Thiol-Activated DNA Damage by α-Bromo-2-cyclopentenone

ARTICLE pubs.acs.org/crt

Thiol-Activated DNA Damage by R-Bromo-2-cyclopentenone )

)

Mostafa I. Fekry,†,‡ Nathan E. Price,† Hong Zang,† Chaofeng Huang,† Michael Harmata,† Paul Brown, J. Scott Daniels,*,^, and Kent S. Gates*,†,§ †

Department of Chemistry, University of Missouri, 125 Chemistry Building Columbia, Missouri 65211, United States Pharmacognosy Department, Faculty of Pharmacy, Cairo University, Kasr El-Aini, Cairo, Egypt 11562 § Department of Biochemistry, University of Missouri, 125 Chemistry Building Columbia, Missouri 65211, United States Pfizer, Inc., 700 Chesterfield Parkway, Chesterfield, Missouri 63017, United States

)

‡

bS Supporting Information ABSTRACT: Some biologically active chemicals are relatively stable in the extracellular environment but, upon entering the cell, undergo biotransformation into reactive intermediates that covalently modify DNA. The diverse chemical reactions involved in the bioactivation of DNA-damaging agents are both fundamentally interesting and of practical importance in medicinal chemistry and toxicology. The work described here examines the bioactivation of R-haloacrolyl-containing molecules. The R-haloacrolyl moiety is found in a variety of cytotoxic natural products including clionastatin B, bromovulone III, discorahabdins A, B, and C, and trichodenone C, in mutagens such as 2-bromoacrolein and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), and in the anticancer drug candidates brostallicin and PNU-151807. Using R-bromo-2-cyclopentenone (1) as a model compound, the activation of R-haloacrolylcontaining molecules by biological thiols was explored. The results indicate that both low molecular weight and peptide thiols readily undergo conjugate addition to 1. The resulting products are consistent with a mechanism in which initial addition of thiols to 1 is followed by intramolecular displacement of bromide to yield a DNA-alkylating episulfonium ion intermediate. The reaction of thiol-activated 1 with DNA produces labile lesions at deoxyguanosine residues. The sequence specificity and salt dependence of this process is consistent with involvement of an episulfonium ion intermediate. The alkylated guanine residue resulting from the thiol-triggered reaction of 1 with duplex DNA was characterized using mass spectrometry. The results provide new insight regarding the mechanisms by which thiols can bioactivate small molecules and offer a more complete understanding of the molecular mechanisms underlying the biological activity of cytotoxic, mutagenic, and medicinal compounds containing the R-haloacrolyl group.

’ INTRODUCTION Many DNA-damaging natural products are relatively stable in the extracellular environment but, upon entering cells, undergo biotransformation to reactive intermediates that covalently modify the genetic material.1,2 For example, DNA damage by mitomycins,3,4 saframycins,5 and dynemicin6 is activated by enzymatic reduction of their quinone moieties. Azomycin and 2-carboxyquinoline 1,4-dioxide are activated by enzymatic reduction of nitro and heterocyclic N-oxide groups, respectively.7,8 Bioactivation of acylfulvene natural products is carried out by NADPH-dependent alkenal/one reductase,9 while DNA alkylation by pyrrolizidine alkaloids and alfatoxins is triggered by cytochrome P450-mediated monooxygenation reactions.10-12 Intracellular thiols13,14 also have the potential to activate DNA-damaging natural products. For example, DNA strand cleavage by calicheamicin, dynemicin, and neocarzinostatin are triggered by reactions with thiols.6,15-17 DNA alkylation by acylfulvenes may be triggered by 1,4-addition of thiols.18 Reaction of the Streptomyces-derived natural product leinamycin r 2011 American Chemical Society

with thiols initiates both DNA alkylation and generation of reactive oxygen species (ROS).19-27 The natural products varacin, lissoclinotoxin A, and thiarubrin C also generate ROS following reactions with thiols.28-31 In addition, thiol-triggered analogues of mitomycin C have been characterized.32,33 The diverse chemical reactions involved in the bioactivation of DNA-damaging agents are both fundamentally interesting and of practical importance in medicinal chemistry and toxicology.1,2,34 For example, elucidation of bioactivation processes can reveal new chemical mechanisms by which small organic molecules can deliver reactive intermediates to the interior of cells. The work described here examines thiol-mediated bioactivation of Rhaloacrolyl-containing molecules. The R-haloacrolyl moiety is found in a variety of cytotoxic natural products including clionastatin B, bromovulone III, discorahabdins A, B, and C, and trichodenone C, in mutagens such as 2-bromoacrolein and 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone (MX), Received: August 17, 2010 Published: January 20, 2011 217

dx.doi.org/10.1021/tx100282b | Chem. Res. Toxicol. 2011, 24, 217–228

Chemical Research in Toxicology

ARTICLE

mass spectrometer (Thermoelectron Corp., San Jose, CA). For direct infusion ESI-MS, the solid phase extracted reaction was infused into the mass spectrometer at a flow rate of 10 μL/min using a stainless steel emitter on a Thermo-Fisher MaxIon source. The heated capillary was maintained at 275 C. For MS/MS analysis, ions of interest were selected with an isolation window of 2.0 Da. Fragmentation was catalyzed via collision-induced dissociation (CID) and high-energy C-trap dissociation (HCD), with normalized collision energies varying from 10 to 40%. All spectra were acquired in the full profile mode with a resolution of 100,000 at m/z 400. Each spectrum was generated by signal averaging for approximately 20 s with a maximum injection time of 1000 ms in MS mode and 2000 ms in MS/MS mode. All the data were acquired using external calibration with a mixture of caffeine, MRFA peptide, and Ultramark 1600, dissolved in a acetonitrile/water (50:50, v/v). The multiply charged, pseudomolecular ions of the alkylated peptides were subjected to CID in the LTQ and HCD in the multipole collision cell adjacent to the Orbitrap mass analyzer. For LC-MS/MS, the reaction mixtures were separated employing an Agilent 1200 nanoflow LC system (Agilent, Wilmington, DE) utilizing a gradient of 0.1% formic acid and acetonitrile (3-45% acetonitrile over 45 min) and coupled to a C-18 reverse phase column (5 μm, 200 Å, Michrom Bioresources, Inc., USA) and a 15-cm fused silica emitter (75 μm i.d., PicoTip EmitterTM/PicoFrit Self/P, New Objective, USA). The LC eluent was introduced into the LTQ Orbitrap mass spectrometer equipped with a nanoelectrospray ion source (Thermoelectron Corp, Bremen, Germany). Full scan MS spectra (m/z 300-2000 Da) were acquired with a resolution of 60,000 at m/z 400. Reaction of 1 with 2-Mercaptoethanol. A mixture of 1 (80.5 mg, 0.5 mmol), 2-mercaptoethanol (35 μL, 0.5 mmol), and triethylamine (70.3 μL, 0.5 mmol) were mixed in methanol (10 mL) and incubated at 37 C for 24 h. Following the removal of solvent, the major product was purified by column chromatography on silica gel eluted with 40% ethyl acetate in dichloromethane. The compound 2-(2-hydroxyethyl)cyclopent-2-enone (6c) was obtained in approximately 40% yield (contaminated with traces of 2-mercaptoethanol and 2-mercaptoethanol disulfide). 1H NMR (300 MHz, CDCl3): δ 7.41 (t, J = 3 Hz, 1H), 3.78 (t, J = 6 Hz, 2H), 3.04 (t, J = 6 Hz, 2H), 2.72-2.68 (m, 2H), 2.54-2.51 (m, 2H).

Figure 1. Bioactive compounds containing an R-haloacrolyl group.

and in the anticancer drug candidates brostallicin and PNU151807 (Figure 1).35-46 We employed R-bromo-2-cyclopentenone as a model compound to examine the thiol-mediated bioactivation of R-haloacrolyl-containing molecules. The evidence presented here suggests that 1,4-addition of thiols to the R-haloacrolyl moiety leads to the formation of an episulfonium ion that can alkylate guanine residues in duplex DNA (Scheme 1).

’ EXPERIMENTAL PROCEDURES Materials. Materials were purchased from the following suppliers: 3-[N-morpholino]propanesulfonic acid (MOPS), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), 1,4-piperazinediethanesulfonic acid (PIPES), ethylenediaminetetraacetic acid (EDTA), N,N,N0 ,N0 tetraethylenediamine (TEMED), sigmacote, L-lysine, glutathione-Stransferase from equine liver, and cysteamine were purchased from Sigma Chemical Company; dimethylsulfate, sodium acetate, 2-cyclopenten1-one, 3-bromo-2-butanone, and N-methylaniline were purchased from Aldrich Chemical; G-25 Sephadex, glutathione, and 2-mercaptoethanol were purchased from Sigma-Aldrich; diethylenetriaminepentaacetic acid (DETAPAC) was purchased from Fluka Chemical Company; piperidine was purchased from Alfa Aesar; xylene cyanol, bromophenol blue, formamide, urea, and ammonium persulfate were purchased from United States Biochemical; herring sperm DNA and TRISborate-EDTA (TBE) were purchased from Roche Molecular Biochemicals; acrylamide was purchased through Fischer Scientific Inc., T4 polynucleotide kinase (T4-PNK) and bovine serum albumin (BSA) were purchased from New England Biolabs; [γ-32P]-dATP was purchased from Perkin-Elmer Life Sciences; and oligonucleotides were purchased from Integrated DNA Technologies.

In Vitro Metabolism of 1 in Human Hepatic S9 Fractions and Hepatocytes . Human Hepatic S9 Fractions. The in vitro metabolism of 1 was investigated using human hepatic S9 fractions.47 A potassium phosphate-buffered reaction (0.1 M, pH 7.4) of 1 (100 μM), hepatic S9 fractions (5 mg/mL), glutathione (2 mM), and MgCl2 (3 mM) was initiated by the addition of NADPH (2 mM) and incubated at 37 C in borosilicate glass test tubes under ambient oxygenation for 50 min. Protein was precipitated by the addition of two volumes of acetonitrile, and the resulting mixture was chilled at 4 C for 20 min followed by centrifugation. The residues were reconstituted in 85:15 (v/v) water/acetonitrile, a solution reflective of the ensuing LC/MS analysis. Hepatocytes. Cryopreserved human hepatocytes (1  106 cells/ mL) were incubated with 1 (100 μM) at 37 C for 4 h under controlled atmospheric conditions (95% air/5% CO2). Cells were suspended in Krebs-Hensleit buffer that was supplemented with glucose (5 mM) and sodium pyruvate (1 mM). Cells were precipitated and reactions stopped by the addition of two volumes of acetonitrile, followed by storage at -20 C. Samples were prepared for LC-MS analysis by decanting the supernatants and drying under a stream of nitrogen. The residues were reconstituted in water/acetonitrile (85:15, v/v) in preparation for LC/ MS/MS analysis. LC/MS/MS characterization was completed employing an Agilent 1100 HPLC system coupled to a Symmetry C18 column (5 μm, 3.8  150 mm; Waters Corporation, Milford, MA). Solvent A was formic acid (0.1%), and solvent B was acetonitrile (fortified with

Reaction of 1 with the Peptide Probe γ-ECGHDRKAHYK.

The peptide probe, γ-ECGHDRKAHYK (200 μM), was incubated with 1 (200 μM) in potassium phosphate (0.1 M, pH 7.4) at 25 C for 0.5 h. Following incubation, the modified peptide(s) was extracted from the solution employing a solid phase (C8 HLB cartridges, Waters) chromatographic workup consisting of a series of acetonitrile-0.1% formic acid mixtures to wash and subsequently elute the peptide. The peptidecontaining eluant resulting from an 80% acetonitrile (0.1% formic acid) wash was subjected to electrospray ionization-tandem mass spectrometry (ESI-MS/MS) and liquid chromatography-tandem mass spectrometry (LC/MS/MS) employing an LTQ-Orbitrap XL high-resolution 218

dx.doi.org/10.1021/tx100282b |Chem. Res. Toxicol. 2011, 24, 217–228

Chemical Research in Toxicology

ARTICLE

Scheme 1

5 min in buffer (25 mM MOPS, pH 7, and 100 mM NaCl), followed by slow-cooling to room temperature overnight (∼20 h). In a typical alkylation reaction, the 50 -32P labeled oligonucleotide duplex (3 μL, approximately 50,000 cpm), HEPES buffer (2 μL of a 500 mM aqueous solution, pH 7), DETAPAC (0.5 μL of a 40 mM aqueous solution), 2-mercaptoethanol (0.5 μL, 2.5 μL, or 5 μL of a 40 mM aqueous solution), and 1 (0.5 μL, 2.5 μL, or 5 μL of a 40 mM solution in acetonitrile) were mixed with an appropriate amount of distilled, deionized water to give a final volume of 20 μL and the solution agitated using a vortex mixer (final concentrations: 50 mM HEPES, pH 7; 1 mM DETAPAC; 1 mM, 5 mM, or 10 mM of 2-mercaptoethanol; 1 mM, 5 mM, or 10 mM of 1). The reaction was quenched by precipitation of the DNA. To the mixture was added herring sperm carrier DNA (3 μL of a 1 mg/mL solution), sodium acetate (2 μL of a 3 M, pH 5 solution), and absolute ethanol (175 μL); (final concentrations: 15 μg/mL of carrier DNA, 30 mM of sodium acetate, 87.5% ethanol, final volume 200 μL). The DNA was precipitated by cooling this mixture on dry ice for 45 min, followed by centrifuging for 45 min at 14,000 rpm in a benchtop microcentrifuge. The supernatant was removed, and the pellet was washed twice with 70% ethanol-water (v/v). The DNA pellet was redissolved in aqueous piperidine (100 μL of a 0.5 M solution) and incubated at 90 C for 25 min.49 The solution was frozen on dry ice and lyophilized for 1.5 h in a SpeedVac Concentrator at 37 C. The oligonucleotides were then taken through two cycles of redissolution in 90 μL of water, freezing, and lyophilization in a SpeedVac Concentrator at 37 C. The dried DNA fragments were dissolved in formamide loading buffer and denatured at 90 C for 4 min, then immersed in ice water. An equal number of counts were loaded in each lane of a 20% denaturing polyacrylamide gel (19:1 cross-linking, containing 5 M urea). The gel was electrophoresed at 800 V for approximately 9 h. The DNA fragments in the gel were visualized by phosphorimager analysis (Molecular Imager FX, Imaging Screen-K, cat 170-7841, Bio-Rad, using Quantity One Version 4.2.3, Bio-Rad). Experiments involving other thiol or amine

0.1% formic acid). The initial mobile phase was 98:2 A/B (v/v) and by linear gradient transitioned to 10:90 A/B over 22 min.

Reaction of 1 with 2-Mercaptoethanol or Glutathione: Qualitative Rate Measurements. A solution containing 1 (0.05 mM), 2-mercaptoethanol (0.5 mM, 1 mM, or 2 mM), HEPES buffer (50 mM, pH 7), DETAPAC (1 mM as a chelator of adventitious trace metals to suppress autoxidation of thiols and the production of radicals that accompanies this process), and acetonitrile (0.06% v/v) was prepared in HPLC grade water, vortex-mixed, and placed in a quartz cuvette (1 cm path length). Absorption spectra were collected at 5 s intervals at room temperature (Figure 3). Alternatively, the reaction of 1 with glutathione was measured using HPLC. A solution containing sodium phosphate buffer (60 μL of a 500 mM aqueous solution, pH 7), 1 (15 μL of a 40 mM solution in acetonitile), and glutathione (150 μL of a 40 mM aqueous solution) were mixed with an appropriate amount of distilled, deionized water to give a final volume of 600 μL, and the solution agitated using a vortex mixer and incubated at 37 C for 2 min (final concentrations: 50 mM sodium phosphate buffer, pH 7; 1 mM of 1; 10 mM of glutathione; 2.5% (v/v) acetonitrile; 600 μL final reaction volume). After incubation, 5 μL of the reaction mixture was analyzed by HPLC using a reverse phase Supilcosil LC-18-S column (particle size, 5 μm; 15 cm L  2.1 mm ID) at a flow rate of 0.2 mL/min using a gradient of increasing methanol in water. The HPLC elution protocol was a continuous gradient of 80-95% methanol over 30 min. The column was then washed with 95% methanol for 10 min, followed by a continuous gradient of 95-80% methanol over 5 min, then 80% methanol for 15 min. Thiol-Triggered DNA Alkylation by 1. The single-stranded 20 deoxyoligonucleotide 50 -GTT CGT ATA TGG GAG GTC GCA TGT G-30 was labeled using [γ-32P]dATP and T4 polynucleotide kinase according to standard protocols48 and were purified on a 20% denaturing polyacrylamide gel.48 The purified single-stranded radiolabeled oligonucleotide was annealed with 1.5 equiv of the complementary strand 50 -CAC ATG CGA CCT CCC ATA-30 by heating to 95 C for 219

dx.doi.org/10.1021/tx100282b |Chem. Res. Toxicol. 2011, 24, 217–228

Chemical Research in Toxicology

ARTICLE

Figure 2. Mass spectra of the products obtained from the reaction of 1 with the peptide γ-ECGHDRKAHYK.

LC/MS/MS Characterization of the Guanine Adducts Generated by the Reaction of 1 and Thiol with Duplex DNA.

activating agents were carried out in an identical manner. Control reactions examining alkylation by 3-bromo-2-butanone and 2-cyclopentenone were carried out as described above except that 1 was replaced with these molecules. Experiments in which the pH was varied (pH 6.1 and 8) employed a buffer consisting of a HEPES (50 mM) and PIPES (50 mM) mixture.

Herring sperm DNA (double-stranded, 5 mM bp) was treated with 1 (5 mM) and 2-mercaptoethanol (5 mM) in HEPES buffer (pH 7, 50 mM) containing DETAPAC (1 mM). The reaction mixture was incubated at 37 C for 72 h. The resulting alkylated DNA was heated at 220

dx.doi.org/10.1021/tx100282b |Chem. Res. Toxicol. 2011, 24, 217–228

Chemical Research in Toxicology

ARTICLE

Table 1. Calculated Exact Mass (calcd) and Measured Accurate Mass (obsvd) for the Native and Adducted Peptides compound

[M þ 2H]2þ calcd

[M þ 2H]2þ obsvd

[M þ 3H]3þ calcd

[M þ 3H]3þ obsvd

ECGHDRKAHYK native peptide

672.3173

672.3170

448.5473

448.5468

4a

721.3356

721.3359

481.2262

481.2255

5a

761.3188

761.3193

507.8817

507.8815

6a

712.3304

712.3308

475.2227

475.2226

product eluting at 25.7 min, producing a [M þ 2H]2þ and a [M þ 3H]3þ at m/z of 721.3359 and 481.2255, respectively (Figure 2B), consistent with a structure such as 4a (Scheme 1). The presence of the y9 fragment ion of 4a (m/z 1111.5743 Da) and the corresponding y102þ fragment ion (m/z 656.8138 Da) were again indicative of attachment of the small molecule to the cysteine residue of the probe peptide. Interestingly, another product eluting at 23.3 min was observed, producing a [M þ 2H]2þ and a [M þ 3H]3þ at m/z 761.3193 and 507.8815 Da (Figure 2C), respectively, consistent with a phosphate adduct such as 5a. Again, both the y9 ion (m/z 1111.5731 Da) and the y102þ ion (m/z 696.7955 Da) were consistent with the aforementioned attachment of the peptide to 1 via the cysteine thiol group. The stereochemistry and regiochemistry of the products identified here (4a-6a) are not defined by the mass spectrometry experiments. In the case of the sulfide, 6a, the results of a model reaction led us to favor a structure in which the sulfur of the attacking thiol is ultimately attached at the R-position adjacent to the carbonyl residue. Specifically, 6c is observed as a major product of the reaction between 1 and 2-mercaptoethanol in methanol containing 1 equiv of triethylamine at 37 C. NMR analysis of this product suggests that the sulfide side chain resides at the R-position adjacent to the carbonyl residue. This assignment is supported by the observation that the vinylic proton in the product appears as a triplet (J = 3 Hz) at 7.41 ppm. This is in line with NMR data reported for structurally analogous compounds.53 The reaction of thiols with 1 in neutral solution is quite facile. We monitored the reaction of 1 with 2-mercaptoethanol in HEPES buffer (50 mM, pH 7) containing acetonitrile (0.06% v/v) using UV-vis spectrometry (Figure 3). A rapid decrease in the absorbance associated with the starting material was observed over the course of several minutes. In a separate HPLC assay, we find that more than 90% of 1 is consumed after incubation for 2 min with glutathione (10 equiv, 10 mM) in sodium phosphate buffer (50 mM, pH 7) containing 2.5% acetonitrile (v/v) at 37 C (Supporting Information). In the absence of thiol, no degradation of 1 is observed within this time frame under the same conditions. These findings are consistent with literature reports showing that conjugate addition of thiols to R,β-unsaturated ketones occurs readily.54-56 Although the data presented above indicates that compound 1 reacts readily with thiol groups, there are a variety of nucleophiles in the intracellular environment that could also, in principle, react with this compound. Accordingly, we set out to investigate whether the principle intracellular thiol, glutathione, reacts with 1 in human hepatic subcellular fractions (S9) and in human hepatocyte cells. We incubated 1 with human hepatic subcellular fractions (S9) that were fortified with glutathione to probe the formation of the corresponding thiol adduct of 1. Indeed, LC/ MS analysis of the reaction supernatant revealed the presence of two isobaric metabolites producing a [M þ H]þ at m/z 388 Da (Figure 4A), consistent with the addition of glutathione to 1 to

90 C for 45 min to release alkylated bases. Large DNA fragments were then removed by filtering the solution through a microconcentrator filter (YM-3, Microcon) by centrifugation at 10,000 rpm for 1.5 h. The resulting solution was lyophilized in preparation for LC/MS/MS analysis. The resulting residue was dissolved in aqueous methanol (25% v/v) and a portion (5 μL) subsequently diluted in 15% aqueous acetonitrile (45 μL). LC/MS/MS characterization was completed employing an Agilent 1100 HPLC system coupled to a Symmetry C18 column (5 μm, 3.8  150 mm; Waters Corporation, Milford, MA). Solvent A was formic acid (0.1%), and solvent B was acetonitrile (fortified with 0.1% formic acid). The initial mobile phase was 95:5 A/B (v/v) and by linear gradient transitioned to 20:80 A/B over 20 min. The flow rate was 0.4 mL/min. The HPLC eluent was introduced via electrospray ionization directly into a Finnigan LCQ Deca XPPLUS ion trap mass spectrometer (Thermoelectron Corporation, San Jose, CA) operated in the positive ionization mode. Ionization was assisted with sheath and auxiliary gases (ultra pure nitrogen) set at 60 and 40 psi, respectively. The electrospray voltage was set at 5 kV with the heated ion transfer capillary set at 300 C and 30 V. Relative collision energies of 32% were used when the ion trap mass spectrometer was operated in the MS/MS mode.

’ RESULTS Reaction of r-Bromo-2-cyclopentenone (1) with the Cysteine-Containing Probe Peptide γ-ECGHDRKAHYK, with 2-Mercaptoethanol, or with Glutathione in Human Hepatic S9 Fractions. Compound 1 was prepared by the reaction of

2-cyclopentenone with Br2 in the presence of triethylamine as described previously.50,51 To examine the reactivity of 1 with a range of nucleophiles, we incubated 1 (200 μM) in a phosphatebuffered (100 mM, pH 7.4) solution containing a peptide (γECGHDRKAHYK, 200 μM) bearing a variety of nucleophilic side chains including carboxylate, thiol, alcohol, imidazole, and amine groups. The peptide is an analogue of glutathione (γ-GluCys-Gly) bearing a H2N-His-Asp-Arg-Lys-Ala-His-Tyr-Lys-CO2H peptide linked to the C-terminal glycine residue.52 Following a 30 min incubation (25 C), the reaction was subjected to solid phase extraction (C18 Sep-Pak) followed by reversed-phase LC-MS/MS analysis employing accurate mass spectroscopy (Supporting Information). Several products arising from the reaction of 1 with this nucleophilic probe peptide were observed (Scheme 1 and Figure 2). Most notably, a product eluting at 26.8 min produced a [M þ 2H]2þ and a [M þ 3H]3þ at the mass-to-charge ratios (m/z) of 712.3308 and 475.2226, respectively (Table 1), consistent with adduct 6a arising from the addition of the peptide to 1 (net addition of 80 Da to the peptide, Scheme 1). Importantly, the presence of the y9 and y102þ fragment ions at m/z 1111.5727 and 647.8070 (Figure 2D) suggest that the peptide was linked to 1 via the cysteine residue. This is indicated by a net 40 Da increase in the y102þ fragment ion compared to the same ion seen for the native peptide (607.7940 Da, Figure 2A), while the y9 ions in both spectra appear at an m/z of 1111.5730 (isobaric). Upon closer examination of the reaction mixture, we observed two additional peptide adducts. Specifically, LC/MS/MS analysis revealed a 221

dx.doi.org/10.1021/tx100282b |Chem. Res. Toxicol. 2011, 24, 217–228

Chemical Research in Toxicology

ARTICLE

Figure 3. Time-dependent change in the UV-vis spectra of the reaction between 1 (50 μM) and 2-mercaptoethanol (1 mM) in 50 mM HEPES buffer, pH 7, at 24 C.

Figure 5. Thiol-activated DNA damage by 1. The 50 -32P-labeled oligonucleotide 50 -GTTCGTATATGGGAGGTCGCATGTG-30 was treated with 1 and 2-mercaptoethanol. Reactions were conducted in HEPES buffer (50 mM), pH 7.0, containing DETAPAC (1 mM) at 37 C for 23 h and 20 min, followed by Maxam-Gilbert workup and analysis using 20% denaturing polyacrylamide gel. Lane G, MaxamGilbert G-sequencing reaction. Lanes 1-8 employed duplex DNA, where the underlined region of the oligonucleotide shown above is double stranded: lane 1, duplex with Maxam-Gilbert workup (no 1 or 2-mercaptoethanol); lane 2, duplex treated with 1 (10 mM); lane 3, duplex treated with 2-mercaptoethanol (10 mM); lane 4, duplex treated with 1 (10 mM) and 2-mercaptoethanol (1 mM); lane 5, duplex treated with 1 (10 mM) and 2-mercaptoethanol (5 mM); lane 6, duplex treated with 1 (10 mM) and 2-mercaptoethanol (10 mM); lane 7, duplex treated with 1 (5 mM) and 2-mercaptoethanol (10 mM); lane 8, duplex treated with 1 (1 mM) and 2-mercaptoethanol (10 mM). Figure 4. Mass spectrometric analysis of products obtained from the reaction of 1 with glutathione in human hepatocytes.

Therefore, we examined the potential for 1 to act as a thioltriggered DNA alkylating agent. Toward this end, the 50 -32Plabeled oligonucleotide 50 -GTTCG8TATATG7G6G5AG4G3TCG2CATG1TG-30 (where the underlined region is double stranded, and the subscripts represent the numbering of guanine residues in Figures 5-7) was treated with various concentrations of 1 and 2-mercaptoethanol in 50 mM HEPES buffer, pH 7.0, at 37 C for 24 h. Maxam-Gilbert workup (500 mM piperidine, 90 C, 25 min)49 and sequencing gel analysis revealed strand cleavage selectively at guanine residues in the reactions that contained both 1 and thiol (Figure 5). Control reactions involving the incubation of DNA with 1 alone or thiol alone showed little or no strand cleavage upon Maxam-Gilbert workup. A control reaction in which 1 was preincubated with 2-mercaptoethanol (37 C, 26 h) prior to the addition of the DNA also generated little or no DNA damage above background. This

yield compounds with the general structure 6b. The fragmentation spectra of the two products were nearly identical, producing m/z 259 as a major fragment ion, corresponding to the loss of glutamate (-129 Da) from the parent ion (Figure 4B). The loss of glycine (-75 Da) produced an ion at m/z 313 and represented an additional fingerprint fragment consistent with glutathione conjugation. We observed the same two glutathione conjugates when cryopreserved human hepatocyte suspensions (1  106 cells/mL) were incubated with 1 (Supporting Information). Thiol-Triggered DNA Alkylation by r-Bromo-2-cyclopentenone (1). A number of compounds containing the R-haloacrolyl fragment (Figure 1) are cytotoxic or mutagenic and may derive biological activity through reactions with DNA.41,42 222

dx.doi.org/10.1021/tx100282b |Chem. Res. Toxicol. 2011, 24, 217–228

Chemical Research in Toxicology

ARTICLE

3-bromo-2-butanone produces no strand cleavage above background levels (Supporting Information). A separate control experiment shows that 2-cyclopentenone also does not generate any lesions that are labile to Maxam-Gilbert workup. Finally, we observed that 2-bromo-2-cyclohexenone generates thioldependent, labile lesions comparable to 1 (data not shown). Overall the structure-activity data is consistent with the involvement of an episulfonium ion in thiol-triggered DNA alkylation by 1, rather than direct alkylation by 1, 2, or 6 (Supporting Information). DNA alkylation by charged alkylators such as episulfonium ions can be inhibited by added salt.24,60-63 We find that the addition of salt decreases the yield of DNA alkylation by 1 and 2-mercaptoethanol. Specifically, the addition of 1 M NaClO4 caused a 27 ( 3% decrease in overall alkylation yield. We also examined the ability of glutathione and cysteamine to trigger DNA alkylation by 1. We find that the use of these different thiols leads to different DNA alkylation yields and different sequence specificities (Figure 6). This observation is consistent with the generation of different intermediates in which the side chain of the attacking thiol has been incorporated into the ultimate DNA-alkylating species. The relative overall yields for thiol-triggered alkylation of duplex DNA by glutathione, cysteamine, and 2-mercaptoethanol are 1:5.7:8 in doublestranded DNA. It is noteworthy that glutathione, the major thiol present in mammalian cells, is a rather weak activator of the DNA alkylation process measured in this particular assay. The yields for the alkylation of single-stranded DNA by 1 using glutathione, cysteamine, and 2-mercaptoethanol were 1:1.9:1.7. Thiol-triggered alkylation of single-stranded DNA by 1 is less efficient than the alkylation of duplex DNA. Previous studies have shown that small, positively charged alkylating agents such as aziridinium ions preferentially alkylate duplex DNA over single-stranded DNA.63 Addition of amines to R-haloacrolyl systems can generate aziridines in organic solvents.64 Aziridines have the potential to alkylate DNA.65,66 Accordingly, we examined whether amines activate DNA alkylation by 1. We found that the addition of N-methylaniline or L-lysine did not activate DNA alkylation by 1 under the conditions identical to those employed for the thioltriggered DNA alkylation by 1 (