Synthesis of Oligodeoxynucleotides Containing a C8‐2′‐Deoxyguanosine Adduct Formed by the Carcinogen 3‐Nitrobenzanthrone

Arindom Chatterjee1, Chanchal K. Malik2, Ashis K. Basu1

1 Department of Chemistry, University of Connecticut, Storrs, Connecticut, 2 Department of Chemistry, College of Arts and Science, Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.73
DOI:  10.1002/cpnc.28
Online Posting Date:  June, 2017
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Abstract

This unit describes the detailed procedure in five parts for the synthesis of the C8‐2′‐deoxyguanosine‐3‐aminobenzanthrone adduct located in a desired site in an oligonucleotide. The synthesis of the protected 2′‐deoxyguanosine, O6‐benzyl‐N2‐DMTr‐3′‐5′‐bisTBDMS‐C8‐Br‐2′‐deoxyguanosine, is described in the first part. The synthesis of the reduced carcinogen 3‐aminobenzanthrone is detailed in part two. The third part outlines the key step of the adduct formation between the reduced carcinogen and the protected nucleoside by a palladium‐catalyzed cross coupling reaction. The final two parts describe phosphoramidite synthesis from the nucleoside‐carcinogen adduct followed by its site‐specific incorporation into DNA by solid‐phase oligonucleotide synthesis. The adducted oligonucleotides are purified by reversed‐phase HPLC and characterized by mass spectrometry. © 2017 by John Wiley & Sons, Inc.

Keywords: cross coupling; C8 guanine adduct; DNA adduct; DNA lesion bypass; environmental carcinogen; palladium‐catalyzed

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Protected 8‐Bromo‐2′‐Deoxyguanosine
  • Basic Protocol 2: Synthesis of the Reduced Carcinogen 3‐Aminobenzanthrone: A Precursor of the Buchwald‐Hartwig Cross‐Coupling
  • Basic Protocol 3: Buchwald‐Hartwig Cross‐Coupling Reaction
  • Basic Protocol 4: Deprotection of N2‐DMT Followed by N2‐Amino Protection, Dimethoxytritylation of 5′‐Hydroxyl, and Conversion to Phosphoramidite
  • Basic Protocol 5: Solid‐Phase Automated Synthesis of Oligonucleotide Containing Modified 2′‐Deoxyguanosine and Its Purification
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Protected 8‐Bromo‐2′‐Deoxyguanosine

  Materials
  • 2′‐Deoxyguanosine monohydrate (1) (Berry & Associates)
  • Acetonitrile (Sigma Aldrich Chemical)
  • N‐Bromosuccinimide (freshly recrystallized from hot water)
  • Acetone (Sigma Aldrich Chemical)
  • Argon
  • Anhydrous N, N′‐dimethylformamide (DMF, Agros Organics)
  • Imidazole (Acros Organics)
  • tert‐Butylchlorodimethylsilane (Agros Organics)
  • Methanol (MeOH)
  • Dichloromethane (DCM)
  • Silica gel (63 to 200 µm, 65 to 250 mesh; Sorbent Technologies)
  • Triphenylphosphine (PPh 3, 99%; Acros Organics)
  • Anhydrous 1, 4‐dioxane (Acros Organics)
  • Benzyl alcohol (PhCH 2OH, Sigma Aldrich Chemical)
  • Diisopropyl azodicarboxylate (DIAD, 94%; Acros Organics)
  • Ethyl acetate (EtOAc)
  • n‐Hexane
  • Anhydrous pyridine (Acros Organics)
  • 4, 4′‐Dimethoxytrityl chloride (DMTr‐Cl, 98%, Chem‐Impex International)
  • Aluminium oxide, neutral (Brockmann I, 50 to 200 µm; Acros Organics)
  • 50‐mL round‐bottom flasks
  • Magnetic stir plate and stir bars
  • Vacuum apparatus for filtration
  • Chromatography columns
  • Thin layer chromatography (TLC) equipment

Basic Protocol 2: Synthesis of the Reduced Carcinogen 3‐Aminobenzanthrone: A Precursor of the Buchwald‐Hartwig Cross‐Coupling

  Materials
  • 7H‐Benz[d,e]anthracene‐7‐one (Benzanthrone, 6, 99%, Alfa Aesar Chemical)
  • Nitrobenzene (Alfa Aesar Chemical)
  • Concentrated nitric acid (HNO 3)
  • n‐Hexane
  • Ethanol
  • Argon source
  • Hydrazine monohydrate (Alfa Aesar Chemical)
  • Palladium, 10% on activated carbon (Pd‐C; Alfa Aesar Chemical)
  • 25‐mL round‐bottom flasks
  • 80ºC oil bath
  • Filtration apparatus
  • Dimroth condenser

Basic Protocol 3: Buchwald‐Hartwig Cross‐Coupling Reaction

  Materials
  • O6‐benzyl‐8‐bromo‐3′, 5′‐O‐bis(tert‐butyldimethylsilyl)‐N2‐dimethoxytrityl‐2′‐deoxyguanosine 5 (see protocol 1)
  • 3‐Aminobenzanthrone 8 (see protocol 2)
  • Argon balloon
  • Toluene (anhydrous, kept dry with molecular sieves)
  • Tris (dibenzylideneacetone)‐dipalladium (0) [(Pd 2(dba) 3] (Aldrich Chemical)
  • Rac‐2, 2′‐bis(diphenylphosphino)‐1,1′‐binaphthyl (rac‐BINAP, 98%, Acros Organics)
  • Sodium tert‐butoxide (98%, Acros Organics)
  • Ethyl acetate (EtOAc)
  • n‐Hexane
  • Aluminium oxide, neutral (Brockmann I, 50 to 200 µm; Acros Organics)
  • 50‐mL round‐bottom flasks
  • Magnetic stir bar and plate
  • Celite bed
  • Filter apparatus attached to vacuum
  • Column chromatography

Basic Protocol 4: Deprotection of N2‐DMT Followed by N2‐Amino Protection, Dimethoxytritylation of 5′‐Hydroxyl, and Conversion to Phosphoramidite

  Materials
  • Buchwald‐Hartwig coupling adduct 9 (see protocol 3)
  • Anhydrous dichloromethane (CH 2Cl 2, Acros Organics)
  • Zinc Bromide (ZnBr 2; Acros Organics)
  • Isopropanol (iPrOH; Aldrich Chemical)
  • Sodium bicarbonate (NaHCO 3, saturated aqueous solution)
  • Silica gel
  • Ethyl acetate (EtOAc)
  • n‐Hexane
  • Anhydrous tetrahydrofuran (THF, Acros Organics)
  • Argon source
  • Triethylamine trihydrofluoride (Et 3N.3HF, 98%, Aldrich Chemical)
  • Triethylamine (Et 3N, Aldrich Chemical)
  • Anhydrous methanol (MeOH, Acros Organics)
  • Palladium, black (Pd‐black, 99.9% metal basis, Alfa‐Aesar Chemical)
  • H 2 gas cylinder or balloon
  • Anhydrous N, N′‐dimethylformamide dimethylacetal (Acros Organics)
  • Anhydrous pyridine (Acros Organics)
  • 4‐N, N′‐Dimethylaminopyridine (DMAP, Chem‐Impex International)
  • 4, 4′‐Dimethoxytrityl chloride (DMTr‐Cl, 98%, Chem‐Impex International)
  • N, N′‐Diisopropylethyl amine (DIPEA, Aldrich chemical)
  • 2‐Cyanoethyl diisopropylchlorophosphoramidite [iPrNP(Cl)O(CH 2) 2CN, Aldrich Chemical]
  • Magnetic stir plate and stir bars
  • Filter vacuum apparatus
  • Column chromatography apparatus
  • 25‐ and 50‐mL round‐bottom flasks
  • Rotary evaporator
  • Reversed‐phase C18 column

Basic Protocol 5: Solid‐Phase Automated Synthesis of Oligonucleotide Containing Modified 2′‐Deoxyguanosine and Its Purification

  Materials
  • Crude adducted oligonucleotides
  • Ammonium acetate (NH 4OAc)
  • Acetonitrile (ACN; HPLC‐grade, Aldrich Chemical)
  • Nanopuro HPLC‐grade water (HPLC‐H 2O)
  • Reversed‐phase HPLC
  • HPLC column (Phenomenex Gemini –NX 5µ C18 (250 × 10–mm) semi‐prep column)
  • 1.5‐mL microcentrifuge tubes
  • Membrane filters (Amicon ultracel 3 K centrifugal filters, 0.5 mL, Merck Millipore)
  • SpeedVac concentrator
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Figures

Videos

Literature Cited

Literature Cited
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