Chemical Research in Toxicology ARTICLE Figure 4. Expansions of NOESY spectra at pH 5.3: (A) NOESY at 30 C, (B) 15N NOESY-HSQC at 30 C, and (C) NOESY at 25 C. NOEs of the major DNA peptide conjugate were assigned to (a) X7 N2H f Hβ, (b) X7 N2H f HR1, (c) X7 N2H f HR2, (d) X7 N2H f A8 H2, (e) X7 N2H f X7 N1H, (f) X7 N1H f C18 N 4 H2, (g) T17 N3H f A8 H2, (h) C18 N4H2 f C18 H5, (i) C18 N4H2 f C18 N4H1, (j) C18 N4H1 f C18 H5, and (k) C18 H6 f C18 H5. occupancy with regard to the formation of a hydrogen bond with K25 CdO. The hydroxyl group in the R-configuration exhibited minimal occupancy of intramolecular hydrogen bonds with A8 O40 or X7 N3. ’ DISCUSSION DNA protein conjugates (DPCs) arise from cellular expo- sures to ionizing radiation, ultraviolet light, and a variety of chemicals and metals.51 If not repaired, DPCs are anticipated to interfere with both DNA replication and transcription; conse- quently, the mechanisms by which cells recognize and repair DPCs are of considerable interest. A possible mechanism for DPC repair involves proteolysis, leading to the formation of DNA peptide conjugates, as substrates for nucleotide excision repair (NER).10 13 Two spectroscopically detectable DNA KWKK conjugates are produced by reaction of the acrolein-derived γ-OH-PdG lesion with a molar excess of the peptide KWKK. These are formed by the N-terminal amine of lysine;14 the lysine side-chain amines are protonated at pH 7.36,52 The existence of imine 9 is surmised because the conjugate can be reduced with NaCNBH3.14 However, these conjugates potentially exist as an equilibrium mixture of carbinolamine 8, imine 9, and pyrimido- purinone 10 (Scheme 1). Carbinolamines are intermediates of imine formation and dissociation;53,54 those arising from primary- or secondary-amines often dehydrate to imines.55 57 Indeed, the N2-dG-lysine conjugate formed by the malondialdehyde-induced M1dG adduct favors the imine structure.58 Formation of Diasteromeric Carbinolamine Linkages. The present data reveal that at equilibrium, these acrolein-derived DNA KWKK conjugates exist predominantly as diastereomeric carbinolamines 8, in equilibrium with trace amounts of the imine 9. NMR resonances attributed to the Hγ and Cγ nuclei of imine 9 are not observed in 13C HSQC experiments. Pyrimidopurinone cross-link 7 has been isolated following enzymatic digestion of DNA interstrand cross-links formed by the γ-OH-PdG lesion.40,59 However, pyrimidopurinone 10 is excluded for the acrolein- derived DNA KWKK conjugates as it lacks an imino proton, whereas the two observable conjugates exhibit imino resonances. The upfield shifts observed for the Cγ and Hγ resonances and the observation of Watson Crick hydrogen bonding of base pair X7 3 C18 in acidic solution therefore lead to the conclusion that these DNA KWKK conjugates exist predominately as diaster- eomeric carbinolamines 8 (Scheme 2). At equilibrium the diastereomeric carbinolamine DNA KWKK conjugates are not present in equal amounts. Their absolute stereochemical conﬁgurations cannot be established from the present NMR data. We anticipate that hydrogen bonding inter- actions are important in stabilizing the formation of these car- binolamine DNA KWKK conjugates and in determining the stereochemistry. Molecular dynamics calculations carried out in explicit solvent suggest that intramolecular hydrogen bonds might form between the carbinol and the proximate X7 N3, A8 O40, G19 O40, and K25 carboxyl oxygen. Molecular dynamics simulations suggest that the S-carbinol is more likely to form an intramolecular hydrogen bond than the R-carbinol (Table 1). Thus, it is anticipated that the S-carbinolamine is the major DNA KWKK conjugate and that the R-carbinolamine is the minor one. In DNA, these KWKK conjugates exist for weeks (Figure 3). However, upon denaturation of the duplex the DNA KWKK conjugates reverse rapidly (Figure 5). This may also explain the MALDI-TOF mass spectrum of the N2-dG:N-Lys conjugates, indicating the presence of the imine 9 and/or pyrimidopurinone 10 linkages. This is attributed to denaturation of the DNA during the MS experiments, allowing carbinolamine DNA peptide conjugates 8 to convert to imine 9 and/or pyrimidopurinone 10. The N2-dG:N2-dG DNA interstrand cross-links arising from the γ-OH-PdG adduct in the 50-CpX-30 sequence also exist predominantly as diastereomers of carbinolamine 5.17,44,60 This is attributed to the observation that carbinolamine 5 is accom- modated within the minor groove, with minimum perturbation 1129 dx.doi.org/10.1021/tx200113n |Chem. Res. Toxicol. 2011, 24, 1123–1133