Chemical Research in Toxicology Scheme 1. Acrolein-Derived γ-OH-PdG Adduct in the Pre- sence of the Peptide KWKK When Placed Opposite 20- Deoxycytosine in the 50-CpX-30 (X = N2-modiﬁed dG) Se- quence Contexta a The asterisks indicate the isotope-edited carbon or nitrogen. the presence of camptothecin, proteolysis occurs in a ubiquitin- dependent fashion, giving rise to a ladder of DNA polypeptide products.13 Kurtz and Lloyd14,15 trapped a DNA KWKK conjugate involving a trimethylene linkage between the N-terminal amine of the peptide and N2-dG upon insertion of γ-OH-PdG adduct 2 into an oligodeoxynucleotide duplex, followed by exposure to a molar excess of the KWKK peptide, and NaCNBH3 treatment. Similar chemistry has been used to link peptides to γ-OH-1, N2-propano-20-deoxyadenosine (γ-OH-PdA).16 The observa- tion that the peptide conjugate is reducible implies the presence of an imine 9, in equilibrium with a conjugated carbinolamine 8. However, carbinolamine 8, imine 9, and potentially the pyrimi- dopurinone 10 conjugates exist in equilibrium (Scheme 1), and monitoring the composition of the mixture in situ was of considerable interest. All three conjugated species may inﬂuence the biological processing of DNA KWKK conjugates. The site-speciﬁc introduction of a 13C label at the γ carbon, and a 15N label at N2-dG enables the equilibrium chemistry of γ-OH- PdG adduct 2 to be monitored, in situ.17 Using this approach, we demonstrate that the native DNA KWKK linkage exists predomi- nantly as a pair of diastereomeric carbinolamines (Scheme 2), in equilibrium with trace amounts of the corresponding imine (Schiﬀ ARTICLE base) linkages. The structures of the diastereomeric DNA KWKK conjugates appear similar to the reﬁned structure of the fully reduced cross-link.18 Molecular dynamics calculations carried out in explicit solvent suggested that the conjugate bearing the S-carbinolamine linkage is the major species due to its potential for intramolecular hydrogen bonding. The native carbinolamine DNA KWKK conjugates stabilize the DNA to thermal denaturation. Upon dissociation in the 50-CpX-30 sequence, the native DNA KWKK conjugates slowly convert to interstrand N2-dG:N2-dG DNA cross-links (5,6) or revert to the N2-dG aldehyde adducts (3, 4). ’ MATERIALS AND METHODS Materials. The oligodeoxynucleotide 50-d(GGACTCGCTAGC)- 30 was synthesized and purified by the Midland Certified Reagent Co. (Midland, TX). It was purified by anion-exchange chromatography. Site- specific 13C- or 15N-labeled γ-OH-PdG adducts were synthesized and incorporated into 50-d(GCTAGCXAGTCC)-30 (X = γ-OH-PdG) using established procedures.17 The oligodeoxynucleotides were char- acterized by MALDI-TOF mass spectrometry; their purity was assessed by capillary gel electrophoresis and HPLC. Oligodeoxynucleotides were desalted on Sephadex G-25. The concentrations of the oligodeoxynu- cleotides were measured by UV absorption at 260 nm.19 The strands were annealed in 10 mM NaH2PO4, 100 mM NaCl, and 50 μM EDTA (pH 7.0). The solutions were heated to 95 C for 10 min, then slowly cooled to room temperature. The duplex oligodeoxynucleotides were purified using DNA grade hydroxyapatite with a gradient from 10 to 200 mM NaH2PO4 (pH 7.0) in 100 mM NaCl, 50 μM EDTA, and desalted by elution from Sephadex G-25. The tetrapeptide KWKK was synthesized using standard solid phase peptide synthesis methods employing Fmoc protecting groups and purified by reverse-phase HPLC. It was characterized by MALDI-TOF MS and NMR spectros- copy. The concentration of the peptide was determined by UV absorp- tion at 280 nm. Preparation of DNA KWKK Conjugates. The DNA duplex containing the site-specific γ-OH-PdG adduct was dissolved at 0.8 mM strand concentration in 280 μL of 10 mM NaH2PO4, 100 mM NaCl, and 50 μM EDTA (pH 7.0). Fifty equivalents of KWKK were added, and pH was adjusted to 7.0 by the addition of dilute HCl or NaOH. The reaction was incubated at 5 C for 8 weeks and was monitored by either 13C or 15N HSQC NMR. Excess KWKK was removed by gel filtration with Sephadex G-25. The product was lyophilized immediately. The DNA KWKK conjugates were characterized by MALDI-TOF mass spectrometry: observed at m/z of 4272.5 (calcd for carbinolamine 8 [M-H2O-1]: 4272.7). The complementary strand was observed at m/z of 3645.6 (calcd for M-1: 3645.2). NMR. Experiments were performed on a Bruker Avance 500 spectro- meter using a cryogenic probe. Samples were at 0.8 mM strand concentration and were dissolved in 280 μL of 10 mM NaH2PO4, 100 mM NaCl, and 50 μM EDTA (pH 7.0). Samples for observation of nonexchangeable protons were exchanged with D2O and suspended in 99.996% D2O. The pH was adjusted with dilute DCl or NaOD solutions. Chemical shifts of protons were referenced to water. Chemical shifts of carbon and nitrogen resonances were not precisely referenced to an internal standard. Data were processed using FELIX (Accelrys Inc., San Diego, CA) on Silicon Graphics workstations (Silicon Graphics Inc., Fremont, CA). For all experiments, a relaxation delay of 1.5 s was used. For assignment of exchangeable protons, NOESY experiments used the WATERGATE sequence.20 Spectra were recorded with 512 real data in the t1 dimension and 2048 real data in the t2 dimension, and were zero- filled during processing to create a matrix of 1024 512 real points. The mixing time was 250 ms. For assignment of nonexchangeable protons, NOESY experiments used TPPI quadrature detection and a mixing time of 250 ms. The spectra were recorded with 512 real data in the t1 1124 dx.doi.org/10.1021/tx200113n |Chem. Res. Toxicol. 2011, 24, 1123–1133