Carbohydrate‐Oligonucleotide Conjugates

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

This unit describes a strategy for attaching two mannose and two galactose residues to an oligonucleotide. This conjugation can be performed at the 5??end of the oligonucleotide sequence, using modified phosphoramidites. First, the oligonucleotide scaffold is synthesized on solid support using a DNA synthesizer, with commercially available and modified phosphoramidites. After the first ?click? reaction with a galactosylated azide derivative on solid support, the bromine atoms are replaced with azides and a second click reaction is performed with propargylated mannose either on solid support or in solution. Additionally, using a monoalkynated solid support, the conjugation with carbohydrate residues can be performed at the 3??end of the oligonucleotide according to a similar protocol. Curr. Protoc. Nucleic Acid Chem. 39:4.38.1?4.38.25. © 2009 by John Wiley & Sons, Inc.

Keywords: carbohydrate; oligonucleotide; click chemistry; conjugate; galactose; mannose

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Introduction
Reagents and Solutions
Commentary
Literature Cited
Figures
Tables

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Materials

Basic Protocol 1:

Materials

1,1,1‐Tris(hydroxymethyl)ethane
Anhydrous pyridine (dried over CaH₂ and distilled)
Argon
Methanol (MeOH)
4,4′‐Dimethoxytrityl chloride
Sulfuric acid stain solution [10% (v/v) sulfuric acid in ethanol]
Anhydrous methylene chloride (CH₂ Cl₂ ; dried over P₂ O₅ and distilled)
Saturated aqueous sodium bicarbonate solution
Solid anhydrous sodium sulfate
Toluene
Silica gel (0.04 to 0.06 nm)
Triethylamine (Et₃ N)
Acetone
Anhydrous acetonitrile (dried over CaH₂ and distilled)
Anhydrous tetrahydrofuran (THF; dried over alumina)
60% (w/v) sodium hydride in oil
80% (w/v) propargyl bromide in toluene
Cyclohexane
Ethyl acetate (EtOAc)
Sodium iodide
1,6‐Dibromohexane
Diisopropylammonium tetrazolide (DIAT)
2‐cyanoethyl N,N,N′,N′ ‐tetraisopropylphosphordiamidite
Molybde stain solution (see unit 3.16 )

50‐mL and 500‐mL round‐bottom flasks equipped with stir bar and CaCl₂ guard
TLC silica plates (0.20‐mm silica gel 60 with fluorescent indicator UV₂₅₄ ; Macherey‐Nagel)
254‐nm UV lamp
Rotary evaporator
Vacuum pump
250‐mL separatory funnel
Cotton
2.5‐cm and 5‐cm diameter chromatography columns

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

NOTE: For each step, starting materials, solvents, and glassware must be as dry as possible to obtain high yields.

Basic Protocol 2:

Materials

1,4‐cyclohexanedimethanol (mixture of cis and trans )
4‐dimethylaminopyridine (DMAP)
Anhydrous pyridine
Anhydrous methylene chloride
p ‐Toluenesulfonyl chloride
Anhydrous triethylamine (dried over CaH₂ and distilled)
Argon
Sulfuric acid stain solution
Saturated aqueous sodium bicarbonate solution
Methylene chloride (CH₂ Cl₂ )
Solid anhydrous sodium sulfate
Silica gel (0.04 to 0.06 nm)
Methanol
N,N ‐dimethylformamide (DMF)
Sodium iodide
Sodium azide
Vanillin stain solution (see recipe )
Saturated aqueous sodium chloride solution (brine)
Ethyl acetate
Cyclohexane
Peracetylated D‐galactose
Anhydrous acetonitrile
Boron trifluoride etherate

50‐mL round‐bottom flask equipped with stir bar and CaCl₂ guard
TLC silica plates (0.20 mm silica gel 60 with fluorescent indicator UV₂₅₄ , Macherey‐Nagel)
254‐nm UV lamp
250‐mL separatory funnel
Cotton
Rotary evaporator
Vacuum pump
2.5‐cm diameter chromatography column
Vacuum evaporator
100‐mL round‐bottom flask equipped with stir bar and condenser
Oil bath and heater

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

Basic Protocol 3:

Peracetylated D‐mannose
Propargyl alcohol
Petroleum ether (Ep)

Basic Protocol 4:

Materials

Long‐chain alkylamine controlled‐pore glass (LCAA‐CPG, 500 Å, 80 to 120 mesh, amino group, 80 to 90 µmol/g)
3% trichloroacetic acid (TCA) in methylene chloride (commercial deblocking solution)
Anhydrous methylene chloride
Phosphorous oxide (P₂ O₅ )
Anhydrous pyridine
Succinic anhydride
4‐dimethylaminopyridine (DMAP)
1‐O ‐(4,4′‐dimethoxytrityl)‐2‐propargyloxymethyl‐2‐methyl‐1,3‐propanediol ( S.3 )
N ‐(3‐dimethylaminopropyl)‐N ′‐ethylcarbodiimide (DEC)
Anhydrous triethylamine
Pentachlorophenol
Anhydrous piperidine (dried over CaH₂ and distilled)
Acetic anhydride in dry pyridine/THF 10/10/80 v/v (commercial Cap A solution)
10% (w/v) N ‐methylimidazole in dry THF (commercial Cap B solution)
0.1 M p ‐toluenesulfonic acid solution in acetonitrile

Frits (porosity 3)
Vacuum desiccator
Sealed tube
Shaker
10‐mL volumetric flasks
Spectrophotometer

Additional reagents and equipment for automated oligonucleotide synthesis and the trityl assay ( appendix 3C )

Basic Protocol 5:

Materials

Reagents recommended for automated solid‐phase oligonucleotide synthesis:
- Standard 2‐cyanoethyl deoxyribonucleoside phosphoramidites (T, C^Bz , A^Bz , and G^iBu )
- Activator: 5‐Benzylthio‐1H ‐tetrazole (BMT)
- Oxidation solution: 0.1 M iodine in 70:10:20 (v/v/v) THF/pyridine/water
Extra dry acetonitrile
Cap A solution: 10:10:80 (v/v/v) acetic anhydride in dry pyridine/THF
Cap B solution: 10% N ‐methylimidazole in dry THF
Deblocking solution: 3% dichloroacetic acid (DCA) in dry methylene chloride
Commercial thymidine‐derived solid support (Link technologies, CPG 500 Å, L=61 µmol/g)
Phosphoramidites S.5 and S.6 ( protocol 1 )
Galactopyranoside S.10 ( protocol 2 )
Methanol
Copper sulfate (CuSO₄ )
Sodium ascorbate (Na Asc)
Degassed water
Dry argon gas cylinder
Dry nitrogen
Sodium azide (NaN₃ )
Sodium iodide (NaI)
Dimethylformamide (DMF)
Methylene chloride
Mannopyranoside S.11 ( protocol 3 )
Concentrated aqueous ammonia solution (30%)
0.05 M triethylammonium acetate, pH 7

ABI 394 DNA Synthesizer (Applied Biosystems)
Empty synthesis columns, TWIST style (Glen Research)
Initiator microwave synthesizer (Biotage)
0.2‐ to 0.5‐mL microwave vials (equipped with Teflon septa, micro stir bar, and aluminum cap)
0.2‐ to 0.5‐mL vial adapters
Crimper
0.5‐ and 2‐mL microcentrifuge tubes
0.5‐ to 10‐µL, 5‐ to 40‐µL, 40‐ to 200‐µL, and 200‐ to 1000‐µL micropipettors with tips
2‐mL syringes
Ultrasound bath
65°C oven
Sealed vials (with Teflon septa and screw top) for HPLC samples preparations
55°C dry bath
Speed vacuum
Vacuum desiccator
Reverse‐phase C₁₈ (5 µm) column (150 × 4.6–mm; for analyses)
Reverse‐phase C₁₈ (15 µm) column (300 × 7.8–mm; for analyses)
NAP 10 column

Additional reagents and equipment for automated oligonucleotide synthesis ( appendix 3C ), synthesis yield (unit 10.3 ), MALDI‐TOF mass spectrometry (unit 10.1 ), and reversed‐phase HPLC chromatography (unit 10.5 )

Basic Protocol 6:

Solid support S.12 ( protocol 4 )

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Figures

Figure 4.38.1 Synthesis of 1‐ O ‐(4,4′‐dimethoxytrityl)‐2‐propargyloxymethyl‐2‐methyl‐3‐ O ‐[(2‐cyanoethoxy)‐( N,N ‐diisopropylamino)phosphinyl]‐1,3‐propanediol (S.5 ) and 1‐ O ‐(4,4′‐dimethoxytrityl)‐2‐(6‐bromohexyloxymethyl)‐2‐methyl‐3‐ O ‐[(2‐cyanoethoxy)‐( N,N ‐diisopropylamino)phosphinyl]‐1,3‐propanediol (S.6 ).

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Figure 4.38.2 Synthesis of 1‐ O ‐[4‐(azidomethyl)cyclohexyl‐1‐methyl]‐2,3,4,6‐tetra‐ O ‐acetyl‐β‐D ‐galactopyranoside (S.10 ).

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Figure 4.38.3 Synthesis of 1‐ O ‐propargyl‐2,3,4,6‐tetra‐ O ‐acetyl‐α‐D ‐mannopyranoside (S.11 ).

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Figure 4.38.4 Preparation of the monoalkynated solid support S.12 .

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Figure 4.38.5 Solid‐phase synthesis of oligonucleotide S.15 bearing 2 azido functions and two galactose residues. Abbreviations: SPOS, solid‐phase oligonucleotide synthesis; N^P , protected nucleoside; MW, microwave.

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Figure 4.38.6 Synthesis of the heteroglycomimic oligonucleotide 5′‐conjugate S.17 using the solid‐phase strategy.

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Figure 4.38.7 Synthesis of the heteroglycomimic oligonucleotide 5′‐conjugate S.17 using the “in‐solution” strategy.

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Figure 4.38.8 Synthesis of the heteroglycomimic oligonucleotide 3′‐conjugate S.21 . Abbreviations: SPOS, solid‐phase oligonucleotide synthesis; MW, microwave.

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Videos

Literature Cited

Literature Cited
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	D'Onofrio, J., de Champdore, M., De Napoli, L., Montesarchio, D., and Di Fabio, G. 2005. Glycomimetics as decorating motifs for oligonucleotides: Solid‐phase synthesis, stability, and hybridization properties of carbopeptoid‐oligonucleotide conjugates. Bioconjug. Chem. 16:1299‐1309.
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Carbohydrate‐Oligonucleotide Conjugates

Abstract

Table of Contents

Materials

Basic Protocol 1:

Basic Protocol 2:

Basic Protocol 3:

Basic Protocol 4:

Basic Protocol 5:

Basic Protocol 6:

Figures

Videos

Literature Cited