrabutylammonium fluoride (1 M in THF)
Sodium sulfate (Na₂ SO₄ )
Hexanes (reagent grade)
Ethyl acetate (EtOAc; reagent grade)
Diisopropylazodicarboxylate (Aldrich Chemical)
Palladium (0) tetrakistriphenylphosphine (Aldrich Chemical)
Butylamine (Aldrich Chemical)
Formic acid (Aldrich Chemical)
Diisopropylethylamine (Aldrich Chemical)
N ,N ‐diisopropylamino cyanoethyl phosphonamidic chloride (ChemGenes)

10‐, 25‐, 50‐ and 100‐mL round‐bottom flasks with a rubber septum
Magnetic stir plate and stir bar
1‐, 2‐, and 10‐mL glass syringes
Rotary evaporator and chemically resistant dry vacuum pump
100‐ and 250‐mL Separatory funnels
Glass funnel and filter paper
Vacuum manifold
Vacuum pump
3 × 70–cm and 4 × 70– cm Flash chromatography columns

Additional reagents and equipment for column chromatography ( appendix 3E )

Basic Protocol 4: Solid‐Phase Synthesis and Deprotection of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine Oligonucleotides

Materials

5′‐O ‐DMT‐2′‐deoxynucleoside‐3′‐O ‐succinate long‐chain alkylamine controlled‐pore glass (LCAA‐CPG, 500 Å; Glen Research)
Protected 2′‐deoxyribonucleoside 3′‐phosphoramidites (Glen Research)
Anhydrous acetonitrile (MeCN; Applied Biosystems)
O⁶ ‐2′‐deoxyguanosine‐alkyl‐O⁶ ‐2′‐deoxyguanosine bis‐ or mono‐phosphoramidite ( S.7 or S.12 ; Basic Protocols protocol 22 and protocol 33 , respectively)
4Å activated molecular sieves
Argon
Ammonium hydroxide, concentrated (conc. NH₄ OH)
Anhydrous ethanol (EtOH)
Triethylamine (TEA)
Triethylamine trihydrofluoride (TEA⋅3HF; Aldrich Chemical)
Protected 2′‐deoxyribonucleoside 5′‐phosphoramidites (ChemGenes)

Screw‐capped columns
Brown bottles
DNA synthesizer (Applied Biosystems)
2‐mL screw‐capped microcentrifuge tubes
55°C sand bath
DNA concentrator
5′‐O‐DMT‐2′‐deoxynucleoside polystyrene columns (Glen Research)
3‐mL disposable syringes
Vacuum pump

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Figures

Figure 5.9.1 Chemical structure of the O⁶ ‐2′‐deoxyguanosine‐alkyl‐O⁶ ‐2′‐deoxyguanosine cross‐link.

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Figure 5.9.2 Preparation of mono‐ tert ‐butyldiphenylsilylated diols (S.1 and S.2 ). Abbreviations: TBDPS‐Cl, tert‐butyldiphenylsilyl; THF, tetrahydrofuran.

View Image

Figure 5.9.3 Preparation of S.7 . (a) S.1 , triphenylphosphine, DIAD, 1,4‐dioxane; (b) TBAF, THF; (c) triphenylphosphine, DIAD, 1,4‐dioxane; (d) triphenylphosphine, Pd(Ph₃ P)₄ , formic acid, butylamine, 1,4‐dioxane; (e) Cl‐P(OCE)N i Pr_2, DIPEA, THF. Abbreviations: DIAD, diisopropylazodicarboxylate; TBAF, tetrabutylammonium fluoride; Pd(Ph₃ P)₄ , palladium (0) tetrakistriphenylphosphine; Cl‐P(OCE)N i Pr₂ , N , N ‐diisopropylamino cyanoethyl phosphonamidic chloride; DIPEA, diisopropylethylamine; DMT, dimethoxytrityl; Alloc, allyloxycarbonyl; Pac, phenoxyacetyl; TBDPS, tert ‐butyldiphenylsilyl; THF, tetrahydrofuran.

View Image

Figure 5.9.4 Synthesis of S.12 . (a) S.2 , triphenylphosphine, DIAD, 1,4‐dioxane; (b) TBAF, THF; (c) triphenylphosphine, DIAD, 1,4‐dioxane; (d) triphenylphosphine, Pd(Ph₃ P)₄ , formic acid, butylamine, 1,4‐dioxane; (e) Cl‐P(OCE)N i Pr_2, DIPEA, THF. Abbreviations: DIAD, diisopropylazodicarboxylate; TBAF, tetrabutylammonium fluoride; Pd(Ph₃ P)₄ , palladium (0) tetrakistriphenylphosphine; Cl‐P(OCE)N i Pr₂ , N , N ‐diisopropylamino cyanoethyl phosphonamidic chloride; DIPEA, diisopropylethylamine; DMT, dimethoxytrityl; Alloc, allyloxycarbonyl; Pac, phenoxyacetyl; TBDPS, tert ‐butyldiphenylsilyl; TBS, tert ‐butyldimethylsilyl; THF, tetrahydrofuran.

View Image

Figure 5.9.5 Bis‐phosphoramidite approach for assembly of oligonucleotides containing an O⁶ ‐2′‐deoxyguanosine‐alkyl‐O⁶ ‐2′‐deoxyguanosine cross‐link by solid‐phase synthesis. (a) Oligonucleotide synthesis with 2′‐deoxyribonucleoside 3′‐phosphoramidites; (b) coupling with bis‐phosphoramidite S.7 ; (c) chain extension with 2′‐deoxyribonucleoside 3′‐phosphoramidites; (d) cleavage from the solid‐support and deprotection with NH₄ OH (29% w/v)/ethanol (3:1).

View Image

Figure 5.9.6 Mono‐phosphoramidite approach for assembly of oligonucleotides containing a O⁶ ‐2′‐deoxyguanosine‐alkyl‐O⁶ ‐2′‐deoxyguanosine cross‐link by solid‐phase synthesis. (a) Oligonucleotide synthesis with 2′‐deoxyribonucleoside 3′‐phosphoramidites; (b) coupling with phosphoramidite S.12 ; (c) chain extension with 2′‐deoxyribonucleoside 3′‐phosphoramidites; (d) removal of the 3′‐ O ‐ tert ‐butyldimethylsilyl group with TEA⋅3HF and extensive washing; (e) chain extension with 2′‐deoxyribonucleoside 5′‐phosphoramidites; (f) cleavage from the solid‐support and deprotection with NH₄ OH (29% w/v)/ethanol (3:1).

View Image

Synthesis of Building Blocks and Oligonucleotides with {G}O6‐Alkyl‐O6{G} Cross‐Links

Abstract

Table of Contents

Materials

Basic Protocol 1: Protection of the Diol with a tert‐Butyldiphenyl Silyl Group

Basic Protocol 2: Synthesis of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine‐3′‐O‐bis‐Phosphoramidites

Basic Protocol 4: Solid‐Phase Synthesis and Deprotection of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine Oligonucleotides

Figures

Synthesis of Building Blocks and Oligonucleotides with {G}O6‐Alkyl‐O6{G} Cross‐Links

Abstract

Table of Contents

Materials

Basic Protocol 1: Protection of the Diol with a tert‐Butyldiphenyl Silyl Group

Basic Protocol 2: Synthesis of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine‐3′‐O‐bis‐Phosphoramidites

Basic Protocol 4: Solid‐Phase Synthesis and Deprotection of O6‐2′‐Deoxyguanosine‐Alkyl‐O6‐2′‐Deoxyguanosine Oligonucleotides

Figures

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