Modeling Triple Conformational Disorder in a New Crystal Polymorph

Article pubs.acs.org/crystal

Modeling Triple Conformational Disorder in a New Crystal Polymorph of cis-Aquabis(L-isoleucinato)copper(II) Marijana Marković,† Dalibor Milić,‡,§ and Jasmina Sabolović*,† †

Institute for Medical Research and Occupational Health, Ksaverska cesta 2, P.O. Box 291, HR-10001 Zagreb, Croatia Department of Chemistry, Faculty of Science, University of Zagreb, Horvatovac 102a, HR-10000 Zagreb, Croatia

‡

S Supporting Information *

ABSTRACT: The X-ray crystal and molecular structure of a new polymorph of cis-aquabis(L-isoleucinato)copper(II), obtained by recrystallization from an acetic acid−water mixture and determined at 120 and 295 K, revealed triple dynamic disorder over one isoleucinato ligand at both temperatures. The complicated multipart disorder was resolved with the help of extensive computational crystal structure simulations, which were undertaken for the first time to interpret disorder in a crystal of bioinorganic compound. The new polymorph (space group C2) is conformationally polymorphic with the already known P212121 crystal form. To discover the conformers that can participate in self-associations in solution, and to rationalize an interplay of intramolecular and intermolecular interactions in the crystallization of different conformers, conformational analyses of cis and trans isomers were performed using the same force field in vacuo, in aqueous solution, and for selected conformers in P212121 and C2 crystals. Three conformers identified in the disorder were estimated to form the most favorable intermolecular interactions in the solid state, and one of them (the most populated conformer in the disorder) also in aqueous solution. The crystal structure reproduction of all possible arrangements of the three conformers in C2 unit cell helped to find the most plausible crystal packing motif.

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the first two complexes had the cis configuration, and Cu(D,LIle)2 the trans one. In an attempt to discover optimal conditions for preparing single crystals of trans-Cu(L-Ile)2, we obtained single crystals for only cis-Cu(L-Ile)2·H2O. Namely, the single crystals identical to already known P212121 crystals11 crystallized from ethanol−water and methanol−water mixtures. Moreover, from a more polar mixture of acetic acid and water, a new polymorph of cis-Cu(L-Ile)2·H2O, which crystallized in space group C2, was obtained. In this paper, we present the results of the X-ray diffraction measurements performed on the same single crystal of the new polymorph first at 120 K and then at ambient temperature. Unexpectedly, the crystal structure refinement revealed triple disorder over all atoms of one L-Ile residue at both temperatures. Positional disorder of L-Ile side chains is not uncommon in the crystal structures of copper(II) isoleucinato complexes. Namely, a survey of the Cambridge Structural Database completed using ConQuest20 (version 5.32, update of November 2011) and limited to bis(isoleucinato-N,O)metal(II) complexes revealed 7 entries,21 two of which had positional disorder at room temperature. The first one is trans-bis(L-N,Ndimethylisoleucinato)copper(II), Cu(L-Me2Ile)2, having reported relatively short Cγ−Cδ bond length of 1.316 Å, and

INTRODUCTION Copper(II) complexes with amino acids are involved in the mediation of physiological copper in biological systems.1,2 Since their complete molecular structure determinations in solutions are rare,3−6 the most usual sources of experimental information have been the X-ray crystal and molecular structures.7 Knowing the extent of the effects of environment (such as crystal and solution) and amino acid side-chain interactions on the changes in the copper(II) coordination sphere is a prerequisite for understanding the impact of noncovalent interactions on the copper binding sites in metalloproteins. Recently we succeeded in preparing single crystals of the trans isomer of bis(L-valinato)copper(II), Cu(L-Val)2,8 although for many years the crystal and molecular structure of only the aqua cis isomer was known.8−10 Equally, for the copper(II) complex with L-isoleucine, the crystal structure of cis-aquabis(Lisoleucinato)copper(II), Cu(L-Ile)2·H2O, with P212121 space group symmetry, has been the only one reported for more than 40 years.11,12 For these orthorhombic single crystals of cis-Cu(LIle)2·H2O, electronic properties and magnetic interactions were studied at temperatures between 1.5 and 293 K,13 and at two microwave frequencies at room temperature14 using EPR techniques. In addition, magnetic susceptibility at temperatures between 0.01 and 4.2 K15 and the intensities of d−d electron absorption bands16 were determined. Infrared17,18 and diffuse reflectance spectra18,19 measured for polycrystalline Cu(LIle)2·H2O, Cu(D,L-Ile)2·H2O, and Cu(D,L-Ile)2 suggested that © 2012 American Chemical Society

Received: May 10, 2012 Revised: June 15, 2012 Published: June 20, 2012 4116

dx.doi.org/10.1021/cg300636w | Cryst. Growth Des. 2012, 12, 4116−4129

Crystal Growth & Design

Article

filtration, the resulting blue powder was washed with water, filtered off, dried at 90 °C overnight and weighed (yield: 75%). Blue plate-like single crystals of cis-Cu(L-Ile)2·H2O (1) were obtained by the recrystallization of the blue powder (0.0498 g) from the ethanol−water (10 mL:0.5 mL) mixture. The mixture was heated at 60 °C in a water bath until the complex dissolved, and then it was left at room temperature. The crystals appeared a few days later and were filtered off. Blue plate-like single crystals of the new polymorph of cis-Cu(LIle)2·H2O (2) with rhomboid-like habitus were obtained after 10 days of recrystallization of the blue powder (0.0825 g) from an acetic acid− water mixture (5 mL:0.75 mL), which occurred on the vessel walls at room temperature. When 0.2825 g of the blue powder was dissolved in the mixture of 5 mL of acetic acid and 0.75 mL of water, the crystals of copper(II) acetate monohydrate appeared in the solution at room temperature after two weeks. After removing these crystals by filtration, the crystals of 2 formed after 6 days (sample A) from the residual mother liquor, and the crystals of 1 started to form after one additional day (sample B). However, when 0.2000 g of the blue powder was dissolved in a mixture of 2 mL of acetic acid and 0.1 mL of water, only sample A formed after a few days. Both cis-Cu(L-Ile)2·H2O crystalline powder samples were characterized by the powder X-ray diffraction (PXRD) experiments on a Philips PW 3710 diffractometer with Cu Kα radiation (40 kV and 40 mA). The patterns (Figures S1 and S2 in the Supporting Information) were collected from flat plate samples on a zero background sample holder in the Bragg−Brentano geometry. The diffraction angle (2θ) range was 4−50° for both samples. Sample A was measured with a step size of 0.01° and exposition of 7.0 s per step, while for sample B the step size of 0.02° and exposition of 1.0 s per step were used. Single-Crystal X-ray Structural Analysis. Single-crystal X-ray diffraction data for the two polymorphs of cis-Cu(L-Ile)2·H2O were collected on an Oxford Diffraction Xcalibur 3 CCD diffractometer with graphite-monochromated Mo Kα radiation (λ = 0.71073 Å). For the monoclinic polymorph, data sets were collected from the same crystal at two different temperatures (2 and 3; Table 1). The data were reduced using the CrysAlis PRO software package.36 The solution, refinement and analysis of the structures were done using the software integrated in the WinGX system.37 The structures were solved by direct methods (SHELXS)38 and refined by the full-matrix leastsquares method based on F2 against all data (SHELXL-97).38 The non-hydrogen atoms were refined anisotropically. The hydrogen atoms in a water molecule were refined using the restraints on the corresponding bond lengths and angle. All other hydrogen atoms were introduced in calculated positions and refined using the appropriate riding model. Electron density maps for structures 2 and 3 were visualized and the corresponding disordered parts built using Coot.39 The disordered parts of 2 and 3 were refined using the restraints on geometrical and atomic displacement parameters. Geometrical calculations were made using PLATON,40 and molecular graphics were prepared using Mercury,41 ORTEP-3,42 and POV-Ray.43

two valence angles significantly deviating from ideal tetrahedral values.22 The second one represents trans Cu(L-Ile)2 and Cu(DIle)2 complexes grown from racemic mixture in a water solution, for which Martino et al. concluded on the basis of single crystal X-ray diffraction pattern that copper(II) had trans coordination, but positional disorder prevented complete molecular structure determination.23 Disorders, experimentally detected in several organic crystal structures, have been successfully rationalized by computational approaches such as (i) crystal structure prediction (crystal energy landscape) studies of eniluracil,24 halobenzenes,25 chlorothalonil,26 cyclopentane,27 and DMSO solvate of carbamazepine;28 (ii) symmetry-adapted ensemble model studies of dichloro/dibromobenzene solid solution,29 eniluracil,29 and form II of caffeine;30 (iii) lattice dynamics calculations based on atom−atom model potential and solidstate density functional theory applied to reproduce crystalline benzoic acid terahertz spectrum;31 and (iv) Monte Carlo simulations of form I of caffeine.32 To the best of our knowledge, systematic computational study of disorder in a crystal of a bioinorganic compound has not been undertaken until now. Nevertheless, such a study is possible thanks to the environment independent FFWa-SPCE force field,7,33 which is suited for modeling trans- and cis-bis(amino acidato)copper(II), Cu(aa)2, complexes by explicitly accounting for the environmental effects such as crystal lattice or aqueous solution. Previous applications of FFWa-SPCE in molecular mechanics (MM) simulations of Cu(aa)2 crystal structures and in molecular dynamics (MD) simulations of Cu(aa)2 complexes solvated in aqueous solution7,8,34 contributed to the understanding of the processes occurring at the molecular level related to the crystallization processes, e.g. self-assembly to crystallization nuclei,7,8 conformational changes,8,34 changes in the copper(II) coordination sphere,34 and cis−trans isomerization.8 In this paper, we assumed that the triple disorder in the new polymorph of cis-Cu(L-Ile)2·H2O could be due to energetic reasons, i.e., several conformers of Cu(L-Ile)2 should have very close stability to partake in the disorder. To examine this hypothesis, conformational analyses were estimated in vacuo, in aqueous solution, and in crystal using FFWa-SPCE. The crystal structure simulations were performed to assist relatively difficult disorder refinement, and to examine different conformer combinations in the unit cell. The purpose of this was to find the packing motif that would result in the triple disorder. The X-ray crystal and molecular structures determined at 295 K and lower temperatures (120 and 183 K for C2 and P212121 crystals, respectively) were compared to understand the effect of temperature on the two polymorphs. Additionally, the relative energies of trans- and cis-Cu(L-Ile)2 in anhydrous and hydrate crystal structures were evaluated with the aim to understand why it has been difficult to obtain trans-Cu(L-Ile)2 crystals.

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MOLECULAR MODELING METHODS

MM Calculations. All MM calculations were performed using the modified version44 of the Lyngby-CFF program,45−47 and the FFWaSPCE force field,7,33 which is suited for modeling anhydrous and aqua Cu(aa)2 complexes with a trans- or a cis-CuN2O2 coordination polyhedron. The MM model is described elsewhere.33,48−52 The FFWa-SPCE force field parametrization procedure for deriving a set of empirical parameters equally applicable to vacuum, crystal, and aqueous solution and the force field testing for the reproduction of crystal and molecular structures of 25 anhydrous and aqua copper(II) complexes with aliphatic amino acids are described elsewhere.7 The basic formula for calculating the conformational (strain) potential energy, Vstrain, is given in the Supporting Information. The potential energy was minimized for an isolated Cu(L-Ile)2 molecule (in vacuo or a gas-phase approximation, Vvacuum = Vstrain), and for an asymmetric unit surrounded by copies of the asymmetric unit in

EXPERIMENTAL SECTION

Synthesis of cis-Cu(L-Ile)2·H2O, Single Crystal Preparations, and Powder X-ray Diffraction. Materials CuCl2·2H2O (Merck, Darmstadt, Germany) and L-isoleucine (Merck-Schuchardt, Hohenbrunn, Germany) were used without further purification. According to the published method,35 the synthesis of cis-Cu(L-Ile)2·H2O was initiated by dissolving L-isoleucine (1.5 g, 11.435 mmol) in 10 mL of 1 M NaOH aqueous solution. The solution was heated at 60 °C until the amino acid dissolved. Water solution of CuCl2 (26 mL, 0.20 M) was then added to the mixture. The reaction mixture was left for one day at room temperature. After removing the mother liquor by 4117



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parameters are used for an assignment of fractional atomic charges by a charge redistribution algorithm.44,47 The algorithm was adjusted to assign the values close to those resulting from the natural population analysis (NPA) estimated for several anhydrous and aqua trans- and cis-Cu(aa)2 systems.48,49 The NPA charges for the Cu, N and O atoms have very similar values in the studied copper(II) chelates with natural α-amino acids.48,49 Accordingly, the same and fixed fractional charge values were assigned to cis- and trans-Cu(LIle)2.53 The potential energy of an asymmetric unit in the simulated crystal environment, Vin‑crystal, was calculated as the sum of the intramolecular strain potential energy, Vstrain, and the intermolecular interactions between the original asymmetric unit atoms and the atoms of surrounding asymmetric unit copies within the cutoff limit (i.e., Vin‑crystal = Vstrain + Vintermolecular). The energy of the crystal lattice was minimized by optimizing the geometry of the asymmetric unit and unit cell dimensions while maintaining space group symmetry. During the in-crystal energy minimization, all atomic coordinates and unit cell dimensions were allowed to vary, except the α and γ unit cell angles in the case of a monoclinic crystal, and all three unit cell angles in the case of an orthorhombic unit cell symmetry were kept equal to 90°. Crystal simulations were performed using the Williams variant of the Ewald lattice summation method54,55 with a spherical cutoff limit of 22 Å, and convergence constants of 0.2 Å−1, 0.2 Å−1 and 0.0 for Coulomb, dispersion, and repulsion lattice summation terms, respectively. The details of the in-crystal calculations are given elsewhere.55 MD Simulations. The simulations were performed using the program package Gromacs, version 4.0.5,56−59 with the force field FFWa-SPCE.7,53 The suitability of FFWa-SPCE for simulations and predictions in aqueous solution is examined elsewhere.7 The MD simulations were performed separately for trans and cis isomers of Cu(L-Ile)2. Periodic boundary conditions were applied. One Cu(L-Ile)2 molecule was solvated in a cubic box containing 3435 water molecules, and equilibrated for 500 ps. 20 ns productive MD phases were accomplished under constant temperature and pressure (298.15 K and 1 bar) using Berendsen T-coupling (τT = 0.1 ps) and Berendsen pcoupling (τp = 0.5 ps).60 The time step was 1 fs. The water molecules' geometry was constrained by the SETTLE procedure,61 whereas all Cu(L-Ile)2 degrees of freedom were relaxed during MD simulations. A cutoff limit of 1.5 nm was applied for the calculations of Coulomb and Lennard-Jones 12-6 interactions. The cutoff distance for the shortrange neighbor list was set to 1.0 nm.

Table 1. X-ray Crystallographic Data for 1, 2 and 3 1 chemical formula Mr cryst color, habit cryst size (mm3) cryst syst space group a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) V (Å3) Z T (K) Dcalc (g cm−3) μ (mm−1) data total/unique Rint obsd data [I > 2σ(I)] data/restraints/ params R1 [I > 2σ(I)] wR2 (all data) S Flack parameter min. and max. resd dens (e Å−3)

2

3

C12H26CuN2O5 341.89 blue, plate 0.08 × 0.18 × 0.28 orthorhombic P212121 7.5799(15) 9.4213(19) 21.537(4) 90 90 90 1538.0(5) 4 183(2) 1.477 1.440 37122/6004 0.032 4802

C12H26CuN2O5 341.89 blue, plate 0.05 × 0.30 × 0.50 monoclinic C2 23.2551(14) 9.4928(4) 7.5418(4) 90 107.560(6) 90 1587.32(15) 4 295(2) 1.431 1.395 23688/4626 0.030 4284

C12H26CuN2O5 341.89 blue, plate 0.05 × 0.30 × 0.50 monoclinic C2 22.5755(5) 9.44266(17) 7.46733(15) 90 106.250(2) 90 1528.24(6) 4 120(2) 1.486 1.449 9653/4451 0.021 4227

6004/0/193

4626/153/264

4451/153/264

0.0221 0.0541 1.00 −0.020(7) −0.37, 0.33

0.0308 0.0812 1.03 0.002(11) −0.32, 0.36

0.0303 0.0738 1.02 −0.010(10) −0.42, 0.54

a crystal lattice. The crystal simulator of the Lyngby-CFF program generates these asymmetric unit copies from the original asymmetric unit by taking into account the unit cell dimensions, Z, and symmetry operations of a specific space group assigned to the crystal lattice. Intermolecular atom−atom interactions, Vintermolecular, were calculated using the Lennard-Jones 12-6 and Coulomb potentials, and empirical parameters the same as for the intramolecular nonbonded interactions.50 In the Lyngby-CFF program, the input charge

Figure 1. Definition of 18 chelate-ring conformations for Cu(L-Ile)2 differentiated by the values of denoted torsion angles in selected conformers with one mixed (defining e7 conformation) and 17 with the same chelate-ring conformations. In the conformer names, “t” and “c” stand for trans and cis configuration, and “a” and “e” denote Cβ in the axial and equatorial positions. 4118



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RESULTS AND DISCUSSION Conformers of Cu(L-Ile)2. From the stereochemical and theoretical points of view, each L-Ile ligand of Cu(L-Ile)2 molecule can have 18 chelate-ring conformations, that is, two conformations of the five-atom chelate ring having Cβ in an axial position or in an equatorial position, and nine conformations of an L-Ile residue characterized by the torsion angle Cγ′−Cβ−Cα−N (≈ 60°, −60°, and 180°), and the torsion angle Cδ−Cγ−Cβ−Cα (≈ 60°, −60°, and 180°). The 18 conformations, whose combinations yield the total of 171 conformers with trans configuration and 171 conformers with cis configuration, are defined in Figure 1. Experimental Crystal and Molecular Structures of cisCu(L-Ile)2·H2O. The structure of the P212121 polymorph of cisCu(L-Ile)2·H2O was redetermined at 183 K (structure 1; Figure 2, Table 2). General characteristics of this structure are in line

Cu−N and Cu−O bond lengths (Table 2). The cis-Cu(L-Ile)2 complex in this polymorph can be denoted as the ce1-ca6 conformer. Crystallization of cis-Cu(L-Ile)2·H2O from the acetic acid− water mixture resulted in a new crystal form with C2 space group symmetry. The diffraction data for this polymorph were collected from the same single crystal at two different temperatures: 295 and 120 K (structures 2 and 3, respectively). In both cases, the asymmetric unit of the C2 structure consisted of one cis-Cu(L-Ile)2·H2O molecule (Figure 2). As in the P212121 polymorph (structure 1 and refs 11 and 12), the coordination sphere around Cu(II) cation was a distorted tetragonal pyramid with the O1W atom of the water molecule in the apical position and the N1, O11, N2 and O21 atoms in the basal plane. The sec-butyl group of one of the L-Ile ligands in the C2 polymorph was triply disordered at both 295 and 120 K (Figure 2, Table 2) resulting in conformational synmorphism.62 Only the major conformer (ce1-ca6, denoted as A in Figure 2) could be unambiguously found from the corresponding electron density maps. The residual electron density corresponding to the other two conformers (ce1-ca9 and ce1-ca8, denoted as B and C, respectively, in Figure 2) could not be reasonably interpreted without using the results of conformational analyses and crystal structure simulations (see further in text). Interestingly enough, the distribution of conformers in the crystal is not identical at different temperatures. At 120 K (3) the ce1-ca6, ce1-ca9, and ce1-ca8 conformers were found in number ratios 0.493(3):0.155(5):0.352(4), while at 295 K (2) these ratios changed to 0.538(3):0.267(7):0.194(7). This indicates the dynamic nature of the observed disorder. As usual, the disorder in the C2 polymorph was accompanied by diffuse scattering streaks, which are visible on the diffraction images and more pronounced for the structure at 120 K (Figure S3a in the Supporting Information). Small and very broad maxima of diffuse-scattering intensity appeared on the diffraction images at the positions where diffraction maxima obtained from a crystal structure with C2 symmetry are systematically absent (reflections hkl, where h + k is an odd number; Figure S3b in the Supporting Information). In a less careful analysis these diffuse-scattering maxima could be easily misinterpreted as diffraction maxima, thus giving rise to the incorrect space-group assignment for the new polymorph of cisCu(L-Ile)2·H2O. All non-carbon hydrogen bond donors and acceptors in the C2 polymorph participate in an extensive hydrogen bonding network (Table S1 in the Supporting Information). Each cisCu(L-Ile)2·H2O molecule is connected by hydrogen bonds to six surrounding molecules thus giving rise to an infinite molecular layer parallel to the crystallographic bc-plane (Figure S4 in the Supporting Information). A completely identical 2D arrangement of cis-Cu(L-Ile)2·H2O molecules, which were connected by hydrogen bonds, was found in the crystal structure of the P212121 polymorph parallel to the crystallographic ab-plane (Figure S5 in the Supporting Information). In both polymorphs, molecular layers were further stacked by van der Waals interactions between the nonpolar aliphatic parts (Figure 3). If the ce1-ca6 conformer is the only one considered in the C2 polymorph, the difference in 3D architectures of the two polymorphs is only in different stacking modes (Figure 3) and the structures can be classified as being polytypic. However, as the disorder in the C2 polymorph involves different conformers, the observed crystal polymorphism of cis-Cu(L-

Figure 2. ORTEP-POV-Ray rendered view of cis-Cu(L-Ile)2·H2O in 1, 2 and 3. Ellipsoids are drawn at the 30% probability level. In 2 and 3, disordered B and C parts are colored in yellow and green, respectively. Hydrogen atoms are presented as spheres of arbitrary small radii, but omitted from the drawing of the B and C parts for clarity.

with the ones measured at 295 K11 and 273 K.12 Compared with the room temperature structure, at 183 K the unit cell contracted along all three axes, reducing the unit cell volume by 1.6%. Copper to water oxygen distance was observed to be shorter [2.427(1) Å compared to 2.474(1) at 295 K, and 2.481(2) at 273 K] without other significant changes in the 4119



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Table 2. Selected X-ray Bond Distances (Å) and Angles (deg) of cis-Cu(L-Ile)2·H2O in 1, 2, and 3a conformer bond distances Cu1−N1 Cu1−N2 Cu1−O11 Cu1−O21 Cu1−O1W valence angles N1−Cu1−O11 N1−Cu1−O21 N2−Cu1−O11 N2−Cu1−O21 N1−Cu1−N2 O11−Cu1−O21 torsion angles O11−Cu1−N1−C12 C13−C12−N1−Cu1 C14−C13−C12−N1 C16−C15−C13−C12 O21−Cu1−N2−C22 C23−C22−N2−Cu1 C24−C23−C22−N2 C26−C25−C23−C22 a

1

2 (A)b

2 (B)

2 (C)

3 (A)

3 (B)

3 (C)

ce1-ca6

ce1-ca6

ce1-ca9

ce1-ca8

ce1-ca6

ce1-ca9

ce1-ca8

−9.2(2) −106.3(2) 147.8(5) 154.5(7)

−15.7(3) −94.3(4) 160.9(5) −71.4(6)

1.991(1) 2.0034(9) 1.9415(8) 1.9535(9) 2.427(1)

1.968(3) 2.003(3) 1.943(3) 1.942(2) 2.426(2)

84.09(4) 162.11(4) 173.33(4) 84.05(4) 98.83(4) 91.35(4)

84.3(1) 163.9(1) 172.8(1) 83.9(1) 98.9(1) 91.3(1)

7.79(7) −139.22(8) 69.2(1) 52.4(2) −3.77(7) −113.84(9) −60.6(1) 162.0(1)

8.3(2) −137.2(2) 65.8(3) 48.5(4) −4.1(2) −116.4(2) −65.7(4) 156.5(4)

1.978(2) 2.010(2) 1.949(2) 1.949(2) 2.366(2) 84.04(8) 162.76(8) 171.24(8) 83.83(8) 99.07(8) 90.83(8)

−11.0(2) −105.1(3) 152.8(5) 159.3(8)

−15.4(6) −95.8(9) 162(1) −84(2)

11.3(1) −140.9(1) 66.3(2) 47.5(2) −3.3(1) −115.7(2) −61.4(3) 156.9(3)

Unlisted bond distances and angles for the B and C parts are the same as for the A part. bDisorder in parentheses.

Figure 3. Molecular layers stack differently in the P212121 (1, left) and C2 (3, right) polymorphs of cis-Cu(L-Ile)2·H2O. Only the conformer ce1-ca6 is shown for the C2 structure. Hydrogen bonds are depicted as green dashed lines. Hydrogen atoms are omitted for clarity.

operator −x, y, 1 − z. Similarly, for the conformer ce1-ca8 (labeled as C in Figure 2) a different conformer (ce1-ca6 or ce1-ca9) should be located at −x, y, −z. Otherwise, in all three cases, there would be steric clashes between the disordered C26 atoms from two adjacent molecules of the same conformer [contacts as short as 2.843(6), 3.04(2) and 2.85(1) Å for C26A, C26B and C26C atoms, respectively]. Conformational Analyses. The conformational analyses were performed using FFWa-SPCE to get an insight into the influence of intermolecular interactions on the energy

Ile)2·H2O is better described as conformational polymorphism.62 It is interesting to note that, if we neglect the disorder of the alkyl groups, the C2 polymorph of cis-Cu(L-Ile)2·H2O and the crystal structure of cis-Cu(L-Val)2·H2O8−10 are isostructural with the cell-similarity indices (Π)63 0.052 and 0.031 for 2 and 3, respectively. Based just on the geometric parameters of refined structures, we can hypothesize that conformers ce1-ca6 and ce1-ca9 (labeled as A and B in Figure 2) should not be found next to an identical neighboring conformer related by a symmetry 4120



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distribution among the conformers of trans- and cis-Cu(L-Ile)2 in different environments: in vacuo without the impact of intermolecular interactions, then in aqueous solution by accounting for the interactions between the complex and water molecules (as well as to assess the conformers that may participate in the self-assembly of a crystallization nucleus), and in the crystal state by considering the intermolecular interactions among the complexes themselves. The aim was to examine the hypothesis that conformers should have similar stability in solution and in the solid state to be able to take part in the crystallization and in the disorder. Conformational Analysis in Vacuo. The equilibrium vacuum geometries were calculated for the trans- and cis-Cu(LIle)2 conformers having the same conformations of the two chelate rings. The minimum Vvacuum values were lower for the trans conformers than for the cis ones. The same relative stability was attained for the trans and cis isomers of bis(glycinato)copper(II), Cu(Gly)2 (by quantum chemical7,64 and MM7,48 calculations) and Cu(L-Val)2 (by MM calculations8). Specifically, the energy difference between the most stable cis and trans conformers of Cu(L-Ile)2, ca1-ca1 and ta3ta3, equals 93 kJ mol−1. Relative to ta3-ta3 and ca1-ca1, other most stable conformers are ta1-ta1, ta9-ta9 (less stable by 5 and 10 kJ mol−1, respectively), and ca9-ca9 (less stable by 11 kJ mol−1), respectively. The Vvacuum values of the most stable equatorial counterparts te1-te1 and te3-te3 are higher by 26 and 22 kJ mol−1 relative to ta3-ta3, and ce9-ce9, ce3-ce3, ce1-ce1 from 29 up to 32 kJ mol−1 relative to ca1-ca1. Conformational Analysis in Aqueous Solution. The MD simulations of solvated Cu(L-Ile)2 in aqueous solution were undertaken in view of the fact that the single crystals of cis-Cu(L-Ile)2·H2O were prepared by recrystallization from ethanol and acetic acid aqueous solutions. Modeling was based on the assumption of the bis-complex stability in aqueous solution. Namely, the stability constant measurements of copper(II) chelates with aliphatic α-amino acids showed that Cu(aa)2 complexes are predominant species in aqueous solutions at ambient temperatures.65,66 The 20 ns MD simulations of the Cu(L-Ile)2·3435H2O system at 298.15 K were performed for the trans and cis conformers having the same starting conformations of both chelate rings, and for four conformers (trans e1-a9, and cis e1-a6, e1-a9, and e1-a8) having mixed different conformations. Generally, a frequent interchange between the conformations with Cβ in axial and equatorial positions was observed. The distribution functions for the Cβ−Cα−N−Cu angles, calculated from the 20 ns MD data, suggested the most prevailing axial or equatorial conformations of the studied systems (Figure 4). During the simulations, both trans and cis a2(e2)-a2(e2) transformed quickly to a8-a8 by rotating around the Cβ−Cα bond, and a4(e4)-a4(e4) and a5(e5)-a5(e5) to e6(a6)-e6(a6) by rotating around the Cγ−Cβ bond. Also, the e7/a7 conformations changed to either e1/a1 or a9 by rotating around the Cβ−Cα or Cγ−Cβ bond, respectively, hence changing trans and cis a7(e7)a7(e7) to a1(e1)-a9. During the 20 ns MD simulations, no transition between the cis- and trans-Cu(L-Ile)2 configurations was obtained. Although the intramolecular potential energy, Vstrain, is lower for the trans than cis conformers, more favorable intermolecular interactions with the water molecules are formed by cis than trans conformers (Figure 4). Consequently, the average MD total energies of the cis- and trans-Cu(L-Ile)2·3435H2O systems are almost identical for the same conformers (Figure 4). These

Figure 4. MD average values of the total energy of the Cu(LIle)2·3435H2O system and several energy contributions (intramolecular Vstrain contribution, electrostatic interactions between the complex and water molecules, and between water molecules themselves) calculated from the values attained during the 20 ns MD simulations at 298.15 K for denoted conformers (prevailing conformers are denoted in magenta). The reference values of the ta9ta9 conformer system are given in Table S2 in the Supporting Information.

findings are in accord with the MD results obtained for the solvated cis and trans isomers of Cu(Gly)27 and Cu(L-Val)28 systems. The solvated trans and cis 9-9 and 1-9 systems had the lowest average total energies (Figure 4). The cis 6-6 and 1-6 conformers formed the most favorable intermolecular interactions with water molecules. Like in vacuo, the resulting average energy of mixed conformer systems (e.g., trans and cis 1-9, cis 1-6 and 1-8 systems in Figure 4) can be considered as half the sum of the average energies of the systems with conformers having the same corresponding chelate ring conformations. Therefore, it can be expected that, just like cis e6(a6)-e6(a6) and a1(e1)-e6(a6), the mixed cis conformers with the e6/a6 chelate-ring conformation form more favorable intermolecular interactions with water molecules than other conformers. During the 20 ns MD simulations, water molecules influenced the dynamic behavior of Cu(L-Ile)2, and vice versa. Different hydrogen bonding patterns formed between the trans and cis conformers and water molecules influenced different organization structures of water molecules around the isomers,7 as well as the interactions among water molecules (VCoulomb, Figure 4). For instance, for two solvated cis a3(e3)-a3(e3) systems, which had different initial water molecules’ positions, two different trajectories were calculated because of stochastic nature of our MD simulations. In the first one, Cu(L-Ile)2 retained the initial 3-3 conformation, while in the second one, at around 15 ns of the 20 ns MD simulation, the conformation changed to cis a1(e1)-a3(e3). Obviously, in the latter case the water molecules’ arrangement and energy fluctuations were such that they made the conformational change possible. Besides, both cis 1-1 and 3-3 systems have very similar average MD energy contributions (Figure 4). On the other hand, for the solvated trans and cis 1-1, 6-6, 8-8, 9-9, trans 3-3, and cis 1-6 systems, we collected a few MD trajectories without any change in the L-Ile side-chain conformations. The average MD total energies of the studied conformers’ systems (Figure 4) differed up to 63 kJ mol−1 and 54 kJ mol−1 4121



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Figure 5. FFWa-SPCE minimum energy values estimated for the Cu(L-Ile)2 (□, thin line) and Cu(L-Ile)2·H2O (●, thick line) systems in the simulated P212121 and C2 unit cell packings: Vin‑crystal (full line) and Vintermolecular (broken line) for denoted conformers with cis configuration (P212121 black; C2 red), and trans configuration (P212121 blue; C2 green). Empty space means that the corresponding conformer had unfavorable starting packing and thus changed to another conformer via energy minimization.

relative to the solvated trans a9-a9 and cis a1(e1)-a9 systems, respectively. The comparison of average MD total energies between the Cu(L-Ile)2·3435H2O systems with cis a1(e1)e6(a6), a1(e1)-a8, and a1(e1)-a9 conformers (i.e., three experimentally determined conformers in the crystal structures 2 and 3) yielded the energy differences of 29 and 20 kJ mol−1 for the cis 1-6 and cis 1-8 systems, respectively, relative to the cis 1-9 one. The root-mean-square, rms, deviations (fluctuations) of the short-range Coulomb interactions between Cu(L-Ile)2 and water molecules (VCoulomb,SR, Table S2 in the Supporting Information), which can trigger conformational changes, were around 45 and 52 kJ mol−1 for trans and cis conformers, respectively. Essentially, they were the same order of magnitude as the differences in average MD total energies calculated for the studied solvated conformers’ systems. Hence, our MD simulations suggest the possibility of conformational interconversions at a longer time scale than 20 ns, and simultaneous presence of different conformers in aqueous solution. Besides, the MD modeling of self-associations of Cu(Gly)27 and Cu(LVal)28 complexes in aqueous solution suggested that the selfassembly from monomers to oligomers could occur practically just upon the dissolvation even for a very dilute solution.8 Thus, we may expect that both trans and cis isomers of Cu(L-Ile)2 with the chelate-ring conformations 1, 3, 6, 8, and 9 can be present in aqueous solution at room temperature and take part in the formation of crystallization nuclei. The population of conformers and the rate constants for conformational changes in aqueous solution remain to be examined by MD calculations of the free energy profile and activation free energy in the forthcoming studies. Partial Conformational Analysis in Crystal. Because the conformation of one L-Ile sec-butyl was the same in the X-ray crystal structures of cis-Cu(L-Ile)2·H2O, trans-Cu(L-Me2Ile)2,22 and trans aqua Cu(L-Me2Ile)267 (i.e., e1/a1 with Cδ−Cγ−Cβ− Cα ≈ Cγ′−Cβ−Cα−N ≈ 60°, also being among the lowerenergy minima in vacuo), we focused crystal structure simulations on the cis and trans conformers having the chelate-ring conformations between e1/a1 and 9 possible axial conformations. The simulations were performed for anhydrous and aqua Cu(L-Ile)2 to examine the effect of

additional hydrogen bonding due to water molecules on the relative energy values of the trans and cis conformers in anhydrous and hydrated crystal structures. An asymmetric unit was either Cu(L-Ile)2 or Cu(L-Ile)2·H2O. We used the atomic coordinates constructed for the 18 cis and 18 trans conformers, the experimental X-ray unit cell lengths and angles, the P212121 and C2 space group symmetry operations, and the molecule orientation defined according to the experimental ones (detailed description is given elsewhere)8 as the starting data for energy minimization in crystalline environment. Figure 5 shows the lowest Vin‑crystal and Vintermolecular values (Table S3 in the Supporting Information) among all attained conformers’ packings. In addition, unit cell simulations were calculated for the aqua ca1-ce6 conformer, as it was seen to be prevailing in aqueous solution over ce1-ca6 (Figure 4). The comparison between P212121 and C2 crystal packings of the two conformers resulted in the Vin‑crystal energy difference of 16 kJ mol−1 and 24 kJ mol−1, respectively, in favor of the experimentally observed ce1-ca6 conformer. The predictions of the unit cell packings for aqua cis conformers yielded the e1-a6, e1-a7, e1-a8, and e1-a9 conformers as the ones with the lowest intermolecular energies (Figure 5). For these conformers, very small energy differences were obtained, as Vintermolecular ranged between −479 kJ mol−1 (e1-a9) and −483 kJ mol−1 (e1-a7) for space group P212121, and between −473 kJ mol−1 (e1-a9) and −478 kJ mol−1 (e1a7) for space group C2. Except for ce1-ca9, these conformers were not among the ones with the lowest calculated Vin‑crystal. The result that experimentally observed ce1-ca6 and ce1-ca8 conformers do not have the lowest Vin‑crystal may be attributed to the force field’s possible imperfections, or to some specific reasons (e.g., solvent effects) that occurred during the selfassembly of the complexes in solutions and the crystallization process. The smallest Vin‑crystal values were obtained for the cis e1(a1)-a1 and e1(a1)-a3 conformers (Figure 5). The energy difference between Vin‑crystal of aqua cis e1-a6 and a1-a1 in P212121 crystal is 35 kJ mol−1 compared to the energy difference of 50 kJ mol−1 in vacuo. Generally, the cis conformers have smaller Vintermolecular values than the trans ones (Figure 5). The aqua cis conformers have 4122



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Table 3. FFWa-SPCE In-Crystal Energy Values (kJ mol−1) and Unit Cell Dimensions Estimated for cis-Cu(L-Ile)2·H2O Conformer Pairs in an Asymmetric Unit, Space Group P21 conformer pair ce1-ca6, ce1-ca6, ce1-ca6, ce1-ca6, ce1-ca3, ce1-ca3, ce1-ca3, ce1-ce6, ce1-ca8,

ce1-ca8 ce1-ca6 ce1-ca9 ce1-ca3 ce1-ca8 ce1-ca3 ce1-ca9 ce1-ca1 ce1-ca8

Vin‑crystal

Vintermolecular

a/Å

b/Å

c/Å

β/deg

V/Å3

927.6 952.3 910.8 935.5 912.5 895.8 902.1 937.6 914.6

−960.1 −958.9 −955.4 −949.6 −949.2 −948.0 −947.2 −945.5 −942.2

7.718 8.043 7.667 8.282 8.144 8.018 8.057 8.302 7.282

9.514 9.408 9.389 9.199 9.133 9.153 9.277 9.216 9.471

21.823 21.934 22.555 21.848 22.154 22.773 22.413 21.282 23.489

90.3 84.7 86.6 93.7 97.5 95.4 94.7 94.8 93.7

1602.4 1652.6 1620.9 1661.2 1633.8 1663.8 1669.7 1622.7 1616.7

Crystal Structure Simulations Assisted in Resolving the Disorder in 2 and 3. As crystallographic modeling of the heavily disordered L-Ile ligand in the X-ray crystal structures 2 and 3 was nontrivial, we assumed that the MM calculated lowenergy crystal structures could help us to interpret the electron density maps and in the refinement of disordered parts in 2 and 3. Preliminary structural models suggested that the disordered parts of the adjacent molecules were in contact. Thus, instead of using the experimentally determined space group C2, we selected space group P21 with two Cu(L-Ile)2·H2O in the asymmetric unit. This lower-symmetry model allowed us to treat independently the atoms of L-Ile moieties in the two neighboring Cu(L-Ile)2 molecules, which interacted via aliphatic−aliphatic interactions. The starting data for the geometry optimization using FFWa-SPCE were as follows: the experimental unit cell dimensions of 3 transformed to suit P21 space group unit-cell setting [a = 7.4678(2) Å, b = 9.4436(2) Å, c = 21.7041(4) Å, β = 93.042°, V = 1528.48(6) Å3]; available experimental Cartesian coordinates of the fiveatom chelate rings, ordered equatorial L-Ile (the e1 chelate-ring conformation) and water oxygen atoms; constructed atomic coordinates for the second (disordered) chelate-ring conformations oriented to fit with the experimental positions of the Cu and donor O and N atoms. We used the information from the conformational analyses, which suggested that only the chelate-ring conformations that according to the MD results potentially exist in aqueous solution (i.e., the 1, 3, 6, 8, and 9 chelate-ring conformations; Figure 4), combined with identified e1 conformation, could be good candidates for crystallization. FFWa-SPCE yielded experimentally observed conformers of cis aqua Cu(aa)2 complexes, that is, ce1-ca6 in 1 and the ones in the previously studied cis-Cu(L-Val)2·H2O,8 as the conformers with the lowest Vintermolecular but not Vin‑crystal. Hence, we considered the predictions of the unit-cell packings of different Cu(L-Ile)2·H2O conformer pairs in P21 to be a good test of whether FFWa-SPCE would produce the same outcome regarding Vintermolecular and Vin‑crystal values. Table 3 presents a list of conformer pairs for which the lowest Vintermolecular values were calculated. The estimated data for other conformer pairs are collected in Table S4 in the Supporting Information. Due to sterical hindrance in the unit-cell packing, several conformer pairs (e.g., ce1-ca9 with ce1-ca9, and ce1-ca9 with ce1-ca8) underwent conformational changes via energy minimization and were not among the predicted crystal structures. Indeed, the conformer pairs for which the lowest Vintermolecular had been calculated were the ones identified in the crystal structures 2 and 3. This confirmed that the intermolecular interactions (calculated using FFWa-SPCE) could be taken as an empirical criterion to predict the most probable conformers

smaller Vin‑crystal values than the aqua trans conformers, and both aqua cis and trans conformers have lower in-crystal energy values than in the corresponding anhydrous crystal packings (Figure 5, Table S3 in the Supporting Information). Conversely, a similar conformational analysis in crystal performed for the anhydrous and aqua Cu(L-Val)2 systems showed that the lowest-energy minima of trans and cis conformers had lower and higher Vin‑crystal values, respectively, in the anhydrous than hydrous Cu(L-Val)2 crystals, in accord with their experimental crystallization outcome.8 We performed additional in-crystal simulations by the energy minimization from the experimental data of trans-Cu(L-Me2Ile)2 complexes,22,67 and with the methyl groups substituted by the hydrogen atoms on the nitrogen atoms. The corresponding conformers were ta1-ta3 for the anhydrous crystal (space group P21) and te1-te3 for the aqua crystal (space group P212121). The estimated Vin‑crystal and Vintermolecular of the P21 minimum ta1-ta3 crystal structure were by 12 and 52 kJ mol−1, respectively, higher than for the anhydrous lowest-energy C2 crystal structure of te1-ta3 conformer (Figure 5, Table S3 in the Supporting Information). For the aqua te1-te3, Vin‑crystal and Vintermolecular were by 33 and 20 kJ mol−1, respectively, higher than for the P212121 lowest-energy crystal structure of trans hydrated te1-ta9 (Figure 5, Table S3 in the Supporting Information). Because of the lowest Vin‑crystal and Vintermolecular energies that te1-ta3 has in a quite dense packed C2 crystal structure (Table S3 in the Supporting Information), this conformer may be the most probable candidate for crystallization of anhydrous transCu(L-Ile)2. Nevertheless, closer examination of the predicted anhydrous Cu(L-Ile)2 crystal packings revealed that when an intermolecular bond between the Cu(II) and axially placed Ocarbonyl from an adjacent complex was formed (expected event in anhydrous Cu(aa)2 crystal structures7), there may be significant sterical crowding due to the branched L-Ile side chains (Figure 1), especially for trans conformers. Thus, not only does the presence of a water molecule in an asymmetric unit lower the in-crystal energy but it also allows a disposition of the Cu(L-Ile)2 molecules far away one from another, and better accommodation of different L-Ile conformations in the crystal packing. As the crystal simulations were done for only several conformers and two space groups, they do not exclude a possibility for the trans isomer to crystallize in a different crystal lattice in which it might be more stable than the cis isomer. It will be challenging to discover if the presented energy and spatial issues (which predict aqueous trans-Cu(L-Ile)2 crystal lattice to be preferred to anhydrous one, and te1-ta3 to crystallize if anhydrous crystal is possible) can be confirmed with future crystallization experiments. 4123



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Table 4. The Root-Mean-Square, rms, Deviations and Differences, Δ, Calculated between the Two Denoted Crystal Structures' Internal Coordinates and Unit Cell Dimensions of cis-Cu(L-Ile)2·H2Oa,b internal coordinates

unit cell dimensions

compared structures

rms(Δb)

rms(Δθ)

rms(Δφ)

rms (Δa,Δb,Δc)

Δα,Δβ,Δγ

100ΔV/V1

1−rtc (X-ray) 3−2 (X-ray) ce1-ca6 ce1-ca9 ce1-ca8 1 (X-ray−MM) 2 (X-ray−MM) ce1-ca6 ce1-ca9 ce1-ca8 minimum (a) minimum (b) minimum (c) minimum (d) minimum (e) minimum (f) minimum (g) minimum (h) minimum (i) 3 (X-ray−MM) ce1-ca6 ce1-ca9 ce1-ca8 minimum (a) minimum (b) minimum (c)

0.009

0.7

1.2

0.084 0.396

0.0,0.0,0.0 0.0,−1.3,0.0

−1.7 −3.9

0.016 0.016 0.018 0.011

1.1 1.2 1.1 2.8

2.2 2.7 3.9 6.0

0.223

0.0,0.0,0.0

−4.4

0.025 0.024 0.025 0.024 0.024 0.025 0.024 0.024 0.025 0.024 0.024 0.025

4.2 4.0 3.8 3.9 3.9 3.8 4.2 4.8 4.4 4.3 4.1 4.3

10.2 10.4 12.3 10.5 9.6 10.5 10.7 10.2 11.1 10.9 10.3 14.8

0.857 2.935 0.292 0.391 0.286 0.092 1.052 0.367 0.286 0.354 0.890 0.168

0.0,−8.5,0.0 0.0,−7.3,0.0 0.0,2.3,0.0 0.0,−3.0,0.0 0.0,−4.2,0.0 0.0,0.0,0.0 0.0,−5.5,0.0 0.0,−3.4,0.0 0.0,−2.9,0.0 0.0,−2.2,0.0 0.0,−4.8,0.0 0.0,−0.3,0.0

−2.7 −14.4 −4.5 −2.4 −2.1 −2.7 −6.1 −5.4 −4.7 −4.0 −5.1 0.0

0.018 0.018 0.016 0.017 0.017 0.017

3.8 4.8 3.0 3.3 3.4 3.2

9.5 11.8 10.1 10.0 9.9 9.1

1.228 0.725 0.666 0.784 0.648 0.419

0.0,−9.8,0.0 0.0,−2.7,0.0 0.0,1.0,0.0 0.0,−4.4,0.0 0.0,−5.6,0.0 0.0,−1.4,0.0

−6.7 −10.3 −8.6 −6.4 −6.1 −6.7

a Internal coordinates: bond lengths, b (in Å), valence angles, θ (in deg), torsion angles, φ (in deg). Hydrogen atoms are not taken into account. Unit cell dimensions: unit cell lengths, a, b, and c (in Å), unit cell angles, α, β, and γ (in deg), and volume, V. bThe minima are defined in Table 5. cRoom temperature structure, ref 11.

to occur in the crystal structure. Namely, after model building in the electron density maps and completed crystal structure refinement in space group C2, the disorder included the ce1ca6, ce1-ca9, and ce1-ca8 conformers. The results of MM calculations concerning the three conformers (Table 3) suggested that ce1-ca6 could be surrounded by any of the three identified conformers whereas ce1-ca8 and ce1-ca9 alone as a conformer pair in the crystal structure were unfavorable neighbors. MM Reproduction of the X-ray Crystal and Molecular Structures. The efficacy of FFWa-SPCE in reproducing the experimental crystal and molecular structures 1, 2 and 3 was measured by the rms deviations and differences between the Xray and MM crystal internal coordinates and unit cell dimensions (Table 4). The experimental structural data were taken as the starting point for geometry optimization. The asymmetric unit was cis-Cu(L-Ile)2·H2O if not otherwise stated. The superpositions of the X-ray and MM derived unit cells and packings are illustrated in Figures 6 and 7. Reproduction of 1 and “Ordered” 2 and 3. The error values for crystal and molecular structure 1 determined at 183 K (Table 4, Table S5 in the Supporting Information, Figure 6) are only slightly different from the values estimated for the corresponding room-temperature structure. Namely, the latter one11 was among 25 Cu(aa)2 complexes on which FFWa-SPCE was previously tested (i.e., the rms values were 0.013 Å in bond lengths, 3.2° in valence angles, 6.7° in torsion angles, and 0.123 Å in unit cell dimensions; Cu to OW1 distance was 2.998 Å).7

Figure 6. Superposition of the X-ray (black) and MM crystal (red) unit cells and packing of cis-Cu(L-Ile)2·H2O for 1 (MM unit cell dimensions: a = 7.731 Å, b = 9.495 Å, c = 21.885 Å, V = 1606.5 Å3).

Better reproduction is obtained for structure 1 than for 2 and 3 (Table 4). The result is connected with the difficulty in building a crystal lattice composed of different conformers for the purpose of simulating real crystalline surroundings in the MM calculations. To examine the effects of different L-Ile sidechain conformations on the crystal packing, we first calculated the MM crystal structures as if only one kind of conformer was present in the crystal lattice. Selected internal coordinates of their minimum structures are given in Table S5 in the Supporting Information. By starting the energy minimization 4124



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Figure 7. Superposition of the X-ray (black, gray) and MM crystal (red, rose) unit cells and packing of cis-Cu(L-Ile)2·H2O for crystal structures 2 and 3 (MM unit cell dimensions for minima (a), (d), and (g) are given in Table 5). Ce1-ca6 conformer is shown in black and red; ce1-ca9 in dark gray and dark rose, and ce1-ca8 in light gray and light rose.

Table 5. FFWa-SPCE In-Crystal Energy Values (kJ mol−1) and Unit Cell Dimensions Estimated for 4 cis-Cu(L-Ile)2·H2O, That Adopted Denoted Conformations and Packing Motifs in the Unit Cell of 2 and 3 conformers' unit-cell symmetry positions

a

motif

x, y, z

1−x, y, 1−z

1/2 + x, 1/2 + y, z

1/2 − x, 1/2 + y, 1 − z

min

1 1 1 1 2 2 2 2 3 3 3 3 min

ce1-ca6 ce1-ca6 ce1-ca6 ce1-ca6 ce1-ca6 ce1-ca6 ce1-ca6 ce1-ca6 ce1-ca6 ce1-ca6 ce1-ca6 ce1-ca6




Vin‑crystal

Vintermolecular

a/Å

b/Å

c/Å

β/deg

(a) (a) (b) (c) (d) (d) (e) (f) (g) (g) (h) (i)a V/Å3

(a) (b) (c) (d) (e) (f) (g) (h) (i)

1839.5 1821.5 1859.5 1875.3 1871.1 1885.4 1850.5 1833.4 1834.2

−1913.4 −1911.1 −1912.3 −1877.1 −1907.9 −1909.4 −1915.7 −1894.8 −1926.9

23.916 24.238 23.316 25.029 23.672 23.614 23.833 24.760 23.327

9.452 9.391 9.598 9.270 9.442 9.539 9.502 9.318 9.272

7.686 7.667 7.645 7.896 8.019 7.881 7.745 7.822 7.716

110.6 111.8 107.6 113.1 111.0 110.5 109.8 112.4 107.9

1626.0 1620.8 1630.8 1684.6 1673.2 1662.6 1650.1 1668.5 1587.7

Initial ce1-ca6 conformers changed to ca1-ce6 during geometry optimization.

positions with carbon atoms at the intermolecular distances greater than 3.5 Å. Consequently, torsion angles in the MM structures significantly deviated from their experimental values (Table S5 in the Supporting Information). The rms(Δθ) values were slightly higher than for the previously studied Cu(aa)2 complexes7 [up to 1.6°; the same result was yielded for the disorder case in cis-Cu(L-Val)2·H2O].8 Particularly, trans N− Cu−O angle bending in the MM structures was more pronounced than in the experimental ones because the N

from experimental structures 2 and 3, the same corresponding minimum was obtained for the ce1-ca6 and ce1-ca8 conformers, but different for the ce1-ca9 conformer. Namely, when only one conformer type was assumed in the crystal lattice, experimental intermolecular close contact between C26 atoms of adjacent molecules in 2 and 3 shortened from 3.008 Å to 2.843 Å for ce1-ca6, from 2.970 Å to 2.853 Å for ce1-ca8, but lengthened from 2.887 Å to 3.035 Å in ce1-ca9. On the other hand, in the MM structures, the L-Ile side chains adopted 4125



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room temperature but the internal coordinates match better the lower temperature data in both polymorphs (Table 4). For the motif 1 minima, the MM reproduction of the experimental bond and unit cell lengths is comparable with the rms deviations calculated between the X-ray data of 2 and 3 (Table 4). Also, motif 1’s rms deviations in valence and torsion angles are smaller than for the “ordered” crystal structures (Table 4) and within the ranges of the FFWa-SPCE reproduction errors7 obtained for the previously studied Cu(aa)2 complexes. Better overall reproduction of experimental structures 2 and 3 (Table 4, Figure 7) suggests that the (a), (b), and (c) unit cell packings are the most plausible candidates to be mostly present in the real crystal lattice. The lowest Vin‑crystal values calculated for the (a) minimum relative to the (d) and (g) ones, as well as for the unit cells (b) and (c) relative to those with the same conformers' compositions in motifs 2 and 3 (Table 5) also corroborate motif 1’s type of packing. In the motif 1 minima, the ce1-ca6 conformers are situated in the same 2D layer bonded via the N−H···O hydrogen bond network, while ce1ca8 and ce1-ca9 are at alternating positions in the neighboring 2D layers. If so, the repeating motif in these neighboring 2D layers would mostly be the combination of (a) and (c) unit-cell packings at 120 K, and (a) and (b) packings at 295 K (Figure 8). The Effect of Temperature Increase on the Polymorphs. In the MM crystal structure reproductions, the ce1ca9 conformer consistently required longer unit cell lengths (especially in the a-direction, Table 5) than the ce1-ca8 conformer. Similarly to relatively small differences between the 183 K crystal and molecular structure 1 and the one determined at room temperature (Table 4), the unit cell volume of cis-bis(L-alaninato)copper(II), with the same P212121 space group symmetry as 1, contracted by 1.9% upon cooling from ambient temperature to 7 K.69 Relative to the P212121 crystals, composed of only one conformer type, more pronounced enlargement of the unit cell volume between 2 and 3 (Table 4) may suggest a connection between the lengthening of the a unit cell length and greater population of the ce1-ca9 conformers in 2 than 3. Therefore, the experimental crystallographic observations combined with the computational results make it evident that dynamic disorder takes place in the real crystal structure.

and O atoms accommodated in a way to form intermolecular O···N−H hydrogen bonds. The Most Plausible Packing Motif of cis-Cu(L-Ile)2·H2O in 2 and 3. According to the 0.493(3):0.155(5):0.352(4) conformer population ratio suggested by the refinement of Xray diffraction data of 3, the numbers of ce1-ca6, ce1-ca9, and ce1-ca8 conformers present in the crystal lattice of 3 are approximately 50, 15, and 35, respectively, if 25 unit cells are accounted. The respective 25 unit-cell approximated conformer numbers in 2 are 54, 26, and 20. Consequently, as most of the unit cells contain two ce1-ca6 conformers in both 2 and 3, we built all combinations of two ce1-ca6 with one or two ce1-ca9 and ce1-ca8 as a unit-cell tetramer according to the C2 space group symmetry operations. We used these tetramers as an asymmetric unit, and P1 symmetry operation as the initial data for in-crystal geometry optimization. There are three different packing motifs of the two ce1-ca6 in the unit cell (Table 5, Figure 7). By combining them with one or two ce1-ca8 and ce1-ca9, nine different minimum crystal structures can be evaluated (Table 5). Similarly, the molecular modeling procedure consisting of the insertion of a given ratio of X molecules into the Y structure, and vice versa, was employed to predict the possibility of solid solution formation between different derivatives of a series of ephedrine− cyclophosphoric acid salts in different ratios.68 During the geometry optimization, the four Cu(L-Ile)2·H2O geometries in the unit cell were not confined by any symmetry operation, thus good match between the experimental and MM structures (Figure 7) confirmed that in general FFWa-SPCE accurately reproduces the crystal lattice effects. In several cases, especially for motif 3, initial equatorial−axial conformation changed during the geometry optimization to either axial−equatorial or axial−axial conformation. Table 5 lists the lowest energy minima with preserved experimental equatorial−axial conformations, except for motif 3's crystal structure with the initial two ce1-ca6 and two ce1-ca8 conformers. For this crystal structure, we were only able to obtain the minimum (i) (Table 5) with ce1-ca6 transformed to ca1-ce6 (the positions of L-Ile side chains remained quite close to their initial positions but the five-atom chelate rings reoriented during the energy minimization). Nevertheless, the Vin‑crystal energies of the (a) and (d) minima can be considered as half the sum of the Vin‑crystal values of (b) and (c) unit cells, and (e) and (f) unit cells, respectively (Table 5). Accordingly, Vin‑crystal of motif 3's minimum with two ce1-ca6 and two ce1ca8, if obtained by energy minimization, would be approximately equal to 1867 kJ mol−1. The errors in the reproduction of X-ray crystal structure 2 for the nine minima are listed in Table 4. Although there were no axial−equatorial conformational changes for motif 2 by the geometry optimization, too close experimental intermolecular contacts between the aliphatic groups (as already discussed) rule out the existence of such a motif. Motif 3 led to axial−equatorial conformational changes, which may be computationally energetically preferred to the experimental conformations without any symmetry restriction within the unit cell, but yielded the largest rms(Δφ) reproduction error (Table 4). It was only for motif 1 that the best reproduction of both internal coordinates and unit cell dimensions was obtained with two and three different conformers in the unit cell [minima (a), (b), and (c), Table 4]. Generally, the estimated unit cell dimensions fit better with the experimental data measured at

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CONCLUSIONS The packing and conformational polymorphism of cis-Cu(LIle)2·H2O obtained by recrystallization from ethanol and acetic acid aqueous solutions suggest that different hydrogen bonding capabilities of solvents might affect the self-assembly of Cu(LIle)2 complexes in solutions, and consequently their different packing in crystal lattices. The 183 K crystal and molecular structure 1 (composed of one conformer type), obtained by recrystallization from a less polar ethanol−water mixture, was essentially the same as the one11 measured at 295 K. As confirmed by the MM crystal structure simulations, relatively more pronounced unit cell enlargement between crystal structures 3 and 2 (composed of three conformer types), recrystallized from a more polar acetic acid−water mixture and measured at 120 and 295 K, respectively, is connected with the conformational changes and dynamic disorder. The crystal simulations of Cu(L-Ile)2·H2O in space groups P212121 and C2 yielded the lowest and very close Vintermolecular values for the cis e1-a6, e1-a7, e1-a8, and e1-a9 conformers. These conformers, except for ce1-ca7, were found to take part 4126



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layers interacting via aliphatic−aliphatic interactions with ce1ca8 and ce1-ca9 conformers situated at alternating positions in the neighboring 2D layers. Although the trans isomer of Cu(L-Ile)2 is energetically preferred to the cis one in vacuo, more favorable intermolecular interactions are formed by the cis than the trans isomer both in crystal and in aqueous solution. Hence, MD simulations yielded that both trans and cis isomers of Cu(L-Ile)2 can be present in aqueous solution at room temperature. A partial conformational analysis in crystal suggests that the investigated crystal packings of cis-Cu(L-Ile)2·H2O are energetically preferred to trans-Cu(L-Ile)2·H2O, and the hydrated Cu(L-Ile)2 system is favored to the anhydrous one for both isomers. Although the computational results corroborate the experimental evidence that single crystals of the aqua cis isomer can easily be obtained, and those with enantiopure trans-Cu(L-Ile)2 have not been isolated, the exact answer on the question if the latter ones are possible requires additional experimental and computational validation.

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ASSOCIATED CONTENT

S Supporting Information *

X-ray crystallographic informational files (CIF) are available for structures 1, 2 and 3. Illustrations of measured and simulated PXRD patterns for polymorphs of cis-Cu(L-Ile)2·H2O (Figures S1 and S2), detail of the reconstructed precession image of hk0 reciprocal layer for 3, and intensity profile h60 extracted from the hk0 reciprocal layer for 3 (Figure S3), hydrogen bonding network in 3 joining the molecules in a layer parallel to the crystallographic bc-plane (Figure S4), molecular layers formed by hydrogen bonding for 1 and 3 polymorphs (Figure S5). Listing of hydrogen bonding in 1, 2 and 3 (Table S1), average values and rms deviations of energy contributions calculated from the values attained during the 20 ns MD simulations at 298.15 K for ta9-ta9 conformer of the Cu(L-Ile)2·3435H2O system (Table S2), Vin‑crystal and Vintermolecular values depicted in Figure 5 (Table S3), FFWa-SPCE in-crystal energy values and unit cell dimensions predicted for asymmetric-unit conformer pairs of cis-Cu(L-Ile)2·H2O in space group P21 (Table S4), selected bond distances and angles of cis-Cu(L-Ile)2·H2O in the MM reproduced experimental crystal structures 1, 2 and 3 with one conformer type in the crystal lattice (Table S5). The basic formula for the calculation of Vstrain. The 3D rendering for FFWa-SPCE crystal structures of Figures 6 and 7 in pdb format. This material is available free of charge via the Internet at http://pubs.acs.org.

Figure 8. The crystal packing motif of cis-Cu(L-Ile)2·H2O proposed on the basis of the MM simulations and experimental conformers' population ratios in 2 and 3. The axially placed L-Ile moieties of ce1ca6 (gray), ce1-ca9 (yellow), and ce1-ca8 (green) correspond to the disordered A, B, and C parts, respectively, in Figure 2.

in disorder in 2 and 3. The a7/e7 chelate-ring conformations were predicted by MD simulations to be unstable in solution, which may cause their absence in the crystallization nuclei formed in solution, and subsequently in the conformational disorder. However, we can only hypothesize about this prediction without proper information on the energy barriers for conformational interchanges that may occur during crystallization. In accordance with experimental crystallization results, the conformational analysis in aqueous solution and the predicted unit-cell packings of different conformers prove that FFWa-SPCE intermolecular energy estimated between the aqua Cu(aa)2 complexes in the solid state, and between Cu(aa)2 and water molecules in aqueous solution, can be used as an empirical criterion to predict conformation(s) occurring in the crystal lattice. The crystal structure simulations suggest the packing motif disguised by the triple disorder in the average experimentally determined structures 2 and 3 as follows: hydrogen bonded ce1-ca6 conformers within the same 2D

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AUTHOR INFORMATION

Corresponding Author

*Institute for Medical Research and Occupational Health, Ksaverska cesta 2, P.O. Box 291, HR-10001 Zagreb, Croatia. Email: [email protected]. Fax: +385 1 4673 303. Tel: +385 1 4682 526. Present Address §

Laboratory of Biomolecular Research, Paul Scherrer Institute, CH-5232 Villigen PSI, Switzerland. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Croatian Ministry of Science, Education and Sports (Project Grants 022-0222148-2822 and 4127



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119-1193079-1084). We thank Professor Dubravka MatkovićČ alogović for the X-ray diffraction measurements and continuous encouragement. The X-ray diffraction measurements and refinement of structure 1 were done during M.M.’s attendance at The Zürich School of Crystallography 2007 Bring Your Own Crystals.

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Modeling Triple Conformational Disorder in a New Crystal Polymorph

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