An Aminooxy‐Functionalized Non‐Nucleosidic Phosphoramidite for the Construction of Multiantennary Oligonucleotide Glycoconjugate实验方法详情页

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An Aminooxy‐Functionalized Non‐Nucleosidic Phosphoramidite for the Construction of Multiantennary Oligonucleotide Glycoconjugate

关键词： phosphoramidite multiantennary oligonucleotide glycoconjugates来源：互联网

Abstract
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Abstract

In this unit, a method is described that allows construction of multiantennary oligonucleotide glycoconjugates on a solid support. A bis(hydroxymethyl)malondiamide?based phosphoramidite that contains two phthaloyl?protected aminooxy groups compatible with normal chain assembly is prepared. The aminooxy functions can be deblocked with a hydrazinium acetate treatment and subsequently oximated on?support with fully acetylated 4?oxobutyl ??D ?mannopyranoside. The resulting reagent is then used to prepare a conjugate containing two non?nucleosidic building blocks (i.e., four ??D ?mannopyranosyl units) close to the 5? terminus of the oligonucleotide.

Keywords: Solid support; oligonucleotides; glycoconjugates; oximation; phosphoramidites

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Basic Protocol 1: Preparation of the Aminooxy‐Functionalized Phosphoramidite from Diethyl 2,2‐Bis(Hydroxymethyl)Malonate
Basic Protocol 2: Preparation of the Mannosyl Aldehyde Ligand
Basic Protocol 3: Synthesis of Oligonucleotide Glycoconjugates by On‐Support Oximation
Commentary
Literature Cited
Figures

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Basic Protocol 1: Preparation of the Aminooxy‐Functionalized Phosphoramidite from Diethyl 2,2‐Bis(Hydroxymethyl)Malonate Materials

Diethyl 2,2‐bis(hydroxymethyl)malonate ( S.1 .), 95% pure (Acros)
Dry tetrahydrofuran (THF), freshly distilled from sodium (store over 4‐Å molecular sieves)
Trimethyl orthoformate, 98% pure (Aldrich)
p ‐Toluenesulfonic acid monohydrate, ≥98.5% pure (Aldrich)
5% (w/v) aqueous sodium hydrogen carbonate (NaHCO 3 )
Diethyl ether
Saturated aqueous sodium chloride (NaCl)
Sodium sulfate (Na 2 SO 4 ), anhydrous
Silica gel: 0.040‐ to 0.063‐mm Fluka Kieselgel 60 (dry overnight in 150°C oven)
Dichloromethane (CH 2 Cl 2 ), ≥99% pure
Bromocresol green indicator: dissolve 0.04 g bromocresol green (Merck) in 100 mL ethanol and add 0.1 M aqueous NaOH until the blue color appears (store up to 1 month at room temperature)
3‐Aminopropanol, 99% pure (Aldrich)
Methanol (MeOH), ≥99.8% pure
Dry benzene, distilled from powdered CaH 2 (store over 4‐Å molecular sieves)
Triphenylphosphine, 99% pure (Aldrich)
N ‐Hydroxyphthalimide, 97% pure (Aldrich)
Diethyl azodicarboxylate (DEAD), ≥97% (Fluka)
2‐Propanol (i ‐PrOH), Baker analyzed
80% (v/v) aqueous acetic acid (AcOH)
Dry pyridine, distilled from powdered CaH 2 (store over 4‐Å molecular sieves)
4,4′‐Dimethoxytrityl chloride (DMTr‐Cl), 97% (Aldrich)
Dry toluene, distilled from powdered CaH 2 (store over 4‐Å molecular sieves)
Dry acetonitrile, HPLC grade (store over 4‐Å molecular sieves)
Phosphorus pentoxide (P 2 O 5 ), 97% pure (Aldrich)
Dry nitrogen (or argon)
Anhydrous triethylamine, distilled from powdered CaH 2 (store over 4‐Å molecular sieves)
2‐Cyanoethyl N,N ‐diisopropylphosphonamidic chloride (TRC)
Ethyl acetate (EtOAc), analytical grade
Hexane, HPLC grade

Separatory funnels
Rotary evaporator equipped with a water aspirator
5 × 35–cm sintered glass chromatography column, porosity 2
TLC plate: silica‐coated aluminium plate with fluorescent indicator (Merck silica gel 60 F 254 )
3 × 20–cm sintered glass chromatography column, porosity 3
254‐nm UV lamp
Vacuum desiccator

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

Basic Protocol 2: Preparation of the Mannosyl Aldehyde Ligand Materials

α‐D‐Mannose pentaacetate ( S.7 ; Sigma)
Dry benzene, distilled from powdered CaH 2 (store over 4‐Å molecular sieves)
Dry nitrogen (or argon)
Dry acetonitrile (MeCN), HPLC grade (store over 4‐Å molecular sieves)
1,4‐Butanediol, 99% pure (Aldrich)
Boron trifluoride etherate (Merck)
Dichloromethane (CH 2 Cl 2 ), ≥ 99% pure
Sodium sulfate (Na 2 SO 4 ), anhydrous
Silica gel: 0.040‐ to 0.063‐mm Fluka Kieselgel 60 (dry overnight in oven at 150°C)
Ethyl acetate (EtOAc), analytical reagent
Hexane, HPLC grade
10% (v/v) H 2 SO 4
Oxalyl chloride, 99% pure (Aldrich)
Dry argon
Dry ice/isopropanol bath
Dry dimethyl sulfoxide (DMSO; store over 4‐Å molecular sieves)
Triethylamine (TEA), freshly distilled from powdered CaH 2
Diethyl ether
1 M aqueous HCl, ice cold
Saturated aqueous sodium hydrogen carbonate (NaHCO 3 )
Saturated aqueous sodium chloride (NaCl)
Methanol (MeOH), ≥99.8% pure

Separatory funnels
Rotary evaporator equipped with a water aspirator
3 × 20–cm sintered glass chromatography column, porosity 3
TLC plate: silica‐coated aluminium plate with fluorescent indicator (Merck silica gel 60 F 254 )
254‐nm UV lamp

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

Basic Protocol 3: Synthesis of Oligonucleotide Glycoconjugates by On‐Support Oximation Materials

Phosphoramidite S.6 (see protocol 1 )
Dry acetonitrile (MeCN), HPLC grade (store over 4‐Å molecular sieves)
2′‐Deoxyribonucleoside phosphoramidites (Glen Research)
0.5 M hydrazinium acetate solution: 0.124:4:1 (v/v/v) mixture of hydrazine monohydrate (>98% pure, Aldrich), analytical‐grade pyridine, and glacial acetic acid
Pyridine, analytical grade
Acetonitrile (MeCN), analytical grade
Mannosyl aldehyde ligand S.9 (see protocol 2 )
33% aqueous ammonia
Mobile phase A: 0.05 M ammonium acetate, pH 7.0, in H 2 O
Mobile phase B: 0.05 M ammonium acetate in 65% (v/v) aqueous MeCN

55°C water bath
Speedvac evaporator
HPLC system with 4.6 × 150–mm analytical ThermoHypersil C18 column and 260‐nm detector
Desalting column: 7.5 mm × 30 cm TSKgel G 2000 size‐exclusion chromatography column, particle size 10 µm (Toso‐Haas)
UV spectrophotometer

Additional reagents and equipment for automated solid‐phase oligonucleotide synthesis ( appendix 3C ) and for purification (unit 10.5 ) and characterization (unit 10.2 ) of oligonucleotides

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Figure 4.26.1 Synthesis of the non‐nucleosidic phosphoramidite S.6 (see ). DMTr, 4,4′‐dimethoxytrityl. Reprinted from Katajisto et al. () with permission from the American Chemical Society.

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Figure 4.26.2 Synthesis of the sugar ligand S.9 (see ). Ac, acetyl. Reprinted from Katajisto et al. () with permission from the American Chemical Society.

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Figure 4.26.3 Synthesis of the oligonucleotide glycoconjugate S.12 utilizing on‐support oximation. Ac, acetyl; Pht, phthaloyl.

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Figure 4.26.4 RP‐HPLC profile of crude S.12 at 260 nm. Reprinted from Katajisto et al. () with permission from the American Chemical Society.

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Literature Cited

Literature Cited
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