Complutense, Ciudad Universitaria, Madrid, 28040, Spain Published online: 03 Jun 2013.

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1 This article was downloaded by: [Biblioteca Universidad Complutense de Madrid], [Mauricio Palafox] On: 05 June 2013, At: 00:43 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: Registered office: Mortimer House, Mortimer Street, London W1T 3JH, UK Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: Molecular structure differences between the antiviral Nucleoside Analogue 5-iodo-2 -deoxyuridine and the natural nucleoside 2 -deoxythymidine using MP2 and DFT methods: conformational analysis, crystal simulations, DNA pairs and possible behaviour M. Alcolea Palafox a a Facultad de Ciencias Químicas, Departamento de Química-Física I, Universidad Complutense, Ciudad Universitaria, Madrid, 28040, Spain Published online: 03 Jun To cite this article: M. Alcolea Palafox (2013): Molecular structure differences between the antiviral Nucleoside Analogue 5-iodo-2 -deoxyuridine and the natural nucleoside 2 -deoxythymidine using MP2 and DFT methods: conformational analysis, crystal simulations, DNA pairs and possible behaviour, Journal of Biomolecular Structure and Dynamics, DOI: / To link to this article: PLEASE SCROLL DOWN FOR ARTICLE Full terms and conditions of use: This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. The publisher does not give any warranty express or implied or make any representation that the contents will be complete or accurate or up to date. The accuracy of any instructions, formulae, and drug doses should be independently verified with primary sources. The publisher shall not be liable for any loss, actions, claims, proceedings, demand, or costs or damages whatsoever or howsoever caused arising directly or indirectly in connection with or arising out of the use of this material.

2 Journal of Biomolecular Structure and Dynamics, Molecular structure differences between the antiviral Nucleoside Analogue 5-iodo-2 - deoxyuridine and the natural nucleoside 2 -deoxythymidine using MP2 and DFT methods: conformational analysis, crystal simulations, DNA pairs and possible behaviour M. Alcolea Palafox* Facultad de Ciencias Químicas, Departamento de Química-Física I, Universidad Complutense, Ciudad Universitaria, Madrid, 28040, Spain Communicated by Ramaswamy H. Sarma (Received 8 February 2013; final version received 21 March 2013) 5-iodo-2 -deoxyuridine Nucleoside Analogue (IUdR) was the first selective antiviral nucleoside against herpes simplex virus type 1 and 2, and it was also a meaningful anticancer drug. Within a full study of this drug and its possible behaviour, previously, a comprehensive theoretical conformational analysis by MP2 and B3LYP was carried out, and all the possible stable structures were determined with full relaxation of all geometrical parameters. The search located 45 stable structures, and in all them, the whole conformational parameters (v, b, c, /, d, P, m max ) were analyzed as well as the NBO natural atomic charges. Comparisons of the conformers with those of the natural Nucleoside 2 -deoxythymidine (dt) were carried out, and the main differences between IUdR and dt were analyzed. The accuracy of the methods used was probed with the simulation of the X-ray crystal data by a tetramer form. Watson-Crick (WC) IUdR/dT 2 -deoxyadenosine pairs were analyzed for the first time using quantum chemical calculations, as well as the mispairing IUdR/dT 2 -deoxyguanosine. As result, it is observed that IUdR give rises to a slightly stronger WC pair and weaker mispairing than those with dt, therefore deforming slightly the DNA axis and difficulting the growth of the DNA virus and consequently, killing it. Keywords: 5-iodo-2 -deoxyuridine; IUdR; thymidine; antiviral; DNA base pairs 1. Introduction Nucleoside analogues have been primarily examined for their ability to inhibit reverse transcriptase, and also the ability to inhibit enzymes necessary for de novo thymidine synthesis, such as thymidylate synthase (TS) and thymidylate kinase (TK). Alternatively, they can be incorporated into a growing strand of DNA, and either cause strand termination, or produce a kink in the DNA that prevents replication (Vortherms, Dang, & Doyle, 2009). Thus, several nucleoside and nucleotide analogues have been identified as potent anticancer and antiviral agents (De Clercq & Neyts, 2004; Taft & Paula da Silva, 2006). Among these compounds, 5-iodo-2 -deoxyuridine nucleoside analogue (IUdR) (Scheme 1) was the first compound identified with antiviral activity against a number of orthopoxviruses, and it has been reported to be active both in vitro and in animal models of infection (Prichard & Kern, 2010). Treatment with IUdR inhibited viral replication of iridovirus (Chen, Zheng, & Jiang, 1999), poxviruses (including variola, vaccinia (Neyts, Verbeken, & De Clercq, 2002; Smee, Humphreys, Hurst, & Barnard, 2008), monkeypox, cowpox, molluscum contagiosum, orf) and ranavirus (Qin et al., 2003). IUdR has antimicrobial activities (Zander et al., 2010) alone and in combination against S. Aureus. When IUdR labelled with 125I (125IUdR) is incorporated into the DNA of mitotic tumour cells, the Auger electrons emitted during iodine decay are highly cytotoxic (Yamasaki, Moritake, & Klein, 2003; Rebischung et al., 2008). Thus, it is used as a clinical radiosensitizer (Atcher et al., 1989; Kinsella, Dobson, Mithcell, & Fornace, 1987; Seo et al., 2006; Zager et al., 2003) for the detection of DNA replication, as a marker for cell kinetic studies (Gratzner, 1982; Speth et al., 1989), and in the studies of early proliferative response to anticancer treatment (Carnochan & Brooks, 1999). The development of novel anti-herpes virus drugs that are more effective for varicella-zoster virus (VZV) and human cytomegalovirus infections and less cytotoxic against host cells is desirable. For this reason, previously we have carried out a previous study (Alcolea, 2013) on IUdR with the aims of establish the * alcolea@ucm.es Ó 2013 Taylor & Francis

3 2 M. Alcolea Palafox Scheme 1. Molecular structure and definition of the exocyclic and endocyclic angles in IUdR nucleoside. relationship between structure, conformational features or physicochemical properties and activity of these drugs. From our understanding, it would be interesting to analyse previously the different conformational possibilities for IUdR and compare the results to the nucleoside natural 2 -deoxythymidine (dt) in order to find the facility to change of conformation during the reaction in the active site. Also an accurate knowledge of the conformational properties of these nucleosides would be an important help for the interpretation of drug target interactions. Different authors have analyzed the conformers of several nucleosides and nucleosides analogues (Baumgartner, Motura, Contreras, Pierini, & Briñón, 2003; Choi et al., 2003; Ponomareva, Yurenko, Zhurakivsky, Mourik, & Hovorun, 2012; Saran & Ojha, 1993a, 1993b; Yurenko, Zhurakivsky, Ghomi, Samijlenko, & Hovorun, 2008; Yurenko, Zhurakivsky, Samijlenko, & Hovorun, 2011; Yurenko, Zhurakivsky, Ghomi, Samijlenko, & Hovorun, 2007a), but no one conformational study appears on IUdR, and no one comparison dt vs. IUdR has been reported. Although calculations have been published for the uracil base (Alcolea, Iza, & Gil, 2002; Alcolea & Rastogi, 2002), thymine base (Brovarets & Hovorun, 2010) and for 5-iodouracil (Alcolea et al., 2010; Rastogi et al., 2010), no similar studies have been carried out previously for IUdR. Another goal of present research is a simulation of the solid-state structure. One of the reasons of this study is to confirm the validity of our calculations performed on IUdR through a comparison with the experimental X- ray data. We have simulated the crystal unit cells in nucleoside analogues (Alcolea & Iza, 2012; Alcolea, 2013) and nucleobase derivatives (Alcolea et al., 2007; 2010; Rastogi & Alcolea, 2011; Ortiz, Alcolea Palafox, & Rastogi, 2012) by dimer and tetramer forms, and in general, the geometric parameters and IR and Raman vibrational spectra were in accordance with those found experimentally. Now, we have carried out the simulation of the crystal unit cell in IUdR with a tetramer form. Nucleosides analogues containing unsaturated ribose ring structure, with lack 2 - and 3 -OH groups, are the most effective compounds because the growth of the DNA viral chain through 3 -OH is impeded. However, IUdR has this 3 -OH group and as consequence the growth is not impeded. Thus, other mechanism of inhibition should be presented in IUdR. Several authors (Camerman & Trotter, 1965; Bailey, Navarro, & Hernanz, 1999) have given a possible general explanation without details. Now, one of the aims of the present work is to carry out a detailed comparison of the molecular structure of IUdR with dt and thus to suggest a possible reason for this inhibition. This is another goal of the present manuscript. For this purpose, with the calculation of the interaction energies in the Watson Crick (WC) pairs and mispairing pairs, the differences with dt were determined, and therefore, a possible reason for this inhibitory activity of IUdR can be given. The molecular mechanism of the spontaneous substitution mutations conditioned by tautomerism of bases has been studied by different authors (Danilov, Anisimov, Kurita, & Hovorun, 2005). 2. Calculations The calculations were carried out by using the MP2 method and by using density functional methods (DFT), such as B3LYP hybrid functional. These methods appear implemented in the GAUSSIAN 03 program package (Frisch et al., 2003). DFT methods provide adequate compromise between the desired chemical accuracy and the heavy demands put on computer time and power. Moreover, DFT methods have been used satisfactory in many studies of drug design (Hoffmann & Rychlewski, 2002; Alcolea et al., 2011). Because of the iodine atom, the DGDZVP basis set was selected for all the calculations. The B3LYP method was chosen because different studies have shown that the data obtained with this level of theory are in good agreement with those obtained by other more costly computational methods as MP2 calculations and it predicts vibrational wavenumbers of DNA bases better than the HF and MP2 methods (Alcolea, 1998, 2000; Alcolea et al., 2002, 2004; Alcolea & Rastogi, 2002). The optimum geometry was determined, by minimizing the energy with respect to all geometrical parameters without imposing molecular symmetry constraints. Berny optimization under the TIGHT convergence criterion was used. Atomic charges were determined with the Natural NBO procedure (Carpenter & Weinhold, 1988; Reed, Curtiss, & Weinhold, 1988). The harmonic wavenumber computations were carried out at the same level of the respective optimization

4 The antiviral Nucleoside Analogue 5-iodo-2 -deoxyuridine (IUdR) 3 process and by the analytic evaluation of the second derivative of the energy with respect to the nuclear displacement. Wavenumber calculations were performed in all the optimized conformers determined by DFT to asses that they correspond to real minimum. All the optimized structures showed positive harmonic vibrations only (true energy minimum). Relative energies were obtained by including zero-point vibrational energies (ZPE). For the calculation of the ZPE, the wavenumbers were retained unscaled. The Gibbs free energy was calculated at K. The potential energy surface of this molecule was determined by rotation of the torsional angles v (glycosidic bond), c (C4 -C5 bond), b (C5 -O5 bond), e (C3 -O3 bond) and the pseudorotation phase P. These dihedral angles simultaneously held fixed at values varying between 0 and 360 in steps of 20 in a detailed second study. All the remaining geometrical parameters were relaxed during these optimizations. All the optimized points were plotted by the SURFER program (SURFER, 1999). Thus, all local minimum were determined. All quantum mechanical computations have been performed on the alpha computer of the Computational Centre from University Complutense of Madrid Interaction energies They were computed on the B3LYP optimized structures with the DGDZVP basis set. In the WC pair and mispairing pair of IUdR and dt with 2 -deoxyadenosine (da) and 2 -deoxyguanosine (dg) the interaction energies obtained were corrected for basis set superposition error (BSSE) using the standard counterpoise (CP) procedure (Boys & Bernardi, 1970; Alcolea et al., 2009) as follows: The total CP corrected interaction energy, DEAB CP ; between the nucleoside A ( IUdR or dt) and the nucleoside B ( da or dg) is calculated according to: DE CP AB ¼ Eint ðabþþe def ðabþ where E int ðabþ is defined as: ð1þ E int ðabþ ¼E AB AB ðabþ EAB A ðabþ EAB B ðabþ ð2þ and where EAB AB ðabþ stands for the electronic energy at the optimized geometry of the whole system, and EA AB (AB) (or EB AB (AB)) is the electronic energy of the isolated subsystem A (or B) in the entire system AB. The deformation energy E def ðabþ is defined as: E def ðabþ ¼E def A ðabþþedef B ðabþ ð3þ where the deformation energy of monomer X ( AorB) is: E def X ðabþ ¼EX X ðabþ EX XðXÞ ð4þ Here, the subscripts denote the molecular system, and the superscripts indicate whether the calculation is done with the basis set of the entire system (AB), or the basis set of the monomer (X). The parenthesis indicates whether the calculation is done at the optimised geometry of the entire system (AB), or at the monomer optimised geometry (X). 3. Results and discussion 3.1. Definition of the conformational angles Following the Saenger s notation (Saenger, 1984), the atomic description of this molecule as well as the most important exocyclic and endocyclic torsional angles is defined in Scheme 1. The different conformations in IUdR can be characterized by the following five important structural parameters: (i) the glycosylic torsional angle, v(o4 C1 N1 C2), which determines the two orientations of the base relative to the furanose ring, denoted as the anti and syn forms. (ii) The exocyclic torsional angle b(c4 C5 O5 H5 ), which describes the orientation of the hydroxyl hydrogen H5 relative to the furanose ring. (iii) The exocyclic torsional angle c(c3 C4 C5 O5 ), which determines the orientation of the 5 -hydroxyl group relative to the furanose ring. This ring is twisted out-of-plane in order to minimize non-bonded interactions between their substituents and located in the north (N) [C2 - exo/c3 -endo] and south (S) [C2 -endo/c3 -exo] conformers. (iv) The torsional angle d(c5 C4 C3 O3 ), which describes the orientation of the CH 2 OH moiety relative to the furanose ring. Finally, (v) the torsional angle /(C2 C3 O3 H3 ) that determines the orientation of hydroxyl hydrogen H In the isolated state Conformers and energetics An extensive conformational study was carried out and the relative calculated energies of the 45 optimum conformers appear collected in Table 1 at the B3LYP and MP2 levels, as well as a detailed collection of the most important conformational parameters of these optimized forms. The conformers were classified according to the three ranges of rotation of v: conformers C (v: ± 11.1 by B3LYP), conformers A (v: ± 6.4 ) and conformers B (v: 74.8 ± 13.3 ), Figure 1. The energy criterion by MP2 was followed for the numbering: firstly,

5 4 M. Alcolea Palafox Table 1. The 45 optimum stable conformers calculated in IUdR molecule, with endocyclic and exocyclic torsional angles in degrees, pseudorotational angle P in degrees, dipole moment l (in Debye), and relative energies ΔE, and Free energies ΔG in (kcal mol 1 ). The values are listed successively at the B3LYP/DGDZVP level, MP2/DGDZVP level (values in parentheses), simulation of the hydration with 20 water molecules and at the B3LYP/DGDZVP level (values in quotation marks and in italic), and with PCM at B3LYP/DGDZVP level (values in quotation marks). Conf v b c / d m0 m1 m2 m3 m4 P a mmax b Sugar conf. l DE DG B (61.7) (64.1) (45.5) (73.3) (143.9) ( 26.37) (38.18) ( 34.78) (20.89) (3.16) (156.38) (37.96) 2 E c 2 E (5.32) 0 d E e 0 f E g 0 h B , T (62.8) (42.6) (46.0) (152.3) (92.6) ( 14.05) ( 6.08) (22.19) ( 31.08) (28.64) (44.75) (31.24) (4.64) (2.130) B E (63.8) (42.2) (48.6) (96.6) (91.0) ( 14.86) ( 4.52) (20.62) ( 29.93) (28.49) (47.30) (30.40) (5.02) (2.168) B T (66.15) (176.5) ( 57.3) ( 178.7) (95.3) ( 8.38) ( 9.49) (22.08) ( 27.86) (23.08) (36.25) (27.38) (3.67) (2.538) B T (66.4) ( 89.1) ( 57.3) (174.8) (94.5) ( 8.71) ( 9.56) (22.50) ( 28.47) (23.66) (36.52) (28.00) (5.08) (3.434) B6 (114.0) (174.7) (54.2) (81.9) (104.8) (32.17) ( 38.67) (30.21) ( 12.10) ( 12.45) (322.56) (38.05) 2 1 T (4.26) (3.454) B T (67.8) ( 59.9) ( 179.6) (87.4) (94.7) ( 6.75) ( 10.43) (22.26) ( 26.75) (21.46) (33.02) (26,55) (4.64) (3.709) B T B E (63.7) (40.8) (49.3) ( 57.2) (87.0) ( 14.65) ( 4.35) (20.07) ( 29.20) (27.87) (47.49) (22.69) (6.75) (4.227) B T B T B T (70.9) (39.3) ( 69.9) (78.2) (81.7) ( 16.41) ( 7.91) (27.24) ( 37.84) (34.29) (43.88) (37.79) (4.85) (4.896) B T B T (67.6) ( 172.7) ( 170.3) (85.2) (91.5) ( 8.24) ( 9.91) (22.72) ( 28.22) (23.29) (35.48) (27.90) (4.17) (6.272) B T (67.1) (66.7) ( 175.1) (86.1) (94.3) ( 4.54) ( 11.31) (21.46) ( 24.78) (18.78) (29.12) (24.56) (6.24) (6.347) B E B T B T B E B E B E B T C E ( 128.5) (175.8) (50.9) (64.2) (145.4) ( 19.42) (33.55) ( 34.15) (23.82) ( 2.90) (165.98) (35.20) 2 E (7.96) (0.086) 3 2 T C E ( 124.1) (174.9) (50.0) ( 179.3) (150.7) ( 18.04) (33.14) ( 34.69) (25.18) ( 4.57) (168.65) (35.38) 2 E (9.24) (0.878) T (Continued)

6 The antiviral Nucleoside Analogue 5-iodo-2 -deoxyuridine (IUdR) 5 Table 1. (Continued) Conf v b c / d m0 m1 m2 m3 m4 P a mmax b Sugar conf. l DE DG T » T C3 ( 123.3) (177.8) (49.4) ( 57.1) (142.6) ( 14.93) (30.78) ( 33.94) (26.08) ( 7.17) (173.20) (34.18) 3 2 T (6.48) (1.499) E T C4 C T g C E ( 135.1) ( 49.6) (173.4) (68.0) (145.3) ( 27.25) (39.25) ( 35.70) (20.79) (3.86) (155.72) (39.16) 2 E (6.23) (3.922) C T C E C T C E C E C T C E A1 ( 164.4) (174.7) (52.9) (148.8) (60.1) ( 10.04) (27.20) ( 32.90) (28.06) ( 11.48) (181.30) (32.91) 2 T (7.31) (0.852) A E ( 160.2) (167.0) (48.0) (153.7) (84.6) (4.70) ( 27.54) (37.99) ( 36.31) (20.16) (11.71) (38.80) (7.48) (1.454) A E ( 174.4) (179.1) (172.8) (61.3) (156.0) (9.91) (12.9) ( 29.11)C (35.62) ( 28.98) (214.52) (35.33) 3 4 T (6.56) (3.562) A E ( 170.6) (59.1) (166.5) (61.6) (155.5) (4.36) (17.79) ( 31.47) (34.92) ( 25.03) (205.66) (34.91) (7.63) (4.006) A E ( 167.3) ( 73.9) ( 65.1) (66.8) (149.6) ( 21.16) (36.31) ( 36.67) (25.52) ( 2.97) (165.60) (37.86) (5.61) (4.116) A E ( 170.4) ( 169.4) ( 64.9) (67.9) (154.4) ( 13.7) (31.8) ( 36.7) (29.9) ( 10.4) (177.36) (36.74) 3 2 T (5.38) (4.189) A A A E E E ( 169.2) (81.0) ( 74.9) (62.1) (149.7) ( 20.90) (36.20) ( 36.92) (25.88) ( 3.34) (166.21) (38.02) (5.60) (5.806) A E a tgp ¼ ðm4þm1þ ðm3þm0þ 2m2ðsinð36Þþsinð72ÞÞ b m max ¼ m2 cosp when m 2 is negative, 180 is added 59 to the calculated value of P. c ΔE = AU. d ΔE = AU. e ΔE = AU. f ΔG = AU. g ΔE = AU. h ΔG = AU. 1 AU = kcal/mol.

7 6 M. Alcolea Palafox Conformers A Conformers B Conformers C Figure 1. Three types of conformers determined in IUdR corresponding to the three ranges of rotation of v, with b and c 60 o. the most stable and also the expected non-active biological syn forms (B) were numbered; secondly, those conformers found in the crystal and, by analogy with related nucleosides (Hoffmann & Rychlewski, 2002; Alcolea et al., 2009; Alcolea & Talaya, 2010; Alcolea & Iza, 2010), the expected active biological anti forms (C); and finally, the high-anti conformers (A) were listed. Conformers A were in general difficult to be optimized, and in many cases, they changed to the equivalent conformer C. Two energy criteria were considered for each conformer: the electronic energy E + ZPE correction and the Gibbs energy G. The conformers differ in general very little in energy. Thus, our 45 optimized conformers were found within (a) Conformer B1 Conformer C1 Figure 2. Calculated parameters in conformers B1 and C1 (global minimum of the syn and anti forms, respectively): (a) optimum bond lengths and angles and (b) natural atomic charges on the atoms. (b) the electronic energy range ΔE = 0 8 kcal/mol and Gibbs energy range ΔG =0 7 kcal/mol related to the global minimum, Figure 2. This range of ΔG values is similar to that reported (Yurenko et al., 2007b) in dt, kcal/mol. The stability order of the five most stable conformers by the E + ZPE criterion is: B1 > C1 > A1 > C2 > A2 (by MP2/dgdzvp) B1 > C1 > C3 > C2 > A2 (by B3LYP/dgdzvp) By Gibbs energy criterion is: C5 > C1 > B1 > C4 > A2 (by B3LYP/dgdzvp)

8 The antiviral Nucleoside Analogue 5-iodo-2 -deoxyuridine (IUdR) 7 Figure 3. Plot with the geometry of the 12 best conformers of IUdR, and with the values of the strongest intramolecular H-bonds determined at B3LY/DGDZVP and MP2/DGDZVP levels (values in parentheses). Six conformers are only found within the electronic energy range DE =0 2.0 kcal/mol by MP2, five of them with v ( to ) anti, mainly S-type furan puckering, with c ( ), b ( ) and with great variation for e angle. The ratio anti/syn form is remarkable decreased from 5.0 in the low-energy group (<2 kcal/mol) to 1.7 in the 0 4 kcal/mol energy group. Figure 3 shows the eleven best conformers selected in each range of the rotational angle v: five of them correspond to conformers C (C1 to C5), two to conformers A (A1 and A2) and four to conformers B (B1 to B4). In each conformer, the values of the most important H- bonds were included in the figure. The distribution of the calculated low-energy conformers (including the global minimum) is similar to that determined (Merchante, Tamara, Alcolea Palafox, & Iza, 2010) in dt. The global minimum by B3LYP and MP2 methods corresponds to the syn-gg-gg form with respect to v, c and b torsional angles, respectively, and with the sugar puckering envelope C2 -endo, 2 E. This conformer is denoted as B1 (Figures 2 and 3) and it appears stabilized by an intramolecular H-bond. The optimized bond lengths and natural NBO atomic charges on this conformer are collected in Figure 2. It is noted that the global minimum by G criterion corresponds to conformer C5, although the difference is very small compared with B1. The second most stable conformer corresponds to anti-gg-gt form, also 2 E, and denoted as C1 (Figures 2 and 3). This anti form is the expected ones for the natural nucleosides and nucleotides in biological systems (Saenger, 1984; Alcolea et al., 2009; Alcolea & Talaya, 2010), and for this reason, it was also analysed in the present manuscript. The difference of energy between both conformers B1 and C1 is very small,.086 kcal/mol by MP2 (.403 kcal/mol by B3LYP).

9 8 M. Alcolea Palafox Conformational angle analysis Figures 4 6 and Figure 1-Sup (of Supplementary Material) show the distribution of the 45 optimized conformers according to their energies, exocyclic torsional angles, and values of P and m max. When it was possible, several of the best conformers are pointed in these figures. An overall examination of the exocyclic torsional angles, defining the conformational space of the IUdR prodrug, leads to conclude the following: (1) The interring dihedral angle v presents a trimodal distribution, conformers A (from to by B3LYP), B (61.5 to 88.2 ) and C ( to ), Table 1 and Figure 5. Conformers C are in general the most stable (Figure 1-Sup (a)) in accordance to the results (Yurenko et al., 2007b; Merchante et al., 2010) in dt and to the mean value of 135 reported in a Figure 4. Distribution of the 45 optimum stable calculated conformers in IUdR, according to their phase angle of pseudorotation P and their: (a) relative electronic energy ΔE + ZPE correction; (b) relative Gibbs energy ΔG; and (c) puckering amplitude, m max. The most stable conformers of each type are pointed in bold type. crystal database of uridine analogues (Allen et al., 1979). Anti forms (conformers A and C, 23 conformers) have similar number that the syn ones (conformers B, 22 conformers), although they cover a wider range of v values. Also they dominate in the low energy range < 2 kcal/mol, anti/ syn = 22/4% by MP2, and 26/14% by B3LYP. This fact can be interpreted by the less sterical restricted by non-covalent interactions between the base and the sugar residue in the anti forms. Moreover, X- ray data of IUdR (Camerman & Trotter, 1965) and related nucleosides (Shishkin, Pelmenschikov, Hovorun, & Leszczynski, 2000a; Hocquet, Leulliot, & Ghomi, 2000) appear with the v angle in anti orientation, and it is also known the preference of the anti form for biological activity (Kovacs et al., 1991; Painter, Aulabaugh, & Andrews, 1993; Painter, Andrews, & Furman, 2000). (2) The b angle has a clear trimodal distribution as in dt (Merchante et al., 2010): b g (17 conformers), b t and b t (15 conformers), and b g (13 conformers), Figure 5. This b angle seems of particular interest because a trans orientation is required for the phosphorylation of the O5 H group in the active site of the TK. (3) The c angle has also a trimodal distribution but with short range of values: c t and c t (13 conformers), c g (18 conformers) and c g (14 conformers). This distribution is similar to that found in AZT and dt nucleosides. Conformers with c g+ are the most stables, Figure 1. -Sup.(c), and confirm that the value of c ca. 50 observed in other nucleosides leads to the most stable conformers. (4) The / angle has a clear trimodal distribution with short range of values: / t and / t (16 conformers), / g (18 conformers) and / g (11 conformers). (5) The d angle has a bimodal distribution with the following range of values: d g (26 conformers) and d t (18 conformers). Correlations between the values of the exocyclic torsional angles c, v and b were not found. It can be explained by the significant flexibility of the structure, which permits many combinations of these torsional angles. The stability areas of the conformers were established, with the following largest ranges without conformers: v , b , b , c , c and c

10 The antiviral Nucleoside Analogue 5-iodo-2 -deoxyuridine (IUdR) 9 Figure 5. Distribution of the 45 optimum stable calculated conformers in IUdR, according to the values of the exocyclic torsional angles: v, b, and c vs. the pseudorotational phase angle P. Figure 6. Distribution of the 45 optimum stable conformers in IUdR according to the values of the exocyclic torsional angle v vs. m max Uracil ring The replacement of the H5 hydrogen with a methyl group or an iodine atom greatly increases the affinity of the nucleoside for the thymidine kinase (Fischer, Fang, Lin, Hampton, & Bruggink, 1988). Also, the substitution of the halogen atom at the 5 position of the pyrimidine ring by an atom with higher molecular weight increases the anti-hsv (herpes simplex virus) and varicella-soster virus (VZV) activities (Shigeta et al., 1999). It has been suggested that the inhibitory effect of IUdR depends not only on the 5-substituted being of a size that avoids steric hindrance, but also on its electron-withdrawing property. The effect of 5-substituents on antiviral activity is frequently related (Molina & Espinosa, 1995) to their ability to donate or withdraw electrons. Electron withdrawing groups shorten the N1 C6, C2=O and C1 O4 and lengthen the C5=C6, N1 C2 and N1 C1 bonds. Due to this feature, and as a first step, a detailed analysis of the geometry of IUdR is presented here, Figure 2. In the six most stable conformers, the base heterocycle appears with a very small nonplanarity, with torsional angles lower than 4, unlike the free base (Rastogi et al., 2010) and the planar uracil molecule (Alcolea et al., 2002). It is arising due to the structural variability of the bases (Govorun et al., 1992; Hovorun, Gorb, & Leszczynski, 1999; Nikolaienko, Bulavin, & Hovorun, 2011) and the anisotropy influence of the sugar residue on the base. Among the six dihedral angles describing the base heterocycle nonplanarity, C5=C6 N1 C2, C6 N1 C2 N3, N1 C2 N3 C4, C2 N3 C4 C5, N3 C4 C5=C6 and C4

11 10 M. Alcolea Palafox C5=C6 N1, the first three are the most dependent on the nucleoside conformation (Yurenko et al., 2007b); that is, they fall by MP2 into the largest ranges of values 1.45 to 3.78, 3.94 to 1.36, and.70 to 3.51, respectively, in the six best conformers. The other three angles cover rather narrower ranges: 1.65 to.21,.74 to.0 and 1.20 to.69, respectively. The highest non-planarity appears in B1, because of its strong H-bond, while the lowest one corresponds to C1. The six concerned torsional angles correlate with each other in such a way that their algebraic sum is close to 0, with standard deviation of.1 and an absolute value no greater than.3. This small nonplanarity in IUdR is due to weak H-bonds/interactions of the uracil base with the CH 2 O5 H moiety. Similar nonplanarity has been reported in dt (Yurenko et al., 2007b; Merchante et al., 2010) and in related nucleosides (Yurenko et al., 2008; Tamara, Merchante, & Alcolea, 2010; Tamara & Alcolea, 2011). It is well known that pyrimidine rings in nucleic acid bases possess high conformational flexibility. Therefore, such small changes of endocyclic torsion angles almost do not require energy. Thus, these heterocycles may adopt slightly non-planar geometry due to any internal influence like hydrogen bonds or steric clashes. Differences in energy between ideal planar structure and slightly nonplanar structure are almost zero or at least they are smaller than.1 kcal/mol (Shishkin, Gorb, Hobza, & Leszczynski, 2000; Shishkin, Gorb, & Leszczynski, 2000; Isayev, Furmanchuk, Shishkin, Gorb, & Leszczynski, 2007). The bond lengths and angles do not differ significantly of similar nucleosides. The lengths in the C N and C C single bonds are intermediate between those of the corresponding aromatic and of the saturated bonds. Hence, there is some aromatic character on the ring structure. Because of the weak O2 H1 interaction in conformers B and C, the C6 N1 C1 angle is slightly more close than C2 N1 C1. In the six most stable conformers B and C, their values vary within the narrow limits. However, in conformers A, the C6 N1 C1 angle is more open than C2 N1 C1, ca. 122 vs These features indicate the significant value of the deformation angle between the glycosidic N1 C1 bond and the N1 C2 C6 plane. In a general comparison of the results, MP2 computes the C=O bond lengths slightly longer, and the C4 C, C4-C5 slightly shorter than B3LYP. Also an opening of the C6 N1 C2, N1 C1 H1 angles, and closing of the N1 C2 N3, C4 C5 O5 and C5 O5 H5 angles by MP2 was observed Ribose ring The ribose ring appears rotated respect to the uracil ring, with an v angle at MP2 level in conformer C1 of 128.5, and with N1 C1 C2 C3 torsional angle of The bulky of this substitution deforms all the neighbour angles. Thus, compared with 5-iodouracil molecule (Alcolea et al., 2010; Rastogi et al., 2010) the ipso angle C2 N1 C6 (122.4 ) is closed 1.6, while the N1 C2 N3 and N1 C6=C5 (122.4 ) angles are opened ca..7. The H 2 C5 O5 H substituent appears out-of-ring plane with a C2 C3 C4 C5 torsional angle of The ribose ring is usually characterized by three structural parameters (Saenger, 1984): (i) the endocyclic torsion angles, m 0 m 4 ; (ii) the pseudorotation phase angle, P, defined in the bottom of Table 1; and (iii) the maximum torsional angle (degree of pucker), m max, Table 1. Figure 4. shows three diagrams with the distribution of the 45 calculated conformers according to the P angle vs. the ΔE, ΔG energies and m max. (i) The endocyclic torsional angles appear in a large range 40 to 40 : m o 6 3.1, m , m , m , and m , indicating a flexible ribose ring. The values of all these angles have in general different sign in conformers B and C. This fact leads to a pseudorotation phase angle P of N-type in conformers B and of S-type in conformers A and C. The algebraic sum of m i dihedral angles (i =0, 1,,4) is close to 0 for all conformers and it fails into the 1.0 to 1.37 range. Similar results were found in dt and in AZT (Alcolea & Talaya, 2010). The ribose pucker in a nucleoside is an important parameter related to the level of activity against HIV-1. Thus, C3 -endo, rather than C3 -exo pucker, or other puckers of the C2 -endo family is reported to be probably the required conformation for antiviral HIV-1 activity (Dijkstra, Benevides, & Thomas, 1991). However, the C2 -endo pucker is the most stable and preferred form in IUdR, and due to the nucleoside phosphorylation does not, in general, change the preferred ribose and deoxyribose puckers of a nucleoside (Dijkstra et al., 1991), it could be the preferred form for both nucleoside kinase and reverse transcriptase recognition. (ii) Three ranges appear for P. The two main groups cover the ranges: P (envelope C 3 -endo, symmetrical twist C 4 -exo-c 3 -endo, N-type) (22 conformers), P (C 2 -endo, C 3 -exo, symmetrical twist, S-type) (19 conformers), while P (O 4 - endo, symmetrical twist C 1 -exo-o 4 -endo, S-type) (3 conformers), values that are in accordance with those found in other nucleosides. Conformers with P values of 70.3 ± 23.4, ± 8.9 and

12 The antiviral Nucleoside Analogue 5-iodo-2 -deoxyuridine (IUdR) ± 78.5 are not stable. The P angle covers a wider range of variation for S-type (22 conformers) and averaging ± 62.4 than for N-type (23 conformers) with an average of 31.9 ± In the IUdR crystal (Camerman & Trotter, 1965) is also S-type. In dt is slightly S-type (Merchante & Alcolea, in preparation) or almost the same (Yurenko, et al., 2007b). The relative energies do not vary in a regular manner with respect to the values of P, Figure 4. In the four lowestenergy conformers S-type prevails, N/S = 0/100%, but in dt is 50/50%. However, in the 0 2 and 2 4kcal/mol ranges, the number of S-type is slightly higher than N-type N/S 42/58% in IUdR, while in dt is 25/75%. Most 3 -substituted dideoxynucleosides with inhibitory activity fall in the southern range (Arissawa, Taft, & Felcman, 2003; Marquez et al., 1998) as in IUdR with P = by X-ray. A S-type is thought to facilitate anabolic phosphorylation of the 5 -OH group. (iii) Although the value of P may differ substantially, the value of m max diminished the importance of these differences and becomes the dominant pseudorotational parameter (Choi et al., 2003). Taking into account that m max represents the radius of the pseudorotational cycle, its impact as structural parameters is quite high. An increase in the ring puckering produces an increment in the flexibility of the molecule (Nikolaienko, Bulavin, & Hovorun, 2012a), which could make easier to adapt to the active site. Choi et al. have reported that ca. 10-fold difference in m max is responsible for the 4 10-fold change in potency seen in the various assays. Moreover, it has been reported (Harte, Starret, Martin, & Mansuri, 1991) that the features that contribute to the variations in biological efficacy are mainly because of differences in conformational flexibility and not due to the small variations in the low-energy conformations. m max has in conformers syn a large range of values , but in conformers anti it is narrow ( ) as well as in high-anti ( ), Figure 6. The four most stable conformers appear by MP2 with m max in the range, slightly shorter than in dt, In resume, the C 2 -endo form of IUdR matches to the preferred conformer for dt and for the saturated analogues C 3 -endo. This orientation together with the high furanose ring puckering and flexibility of the molecule (although slightly lower than in dt) could make IUdR similar amenable that dt for both hydration and phosphorylation on the C5 O5 bond H-bonds The syn and anti orientations of the uracil ring relative to the ribose ring provide opportunities for H-bonding involving the C5 H 2 OH moiety with either uracil s carbonyl oxygen atom at position 2 or its hydrogen atom at position 6, respectively. Several authors have studied the intramolecular H-bonds in related nucleosides, in special using AIM method (Mishchuk, Potyagaylo, & Hovorun, 2000; Fidanza, Sosa, Lobayan, & Peruchena, 2005; Yurenko, Zhurakivsky, Samijlenko, Ghomi, & Hovorun, 2007; Yurenko et al., 2011). In the discussion of the results here, we have considered the classification of the H-bonds according to Desiraju et al. (Desiraju & Steiner, 1999; Panigrahi & Desiraju, 2007). Thus, five intramolecular H-bonds/interactions may be observed in the main conformers of IUdR, Figure 3: (i) hydroxyl oxygen O5 and uracil s hydrogen at position 6, H6 O5, (ii) hydroxyl hydrogen H5 and O4, (iii) H6 and O4, H6 O4, (iv) uracil s carbonyl oxygen atom at position 2 and H1, O2 H1, and (v) hydroxyl hydrogen H5 and O2, O2 H5. Mainly, anti forms involve the H-bonds/interactions (i), (ii) and (iv), and high-anti the (ii) and (iii). By contrast, syn forms involve only the H-bond (v), the strongest one observed in IUdR, Figure 3. H-bond (i) is observed in the three most stable conformers: C1 C3. Their values indicate that this H-bond is weak, although it is stronger than (ii) to (iv), but weaker than (v). In (ii) the large value O4 H5 of Å by MP2 in conformer C1, and the O4 H5 O5 angle of 103.2, indicate a very weak H-bond/interaction. The H-bond O4 H6 is not observed in IUdR, although it is reported in other nucleosides. H-bond (iii) appears only in high-anti forms and its value indicates a weak H-bond/interaction, as it has been also reported in related nucleosides by Yekeler (Yekeler, 2004). The O4 H6 distance appears to have some influence in the value of m max. For example, this distance is long in A2, conformer with the highest m max by MP2 in the high-anti forms. H-bond (iv) is also weak and it appears only in anti forms. H-bond (v) appears to give a great stability to the structure of conformers B1 and B2, especially in B1, the strongest one. The strength of this H-bond in B1 is responsible of a high m max. A shortening of the H-bond appears something correlated with an increase of m max. We are also interested in whether the different intramolecular H-bonds in IUdR make significant contributions to its conformational behaviour. In this sense, the importance of H-bonds has been determined in 6-azaCyd molecule (Mishchuk et al., 2000) where the presence of N6 nitrogen atom leads to the formation of additional H-bonds between the base and sugar ring atoms, as compared to Cyd, and allows the molecule to adopt the

13 12 M. Alcolea Palafox additional conformations stabilized by these H-bonds. The variety of conformations made this molecule very different from the Cyd (Samijlenko et al., 1999a, 1999b). Another study on DNA-related molecules (Nikolaienko et al., 2012b) using DFT calculations, Bader s quantum theory (AIM) and spectroscopic vibrational analysis reveals strong H-bonds with red-shifts over 40 cm 1 in the majority of the cases. MP2 and DFT calculations and analysis AIM (Yurenko et al., 2011) conduct to five types of the CH O H-bonds involving bases and sugar residues: C2 H O5, C1 H O2, C6H O5, C8H O5, and C6H O4, in accordance with our results in IUdR. The energy values of H-bonds are in the range of kcal mol 1, exceeding the kt value (.62 kcal mol 1 ). The interactions in the da-t nucleoside pair gives evidence, that additional to the N6H O4 and N1 N3H canonical H-bonds between the bases adenine and thymine, the third one C2H O2 is formed, and although it is rather weak (about 1 kcal mol 1 ) (Brovarets & Hovorun, 2013a, 2013b; Brovarets, Yurenko, Dubey, & Hovorun, 2012), it satisfies the AIM criteria and may be classified as a true H-bond. The CH O H-bond appears survive turns of bases against the sugar ring in rather large ranges of the angle v values, pertinent to certain conformations. This fact points out to the source of the DNA characteristic ability necessary for the conformational adaptation in processes of its functioning Natural NBO atomic charges The enhanced stability with iodine atom has been explained (Camerman & Trotter, 1965) in terms of several effects, the most plausible of which is that replacement of CH 3 in dt by Br or I alters the electron density in the pyrimidine ring so as to strengthen the hydrogen bonding to the purine base in the complementary DNA chain. For this reason, we have also analysed the NBO charges in IUdR. The optimized values in B1 and C1 forms are collected in Figure 2 at the B3LYP and MP2 levels. In general, MP2 calculates larger charges on the atoms than B3LYP. The largest negative value in all the conformers and levels is on the hydroxyl oxygen O5 as well as on O3. The value in C1 is very close to that calculated in dt (Alcolea et al., 2009; Alcolea & Iza, 2010). The next atom with large negative charge is the thymine nitrogen N3 and oxygen O2. O4 and O4 have similar charge, but the value on O2 is slight higher ca..05 e (where e is the charge of an electron) than on O4. The negative charge on N3 is larger, ca..15 e, than on N1. These features were also found in dt (Alcolea et al., 2009) and in AZT (Alcolea & Talaya, 2010). H5 is the hydrogen atom with the highest positive charge, that is, it is the most reactive. Closely appears H3 and H3 hydrogens, only e lower than H5. The remaining hydrogen atoms have much less positive charge, with values in general between.15 and.24 e. The highest positive charge is on C2 and C4 atoms, in concordance with the high negative value on O2 and O4, respectively. Because O2 has slight higher negative charge than O4, C2 has higher positive charge than C Intramolecular distances The intramolecular distances among the most reactive atoms, like the oxygens in the 9 best syn and anti conformers appear collected in Table 2. The interest of these Table 2. Intramolecular distances (in Å) of interest among the reactive oxygen atoms calculated in the nine most stable B and C conformers of IUdR at B3LYP/DGDZVP and MP2/DGDZVP levels (values in parentheses). Conformer O5 O2 O5 O4 O5 O4 B (2.906) (7.440) (3.005) B (3.050) (7.243) (2.975) B (3.055) (7.213) (2.950) B (4.510) (8.816) (3.620) B (4.559) (8.843) (3.670) B6 (3.384) (4.532) (2.892) B (4.559) (8.843) (3.670) B B (3.040) (7.120) (2.944) C (6.054) (6.776) (2.889) C (6.089) (6.794) (2.927) C (6.027) (6.716) (2.931) C C C (6.683) (7.791) (2.780) C C C

14 The antiviral Nucleoside Analogue 5-iodo-2 -deoxyuridine (IUdR) 13 values is its relation with the possible acceptor sites in the enzyme. Small differences in the structure of conformer C1 appear between IUdR and dt: a significant closing of the structure in IUdR, with shorter O2 O5 and O4 O5 intramolecular distances than in dt, ca..05 Å, and similar value in O4 O Low-frequency vibrations Low-lying molecular vibrations have been studied by different authors (Martel, Hennion, Durand, & Calmettes, 1994; Shishkin et al., 2000b; Pelmenschikov, Hovorun, Shishkin, & Leszczynski, 2000) and its presence indicates a high flexibility in the molecule that may be caused by various factors. This kind of vibrations may play an important role in some biological functions of DNA, such as the melting of the double helix, transcription and replication, specific interactions with proteins and drugs, and gene expression (Shishkin et al., 2000b). Due to the importance of this matter, we are preparing a special work with IUdR (Merchante & Alcolea) Differences between IUdR and dt Although in general the substitution of CH 3 for I does not significantly alter the molecular structure of the nucleoside, we have observed several changes, Scheme 2, that are summarized as follows: (1) A difference that exceed 1 appears only on C4 and C5 angles, while the remaining bond angles are almost identical that in dt. Similarly, in the torsional angles, a difference higher than 1 is only observed in b, d and /. (2) The symmetry through the C4=O axis is higher in dt than in IUdR. For example, the geometric parameters at both sides of the C4=O bond are more similar in dt than in IUdR. (3) Significant changes in the bond lengths are only observed in C=C and C4=O, and of course, in C X. The slight lengthening of N1 C1 (.005 Å) in conformer C1 of IUdR leads to a weakening of the O2 H1 H-bond, and as consequence, a small closing of the N1 C1 O4 angle and a small strengthening of the H6 O5 H-bond (2.249 Å vs Å in dt). This strengthening has been observed in the other anti forms. (4) A noticeable reduction in the negative charge on O4 and O2, and a slight increment in the positive charge on H3 atom is observed in IUdR, that is, the reactivity of O4 and O2 is reduced and by contrast that of H3 is incremented. This fact leads to a strengthening of N3 H3 N DNA base pair with deoxyadenosine, and to a weakening of O4 H N. (5) A slight easier tautomerization to T3 in IUdR than in dt, Figure 7, which facilitate the mispairing with IUdR and as consequence, the inhibition of the DNA viral In the solid state Four molecules appear in the unit cell of IUdR as reported by X-ray (Camerman & Trotter, 1965). The crystal data of this unit cell (the tetramer form) was the starting point used for the simulation of the solid state. The orientations of the four molecules in the unit cell are in agreement to our computations, Figure 8. In the crystal, the atoms attached to the pyrimidine ring show small deviations from exact planarity, and our simulation Scheme 2. Changes accompanying the conversion of thymidine into IUdR. The MP2/DGDZVP values appear as difference respect to dt and in conformer C1: the bond lengths in black colour, the bond angles in orange colour, the torsional angles in violet, and the NBO natural atomic charges in green. Figure 7. Comparison of the T1 T3 tautomerism in IUdR and dt nucleosides. The calculated energy at the MP2/ DGDZVP level, in kcal/mol, is shown in bold type, while at the B3LYP/DGDZVP level appears in parentheses.