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Watson–Crick Base-Pair Radical Cation as a Model for Oxidative Damage in DNA Linda Feketeova, Bun Chan, George N. Khairallah, Vincent Steinmetz, Philippe Maître, Leo Radom, and Richard A. J. O'Hair J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.7b01251 • Publication Date (Web): 19 Jun 2017 Downloaded from http://pubs.acs.org on June 26, 2017

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The Journal of Physical Chemistry Letters

Watson–Crick Base-pair Radical Cation as a Model for Oxidative Damage in DNA Linda Feketeová1-4*, Bun Chan2,5,6*, George N. Khairallah1,2, Vincent Steinmetz7, Philippe Maitre7, Leo Radom2,5, and Richard A. J. O’Hair1,2* 1

School of Chemistry and Bio21 Institute of Molecular Science and Biotechnology, The University of Melbourne, Parkville, 3010 Victoria, Australia 2

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ARC Centre of Excellence in Free Radical Chemistry and Biotechnology Australia

Institut de Physique Nucléaire de Lyon, Université de Lyon, and CNRS/IN2P3, UMR5822, 69622 Villeurbanne, France 4

Institut für Ionenphysik und Angewandte Physik and Center for Molecular Biosciences,

Innsbruck (CMBI), Leopold Franzens Universität Innsbruck, Technikerstrasse 25, 6020 Innsbruck, Austria 5

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School of Chemistry, The University of Sydney, NSW 2006, Australia

Graduate School of Engineering, Nagasaki University, Bunkyo 1-14, Nagasaki 852-8521, Japan

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Laboratoire de Chimie Physique, Bâtiment 349, Université Paris-Sud, CNRS, Université ParisSaclay, F-91405 Orsay, France

ACS Paragon Plus Environment

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*Corresponding Authors [email protected], [email protected], [email protected]


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ABSTRACT The deleterious cellular effects of ionizing radiation are well known but the mechanisms causing DNA damage are poorly understood. The accepted molecular events involve initial oxidation and deprotonation at guanine sites, triggering hydrogen-atomabstraction reactions from the sugar moieties, causing DNA strand breaks. Probing the chemistry of the initially formed radical cation has been challenging. Here we generate, spectroscopically characterise and examine the reactivity of the Watson-Crick nucleobase-pair radical cation in the gas phase. We observe a rich chemistry, including proton transfer between the bases and propagation of the radical site in deoxyguanosine from the base to the sugar, thus rupturing the sugar. This first example of a gas-phase model system providing molecular-level details on the chemistry of ionized DNA base pair, paves the way towards a more complete understanding of molecular processes induced by radiation. It also highlights the role of radical propagation in chemistry, biology and nanotechnology.

TOC GRAPHICS


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Within seven years of Roentgen’s discovery of X-rays, the first radiation-induced cancer was reported1. By 1911, five radiation workers were found to have developed radiation-induced leukemia, a disease to which Marie Curie and her daughter Irene are thought to have eventually succumbed1. Although research on the effects of ionizing radiation2–3 predate Watson and Crick’s discovery of the structure of DNA4, it is now generally accepted that the molecular basis for oxidative damage to DNA involves nucleobase modifications and the formation of strand breaks, which can cause mutagenesis and cancer, and is involved in the aging process5–8. Guanine (G) has the lowest ionization energy of the four DNA bases, in both the gas phase9,10 and in solution11. Moreover, base pairing further lowers the oxidation potential of guanine12–14. While DNA charge transport can occur over long distances, it is well established that electron loss during ionization of DNA mostly occurs from G sites, producing radical cations (G•+•••C) of guanine–cytosine base pairs.15,16 This one-electron oxidation of DNA profoundly affects the acid/base properties of the base pair, with the guanine radical cations having greatly enhanced acidities17–19. In solution, deoxyguanosine (dG) has an enhanced acidity of ~5.6 pKa units17, while in the gas phase, 9-methylguanine radical cation 9MeG•+ is ~470 kJ mol−1 more acidic than 9MeG18, and dG•+ is ~415 kJ mol−1 more acidic than dG19. Indeed, proton-coupled electron and hole transfer becomes an important feature in radiation-damage processes9-14, with one proposed pathway for strand breakage proceeding via deprotonation to give [G−H]•, which triggers specific hydrogen-atom-abstraction reactions from the sugar moiety20. Deprotonation of the oxidized guanine in duplex DNA has been directly observed in pulse radiolysis experiments21. Such proton transfer from guanine to the complementary nucleobase cytosine (C) can lead to relocation of the radical site to the sugar22.


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Since Steenken has suggested that gas-phase acidity data may be more appropriate to characterize the equilibrium position of the proton in oxidized GC base pairs23, it is desirable to form simple functional gas-phase models to probe the fundamental chemistry of relevant radical cations. Previous work blended a combination of gas-phase ion–molecule reactions (IMR), collision-induced dissociation (CID), gas-phase infrared (IR) spectroscopy measurements and computational chemistry to show that the monomer radical cations 9MeG•+ (9-methylguanine) and dG•+ maintained the keto tautomeric form found in DNA in the gas phase, and that they had sufficiently enhanced acidities to theoretically transfer a proton to their base-pair partner18,19. We note that gas-phase studies on base pairing of neutral free nucleobases and mononucleosides demonstrated that complementary nucleobase pairing in DNA is independent of the phosphate– sugar chain24. Here we present the first gas-phase study of the structure and reactivity of the deoxyguanosine–deoxycytidine dimer radical cation dGdC•+, showing that it has a rich unimolecular chemistry, including proton transfer from dG•+ to dC. Electrospray ionization (ESI) of methanolic solutions containing a mixture of the nucleosides, deoxyguanosine (dG) and deoxycytidine (dC), incubated with Cu(NO3)2, resulted in the formation of a range of ions (see the Supporting Information (SI) and Figure S1). Multistage mass spectrometry (MSn) experiments were used to generate dGdC•+ radical cations via lowenergy CID involving multiple collisions of the doubly-charged copper nucleoside complexes [CuIIdGmdCn]2+ (with m + n = 4 and m, n ≠ 0) with the He bath gas. The abundance of dGdC•+ was significantly higher than dG2•+ expected from a statistical distribution, as shown for m = n = 2 (Figure 1a), suggesting enhanced stability for dGdC•+ as expected from a Watson–Crick base pair. This contrasts with [CuIIdG2Gs2]2+, which dissociates into both the homo (dGdG•+ and GsGs•+) and hetero (dGGs•+) dimer radical cations in close to the expected statistical


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distribution25. To confirm that a Watson–Crick base pair was indeed formed, a range of structures were surveyed using computational quantum chemistry procedures (see below), and the gas-phase IR spectrum was measured and matched to those determined from the computational chemistry survey (for a review on the gas-phase spectroscopy of nucleic acids see ref. 26.).


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Figure 1: Mass spectra showing the formation and fragmentation reactions of the deoxyguanosine–deoxycytidine dimer radical cation: (a) MS/MS of the copper(II) complex CuIIdG2dC22+ m/z 525.5. (b) MS3 of dGdC•+ m/z 494. The mass-selected precursor ion is designated with an asterisk (*).

Density functional theory (DFT) calculations at the B3LYP/6-31+G(d,p) level led to 22 structures of moderately low energy, and their structures and IR spectra are displayed in Figures S2 and S3 in the SI. The two lowest-energy structures contain the Watson–Crick hydrogenbonding motif, but differ in the location of the central H. The lowest-energy Watson–Crick structure (1A, designated as dGdC•+, with the radical site located on the dG) has the H bound...) has the H bound to the N1 position of the dG, while in isomer 1B (designated as [dG−H]•dCH+), lying 3 kJ mol−1 higher in energy, proton transfer has occurred to relocate the proton to position N3 of the dC. Several other Watson–Crick base-paired structures were found with different sugar conformations, but these lie higher in energy (within 41 kJ mol−1 of 1A). All non-Watson–Crick structures were found to lie considerably higher in energy, with the lowest of them lying 65 kJ mol−1 higher in energy than 1A and involving hydrogen-bonding interactions between the N−H of the guanine base and the O of the cytosine base. Figure 2a shows the measured IR spectrum of the dGdC•+ radical cation dimer in the regions 900–1750 cm−1 and 3200–3700 cm−1, while Figures 2b and 2c display the spectra calculated for the dGdC•+ and [dG−H]•dCH+ isomers, respectively, where in both cases the radical site is located on the dG nucleobase. In the [dG−H]•dCH+ structure, the N1 proton of the dG has been transferred to N3 of the dC (Figure 2c). Table S1 (SI) lists the experimental band positions. Four fragment ions, discussed further below (m/z 464, 405, 267 and 228, eqs. 1-4), were used to generate the IRMPD spectrum. In the fingerprint region (900–1800 cm−1), the vibrational bands measured experimentally (Figure 2a) show good agreement with the frequencies calculated for


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the lowest energy Watson–Crick structure (1A) (Figure 2b), especially, below 1500 cm−1. The assignment of the broad and unresolved feature between 1500 and 1750 cm−1 is more difficult and we cannot exclude a population of 1B (Figure 2c). In the higher energy region (3200–3700 cm−1), a doublet is observed with maxima at 3470 and 3520 cm−1, as well as a feature at 3655 cm−1. This IR pattern is a clear signature of the Watson–Crick hydrogen-bonded motif, in particular, the doublet that is a signature of the free amino NH stretches, as in 1A and 1B. The observation of only one OH stretching band is a signature of the free sugar OH as predicted for 1A and 1B. As expected, a broad band is observed at lower frequencies corresponding to the three strongly red-shifted hydrogen-bonded NH stretches. Unfortunately, this cannot be resolved by comparison with experiment27,28, given that theory predicts very similar spectra for 1A and 1B. However, the IR spectrum clearly confirms the existence of the Watson-Crick hydrogenbonded nucleoside base-pair.


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Figure 2: (a) IR spectrum of dGdC•+ measured experimentally. Experimental IRMPD spectra correspond to the IRMPD efficiency (–ln[Parent/(Parent+Fragment)]). Two different scales are used for the CLIO and OPO ranges. (b) IR spectrum calculated using B3LYP/6-31+G(d,p) for dGdC•+ (1A). (c) IR spectrum calculated using B3LYP/6-31+G(d,p) for [dG−H]•dCH+ (1B). The experimental spectrum is included for comparison in dashed blue in (b) and (c).

The gas-phase unimolecular chemistry of dGdC•+ was examined in detail via CID involving multiple collisions with the helium bath gas of the linear ion trap mass spectrometer. Three main


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types of fragmentation reactions were observed (Figure 1b): (i) monomer loss to form the radical cation of dG at m/z 267 (eq. 1); (ii) monomer loss coupled with proton transfer to form dCH+ at m/z 228 (eq. 2); and (iii) radical-induced sugar fragmentation from the dG site, i.e., loss of CH2O (m/z 464) (eq. 3), followed by further loss of C2H3O2 (m/z 405) (eq. 4) (see discussion below). Cleavage of the N−C glycosidic bond is a minor channel (eq. 5). dGdC•+ → dG•+ + dC

(1)

dGdC•+ → [dG−H]• + dCH+

(2)

dGdC•+ → [dGdC−CH2O]•+ + CH2O

(3)

dGdC•+ → [dGdC−C3H5O3] + + C3H5O3•

(4)

dGdC•+ → [dGdC−C5H8O3]•+ + C5H8O3

(5)

For the radical-induced sugar fragmentation, eq. 3 and eq. 4, the key question is: ‘Is the sugar damage nucleobase specific?’. Further multistage mass spectrometry experiments, shown in Figure S4 (SI), revealed that the radical-induced damage is nucleobase specific, and occurs on the sugar moiety of the deoxyguanosine nucleoside. CID reactions of [dGdC−CH2O]•+ (m/z 464 in Figure S4a in the SI) show that the fragment ion at m/z 405 is associated with the further loss of C2H3O2 forming [dGdC−C3H5O3]•+, rather than from an immediate loss of C3H5O3 directly from the dGdC•+ dimer. Further CID of [dGdC−C3H5O3]•+ m/z 405 gives the protonated nucleoside dCH+ m/z 228 with 100% relative abundance, which highlights that the deoxycytidine is intact (see Figure S4c in the SI). The fragment ion [dGdC−C5H8O3]•+ m/z 378, which is associated with the loss of the sugar C5H8O3, i.e., the cleavage of the N−C glycosidic bond (eq. 5), also brings up the same question ‘Is the loss of the sugar moiety nucleobase specific?’. To address this point, the fragment ion [dGdC−C5H8O3]•+ m/z 378 was also isolated and subjected to further CID (Figure S4b in the SI).


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The observation of intact deoxyguanosine, dG•+ m/z 267, as well as intact deoxycytidine, dCH+ m/z 228, shows that loss of the sugar through N−C glycosidic bond cleavage is not nucleobase specific. Experiments using deuterated solvents result in H/D exchange of all the labile protons in the dGdC•+. The total number of H replaced by D is 9 (5 for dG and 4 for dC), which causes a shift from m/z 494 to m/z 503. CID of the ion at m/z 503 (see Figure S5 in SI) confirmed the loss of CH2O m/z 473 (eq. 3) and showed that the C3H5O3• loss (eq. 4) involves one labile H (as C3H4DO3 is lost to give an ion at m/z 413). These isotope labelling experiments are consistent with the labile H from an OH group of the sugar being transferred to the nucleobase site prior to the release of CH2O or C3H5O3•.


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Figure 3: (a) M06-2X/6-311+G(3df,2p) schematic free energy profile (298 K, kJ mol−1) associated with the fragmentation reactions of dGdC•+: loss of CH2O (3–7); loss of sugar C5H8O3


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(8–10) followed by proton transfer to the complementary base (11-12); and proton transfer (1A– 1B). The detailed structures 1–12 associated with the free energy profile are displayed in Figure S6 (SI). (b) Key structures in (a) highlighting (top) guanine specific sugar damage irrespective of the position of the charge on either of the two nucleobases, and (bottom) sugar cleavage is a charge-directed fragmentation reaction that requires the charge to be present on the corresponding nucleobase.

The reactions of the dGdC•+ were investigated using computational quantum chemical calculations. A schematic energy profile is shown in Figure 3a, while the corresponding detailed structures are shown in Figure S6 (SI). An examination of our computational results reveals the following: (i) Proton transfer within the dimer to isomerize dGdC•+ (1A) into [dG−H]•dCH+ (1B) is almost thermoneutral, with a reaction free energy of 3 kJ mol−1 and a barrier of 7 kJ mol−1. (ii) Separation of the nucleosides in [dG−H]•dCH+ (1B) to give [dG−H]• and dCH+ (Figure 3a) is endothermic by 97 kJ mol−1, which sets the energy cost of this reaction (eq. 2) at 7 kJ mol−1 above the endothermicity of nucleoside separation in dGdC•+ (to give dG•+ and dC, Figure 3a), as in eq. 1. The calculations are consistent with the experimental observation of radical cation dG•+ m/z 267 and protonated dCH+ m/z 228 with relative abundances of 100% and 16%, respectively (see Figure 1b). (iii) There are two pathways for the elimination of formaldehyde from the sugar moiety of dG within dGdC•+ (1A) (Figure 3a, eq. 3). The first involves direct loss of CH2O from the dGdC•+ isomer and is calculated to have a barrier (via 3A) of 117 kJ mol−1. The second involves initial proton transfer to form [dG−H]•dCH+ (1B) followed by the loss of CH2O with a barrier (via 3B) of 118 kJ mol−1. Thus, both pathways are possible, although the loss of formaldehyde requires 24 kJ mol−1 more energy than the formation of radical cation dG•+ m/z 267, eq. 1. This is consistent


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with the observed 3% relative abundance of [dGdC−CH2O]•+ m/z 464 (see Figure 1b). The guanine specific sugar damage is highlighted by the key structures depicted in Figure 3b. (iv) The loss of the sugar C5H8O3, which was experimentally shown to be nucleobase nonspecific, requires two pathways for N−C glycosidic bond cleavage (eq. 5), both of which were found from our DFT calculations to be triggered by the presence of a charge on the nucleoside losing the sugar. Both of these have higher barriers than the other fragmentation pathways, which is consistent with the experimental observation of < 1% of relative abundance of fragment corresponding to this cleavage at m/z 378, loss of C5H8O3. The barrier for N−C glycosidic bond cleavage in the dGdC•+ isomer (1A) is 131 kJ mol−1 (via 9A, Figure 3a) and the sugar is cleaved from the guanine base. In the case of N−C glycosidic bond cleavage in the [dG−H]•dCH+ isomer (1B), there is a barrier of 140 kJ mol−1 (via 9B, Figure 3a) and the sugar is cleaved from the cytosine base. These results are consistent with the abundance of 100% for the dCH+ formed at m/z 228 relative to dG•+ m/z 267 at 43% (see Figure S4b in the SI). The charge-directed sugar cleavage is highlighted by the key structures shown in Figure 3b. In order to probe the types of bimolecular reactions that a dGdC•+ site in DNA might undergo, the mass-selected dGdC•+ radical cations were allowed to undergo an ion–molecule reaction with diisopropylethylamine (iPr2NEt, Figure S7a in the SI). Proton transfer to iPr2NEt to give [dG−H]•dC, represents a minor channel, consistent with our DFT calculations, which also suggests the loss of the proton from N2 of dG (Figure S8 in SI). The major product arises via Hatom abstraction by the radical site on dG to give [dG+H]+dC. Based on the relative energies from the DFT calculations (Figure S9 in SI), H-atom transfer is likely to occur to N7 of dG. Related reactions are observed for diisopropylamine, iPr2NH (Figure S7b in SI). Importantly, the calculated homolytic bond dissociation enthalpy of 401 kJ mol–1 for [dG+H(N7)]+ is comparable


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to that for a typical C–H bond, suggesting that the generation of dG•+ may initiate formation of other radicals via intra- or inter-molecular hydrogen-abstraction reactions. One electron oxidation of DNA has been widely examined in a number of environments, ranging from in vivo cellular studies to in vitro studies in solution, on surfaces, in carbon nanotube single molecule devices and in DNA crystals.16,29 This work provides the first example of the determination of the gas-phase structure of the Watson–Crick hydrogen-bonded, nucleobase-paired radical cation dGdC•+, and provides mechanistic insights into its fundamental reactivity. We find that proton transfer can be in competition with both selective and nonselective nucleobase-induced cleavage reactions involving the sugar moieties.

METHODS All reagents were used as supplied: Cu(NO3)2 (Ajax chemicals, 99 %), deoxyguanosine, dG, (Sigma, 99 %), and deoxycytidine, dC, (Sigma, 99 %). Complexes were prepared by mixing 2:1 mM solutions of the nucleosides:Cu(NO3)2, dissolved in 3:1 methanol:water, directly before infusing the reaction mixture into the mass spectrometer.

Mass spectrometry experiments All experiments were carried out using a commercially available Finnigan- LTQ-FT (Thermo, Bremen, Germany) mass spectrometer equipped with an ESI source described in detail elsewhere30. The samples were introduced into the mass spectrometer at 5.0 µL/min via ESI. Typical ESI conditions used were: spray voltage, 3.3–5.0 kV; capillary temperature, 250 °C; nitrogen sheath pressure, 8–40 (arbitrary units). The capillary voltage and the tube lens offset were tuned to maximise the desired peak. The injection time was set using the automatic gain


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control function. The LTQ-FT mass spectrometer consists of: (i) a linear ion trap (LTQ); (ii) iontransfer optics; and (iii) an FT-ICR mass analyser. For the multistage mass spectrometry (MSn) experiments, the desired ions produced via ESI were trapped in the LTQ and subjected to CID at a He bath gas pressure of ca. 5 x 10−3 Torr. Low-energy CID was carried out by mass selecting the desired ions with a 1.5–6 m/z units window and subjecting them to multiple collisions with the He bath gas using the following typical conditions: normalised collision energy between 16% and 40%, which determines the translational kinetic energy of the ions; activation (Q), 0.25– 0.35, which assigns the radio-frequency (RF) used to fragment ions, and activation time of 30 ms that is the time set to excite the ions via CID. The high resolution of the FT-ICR mass spectrometer was used to confirm the charge states of the mass-selected precursor ions. For highresolution mass analysis, the ions were transferred via the ion optics transfer region (2 x 10−7 Torr) into an FT-ICR cell at a pressure below 1.5 x 10−9 Torr. The ion-molecule reactions were carried out in the ion trap using a modification previously described.31 The ions stored in the ion trap during the ion-molecule reactions are quasi thermalized by the helium bath gas and are at near room temperature 32.

Gas-phase Infrared Spectroscopy Experiments IRMPD spectroscopy was performed employing a commercial 7 T FT- ICR mass spectrometer (Bruker, Apex Qe) into which pulsed IR light is coupled. The detailed layout of this experimental apparatus is described elsewhere33. IR spectroscopy in the 1000–2000 cm−1 spectral range was carried out using the free electron laser (FEL) at CLIO34. The 3200–3700 cm−1 wavenumber range is explored using an IR OPO/OPA laser (LaserVision), pumped by a 25Hz Nd:YAG laser (Innolas Spitlight 600, 550 mJ per pulse, 1064 nm, 4–6 ns pulse duration). In the


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presently covered wavenumber range, the typical output energy slowly decreases going from 3700 cm−1 to 3200 cm−1 with a spectral bandwidth of ~5 cm−1. Desired mass-selected ions are trapped in a ~5 cm long hexapole ion-trap contained within a collision cell where ions normally are collisionally thermalised using a flow of highpurity argon buffer gas. The ions are pulsed driven into the ICR cell, where they are irradiated. Upon resonant vibrational excitation, dissociation of the selected radical ion was monitored via its fragment peaks. The abundances of these photofragments and their corresponding precursors were recorded as a function of the IR wavelength in order to derive the IR action spectra, where the IRMPD efficiency is plotted against the photon energy. In order to check the validity of comparing the measured versus the calculated IR spectra of the dGdC system, a smaller molecule, i.e., 9-methyl guanine was used as the test18. It was clear from the spectra that the calculated minimum energy structure displays a band that corresponds closely with the one that is experimentally observed. This further confirms that the calculation method used is appropriate.

Computational Methods Standard DFT calculations were carried out with Gaussian 0935. The literature structures for the neutral dGdC dimers were used as our initial guesses for the optimization of the dGdC•+ radical cation dimers.

Geometries for the dGdC•+ dimer and its fragmentation pathways,

including transition structures, were optimized with the M06-2X procedure36 with the 631+G(d,p) basis set. Following each geometry optimization, harmonic frequency analysis was carried out to confirm the nature of the stationary point as an equilibrium structure or a transition structure. Zero-point vibrational energies (ZPVEs), thermal corrections for enthalpies (∆H298),


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and entropies (S298) at 298 K under a pressure of 1 mTorr were calculated using M06-2X/631+G(d,p) harmonic vibrational frequencies. Literature scale factors for the closely-related M05-2X/6-31+G(d,p) procedure37 were employed in the evaluation of ZPVEs (0.9631), ∆H298 (0.9504) and S298 (0.9445). Improved single-point energies were obtained at the M06-2X/6311+G(3df,2p) level. The M06-2X procedure has been shown to yield accurate energies for barrier heights as well as thermochemistry for a diverse set of systems38. All relative energies correspond to free energies at 298 K in kJ mol–1. The IR spectra of possible dGdC•+ dimer radical cations were computed using the B3LYP procedure with the 6-31+G(d,p) basis set. The calculated vibrational frequencies were scaled using scale factors used previously18.

ASSOCIATED CONTENT Supporting

Information.

The

following

files

are

available

free

of

charge.

ESI mass spectrum of a mixture of Cu(NO3)2 and nucleosides, deoxyguanosine and deoxycytidine; Experimental and theoretical IR spectra of dGdC radical cations; Experimental and theoretical IR spectra of (Watson-Crick) dGdC radical cations; Assignment of experimentallyobserved vibrational bands; CID spectra related to dGdC radical cation; CID of deuterated dGdC radical cation; Calculated structures (including cartesian coordinates) related to fragmentation of dGdC radical cation; IMR of dGdC radical cation; Calculated deprotonation enthalpies of dGdC radical cation; Calculated exothermicities for hydrogen abstraction by dGdC radical cation (including cartesian coordinates).

AUTHOR INFORMATION The authors declare no competing financial interests.


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ACKNOWLEDGMENT We thank the Australian Research Council (ARC) for financial support through the ARC Centre of Excellence and Discovery programs. LF thanks the ARC for the award of an APD. GNK thanks UPSud for a Visiting Scholar Fellowship. Financial support from the French FTICR network (FR3624 CNRS) is gratefully acknowledged. The authors gratefully acknowledge the generous allocation of computing time from the National Computational Infrastructure (NCI) National Facility, Intersect Australia Ltd, RIKEN ACCC, the Institute for Molecular Science, and the Victorian Partnership for Advanced Computing (VPAC).

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