Theoretical Study of Incorporating 6-Thioguanine Into a Guanine Tetrad and Their Influence on the Metal Ion–Guanine Tetrad

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Chemical Physics Letters 389 (2004) 421–426

Theoretical study of incorporating 6-thioguanine into a guanine tetrad and their inﬂuence on the metal ion–guanine tetrad

Fancui Meng a,*, Weiren Xu a,b, Chengbu Liu a,*

a Institute of Theoretical Chemistry, Shandong University, Jinan 250100, People’s Republic of China b Tianjin Institute of Pharmaceutical Research, Tianjin 300193, People’s Republic of China

Received 15 January 2004; in ﬁnal form 28 March 2004 Available online 20 April 2004

Abstract

G-tetrad and other 6-thioguanine (SG) incorporated tetrads have been studied in this Letter. The geometries, energies, charge distributions have been discussed. The effects of different cations (Kþ and Naþ) on the various tetrads have also been studied. The outcomes show that as the SG number increases the tetrad becomes more and more unstable. The Naþ binds more tightly with the tetrad than that of Kþ without hydration correction, while considering hydration effects the stability sequence changes to Kþ >Naþ. Electrostatic potential map of the tetrads have been plotted and the binding sites of cations have been also shown. Ó 2004 Elsevier B.V. All rights reserved.

1. Introduction 6-Thioguanine (SG) is a known anti-cancer agent that is readily incorporated in place of guanine during DNA Guanine-rich oligonucleotides are inhibitors for fi- replication. The incorporation of SG nucleotides into brinogen action in thrombin and HIV viral mediated cell DNA and RNA by polymerases can lead to cell death. fusion [1]. Guanine-rich sequences, which occur in The replacement of oxygen by sulfur at the O6 position telomeres [2] at the ends of linear chromosomes, can of guanine has been proposed to hinder hydrogen form G quartets where multiple guanines are organized bonding necessary for normal G-tetrad formation and around a central cation in a four-stranded structure [3]. could lead to changes in telomere stability. Stefl et al. [15] The structural uniqueness of G-quadruplex DNA makes have performed large-scale molecular dynamics simula- it an ideal target for drug design [4]. Various small tions to investigate the ability of the four-stranded molecules have been found to bind to G-quadruplex guanine-DNA motif to incorporate nonstandard guan- DNA. Monovalent cations such as Kþ and Naþ have ine analogue bases, and they found that the incorpora- been shown to stabilize G-quadruplex structures, pre- tion of 6-thioguanine and 6-thiopurine sharply sumably by coordination with eight carbonyl oxygen destabilized four-stranded G-DNA structures. The metal atoms present between stacked tetrads. However, it is binding properties of thio and oxo guanines are different also known that Naþ and Kþ stabilize different G-tetrad [16]. structures in telomere sequences [5–7]. G-tetrads are structure subsets of G-quadruplex and have attracted considerable interests in both experimental [8,9] and 2. Calculation methods theoretical [10–14] chemistry. Perhaps the most inter- esting characteristic of these structures is their selective The initial structure of the G-tetrad has been gener- interaction with certain cations that fit well in the cavi- ated from the coordinates of the DNA telomere with ties formed by stacking of guanine tetrads. Protein Data Bank code 1KF1 [17]. The backbone has been deleted and the bases have been capped with * Corresponding authors. Fax: +86-531-856-4464 (Chengbu Liu). hydrogen atoms. It has been shown previous that E-mail address: [email protected] (C. Liu). the capping of bases has no significant influence on

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422 F. Meng et al. / Chemical Physics Letters 389 (2004) 421–426

H-bonding between the bases [18]. G-tetrad and SG- H H H2N N tetrad have been arranged to have C4h symmetry. The N H NN initial structures of the other tetrads have been obtained H N by replacing the O6 atom with sulfur atom, thus they all H N7 have Cs symmetry. N X 6 X6 H2 Conventional ab initio and DFT methods have been N + N H M H1 2 used to predicate the interactions between nucleic acid H2N H N1 bases [19–21] and have been proved to be useful since X 6 X6 they can get data that are not available by experiments, N N H such as information about the interaction energies and N H cooperative effects. Base tetrads are relatively large N N H N systems for quantum chemical calculations with inclu- N NH H 2 sion of electron correlation. Due to computer time and H size limitations, such calculations are limited to small Fig. 1. Structure and atom labels of the cation-G-tetrad complex. fragments without considering environment. DFT method has proved to be successful in the close agree- ment between calculated and experimental geometrical parameters and less time-consuming. In G-tetrad the donor groups are O6 and N7 atoms, The tetrad and also cation–tetrads have been opti- and the acceptor groups are H1 and H2. From Table 1 mized by analytic gradient techniques. The method used we could see that the two inter-base hydrogen bond is B3LYP, which is Becke’s three parameter hybrid lengths in G-tetrad are 1.802 and 1.990 A, respectively. functional using the Lee–Yang–Parr correlation func- As one of the Gs replaced by SG the hydrogen bond tional. The basis set used is the standard double-zeta with lengths changed. The most significant variation takes polarization functions, 6-31G*. All the calculations have place in the S6...H1 bond distance with a bond distance been performed using GAUSSIAN 98 package. The total of 2.234 A, which is increased by about 0.4 A. One of binding energy has been defined as the energy difference the O6 atoms forms a bifurcated structure with H1 and between the complex and the sum of the monomers. H2 atoms, and the bond distances are 1.885 and 2.060 A, respectively. The other two O6 atoms form hydrogen bonds that are shorter than that in G-tetrad. The bond 3. Results and discussions distances of N7...H2 are all increased in 3G-1SG as compared with those in G-tetrad. In this Letter, G-tetrad refers to guanine tetrad, 3G- 2G-2SG has two conformers, one is the tetrad with SG refers to guanine tetrad with one of the guanines two SGs in diagonal position and the other one is that replaced by 6-thioguanine, 2G-2SG-1 refers to the tetrad with two SGs in adjacent position. As to the adjacent that the two adjacent guanines changes to 6-thiogua- one, 2G-2SG-1, one of the S6 atom forms a bifurcated nines, 2G-2SG-2 refers to the two diagonal guanines structure and the two hydrogen bond lengths are 2.566 changed to 6-thioguanines, G-3SG is the tetrad that and 2.348 A, respectively. The other hydrogen bond three of guanines have been replaced by 6-thioguanines involving sulfur atom is 2.335 A. While as the two SGs and SG-tetrad refers to 6-thioguanine tetrad. in diagonal position, 2G-2SG-2, there exist two bifurcated hydrogen bonds. Both of the two O atoms form a 3.1. Tetrads bifurcated structure between O6 and H1 or H2 atoms, and the bond distances are 1.949 and 1.939 A, respec- All of the aforementioned tetrads have been fully tively, while the hydrogen bonds involving sulfur are optimized and the atom labels are shown in Fig. 1. The 2.274 A long. optimized structures and geometric parameters are There are two bifurcated structures in G-3SG and the shown in Fig. 2 and Table 1, energy properties are listed distances of hydrogen bonds O6...H1 and O6...H2 are in Table 2, electrostatic potential maps are in Fig. 3. 2.512 and 2.394 A in one bifurcated H-bond structure and 2.029 and 1.901 A in the other. 3.1.1. Geometries As to SG-tetrad, the S6...H1 distance is 2.244 A and The N2–H2, N1–H1, C@O bond lengths of guanine N7...H2 distance is 2.627 A, while the corresponding monomer are 1.006, 1.014 and 1.218 A at B3LYP/6- distances in G-tetrad are 1.802 and 1.990 A. We could 31G* level, while the corresponding bond lengths in G- see that as O replaced by S the inter-base distances tetrad are all increased by about 0.02 A. The N2–H2, increased. The differences between the geometries of N1–H1, C@S bond lengths of SG monomer are 1.006, G-tetrad and SG incorporated tetrad all are due to the 1.015 and 1.665 A and the corresponding bond lengths larger radius of sulfur and thus we get less compact in tetrad are all elongated. structures. 中国科技论文在线 http://www.paper.edu.cn

F. Meng et al. / Chemical Physics Letters 389 (2004) 421–426 423

Fig. 2. Optimized geometries of various tetrads.

Table 1 The optimized geometrical parameters of complexes at B3LYP/6-31G* level X6...H1 N7...H2 N2–H2 N1–H1 C@X6 G-tetrad 1.802 1.990 1.024 1.034 1.232 3G-1SG 2.234a 2.051 1.024 1.034 1.677 2.060/1.885b 1.022 1.022 1.241 1.723 2.117 1.022 1.034 1.232 1.789 2.000 1.025 1.035 1.232 2G-2SG-1 2.335 1.995 1.026 1.033 1.678 2.566/2.348 1.019 1.021 1.683 1.811 2.020 1.023 1.032 1.232 1.797 2.063 1.021 1.032 1.232 2G-2SG-2 2.274 2.103 1.023 1.031 1.675 1.949/1.939 1.022 1.024 1.241 2.274 2.103 1.023 1.031 1.675 1.949/1.939 1.022 1.031 1.673 G-3SG 2.358 2.027 1.025 1.030 1.676 2.512/2.394 1.019 1.021 1.683 2.275 2.054 1.024 1.032 1.672 2.029/1.901 1.022 1.023 1.241 SG-tetrad 2.244 2.627 1.016 1.031 1.673 a The hydrogen bonds are listed in clockwise sequence from the up one as shown in Fig. 1. b The two values are bond distances of X6...H1 and X6...H2 in the bifurcated structures.

3.1.2. Energies with the two SGs in diagonal position is )4.25 kcal/mol The complex becomes more and more unstable with more stable than that with the two SGs in neighbor the increasing of the SG number and the binding energy position. Olivas and Maher [22] and others [23,24] in- increases from )77.79 to )48.44 kcal/mol as from dicate that 6-thioguanine alters the structure and sta- G-tetrad to SG-tetrad, which could be seen in Table 2. bility of duplex DNA and inhibits quadruplex DNA As to the two diﬀerent conformers of 2G-2SG, the one formation, due to the increased radius and the decreased 中国科技论文在线 http://www.paper.edu.cn

424 F. Meng et al. / Chemical Physics Letters 389 (2004) 421–426

Table 2 Binding energies calculated at B3LYP/6-31G* level G-tetrad 3G-1SG 2G-2SG-1 2G-2SG-2 G-3SG SG-tetrad Tetrada )82.31 )77.79 )68.13 )72.38 )63.13 )48.44 Kþ–tetradb -80.85 )71.51 )69.77 )62.14 )64.06 )69.32 )39.31c )29.97 )28.23 )20.6 )22.52 )27.78 Naþ–tetrad )115.56 )102.90 )101.43 )89.94 )93.58 )98.23 )31.17 )18.51 )17.04 )5.55 )9.19 )13.84 a The energy is calculated using Ebinding ¼ EðTetradÞmEðGÞnEðSGÞ, where m and n refer to the number of Guanine and Thioguanine, respectively. b The energy is calculated using Ebinding ¼ Eðcation–TetradÞEðTetradÞEðcationÞ. c Hydration corrected binding energy according to equation DEð1Þ¼DEð2ÞDEð3Þ.

Fig. 3. Electrostatic potential of the tetrads mapped onto the surface of the electron density of 0.002 unit. Note. The blue line represents the positive part of the electrostatic potential, and the red line is the negative part. The contour spacing is 0.1au for the positive part and 0.1au for the negative part. Electrostatic potential of the tetrads are mapped onto the surface of the electron density of 0.002 unit.

electronegativity of the sulfur, which would destabilize of this charge [14]. The projection of the electrostatic the Hoogsteen hydrogen bonding of guanine quartets potential on the 0.002 electron density surface clearly relative to Watson–Crick pairing. clarifies that the central area is the only location to host a cation for the G-tetrad. From the contour plot we 3.1.3. Electrostatic potential could see that the electrostatic potential remains almost Electrostatic properties of nucleotides govern the constant inside the cavity. orientational dependence of stacking interactions and As to SG-tetrad the regions with the most negative may also be important for the fidelity of DNA poly- electrostatic potential also exist in the central cavity of merase. Chin et al. [25] proposed that the electrostatic the four sulfur atoms in the SGs, while the cavity is potential might play an important role in RNA recog- much smaller than that of G-tetrad. As compared with nition and stabilization. Sites of unusual electrostatic carbonyl oxygen, sulfur atom has increased radius and features correspond to functionally important regions, decreased electronegativity, which causes the electron of for example, binding sites for metal ions characterized sulfur diffuse more widely than carbonyl oxygen, thus by an unusually negative electrostatic potential. Hold the central cavity of SG-tetrad is smaller than that of this idea in mind we plotted the electrostatic potential G-tetrad. Also, H-bonds involving SG show a slightly for all of the tetrads and listed them in Fig. 3. reduced electrostatic contribution whereas the disper- The electrostatic potential map of G-tetrad shows sion attraction is enhanced. The electrostatic potential significant concentration of the negative charge in the map of 3G-SG nearly has no negative part and its cavity central area of G-tetrad (Fig. 3). The effect of cation in is smaller than G-tetrad. At the same time, the shape of stabilizing G-tetrad is clearly through the neutralization its cavity is not as regular as G-tetrad due to the 中国科技论文在线 http://www.paper.edu.cn

F. Meng et al. / Chemical Physics Letters 389 (2004) 421–426 425 incorporation of SG. The cavity of 2G-2SG-1 is much The cation binds more tightly with 2G-2SG-1 than larger than that of 2G-2SG-2. It could be seen that both with 2G-2SG-2, which is contrary to the stability se- the shape and the size of the negative cavity affect the quence of 2G-2SG-1 and 2G-2SG-2 monomers. This binding of the cation. could be explained by the ESP maps shown in Fig. 3, the negative cavity of 2G-2SG-2 is smaller than that of 2G- 3.2. Cation–tetrads 2SG-1. The negative cavity plays a key role in the binding of the tetrad with the cation. Therefore, 2G- In addition to interactions within the base tetrad 2SG-2 cannot interact favorably with the metal cations studied in the first part, stacking interactions with other in the central cavity. bases, the backbone, the presence of metal ions and solvent effects do affect the nucleic acid structure and þ stability [26]. Therefore, in this part the metal ions (Na 4. Conclusions and Kþ) and the solvent effects on the tetrad have been studied. In the presence of metal ions the tetrad con- Various SG incorporated tetrads have been optimized formation may be changed and different metal ions may at B3LYP/6-31G* level and the geometries and energy induce different conformations. properties have been discussed in this Letter. From the As the cation added in the cavity of the tetrad, the outcomes we can draw the following conclusions: hydrogen bond distances are elongated as compared (1) The SG incorporating tetrad has less compact struc- with the corresponding distances in tetrad. The X6...ion ture than G-tetrad due to the larger radius of sulfur þ þ distance in K –tetrad is longer than that in Na –tetrad atom. due to the larger radius of metal ions. (2) The tetrad becomes more and more unstable with From Table 2 it could be seen that the interaction of the increasing of the SG number. þ Na –tetrad complexes are much stronger than that of (3) The sodium binds more tightly with the tetrad than þ the corresponding K –tetrad complexes, which is not potassium without hydration correction, while after coincide with that of the experiments [27]. Metal ions in hydration correction it is in a quite reverse order. vivo are surrounded by water solutions and hydration energy of the ions affects the binding energy between the ion and the tetrad. At the same time, the G-tetrad in vivo is stacked and the cation could be positioned be- Acknowledgements tween the stacked tetrads, while the binding energy we calculated is that the cation in the central cavity of This work was supported by the National Natural tetrad. Science Foundation of China (Grant No. 20133020). The interaction energy of cation–tetrad complex (DE) is defined as the energy difference between the energy of the cation–tetrad complex and that of the ion and the References optimized tetrad. The hydration free energies of Naþ and Kþ are )84.39 and )41.54 kcal/mol at room tem- [1] K.Y. Wang, S. McCurdy, R.G. Shea, S. Swaminathanan, P.H. perature (Calculated using PCM method at B3LYP/ Bolton, Biochemistry 32 (1993) 1899. [2] E.H. Blackburn, Nature 350 (1991) 569. 6-31G* level). The interaction energy considering [3] J.R. Williamson, M.K. Raghuraman, T.R. Cech, Cell 59 (1989) hydration effect could be evaluated according to the free 871. energies of the following process: [4] W. Tuntiwechapikul, J.T. Lee, M. Salazar, J. Am. Chem. Soc. 123 (2001) 5606. þ þ G-tetrad þ K ðhydratedÞ!K –G-tetrad þ nH2O ð1Þ [5] T. Miura, J.M. Benevides, G.J. Thomas, J. Mol. Biol. 248 (1995) 233. G-tetrad þ Kþ ! Kþ–G-tetrad ð2Þ [6] N.V. Hud, F.W. Smith, F.A.L. Anet, J. Feigon, Biochemistry 35 (1996) 15383. þ þ [7] S. Bouaziz, A. Kettani, D.J. Patel, J. Mol. Biol. 282 (1998) 637. K þ nH2O ! K ðhydratedÞð3Þ [8] V.A. Szalai, H.H. Thorp, J. Am. Chem. Soc. 122 (2000) 4524. From equations above the interaction energy con- [9] P.K. Patel, A.S.R. Koti, R.V. Hosur, Nucleic Acids Res. 27 (1999) sidering hydration effect could be calculated using the 3836. [10] J.-D. Gu, J. Leszczynski, J. Phys. Chem. B 107 (2003) 6609. following equation: [11] J.-D. Gu, J. Leszczynski, J. Phys. Chem. A 106 (2002) 529. DE 1 DE 2 DE 3 : 4 [12] J.-D. Gu, J. Leszczynski, J. Phys. Chem. A 104 (2000) 6308. ð Þ¼ ð Þ ð Þ ð Þ [13] M. Meyer, M. Brandl, J. Suhnel, J. Phys. Chem. A 105 (2001) After hydration correction, Kþ–tetrad complexes are 8223. þ [14] J.-D. Gu, J. Leszczynski, M. Bansal, Chem. Phys. Lett. 311 (1999) more stable than that of the corresponding Na –tetrad 209. complexes. Thus it can be concluded that the cation [15] R. Stefl, Nad’a Spackova, I. Berger, J. Koca, J. Sponer, Biophys. hydration affects the stability of the complexes. J. 80 (2001) 455. 中国科技论文在线 http://www.paper.edu.cn

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