Release of DNA Oligonucleotides and Their Conjugates from Controlled‐Pore Glass Under Thermolytic Conditions

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

The sequential functionalization of long?chain alkylamine controlled?pore glass (CPG) with a 3?hydroxypropyl?(2?cyanoethyl)thiophosphoryl linker and a dinucleoside phosphorotetrazolide leads to a uniquely engineered support for solid?phase synthesis. Unlike conventional succinylated?CPG supports, this support is designed to allow oligonucleotide deprotection and elimination of deprotection side?products to proceed without release of the oligonucleotide. When needed, the DNA oligonucleotide can be thermolytically released in 2 hr under essentially neutral conditions. The modified CPG support has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20?mers. On the basis of reversed?phase HPLC and electrophoretic analyses, the purity of the released oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated?CPG supports, in terms of both shorter?than?full?length oligonucleotide contaminants and overall yields. The detailed preparation of DNA oligonucleotides conjugated with exemplary reporter or functional groups, either at the 3??terminus or at both 3?? and 5??termini, is also described. Curr. Protoc. Nucleic Acid Chem. 35:3.17.1?3.17.21. © 2008 by John Wiley & Sons, Inc.

Keywords: deoxyribonucleoside phosphorodiamidites; solid?phase synthesis; modified CPG support; thermolytic conditions; DNA oligonucleotides conjugates

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Introduction
Basic Protocol 1: Functionalization of CPG with Covalently Linked Dinucleotides and Its Application to Solid‐Phase Synthesis of Native and Modified DNA Oligonucleotides
Support Protocol 1: Preparation of Deoxyribonucleoside Phosphorodiamidites
Basic Protocol 2: Application of the Dinucleotide‐Bound Support for Synthesis of a DNA Oligonucleotide with 5′‐ and 3′‐Functional Groups
Alternate Protocol 1: Application of the Hydroxylated CPG Support for Synthesis of a DNA Oligonucleotide with a 3′‐Reporter Group
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Functionalization of CPG with Covalently Linked Dinucleotides and Its Application to Solid‐Phase Synthesis of Native and Modified DNA Oligonucleotides

Materials

Long‐chain alkylamine controlled‐pore glass, 500 Å (CPG)
Triethylamine (TEA)
Anhydrous acetonitrile (MeCN; Glen Research)
Dry argon gas cylinder
O ‐(2‐Cyanoethyl)‐O ‐[3‐(4,4′‐dimethoxytrityl)oxy‐1‐propyl]‐N ,N ‐diisopropylphosphoramidite (S.1 ; Glen Research)
0.25 M 5‐ethylthio‐1H ‐tetrazole in dry acetonitrile (ETT/MeCN; Glen Research)
3H ‐1,2‐Benzodithiol‐3‐one 1,1‐dioxide (Glen Research)
Reagents recommended for automated solid‐phase oligonucleotide synthesis (Glen Research):
- Standard 2‐cyanoethyl deoxyribonucleoside phosphoramidites (T, C^Bz , A^Bz , and G^iBu )
- Activator solution: 1H ‐tetrazole in acetonitrile
- Oxidation solution: 0.02 M iodine in THF/pyridine/water
- 3H ‐1,2‐Benzodithiol‐3‐one 1,1‐dioxide
- Cap A solution: acetic anhydride in THF/pyridine
- Cap B solution: 1‐methylimidazole in THF
- Deblocking solution: trichloroacetic acid (TCA) in dichloromethane
Deoxyribonucleoside phosphorodiamidites (S.4 ; see protocol 2 ):
- 5′‐O ‐(4,4′‐Dimethoxytrityl)‐3′‐O ‐bis(N ,N ‐diisopropylamino)phosphinyl‐2′‐deoxythymidine
- N⁴ ‐Benzoyl‐5′‐O ‐(4,4′‐dimethoxytrityl)‐3′‐O ‐bis(N ,N ‐diisopropylamino)phosphinyl‐2′‐deoxycytidine
- N⁶ ‐Benzoyl‐5′‐O ‐(4,4′‐dimethoxytrityl)‐3′‐O ‐bis(N ,N ‐diisopropylamino)phosphinyl‐2′‐deoxyadenosine
- N² ‐Isobutyryl‐5′‐O ‐(4,4′‐dimethoxytrityl)‐3′‐O ‐bis(N ,N ‐diisopropylamino)phosphinyl‐2′‐deoxyguanosine
3′‐O ‐Levulinyl‐2′‐deoxyribonucleosides (S.5 ; Rasayan):
- 3′‐O ‐Levulinyl‐2′‐deoxythymidine
- N⁴ ‐Benzoyl‐3′‐O ‐levulinyl‐2′‐deoxycytidine
- N⁶ ‐Benzoyl‐3′‐O ‐levulinyl‐2′‐deoxyadenosine
- N² ‐Isobutyryl‐3′‐O ‐levulinyl‐2′‐deoxyguanosine
Anhydrous ammonia cylinder (Aldrich)
Phosphate‐buffered saline
2 M triethylammonium acetate (TEAA) buffer (Applied Biosystems)
Loading buffer: 1:4 (v/v) 10× TBE, pH 8.3 ( appendix 2A ) in formamide, containing 2 mg/mL bromphenol blue
20 × 40–cm 7 M urea/20% polyacrylamide gel (unit 10.4 and appendix 3B )
Stains‐all (Aldrich)

4‐ and 8‐mL screw‐thread glass vials (Wheaton)
15‐mL glass sintered funnel (coarse and medium porosities)
Rubber septa of assorted sizes
16‐ and 21‐G hypodermic needles
0.5‐, 1‐, 5‐, and 10‐mL glass syringes
Spectrophotometer
1‐mL Luer‐tipped syringe
Empty synthesis columns (Glen Research)
DNA/RNA synthesizer
250‐mL stainless steel pressure vessel (Parr Instrument)
90°C heating block (VWR)
0.45‐µm syringe filters
5‐µm Supelcosil LC‐18S HPLC column (25 cm × 4.6 mm, Supelco)
1.5‐mL microcentrifuge tubes
Platform agitator

Additional reagents and equipment for quantitative trityl assay (unit 3.2 ), automated oligonucleotide synthesis ( appendix 3C ), and polyacrylamide gel electrophoresis (unit 10.4 and appendix 3B )

NOTE: All filtrations, unless otherwise indicated, are performed using a water aspirator as a vacuum source.

Support Protocol 1: Preparation of Deoxyribonucleoside Phosphorodiamidites

Materials

5′‐O ‐Protected deoxyribonucleosides (Chem‐Impex International):
- 5′‐O ‐(4,4′‐Dimethoxytrityl)‐2′‐deoxythymidine
- N⁴ ‐Benzoyl‐5′‐O ‐(4,4′‐dimethoxytrityl)‐2′‐deoxycytidine
- N⁶ ‐Benzoyl‐5′‐O ‐(4,4′‐dimethoxytrityl)‐2′‐deoxyadenosine
- N² ‐Isobutyryl‐5′‐O ‐(4,4′‐dimethoxytrityl)‐2′‐deoxyguanosine
Anhydrous pyridine
Anhydrous methylene chloride (CH₂ Cl₂ ; Aldrich)
N ,N ‐Diisopropylethylamine (DIPEA; Aldrich)
Argon source
Bis(N,N ‐diisopropylamino)chlorophosphine (Aldrich)
Benzene (Aldrich)
Triethylamine (TEA; Aldrich)
Silica gel (60Å, 230 to 400 mesh; Merck)
2.5 × 7.5–cm EMD TLC plates precoated with a 250‐µm layer of Silica Gel 60 F₂₅₄
100‐mL round‐bottom flasks
High vacuum oil pump
Stir bars
Rubber septa
5‐ and 10‐mL glass syringes
Separatory funnels
Rotary evaporator connected to a vacuum pump
2.5 × 20–cm disposable Flex chromatography columns (Kontes)
Fraction collector
Chromaflex TLC developing jars (Kontes)

Basic Protocol 2: Application of the Dinucleotide‐Bound Support for Synthesis of a DNA Oligonucleotide with 5′‐ and 3′‐Functional Groups

Materials

CPG‐linked dinucleotide S.7 (see protocol 1 )
0.5 M hydrazine monohydrate (Aldrich)
Pyridine
Acetic acid
Dry MeCN
Argon
O ‐(2‐Cyanoethyl)‐O ‐(5‐hexyn‐1‐yl)‐N ,N ‐diisopropylphosphoramidite (Glen Research)
0.45 M 1H ‐tetrazole in dry MeCN
Cap A solution (see protocol 1 )
Cap B solution (see protocol 1 )
0.02 M iodine in THF/pyridine/H₂ O
O ‐(2‐Cyanoethyl)‐O ‐[6‐(4‐methoxytrityl)‐amino‐1‐hexyl]‐N ,N‐ diisopropylphosphoramidite (Glen Research)
3% TCA in CH₂ Cl₂
1:1 (v/v) TEA/MeCN
1:1:8 (v/v/v) azidoacetic anhydride/pyridine/THF

Synthesis columns
1‐, 3‐, and 10‐mL syringes
Rubber septa
4‐mL glass vials
Luer‐tipped syringes
16‐G hypodermic needles
DNA/RNA synthesizer

Alternate Protocol 1: Application of the Hydroxylated CPG Support for Synthesis of a DNA Oligonucleotide with a 3′‐Reporter Group

5′‐O ‐(4,4′‐Dimethoxytrityl)‐3′‐O ‐bis(N ,N ‐diisopropylamino)phosphinyl‐2′‐deoxythymidine (S.4 , see protocol 2 )
1‐Pyrenebutanol (Aldrich)

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Figures

Figure 3.17.1 Preparation of CPG support S.3 . Cap A, acetic anhydride/pyridine/THF; Cap B, 10% 1‐methylimidazole in THF; CPG, 500 Å long‐chain alkylamine controlled‐pore glass; DMTr, 4,4′‐dimethoxytrityl; ETT, 5‐ethylthio‐1 H ‐tetrazole. Adapted and reprinted with permission from Grajkowski et al. (). Copyright 2008 American Chemical Society.

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Figure 3.17.2 Preparation of the CPG‐linked dinucleotide S.7 . Note that the capping reaction should be performed before oxidation, but after sulfurization. B^P , thymin‐1‐yl, N ⁴ ‐benzoylcytosin‐1‐yl, N ⁶ ‐benzoyladenin‐9‐yl, or N ² ‐isobutyrylguanin‐9‐yl; Cap A, acetic anhydride/pyridine/THF; Cap B, 10% 1‐methylimidazole in THF; CPG, 500 Å long‐chain alkylamine controlled‐pore glass; DMTr, 4,4′‐dimethoxytrityl; Lev, levulinyl. Adapted and reprinted with permission from Grajkowski et al. (). Copyright 2008 American Chemical Society.

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Figure 3.17.3 Gas‐phase deprotection and thermolytic release of dinucleoside or oligonucleoside phosphate/thiophosphate diesters from the CPG support. B, thymin‐1‐yl, cytosin‐1‐yl, adenin‐9‐yl, or guanin‐9‐yl; Cap A, acetic anhydride/pyridine/THF; Cap B, 10% 1‐methylimidazole in THF; CPG, 500 Å long‐chain alkylamine controlled‐pore glass; PBS, phosphate‐buffered saline; TCA, trichloroacetic acid. Adapted and reprinted with permission from Grajkowski et al. (). Copyright 2008 American Chemical Society.

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Figure 3.17.4 RP‐HPLC analysis of unpurified 5′‐d(CGACTGTGAATCGATGCCAT) and 5′‐d(C_PS G_PS A_PS C_PS T_PS G_PS T_PS G_PS A_PS A_PS T_PS C_PS G_PS A_PS T_PS G_PS C_PS C_PS A_PS T). (A,B ) Native 20‐mer synthesized on commercial CPG (A ) or CPG support S.7 (B ) under standard conditions. (C,D ) Fully phosphorothioated 20‐mer synthesized on commercial CPG (C ) or CPG support S.7 (D ) under standard conditions. Synthesis, nucleobase and phosphate deprotection, thermolytic cleavage from the support, and RP‐HPLC analyses were performed as described in . Peak heights are all normalized to the highest peak, which is set to 1 arbitrary unit. Adapted and reprinted with permission from Grajkowski et al. (). Copyright 2008 American Chemical Society.

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Figure 3.17.5 Polyacrylamide gel electrophoresis of unpurified 5′‐d(CGACTGTG‐AATCGATGCCAT) and 5′‐d(C_PS G_PS A_PS C_PS T_PS G_PS T_PS G_PS A_PS A_PS T_PS C_PS G_PS A_PS T_PS G_PS C_PS C_PS ‐A_PS T) under denaturing conditions. (A ) Native 20‐mer synthesized on unmodified CPG (left lane) and CPG support S.7 (right lane). (B ) Fully phosphorothioated 20‐mer synthesized on unmodified CPG (left lane) and CPG support S.7 (right lane). Synthesis, nucleobase and phosphate deprotection, thermolytic cleavage from the support, and RP‐HPLC analyses were all performed as described in . Adapted and reprinted with permission from Grajkowski et al. (). Copyright 2008 American Chemical Society.

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Figure 3.17.6 Preparation of deoxyribonucleoside phosphorodiamidite S.4 . B^P , thymin‐1‐yl, N ⁴ ‐benzoylcytosin‐1‐yl, N ⁶ ‐benzoyladenin‐9‐yl, or N ² ‐isobutyrylguanin‐9‐yl; DMTr, 4,4′‐dimethoxytrityl.

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Figure 3.17.7 Preparation of a DNA oligonucleotide conjugated with 5′‐ and 3′‐functional groups. Cap A, acetic anhydride/pyridine/THF; Cap B, 10% 1‐methylimidazole in THF; CPG, 500 Å long‐chain alkylamine controlled‐pore glass; DMTr, 4,4′‐dimethoxytrityl; MMTr, 4‐monomethoxytrityl; PBS, phosphate‐buffered saline; TCA, trichloroacetic acid; Bz, benzoyl; iBu, Isobutyryl; p, 2‐cyanoethyl phosphoryl. Adapted and reprinted with permission from Grajkowski et al. (). Copyright 2008 American Chemical Society.

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Figure 3.17.8 Preparation of a DNA oligonucleotide conjugated with a 3′‐reporter group (R). Cap A, acetic anhydride/pyridine/THF; Cap B, 10% 1‐methylimidazole in THF; CPG, 500 Å long‐chain alkylamine controlled‐pore glass; DMTr, 4,4′‐dimethoxytrityl; PBS, phosphate‐buffered saline; R, 1‐pyrenebutyl; TCA, trichloroacetic acid; p, 2‐cyanoethyl phosphoryl. Adapted and reprinted with permission from Grajkowski et al. (). Copyright 2008 American Chemical Society.

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Figure 3.17.9 RP‐HPLC analysis of unpurified 3′‐pyrenylated DNA oligonucleotide S .18 . Nucleobase and phosphate deprotection, thermolytic cleavage, and RP‐HPLC were performed as described in . (A ) Chromatogram of S.18 recorded at 260 nm. (B ) Chromatogram of S.18 recorded at 340 nm. Adapted and reprinted with permission from Grajkowski et al. (). Copyright 2008 American Chemical Society.

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Figure 3.17.10 Thermolytic cyclodeesterification of thymidylyl‐(3′ → 5′)‐thymidine thiophosphate 3‐thiophosphato‐1‐propyl ester or its 4‐thiophosphato‐1‐butyl ester. Adapted and reprinted with permission from Grajkowski et al. (). Copyright 2007 American Chemical Society.

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Figure 3.17.11 RP‐HPLC chromatograms of exemplary dinucleoside phosphate diesters (A ) and dinucleoside phosphorothioate diesters (B ) thermolytically released from modified CPG support S.8 (Fig. ). Adapted and reprinted with permission from Grajkowski et al. (). Copyright 2008 American Chemical Society.

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Literature Cited

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