Chlorine Dioxide Oxidation of Guanosine 5'-Monophosphate

Carlos Alberto Huerta Aguilar , Jayanthi Narayanan , Mariappan Manoharan , Narinder Singh , Pandiyan Thangarasu. Australian Journal of Chemistry 2013 ...
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Chem. Res. Toxicol. 2006, 19, 1451-1458

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Chlorine Dioxide Oxidation of Guanosine 5′-Monophosphate Michael J. Napolitano, David J. Stewart, and Dale W. Margerum* Department of Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907-2084 ReceiVed June 8, 2006

The reactions between aqueous ClO2 and guanosine 5′-monophosphate (5′-GMP) are investigated from pH 5.96 to 8.30. The decay of ClO2 follows mixed first-order and second-order kinetics. The addition of chlorite (0.01-0.05 M) to the reaction mixture suppresses the reaction rate and changes the observed decay of ClO2 to second-order. The reaction rates increase greatly with pH to give oxidized products. The second-order rate constant for the guanosine anion is 4.7 × 105 M-1 s-1 and comprises a mixture of rate constants, k1k2/k-1. The ratio k1/k-1, with a calculated value of 2.4 × 10-4, corresponds to the reversible reaction between ClO2 and the guanosine anion to generate ClO2- and the guanosyl radical. To determine k1/k-1 and k2, E° values for guanosine and ClO2 are used as well as acid dissociation constants for guanosine and its radical. The value of k1 (1.1 × 105 M-1 s-1) represents the reaction between ClO2 and the guanosine anion as determined by initial rates. The second-order rate constant k2, with a value of 1.8 × 109 M-1 s-1, represents the reaction between the guanosyl radical with a second molecule of ClO2 to generate a guanosyl-OClO adduct. The consumption of two mol of ClO2 per mol of 5′-GMP corresponds to a fourelectron oxidation that gives ClO2- in the first step and HOCl in the second step. The 2′,3′,5′-tri-Oacetylated derivative of guanosine is used to more easily separate guanosine from its ClO2 oxidation products. Imidazolone and monochlorinated imidazolone are identified as products of the reaction between ClO2 and guanosine. Introduction The use of aqueous ClO2 as a sanitizing agent is growing in various food industries with applications in meat (1), poultry (2, 3), fish (4-6), and vegetable processing (7-9). Other utilizations of ClO2 as a sanitizer are in water treatment plants in the United States and Europe (10, 11), as a disinfectant against biological hazards such as anthrax (12), and in chemosterilization (13). In order to gain mechanistic insight about the disinfecting properties of ClO2, amino acids have been target molecules of interest (14, 15). In this article, another biomolecule is targeted for study, guanosine 5′-monophosphate (5′-GMP), as a model compound of guanine in nucleic acids. In Scheme 1, structures of guanosine 5′-monophosphate and guanine nucleosides are shown. Guanosine has the lowest reduction potential of all of the nucleosides (16, 17) with a reduction potential E° ) 1.58 V (17). It has been demonstrated that guanosine is a thermodynamic sink upon oxidation (18-21). Oxidation of the guanine base forms mutagenic lesions in DNA and has been implicated in carcinogenesis and aging (22, 23). Experimental work (20, 21) and MO calculations (24, 25) have shown that the preferential site of oxidation in DNA occurs at the guanine base. However, the oxidized guanine is not randomly distributed throughout the DNA strand. It has been shown that GG stacked pairs are the most easily oxidized (21). Because the guanine base in DNA is an oxidative thermodynamic sink, other oxidized nucleotide bases such as pyrimidine and adenine radical cations, albeit short-lived, are usually destined to oxidize a guanine base. This has been proposed to occur via electron hopping through the base pairs facilitated by π stacking (18). Previous one-electron oxidations of guanosine have been accomplished with oxidants, such as the thioanisole radical (17), * To whom correspondence should be addressed. Tel: (765) 494-5268. Fax: (765) 494-0239. E-mail: [email protected].

Scheme 1. Structures of Guanine and Guanine Nucleosides/ Nucleotides

Br2- radical (26), OH radical (18, 27, 28) SO4- radical (26), CO3- radical (29), and Tl(II) (30). All of the 1-electron oxidants listed above generate the same guanosyl radical with absorption bands at ∼312 and 540 nm (except the OH radical, which also adds to the guanine ring). Other oxidations of the guanine ring have been carried out by photooxidation (31), with singlet oxygen (32), and by photosensitization mediated with benzophenone (28) or riboflavin (33). A multitude of oxidation products have been reported for the guanosine system, and the product 8-oxo-7,8-dihydro-2′-deoxyguanosine has gained considerable attention. Although 8-oxo7,8-dihydro-2′-deoxyguanosine is primarily found in DNA and is used as a marker for DNA oxidation, it is a minor product of

10.1021/tx060124a CCC: $33.50 © 2006 American Chemical Society Published on Web 10/31/2006

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Scheme 2. Selected Oxidation Pathways of Guanosine (A) and 8-Oxo-7,8-dihydroguanosine (B)a

a (A) Guanosyl radical formation by the reaction between guanosine and either oxidant CO •- or OH• and the guanosyl adduct formation of O and NO 3 2 2 to generate an imidazolone (27-28), 8-nitroguanosine, and 5-guanidino-4-nitro-imidazole (29). (B) Two one-electron oxidations of 8-oxo-7,8-dihydroguanosine to form the pH-dependent products spiroiminohydantoin and guanidinohydantoin (34-37).

the free nucleoside (33). The bulk of oxidation products have been shown to be ring-opened structures (27, 28, 34-37). Selected pathways and products are shown in Scheme 2. Compared to the parent guanosine, 8-oxo-7,8-dihydro-2′-guanosine has a lower oxidation potential (19, 32, 38) and reacts further with one-electron oxidants to generate spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh) (34-37). The formation of Sp and Gh are pH dependent. At pH > 7, Sp is the dominant oxidation product of 8-oxo-7,8-dihydro-2′-guanosine. Below pH 7, Gh is the predominant species formed (35). Oxidation of 2′-deoxyguanosine by OH radical- or benzophenone-mediated photooxidation leads to the imidazolone product (Iz) (27, 28), as shown in Scheme 2. This product is proposed to form from the reaction between the guanosyl radical and either molecular oxygen (27) or superoxide (39) to generate an intermediate that hydrolyzes and loses CO2. Other reactions with guanine have shown that guanine radical-radical adduct formation occurs. The oxidation of guanosine by the CO3- radical in the presence of NO2 generates two NO2 adducts, 8-nitroguanosine and 5-guanidino-4-nitro-imidazole (29), as shown in Scheme 2. In the present work, we determine the kinetics, mechanisms, and products of the ClO2 oxidation of 5′-GMP. This permits a comparison of this system to highly reactive amino acids with ClO2.

Experimental Procedures Reagents. All solutions were prepared with doubly deionized distilled water. NaClO4 was recrystallized and standardized gravimetrically prior to use. Guanosine 5′-monophosphate (5′-GMP) (Acros Organics) was used without further purification, and stock solutions (25.0 mM) were freshly prepared to prevent microbial growth. ClO2 was prepared and standardized as previously reported (40) and stored in a dark refrigerator to hinder decomposition. Commercially available NaClO2 was recrystallized and standardized before use (41). All other chemicals were of reagent grade quality and were used as received.

Methods. The pH measurements were corrected to give p[H+] values ()-log[H+]) based on electrode calibration at 1.0 M ionic strength controlled by the addition of NaClO4 (40). UV-vis spectra and kinetic spectra were obtained from a PerkinElmer Lamda-9 UV-vis-NIR spectrophotometer (1.00 cm path length) or an Applied Photophysics Stopped-Flow SX.18 MV (APPSF) spectrophotometer (0.962 cm path length) with a PD.1 photodiode array or a single-wavelength detector. Photodiode array difference spectra as a function of time were obtained by the subtraction of the spectrum of 5′-GMP from that of the reaction mixture of these species with ClO2. Analyses of kinetic data were performed by using Sigma Plot 8.0 software. Kinetic traces of absorbance versus time obtained from the APPSF were fit to first, second, or mixed order equations with this software. Under pseudo first-order conditions, the overall second-order rate constants were determined by dividing kobs by the concentration of the reactant in excess. Chromatographic analysis was performed by the use of a Varian 5020 HPLC with a Hewlett-Packard 1050 diode array detector. A Whatman Partisil 5 ODS-3 C18 column was used for the reversedphase (RPLC) determination of the 2′,3′,5′ tri-O-acetylguanosine. 2′,3′,5′ Tri-O-acetylguanosine was synthesized by a literature procedure (42). A Dionex DX-500 chromatograph was used to determine the amount of ClO2- and Cl- generated from the ClO2/5′-GMP reaction. Samples were injected by an auto sampler (AS 40) through a 25 µL injection loop onto a quaternary amine anion exchange guard (AG9 HC) and separation (AS9 HC) columns. Analytes were eluted with 9 mM Na2CO3 with a flow rate of 1 mL/min. The detection of analytes was accomplished by gas-assisted suppressed-conductivity detection (ED 40) with an ASRS-Ultra suppressor in selfregenerating mode and a current of 100 mA. Samples for electrospray ionization mass spectrometry (ESI-MS) were dried via lyophilization on a VirTis Freezemobile 12 lyophilizer. The lyophilized reaction mixture was dissolved in eluent and separated via a Whatman Partisil 5 ODS-3 C18 column with 10% CH3CN as eluent at 1 mL/min. Mass spectra of the products were taken as they eluted from the column. The ESI-MS analyses were carried out at the Purdue Medicinal Chemistry and Molecular Pharmacology Department with a ThermoFinnigan MAT LCQ mass

Guanosine 5′-Monophosphate

Chem. Res. Toxicol., Vol. 19, No. 11, 2006 1453 Table 1. Ion Chromatographic Measurements of [ClO2-] and [Cl-] Formation after Flow Mixing of 10 mL Each of ClO2 and 5′-GMP Solutionsa [5′-GMP]i (mM)

[ClO2]i (mM)

[ClO2-] (mM)

[Cl-] (mM)

1.00 1.00 1.00 1.00 1.00

0.10 0.20 0.30 0.50 1.00

0.05 0.11 0.18 0.31 0.73

0.03 0.05 0.08 0.11 0.22

a Reaction conditions: [Na CO ] ) 9.0 mM. Separation conditions: 2 3 T eluent, 9.0 mM [Na2CO3] in H2O; flow rate, 1.0 mL/min conductivity detection. Analyses were performed 1 h after mixing.

Figure 1. Photodiode array difference spectra for the reaction between ClO2 and 5′-GMP. The spectra show the decay of ClO2 (λmax ) 360 nm) and the formation of products at 300 nm. Spectra were collected every 20 ms. Conditions: [5′-GMP] 1.00 mM, [ClO2] 0.10 mM, p[H+] 7.60, [PO4]T 25.0 mM, µ ) 1.0 M (NaClO4), 25.0 °C, and cell path ) 0.962 cm.

Scheme 3. Mechanism of ClO2 Decay for the Reaction between ClO2 and 5′-GMP and the Formation of the Guanosyl-OClO Adducts

spectrometer system. The electrospray needle voltage was set at 3.5 kV, the heated capillary voltage was set to 23 V, and the capillary temperature at 213 °C. The typical background source pressure was 1.2 × 10-5 Torr as read by an ion gauge. The sample flow rate was approximately 8 µL/min. The drying gas was nitrogen. The LCQ was scanned to 2000 amu for these experiments. The MS/MS results were obtained by selecting the ion of interest (the precursor ion). The precursor ion was then subjected to collision-induced dissociation (CID), resulting in the formation of product ions. Helium was introduced into the system to an estimated pressure of 1 millitorr to improve trapping efficiency and also acted as the collision gas during the collisionally induced dissociation (CID) experiments. The collision energy was set to 40% of the maximum available from the 5 V tickle voltage, with a 2 mass unit isolation window.

Results and Discussion Observed Spectral Changes. The photodiode array difference kinetic spectrum is given in Figure 1 for the reaction between 1.00 mM 5′-GMP and 0.10 mM ClO2 at p[H+] 7.60. Analysis of these data showed that under the above conditions, the decay of ClO2 follows mixed-order kinetics. An absorption band at ∼300 nm forms after ClO2 decays. It is fitted to a double-exponential function that corresponds to the formation of two products with similar overlapping absorption spectra. The absence of an isobestic point, the decay of ClO2, and the growth of products that are not concurrent suggests the existence of a nonobservable intermediate or intermediates that decay to form the observed 300 nm absorbance. Reaction Stoichiometry. The stoichiometry of the reaction between 5′-GMP and ClO2 was determined by flow mixing 1.00 mM 5′-GMP with increasing concentrations of ClO2 (0.101.00 mM). The inorganic products generated are ClO2- and Clas measured by ion chromatography. (The results are given in Table 1.) The results show that for ClO2 concentrations from 0.10 to 0.30 mM, the concentrations of ClO2- formed correspond to one-half of the starting ClO2 concentration ([ClO2]i); therefore, the stoichiometric ratio of ClO2/5′-GMP is 2:1. The stoichiometry of the reaction between ClO2 and 5′-GMP is given in eqs 1 and 2. The anionic form of 5′-GMP reacts with 2 mol of ClO2. The first mol of ClO2 reacts with 5′-GMP(-) to form ClO2-, and the guanosyl radical reacts further with a second mol of ClO2 to form a 5′-GMP-OClO adduct (eq 1). The 5′GMP-OClO adduct decays to form an imidazolone derivative (Iz) and HOCl (eq 2). HOCl can then further oxidize the products to form the observed Cl- or can chlorinate the products,

as discussed later. Structures of reactants and products are shown in Schemes 3 and 4.

5′-GMP(-) + 2 ClO2 f 5′-GMP-OClO + ClO2

(1)

5′-GMP-OClO f Iz + HOCl

(2)

At higher initial ClO2 concentrations (0.50-1.00 mM), the resultant ClO2- concentrations were found to be greater than 1/2[ClO2]i. We propose that the higher ClO2- levels at higher [ClO2]i are due to the oxidation of initial 5′-GMP products. These secondary oxidation products can be formed by two oneelectron oxidations by ClO2. The Burrows group (34-37) has shown that the guanosine oxidation product, 8-oxo-7,8-dihydroguanosine, generates the pH-dependent products (spiroiminodihydantoin and guanidinohydantoin) when reacted with a variety of one-electron oxidants. They proposed that these products form as the result of two one-electron transfers and the addition of H2O. In addition, ClO2 has also been shown to be only a 1-electron oxidant, such as when ClO2 reacts with hydroquinone (43) and dopa (15). The Cl- concentrations detected were never equal to or greater than 1/2 [ClO2]i. We assign the formation of Cl- as a result of a HOCl oxidation. Suzuki et al. have shown that 2′deoxyguanosine is oxidized and chlorinated by HOCl (44). The reaction between 2′-deoxyguanosine and HOCl generates the oxidized products, diimino-imidazole and imidazolone, that can react further with HOCl to generate the chlorinated diiminoimidazole and imidazolone derivatives. The other product

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Scheme 4. Proposed Formation of the Guanosyl-OClO Adduct at the 5-Position of Guanosine and Subsequent Decays of Intermediates to Generate the Imidazolone and Chloroimidazolone Products

observed for the reaction between HOCl and 2′-deoxyguanosine is the chlorinated product 8-chloro-2′-deoxyguanosine. Imidazolone Product Identification. Our results show that HOCl can act as an oxidant to generate Cl- and can also act as a chlorinating agent with the 5′-GMP system. Monochlorination products are detected when ClO2 is reacted with 2′,3′,5′tri-O-acetylguanosine (TAG). In order to separate and identify products in the ClO2/guanosine system, TAG was reacted with ClO2 to take advantage of the ease of separation of the parent 2′,3′,5′-tri-O-acetylguanosine and its oxidation products (28). We have chromatographic and UV-vis evidence for the formation of chloroimidazolone and its parent imidazolone from the reaction between ClO2 and guanosine. In Figure 2A, the C18 HPLC chromatogram is shown for the reaction between 0.30 mM ClO2 and 1.00 mM TAG at pH 6.10 in the presence of 0.100 M [OAc]T. In Figure 3, the UV-vis spectra for the chloroimidazolone (3A) and its parent imidazolone (3B) are shown. These spectra are similar to those previously reported for these types of compounds. For the reaction between HOCl

Figure 2. HPLC chromatogramic evidence for the formation of a chloroimidazolone (Iz-Cl) from the reaction between ClO2 and 2′,3′,5′tri-O-acetylguanosine (TAG). Reaction Conditions: (A) [TAG] 1.00 mM, 0.3 mM ClO2, [OAc]T 0.100 mM, pH 6.10, and injection after 15 min of reaction. (B) 2.00 mM TAG was reacted with 0.60 mM ClO2, 0.200 mM [OAc]T at pH 6.10 for 15 min. Then 5 mL of 2.00 mM TAG/0.60 mM ClO2 was reacted with 5 mL of 2.00 mM cysteine for 15 min. Separation conditions: eluent 10% (v/v) CH3CN, flow rate 1 mL/min, and detection at λ ) 280 nm.

Figure 3. UV-vis spectra of Iz-Cl (A) and the imidazolone product (B). Spectra were obtained from the chromatograms given in Figure 2 or the reaction between 0.30 mM ClO2 and 1.00 mM TAG with 10% CH3CN as eluent; cell path ) 1.00 cm.

and 2′-deoxyguanosine, Suzuki et al. report the formation of isomeric chlorinated imidazolones (44) that have UV-vis spectra similar to ours and have m/z values that correspond to Iz-Cl. Our electrospray ionization (ESI) results confirm the molecular weight of Iz and IzCl. The positive ion ESI spectrum of the IzCl peak in the HPLC has an ion doublet m/z 449/451 that indicates the presence of a chlorine atom. This ion [IzCl + 2Na-H]+ is consistent with the compound IzCl. Further evidence indicating compound IzCl is found in the negative ion ESI mass spectrum, which shows the ion doublet m/z 403/405, consistent with the (IzCl-H)- ion. MS/MS spectra of these ions show the losses of multiple acetic acid molecules. An observed ion doublet at m/z 427/429 is consistent with the [IzCl+Na]+ ion. The next most abundant ions in the positive ion ESI are m/z 393 and m/z 415, [Iz+Na]+ and [Iz+2Na-H]+, respectively, consistent with compound Iz. Not surprisingly, Iz, without the electronegative chlorine atom, does not produce an ion in the negative ion ESI mass spectrum. Suzuki et al. confirmed their assignments by reacting Nacetylcysteine with the chlorinated imidazolone to generate the parent imidazolone that was identified by its UV-vis and mass spectra (44). To further identify our compound as a chloroimidazolone from the ClO2/TAG reaction, we used a method similar to that used by Suzuki et al. TAG (2.00 mM) was reacted with ClO2 (0.6 mM), with 0.200 M [OAc]T at pH 6.10 for 15 min. After 15 min, 5 mL of the TAG/ClO2 mixture was reacted with 5 mL of 2.00 mM cysteine for 15 min. The TAG/ClO2/cysteine reaction mixture was injected into a C18 column under the same conditions as the 1.00 mM TAG/ 0.30 mM ClO2 system. The chromatogram (Figure 2B) shows that the Iz-Cl peak disappears because of its reaction with cysteine, and the Iz peak increases. The parent imidazolone peak was identified by its UV-vis (27, 28, 31, 44) spectrum. Kinetic Analysis of ClO2 Loss. The reaction between ClO2 and guanosine 5′-monophosphate (5′-GMP) was studied at a

Guanosine 5′-Monophosphate

Chem. Res. Toxicol., Vol. 19, No. 11, 2006 1455

Figure 4. Second-order kinetic trace of ClO2 loss for the reaction between ClO2 and 5′-GMP in the presence of added ClO2-. Conditions: [5′-GMP] 5.00 mM, [ClO2] 0.10 mM, [ClO2-] 0.200 M, p[H+] 6.30, [PO4]T 0.100 M, λ ) 390 nm, µ ) 1.0 M (NaClO4), 25.0 °C, and cell path ) 0.962 cm.

Figure 5. Second-order rate constants vs p[H+] for the reaction between ClO2 and 5′-GMP. Conditions: [5′-GMP] 5.00 mM, [ClO2] 0.10 mM, [ClO2-] 0.020 M, [PO4]T and [CO3]T 25.0 mM, λ ) 390 nm, µ ) 1.0 M (NaClO4), 25.0 °C, and cell path ) 0.962 cm.

p[H+] range of 5.96-8.30. The effects of chlorite ion concentration as well as the total 5′-GMP concentrations on observed rate constants were investigated. The loss of ClO2 was studied at 390 nm rather than at its λmax of 359 nm because at lower wavelengths, the growth of products interferes with the ClO2 absorption band. At the pH range studied, the decay of ClO2 follows mixed-order kinetics when no ClO2- is added to the reaction. The addition of ClO2(0.01-0.050 M) to the reaction mixture suppresses the rate and alters the decay of ClO2 from mixed-order to second-order kinetics. Figure 4 shows the second-order decay of ClO2 in presence of added chlorite. In Figure 5, the second-order kobs values versus p[H+] data are shown at µ ) 1.0 M and [ClO2-] ) 0.0200 M, where the data are fit to eq 3. Ka2 is the second acid dissociation constant for 5′-GMP as defined in Table 2 and Scheme 3.

kobs )

k′[5′-GMP]TKa2 + [ClO2 ][H ]

(3)

These data show that the observed rate constant increases with pH, and the guanosine anion is the reactive species with ClO2. Our measured pKa2 (for 5′-GMP) of 9.40(4) is in agreement with other pKa determinations for guanosine (18). The value for pKa2 was determined by a spectrophotometric titration (Supporting Information, Figure S2) of 5′-GMP at λ ) 220 nm at µ ) 1.0 M (NaClO4). The dependence of kobs on [ClO2-] is

given in Figure 6. The second-order kobs values are linearly dependent on 1/[ClO2-], which shows that ClO2- suppresses the rate of ClO2 decay. The reaction is first order in 5′-GMP as shown by the linear dependence of kobs on [5′-GMP]T at pH 6.30 and 0.0200 M [ClO2-] (Figure 7). From the pH, [ClO2-], and [5′-GMP] data, the second-order kobs is given in eq 3, where k′ equals k1k2/k-1 (Scheme 3). The k′ value is calculated to be 4.7(1) × 105 M-1 s-1 (Table 2). Proposed Mechanism. The proposed mechanism of ClO2 decay is shown in Scheme 3. The first step in the mechanism is the pre-equilibrium between 5′-GMP and its conjugate base. Evidence for this is the kobs (M-1 s-1) dependence on pH in Figure 5. The second step in the mechanism is the reversible ClO2 one-electron oxidation of the guanosine anion to generate the guanosyl radical and chlorite ion. Previous studies have shown that the guanosyl radical is an oxidizing radical at similar pH values (17). Support for the reversible k1/k-1 step is found by the fact that the addition of ClO2- to the reaction suppresses the rate of loss of ClO2 and changes the decay of ClO2 from mixed to second-order kinetics. The change in the order of ClO2 loss also indicates that a second molecule of ClO2 reacts with the guanosyl radical (k2 step) after the reversible one-electron ClO2 oxidation of the guanosine anion. Furthermore, it is the k2 step that drives this reaction to completion. Otherwise, the reaction would not proceed because of the unfavorable difference of the guanosine and ClO2 redox potentials; see Table 2. We propose that the radical-radical reaction (the k2 step in Scheme 3) generates a guanosyl-OClO adduct. Other ClO2 radical adducts have been observed in studies of the reactions between ClO2 and tyrosine and ClO2 and N-acetyltyrosine (15). Additionally, ClO2 adducts have been proposed in other aromatic/ClO2 systems (43, 45). The linear dependence of kobs on 5′-GMP concentration (Figure 7) shows that the rate expression for ClO2 loss has a first-order dependence on [5′-GMP]T. From Scheme 3, the rate expression for the loss of ClO2 is given in eq 4, when the guanosyl radical is a steady-state intermediate.

-

d[ClO2] 2k1k2[5′-GMP]TKa2 ) [ClO2]2 + dt (k-1[ClO2 ] + k2[ClO2])(Ka2 + [H ]) (4)

Equation 4 shows that the loss of ClO2 is mixed order when ClO2- is not added to the reaction. However, when ClO2- is added to the reaction mixture such that k-1[ClO2-] >> k2[ClO2] and [H+] >> Ka, eq 4 simplifies to a second-order rate equation, given in eq 5.

-

d[ClO2] 2k1k2[5′-GMP]TKa2 ) [ClO2]2 + dt k [ClO ][H ] -1

(5)

2

Determination of the Second-Order Rate Constants k1, k-1, and k2. From the kinetic data at varying pH as well as different 5′-GMP and ClO2- concentrations (Figures 5-7), the mixed second-order rate constant k1k2/k-1 was determined. We have also determined the rate constants k1, k-1, and k2 from initial rates, standard reduction potentials, and acid dissociation constants. To obtain k1, freshly prepared 0.10 mM ClO2 was reacted with 5′-GMP at concentrations of 1-5.00 mM at constant pH and without the addition of ClO2-. Data at 5-20% reaction was used to determine ∆[ClO2]/∆t. The midpoint [ClO2] of these data was used as the tangent concentration ([ClO2]tan) in order to determine the pseudo firstorder rate constants; see eqs 6 and 7. These data are shown in

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Table 2. Equilibrium and Rate Constants for the Reaction between ClO2 and 5′-GMPa

a Conditions: 25.0 °C. b Conditions: µ ) 1.0 M (NaClO ). c Ref 17 (17), pK ) 1.9, pK ) 3.9. d Ref 48 (48). e Calculated. f Present work. g pK ) 4 a1 r1 a2 9.40(4).

Figure 8 and the resolved second-order rate constant for k1 is given in Table 2.

kobs ) ∆[ClO2] ∆t[ClO2]tan

∆[ClO2] ∆t[ClO2]tan

)

k1Ka2[5′-GMP] [H+]

(6)

(7)

To obtain a value for k1/k-1, standard reduction potentials for guanosine and ClO2 were used to calculate the equilibrium constant K for reaction R(1 + 2); see Table 2. The addition of reactions R1-5 in Table 2 corresponds to the equilibrium

expression in R6, where the equilibrium constant is represented by k1/k-1. The acid dissociation constants employed to determine k1/k-1 are Ka1 for guanosine, Ka2 for 5′-GMP, and Kr1 for guanosine. The value of Kr1 has been shown to be insensitive to the ribose group. The rate constants k-1 and k2 were also determined from our measured rate constants k1k2/k-1, k1, and the calculated k1/k-1 values. To test the validity of our proposed mechanism and determined rate constants, the ratio of k2[ClO2]/k-1[ClO2-] from 1 to 30% of reaction as well as the ratio of k1k2[ClO2]/(k-1[ClO2-] + k2[ClO2]) was calculated. The ratio of k2[ClO2]/k-1[ClO2-] ranges from 851 at 1% of reaction to 20.1 at 30% of reaction (Supporting Information, Table S1). Additionally, the ratio of

Guanosine 5′-Monophosphate

Chem. Res. Toxicol., Vol. 19, No. 11, 2006 1457 Table 3. Comparison of Second-Order Rate Constants at pH 7 of Cysteine, Tyrosine, Tryptophan, and 5′-GMP for the Reaction X- + ClO2 f X• + ClO2-a substrate (X)

second-order rate constant at pH 7 (k) M-1 s-1 6.9 × 106b 1.3 × 105c,d 3.4 × 104f 4.5 × 102c

cysteine (Cys) tyrosine (Tyr) tryptophan (Trp)e guanosine 5′-monophosphate (5′-GMP)

a Conditions: 25.0 °C and µ ) 1.0 M (NaClO ). b Ref 47 (47). 4 Calculated from the expression k)(k1Ka)/[H+]. d Ref 15 (15). e The reactive site is the indolyl group that is uncharged. f Ref 46 (46).

c

Figure 6. Second-order rate constants vs 1/[ClO2-] for the reaction between ClO2 and 5′-GMP. Conditions: [5′-GMP] 5.00 mM, [ClO2] 0.10 mM, [ClO2-] 0.02-0.0500 M, p[H+] 6.30, [PO4]T 50.0 mM, λ ) 390 nm, µ ) 1.0 M (NaClO4), and 25.0 °C.

acetamide. Cadet has also proposed a similar pathway for the type I photo-oxidation and OH radical-mediated oxidation of 2′-deoxyguanosine, where molecular oxygen adds to the 5-position of the guanosyl radical that generates an imidazolone product (27, 28). Another adduct formation at the 5-position of the guanosyl radical has been proposed for NO2 (29). The reaction of NO2 with the guanosyl radical at the 5-position ultimately generates 5-guanidino-4-nitroimidazole. The HOCl, via ClO2 adduct decay, can react with the imidazolone to form Iz-Cl (44).

Conclusions

Figure 7. Second-order rate constants vs [5′-GMP]T for the reaction between ClO2 and 5′-GMP. Conditions: [ClO2] 0.10 mM, [ClO2-] 0.0200 M, p[H+] 6.85, [PO4]T 50.0 mM, λ ) 390 nm, µ ) 1.0 M (NaClO4), and 25.0 °C.

Figure 8. Pseudo first-order rate constants obtained from initial rates vs [5′-GMP]T for the reaction between ClO2 and 5′-GMP. Conditions: [ClO2] 0.10 mM, no added ClO2-, p[H+] 6.34, [PO4]T 50.0 mM, λ ) 390 nm, µ ) 1.0 M (NaClO4), and 25.0 °C.

k1k2[ClO2]/(k-1[ClO2-] + k2[ClO2]) is equal to the value of k1 within 3% error. These calculations show that upto 30% of the decay of ClO2 is first order and that our proposed mechanism and determined rate constants are viable. Product Formation. The pathway shown in Scheme 4 is proposed for the formation of the imidazolone (Iz) and the chlorinated imidazolone (Iz-Cl). A second molecule of ClO2 adds to the 5-position of the guanosyl radical, and this guanosylOClO adduct decomposes to generate the carboxyl-guanidino intermediate (Int1) and HOCl. Subsequent loss of CO2 and hydrolysis of this intermediate generates the imidazolone and

Our results show that 2 mol of ClO2 reacts with 1 mol of 5′-GMP to form a guanosyl-OClO adduct and one mol of ClO2-. At high [ClO2-], second-order ClO2 decay is observed. Evidence is given for the formation of imidazolone and chlorinated imidazole products. The one-electron oxidant ClO2 reacts by both electron transfer and radical-radical bond formation. Evidence for this is the formation of chlorite, chloride, and chlorinated oxidation products. The formation of ClO2- is due to the one-electron oxidation of the 5′-GMP anion by ClO2 that generates the corresponding guanosyl radical. The guanosyl radical reacts with a second molecule of ClO2 to form a guanosyl-OClO adduct that rapidly decays to form HOCl and oxidized products. HOCl is known to act as an oxidizer as well as a chlorinating agent. We observe the formation of Cl- and a chlorinated imidazolone (Iz-Cl). The chlorinated imidazolone product was identified by its UV-vis spectrum, mass spectral data, and its reaction with cysteine to form the parent imidazolone. This study shows that ClO2 oxidation of 5′-GMP is rapid at pH 7 (Table 3). However, our previous study (15) shows that the reaction between ClO2 and the amino acid tyrosine is a factor of 290 times faster at pH 7. Other studies show that at pH 7, tryptophan is 84 times faster (46), and cysteine is 1.5 × 104 times faster (47) to react with chlorine dioxide than 5′-GMP. Thus, the use of ClO2 as a sanitizing agent may be more closely tied with the reaction of these amino acid linkages in proteins, rather than the reaction with nucleic acids. Acknowledgment. This work was supported by National Science Foundation Grant CHE-0139876. We thank Karl V. Wood for his aid in the interpretation of the mass spectral data. Supporting Information Available: Table of calculated rates of reaction between ClO2 and guanosine as a function of ClO2formed, kinetic traces of ClO2 decay for the reaction with 5′-GMP in the absence of added ClO2-, spectrophotometric determination of pKa2 for 5′-GMP, ESI positive ion and negative ion mass spectra for reaction products, and MS/MS spectra of imidazolone deriva-

1458 Chem. Res. Toxicol., Vol. 19, No. 11, 2006 tives. This material is available free of charge via the Internet at http://pubs.acs.org.

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