Synthesis of G‐N2‐(CH2)3‐N2‐G Trimethylene DNA Interstrand Cross‐Links

Francesca Gruppi1, Tracy L. Johnson Salyard1, Carmelo J. Rizzo1

1 Departments of Chemistry and Biochemistry, Center for Molecular Toxicology, Vanderbilt‐Ingram Cancer Center, Vanderbilt University, Nashville, Tennessee
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 5.14
DOI:  10.1002/0471142700.nc0514s56
Online Posting Date:  March, 2014
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Abstract

The synthesis of G‐N2‐(CH2)3N2‐G trimethylene DNA interstrand cross‐links (ICLs) in a 5′‐CG‐3′ and 5′‐GC‐3′ sequence from oligodeoxynucleotides containing N2‐(3‐aminopropyl)‐2′‐deoxyguanosine and 2‐fluoro‐O6‐(trimethylsilylethyl)inosine is presented. Automated solid‐phase DNA synthesis was used for unmodified bases and modified nucleotides were incorporated via their corresponding phosphoramidite reagent by a manual coupling protocol. The preparation of the phosphoramidite reagents for incorporation of N2‐(3‐aminopropyl)‐2′‐deoxyguanosine is reported. The high‐purity trimethylene DNA interstrand cross‐link product is obtained through a nucleophilic aromatic substitution reaction between the N2‐(3‐aminopropyl)‐2′‐deoxyguanosine‐ and 2‐fluoro‐O6‐(trimethylsilylethyl)inosine‐containing oligodeoxynucleotides. Curr. Protoc. Nucleic Acid Chem. 56:5.14.1‐5.14.15. © 2014 by John Wiley & Sons, Inc.

Keywords: interstrand cross‐links; DNA; N2‐(3‐aminopropyl)‐2′‐deoxyguanosine

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Synthesis of N2‐{3‐[(Trifluoroacetyl)Amino]Propyl}‐5′‐O‐Dimethoxytrityl‐O6‐(Trimethylsilylethyl)‐3′‐O‐[(2‐Cyanoethyl –N,N′‐Diisopropyl)Phosphoramidite]‐2′‐Deoxyguanosine from N2‐(3‐Aminopropyl)‐O6‐(Trimethylsilylethyl)‐2′‐Deoxyguanosine (4)
  • Basic Protocol 2: Synthesis of Modified Oligodeoxynucleotides Containing 2‐Fluoro‐O6‐(Trimethylsilylethyl)‐2′‐Deoxyinosine and N2‐(3‐Aminopropyl)‐O6‐(Trimethylsilylethyl)‐2′‐Deoxyguanosine
  • Alternate Protocol 1: Synthesis of the Oligodeoxynucleotide Containing the 2‐Fluoro‐O6‐(Trimethylsilylethyl)‐2′‐Deoxyinosine Modified Base
  • Basic Protocol 3: Synthesis of Oligodeoxynucleotides with Aminopropyl Cross‐Link
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Synthesis of N2‐{3‐[(Trifluoroacetyl)Amino]Propyl}‐5′‐O‐Dimethoxytrityl‐O6‐(Trimethylsilylethyl)‐3′‐O‐[(2‐Cyanoethyl –N,N′‐Diisopropyl)Phosphoramidite]‐2′‐Deoxyguanosine from N2‐(3‐Aminopropyl)‐O6‐(Trimethylsilylethyl)‐2′‐Deoxyguanosine (4)

  Materials
  • N2‐(3‐aminopropyl)‐O6‐(trimethylsilylethyl)‐2′‐deoxyguanosine (Harris and Harris, )
  • Methanol, anhydrous
  • Ethyl trifluoroacetate
  • Dichloromethane, anhydrous
  • Triethylamine
  • 63‐ to 200‐mesh silica gel
  • Pyridine, anhydrous
  • Argon
  • Dimethoxytrityl chloride
  • Tetrazole (0.45 M in acetonitrile)
  • 2‐cyanoethyl‐N,N,N,N′‐tetraisopropylphosphane
  • Abderhalden apparatus
  • 100‐mL round‐bottom flasks with 24/40 joint and septum
  • 10‐mL, 50‐, and 100‐µL glass syringes
  • UV light source for visualizing TLC plates
  • Rotary evaporator
  • Heavy‐walled glass columns for flash chromatography: 3.6 × 40–cm and 1.5 × 20–cm
  • Vacuum system (oil pump) capable of <1 mm Hg pressure, with manifold and cold trap
  • Additional reagents and equipment for thin‐layer chromatography (TLC; appendix 3D) and column chromatography ( appendix 3E)

Basic Protocol 2: Synthesis of Modified Oligodeoxynucleotides Containing 2‐Fluoro‐O6‐(Trimethylsilylethyl)‐2′‐Deoxyinosine and N2‐(3‐Aminopropyl)‐O6‐(Trimethylsilylethyl)‐2′‐Deoxyguanosine

  Materials
  • 5′‐O‐(DMT)‐N2‐[3‐[(trifluoroacetyl)amino]propyl]‐O6‐(trimethylsilylethyl)‐3′‐O‐[(2‐cyanoethyl‐N,N′‐diisopropyl)phosphoramidite]2′‐deoxyguanosine (4)
  • Acetonitrile (Glen Research)
  • 5′‐O‐(DMT)‐2‐fluoro‐O6‐(trimethylsilylethyl)‐3′‐O‐[(2‐cyanoethyl‐N,N′‐diisopropyl)phosphoramidite]2′‐deoxyinosine (Harris and Harris, )
  • UltraMild phosphoramidites (Glen Research)
  • 1‐methylimidazole, 10% solution in tetrahydrofuran/pyridine (Glen Research, CapB solution)
  • Tetrazole, 0.45 N in acetonitrile (Glen Research)
  • Phenoxyacetyl chloride, 5% solution in tetrahydrofuran (Glen Research, CapA solution)
  • NH 4OH
  • 0.1 M Ammonium formate buffer (see recipe)
  • Deionized water
  • 2% Trifluoroacetic acid
  • 3‐mL Snap‐cap vials
  • Perseptive Biosystems Model 8909 DNA synthesizer
  • Synthesis column
  • 1‐mL syringes
  • Centrifugal vacuum evaporator
  • Phenomenex Luna‐C18 column (250 × 10 mm)
  • Lyophilizer

Alternate Protocol 1: Synthesis of the Oligodeoxynucleotide Containing the 2‐Fluoro‐O6‐(Trimethylsilylethyl)‐2′‐Deoxyinosine Modified Base

  Additional Materials protocol 2)
  • 5′‐O‐(DMT)‐2‐fluoro‐O6‐(trimethylsilylethyl)‐3′‐O‐[(2‐cyanoethyl‐N,N′‐diisopropyl)phosphoramidite]‐2′‐deoxyinosine reagent (Harris and Harris, )
  • 0.1 M NaOH solution

Basic Protocol 3: Synthesis of Oligodeoxynucleotides with Aminopropyl Cross‐Link

  Materials
  • 5′‐TCAGCATGCCXCGGGT‐3′ [X = N2‐(3‐aminopropyl)‐2′‐deoxyguanosine]
  • Borate buffer
  • 3′‐CGTACGGCXCCCAGA‐5′ [X = 2‐fluoro‐O6‐(trimethylsilylethyl)inosine]
  • 5% Acetic acid
  • 0.1 M Ammonium formate buffer (see recipe) Acetonitrile
  • Deionized water
  • 0.05 M TEAA buffer, pH 7.0
  • 3‐hydroxypicolinic acid matrix containing ammonium hydrogen citrate
  • Tris·Cl buffer (50 mm, pH 7, with 5 mm MgCl 2)
  • DNase I
  • Phosphodiesterase I
  • Nuclease P1,
  • Alkaline phosphatase
  • Temperature‐controlled water bath
  • HPLC column: YMC ODS‐AQ 250 mm × 1.0 mm; 1.5 mL/min flow
  • Lyophilizer
  • Desalting column: BioSpin 6‐Tris column (Bio‐Rad)
  • HPLC column: Luna 5 µm C18(2) 250 × 1.00 mm (for LC‐MS analysis)
  • UV lamp
  • Acquity ultraperformance liquid chromatography (UPLC) system (Waters Corp)
  • Finnigan LTQ mass spectrometer (Thermo Scientific)
  • Acquity UPLC system BEH octadecysilane (C18) column (1.7 µm, 2.1 mm × 50 mm)
  • Voyager Elite DE instrument (Perseptive Biosystem)
  • 1‐mL microcentrifuge tubes
  • Additional reagents and equipment for PAGE ( appendix 3B)
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Figures

Videos

Literature Cited

Literature Cited
  Angelov, T., Guainazzi, A., and Schärer, O.D. 2009. Generation of DNA interstrand cross‐links by post‐synthetic reductive amination. Org. Lett. 5:661‐664.
  DeCorte, B.L., Tsarouhtsis, D.S.K., MD, C., CM, H., and Harris, T.M. 1996. Improved strategies for postoligomerization synthesis of oligodeoxynucleotides bearing structurally defined adducts at the N2 position of deoxyguanosine. Chem. Res. Toxicol. 9:630‐637.
  Dooley, P.A., Tsarouhtsis, D., Korbel, G.A., Nechev, L.V., Shearer, J., Zegar, I.S., Harris, C.M., Stone, M.P., and Harris, T.M. 2001. Structural studies of an oligodeoxynucleotide containing a trimethylene interstrand cross‐link in a 5′‐(CpG) motif: Model of a malondialdehyde cross‐link. J. Am. Chem. Soc. 123:1730‐1739.
  Dooley, P.A., Zhang, M.Z., Korbel, G.A., Nechev, L.V., Harris, C.M., Stone, M.P., and Harris, T.M. 2003. NMR determination of the conformation of a trimethylene interstrand cross‐link in an oligodeoxynucleotide duplex containing a 5′‐d(GpC) motif. J. Am. Chem. Soc. 125:62‐72.
  Elmquist, C.E., Stover, J.S., Wang, Z., and Rizzo, C.J. 2004. Site‐specific synthesis and properties of oligonucleotides containing C8‐deoxyguanosine adducts of the dietary mutagen IQ. J. Am. Chem. Soc. 126:11189‐11201.
  Harris, T.M. and Harris, C.M. 2000. Synthesis of N2‐substituted deoxyguanosine nucleosides from 2‐Fluoro‐6‐O‐(trimethylsilylethyl)‐2′‐deoxyinosine. Curr. Prot. Nucleic Acid Chem. 1.3.1‐1.3.19.
  Huang, H., Dooley, P.A., Harris, C.M., Harris, T.A., and Stone, M.P. 2009. Differential base stacking interactions induced by trimethylene interstrand DNA cross‐links in the 5′‐CpG‐3′ and 5′‐GpC‐3′ sequence contexts. Chem. Res. Toxicol. 22:1810‐1816.
  Kozekov, I.D., Nechev, L.V., Moseley, M.S., Harris, C.M., Rizzo, C.J., Stone, M.P., and Harris, T.M. 2003. DNA interchain cross‐links formed by acrolein and crotonaldehyde. J. Am. Chem. Soc. 125:50‐61.
  Kozekov, I.D., Turesky, R.J., Alas, G.R., Harris, C.M., Harris, T.M., and Rizzo, C.J. 2010. Formation of deoxyguanosine cross‐links from calf thymus DNA treated with acrolein and 4‐hydroxy‐2‐nonenal. Chem. Res. Toxicol. 23:1701‐1713.
  Manoharan, M., Rasasamy, K., Mohan, V., and Cook, P. 1996. Oligonucleotides bearing cationic groups: N2‐(3‐Aminopropyl)deoxyguanosine. Synthesis, enhanced binding properties and conjugation chemistry. Tetrahedron Lett. 37:7675‐7678.
  Micheli, F., Fabio, R.D., Benedetti, R., Capelli, A.M., Cavallini, P., Cavanni, P., Davalli, S., Donati, D., Feriani, A., Gehanne, S., Hamdan, M., Maffeis, M., Sabbatini, F.M., Tranquillini, M.E., Valeria, M., and Viziano, A. 2004. 3‐Methyl pyrrole‐2,4‐dicarboxylic acid 2‐propyl ester 4‐(1,2,2‐trimethyl‐propyl) ester: An exploration of the C‐2 position. Part I. Farmaco (Lausanne) 59:175‐183.
  Minko, I.G., Harbut, M.B., Kozekov, I.D., Kozekova, A., Jakobs, P.M., Olson, S.B., Moses, R.E., Harris, T.M., Rizzo, C.J., and Lloyd, R.S. 2008. Role for DNA polymerase κ in the processing of N2‐N2‐guanine interstrand cross‐links. J. Biol. Chem. 283:17075‐17082.
  Noll, D., Mason, T., and Miller, P. 2006. Formation and repair of interstrand cross‐links in DNA. Chem. Rev. 106:277‐301.
  Perrin, D.D. and Armarego, W.L.F. 1988. Purification of Laboratory Chemicals, 3rd Ed. Butterworth Heinemann, Oxford.
  Stone, M., Cho, Y.‐J., Huang, H., Kim, H.‐Y., Kozekov, I.D., Kozekova, A., H., W, G, M.I., S, L.R., M, H.T., and Rizzo, C.J. 2008. Interstrand DNA cross‐links induced by α,β‐unsaturated aldehydes derived from lipid peroxidation and environmental sources. Acc. Chem. Res. 793‐804.
  Wilds, C.J., Booth, J.D.M., and Noronha, A.M. 2011. Synthesis of building blocks and oligonucleotides with {G}O6‐alkyl‐O6{G} cross‐links. Curr. Protoc. Nucleic Acid Chem. 5.9.1‐5.9.19.
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