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. 2008 Jul;41(7):793-804.
doi: 10.1021/ar700246x. Epub 2008 May 24.

Interstrand DNA cross-links induced by alpha,beta-unsaturated aldehydes derived from lipid peroxidation and environmental sources

Michael P Stone  1 , Young-Jin Cho, Hai Huang, Hye-Young Kim, Ivan D Kozekov, Albena Kozekova, Hao Wang, Irina G Minko, R Stephen Lloyd, Thomas M Harris, Carmelo J Rizzo
Affiliations

Interstrand DNA cross-links induced by alpha,beta-unsaturated aldehydes derived from lipid peroxidation and environmental sources

Michael P Stone et al. Acc Chem Res. 2008 Jul.

Abstract

Significant levels of the 1, N(2)-gamma-hydroxypropano-dG adducts of the alpha,beta-unsaturated aldehydes acrolein, crotonaldehyde, and 4-hydroxy-2E-nonenal (HNE) have been identified in human DNA, arising from both exogenous and endogenous exposures. They yield interstrand DNA cross-links between guanines in the neighboring C.G and G.C base pairs located in 5'-CpG-3' sequences, as a result of opening of the 1,N(2)-gamma-hydroxypropano-dG adducts to form reactive aldehydes that are positioned within the minor groove of duplex DNA. Using a combination of chemical, spectroscopic, and computational methods, we have elucidated the chemistry of cross-link formation in duplex DNA. NMR spectroscopy revealed that, at equilibrium, the acrolein and crotonaldehyde cross-links consist primarily of interstrand carbinolamine linkages between the exocyclic amines of the two guanines located in the neighboring C.G and G.C base pairs located in 5'-CpG-3' sequences, that maintain the Watson-Crick hydrogen bonding of the cross-linked base pairs. The ability of crotonaldehyde and HNE to form interstrand cross-links depends upon their common relative stereochemistry at the C6 position of the 1,N(2)-gamma-hydroxypropano-dG adduct. The stereochemistry at this center modulates the orientation of the reactive aldehyde within the minor groove of the double-stranded DNA, either facilitating or hindering the cross-linking reactions; it also affects the stabilities of the resulting diastereoisomeric cross-links. The presence of these cross-links in vivo is anticipated to interfere with DNA replication and transcription, thereby contributing to the etiology of human disease. Reduced derivatives of these cross-links are useful tools for studying their biological processing.

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Figures

Figure 1
Figure 1
Phosphoramidite reagents for the site-specific synthesis of oligodeoxynucleotides containing 1,N2-dG enal adducts.
Figure 2
Figure 2
Amino alcohols for synthesis of crotonaldehyde and 4-HNE-modified oligodeoxynucleotides.
Figure 3
Figure 3
Cross-linking of γ-OH-PdG-adducts in the 5′-CpG-3′ sequence, monitored by CGE. The adducted and complementary strands are identified by the letters A and C, respectively; the arrows indicate interstrand cross-links.
Figure 4
Figure 4
Thermal melting analysis of and acrolein modified oligonucleotide in a CpG sequence after incubation for five day. The higher melting transition is assigned to the interstrand cross-link.
Figure 5
Figure 5
Digestion of the cross-linkedγ-OH-PdG-adducted duplex. The pyrimidopurinone bis-nucleosides were identified by comparison with authentic standards.
Figure 6
Figure 6
N2-dG:N2-dG trimethylene cross-link derived from the reduction of 18.
Figure 7
Figure 7
Data from an isotopically enriched sample containing 13C-γ-OH-PdG. The imine linkage remained below the level of NMR detection. The top spectrum shows a 13C resonance assigned as the diastereomeric carbinolamine forms of the cross-link. Assignments of resonances: a, aldehyde 1; b, hydrated-aldehyde; c, diastereomeric carbinolamines 17. An imine resonance would be expected at ~130 ppm. Copyright American Chemical Society 2005.
Figure 8
Figure 8
Isotope-edited NMR identified the carbinolamine linkage for the γ-OH-PdG cross-link. A. 15N- HSQC NOESY spectrum for oligodeoxynucleotide 39 annealed with oligodeoxynucleotide 31. Nucleotides are numbered 5′-d(G1C2T3A4G5C6X7A8G9T10C11C12)-3′•5′-d(G13G14A15C16T17C18Y19C20T21A22G23C24)-3′, X7=γ-OH PdG; Y19=15N2dG. Crosspeaks a, Y19 15N2H→X7 N1H (weak); b, Y19 15N2H→Y19 N1H (strong). B. 15N-HSQC NOESY spectrum for γ-OH-15N2-PdG labeled oligodeoxy-nucleotide 43 annealed with its complement. Crosspeaks c, X7 15N2H→X7 N1H (strong); d, X7 15N2H→G19 N1H (weak). Copyright American Chemical Society 2005.
Figure 9
Figure 9
Modeling the 8R and 8S epimers of the 5′-CpG-3′ acrolein-induced cross-links. A C•G pair is 5′ and a T•A pair is 3′ to the 5′-CpG-3′ sequence. A. 8R-diastereomer of carbinolamine cross-link 17, minor groove view. B. 8R-diastereomer of cross-link 17, base-stacking. C. 8S-diastereomer of cross-link 17, minor groove view. D. 8S-diastereomer of cross-link 17, base-stacking. E. 8R-diastereomer of pyrimidopurinone cross-link 19, minor groove view. F. 8R-diastereomer of cross-link 19, base-stacking. G. 8S-diastereomer of cross-link 19, minor groove view. H. 8S-diastereomer of cross-link 19, base-stacking. Copyright American Chemical Society 2005.
Figure 10
Figure 10
Cross-linking reactions of the 6R and 6S crotonaldehyde-modified duplexes in the 5′-CpG-3′ sequence monitored by CGE. The adducted and complementary strands are identified by the letters A and C, respectively; the arrows indicate the interstrand cross-links.
Figure 11
Figure 11
Structures of reduced cross-links arising from crotonaldehyde. A. The 6R cross-link (red) oriented in the center of the minor groove. B. The 6S cross-link (blue) interfered sterically with the DNA and exhibited lower stability. Nucleotides are numbered 5′-d(G1C2T3A4G5C6X7A8G9T10C11C12)-3′•5′-d(G13G14A15C16T17C18Y19C20T21A22G23C24)-3′, X7= 6R or 6S-crotonaldehyde-adducted dG in the 5′-CpG-3′ sequence; Y19=cross-linked dG in the complementary strand. Copyright American Chemical Society 2007.
Figure 12
Figure 12
Base pairs C6•G19, X7•C18, and A8•T17 in the oligodeoxynucleotide containing the N2-(3-oxo-1S-methyl-propyl)-dG adduct 12. The orientation of the aldehyde does not favor cross-linking to the target G19 N2-dG. Nucleotides are numbered 5′-d(G1C2T3A4G5C6X7A8G9T10C11C12)-3′ 5′-d(G13G14A15C16T17C18Y19C20T21A22G23C24)-3′, X7= N2-(3-oxo-1S-methyl-propyl)-dG adduct 12. Copyright American Chemical Society 2006.
Figure 13
Figure 13
Cross-linking of the (6S,8R,11S)-4-HNE-containing oligodeoxynucleotide.
Scheme 1
Scheme 1
1,N2-dG cyclic adducts arising from Michael addition of enals to dG.
Scheme 2
Scheme 2
Enal-dG adducts mediate DNA interstrand cross-link formation.
Scheme 3
Scheme 3
Formation and Chemistry of the Malondialdehyde-derived M1dG Adduct.
Scheme 4
Scheme 4
Synthesis of 1,N2-γ-OH-PdG in oligodeoxynucleotides by the post-synthetic modification strategy.
Scheme 5
Scheme 5
Preparation of oligodeoxynucleotides containing 13C (red) and 15N (blue) isotopes in the γ-OH-PdG adduct (41,43), and an 15N isotope (blue) in the complementary strand (39).
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