NMR spectra of salicylohydroxamic acid in DMSO-d 6 solution: a DFT study

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1 Journal of Molecular Structure (Theochem) 640 (2003) NMR spectra of salicylohydroxamic acid in DMSO-d 6 solution: a DFT study Agnieszka Kaczor a, Leonard M. Proniewicz a,b, * a Department of Chemistry, Jagiellonian University, ul. Ingardena 3, Krakow, Poland b Regional Laboratory of Physicochemical Analysis and Structural Research, Jagiellonian University, ul. Ingardena 3, Krakow, Poland Received 27 March 2003; revised 11 August 2003; accepted 21 August 2003 Abstract The 1 H, 13 C and 1 H, 13 C COSY NMR spectra of salicylohydroxamic acid (sha) were measured in DMSO-d 6 solution. The B3LYP GIAO method with the 6-311þþG(d,p) basis set was chosen to reproduce the experimental spectra. All possible zusammen and entgegen conformers of monomeric sha were computed. After geometry optimisation (B3LYP/6-311þþG(d,p)) only nine independent models of the molecule were shown to be stable. Additionally, the NMR chemical shifts of the Onsager model of the most stable monomer were calculated. The computed chemical shifts for the labile protons for all aforementioned geometries meaningfully underestimated experimental results suggesting the existence of the H-bonded structure of sha in DMSO solution. The most probable two dimeric structures along with two solvent-bounded aggregates were subsequently calculated at the same level of theory. The best agreement was obtained for sha H-bonded with two DMSO molecules (confirmed by the absence of concentration effect). The relative error not exceeding 10 and 4% for chemical shifts in 1 H and 13 C NMR spectra of sha (DMSO) 2, respectively, showed that the applied method with the B3LYP/6-311þþG(d,p) basis set was efficient to predict the NMR shifts of a compound with strong H-bonds. Thus, this allows to assign properly NMR resonances to specific structure formed in DMSO solution. q 2003 Elsevier B.V. All rights reserved. Keywords: Salicylohydroxamic acid; NMR; DFT calculations 1. Introduction The hydroxamic acids are well-known family of compounds due to their strong chelating properties. Therefore they are involved in many biochemical processes such as iron transport phenomena [1], inhibition of the enzymatic activity of urease [2,3], Alzheimer s Amyloid Precursor Protein a-secretase * Corresponding author. Tel.: þ x2253; fax: þ address: proniewi@chemia.uj.edu.pl (L.M. Proniewicz). [4] or some matrix metalloproteinases [5]. The chelating phenomena are usually increased if two donor groups are present in the same molecule, as it was noticed for dihydroxamic in comparison with monohydroxamic acids [6]. In analogy, salicylohydroxamic acid (sha) containing the hydroxyl moiety in the ortho position to the hydroxamic function was shown to present increased chelating ability compared to benzohydroxamic acid [7,8]. Interesting photochemical properties were also demonstrated for sha molecule isolated in the low-temperature matrix [9] /$ - see front matter q 2003 Elsevier B.V. All rights reserved. doi: /j.theochem

2 134 A. Kaczor, L.M. Proniewicz / Journal of Molecular Structure (Theochem) 640 (2003) The structures of sha in the solid state [10] and the molecules isolated in the argon matrix [9] are different suggesting that the intermolecular hydrogen bond formation in the solid state forces the molecules to change the conformation. The presence of three labile protons in both the hydroxamic and the hydroxyl groups in sha imposes that its structure is stabilized by the H-bonds in the solid state as well as in solution. Therefore, to elucidate the structure of sha in the solution, the consideration of associated species is necessary in order to understand its structure and bonding. The aim of this work is to sha molecular structure formed in DMSO solution based on proper assignment of observed 1 H and 13 C NMR resonances by using a proper computational method and basis set. Without calculations the unambiguous assignment of 1 H NMR spectrum is impossible. Therefore, the DFT (B3LYP/6-311þþG(d,p)) calculations of NMR chemical shifts of possible monomers, dimers and solvent-bounded aggregates of sha are performed on the structures optimised at the same level of theory. The obtained chemical shifts are then compared with the experimental 1 H, 13 C and 1 H, 13 C COSY spectra of sha measured in DMSO-d 6 solution. This procedure allows to find the proper structure of the compound in the studied solvent. Additionally, usefulness of the Onsager model of the most stable monomeric species is tested and discussed. 2. Computational methods Calculations were performed in GAUSSIAN 98 package of programs [11]. Although Bauer and Exner considered oximino tautomers as possible for hydroxamic acids [12], according to our knowledge, they have not been so far proved experimentally for primary hydroxamic acids. Some calculation made at the MP2(FC)/cc-pVDZ theory level suggested the 0.9 kcal/mol difference of the imino- to the most stable amide form for acetohydroxamic acid [13]. Also some NMR solution studies concluded the presence of the imino form for some silylated derivatives [14 16]. However, we do not consider the latter tautomer as possible for the studied compound. Sha conformers with all possible entgegen and zusammen conformations were optimised at the DFT level with the B3LYP functional [17] and 6-31G(d) basis set. The 16 input structures of keto forms of sha yielded 10 independent geometries after B3LYP/6-31G(d) optimisation which were potential energy minima. The obtained geometries were subsequently recomputed using triple-zeta basis set with two sets of diffuse and polarization functions (6-311þþG(d,p)) that allowed to reduce the number of structures from 16 to 9. The two energetically most stable geometries were chosen and optimised as C 2 dimers (B3LYP/6-311þþG(d,p)). For the energetically lowest geometry, also two DMSO-H-bonded structures were assumed and optimised (B3LYP/6-311þþG(d,p)). The harmonic frequencies were calculated for all structures in order to confirm they are potential energy minima, apart from the structure XIV, due to the size (number of atoms) of the latter. For this model the frequencies were computed using the 6-31G(d) basis set. The NMR chemical shifts of all aforementioned models were subsequently computed by B3LYP/6-311þþG(d,p) using GIAO approximation. The Onsager model [18 20] was used to calculate the geometry of the molecule in DMSO solution (1 ¼ 46:7) in order to evaluate the solvent effect. The input structure was the energetically most stable monomer with the radius of the cavity estimated for a 0 ¼ 4:42 Å by volume calculations (B3LYP/6-311þþG(d,p)). NMR keyword is not implemented into the Onsager model in GAUSSIAN 98. Therefore the NMR chemical shifts in this case were computed by NMR keyword on the geometry, optimised previously using SCRF ¼ Dipole and Opt keywords. 3. Experimental methods Sha was synthesized as described elsewhere [21]. The elemental analysis: C, 54.60%; H, 4.69%; N, 8.88%, found; and C, 54.91%; H, 4.61%; N, 9.09%, calculated. NMR spectra were measured on a Mercury VX 300 MHz spectrometer (Varian) at room temperature. Sha samples were dissolved in DMSO-d d containing 0.03% TMS as the internal standard (99.9% purity, Sigma-Aldrich Sp. Z o. o., Poland). The 1 HNMR spectra of , , and M concentrations were measured with the resonance

3 A. Kaczor, L.M. Proniewicz / Journal of Molecular Structure (Theochem) 640 (2003) frequency of MHz. The 13 C NMR spectrum with the resonance frequency of 75.5 MHz and the 13 C, 1 H COSY spectrum with the aforementioned frequencies were obtained for the dilution of M. 4. Results and discussion H NMR spectra There are six groups of signals observed in the 1 H NMR spectrum of sha in the DMSO-d 6 solution. Three singlets at 9.32, and ppm can be assigned to the H 15 hydroxyl proton, the H 17 proton from the NH moiety and the H 18 proton from the OH of the hydroxamic group (for the numeration scheme c.f., Fig. 1). Based on the obtained experimental spectra, the unambiguous assignment of these three protons is impossible. Brown et al. [22] proposed that the proton from the NH group gave the signal around 11 ppm for monosubstituted benzohydroxamic acids (11.2, 11.1, 11.6 for p-methyl-, p-methoxy- and p- nitrobenzohydroxamic acid, respectively, in DMSOd 6 solution). For dihydroxamic acids (CH 2 ) n (CONHOH) 2 with the zusammen conformation around the C N bond, the resonances from the protons of the N H group were observed at ppm for n ¼ 0; ppm for n ¼ 1; and about ppm for n ¼ 2 8 and 10 [22,23]. The chemical shift at ca ppm was also assigned Fig. 1. The numeration scheme for the sha molecule. to the H of the NH group of the z conformer of acetohydroxamic acid [24]. The signals at 9.02, 8.94 and 9.40 ppm were assigned to the proton of the hydroxamic OH moiety for p-methyl-, p-methoxyand p-nitrobenzohydroxamic acids, respectively, and at about 8.7 ppm for all studied z-dihydroxamic acids apart from oxalodihydroxamic acid (n ¼ 0) for which this resonance was observed at 9.2 ppm [22,23]. The assignment proposed in the literature was confirmed by some experimental evidences: the limited concentration studies [22] and by selective decoupling [23]. The previous experimental results suggested then that the broad signals at 9.32, and ppm should be attributed to H 18,H 17 and H 15, respectively, in the sha molecule. The fact that these resonances appeared far downfield imposed the existence of the hydrogen bonding. To solve this problem, obviously the influence of dilution of the sample on 1 H NMR spectrum had to be studied. The present studies showed that no change in the proton chemical shifts of sha was observed for concentrations of , , and M. This clearly suggested that the H-bonds were formed either by sha and solvent molecules or by internal hydrogen bonding. Thus, intermolecular H-bonds between sha molecules can be ruled out from the discussion. The comparison of the experimental and calculated values of 1 H NMR chemical shifts is listed in Table 1 while the optimised geometries are presented in Fig. 2. It is noticed that there is no match between the computed values of H 17 and H 18 resonances for all monomeric structures (I IX). The H 15 proton chemical shift is well reproduced only for the monomers I and IV and the Onsager model (X), indicating the presence of the intramolecular H-bond linking H 15 with the oxygen from the carbonyl group. Therefore, in order to reproduce properly the unexpectedly high chemical shifts for protons, dimers and associates with the solvent molecules were proposed and studied. The dimers were made based on the two most stable monomeric geometries (I) while solventassociates by linking DMSO to the N H group or both the hydroxamic O H and N H moieties of structure I. The results of computations of structures XI XIV, being in agreement with the dilution experiment, impose the sha (DMSO) 2 aggregate (XIV) to be the only species present in DMSO-d 6 solution. The chemical shifts of 12.20, and

4 136 A. Kaczor, L.M. Proniewicz / Journal of Molecular Structure (Theochem) 640 (2003) Table 1 Experimental (exp.) and calculated 1 H NMR chemical shifts of sha relative to TMS Proton Exp. (ppm) Calculated a (ppm) I II III IV V VI VII VIII IX X XI XII XIII XIV H (d d) H (d d d) H (d d d) H (d d) H (s) H (s) H (s) a The geometries of the sha structures relative to TMS optimised in B3LYP/6-311þþG(d,p). NMR computed at the B3LYP/6-311þþG(d,p), GIAO method. The numeration scheme and structures are presented in Figs. 1 and 2, respectively ppm for H 15,H 17 and H 18, respectively, very well agree with the computed values of 12.74, and ppm obtained for the structure XIV. Additionally, the latter is the only geometry with the correct sequence of the proton chemical shifts, being in agreement with the previous findings of Brown et al. [22,24] and Schraml et al. [23], namely the aforementioned resonances of the protons from the hydroxamic N H and O H groups. The relative deviations of 4, 2 and 10% for H 15,H 17 and H 18, respectively, indicate that the proposed model fits well to the experimental data and allows to estimate the geometrical parameters of the molecule. As is shown here, the Onsager model (X), representing the solvent by a dielectric continuum with dielectric constant 1 and the solute molecule placed in a spherical cavity with a given radius [18 20] produced results similar to the most stable monomer I and therefore was not able to reproduce the sha DMSO interaction, based on the H-bonding formation. The H 11 proton signal is manifested in the sha spectrum as doublet of doublets at ca ppm. It is coupled with the H 12 and H 13 protons with the coupling constants of 7.8 and 1.5 Hz (c.f. Table 2), respectively. Similar splitting, with the coupling constants of 8.7 and 1.2 Hz for the ortho H 13 and the meta H 12 is observed for the H 14 signal at ca ppm. Both H 12 at ca ppm and H 13 at ca ppm are coupled to two protons in the ortho and one proton in the meta positions, the latter with much smaller coupling constants. Therefore, the resulting signals for the H 12 and H 13 protons are doublet of doublets of doublets. Due to similar coupling constants with the protons in the ortho position (J ¼7:8; J ¼8:1; J ¼8:7) the signals are observed as split pseudotriplets, additionally superimposed with the H 14 signal in the case of H 12. The computed values of the chemicals shifts for the ring protons are quite conserved in all models, particularly for H 12 and H 13, where the differences are ^0.2 ppm relative to the experimental values. Although the structure XIV presents the best overall agreement with the experimental data, the resonance of H 11 is overestimated about 0.6 ppm due to the fact that the model assumes the formation of the weak H- bond between the latter proton and the oxygen of the DMSO molecule with the distance of 2.36 Å. Other ring proton chemical shifts are excellently reproduced in the calculations (6.60, 7.19 and 6.80 for H 12,H 13 and H 14, respectively) C and 1 H, 13 C COSY NMR spectra The comparison of the experimental and calculated results of 13 C NMR chemical shifts is presented in Table 3. The most downfield value at ppm is assigned to the carbonyl carbon. This value is typical and usually observed at ppm in the zusammen (around the C N bond) conformers of monosubstituted primary derivatives of benzohydroxamic acids, while their secondary derivatives, substituted with the methyl group showed this resonance at ppm [22]. The signal at ppm is

5 A. Kaczor, L.M. Proniewicz / Journal of Molecular Structure (Theochem) 640 (2003) Fig. 2. Geometry of the optimised sha models. Values in parenthesis denote the difference of corrected energy (in kj/mol) relative to the most stable monomer. assigned to the C 7 carbon, being slightly shifted in comparison to the ipso phenyl carbon (155.4 ppm) [25]. The other ring carbon atoms are observed at , , , and ppm. The 1 H, 13 C COSY spectrum of sha, presented in Fig. 3, allows for their unambiguous assignment. Therefore, the signal at ppm, not coupled in the COSY spectrum, is assigned to the quaternary C 6 carbon and resonances at , , and are due to the C 2, C 3, C 4 and C 5 carbon atoms,

6 138 A. Kaczor, L.M. Proniewicz / Journal of Molecular Structure (Theochem) 640 (2003) Table 2 The values of 1 H, 1 H coupling constants for the sha molecule Parameter J value (Hz) Position J ortho J meta J ortho J meta J ortho respectively, on the basis of coupling with H 11,H 12, H 13 and H 14 protons, respectively. Alternatively, the assignment of the carbon signals is possible on the base of performed calculations. In the computed spectra the sequence of the carbon atoms is the same in all discussed models except the structure XIII, where the C 7 and C 8 resonances are reversed. The best agreement is again obtained for the structure XIV. Particularly the C 2 C 5 signals are reproduced more accurately relative to monomeric and dimeric structures (calculated values are in brackets: (128.83), (117.42), (134.10) and (118.57) ppm, for C 2,C 3,C 4 and C 5, respectively). Also the match of the C 6 resonance is better than in other discussed models (exp.: ppm, calc.: ppm). The C 7 resonance is overestimated. It is shifted too far downfield, although the agreement with the experimental data is still very good (exp.: ppm, calc.: ppm, the relative deviation is about 4%). For the other carbon atoms the agreement is nearly perfect with the relative deviation below 1% Comparison of the calculated models The values of the electronic and zero-point corrected energy computed at the B3LYP/6-311þþG(d,p) level of theory, relative to the most stable monomer (structure I) are given in Table 4. The presented values are each time recomputed per one sha molecule. The models X XV are energetically stabilized compared to monomers I IX. In the case of the Onsager model, the energetic advantage is due to including into calculations the interactions solvent solute, thus giving the solvation effect equal to about 24.7 kj/mol for the sha DMSO aggregate. It should be emphasized, however, that the results of solvation energy, obtained by using the simple Onsager model is a rough estimation [26] and cannot be treated quantitively. Additionally, it is worth to mention, that polarization effect is not included in the computation of the shielding constants. However, inclusion of polarization of the solvent in the geometry optimisation (model X) does not make any striking changes of the parameters of the structure in comparison to the most stable monomer. Therefore, the fact that wavefunction is not polarized during calculations of NMR spectra cannot fully explain the inconsistency of the chemical shifts obtained by using the Onsager model. This is predominantly caused by the inability of the applied model to reproduce directional interactions, such as the existing hydrogen bonding. In the case of a solute dissolved in a polar solvent, the competition between the formation of dimeric species and solvent-bounded aggregates is possible. Table 3 Experimental (exp.) and calculated 13 C NMR chemical shifts of sha relative to TMS Carbon Exp. (ppm) Calculated a (ppm) I II III IV V VI VII VIII IX X XI XII XIII XIV C C C C C C C a The geometries of the sha structures relative to TMS optimised in B3LYP/6-311þþG(d,p). NMR computed at the B3LYP/6-311þþG(d,p), GIAO method. The numeration scheme and structures are presented in Figs. 1 and 2, respectively.

7 A. Kaczor, L.M. Proniewicz / Journal of Molecular Structure (Theochem) 640 (2003) Fig. 3. The 1 H, 13 C COSY NMR spectrum of sha in DMSO-d 6. The chemical shifts in ppm. However, according to the calculations, the stabilization of DMSO aggregates predominates over dimers, excluding the presence of the latter in the studied solution. Thus, the energy difference between dimer and the most stable monomer equals, and kj/mol for the structures X and XI, respectively, while this difference is much larger for the DMSO associates, even for the aggregate including only one DMSO molecule (247.9 kj/mol). Although, due to the number of atoms that form the large DMSO associate XIV, the zero-point corrected energy was not computed for this structure. However, the value of the electronic energy allows to estimate that it is stabilized by about 90 kj/mol relative to the structure I. The comparison of the electronic energy values of structures I, XIII and XIV allows to estimate roughly the strength of the H-bonds at about 50 and 40 kj/mol for N 1 H 17 O DMSO and O 16 H 18 O DMSO, respectively. The most striking feature of the structure XIV is disappearance of the interaction between the hydrogen of the hydroxamic O H group and the oxygen of the carbonyl group. This competition between the intraand intermolecular bonds results in the creation of the latter and, therefore, changes in the geometry of the O 9 C 8 N 1 O 16 H 18 pseudo-ring are observed such as significant lengthening of the H 18 O 9 distance (2.641 Å in XIV, compared to Å in I and Å in X) or slight opening of the C 8 N 1 O 16 and C 8 N 1 H 18 angles ( and for the C 8 N 1 O 16 and N 1 O 16 H 18 angle in XIV, respectively, compared to about 1168 and 1028 in I and X). The slight shortening

8 140 A. Kaczor, L.M. Proniewicz / Journal of Molecular Structure (Theochem) 640 (2003) Table 4 The electronic energies (DE) and the sum of electronic and zeropoint energies (DE corr ) per molecule relative to the most stable monomer (I) for optimised structures of sha (B3LYP/6-311þþG(d,p)) Structure DE (kj/mol) a DE corr (kj/mol) b I c II c III c IV c V c VI c VII c VIII c IX c X c XI d 27.8 e 25.7 e XII d e e XIII c f f XIV c g a The values of the electronic energy relative to the electronic energy of structure I ( Hartree/molecule, the conversion factor 1 Hartree/molecule ¼ kj/mol). b The values of the zero-point corrected energy relative to the zero-point corrected energy of structure I ( Hartree/ molecule, the conversion factor 1 Hartree/molecule ¼ kj/mol). c Optimised in symmetry C 1 : d Optimised in symmetry C 2 : e The values of energy obtained by dividing by two the values of the electronic or zero-point corrected energy. f The values of energy obtained bysubtracting the electronic or zeropointcorrectedenergyfordmso(c s,optimisedbyb3lyp/6-311þþg (d,p), E ¼ 2553: Hartree, E corr ¼ 2553: Hartree). g The values of energy obtained by subtracting the energy for DMSO doubled (E ¼ 2553: Hartree, E corr ¼ 2553: Hartree). of the intramolecular O 10 H 15 O 9 H-bond for the Onsager model (1.730 Å) and particularly for the sha (DMSO) 2 associate (1.641 Å) is noticed relative to monomer I (1.740 Å). The shortening of the latter bond in the case of XIV is the result of breaking of the intramolecular O 16 H 18 O 9 bond that makes the oxygen from the carbonyl moiety the single acceptor in the O 10 H 15 O 9. This causes increase in the H-bond strength. The existence of the intermolecular H-bonds with DMSO molecules in XIV (1.664 and Å for the O 16 H 18 O DMSO and N 1 H 17 O DMSO, respectively) and the strengthening of the O 10 H 15 O 9 bond result in the concomitant lengthening of O 10 H 15, O 16 H 18 and N 1 H 17 bonds (0.993, and Å, respectively, compared to 0.983, and in I). The values of dihedral angles are quite conserved in all molecules apart from the changes in the O 9 C 8 N 1 O 16 H 18 pseudo-ring. Generally, the parameters of the Onsager model are very similar to the original monomer I. However, the changes are observed in the structure of the aggregate XIV, influencing meaningfully the agreement between the experimental and computed NMR chemical shifts for the latter. 5. Conclusions Monomeric, dimeric and aggregates with the solvent models of sha were studied in order to determine the structure of the compound in DMSO. The calculations (B3LYP/6-311þþG(d,p)) of NMR chemical shifts produced the best agreement with the sha (DMSO) 2 associate and allowed estimating the bond lengths and angles of sha in the investigated solution. This associated molecule is H-bounded to two oxygen atoms of DMSO molecules by the hydrogen atoms from the hydroxamic group. The third labile proton, namely the hydrogen of the hydroxyl group, forms the intramolecular H-bond with the oxygen atom of the carbonyl moiety. The molecule adopts the zusammen conformations around all free-rotating bonds (C 6 C 8, C 7 O 10, C 8 N 1, N 1 O 16 ), as it is shown for sha in the argon matrices [9] or in a model for a peroxidase-inhibitor complex [7]. The z-conformations in the DMSO solution with the conserved strong intramolecular bond suggest that sha rearranges to form a complex with myeloperoxidase in the solid state [27] or crystallising (entgegen conformation around the C 6 C 8,C 7 O 10 bonds) [10]. It is possible, though, that in the solution, which means under the physiological conditions, the full zusammen geometry is more energetically stable than the rearrangement leading to a different conformer. Therefore, the crystalline structures must be treated with some uncertainty as a model of sha interactions with the metalloproteinases under the physiological conditions. The proper modelling of the NMR chemical shifts of a polar solute in a polar solvent imposes the use of the model in which solute molecule(s) interacts with solvent molecule(s). The possible competition

9 A. Kaczor, L.M. Proniewicz / Journal of Molecular Structure (Theochem) 640 (2003) between dimeric and solvent-bonded aggregates was investigated for sha. The results of both comparisons of values of energies as well as the agreement of the experimental and calculated chemical shifts strongly suggest the aggregates with DMSO to be the only species present in the solution. This is confirmed by the absence of the concentration effect. Additionally, the applicability of the Onsager model to the computation of the sha NMR resonances in DMSO has been tested. The lack of significant changes in the geometry and, therefore, in the values of both 1 H and 13 C chemical shifts relative to the original monomer showed that the model cannot reproduce the experimental data in the case of sha, since the H-bond interactions with the solvent are the most stabilizing factor of the structure. Generally, for the studied compound the B3LYP GIAO method with the 6-311þþG(d,p) basis set was shown to reproduce experimental spectra suggesting that sha forms hydrogen bonds with two DMSO molecules. Acknowledgements The authors wish to thank Mr Z. Urbanek for the synthesis of the compound. Calculations were done at the Academic Computer Centre Cyfronet, Krakow, Poland and the Interdisciplinary Centre for Mathematical and Computational Modelling, Warsaw University, Warsaw, Poland, which are acknowledged for computing time. Part of this research was supported by the grant 4T09A from the Polish State Committee for Scientific Research (to LMP). References [1] K.N. Raymond, Coord. Chem. Rev. 105 (1990) 135. [2] A.J. Stemmler, J.W. Kampf, M.L. Kirk, V.L. Pecoraro, J. Am. Chem. Soc. 117 (1995) [3] M.A. Pearson, L.O. Michel, R.P. Hausinger, P.A. Karplus, Biochemistry 36 (1997) [4] S. Parvathy, I. Hussain, E.H. Karran, A.J. Turner, N.M. Hooper, Biochemistry 37 (1998) [5] S. Pikul, K.L. McDow Dunham, N.G. Almstead, B. De, M.G. Natchus, M.V. Anastasio, S.J. McPhail, C.E. Snider, Y.O. Taivo, L. Chen, C.M. Dunaway, F. Gu, G.E. Mieling, J. Med. Chem. 42 (1999) 87. [6] B. Kurzak, L. Nakonieczna, G. Rusek, H. Kozlowski, E. Farkas, J. Coord. Chem. 38 (1993) 17. [7] E.C. O Brien, E. Farkas, M.J. Gil, D. Fitzgerald, A. Castineras, K.B. Nolan, J. Inorg. Biochem. 79 (2000) 47. [8] M. Ikeda-Saito, D.A. Shelley, L. Lu, K.S. Booth, W.S. Caughey, S. Kimura, J. Biol. Chem. 266 (1991) [9] A. Kaczor, J. Szczepanski, M. Vala, L.M. Proniewicz, Phys. Chem. Chem. Phys. 5 (2003) [10] I.K. Larsen, Acta Cryst. B34 (1978) 962. [11] M.J. Frisch, G.W. Trucks, H.B. Schlegel, G.E. Scuseria, M.A. Robb, J.R. Cheeseman, V.G. Zakrzewski, J.A. Montgomery Jr., R.E. Stratmann, J.C. Burant, S. Dapprich, J.M. Millam, A.D. Daniels, K.N. Kudin, M.C. Strain, O. Frakas, J. Tomasi, V. Barone, M. Cossi, R. Cammi, B. Mennucci, C. Pomelli, C. Adamo, S. Clifford, J. Ochterski, G.A. Petersson, P.Y. Ayala, Q. Cui, K. Morokuma, P. Salvador, J.J. Dannenberg, D.K. Malick, A.D. Rabuck, K. Raghavachari, J.B. Foresman, J. Cioslowski, J.V. Ortiz, A.G. Baboul, B.B. Stefanov, G. Liu, A. Liashenko, P. Piskorz, I. Komaromi, R. Gomperts, R.L. Martin, D.J. Fox, T. Keith, M.A. Al-Laham, C.Y. Peng, A. Nanayakkara, M. Challacombe, P.M.W. Gill, B. Johnson, W. Chen, M.W. Wong, J.L. Andres, C. Gonzalez, M. Head- Gordon, E.S. Replogle, J.A. Pople, GAUSSIAN 98, Revision A.11, Gaussian, Pittsburgh, PA, [12] L. Bauer, O. Exner, Angew. Chem. Int. Ed. Engl. 13 (1974) 376. [13] C. Muńoz-Caro, A. Nińo, M.L. Senent, J.M. Leal, S. Ibeas, J. Org. Chem. 65 (2000) 405. [14] J. Schraml, M. Kvíčalová, L. Soukupová, V. Blechta, Magn. Reson. Chem. 37 (1999) 427. [15] J. Schraml, M. Kvíčalová, L. Soukupová, V. Blechta, O. Exner, J. Phys. Org. Chem. 12 (1999) 668. [16] J. Schraml, M. Kvíčalová, L. Soukupová, V. Blechta, O. Exner, J. Organomet. Chem. 597 (2000) 200. [17] A.D. Becke, J. Chem. Phys. 98 (1993) [18] M.W. Wong, M.J. Frisch, K.B. Wiberg, J. Am. Chem. Soc. 113 (1991) [19] M.W. Wong, M.J. Frisch, K.B. Wiberg, J. Am. Chem. Soc. 114 (1992) 523. [20] M.W. Wong, M.J. Frisch, K.B. Wiberg, J. Am. Chem. Soc. 114 (1993) [21] W. Ruiqin, Y. Xiaolan, F. Zhenye, M.M.I. Haq, M.M.P. Khurshid, T.J. Rayner, J.A.S. Smith, M.H. Palmer, J. Am. Chem. Soc. 111 (1989) 114. [22] D.A. Brown, R.A. Coogan, N.J. Fitzpatrick, W.K. Glass, D.E. Abukshima, L. Shiels, M. Ahlgren, K. Smolander, T.T. Pakkanen, T.A. Pakkanen, M. Peräkylä, J. Chem. Soc., Perkin Trans. 2 (1996) [23] J. Schraml, M. Kvíčalová, V. Blechta, L. Soukupová, O. Exner, H.-M. Boldhaus, F. Erdt, C. Bliefert, Magn. Reson. Chem. 38 (2000) 795. [24] D.A. Brown, W.K. Glass, R. Mageswaran, B. Girmay, Magn. Reson. Chem. 26 (1988) 970. [25] H.-O. Kalinowski, S. Berger, S. Braun, Carbon-13 NMR Spectroscopy, Wiley, Chichester, 1991, (Chapter 4). [26] T. Yasuda, S. Ikawa, Chem. Phys. 238 (1998) 173. [27] C.A. Davey, R.E. Fenna, Biochemistry 35 (1996)