Synthesis, Characterization, and Application of Substituted Pyrazolopyrimidine Nucleosides

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Abstract
Table of Contents
Materials
Figures
Literature Cited

Abstract

This unit describes, in detail, the preparation of 3?aminopropyl?substituted pyrazolo[3,4?d]pyrimidine analogs of the purines deoxyadenosine (dA) and deoxyguanosine (dG). Phosphoramidite reagents of these so?called aminopropyl?PPA and ?PPG nucleosides (AP?PPA and AP?PPG, respectively) allow introduction of amino linkers into internal positions of synthetic DNA strands. Synthesis of suitably protected AP?PPA and AP?PPG phosphoramidites are described. The stepwise alkynylation, hydrogenation, selective protection, and phosphoramidite synthesis is similar for both the PPA and PPG analogs. To demonstrate the application of these reagents, a protocol is given in which a simple DNA strand is synthesized and conjugated to a lipophilic activated ester (dabcyl?SE) to form a stable amide linkage. Utility of this chemistry for preparing internally modified DNA conjugates is discussed.

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Basic Protocol 1: Preparation of Protected Aminopropyl‐PPG Phosphoramidite
Basic Protocol 2: Preparation of Protected Aminopropyl‐PPA Phosphoramidite
Basic Protocol 3: Synthesis of DNA Conjugates Using AP‐PPA and AP‐PPG Phosphoramidites
Reagents and Solutions
Commentary
Literature Cited
Figures

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Materials

Basic Protocol 1: Preparation of Protected Aminopropyl‐PPG Phosphoramidite

Materials

6‐Amino‐1‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)‐3‐iodopyrazolo[3,4‐d ]pyrimidin‐4(5H )‐one (Seela and Zulauf, )
CuI
Tetrakis[triphenylphosphine]palladium[0] (Aldrich)
Argon
Anhydrous dimethylformamide (DMF), deoxygenated (i.e., argon purged)
Anhydrous triethylamine
N ‐Trifluoroacetylpropargylamine (Cruickshank and Stockwell, )
Chloroform
230‐ to 400‐mesh (40‐ to 63‐µm) silica gel (EM Sciences)
Methanol
Ethyl acetate
Diethyl ether
recipe20% (w/v) palladium hydroxide on carbon, preactivated (see reciperecipe )
4 M triethylammonium formate buffer, pH 6.5 (see recipe )
Hydrogen gas
Celite (1.5‐cm pad)
N ,N ‐Dimethylformamide dimethylacetal (Aldrich)
Xylenes
Anhydrous pyridine
4,4′‐Dimethoxytrityl chloride
5% (w/v) sodium bicarbonate
Sodium sulfate
Anhydrous methylene chloride
N ,N ‐Diisopropylethylamine
2‐Cyanoethyl‐N ,N ‐diisopropylchlorophosphoramidite
Hexane
100‐mL round‐bottom flask
Rotary evaporator equipped with a vacuum pump
5 × 35–cm chromatography columns
Silica gel 60 F254 aluminum‐backed TLC plates (EM Science)
254‐nm UV lamp
Oil pump (>1 mmHg)
500‐mL Parr hydrogenation bottle
250‐mL separatory funnel
Additional reagents and equipment for column chromatography ( appendix 3E ) and thin‐layer chromatography (TLC; appendix 3D )

NOTE : ¹ H NMR spectra were obtained at 300 MHz on a Varian VXR‐300 spectrometer. Two‐dimensional COSY and NOE experiments assisted in the assignment of proton resonances. Elemental analyses were performed by Quantitative Technologies.NOTE : Like most phosphoramidite reagents, AP‐PPA and ‐PPG are sensitive to hydrolysis and oxidation and must be handled with care after isolation. Desiccated storage at −20°C is recommended. Performance in DNA synthesis depends on good quality reagents; only reagent‐grade chemicals should be used to synthesize these phosphoramidites. Characterization of the compounds as described here helps ensure purity.

Basic Protocol 2: Preparation of Protected Aminopropyl‐PPA Phosphoramidite

Materials

4‐Amino‐1‐(2‐deoxy‐β‐D‐erythro‐pentofuranosyl)‐3‐iodo‐1H ‐pyrazolo[3,4‐d ]pyrimidine (Seela and Zulauf, )
Celite (1.5–cm pad)
N ,N ‐Dimethylacetamide dimethylacetal (TCI America)
Anhydrous triethylamine
Anhydrous dimethylformamide (DMF)
Xylenes
4,4′‐Dimethoxytrityl chloride
Anhydrous pyridine
230‐ to 400‐mesh (40‐ to 63‐µm) silica gel (EM Sciences)
Ethyl acetate
Methylene chloride
Methanol
5 × 35–cm chromatography column
Additional reagents and equipment for synthesizing AP‐PPG (see protocol 1 )

Basic Protocol 3: Synthesis of DNA Conjugates Using AP‐PPA and AP‐PPG Phosphoramidites

Materials

AP‐PPA‐modified 9‐mer oligonucleotide from a 1‐µmol‐scale synthesis, trityl‐on (e.g., Epoch Biosciences)
3 M sodium acetate
Butanol
Ethanol
Solvent A: 0.1 M triethylammonium acetate, pH 7.5
Solvent B: acetonitrile
Anhydrous dimethylsulfoxide (DMSO; e.g., Aldrich Sure‐seal; store over calcium hydride)
Anhydrous triethylamine (Fluka; store over calcium hydride)
N ‐Hydroxysuccinimidyl ester of dabcyl (dabcyl‐SE; Molecular Probes)
2% (w/v) sodium perchlorate in acetone
Acetone
Centrifugal evaporator (i.e., Savant Speedvac) with high vacuum source (<1 mmHg)
High‐performance liquid chromatograph (HPLC) with:
4.6 × 250–mm C18 analytical column and appropriate guard column (Varian Dynamax)
UV‐Vis or photodiode array detector
14‐mL polypropylene tubes
UV‐Vis spectrophotometer
Additional reagents and equipment for RP‐HPLC and detritylation (unit 10.5 )

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Figures

Figure 1.8.1 Preparation of AP‐PPG phosphoramidite (S.1 ). Abbreviations: DMF, N , N ‐dimethylformamide; DMTr⋅Cl, N , N ′‐dimethoxytrityl chloride.

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Figure 1.8.2 Preparation of AP‐PPA phosphoramidite (S.2 ). Abbreviations: DMF, N , N ‐dimethylformamide; DMTr⋅Cl, N , N ′‐dimethoxytrityl chloride.

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Figure 1.8.3 Synthesis of dabcyl conjugate at internal AP‐PPA linker position in a 9‐mer oligonucleotide.

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Figure 1.8.4 Reversed‐phase HPLC chromatograms and UV‐Vis spectra of AP‐PPA‐modified oligonucleotides (S.9 ; dashed lines) and dabcyl conjugate (S.10 ; solid lines). The large difference in retention time simplifies isolation and analysis of the lipophilic conjugate.

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Videos

Literature Cited

Literature Cited
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	Cruickshank, K.A. and Stockwell, D.L. 1988. Oligonucleotide labeling: A concise synthesis of a modified thymidine phosphoramidite. Tetrahedron Lett. 29:5221‐5224.
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	Seela, F. and Zulauf, M. 1999. Synthesis of oligonucleotides containing pyrazolo[3,4‐d]pyrimidines: The influence of 7‐substituted 8‐aza‐7‐deazaadenines on the duplex structure and stability. J. Chem. Soc. Perkin Trans. 1(4):479‐488.
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