Electrochemical oxidation of guanosine and adenosine: Two convergent pathways

Electrochemistry Communications 9 (2007) 1862–1866 www.elsevier.com/locate/elecom

Electrochemical oxidation of guanosine and adenosine: Two convergent pathways ´ lvarez, Patricia de-los-Santos-A ´ lvarez, Marı´a Jesu´s Lobo-Castan˜o´n, Noemı´ de-los-Santos-A Ramo´n Lo´pez, Arturo J. Miranda-Ordieres, Paulino Tun˜o´n-Blanco * Departamento de Quı´mica Fı´sica y Analı´tica, Universidad de Oviedo, Julia´n Claverı´a 8, 33006 Oviedo, Spain Received 27 March 2007; received in revised form 13 April 2007; accepted 20 April 2007 Available online 30 April 2007

Abstract We herein explore a novel route for oxidative stress in DNA by using electrochemistry as mimicking tool. Essentially, the electrochemical oxidation of guanine and adenine nucleosides and oligo-homo-nucleotides at pyrolytic graphite electrodes in neutral and alkaline aqueous solutions was studied. Under these experimental conditions all these compounds give rise to an adsorbed product not previously described, which was electrochemically and kinetically characterized. The virtually identical kinetic and electrochemical features exhibited by the oxidized compounds generated from all precursors strongly suggest a common species arising from both adenine and guanine derivatives. Supported by DFT calculations, we propose two convergent pathways for the electrochemical oxidation of adenosine and guanosine. Those calculations indicate that the common oxidized base product forms stable H-bond base pairs with both thymine and cytosine.  2007 Elsevier B.V. All rights reserved. Keywords: Cyclic voltammetry; Density functional calculations; Electrochemistry; Guanosine; Substitution mutation

1. Introduction Oxidative lesions in DNA are the primary risk factor for gene mutations which plays a key role in carcinogenesis and aging. Oxidation of guanine nucleobase to 7,8-dihydro-8-oxo-2 0 deoxyguanosine (8-oxoG), is one of the most widely studied lesions in cellular systems [1,2]. However, recent studies indicate that this modified purine base is readily subjected to further oxidation reactions leading to different products depending on the oxidation conditions [3,4]. Some of these modified bases have been detected in vivo [5] but in many cases the types and biological significance of oxidative lesions in DNA needs further investigations. In this context, understanding the electro-oxidation of DNA bases may shed light on the mechanism of oxida-

*

Corresponding author. Fax: +34 98 5103125. E-mail address: [email protected] (P. Tun˜o´n-Blanco).

1388-2481/$ - see front matter  2007 Elsevier B.V. All rights reserved. doi:10.1016/j.elecom.2007.04.018

tive DNA damage. Electrochemical oxidation of guanosine [6–9] passes through an 8-oxoderivative intermediate under any conditions thus mimicking the primary effects of oxidative stress in biological systems. The 8-oxoderivative is immediately oxidized at potentials where the oxidation of guanosine occurs to an intermediate species with diimine structure [6–9]. In these and similar works, only stable soluble products resulting from exhaustive electrolysis of guanine derivatives were identified, which would correspond to products obtained under extreme degradative conditions leading to depurination. To the best of our knowledge, no previous studies searching for electrochemically oxidized products of guanine derivatives adsorbed on the electrode surface have been reported so far. The identification of this kind of products would facilitate the understanding of other oxidative damage that leads to chemically modified bases, which could be found naturally. We have extensively studied the oxidation of N9-substituted adenine derivatives [10–12] on pyrolytic graphite electrodes (PGE).

N. de-los-Santos-A´lvarez et al. / Electrochemistry Communications 9 (2007) 1862–1866

Their oxidation takes place at the adenine moiety yielding adsorbed compounds with a common structure that act as very efficient catalysts of the oxidation of NADH at low potentials. In this work, the electrochemical behaviour of guanosine and its oligonucleotide, dG20, was investigated at PGE and the results compared to that obtained for adenosine and dA20. 2. Experimental and theoretical details

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tionary points was verified by analytical computations of harmonic vibrational frequencies, showing that all the located TSs have only one imaginary frequency. DG values in gas phase were calculated within the ideal gas, rigid rotor, and harmonic oscillator approximations at a pressure of 1 atm and a temperature of 298.15 K [19]. To take into account condensed-phase effects, we evaluated the DG values in solution using the polarizable continuum model (PCM) [20,21] with the united atom Hartree–Fock (UAHF) parametrization [22].

2.1. Materials and equipment 3. Results and discussion Guanosine was purchased from Sigma (Spain). Deoxyguanylic acid icosanucleotide (dG20) was obtained as desalted products from Sigma-Genosys (London–UK). Stock solutions of 1 g L1 dG20 in water were stored at 4 C and diluted to the appropriate concentration with 2 · SSPE buffer solution (dilution 1:10 of concentrated saline sodium phosphate EDTA, 20 · SSPE (0.2 M sodium phosphate + 3 M NaCl + 0.02 M EDTA) DNase-free adquired from Sigma). Other chemicals employed were of analytical grade. Cyclic voltammetry measurements were carried out with a conventional three-electrode electrochemical cell driven by a computer-controlled Autolab PGstat10-potentiostat (EcoChemie, The Netherlands). A platinum wire acted as auxiliary electrode. All the potentials are referred to AgjAgCljKCl saturated reference electrode. The working electrode was home-made using a 3 mm diameter pyrolytic graphite rod (Goodfellow, UK). Reproducible surfaces were achieved by polishing on sandpaper (600 grit, Buehler, Germany) and by washing with purified water in an ultrasonic bath for a few minutes. 2.2. Preparation of the modified electrodes The oxidized guanosine-modified electrodes were prepared by the following procedure: a freshly polished pyrolytic graphite electrode (PGE) was immersed in 0.1 M phosphate solution pH 12 containing 0.5 mM guanosine, and 10 potential scans were carried out between 0.2 V and +1.4 V at 7 V s1. For the preparation of oxidized dG20-modified electrodes a 10 lL droplet of 0.5 g L1 oligonucleotide in 2 · SSPE solution was placed on the electrode surface and evaporated till dryness at room temperature. The electrode was then immersed into 0.1 M phosphate solution pH 12 containing no dG20, and the electrochemical oxidation was carried out at 7 V s1. 2.3. Computational details Quantum chemical computations were carried out with the Gaussian 03 series of program [13]. Full geometry optimizations of stable species and transition states were performed by using the hybrid density functional B3LYP [14–16] along with the 6-31+G(d) basis set [17] and the standard Schlegel’s algorithm [18]. The nature of the sta-

When the oxidation of guanosine or dG20 was carried out at high scan rates on PGE at physiological pH and then the electrode was transferred into a fresh solution, a quasireversible redox process, not previously described, was obtained. The formal potential of this process, 0 E0 = 0.099 ± 0.003 V, is virtually identical for both guanosine and dG20, and, surprisingly, identical to those reported for the oxidation products of adenosine [11] and dA20 [12]. This process is observed when the oxidation is performed in neutral and alkaline media, with an increase in the surface concentration as the pH of the oxidation media increases. In consequence a phosphate solution of pH 12 was used to generate larger amounts of the surface products, and hence, to facilitate its electrochemical characterization. While surface coverage obtained from the oxidation of adenine derivatives in alkaline solution is near monolayer (mean concentration values between 5 and 6 · 1010 mol cm2), those obtained from guanine derivatives are much lower (mean concentration values of 1 · 1011 mol cm2 for guanosine and 3 · 1011 mol cm2 for dG20). The global apparent electron transfer rate constant (ks) for the above surface electrochemical reaction was determined by measuring the square-wave peak currents at different frequencies [23]. All measurements were carried out at 25 C and pH 10. We found identical ks values, within the experimental error, for guanosine and dG20 oxidation products. Moreover, the ks values obtained for oxidized guanine derivatives are also identical to the values obtained for the products derived from adenosine and dA20 (Table 0 1). The identical thermodynamic (E0 ) and kinetic constant values obtained for the electrochemical reaction of the oxidized guanine and adenine derivatives strongly suggests a common redox active structure for both of them. Table 1 Formal potential and electron transfer rate constant for oxidation products of purine nucleosides and oligo-homo-nucleotides 0

Precursor

E0 /V

Guanosine dG20 Adenosine dA20

(0.55 ± 0.02)  (0.061 ± 0.003) (0.56 ± 0.01)  (0.060 ± 0.001) (0.51 ± 0.01)  (0.056 ± 0.003) (0.51 ± 0.01)  (0.056 ± 0.001)

a

From Ref. [10].

ks/s1 pH pH pH pH

26 ± 3 24 ± 2 24 ± 2a 24 ± 3

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In addition, as we have previously reported for oxidized adenine derivatives [10–12], the oxidized guanine derivatives exhibit catalytic activity towards the oxidation of NADH. Fig. 1 shows a great enhancement in the anodic 0 current at potentials close to the E0 of the corresponding redox couple along with a significant decrease in the cathodic one for oxidized dG20 modified PGE in the presence of NADH; similar results are obtained for oxidized guanosine modified electrodes. This result indicates that the common oxidation product would be a diimine structure, which is known to be a fast NADH oxidant [24]. On the basis of the above-mentioned findings, we propose a mechanism (Scheme 1) that implies the elimination of an amidure group at C2 of the guanine derivative and at C6 of adenine derivative. The accepted primary products of the electro-oxidation of N9-substituted purine derivatives i.e. 2,8-dihydroxyadenosine (3) and 8-oxo-guanosine (5) could evolve in two different pathways: one initiated by a tautomerization followed by nucleophilic attack of OH, and then a proton transfer (PT) with further elimination of NH 2 , and the other one initiated by the nucleophilic attack of OH. Both pathways yield a diimine structure (7) that could reduce the cofactor NADH giving rise to 9-b-ribofuranosyluric acid (8). The redox pair 7/8 would thus be the electrocatalyst for the oxidation of NADH. But, is this scheme of reactions kinetically feasible? DFT calculations of N9-ethyl derivatives (R = ethyl in Scheme 1) were carried out in order to determine the viability of the proposed reaction sequence. Recent theoretical studies have reported that the energy barrier for the direct waterassisted tautomerization of different guanine derivatives [25–27] ranges from 5 to 9.2 kcal mol1. These results are in good agreement with the experimental evidence that the proton transfer assisted by water presents low energy barriers. Taking into consideration these foregoings the nucleophilic attack should be the most decisive step, so we undertook a theoretical study of this part of reactive processes at the PCM-UAHF/B3LYP/6-31+G(d)// B3LYP/6-31+G(d) level of theory. Due to the taumeriza-

NH

NH2

NH2

3 H2O -6e-- 6H+

N

N

N

O

O

O

N

N

N

N

tautomerization HN

(3)

N

N

O

N

R (4) - 1. OH 1. OH- 3. -NH2 2. PT 3. -NH22. PT

R N9-substituted adenine

N R

O

(1)

O O

O N

HN

O N

N

(7)

R

H N

2e- 2H+ HN -2e-- 2H+ O

1. OH3. -NH2HN 2. PT 2 H2O 1. OHO 3. -NH2-4e-- 4H+ O 2. PT N H2N N N tautomerization N HN R O N9-substituted guanine N H2N HN N N (2) R (5) (6)

O N

N (8) H

R

N

N O N R

Scheme 1. Reaction pathways proposed for the electrochemical oxidation of purine nucleosides and oligonucleotides.

tion step (Scheme 1, 3 ! 4 and 5 ! 6) for each oxidized species we located two different starting complexes (Figs. 2 C1-A,C2-A and 3 C1-G,C2-G) corresponding to the addition of OH to C5 of the substrates. For the oxidized adenosine derivative the most favourable way starts with the formation of the complex C1-A, which in turn evolves through the transition state (TS) TS1-A for the formation of a new bond between the hydroxyl O atom and the C atom bearing the amino group. This TS presents a Gibbs energy barrier in solution of 26.5 kcal mol1. In the case of the oxidized guanine derivative the most favourable route starts with the tautomerization step (5 ! 6), followed by the formation of the complex C2-G, 0.5 kcal mol1 in Gibbs energy in solution less stable than its tautomer

1.460 Å 1.845 Å

0.75 C1-A (0.0)

TS1-A (26.5)

0.50 1.465 Å

I / µA

1.669 Å

0.25

0 C2-A (-3.3) -0.25

C

-0.3

0

0.3

TS2-A (28.6) N

O

0.6

E/V Fig. 1. Representative cyclic voltammogram (10 mV s1) of an oxidized dG20-modified-PGE in 0.1 M Tris/HCl pH 9 solution containing 20 mM CaCl2 (solid) and NADH 1 · 104 M (dashed).

Fig. 2. B3LYP/6-31+G(d) optimized structures of the most relevant critical points involved in the nucleophilic attack of the hydroxide ion to oxidized 9-ethyladenine. Relative Gibbs energies in solution, in kcal mol1, are shown in parentheses. The imaginary vibrational frequencies of TS1-A, and TS2-A are 312i, and 230i cm1, respectively.

N. de-los-Santos-A´lvarez et al. / Electrochemistry Communications 9 (2007) 1862–1866

1.470 Å 2.010 Å

C1-G (0.0)

TS1-G (41.8)

1.472 Å

2.162 Å

C2-G (0.5)

TS2-G (30.3) C

N

O

Fig. 3. B3LYP/6-31+G(d) optimized structures of the most relevant critical points involved in the nucleophilic attack of the hydroxide ion to oxidized 9-ethylguanine. Relative Gibbs energies in solution, in kcal mol1, are shown in parentheses. The imaginary vibrational frequencies of TS1-G, and TS2-G are 213i, and 267i cm1, respectively.

C1-G. The complex undergoes a nucleophilic attack through the TS TS2-G with a Gibbs energy barrier in solution of 30.3 kcal mol1 to form a new bond between the hydroxyl O atom and the C2 atom of the substrate (Fig. 3). The computed reaction barriers described above give theoretical support to the mechanistic proposal for oxidative transformation of both purine nucleobases into a common diimine structure, explaining thus that a common catalyst for the oxidation of NADH is obtained from both adenine and guanine nucleosides and oligo-homo-nucleotides. Furthermore, the difference between the two com-

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puted energy barriers also matches the observed higher reactivity of adenine derivatives over guanine ones. An important implication of the proposed guanosine and adenosine oxidative pathways is that the formation of this diimine derivative as a consequence of DNA oxidative damage could produce miscoding during replication. B3LYP/6-31+G(d) calculations indicate that (7) can potentially form a double H-bond base pair with both thymine and cytosine (Fig. 4) with stabilities of 11.1 and 11.9 kcal mol1, respectively (B3LYP/6-31+G electronic energies in hartree for (7)-cytosine, (7)-thymine, cytosine, thymine, and (7) are 1110.049741, 1169.256722, 394.949754, 454.158107, and 715.080974, respectively). Although the stability of both complexes is comparable to the Watson Crick A–T base pair (13 kcal mol1) [28] (7)-cytosine adopts a non-planar conformation thus disturbing the stacking interactions between the bases. The proposed mechanism could be investigated as a possible alternative route to identify the genesis of GC M AT substitution mutations. 4. Conclusions The electrochemical oxidation of guanine and adenine nucleosides and oligo-homo-nucleotides at pyrolytic graphite electrodes gives rise to a common adsorbed product not previously described. Nucleophilic attack of OH to purine bases followed by proton transfer and loss of amidure at C2 of guanine derivatives and C6 of adenine derivatives provides a suitable rationalization of the experimental results. DFT calculations support the proposed reaction paths and indicate that the common oxidized base product forms stable H-bond base pairs with both thymine and cytosine, raising a possible chemical explanation for substitution mutations GC M AT. Acknowledgement This research was supported by Principado de Asturias (FICYT, Project IB05-048).

a 1.753 Å

References 1.874 Å

b

1.826 Å

1.870 Å

C

N

O

Fig. 4. B3LYP/6-31+G(d) optimized structures of the base pair complexes: (a) product (7)-cytosine and (b) product (7)-thymine. Only the most relevant distances are shown in angstroms.

[1] K.A. Freidman, A. Heller, J. Am. Chem. Soc. 126 (2004) 2368. [2] M.L. Wood, A. Esteve, M.L. Morningstar, G.M. Kuziemko, J.M. Essigmann, Nucleic Acids Res. 20 (1992) 6023. [3] G. Pratviel, B. Meunier, Chem. Eur. J. 12 (2006) 6018. [4] T. Gimisis, C. Cismas, Eur. J. Org. Chem. (2006) 1351. [5] M.K. Hailer, P.G. Slade, B.D. Martin, T.A. Rosenquist, K.D. Sugden, DNA Repair 4 (2005) 41. [6] P. Subramanian, G. Dryhurst, J. Electroanal. Chem. 224 (1987) 137. [7] R.N. Goyal, N. Jain, D.K. Garg, Bioelectrochem. Bioenerg. 43 (1997) 105. [8] A.M.O. Brett, F.M. Matysik, Bioelectrochem. Bioenerg. 42 (1997) 111. [9] E.E. Ferapontova, Electrochim. Acta 49 (2004) 1751. [10] M.I. Alvarez-Gonzalez, S.A. Saidman, M.J. Lobo-Castanon, A.J. Miranda-Ordieres, P. Tunon-Blanco, Anal. Chem. 72 (2000) 520. ´ lvarez, P.M. Ortea, A.M. Pan˜eda, M.J.L. Castan˜o´n, A.J.M. [11] N.D.S. A Ordieres, P.T. Blanco, J. Electroanal. Chem. 502 (2001) 109.

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´ lvarez, M.J. Lobo-Castan˜o´n, A.J. Miranda-Ordi[12] P. de-los-Santos-A eres, P. Tun˜o´n-Blanco, Anal. Chem. 74 (2002) 3342. [13] M.J. Frisch et al., Gaussian 03, Revision C.02, Gaussian, Inc., Wallingford, CT, 2004. [14] A.D. Becke, Phys. Rev. A 38 (1988) 3098. [15] C.T. Lee, W.T. Yang, R.G. Parr, Phys. Rev. B 37 (1988) 785. [16] A.D. Becke, J. Chem. Phys. 98 (1993) 5648. [17] W.J. Hehre, L. Radom, J.A. Pople, P.v.R. Schleyer, Ab Initio Molecular Orbital Theory, Wiley, New York, 1986. [18] H.B. Schlegel, J. Comput. Chem. 3 (1982) 214. [19] D.A. McQuarrie, Statistical Mechanics, Harper & Row, New York, 1986.

[20] J. Tomasi, M. Persico, Chem. Rev. 94 (1994) 2027. [21] R. Cammi, J. Tomasi, J. Comput. Chem. 16 (1995) 1449. [22] V. Barone, M. Cossi, J. Tomasi, J. Chem. Phys. 107 (1997) 3210. [23] S. Komorsky-Lovric, M. Lovric, J. Electroanal. Chem. 384 (1995) 115. [24] A. Kitani, Y.H. So, L.L. Miller, J. Am. Chem. Soc. 103 (1981) 7636. [25] C. Chatgilialoglu, C. Caminal, M. Guerra, Q.G. Mulazzani, Angew. Chem. Int. Ed. 44 (2005) 6030. [26] L. Gorb, J. Leszczynski, J. Am. Chem. Soc. 120 (1998) 5024. [27] J. Llano, L.A. Eriksson, Phys. Chem. Chem. Phys. 6 (2004) 4707. [28] L.F. Sukhodub, Chem. Rev. 87 (1987) 589.