Synthesis of DNA Oligodeoxynucleotides Containing Site‐Specific 1,3‐Butadiene‐ Deoxyadenosine Lesions

Susith Wickramaratne1, Christopher L. Seiler1, Natalia Y. Tretyakova1

1 Department of Medicinal Chemistry and the Masonic Cancer Center, University of Minnesota, Minneapolis, Minnesota
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.61
DOI:  10.1002/0471142700.nc0461s61
Online Posting Date:  June, 2015
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Abstract

Post‐oligomerization synthesis is a useful technique for preparing site‐specifically modified DNA oligomers. This approach involves site‐specific incorporation of inherently reactive halogenated nucleobases into DNA strands using standard solid‐phase synthesis, followed by post‐oligomerization nucleophilic aromatic substitution (SNAr) reactions with carcinogen‐derived synthons. In these reactions, the inherent reactivities of DNA and carcinogen‐derived species are reversed: the modified DNA nucleobase acts as an electrophile, while the carcinogen‐derived species acts as a nucleophile. In the present protocol, we describe the use of the post‐oligomerization approach to prepare DNA strands containing site‐ and stereospecific N6‐adenine and N1,N6‐adenine adducts induced by epoxide metabolites of the known human and animal carcinogen 1,3‐butadiene (BD). The resulting oligomers containing site‐specific, structurally defined DNA adducts can be used in structural and biological studies to reveal the roles of specific BD adducts in carcinogenesis and mutagenesis. © 2015 by John Wiley & Sons, Inc.

Keywords: post‐oligomerization synthesis; 1,3‐butadiene‐induced DNA adducts; adenine; site‐ and stereospecific oligodeoxynucleotides

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of Stereoisomeric N‐Fmoc‐1‐Amino‐2‐Hydroxy‐3,4‐Epoxybutane
  • Basic Protocol 2: Synthesis, Purification, and Characterization of Oligodeoxynucleotides Containing 6‐Chloropurine
  • Basic Protocol 3: Synthesis of Oligodeoxynucleotides Containing N6‐(2‐Hydroxy‐3‐Buten‐1‐Yl)‐Adenine Adducts on CPG Support
  • Alternate Protocol 1: Synthesis of Oligodeoxynucleotides Containing N6‐(2‐Hydroxy‐3‐Buten‐1‐Yl)‐Adenine Adducts in Solution
  • Basic Protocol 4: Synthesis of Oligodeoxynucleotides Containing 1,N6‐(2‐Hydroxy‐3‐Hydroxymethylpropan‐2,3‐Diyl)‐Adenine Adducts on CPG Support
  • Alternate Protocol 2: Synthesis of Oligodeoxynucleotides Containing 1,N6‐(2‐Hydroxy‐3‐Hydroxymethylpropan‐2,3‐Diyl)‐Adenine Adducts in Solution
  • Basic Protocol 5: Synthesis of Oligodeoxynucleotides Containing N6,N6‐(2,3‐Dihydroxybutan‐1,4‐Diyl)‐Adenine Adducts on CPG Support
  • Alternate Protocol 3: Synthesis of Oligodeoxynucleotides Containing N6,N6‐(2,3‐Dihydroxy‐Butan‐1,4‐Diyl)‐Adenine Adducts in Solution
  • Support Protocol 1: Synthesis of (3R,4R)‐Pyrrolidine‐3,4‐Diol (9)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of Stereoisomeric N‐Fmoc‐1‐Amino‐2‐Hydroxy‐3,4‐Epoxybutane

  Materials
  • Acrolein (1; Fluka; cat. no. 01680)
  • Zinc iodide (ZnI 2)
  • Trimethylsilyl cyanide (TMSCN; Sigma‐Aldrich; cat. no. 212849)
  • Lithium aluminum hydride (LiAlH 4; Sigma‐Aldrich; cat. no. 199877)
  • Anhydrous diethyl ether
  • 4 Å molecular sieves
  • N 2 or Ar
  • Triethanolamine
  • Celite (optional)
  • Anhydrous magnesium sulfate (MgSO 4)
  • Ammonium salt of [(1R)‐(endo,anti)]‐(+)‐3‐bromocamphor‐8‐sulfonic acid (TCI; cat. no. B1125)
  • Methanol (MeOH)
  • Ethyl acetate (EtOAC)
  • Barium hydroxide [Ba(OH) 2·8H 2O]
  • Hydrochloric acid (HCl)
  • Sodium carbonate (Na 2CO 3)
  • 9‐fluorenylmethoxycarbonyl chloride (FmocCl; Sigma‐Aldrich; cat. no. 160512)
  • 1,4‐dioxane (Sigma‐Aldrich; cat. no. 296309)
  • Hexanes
  • Silica gel (230‐400 Mesh, Grade 60; Fisher Scientific; cat. no. S825‐1)
  • Anhydrous dichloromethane (CH 2Cl 2)
  • meta‐chloroperbenzoic acid (mCPBA; Sigma‐Aldrich; cat. no. 273031)
  • Chloroform (CHCl 3; Fisher Scientific, cat. no. 607)
  • 100‐, 250‐, and 500‐mL round‐bottom flasks
  • 100‐ and 250‐mL dropping funnels
  • Magnetic stir bars
  • Magnetic stirrer/hotplate
  • Reflux condensers
  • Silicone/paraffin oil bath
  • Reduced‐pressure distillation setup
  • 500‐mL three‐necked round‐bottom flask
  • Calcium chloride drying tube
  • Rubber septa
  • Heating mantle
  • Syringe needles
  • Buchner funnel setup with vacuum source
  • Rotary evaporator equipped with a dry ice condenser and connected to an oil pump
  • Whatman Partisil 10 (9.5 × 500 mm) column
  • Additional reagents and equipment for column chromatography ( appendix 3E) and HPLC (unit 10.5)

Basic Protocol 2: Synthesis, Purification, and Characterization of Oligodeoxynucleotides Containing 6‐Chloropurine

  Materials
  • 5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropylphosphoramidite of 6‐chloropurine‐2′‐deoxyriboside (6‐Cl‐Pu phosphoramidite, ChemGenes Corp.)
  • 5′‐O‐(4,4′‐dimethoxytrityl)‐3′‐O‐(2‐cyanoethyl)‐N,N‐diisopropyl phosphoramidites (Glen Research):
    • Phenoxyacetyl‐2′‐deoxyadenosine (PAC‐dA‐CE) phosphoramidite
    • N‐Acetyl‐2′‐deoxycytidine (Ac‐dC‐CE) phosphoramidite
    • Np‐isopropyl‐phenoxyacetyl‐2′‐deoxyguanosine (piPr‐PAC‐dG‐CE) phosphoramidite
    • Thymidine (dT‐CE) phosphoramidite
  • Argon source
  • DNA controlled‐pore glass (CPG) supports (Glen Research):
    • PAC‐dA‐CPG ABI
    • Ac‐dC‐CPG ABI
    • piPr‐PAC‐dG‐CPG ABI
    • dT‐CPG ABI
  • Reagents for DNA synthesis (Glen Research; also see unit 3.1 and other units of Chapter 3):
    • Phenoxyacetic anhydride in THF/pyridine
    • 16% 1‐methylimidazole in THF
    • 0.02 M iodine in THF/pyridine/water
    • 1H‐tetrazole in anhydrous ACN
    • 3% trichloroacetic acid in CH 2Cl 2
    • Anhydrous CH 2Cl 2
    • Concentrated ammonia
  • Anhydrous acetonitrile (ACN; Fisher Scientific cat. no. BP1170)
  • 1H‐Tetrazole in anyhydrous ACN (Glen Research, prod. no. 30–3100–45)
  • Sodium hydroxide (NaOH)
  • Acetic acid (AcOH)
  • 100 mM triethylammonium acetate (see recipe)
  • Ammonium acetate
  • 10 mM Tris.Cl, pH 7, containing 5 mM MgCl 2
  • Deoxyribonuclease I (DNase I; Worthington Biochemical; cat. no. LS006331)
  • Phosphodiesterase I (PDE I; Worthington Biochemical; cat. no. LS003926)
  • Phosphodiesterase II (PDE II; Worthington Biochemical; cat. no. LS003603)
  • Alkaline phosphatase (New England BioLabs Inc.; cat. no. M0290S)
  • Glove box or glove bag
  • DNA synthesizer (e.g., Applied Biosystems Model 394)
  • 1‐mL syringes with needles
  • 3‐mL graduated V‐vials (Wheaton)
  • Spin‐X centrifuge tube filters (Corning Life Sciences)
  • Jupiter Proteo 90 Å C12 HPLC column (250 mm × 10.0 mm, Phenomenex)
  • SpeedVac concentrator
  • Illustra NAP‐25 columns (GE Healthcare)
  • Zorbax 300SB‐C18 column (150 mm × 0.5 mm, 5 μm, Agilent Technologies, Inc.)
  • Heating block
  • Additional reagents and equipment for solid‐phase synthesis of oligodeoxynucleotides (see unit 3.1 and other units of Chapter 3), HPLC (unit 10.5), electrospray ionization mass spectrometry of oligonucleotides (unit 10.2), and quantitation of DNA by UV absorbance (unit 10.3)

Basic Protocol 3: Synthesis of Oligodeoxynucleotides Containing N6‐(2‐Hydroxy‐3‐Buten‐1‐Yl)‐Adenine Adducts on CPG Support

  Materials
  • (2S)‐N‐Fmoc‐1‐amino‐3‐buten‐2‐ol (6; protocol 1)
  • Anhydrous dimethylsulfoxide (DMSO)
  • Diisopropylethylamine (DIPEA)
  • ABI column containing the site‐specific 6‐Cl‐Pu oligodeoxynucleotide on CPG support (from step 9 of protocol 2)
  • Anhydrous acetonitrile (ACN)
  • Sodium hydroxide (NaOH)
  • Acetic acid (AcOH)
  • 100 mM triethylammonium acetate (see recipe)
  • Concentrated ammonia
  • Triethylamine
  • Heat block
  • Table‐top centrifuge (e.g. Midwest Scientific, C1301R)
  • 3‐mL graduated V‐vials (Wheaton)
  • Spin‐X centrifuge tube filters (Corning Life Sciences)
  • Jupiter Proteo 90 Å C12 column (250 mm × 10.0 mm, Phenomenex)
  • SpeedVac concentrator
  • Illustra NAP‐25 columns (GE Healthcare)
  • Additional reagents and equipment for HPLC (unit 10.5)

Alternate Protocol 1: Synthesis of Oligodeoxynucleotides Containing N6‐(2‐Hydroxy‐3‐Buten‐1‐Yl)‐Adenine Adducts in Solution

  Additional Materials (also see protocol 3)
  • Synthetic oligodeoxynucleotides containing site‐specific 6‐Cl‐Pu (from step 13 of protocol 2)

Basic Protocol 4: Synthesis of Oligodeoxynucleotides Containing 1,N6‐(2‐Hydroxy‐3‐Hydroxymethylpropan‐2,3‐Diyl)‐Adenine Adducts on CPG Support

  Materials
  • (2R,3S) or (2R,3R)‐N‐Fmoc‐1‐amino‐2‐hydroxy‐3,4‐epoxybutane (7a or 7b; protocol 1)
  • Anhydrous DMSO
  • Diisopropylethylamine (DIPEA)
  • ABI column containing the site‐specific 6‐Cl‐Pu oligodeoxynucleotide on CPG support (from step 9 of protocol 2)
  • Anhydrous acetonitrile (ACN)
  • 100 mM triethylammonium acetate (see recipe)
  • Jupiter Proteo 90 Å C12 column (250 mm × 10.0 mm, Phenomenex)
  • Additional reagents and equipment for purification of oligodeoxynucleotide ( protocol 3, steps 5 to 7) and characterization and quantification of oligodeoxynucleotides ( protocol 2, steps 14 to 17)

Alternate Protocol 2: Synthesis of Oligodeoxynucleotides Containing 1,N6‐(2‐Hydroxy‐3‐Hydroxymethylpropan‐2,3‐Diyl)‐Adenine Adducts in Solution

  Additional Materials (also see protocol 5)
  • Synthetic oligodeoxynucleotides containing site‐specific 6‐Cl‐Pu (from step 13 of protocol 2)

Basic Protocol 5: Synthesis of Oligodeoxynucleotides Containing N6,N6‐(2,3‐Dihydroxybutan‐1,4‐Diyl)‐Adenine Adducts on CPG Support

  Materials
  • (3R,4R)‐Pyrrolidine‐3,4‐diol (9, see protocol 9Support Protocol)
  • Anhydrous DMSO
  • Diisopropylethylamine (DIPEA)
  • ABI column containing the site‐specific 6‐Cl‐Pu oligodeoxynucleotide on CPG support (from step 9 of protocol 2)
  • Anhydrous acetonitrile (ACN)
  • Jupiter Proteo 90 Å C12 column (250 mm × 10.0 mm, Phenomenex, Torrance, CA)
  • Illustra NAP‐25 columns (GE Healthcare)
  • Additional reagents and equipment for cleaving oligonucleotide for CPG support ( protocol 3, step 9) and characterizing and quantifying oligonucleotide ( protocol 2, steps 14 to 17), HPLC (unit 10.5), and electrospray ionization mass spectrometry (unit 10.2)

Alternate Protocol 3: Synthesis of Oligodeoxynucleotides Containing N6,N6‐(2,3‐Dihydroxy‐Butan‐1,4‐Diyl)‐Adenine Adducts in Solution

  Additional Materials (also see protocol 7)
  • Synthetic oligodeoxynucleotides containing site‐specific 6‐Cl‐Pu (from step 13 of protocol 2)

Support Protocol 1: Synthesis of (3R,4R)‐Pyrrolidine‐3,4‐Diol (9)

  Materials
  • (3R,4R)‐(−)‐1‐Benzyl‐3,4‐pyrrolidinediol (8; Sigma Aldrich)
  • 10% Pd/C (Sigma‐Aldrich; cat. no. 205699)
  • Anhydrous methanol (MeOH)
  • H 2 gas
  • Celite (Sigma‐Aldrich; cat. no. 419931)
  • Magnetic stir plate
  • Small magnetic stir bar
  • 50‐mL round‐bottom flask
  • Heating mantle
  • Rubber septum
  • Balloon
  • Syringe needles
  • Fritted‐glass Buchner funnel setup
  • Rotary evaporator equipped with a dry ice condenser and connected to an oil pump
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Figures

Videos

Literature Cited

Literature Cited
  Carmical, J.R., Nechev, L.V., Harris, C.M., Harris, T.M., and Lloyd, R.S. 2000. Mutagenic potential of adenine N6 adducts of monoepoxide and diolepoxide derivatives of butadiene. Environ. Mol. Mutagen. 35:48‐56.
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  Stone, M.P., Cho, Y.J., Huang, H.A.I., Kim, H.Y., Kozekov, I.D., Kozekova, A., Wang, H.A.O., Minko, I.G., Lloyd, R.S., Harris, T.M., and Rizzo, C.J. 2008. Interstrand DNA crosslinks induced by α,β,‐unsaturated aldehydes derived from lipid peroxidation and environmental sources. Acc. Chem. Res. 41:793‐804.
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