A covalent thymine-tyrosine adduct involved in DNA-protein crosslinks: synthesis, characterization, and quantification

Free Radical Biology & Medicine, Vol. 27, Nos. 3/4, pp. 254 –261, 1999 Copyright © 1999 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/99/$–see front matter

PII S0891-5849(99)00030-1

Original Contribution A COVALENT THYMINE-TYROSINE ADDUCT INVOLVED IN DNA-PROTEIN CROSSLINKS: SYNTHESIS, CHARACTERIZATION, AND QUANTIFICATION TIMOTHY S. CHARLTON,* BENNO A. INGELSE,* DAVID STC. BLACK,† DONALD C. CRAIG,† KERRYN E. MASON,* and MARK W. DUNCAN* *Ray Williams Biomedical Mass Spectrometry Facility; and †School of Chemistry, The University of New South Wales, Sydney, Australia (Received 15 July 1998; Revised 11 January 1999; Accepted 13 January 1999)

Abstract—A thymine-tyrosine adduct, (3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]-L-tyrosine), was synthesized using a simple, single-step condensation between 5-(hydroxymethyl)uracil and L-tyrosine. This approach provides access to useful quantities (mg-g) of analytically pure reference material, and with minor modification, to stable isotope-labeled analogues (isotopomers). With reference material and a suitable internal standard available, isotopedilution liquid chromatography— electrospray ionization—tandem mass spectrometry (LC/MS/MS) was used to assay the adduct in a model system purged of oxygen, i.e., a ␥-irradiated N2O-saturated aqueous solution of thymine and tyrosine. The convenient synthetic route to standards and the method for quantification reported here will prove useful in assessing the significance of the adduct in biological systems. These studies also highlight the potential for artefactual adduct formation if the appropriate substrates are present under acidic conditions. © 1999 Elsevier Science Inc. Keywords—DNA-protein crosslinks, Thymine-tyrosine adduct, DNA damage, ␥-Irradiation, Liquid chromatography, Electrospray ionization mass spectrometry, Free radicals

INTRODUCTION

levels of DPCs have also been reported in untreated mammalian cells [7]. Covalent cross-linking between DNA and protein could involve several adducts. A thymine-tyrosine adduct, (3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl)methyl]L-tyrosine; 3) has been identified as a possible DPC and has been reported to be present in ␥-irradiated calfthymus nucleohistone [9], a model system containing thymine and angiotensin [10,11], isolated chromatin [12], cultured mammalian cells [13,14] and rat renal chromatin [15]. The adduct was first prepared, albeit in low yield, by ␥-irradiation of an equimolar solution of thymine and tyrosine [16,17], and although characterized by both mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy [18], the material was never isolated in crystalline form. The adduct has not since been prepared by more conventional (and practical) synthetic methods, nor has it been fully characterized. Consequently, of the limited analytical work undertaken, most has been performed without reference to a suitable standard, and perhaps without due concern for the potential of the adduct to form artefactually. Consequently,

When exposed to ionizing radiation, DNA can undergo any number of reactions, but of particular importance is the possibility that covalent bonds can form between DNA and protein. DNA-protein crosslinks (DPCs) are postulated to have a deleterious effect on cell function because these modifications are sometimes inefficiently repaired [1], and can accumulate, compromising the function of DNA, and ultimately contributing to degenerative disorders including cancer [2– 4]. In cell culture studies, DPCs have also been implicated in an increase in cell lethality [5] and mutagenicity [6]. DPCs have been detected in isolated chromatin in vitro, and in cultured cells either after irradiation (e.g., ␥-irradiation [7]), or exposure to a range of chemicals such as 4⬘-(9-acridinylamino)-methanesulfon-m-anisidide (m-AMSA) [8] and hexavalent chromate and vanadate [6]. Background Address correspondence to: Mark W. Duncan, Ph.D., Department of Pharmaceutical Sciences, School of Pharmacy, C238, University of Colorado Health Sciences Center, Denver, CO 80262, USA; Tel: (303) 315-0239; Fax: (303) 315-0274; E-Mail: [email protected]. 254

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Fig. 1. The synthetic approach employed to prepare the tyrosine-thymine adduct, 3, from 5-(hydroxymethyl)uracil and L-tyrosine.

there are few data available on which to base an assessment of its significance in biological systems. Here we report a simple one-step synthesis of the thymine-tyrosine adduct, 3, via an acid-catalyzed condensation of 5-(hydroxymethyl)uracil (5-OHmUra, 1) with L-tyrosine (2) (Fig. 1). The adduct was isolated as a crystalline solid and was thoroughly characterized by mass spectrometry, NMR spectroscopy and x-ray crystallography. By employing [13C6]-tyrosine as a starting material in the same synthetic scheme, stable-isotope labeled adduct was also prepared. Once the appropriate standards were available to us, we employed liquid chromatography tandem mass spectrometry (LC/MS/MS) to quantify the adduct in a ␥-irradiated, equimolar solution of thymine and tyrosine. The samples were also subjected to protein hydrolysis conditions to assess the possibility of artefactual formation of 3. The samples were analyzed both before and after acid hydrolysis, a step routinely incorporated when recovering the thymine-tyrosine adduct from intact nucleoprotein [12,15]. The LC/MS/MS approach we have adopted offers sensitive and selective detection of the adduct and can be applied to the analysis of biologic samples. We draw special attention to the issue of artefactual formation: this can occur under certain conditions and compromise identification and quantification. MATERIALS AND METHODS L-tyrosine

and 5-OHmUra were purchased from Aldrich (Milwaukee, WI, USA). Ring labeled [13C6 ]-L-tyrosine was purchased from Cambridge Isotope Laboratories (Andover, MA, USA). The derivatizing reagents, bis(trimethylsilyl)trifluoroacetamide (BSTFA) and N-methylN-(tert-butyldimethylsilyl)trifluoroacetamide (MTBSTFA), were obtained from Pierce Inc. (Rockford, IL, USA). All other reagents were purchased from commercial suppliers (analytical grade) and were used without purification. Deionized water (resistivity ⬎18 M/cm) was used in all experiments.

Synthesis of 3-[(1,3-dihydro-2,4-dioxopyrimidin-5-yl) methyl]-L-tyrosine (3) Hydrated 5-OHmUra (0.50 g), L-tyrosine (0.65 g), glacial acetic acid (14.0 mL) and hydrochloric acid (6 M; 0.5 mL) were refluxed at 140 –150oC for 30 min. After cooling, the reaction mixture was evaporated to dryness under vacuum and then reconstituted in boiling water (100 mL). The solution was left at 4oC overnight, and the precipitate removed by filtration. The supernatant was concentrated to half its volume, and left at 4oC, again overnight. The white crystalline precipitate was collected (0.3 g, 30%; melting point noncalibrated, (dec) 285– 289oC) and recrystallized from water to give 3 (C14H15N3O5•0.3H2O2O requires C, 54.1; H, 5.1; N, 13.5%; found: C, 54.1; H, 5.2; N, 13.2). Characterization of the product and its volatile derivatives Gas chromatography-mass spectrometry (GC/MS), 1H and 13C NMR spectroscopy, elemental analysis, x-ray crystallography were all performed on a single batch of the recrystallized material. Preparation of derivatives and characterization by GC-MS Trimethylsilyl (TMS) and tert-butyldimethylsilyl (tBDMS) derivatives were prepared according to methods detailed in the literature [18,19]. Aliquots (0.5–1.0 ␮L) of the derivatized sample were injected (split mode) onto a fused silica capillary column (12 m, 0.22 mm inside diameter, 0.25 ␮m film thickness, 5% phenylmethylsilicone stationary phase (BPX5; SGE, Melbourne, Australia). The gas chromatograph (HP5890, Hewlett-Packard, Palo Alto, CA, USA) was temperature programmed from 50 –300oC at 10oC min-1. The capillary column was directly interfaced (transfer line 300oC) to the mass spectrometer (Autospec-Q, Micromass, UK). The source temperature was 220oC. Mass spectra were

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acquired in positive electron ionization mode (70 eV) over the mass range m/z 50 to 1000 at a scan rate of 1 s per decade and a resolution of ca. 1000 (10% valley definition). NMR spectroscopy Spectra were acquired using a Bruker Instruments Model AM-500 spectrometer. The product (0.2 M, DCl in D2O) was examined by 1H and 13C NMR. 1H NMR spectra were recorded using a 45o pulse (5– 8 ␮s), a spectral width of 5.5 kHz, 16 scans and 8000 data points acquired and zero filled to 64,000 data points on processing. 1 H-decoupled 13C NMR spectra were acquired using a 45o pulse (3 ␮s), a spectral width of 28 kHz, 2,500 scans and 16,000 data points zero filled to 32,000 for data processing, with 3 Hz line broadening. A 135o distortionless enhancement by polarization transfer (DEPT) spectrum was used to determine the multiplicity of carbons in the 1H-decoupled 13C spectrum. The same acquisition conditions were used as for 1H-decoupled 13C NMR. X-ray crystallography The crystal used for x-ray crystallography was prepared by dissolving 3 (0.082 g) in water (90oC; 15 mL) and filtering the solution through silica wool into a Petri dish. The dish was then placed on a covered water bath (90oC), and the temperature of the water bath was immediately set to 50oC. The resulting crystals were dried over activated silica at ambient temperature and pressure. Structure determination. Reflexion data were measured with an Enraf-Nonius CAD-4 diffractometer in q/2q scan mode using nickel-filtered copper radiation (l 1.5418Å). Absorption corrections were not applied and reflexions with I ⬎ 3s(I) were considered observed. The structure was determined by direct phasing and Fourier methods indicating the water molecules were poorly defined and considerably disordered. Hydrogen atoms were included in calculated positions and were assigned thermal parameters equal to those of the atom that they were bonded. Amine, hydroxyl and water hydrogen atoms were not located. Positional and anisotropic thermal parameters for the non-hydrogen atoms were refined using full matrix least squares. Constraints were applied to make corresponding distances in the two independent molecules in the asymmetric unit approach equality, thus reducing the impact on the refinement of the relatively small number of observed reflexions. Reflexion weights used were 1/s2(Fo), with s(Fo) being derived from s(Io) ⫽ [s2(Io) ⫹ (0.04Io)2]1/2. The weighted residual is defined

as Rw ⫽ (SwD2/SwFo2)1/2. Atomic scattering factors and anomalous dispersion parameters were obtained from literature sources [20]. Structure solution was by MULTAN80 [21] and refinement used RAELS [22]. ORTEP-II [23] running on a Macintosh IIcx was used for the structural diagram, and a DEC Alpha-AXP workstation was used for calculations. Liquid chromatography–mass spectrometry Liquid chromatography-tandem mass spectrometry using electrospray ionization was used for the quantification of 3. The electrospray LC/MS/MS system consisted of an HP1090 liquid chromatograph (Hewlett Packard, Palo Alto, CA, USA) coupled to a Finnigan TSQ7000 mass spectrometer (Finnigan, San Jose, CA, USA) via a Finnigan electrospray interface. Liquid chromatographic separations were performed in an Alltima C18 column (150 ⫻ 1 mm, 5 ␮m, [Alltech, Deerfield, IL, USA]). A gradient was applied to optimize efficiency: i.e., for 0 – 4 min, a linear ramp from 98% water (solvent A) and 2% acetonitrile (solvent B) to 70% solvent A and 30% solvent B. The flow rate was 50 ␮L min-1. The ESI source conditions were: spray voltage 5.0 kV; capillary temperature 250°C; sheath gas pressure 70 psi and the auxiliary gas turned off. To obtain maximum sensitivity and selectivity the MS was operated in the selective reaction monitoring (SRM) mode. Two precursor-product reactions were monitored continuously: m/z 306 3 m/z 260 for the thymine-tyrosine adduct and m/z 312 3 m/z 266 for the corresponding [13C6]-labeled adduct (these transitions correspond to the loss of HCOOH from the corresponding [M⫹H]⫹ ion). The collision gas (argon) was used at a pressure of ca. 2.2 mTorr and a collision energy of 20 eV. Analysis and quantification of irradiated mixed solutions of thymine and tyrosine Mixed aqueous solutions of thymine and tyrosine (1 mM) were prepared and purged with 100% nitrous oxide for 30 min before and during the irradiation experiments. Solutions were irradiated at 18.0 Gy min-1 for five time intervals: i.e., 0, 6, 17, 28, 39 min corresponding to 0, 108, 306, 504, 702 Gy respectively. This allowed us to obtain a dose-response curve for the amount of 3 formed vs. irradiated dose. The internal standard (20 ␮L of a 500 pg/␮L solution of [13C6]-adduct) was added to aliquots (50 ␮L) of each of the irradiated solutions, these were mixed thoroughly, and then injected (5 ␮L) directly into the LC-system. Quantification was performed by reference to a calibration curve prepared from samples containing a fixed amount of the internal standard (10 ng of

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Fig. 2. GC/MS analysis of the t-BDMS derivative of 3, the tyrosine-thymine adduct. (A) The EI total ion chromatogram; (B) the mass spectrum of the peak assigned to the penta t-BDMS derivative of 3. The proposed fragmentation scheme for the penta t-BDMS derivative under EI conditions is shown as a insert on the figure.

the [13C6]-labeled analogue) and serial dilutions of a stock solution of the adduct. The samples were also subjected to protein hydrolysis conditions to assess the possibility of artefactual formation of 3. Gas-phase hydrolysis was performed according to the procedure we described previously [24]. After hydrolysis, samples were evaporated to dryness and reconstituted in water (70 ␮L) before analysis. RESULTS AND DISCUSSION

Synthesis The one-step reaction described here (Fig. 1), followed by isolation and recrystallization, delivers a 30% yield of analytically pure product. This is a practical alternative to ␥-irradiation that proves cumbersome, very low yielding, and requires elaborate isolation and purification of 3 [18]. Product characterization Gas chromatography–mass spectrometry. Analysis of the product by GC/MS after trimethylsilylation provided evidence for two sample-derived peaks (retention time (tR) 23:28 and 23:41 min; data not shown). The mass

spectra of these components are consistent with the formation of two separate TMS derivatives of compound 3: the tetra-TMS and penta-TMS derivatives. Relative abundances of the major ions of these two derivatives are: (a) penta-TMS; m/z 665 (0.4%), 664 (0.6%), 650 (10%), 548 (11%), 448 (100%) and (b) tetra-TMS; m/z 593 (0.5%), 592 (0.8%), 578 (12%), 476 (7%), 448 (100%). The m/z 448 ion is the base peak in the mass spectra of both derivatives and is likely due to cleavage between the carbon and the substituted phenol of the tyrosyl moiety. The molecular ions for both species were detected (m/z 665 and m/z 593), but in each instance the [M-H]⫹ ion was marginally more intense. The [M-CH3]⫹ ions (i.e., m/z 650 and m/z 578) are significant in the mass spectra of both derivatives. GC/MS analysis of 3 after t-BDMS derivatization yielded only one sample-derived peak, tR 29:56 min (Fig. 2A). The mass spectrum of this peak (Fig. 2B) is consistent with formation of a penta-tBDMS derivative of 3 (see insert on Fig. 2). Abundances for selected ions in the EI mass spectrum of this derivative are as follows: m/z 875 (1%), 874 (1%), 860 (9%), 818 (42%), 790 (17%), 716 (15%) and 574 (100%). The ions corresponding to m/z 875 and 874 are the molecular ion, M⫹ and the [M-H]⫹ ion respectively. Identification of m/z 875 as the molecular ion is supported by the presence of an ion at

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Fig. 3. Two conformers (assigned A and B) of 3 were found in the crystal selected for x-ray crystallographic analysis. A scale diagram of conformer A is shown here (Insert shows structural formula of the adduct in the same conformation).

m/z 860, corresponding to the loss of a methyl radical. The mass spectrum is characteristic of electron ionization (EI) spectra of the tBDMS derivatives of the naturally occurring amino acids [19].

tyrosine molecule in the crystal lattice is due to rotation about the methylene bridges between the two ring structures and between the phenolic and amino acid moieties.

NMR data and interpretation. The 1H and 13C NMR spectra of 3 (not shown) were compared to the spectra of the reference compounds 5-OHmU and tyrosine, and to the values previously reported by Margolis et al. [18]. The results are consistent with the formation of a single covalent bond between the methylene group of 5-OHmU and C-3 carbon of tyrosine to form 3. There is, however, one disparity in the 13C chemical shift measurements: Margolis et al. [18] reported a shift of 183.8 ppm for the C-2⬘ carbon whereas the value determined here is 155.7 ppm. The latter value correlates well with the shift value obtained for the corresponding carbon atom in thymine, i.e. 156.1 ppm [18], and is in agreement with 13C chemical shift value reported for the C⫽O function of normal and modified pyrimidine bases [25].

Mechanism of formation and quantitative analysis of the adduct

X-ray crystallography. Two conformers (assigned A and B) of 3 were found in the crystal selected for x-ray crystallographic analysis. A scale diagram of conformer A is shown in Fig. 3. Conformation of the thymine-

Formation of 3 in the series of irradiated samples was quantified by LC/MS/MS with reference to a calibration curve. Calibration curves were generated from the ratio of peak areas for the appropriate transitions and were linear (r2 ⫽ 0.999) and reproducible across the range 5–5000 pg ␮L-1. The limit of quantification for the adduct was 1 pg on column at a S/N⫽10. A plot of the amount of the adduct formed in the samples vs. irradiated dose is shown in Fig. 4. The amount of adduct varied between 107 ng mL-1 (108 Gy) and 417 ng mL-1 (702 Gy) for the nonhydrolyzed samples. This corresponds to a molar yield, relative to thymine (or tyrosine), of between 0.03% and 0.14%. Figure 5 shows a typical mass chromatogram of an irradiated sample. Two clear peaks can be distinguished with a retention time of ca. 6 min. The first of these corresponds to 3, whereas the second (minor) peak is likely to be a closely related structure (note that the second peak is not

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Fig. 4. A plot of the amount of the adduct formed in the samples vs. irradiated dose, both before and after acid hydrolysis (note the different scales for the results before and after hydrolysis).

seen in the chromatograms of the hydrolyzed samples). After acid hydrolysis, the concentration of 3 increased by ca. 10-fold to yield up to 1.2% of the adduct (i.e., at 702 Gy). The adduct was also detected at a concentration of 46 ng mL-1 (0.015%) in a nonirradiated sample consisting of an equimolar solution of thymine and tyrosine that was subjected to the same hydrolysis conditions. No adduct was detectable in this sample before the hydrolysis. Dose-dependent formation of the thymine-tyrosine adduct with increasing irradiation was observed (Fig. 4). There are two possible mechanisms for the formation of 3 during acid hydrolysis. The first mechanism has been described previously [16,18] and involves formation of a hydrated analogue in situ during ␥-irradiation, followed by its dehydration under acid hydrolysis to form 3 (see Fig. 6). The hydrated analogue of 3 has a molecular weight of 323, ([M⫹H]⫹, m/z 324) and in the full scan LC-ESI-MS analysis of the nonhydrolyzed sample, two major peaks are present in the m/z 324 ion chromatogram, one of which could be the hydrated adduct. In the hydrolyzed samples, however, these peaks are no longer present. In the second mechanism, 5-(hydroxymethyl)uracil

(5-OHmUra)—a known irradiation product of thymine [16] and starting material in the synthesis presented here— could be formed during irradiation and subsequently react with tyrosine under hydrolysis conditions to give 3 (as shown in Fig. 1). Full scan LC/MS analysis again provided support for this proposal: a peak was observed in the m/z 143 ion chromatogram of the nonhydrolyzed sample consistent with the presence of 5-OHmUra ([M⫹H]⫹, m/z 143.1) that was absent in the hydrolyzed sample. In addition, the reaction of 5-OHmUra with tyrosine to give 3 under the above hydrolysis conditions was confirmed by LC/MS in a separate experiment. The acid-catalyzed reaction between 5-OHmUra and tyrosine would also explain the presence of 3 in the hydrolyzed sample of the nonirradiated (control) solution of thymine and tyrosine, i.e., 46 ng mL-1. LC/MS/MS established 5-OHmUra to be a trace contaminant of the thymine standard. After hydrolysis of the thymine-tyrosine control sample, 5-OHmUra was no longer detected. This work also demonstrates that the adduct described here can form artefactually if the appropriate precur-

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Fig. 5. A typical set of selected reaction mass chromatograms for an irradiated sample. Two precursor-product reactions were monitored continuously: m/z 306 m/z 260 for the thymine-tyrosine adduct (bottom) and m/z 312 m/z 266 for the corresponding [13C6]-labeled adduct (top).

sors—5-(hydroxymethyl)uracil and tyrosine—are present in solution before acid treatment. Because acidic conditions are frequently employed to hydrolyze the constituent biopolymers in advance of chemical analysis, this finding has important implications. Irradiated samples of DNA-nucleohistone would be expected to contain both substates, and the acidic conditions employed during work up would yield the adduct. Consequently, we stress the need to take elaborate precautions to ensure that the substrates are not available when monitoring the formation of the adduct in biological systems. Further, our data lead us to conclude that some previously reported levels of the adduct may be artefactually elevated.

Development of accurate assays for the thymine-tyrosine adduct have been hampered by several factors, including a failure to obtain sufficient quantity of 3 as a reference standard. A convenient synthetic method for obtaining 3 is reported here involving a single-step reaction between 5-OHmU and L-tyrosine. The thymine-tyrosine adduct (3) is a major product of this reaction and has been fully characterized. Modifications to the synthesis allow the preparation of stable-isotope labeled forms of the compound and enable robust and precise analysis based on LC/MS/MS. LC/MS/MS was applied to the quantification of the adduct in an equimolar solution of tyrosine and thymine irradiated at several doses. The adduct was detected in each of these, albeit at low yield. Hydrolysis of the samples resulted in a 10-fold increase in the abundance of the thymine-tyrosine adduct. This work also demonstrates that the adduct described here can form artefactually under appropriate conditions: i.e., where 5-(hydroxymethyl)-uracil and tyrosine are present in solution before acid treatment. Future studies aimed at quantifying the adduct in biological systems must pay particular attention to the facile nature of the reaction between trace quantities of 5-(hydroxymethyl) uracil and tyrosine, and we are led to question previously reported values for the adduct that have not accounted for this issue. Acknowledgements — The authors are grateful to Assoc. Prof. J. Gebicki (Macquarie University, Sydney) who undertook the irradiation work and also provided advice on the calculation of radiation yield. NMR spectra were obtained by Ms. H. Stender and Dr. J. Hook (School of Chemistry, University of New South Wales). Drs. Bruce Ames and Tory Hagen are thanked for originally introducing the authors to this problem, and for helpful discussions relating to the work. This research was supported in part by the National Health and Medical Research Council, Australia. Mark W. Duncan also thanks Mr. Ray Williams for his ongoing support of our activities.

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Fig. 6. A possible mechanism for formation of the adduct involving formation of a hydrated analogue in situ during ␥-irradiation, followed by its dehydration under acid hydrolysis to form 3.

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BSTFA—bis(trimethylsilyl)-trifluoroacetamide DPC—DNA-protein crosslink EI— electron ionization 5-OHmUra—5-(hydroxymethyl)uracil GC/MS— gas chromatography-mass spectrometry LC/MS/MS—liquid chromatography-tandem mass spectrometry MTBSTFA—N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide NMR—nuclear magnetic resonance tBDMS—tert-butyldimethylsilyl TMS—trimethylsilyl tR—retention time