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Reactivity Pattern of Bromonucleosides Induced by 2‑Hydroxypropyl Radicals: Photochemical, Radiation Chemical, and Computational Studies Magdalena Zdrowowicz,† Lidia Chomicz,† Justyna Miloch,† Justyna Wiczk,† Janusz Rak,*,† Gabriel Kciuk,‡ and Krzysztof Bobrowski‡ †

Faculty of Chemistry, University of Gdañsk, Wita Stwosza 63, 80-308 Gdańsk, Poland Centre of Radiation Research and Technology, Institute of Nuclear Chemistry and Technology, Dorodna 16, 03-195 Warsaw, Poland

‡

S Supporting Information *

ABSTRACT: The bromonucleosides (BrdX’s) 5-bromo-2′deoxyuridine (BrdU), 5-bromo-2′-deoxycytidine (BrdC), 8bromo-2′-deoxyadenosine (BrdA), and 5-bromo-2′-deoxyguanosine (BrdG) may substitute for ordinary nucleosides in DNA. As indicated by electron-stimulated desorption experiments, such a modified biopolymer is greater than 2−3-fold more sensitive to damage induced by excess electrons. The other major product of water radiolysis, the •OH radical, may form a number of other radicals in chemical reactions with the complex content of the cell. Thus, the well-proved BrdUlabeled DNA radiosensitivity may be, at least in part, related to secondary organic radicals. Therefore, in the current study, the propensity of BrdX’s to damage induced by 2-hydroxypropyl radical (OHisop•)a prototype radical specieswas investigated. The HPLC and LC-MS analyses revealed the formation of two major products from the brominated pyrimidine nucleosides, a native nucleoside and an adduct of BrdX and OHisop• , and only an adduct of BrdX from the bromopurine nucleosides. Quantum chemical calculations ascribed this evident difference between purines and pyrimidines to the electron transfer from OHisop• to BrdX that is especially favorable in pyrimidines.

1. INTRODUCTION Hypoxia is a hallmark of solid tumors,1 and the increased radioresistance of hypoxic cells2 results in a higher dose of radiation being needed for effective radiotherapy. As a consequence, a risk of secondary cancer and other early and late side effects is unavoidable in patients with tumors. One approach enabling hypoxia of tumor cells to be overcome is to deliver sensitizing substances dubbed radiosensitizers to a tumor. Such species may be grouped into five categories:3,4 (i) suppressors of the natural radioprotectors occurring in cells (e.g., thiols), (ii) substances with radiation-induced cytotoxicity, (iii) inhibitors of DNA repair processes, (iv) modified nucleosides that are incorporated into intracellular DNA, and (v) electron affinity substances that work as oxygen mimetics. Modified nucleosides seem to be especially well suited for radiotherapy, since DNA is one of the main targets of such treatment. Indeed, in an elegant radiolytic study, Warters et al.5 demonstrated that only radiation that reaches the nucleus leads to cellular death. Thus, a suitable modification of DNA should have effects that lead to increased sensitivity of cells to ionizing radiation. In the past, it was shown that 5-bromo-2′-deoxyuridine (BrdU) is a good substrate for thymidine kinase6 and, after © 2015 American Chemical Society

phosphorylation, is easily incorporated into cellular DNA by human polymerases.7 Several in vitro trials on tumor cell lines demonstrated that up to 50% thymidine could be substituted with bromouridine during DNA replication.8−10 Such modified DNA is effectively destroyed by the action of ionizing radiation, even under hypoxic conditions. This is due to a high electron affinity of 5-bromouracil (BrU) molecules. These molecules are prone to the so-called dissociative electron attachment (DEA)a process that leads to the reactive uracil-5-yl radical (dU•) via the unstable 5-bromouracil anion (BrdU•−).11−13 Thus, hydrated electrons (e−aq), the reducing counterpart of hydroxyl radicals (•OH) (major products of water radiolysis),14 become a damaging factor to the BrdU-labeled DNA, although they are inactive toward native DNA, especially when it comes to strand breaks.15 Here, it is worth emphasizing that not only BrU but also the bromo derivatives of the remaining nucleobases, i.e., 5bromocytosine (BrC), 8-bromoadenine (BrA), and 8-bromoguanine (BrG), are prone to the DEA process.13 In an aqueous Received: February 26, 2015 Revised: May 8, 2015 Published: May 14, 2015 6545

DOI: 10.1021/acs.jpcb.5b01904 J. Phys. Chem. B 2015, 119, 6545−6554

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The Journal of Physical Chemistry B

BrdX’s. In this work, we investigate the radical processes involving all four BrdX’s with the selected organic radical (OHisop•) formed in the reactions of either photochemically generated triplet state of acetone (reactions 1 and 2) or radiationally induced •OH radicals with isopropanol (reactions 3−5):

solution, the adiabatic electron affinities (AEAs) of bromonucleobases (BrNB’s) are at most a few tenths of an electronvolt larger than those of their parent nucleobases (cf. the respective AEAs values gathered in refs 13 and 16). However, an electron attachment to BrNB’s leads to a swift release of the bromide anion (Br−), which leaves behind a reactive radical of the nucleobase (NB•), a potent damaging factor, and makes the electron attachment to BrNB’s an irreversible process. Low-temperature EPR measurements showed that 5-bromo-2′-deoxycytidine (BrdC) in DNA was a substantially more efficient electron scavenger than 2′-deoxycytidine (dC) itself.17 Moreover, it was demonstrated that BrdC and BrdU trigger a similar degree of radiosensitization while incorporated into cell lines.18 Furthermore, a rapid Br− release from the anions of bromopurine nucleosides formed in the reaction with e−aq was shown in radiolytic experiments by Chatgilialoglu’s group.19−23 The same group, in excellent studies on photoinduced electron transfer24,25 from a reduced and deprotonated flavin donor to a distant brominated nucleobase, demonstrated the occurrence of efficient Br− release in the BrNB-labeled double-stranded DNA. Although the yield of debromination was lower for the brominated purines,24 the DEA process proceeded even though BrU and a brominated purine were simultaneously present in the same duplex.25 Recently, the radiosensitizing properties of all four bromonucleosides in a DNA context were also studied by the Sanche group and our group26,27 by bombarding a thin layer of single-stranded trinucleotides TYT (where Y = BrdU, BrdC, BrdA, or BrdG), adsorbed on an Au substrate, with low energy electrons (LEEs) under ultrahigh vacuum. The electronstimulated desorption (ESD) to the gas phase by electrons of 0−20 eV indicated that one of the most abundant ESD signals for all of the studied TYT trimers was associated with desorption of Br−.26 Moreover, a 2−3-fold increase in the sensitivity of TYT oligonucleotides compared to the susceptibility of native trimers to be damaged by LEEs was also demonstrated.27 Hence, it seems understandable that the radiosensitizing properties of bromonucleobases are related to the direct reaction between BrNB’s and solvated electrons. Another, less evident process leading to the labeled DNA damage may concern the oxidation of organic radicals, abundant in cellular environment irradiated with ionizing radiation, by BrNB’s. Such an electron-transfer process, from a radical to BrNB molecule, was postulated by Zimbrick et al.28 in their work devoted to the pulse and stationary radiolysis of aqueous solutions containing BrdU or thymidine. Interestingly, organic radicals can be generated not only within the radiolysis of aqueous solutions containing biomolecules but also with the help of much less energetic radiation. For instance, in an aqueous, deoxygenated solution of isopropanol with a small amount of acetone as a photosensitizer, 2-hydroxypropyl radicals (OHisop•) are formed very efficiently as a result of irradiation with near-UV photons.29 Indeed, Görner’s29 as well as our own studies30 demonstrate that, under the conditions described above, BrdU is efficiently converted to 2′-deoxyuridine (dU), which mirrors the processes described by Zimbrick et al.28 for reaction between BrdU and e−aq generated radiolytically. The current paper is dedicated not only to an extension of our previous studies on the photoinduced reactivity of BrdU,30 including in addition three remaining BrdX (where X = C, A, and G), but also to the γ-radiation-induced reactivity of all

H3C−C(O)−CH3 → 3[H3C−C(O)−CH3]*

(1)

[H3C−C(O)−CH3]* + (CH3)2 −CH−OH

3

→ 2H3C−C•(OH)−CH3

H 2O ⇝ •OH,

•

(2)

H, e−aq

(3)

e−aq + N2O + H 2O → N2 + •OH + OH−

(4)

•

OH + (CH3)2 −CH−OH → (CH3)2 −C•−OH + H 2O (5)

In photochemical studies, the aqueous solutions of all four BrdX’s were irradiated in the presence of acetone as a sensitizer and isopropanol as a radical source. In radiation chemical studies, the aqueous solutions of all four BrdX’s were irradiated in N2O-saturated solutions containing only isopropanol. Under the employed experimental conditions, the reactivity of the BrdX studied is significant. The LC-MS experiments show that a native nucleoside and an adduct of the nucleoside and OHisop• are formed in comparable amounts in the irradiated bromopyrimidine solutions while the adduct is a major product in the irradiated bromopurine solutions. This striking difference between brominated nucleosides correlates well with their vertical electron attachment energies (VAEs). All these findings lead to the reaction mechanism in which the formation of native nucleosides is due to electron transfer (ET) between a bromonucleoside and the OHisop• radical. On the other hand, the adduct is generated via radical substitution. Since only in the ET process is a reactive nucleobase radical formed as an intermediate species, our findings suggest the brominated pyrimidines to be better sensitizers of radical DNA damage than the brominated purines.

2. EXPERIMENTAL SECTION 2.1. Materials. The brominated nucleosides (BrdU, BrdC, BrdA, and BrdG) of >99% purity and acetonitrile were purchased from Sigma-Aldrich. Aqueous solutions (ultrapure water obtained using a Milli-Q system from Hydrolab, Poland HLP) containing ca. 1.0 × 10−4 M of a brominated nucleoside, 1.4 M isopropanol (Chempur, Poland), and 0.15 M acetone (Chempur, Poland) were prepared for photolysis, while the analogous solutions devoid of acetone were used for radiolysis. Formic acid and acetonitrile used in HPLC analysis were purchased from POCH S.A., Poland, and Sigma-Aldrich, Poland, respectively. 2.2. Steady-State Photolysis. Literature reports suggest removing oxygen by purging with argon prior to UV irradiation since the triplet of acetone is the species responsible for the studied reaction progress.29,30 Since the flow of Ar in a capillary is not easy to control without a special microdosimeter, deoxygenated samples may differ substantially one from another with the concentration of acetone. Therefore, all measurements have been performed in non-deoxygenated solutions being in equilibrium with air. 6546


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Figure 1. HPLC and MS analysis of the UV-irradiated solutions of bromopyrimidines: BrdU (a) and BrdC (b). Middle panel: HPLC traces of water solutions containing 10% of isopropanol, 0.15 M acetone and 1 × 10−4 M BrdX before (black) and after (pink) UV irradiation. Upper panel: MS spectra (in the positive ionization mode) of HPLC signals. Lower panel: MS/MS spectra of HPLC signals and ion identities.

Photolysis was carried out in quartz capillaries (3 × 3 mm) containing 50 μL of solution with a 500 W high-pressure mercury lamp for 15 min. The 300 nm wavelength of incident

light (half−width 2.5 nm) was selected using a grating monochromator (M250, Optel, Opole, Poland) and focused onto a 3 × 5 mm area of the quartz capillary. 6547


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Figure 2. HPLC and MS analysis of the UV-irradiated solution of bromopurines: BrdA (a) and BrdG (b). Middle panel: HPLC traces of the water solutions containing 10% isopropanol, 0.15 M acetone, and 1 × 10−4 M BrdX before (black) and after (pink) UV irradiation. Upper panel: MS spectra (in the positive ionization mode) of HPLC signals. Bottom panel: MS/MS spectra of HPLC signals and ion identities.

2.3. Steady-State Radiolysis. Stationary γ-radiolysis was performed using a 60Co source (Issledovatel, USSR) in the Institute of Nuclear Chemistry and Technology (Warsaw, Poland). The dose rate equal to 1.05 Gy min−1 was determined

using the Fricke chemical dosimeter.31 All γ-irradiations were performed in quartz capillaries (3 × 3 mm) at room temperature with the radiation dose of 140 Gy. They were filled with the 1 × 10−4 M solution of each BrdX containing 6548


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The Journal of Physical Chemistry B 10% isopropanol in the total volume of 50 μL. Each solution was saturated with N2O before irradiation in order to convert the solvated electrons into hydroxyl radicals (see reaction 4). 2.4. HPLC and LC-MS/MS. All HPLC separations were performed using a Dionex UltiMate 3000 apparatus with diode array detector, which was set at 260 nm for monitoring the effluents. An Atlantis reverse-phase dC18 column (4.6 × 150 mm; 5 μm in particle size) was used for all separations. The separations of BrdC and BrdU solutions were done using an isocratic mobile phase A: a buffer containing deionized water, acetonitrile, and 1% formic acid (pH 2.55; 87.7:2:10.3, v/v/v). An isocratic elution system of 99% phase A/1% phase B (80% acetonitrile) was employed for BrdA analysis, and in the case of BrdG, the separation conditions were as follows: isocratic elution for 40 min with the use of mobile phase A, then the linear gradient elution from 0% B to 10% B for 15 min. Each analysis was performed at the same flow rate of 1 mL/min and column temperature of 30 °C. An Agilent 1200 HPLC system was employed for the LCMS/MS experiments. The effluent was coupled to an HCTultra ion-trap mass spectrometer, which was operated in the positiveion mode. The spray voltage was set at −4.0 kV, and drying gas (N2) pressure, flow, and temperature were 50 psi, 11 L/min, and 360 °C, respectively. Each spectrum was obtained by averaging three scans. 2.5. Computational Details. All calculations were performed with the density functional theory (DFT) methods, using the M06-2x32 hybrid meta exchange correlation functional and the 6-31++G(d,p) basis set.33 Very recently, it has been shown that this level of theory leads to energetic characteristics (activation barriers for bond cleavage in particular) of the anion of 3′ and 5′ thymidine monophosphate differing by no more than 2−3 kcal/mol (ca. 0.1 eV) from the reference CCSD(T) values.34 The Polarizable Continuum Model (PCM)35 was employed, with the generic solvent (denoted “mix” in this report) with the static dielectric constant (ε) equal to 72.446 in order to account for the effect of polar environment. The latter value is a weighted average calculated using ε = 78.355 for water and ε = 19.264 for isopropanol at 298 K as well as the actual constitution of the mixed solvent employed in the experiment (10% isopropanol in water). The unconstrained geometry optimizations for stationary points (minima and transition states) were carried out in both the gas phase and mixed water/isopropanol solvent (“mix”). The excited-state calculations were performed using the TD-DFT method, with the B3LYP36 functional and 6-31++G(d,p) basis set, to be consistent with our previous report.30 The energy changes (ΔE) calculated for particular reaction steps are the differences between the electronic energies of products and substrates. The corresponding Gibbs free energies changes (ΔG) result from ΔE values corrected for zero-point energies, thermal contributions to energies, and the pV and entropy terms. These terms were computed in the rigid rotor− harmonic oscillator approximation37 at T = 298 K and p = 1 atm. Solvent ΔG values were obtained in the same manner as the gas-phase reaction Gibbs free energies.38 The Gaussian0939 code was used for all computations, while the molecule structures were visualized with the GaussView package.40

reactive uracil-5-yl radical which may damage DNA, providing that BrdU constitutes a part of the biopolymer. In the following, we will discuss reactions between bromonucleosides, i.e., BrdU, BrdC, BrdA, and BrdG, and the OHisop• radical. In these systems, the excited triplet of acetone, that is formed very efficiently in UV irradiation (see reaction 1),41 abstracts a hydrogen atom from isopropanol, producing the OHisop• radical (see reaction 2), which, in the next step, interacts with BrdX (reaction 6): OHisop• + BrdX → products

(6)

This same radical reaction is also triggered radiolytically by γirradiation of N2O-saturated aqueous solutions of BrdX containing isopropanol (see reactions 3−6). 3.1. HPLC and MS Analyses. 3.1.1. UV-Irradiated BrdU, BrdC, BrdG, and BrdA (BrdX) Solutions. The HPLC traces for water/isopropanol solutions, containing 1 × 10−4 M BrdX and 0.12 M acetone, irradiated for 150 min with 300 nm photons (the dose absorbed by acetone amounted to ca. 1400 J/m2), are depicted in the middle panels of Figures 1 and 2. The most intense signal, with the retention time ∼4 min, observed in all chromatograms is due to acetone. It is worthy to note that most of HPLC signals either are not characterized by a Gaussian shape (see, e.g., those of BrdU or BrdC in Figure 1a and 1b, respectively) or they are observed as double peaks (see, e.g., the dU, dA, and dG signals in Figures 1a, 2a, and 2b, respectively). This effect, already described in our earlier investigation,30 results from a disturbance in the chromatographic process by the presence of significant amount (10%) of isopropanol in the analyzed samples, which makes the elution capability of the analyzed samples to be larger than that of the mobile phase used for the separation. The identity of particular products was confirmed by LC-MS analysis. The MS spectra (in a positive ionization mode) for the HPLC signals depicted in the middle chromatogram are shown (note the +MS signals marked with a circle corresponding to the respective parent ions) in the upper part of Figures 1 and 2. In the case of BrdU, the MS picture recorded in positive ionization is somewhat confusing due to the fact that particular signals originate from the adducts with sodium cation (Na+) (see Figure 1a). Therefore, both the MS and MS/MS spectra for the BrdU photolyte were recorded in negative ionization (see Figure S1). The MS/MS spectra, (where the fragmentation of particular ions is indicated), are presented in the bottom part of Figures 1 and 2. For instance, the parent ions of the adduct of dC and OHisop• (add-dC), BrdC and dC with the m/z ratio equal to 286.1, 307.2, and 228.1, respectively (positive ionization), appear in the respective MS spectra and correspond to the most or second most intensive signal (see the upper part of Figure 1b). The fragmentations of the cations mentioned above (depicted in the bottom part of Figure 1b) show that both dC and BrdC loose sugar. On the other hand add-dC undergoes a more complex fragmentation since the ions corresponding to the elimination of the water molecule or/and sugar residue are present. Similar analysis of the HPLC and LC-MS(MS/MS) results for the remaining photolytes (Figures 1a, 2a, and 2b) enable identification of the main photoproducts. Hence, the data presented in Figures 1 and 2 demonstrate unequivocally that UV irradiation of the systems studied leads to two types of products: debrominated (native) nucleoside (dX), and the adduct of dX and OHisop• (add-dX). 3.1.2. γ-Irradiated BrdU, BrdC, BrdG, and BrdA (BrdX) Solutions. The solutions containing 1 × 10−4 M BrdX and 10%

3. RESULTS AND DISCUSSION Already in the late 1960s, it was demonstrated that BrdU is able to react with organic radicals.28 These reactions lead to a 6549


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The Journal of Physical Chemistry B isopropanol were saturated with N2O prior to radiolysis which converted all e−aq into the •OH radicals (see reaction 4). The • OH radicals, in turn, reacted with isopropanol producing the OHisop• radicals (see reaction 5). This way, the reactions studied proceed without the involvement of the triplet of acetone. The chromatograms corresponding to the experiments described above, performed for BrdU, BrdC, BrdG, and BrdA are presented in Figures 3a, 3b, 4a, and 4b, respectively.

Figure 4. HPLC chromatograms recorded in aqueous solutions containing 10% isopropanol and 1 × 10−4 M BrdG (a) and BrdA (b) before (black) and after (red) γ-radiolysis (140 Gy).

Table 1. Yield of BrdX Decomposition (in %), Its Standard Deviation (SD), and Decay Corrected for Internal Filter Effect (NYield, in %) for Irradiations Done in Triplicate Figure 3. HPLC chromatograms recorded in N2O-saturated aqueous solutions containing 10% isopropanol and 1 × 10−4 M BrdU (a) and BrdC (b) before (black) and after (red) γ-radiolysis (140 Gy).

Comparing these chromatograms with those shown in Figures 1 and 2, respectively, one can conclude that qualitatively the studied reactions proceed in the same manner in the UV and γirradiated solutions. 3.2. Yield of the Acetone-Sensitized Degradation of BrdX. Qualitatively, the photodegradation of the BrdX’s studied proceeds in a very similar manner. However, comparing HPLC peak areas (see the middle part of Figures 1 and 2), one can note a striking difference between the brominated pyrimidines and purines. Although, from both types of the modified nucleosides two photoproducts are generateda debrominated 2′-deoxynucleoside, dX (where X stands for U, C, A, or G), and the adduct of dX and the OHisop• radical, add-dXtheir relative amounts are very different. Indeed, the irradiation of bromopyrimidines mainly leads to dX with a smaller amount of the adduct (see Figure 1), while bromopurines produce the adducts and almost negligible amount of dX (see Figure 2). The yields of the UV-induced decomposition of BrdX’s are compared for all the studied systems in Table 1. These quantities were calculated on the basis of the peak area of substrate (BrdX) in the irradiated and non-irradiated solutions of the same initial concentration of BrdX. Since not only acetone but also particular BrdX’s are excited at 300 nm, one has to take into account internal filter effect (IFE)42 in order to

system

yield

SD

NYield

BrdU BrdC BrdA BrdG

65.7 66.7 51.2 25.0

5.1 4.9 3.4 3.0

65.7 72.6 46.7 23.9

compare the particular yields of photochemical degradation. The yields corrected for IFE are displayed in Table 1 and in Table S1 in the Supporting Information (SI). First, we assumed that the photons absorbed by BrdX did not give rise to the photoreaction studied. Indeed, the degradation quantum yield of BrdU in solution triggered by the direct homolysis of the Br−C5 bond is pretty small and ca. 1000-fold lower than that associated with the acetone-sensitized process.29 Then, employing the absorbance of the particular BrdX samples and of the reference solution (devoid of BrdX, i.e., containing only water, isopropanol, and acetone in appropriate proportions), we estimated the percentage of photons that excites exclusively acetone (see Table S1 in SI). The IFE-corrected decomposition yields normalized to the number of photons absorbed by acetone in the BrdU solution are collected in Table 1. The comparison of the normalized yields of degradation indicates that BrdC is the most photoreactive nucleoside while BrdG is the most photoresistant (see Table 1). It is also evident that bromopyrimidines are more photoreactive than bromopurines. This observation demonstrates, similarly to variations in the formation of particular photoproducts, the chemical difference between pyrimidines and purines. Nevertheless, the actual yields of degradation collected in Table 1 demonstrate that all four modified nucleosides are reactive toward OHisop• 6550


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Scheme 1. Degradation Paths of BrdX Leading to the Adduct-Type Products (Circled Green, Path a) and Debrominated Native dX Products (Circled Red, Paths b−d), Illustrated for the BrdU Case: (a) Radical Substitution, (b) Debromination via Br• Transfer, (c) Debromination via Electron Transfer in Triplet State, and (d) Debromination via Electron Transfer

and may probably lead to serious DNA photodamage when incorporated into this biopolymer and exposed to radical species. 3.3. Degradation Mechanism. The occurrence of dU due to the acetone-sensitized photochemical degradation of BrdU and IdU has been reported by Görner et al.29,43 However, the generation of an adduct (add-dU) was reported only recently by our group.30 These two products are formed in at least two competitive mechanisms depicted in Scheme 1. Thus, the recently characterized adduct,30 add-dX, is a product of radical substitution of BrdX by OHisop• (see Scheme 1a). The formation of add-dX can be rationalized with the help of the frontier molecular orbital theory (FMO).44 In case of the considered radical process one should analyze interactions between the SOMO of a radical species (unpaired electron localized on the C2 atom of OHisop•) and the LUMO of BrdX. The surfaces of a constant (0.05 e/Å3) density (corresponding to the shape of a molecule) on which the values of BrdX’s LUMO were encoded with a color changing gradually from positive (blue) to negative (red) extremes are depicted in Figure 5. The most positive values of the LUMO are located near the C5 and C8 sites for bromopyrimidine and bromopurine nucleosides, respectively (see Figure 5). Although the blue and red patches are also visible near the other molecular sites (e.g., near C4 in all nucleosides and C6 in pyrimidines), the bromine atom (Br•) can be easily eliminated only if the OHisop• radical attacks the C5/C8 center directly. In Table 2, the thermodynamic driving forces calculated at the M06-2x/631++G(d,p) level for this substitution reaction are collected. All four reactions are associated with significant free energies of reactions that vary between −17 and −22 kcal/mol, thus suggesting their high thermodynamic probability. Similarly, activation barriers for the process shown in Scheme 1a are relatively low, as they do not exceed 12 kcal/mol (see Table 2). Therefore, they should be easily overcome at ambient temperature of the photochemical experiment. Hence, both

Figure 5. Neutral BrdX structures, along with electron density surfaces (density isovalue = 0.05), and LUMO values encoded with colors: maximum negative (red) and maximum positive (navy blue). At the left, surfaces are faded to uncover the molecule backbones.

Table 2. Thermodynamic (ΔG) and Kinetic (ΔG*) Barriers for Radical Substitution Mechanism Calculated at the M062x/6-31++G(d,p) Level; All Values Given in kcal/mol BrdX

ΔG

ΔG*

BrdA BrdG BrdC BrdU

−20.7 −18.3 −17.3 −22.4

5.5 10.5 11.5 10.3

thermodynamic and kinetic arguments suggest that add-dX should be formed in all four BrNB’s. Indeed, they are observed in the experiments performed (see Figures 1 and 2 and the discussion above). Formation of the other product, a debrominated nucleoside although intuitively more probable, is less obvious. It can, for instance, occur as a result of a bromine atom (Br•) transfer between BrdX and the OHisop• radical, to give dX• radical that in the following step abstracts H atom from isopropanol (isop, see Scheme 1b). Such a mechanism was invoked previously to 6551


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The Journal of Physical Chemistry B explain the formation of dU in γ-irradiated aqueous solutions containing BrdU.28 In order to verify, this mechanism, we calculated thermodynamic parameters for the first step of reaction (b) (see Scheme 1) for all four bromo derivatives (see Table 3). The Gibbs free energy of this process ranges from 15

One should, however, remember that the flash photolysis experiments described by Wood and Redmond45 were initiated by laser excitation, which led to quite a large amount of the triplet states of nucleobases. Since in our experiment a conventional high-pressure mercury lamp was used, the stationary concentration of triplets was 1−2 orders of magnitude lower. Moreover, the concentration of BrdX’s in our experiments was ca. 20-fold lower than that in ref 45. Since triplet energy transfer is governed by the short-range Dexter mechanism46 (the Förster mechanism is doubly forbidden here due to spin limitations for the triplet−triplet energy transfer47), the interacting species has to collide (to form a collision complex) before the energy transfer will take place. However, the concentrations of both BrdX and the acetone triplet seem to be too small to enable an efficient formation of pyrimidine 3 [BrdX]*. Therefore, in order to adjudicate the role of triplet BrdX’s in the dX formation, we carried out additional radiolytic experiments where the OHisop• radicals were produced directly within the radiolysis of a water solution containing 10% isopropanol (see section 3.1.2). One can note that the substitution of triplet acetone with the γ-generated •OH radicals did not change the overall picture (compare Figures 1 and 2 with Figures 3 and 4, respectively). If the triplet BrdX were indeed necessary for the formation of dX, only adduct would be observed in the experiments depicted in Figures 3 and 4. Since the general picture did not change in the radiolysis, one can state with a high probability that the triplet states of BrdX’s are not involved in the photolysis, and dX’s are probably formed on the ground-state PES. The analysis carried out above suggests the pathway shown in Scheme 1d as the mechanism responsible for the observed photoproducts. Hence, a collision between the BrdX molecule and OHisop• radical should, in principle, lead to two products. A direct attack of the radical on the C5/C8 sites produces the adduct, while ET from OHisop• to BrdX results in formation of an ion-pair: the OHisop+ cation and the BrdX•− radical anion. The latter species swiftly dissociates, releasing Br− and dX• radical. The radical, in the subsequent step, reacts with isopropanol, resulting in formation of dX. Therefore, the final outcome of the process studied should depend on the rates of ET and radical substitution. Since dX’s are formed in negligible amounts during the photolysis of the brominated purine nucleosides, therefore ET is significantly less probable than for BrdU and BrdC photolysis. On the other hand, the rates for ET and radical substitution have to be comparable for the brominated pyrimidine nucleosides. The activation barriers for the radical substitution collected in Table 2 suggest that the process should be possible for all studied compounds at the ambient temperature. Indeed, the adducts are formed in all irradiated BrdX solutions. Furthermore, thermodynamic driving force is very similar for all bromonucleobases as indicated by our recent B3LYP/6-31++G(d,p) calculations on DEA to the brominated nucleobases13 and ab initio MD study48 on those systems. Since the activation barriers can be readily overcome at ambient temperature for radical substitution in both brominated pyrimidine and purine nucleosides (see Table 2), the similarity in DEA driving force cannot account for the fact that dX are formed only in solutions containing BrdU and BrdC. However, this situation is completely different when, instead of a total driving force for DEA, one considers its first step, i.e., electron attachment. These characteristics, the vertical attachment energies (VAEs), calculated at the M06-2x level, are collected in Table 5. Since ET is a rapid process, the geometries

Table 3. Reaction Energy (ΔE) and Gibbs Free Energy (ΔG) for OHisop• + BrdX → dX• + Br-isop, Calculated with the PCM Model of Solvent and the M06-2x/6-31++G(d,p) Method; All Values Given in kcal/mol BrdX

ΔE

ΔG

BrdA BrdC BrdG BrdU

14.2 16.6 15.5 17.7

14.6 16.5 15.4 17.7

to 18 kcal/mol for BrdA and BrdU, respectively (see Table 3). Such high thermodynamic barriers exclude, thus, the formation of dU via Br• transfer between BrdX and the OHisop• radical. Another reaction pathway which cannot be ignored without a thorough consideration is the possibility of the involvement of BrdX’s triplet state (see Scheme 1c). Indeed, an efficient population of the BrdX triplet should enable ET from the species with the most electron-donating properties (i.e., from OHisop•) to the electronically excited 3[BrdX]*. In a laser flash photolysis study, Wood and Redmond45 demonstrated that the triplet states of all nucleic acid bases are efficiently sensitized by the triplet of acetone. It is worth noting that under these circumstances the thermodynamic driving force for the considered ET process would be increased by the triplet excitation energy in comparison to the same process undergoing on the ground-state potential energy surface (PES). The triplet-state energies of BrdX’s along with that of acetone, all calculated at the B3LYP/6-31++G(d,p) level, are compiled in Table 4. The triplet energy data shown in Table 4 suggest that Table 4. Energy Difference between T1 and S0 (in kcal/mol) for BrdX’s Calculated at the TD-DFT B3LYP/6-31++G(d,p) Level, PCM Model (“Mix” Type Solvent), Compared to Experimental Value for Acetonea system

E(T1−S0)

acetone BrdU BrdC BrdA BrdG

78.045 77.2 79.2 83.8 85.4

For BrdX’s, the T1−S0 energy difference was calculated for equilibrium geometry of the singlet state (vertical excitation).

a

only the brominated pyrimidine nucleosides can be populated by the triplet transfer from the excited acetone (3[Ac]*). This finding remains in a good accordance with our observation that dX are formed from the brominated pyrimidine nucleosides while only negligible amounts of them are observed for the irradiated derivatives of purine nucleosides (see Figures 1 and 2). Such a mechanism seems to be also consistent with the findings of ref 45, showing that excitation to the triplet state in a purine/pyrimidine pair triggers ET from purine to the triplet state of pyrimidine. The latter process produces the purine radical-cation which easily deprotonates, giving the neutral purine radical, eventually.45 6552


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Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.5b01904.

Table 5. Vertical Attachment Energies (VAE, in eV) Calculated for Brominated Nucleosides (BrdX) in PCM Model (“Mix” Type Solvent) at the M06-2x/6-31++G(d,p) BrdX

VAE

BrdA BrdC BrdG BrdU

1.21 1.66 0.67 1.75

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone: +4858 523 5118. Fax: +4858 523 5771. Notes

The authors declare no competing financial interest.

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of the involved species have no time to relax. Therefore, the vertical rather than adiabatic electron attachment energies are more informative. A striking difference between pyrimidines and purines becomes finally visible, based on the data shown in Table 5. Actually, the VAEs of brominated purines are at least by 0.5 eV lower than those of pyrimidines. Clearly, this 11.5 kcal/mol difference is sufficient to hamper the ET process, almost completely, in the brominated purines. The fact that ET from organic radicals to BrdU/IdU was postulated to be a reason for dU formation28,29 is another premise suggesting the pathway shown in Scheme 1d to be operative in the studied systems.

ACKNOWLEDGMENTS Kamila Kiryluk is gratefully acknowledged for her valuable assistance at the initial stage of the project. This work was supported the Polish National Science Centre under Grant Nos. N N204 156040 and 2012/2012/05/B/ST5/00368 (J.R.) and by Foundation for Polish Science (L.C.). The support by the CMST COST Action CM1201 “Biomimetic Radical Chemistry” is kindly acknowledged (K.B. and J.R.). The calculations were performed at the Academic Computer Centre in Gdańsk (TASK).

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4. SUMMARY The reactivity of four bromonucleosides (BrdU, BrdC, BrdA, and BrdG) toward a model organic radical (OHisop•), generated either photochemically or radiationally, was studied in aqueous solutions by means of HPLC, mass spectrometry, and quantum chemical modeling. Since the compounds under consideration are modified nucleosides, they may potentially be introduced into DNA. If such modified biopolymers turn out to be sensitive to radical species generated in the cell during radiotherapy, the studied bromonucleosides will expand the modest pool of contemporary DNA radiosensitizers employed in clinics. OHisop• was generated photochemically from isopropanol via hydrogen atom transfer to the triplet acetone or via radiolysis of aqueous isopropanol solution. The HPLC analyses of photolytes and radiolytes revealed that two stable products, debrominated nucleoside and the adduct of OHisop• and BrdX, were characteristic for the degradation of brominated pyrimidines. On the other hand, only the adduct was observed in the irradiated solutions of bromopurine nucleosides. The results of our DFT investigations, especially the heights of activation barriers for formation of adducts, confirm that these products can be formed at ambient temperature due to interactions between OHisop• and all BrdX’s. The mechanism leading to dX is, however, completely different. As indicated by the calculated VAEs of BrdX’s, ET from OHisop• to the brominated pyrimidines is responsible for dX formation. The BrdX radical anion resulting from the ET process swiftly dissociates, leaving behind a reactive pyrimidine nucleoside radical localized on the nucleobase. In a double-stranded DNA, such a radical abstracts a hydrogen atom from the sugar residue or interacts with the adjacent bases, leading to serious DNA damage as strand breaks and intra- or interstrand crosslinks.49,50

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ASSOCIATED CONTENT

S Supporting Information *

Estimation of internal filter effect, HPLC and MS analysis of the UV-irradiated solution of BrdU in the negative ionization mode, and complete refs 30 and 39. The Supporting 6553


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