ARTICLE pubs.acs.org/crt 2 γ-Hydroxy-1,N -propano-20-deoxyguanosine DNA Adduct Conjugates the N-Terminal Amine of the KWKK Peptide via a Carbinolamine Linkage Hai Huang, Hao Wang, Markus W. Voehler, Albena Kozekova, Carmelo J. Rizzo, ‡ ‡ Amanda K. McCullough, R. Stephen Lloyd, and Michael P. Stone* Department of Chemistry, Center in Molecular Toxicology, Center for Structural Biology, Vanderbilt University, Nashville, Tennessee 37235, United States ‡Center for Research in Occupational and Environmental Toxicology, Oregon Health & Science University, 3181 SW Sam Jackson Park Road, L606, Portland, Oregon 97239-3098, United States bS Supporting Information ABSTRACT: The γ-hydroxy-1,N2-propano-20-deoxyguano- sine adduct (γ-OH-PdG) was introduced into 50-d- (GCTAGCXAGTCC)-30 3 50-d(GGACTCGCTAGC)-30 (X = γ-OH-PdG). In the presence of excess peptide KWKK, 13C isotope-edited NMR revealed the formation of two spectro- scopically distinct DNA KWKK conjugates. These involved 2 the reaction of the KWKK N-terminal amino group with the N - dG propylaldehyde tautomer of the γ-OH-PdG lesion. The guanine N1 base imino resonance at the site of conjugation was observed in isotope-edited 15N NMR experiments, suggesting that the conjugated guanine was inserted into the duplex and that the guanine imino proton was protected from exchange with water. The conjugates could be reduced in the presence of NaCNBH3, suggesting that they existed, in part, as imine (Schiﬀ base) linkages. However, 13C isotope-edited NMR failed to detect the imine linkages, suggesting that these KWKK conjugates existed predominantly as diastereomeric carbinolamines, in equilibrium with trace amounts of the imines. The structures of the diastereomeric DNA KWKK conjugates were predicted from potential energy minimization of model structures derived from the reﬁned structure of the fully reduced cross-link [Huang, H., Kozekov, I. D., Kozekova, A., Rizzo, C. J., McCullough, A., Lloyd, R. S., and Stone, M. P. (2010) Biochemistry, 49, 6155 6164]. Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the S-carbinolamine linkage was the major species due to its potential for intramolecular hydrogen bonding. These carbinolamine DNA KWKK conjugates thermally stabilized duplex DNA. However, the DNA KWKK conjugates were chemically reversible and dissociated when the DNA was denatured. In this 50-CpX-30 sequence, the DNA KWKK conjugates slowly converted to interstrand N2-dG:N2-dG DNA cross- links and ring-opened γ-OH-PdG derivatives over a period of weeks. ’ INTRODUCTION Acrolein (Scheme 1, 1), a potential mutagen and carcinogen,1,2 reacts with dG to form 3-(2-deoxy-β-D-erythro-pentofuranosyl)- 5,6,7,8-tetrahydro-8-hydroxypyrimido[1,2a] purin-10(3H)-ones, γ-OH-PdG diastereomeric adduct 2.2,3 Adduct 2 has been detected in animal and human tissues2 and have been suggested to be involved in mutagenesis and carcinogenesis from exposure to acrolein.4 Methods for site-speciﬁc synthesis of 2 in oligodeox- ynucleotides have been developed.5,6 When placed into DNA opposite dC at neutral pH, 2 opens spontaneously to aldehyde 3 and the corresponding aldehydrol 4.7 Aldehyde 3 potentially reacts with primary amines in proteins to yield DNA protein conjugates (DPCs); DNA containing the structurally related 1, N2-dG adduct arising from crotonaldehyde exposures yields DNA topoisomerase I conjugates.8 Indeed, DPCs have been detected in cultured human lymphoma cells exposed to acrolein.9 If not repaired, such DPCs are anticipated to interfere with both DNA replication and transcription. The mechanisms by which cells repair DPCs are of consider- able interest. One potential mechanism involves partial proteo- lysis, leading to the formation of DNA peptide conjugates, as substrates for nucleotide excision repair (NER). This model is consistent with several observations. Inhibition of nuclear pro- teosomal protein degradation greatly reduces the repair of DPCs.10 The proteolytically active 20S core of the 26S protea- some localizes to the nucleus,11,12 and inhibition of proteasome function with lactacystin results in the inhibition of the repair of formaldehyde-induced DPCs, in normal, XP-A, and XP-F ﬁbroblasts.10 When topoisomerase I is conjugated to DNA in Received: Published: March 12, 2011 May 11, 2011 r 2011 American Chemical Society 1123 dx.doi.org/10.1021/tx200113n | Chem. Res. Toxicol. 2011, 24, 1123–1133