Controlled Crystallization, Structure, and Molecular Properties of

Article pubs.acs.org/crystal

Controlled Crystallization, Structure, and Molecular Properties of Iodoacetylamphotericin B Katarzyna N. Jarzembska,†,# Daniel Kamiński,‡,# Anna A. Hoser,† Maura Malińska,† Bogusław Senczyna,‡ Krzysztof Woźniak,*,† and Mariusz Gagoś*,§,⊥ †

Department of Chemistry, University of Warsaw 02-093 Warsaw, Pasteura 1, Poland Department of Chemistry, University of Life Sciences in Lublin, Akademicka 15, 20-950 Lublin, Poland § Department of Biophysics, University of Life Sciences in Lublin, Akademicka 13, 20-950 Lublin, Poland ⊥ Department of Cell Biology, Institute of Biology and Biochemistry, Maria Curie-Skłodowska University, 20-033 Lublin, Poland ‡

S Supporting Information *

ABSTRACT: A new crystallization method of N-iodoacetylamphotericin B derivative is reported. The crystallization process in the presence of different quantities of amphotericin B additives was extensively studied and its mechanism was proposed. It also resulted in good quality single crystals suitable for X-ray structure determination (100 K). The structural information obtained allowed for periodic and dimer single point computational studies at the B3LYP/6-31G** level of theory. These confirmed the proposed controlled crystallization mechanism and the stabilization of crystals formed by the iodoacetyl derivative and parent amphotericin B. The calculation results indicate the strength of different intermolecular interactions and reveal the great contribution of the solvent molecules to the crystal lattice formation, with the total energy gain of about 335 kJ·mol−1, which almost doubles the cohesive energy value. Hirshfeld surface analysis shows the more efficient crystal packing of N-iodoacetylamphotericin versus amphotericin B and the effect of the iodoacetyl group on the intermolecular contacts. The generated electrostatic potential maps reveal the impact of the iodoacetyl substituent on the nitrogen atom basicity and thus confirm the stronger hydrogen bonding created via nitrogen atom in the case of N-iodoacetyl amphotericin B, and higher drug activity of amphotericin B related to the ability of the zwitterion formation.

1. INTRODUCTION In the last few decades, an alarming rise in life-threatening fungal infections due to the appearance of opportunistic pathogens has intensified the development of new drugs and new formulations of the old antifungal agents.1 Amphotericin B (hereafter abbreviated AmB; Scheme 1) constitutes a macrocyclic lactone,2 which gained medical and scientific attention because of its broad range of activity, especially against most pathogenic fungi.3 Although this macrolide polyene (also

referred to as macrolide poliketide) antibiotic was isolated over half a century ago4 from the Streptomyces nodosus culture,5 it still remains the golden standard in the clinical treatment of serious systemic mycoses, despite its nephrotoxicity and several adverse effects.6 Because of the lack of better alternatives, AmB is the drug of choice in the case of invasive fungal infections, especially frequent in immune-compromised individuals.7 This is of particular importance in the therapy of such diseases as AIDS and cancer, or in the case of patients with transplanted organs.8 Up to now, many experimental and theoretical studies,6b including biosynthesis, spectroscopy,9 and molecular dynamics, were carried out to understand the mechanism of the AmB action and reduce its side effects. The most popular concept regarding the influence of AmB on biomembranes is directly associated with the formation of ion-permeable pores across the lipid bilayers.10 AmB moieties aggregate in such a way that they form a barrel with their polyhydroxy chain fragment pointing inward, while the heptane part pointing outward.9e,10,11 Such a

Scheme 1. The N-Iodoacetylamphotericin B Moleculea

Received: December 30, 2011 Revised: March 7, 2012 Published: March 22, 2012

a

The attached iodoacetate group (red) is bonded to mycosamine fragment through amine group. © 2012 American Chemical Society

2336

dx.doi.org/10.1021/cg2017227 | Cryst. Growth Des. 2012, 12, 2336−2345

Crystal Growth & Design

Article

the growth of high quality crystals, as it stabilizes the proper crystal structure. Pure THF crystallization environment leads to the formation of aggregates and crystals of poor quality. THF was slowly evaporated from the crystallization solution next to the saturation edge, by N2 stream when stirring. Then the solution was centrifuged at 15000g for 3 min prior to crystallization. For the purpose of a single crystal growth induction, AmB-I seeds obtained by spontaneous nucleation were inserted into the saturated solution. Supersaturation was achieved by slow evaporation. In this method, the concentration is increased by removing the solvent molecules at constant temperature. The seed was placed at the bottom of 2 mL vial with an evaporation rate of 0.05− 0.15 mL per day at 22 °C. In order to avoid the side effects of AmB-I degradation due to its instability, the growth experiments were conducted for no longer than 1 month in a dark room. All solvents used were purchased from Sigma Co. 2.2. Computational Details. The size of the studied systems shortens the possibility of applying very sophisticated quantum chemical methods for the purpose of deriving molecular properties. A single molecule of AmB-I consists of 143 atoms, which is at the edge of rational level for density functional approach. All energy computations were performed within the CRYSTAL0920 program package at the B3LYP21 level of theory. 6-31G**(6-311G** for iodine atom) molecular all-electron basis22 set constituted a reasonable compromise between the accuracy and computational cost. Both Grimme dispersion correction and correction for basis set superposition error were applied.20,23 Ghost atoms were selected up to 5 Å distance from the studied molecule in a crystal lattice and were used for the basis set superposition error estimation. The evaluation of Coulomb and exchange series was controlled by five thresholds, set arbitrary to values of 10−6, 10−6, 10−6, 10−6, 10−14. The condition for the SCF convergence was set to 10−7 on the energy difference between two subsequent cycles. Shrinking factor was equal to 4, which refers to 30 k-points in the irreducible Brillouin zone and should ensure the full convergence of the total energy. AmB-I molecular geometry was taken directly from our X-ray structural analysis, while all H−X bonds were fixed at standard neutron distances.24 These coordinates were also utilized to model the AmB parent molecule; however, in that case the iodoacetyl group was changed to hydrogen atom (at a proper N−H distance and angle). Cohesive energy was estimated for AmB-I crystal, AmB-I crystal with no solvent molecules included, and for AmB hypothetical crystal created on the basis of its iodoacetyl derivative, also with no solvent involved. The cohesive energy (Ecoh) was calculated as follows:23a

molecular arrangement changes the membrane potentials and increases ion permeability, thus causing the cell’s death.10,12 Structural determination, total synthesis, and reactivity analysis of AmB became an objective immediately after its isolation. Difficulties associated with crystallization and, consequently, limited access to structural information, implied increased efforts toward the chemical modifications of the parent compound in the context of structure−activity studies. AmB was first fully synthesized by Nicolaou et al.13 and its absolute stereochemical configuration was derived almost 40 years ago from the N-iodoacetyl derivative in X-ray singlecrystal diffraction experiment by Ganis et al.14 1H NMR stereochemical data obtained for AmB derivatives by Sowiński et al.15 revealed the identity of the drug conformation in the solution (mixture of pyridine and methanol) with that one found in the crystal of its studied modification. Commercial formulations of AmB contain a multiple polyene component, especially rich in amphotericin A, crystal growth impurity. Molecular structure of amphotericin A is identical to that of amphotericin B except for the presence of one single bond in the polyene carbon chain. As a consequence, regarding the purification difficulties16 and chemical instability,4c the parent compound has not been successfully crystallized for a single crystal X-ray diffraction yet. Furthermore, crystals of pure AmB, free of any modifications, are very sensitive to any kind of contamination and grow as spherulites with a size of about 1− 10 μm (Figure S.1. in the Supporting Information). However, certain alterations applied to the parent molecule may increase binding properties, and thus induce a better crystal growth. N-Iodoacetylamphotericin B (AmB-I), being a biologically active heavy atom derivative of AmB, has already been a matter of scientific interest.14 The particularity of this compound is its capability to form good quality crystals, when grown from a solution of tetrahydrofuran (THF) and water. In this contribution, we present a crystal growth method of AmB-I and crystal shape control with the use of a “tailor-made” additive. Our crystallization technique resulted in good quality single crystals which were then studied by X-ray diffraction methods. Hence, we report the details of the obtained threedimensional (3D) AmB-I structure, which opens up a discussion on several related topics. The present study is devoted to the extended analysis of AmB-I crystal lattice features and intermolecular interactions in the context of crystal growth mechanism and its comparison with the parent AmB. Good quality structural data allow us to perform a series of computational studies of intermolecular interactions of AmB-I and also AmB. We expect that the results would support a better stability of AmB-I crystals in comparison with AmB and indicate the main interactions and mechanism responsible for such a behavior.

Ecoh =

1 E bulk − Emol Z

where Ebulk is the total energy of a system (calculated per unit cell) and Emol is the energy of a molecule extracted from the bulk. Z stands for the number of molecules in the unit cell. This formula was slightly modified for the purpose of AmB-I crystal cohesive energy calculation, so as to include solvent moieties. Additionally, the same geometries were employed to estimate the interaction energy of selected dimers extracted from the crystal lattice. The computations were performed in the supermolecular approach within the CRYSTAL09 package, taking into account both BSSE and dispersive corrections (Grimme dispersion correction included in the CRYSTAL09 code). Interaction energy was estimated for the chosen AmB-I···AmB-I, AmB···AmB dimers, and also for the most significant AmB···solvent contacts.

2. METHODS AmB-I synthesis17 and X-ray data collection18 (at 100 K) were carried out following standard procedures. Therefore, all the experimental details are described in the Supporting Information (see paragraphs 1.1 and 1.2). The theory concerning Hirshfeld surfaces19 is outlined in the Supporting Information (paragraph 1.3) together with molecular electrostatic potential determination (paragraph 1.4). Crystallization, crystal growth kinetics, and also selected computational details, particularly important for the understanding of the subsequently presented results, are discussed below. 2.1. Crystallization and Growth Kinetics (Seed Growth). All crystals were grown from tetrahydrofuran (THF)/water solution with the ratio of 100 to 1, respectively. A small water fraction is essential for

3. RESULTS AND DISCUSSION 3.1. Controlled Crystallization. AmB-I has been successfully crystallized from tetrahydrofuran solution with a small water fraction essential for crystal formation. Our studies revealed a significant influence of AmB additives on AmB-I crystal growth and shape. The crystallization process strongly depends on the concentration of AmB in AmB-I solution, as some of crystal growth directions become inhibited, while 2337



Article

Figure 1. Schematic illustration of the growth inhibition mechanism of N-iodoacetylamphotericin B crystals. The round shape of crystals is caused most likely by AmB kink-poisoning effect.26

others favored. “Tailor-made” impurities very often resemble host molecules. The only difference might be, for instance, addition, or lack, of a certain functional group. This is a matter of a well-known example of alanine blocking the (010) face growth of glycine crystals.25 In the case of AmB-I crystals, a similar effect is achieved by the absence of the iodoacetyl substituent in the structure of added AmB molecules. The mechanism of AmB moieties inhibiting the growth of AmB-I crystals is schematically illustrated in Figure 1. The vast part of AmB molecule is identical to its iodoacetyl derivative, which enables AmB to be easily attached to the AmB-I crystal faces, especially when the iodoacetyl group does not participate in this binding. Consequently, AmB species freely bind to AmB-I crystal in the [010] direction. However, while AmB is already built in, the absence of the external iodoacetyl substituent hinders further attachment of the incoming AmB-I molecules, thus restraining crystal growth in this direction. A different process is observed in the [01̅0] direction. Because of the absence of the iodoacetyl fragment in AmB molecules approaching the AmB-I surface, the bond between AmB impurity and host AmB-I molecules is weaker. Therefore, even if an AmB molecule is already bonded to the surface, it can be relatively easily replaced by an incoming AmB-I species. As a consequence, the adsorption of AmB is limited and so the crystal can grow freely in this direction. Figure 2 shows all observed habit changes, from the needle-like to heart-like crystal morphologies, formed at different AmB concentrations. The supersaturation shown in the morphodrome was calculated as described in the literature.27 For high AmB-I supersaturation and small AmB concentration, the growth in the [010] direction is limited. High supersaturation and moderate concentration of AmB (1−2%) lead not only to the growth inhibition, but also to facet roughening in the [010] direction (see Figure 2). Around point 2 in Figure 2, surfaces (−1, −1, L) and (−1, −1, −L) of the observed crystals are curved in the

Figure 2. Morphodrome showing the changes of AmB-I crystal habit. (Δμ/kT) stands for supersaturation, k denotes the Boltzmann constant, and T stands for the growth temperature. Crystals of AmB-I were grown from THF−water solution with an addition of AmB as a “tailor-made” additive. The concentration of AmB is expressed in weight percentage of AmB-I. Structural data were obtained from crystals (2). Roughening of AmB-I crystals in the [010] direction is caused by AmB at high supersaturation (3).

[010̅ ] direction. This is associated with the non-integer L indexes in the symbol of the surfaces. A similar phenomenon occurs in the opposite [010] direction, though the effect is less pronounced. This morphological instability might result from kinetic roughening of (−1, −1, L) and (−1, −1, −L) facets.27 By decreasing the supersaturation below the kinetic roughening level, we should obtain faceted crystals again. In this case, however, the curvedness of facets in the [01̅0] direction is still 2338



Article

Figure 3. Atom labeling and ORTEP representation of atomic displacement parameters (ADPs) at 50% probability level for Niodoacetylamphotericin B (AmB-I). Solvent molecules, shown in a ball-and-stick representation, located as in the crystallographic structure of AmB-I.

Figure 4. Packing of molecules in the crystal lattice − projection along: (a) the Y-axis, (b) the Z-axis, (c) the X-axis, (d) perspective view of a molecular channel across the crystal lattice in the [010] direction. Water oxygen atoms are in red, THF molecules in green, and the hydrogen atoms are omitted for clarity. In the case of (c) all solvent molecules are skipped.

shaped crystals are observed, but rather AmB-I aggregates. Successful crystallization provided single crystals suitable for Xray structural investigations, and reliable structural data opened new computational opportunities. 3.2. Crystal and Molecular Structure of AmB-I. AmB-I crystallizes in the polar monoclinic P21 space group with one molecule of AmB-I, three molecules of THF, and one water

visible at low AmB-I supersaturation, when the crystallization solution contains some AmB additive. It suggests that the effect is caused by the presence of AmB moieties. At high concentrations of AmB all crystal corners and edges are rounded (point 3, Figure 2). Both of the described phenomena might be related to the kink-poisoning mechanism.26 When the concentration of AmB (impurity) exceeds 6%, no regular 2339



Article

Table 1. Details of H-Bonding in the Crystal Lattice of AmB-I D−H···A

symm

O3−H3O···O2 O4−H4O···O3 O7−H7O···O6 O8−H8O···O7 O3−H3O···O1C O14−H14O···O1W O1W−H2W···O8 O1W−H1W···O5 O16−H16O···O17 O12−H12O···O16 O5−H5O···O10 O18−H18O···O15 O10−H10O···O1A N1−H1N···O11 O6−H6O···O10

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

d(D−H)/Å 0.84 0.84 0.84 0.84 0.84 0.84 0.81(4) 0.82(2) 0.84 0.84 0.84 0.84 0.84 0.88 0.84

d(H···A)/Å 2.22 1.850(4) 1.94 2.01 2.34 1.84 2.04(5) 2.01(3) 1.91 1.91 2.09 2.17 1.80 2.05 2.060(3)

d(D···A)/Å 2.841(5) 2.702(5) 2.694(4) 2.673(4) 2.973(10) 2.655(5) 2.815(5) 2.802(5) 2.678(5) 2.690(4) 2.911(5) 2.976(5) 2.632(4) 2.870(6) 2.912(4)

∠DHA/° 130.5 179.2(4) 148.1 135.4 132.2 163.5 158(7) 162(8) 152.2 154.8 165.6 160.4 169.9 155.4 179.8(2)

3.3. Packing and Intermolecular Interactions. The 3D packing of AmB-I molecules and the most significant weak interactions in the crystal lattice are illustrated in Figure 4. AmB-I species form a layered architecture in the crystal lattice, parallel to the (001) crystal planes. Within the layer, molecules interact via polyene chains and the so-called, poliketide subunits. Interlayer interactions are based on polar “headgroup” fragments of molecules, pointing directly to each other, being complementarily tangled together. Additionally, AmB-I species create molecular planes perpendicular to the (100) crystallographic plane. The distance between the closest corresponding motifs equals ca. 4.0 Å. In each layer, the hydroxyl groups from AmB-I are directed toward the carbon chains of the neighboring upper layer (Figure 4c). An interesting crystal architecture feature is also seen in the structure projection along the Y-axis (Figure 4a). Here, the mycosamine groups of AmB-I moieties are mutually arranged in space in such a way that they form molecular channels across the crystal lattice in the [010] direction (Figure 4d). These channels are tightly filled with the THF molecules contributing significantly to the crystal lattice stability. One can see in Figure 4a that all THF molecules are arranged in a T-shape manner, which results from the nature of their mutual interactions. A great variety of different hydrogen bonds present in the AmB-I crystal structure undoubtedly constitutes one of the most relevant factors stimulating and stabilizing the crystal lattice formation (see Table 1). One AmB-I molecule is involved in hydrogen bonding with eight AmB-I, two THF, and two water molecules. The majority of hydrogen bonds between two different AmB-I moieties is formed within polar “headgroup” mediation (e.g., O16−H16O···O17, O12−H12O···O16, N1−H1N···O11, O18−H18O···O15). Nevertheless, the hydroxyl groups from the polyol subunit also reveal the ability to form hydrogen bonds. Four hydrogen atoms from those hydroxyl groups participate in strong intramolecular hydrogen bond contacts and, therefore, cannot be involved in intermolecular hydrogen bonding (O3− H3O···O2, O4−H4O···O3, O7−H7O···O6, O8−H8O···O7), whereas, O5−H5O and O6−H6O form intermolecular two centered hydrogen bonds with the O10 oxygen atom. Despite hydrogen bonds formed between AmB-I molecules described above, the crystal structure is stabilized via hydrogen bonds with solvent molecules. The water molecule is particularly important in the crystal structure stabilization as it is involved

molecule in the asymmetric part of the unit cell. The unit cell has two almost equal dimensions (i.e., a and c) close to 20 Å, and the third one significantly shorter (b = ca. 8.6 Å). The AmB-I molecule (see Scheme 1 and Figure 3) consists of three basic subunits, including polyene and polyol fragments and the top headgroup. The polyene part together with the socalled poliketide chain constitute a 38-component macrolactone ring. Within this macrocycle, a smaller six-member ketal ring can be distinguished. It is formed by the carbon atoms starting from C13 up to C17 linked together via the O9 oxygen atom (Figure 3). Two hydroxyl groups are also attached at the C13 and C15 carbon atom positions. The D-mycosamine sugar moiety is β-glycosidically bonded to the macrolide ring at the C19 hydroxyl group. In general, the conformation and symmetry of the AmB-I molecule and its crystal structure, presented in our study, is the same as the one found in the pioneering work of Ganis et al. in 1971.14 However, more detailed comparison of both structures shows that bond lengths and valence angles between nonhydrogen atoms differ significantly. This is due to different data collection temperatures, that is, the current experiment was carried out at 100 K, whereas the prior measurement by Ganis et al. was performed at room temperature. The relative quality of both measurements is also substantially different. In the present study, apart from heavy atom position and atomic displacement parameter (ADP) refinement (Figure 3), it was possible to derive approximate positions of the hydrogen atoms. Hydrogen atom arrangement is crucial for intermolecular interaction elucidation. Our X-ray structural analysis revealed high rigidity of the chromophore region (i.e., polyene subunit) of AmB-I species, regarding the small ADP values observed for this molecular fragment. On the contrary, the mycosamine moiety and THF solvent molecules exhibit considerably larger ADP values. The ADPs of the oxygen atoms (O2, O3, O4) involved in the intra- and intermolecular hydrogen bonds are elongated perpendicularly to the bond directions and to the plane of macrolactone ring. This plane consists of seven conjugated double bonds in all-trans conformation, and a more flexible hydrophilic subunit containing seven hydroxyl groups.6a,28 Thus, AmB-I, and further AmB, can be regarded as semirigid molecules consisting of two well-distinguished fragments.6a,29 The full geometry of the AmB-I molecule, including atomic coordinates, can be retrieved from the cif file in the Supporting Information. 2340



Article

AmB (the same crystal lattice and packing as for AmB-I). For the purpose of this study, we also assumed similar packing of the solvent molecules. It is, however, quite probable that some additional solvent molecules could be located in the voids present in the AmB structure due to the absence of a bulky iodoacetyl substituent. As it is not straightforward how these supplementary water or THF molecules are positioned, we examined the analogous AmB-I and AmB crystal lattices. Differences between AmB-I and imaginary AmB 3D crystal networks, especially between hydrogen bonding patterns, can be visualized by Hirshfeld surface evaluation.19a,31 Comparison of dnorm mapped on Hirshfeld surfaces for AmB-I and imaginary AmB leads to a few interesting conclusions (Figure 7). First of all, both Hirshfeld surfaces look exactly the same in the area of the polyene and polyol subunits, except for the region between the O5 and C10 atoms which are in the vicinity of the iodine atom from the neighboring molecule. As expected, the main differences are observed for the “headgroup” fragment. Secondly, the presence of the iodoacetyl group creates a number of intermolecular hydrogen bonds, which are not present in the case of modeled AmB (see region 1 in Figure 7a,b). The closest intermolecular interactions from the iodoacetyl group to the neighboring molecules in the crystal lattice of AmB-I are illustrated in Figure 7c (for the details of geometry of these interactions see Table 1S in the Supporting Materials). The absence of iodoacetyl group in AmB leads to its much weaker bonding to the surface in the [01̅0] direction as compared to the [010] one. Further analysis of dnorm on HS implies that in the predicted AmB network, the N−H···O hydrogen bond with the carboxyl O11 atom from another AmB molecule (x, 1 + y, z) still exists (see region 2 in Figure 7). Obviously, for the imaginary AmB, contrary to AmB-I, the O16−H16O···O17 hydrogen bond, which involves O17 from the iodoacetyl group, is not present. This causes the discrepancies in dnorm mapped on HS, in region 3 (Figure 7a,b). Furthermore, modern HS analysis allows for quantification of intermolecular interactions by calculating the percentage contribution of particular interaction types on HS. It turns out that for the hypothetical AmB network the percentage of H···H interactions is slightly higher (70% vs 64%, respectively), whereas for O···H contacts slightly lower (22.6% vs 24.3%) than for AmB-I. These small differences in the percentage of the H···H and H···O interactions are likely to result from the extra atoms present in the AmB-I molecule compared to AmB.

in three different hydrogen bonds (O14−H14O···O1W, O1W−H2W···O8, O1W−H1W···O5). The significance of these water-mediated intermolecular contacts explains the fact that AmB-I does not crystallize from anhydrous solvents. Our studies show that two of the THF moieties form hydrogen bonds with AmB-I (O10−H10O···O1A, O3−H3O···O1C), whereas Ganis et al. suggested that only one of the THF molecules is involved in such a bond formation (see Figure 5).

Figure 5. Hydrogen bonds present in AmB-I crystal lattice.

3.4. Structure of AmB-I vs Assumed Structure of AmB. As it was already mentioned, AmB is much less willing to form single crystal suitable for X-ray diffraction experiments than its iodoacetyl derivative to form single crystals suitable for X-ray diffraction experiments. On the other hand, good quality structural data constitute a good starting point for further analysis. It is especially essential for the purpose of quantum chemical calculations and molecular dynamics.30 Fortunately, the major part of molecules, that is, the polyene and polyole subunits, and so, the great percentage of the head fragment, are preserved between AmB and AmB-I. Moreover, powder diffraction indicated very similar elementary cell dimensions in both cases (Figure 6). Therefore, one may assume that the AmB and AmB-I crystal lattices are very much alike. Regarding the vast similarities of the studied systems, the difference in the crystal stability and formation must come from the presence of the iodoacetyl substituent in the AmB-I moiety. So, in order to understand better the impact of iodoacetyl group in the AmB-I molecule on crystal formation, we decided to compare the 3D crystal network of AmB-I with the crystal network of a hypothetical

Figure 6. AmB powder diffractogram (Sigma Co. 80%, data collected on Bruker D8 Discovery diffractometer) − blue line. The red line stands for the AmB-I spectrum simulated using single crystal unit cell data. AmB crystallizes in the P21 space group with the unit cell dimensions of a = 21.203 Å, b = 8.509 Å, c = 19.042 Å, and β = 103.97°. 2341



Article

Figure 7. (a) dnorm mapped on Hirshfeld surface for AmB-I; (b) and for imaginary AmB, − donors and acceptors of hydrogen bonds − red color,32 (c) the closest intermolecular contacts between the N-iodoacetate moiety and the neighboring molecules in the crystal lattice.

Figure 8. Electrostatic potential mapped on the electron density isosurface of (a) AmB and (b) AmB-I, contoured at a ρ(r) value of 0.1 e bohr−3 (≈0.67 e Å−3); single point B3LYP/6-31G** (6-311G** for iodine atom) calculation.36

Information (paragraph 1.4). Crystallographic geometry and conformation of N-iodoacetylamphotericin B derivative was used as a prototype of AmB. The AmB and AmB-I electrostatic potential maps derived at the B3LYP/6-31G** (6-311G** in the case of iodine atom) level of theory are alike (Figure 8). The only discrepancy appears in the N-fragment. The iodoacetyl substituent introduced into AmB moiety influences

Although they are relatively small, they may have significant biological consequences. 3.5. Electrostatic Potential. To investigate and compare the chemical character and nature of potential interactions concerning AmB and AmB-I molecules, electrostatic potential was modeled in three different ways.33 The calculation details and all the results are available from the Supporting 2342



Article

and A6) are both about two times stronger than the corresponding ones in the N5 and N6 AmB dimers. In the case of the A5 dimer, this is caused by the presence of a carbonyl group in the AmB-I moiety which is involved in an additional hydrogen bond, as compared to AmB dimer. In turn, A6 is more strongly stabilized than N6 as a consequence of the more acidic character of the nitrogen atom in the AmB-I molecule. This supports the formation of stronger hydrogen bond contact. The N8 intermolecular interaction is also substantially weaker than in the case of A8, which can be explained by the bulky iodoacetyl substituent and the positive potential of the iodine atom in contact with the polyol chain. The results obtained for A7 and N7 dimers are comparable. The interatomic contacts involve the preserved molecular fragments and so there is not much interaction energy difference. This motivates the predicted AmB-I crystallization mechanism in the presence of AmB additives. If in the crystallization direction macrolide polyene contact plays a crucial role, both AmB and AmB-I are bonded with similar strength and, thus, probability. When the headgroup contact is more emphasized, the binding becomes significantly weaker in the case of AmB molecules. Therefore, in one direction AmB does not affect the crystallization process, while in the opposite direction it may hamper further stable attachment of the incoming molecules. Computational results also show the importance of the solvent molecules for the crystal lattice formation. The most significant AmB-I-solvent contacts are grouped in Table 3 and visualized in Figure 9. The presence of the water molecule is particularly essential, as it is involved in three hydrogen bonds of the total stabilization energy equal to almost −65 kJ·mol−1. This is the reason for the indispensability of the water fraction in the crystallization process. The total energy difference between the nonsolvent and full experimental structure of AmB-I amounts to 335 kJ·mol−1. Moreover, interaction energy computations gave the final answer on the O5−H5 geometry. During the structure refinement, there were two almost equally probable hydrogen atom positions. Although it was not clear from the charge density differential maps which solution is right, the stabilization energy difference for the A2 dimer equal to 30 kJ·mol−1 implied the chosen H5 position. The energetic gain due to the selected hydrogen atom location was also significant in the case of A8, and corresponding N8, dimer. On the whole, the obtained crystal structure of AmB-I was over 60 kJ·mol−1 more stable than its analogue structure with the other O5− H5O geometry. Such result exhibits a great importance of the hydrogen atom positions for further analysis and concluding.

the electrostatic potential of the N atom, shifting its potential toward more positive values. A higher basicity of the nitrogen atom in AmB compared AmB-I is also confirmed by Mulliken atom charges.34 The zwitterion AmB form, with the protonated N-group and negatively charged carboxyl group, was found to be crucial for its biological activity.9a,b,30,35 The electrostatic potential values suggest that the N+ group would be more favorably created in the case of AmB, indicating its better properties as a drug. There is, of course, a significant steric difference between the two compounds in the headgroup region, and subsequently different charge distributions occur. This may also affect the overall packing preferences, leading to more stable crystals in the case of AmB-I. Nucleophilic carbonyl group opens up a possibility to form additional hydrogen bonds as described in the former paragraph. In turn, more acidic nitrogen atoms more easily share attached hydrogen atoms in hydrogen bonds, therefore increasing their strength. In all the other derived electrostatic potential models main qualitative features are preserved (Figure S.4. in the Supporting Information). However, the UBDB databank33b,37 is not accurate and flexible enough to properly describe iodine atom and the polyene fragment consisting of easily polarizable double bond carbon chain. The specificity of the polyene part is worth stressing as within this molecular fragment the relative electrostatic potential might be modified so as to better adapt the whole molecule to its surroundings. 3.6. Computational Studies. A number of computations were carried out to confirm the predicted crystallization mechanism and the importance of solvent molecules for crystal stabilization, and to compare AmB and AmB-I binding properties. Therefore, cohesive energy was calculated for AmB-I experimentally derived crystal structure, and also for AmB-I and AmB crystal packing with no solvent. Additionally, selected dimer interaction energies were estimated in order to verify the relative interaction strength of the crucial intermolecular contacts. As in the previously described analysis, it was assumed that AmB and AmB-I geometries and crystal packing are the same. Computational results clearly show that the AmB-I crystal structure is better stabilized than the structure of parent AmB, providing the primary assumption, concerning the similarity of the two molecular geometries and crystal lattices, was correct (see Tables 2 and 3). The cohesive energy23a calculated per one Table 2. Cohesive Energy Values Computed for the Analyzed Systems chemical system

cohesive energy per asymmetric unit/ kJ·mol−1

AmB with no solvent AmB-I with no solvent AmB-I experimental structure

−325 −415 −750

4. CONCLUSIONS In this contribution, we have presented a new efficient way of obtaining N-iodoacetylamphotericin B crystals and controlling their growth and shape. The observed curve-facet growth at different supersaturations is most probably related to a kinkpoisoning mechanism of AmB. Additionally, our study provides a much better evaluated crystal structure of N-iodoacetylamphotericin, when compared to the previous investigations. It was possible to refine anisotropic temperature factors (for non H-atoms) and hydrogen atom positions. In consequence, we have found a new hydrogen bonding of the AmB-I with THF solvent molecule. Our calculations have allowed for correcting the position of the H5O atom in hydrogen bonding. Furthermore, the computational results confirm the predicted

molecule is 90 kJ·mol−1 lower in the case of the iodoacetyl derivative. When all solvent molecules are neglected, the whole difference in the cohesive energy values comes from the nature of the terminal amino group. The total energy mismatch is equal to about three medium-strength hydrogen bonds. Careful examination of crystal structures shows that there are two main dimers interacting via head fragments, which is reflected in the biggest energy discrepancies in the dimer interaction energy table (Table 3). For AmB-I these two interactions (dimers A5 2343



Article

Table 3. Interaction Energy Values Computed for the Selected Dimersa

AmB-I-solvent dimer

interaction energy/kJ·mol−1

AmB-I−AmB-I dimer


AmB−AmB dimer


A1 A2 A3 A4

−54 −10 −19 −37

A5 A6 A7 A8

−90 −39 −154 −81

N5 N6 N7 N8

−44 −20 −152 −74

a

Dimers A1−A4 are defined in Figure 9. The other AmB-I dimers (also corresponding AmB dimers) are defined in the figure above this table.

note on Hirshfeld surfaces, and computation details including electrostatic potential evaluation. This information is available free of charge via the Internet at http://pubs.acs.org/. The CCDC 866798 entry contains the supplementary crystallographic data. These data can be obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected] (K.W.); mariusz.gagos@ up.lublin.pl (M.G.).

Figure 9. The most important iodoacetylamphotericin B interactions with solvent molecules A1(O14−H14O···O1W; O1W−H2W···O8), A2(O1W−H1W···O5), A3(O10−H10O···O1A), and A4(O3− H3O···O1C).

Author Contributions #

These authors have contributed equally.

Notes

The authors declare no competing financial interest.

■

crystallization mechanism and indicate the crucial interactions for the crystal lattice formation. It appears that there is a very significant contribution of the interactions with the solvent molecules to the crystal stability − the cohesive energy gain, relative to the nonsolvent case, amounts to ca. 335 kJ·mol−1. The presence of water is particularly important for crystal lattice formation and stabilization, as a single water molecule is involved in three hydrogen bonds of the total stabilization energy equal to about −65 kJ·mol−1. This explains the water fraction essentiality for the growth of appropriate quality single crystals. Computational studies have also revealed a better stability of AmB-I crystals in comparison with the parent AmB.

■

ACKNOWLEDGMENTS The authors would like to thank Dr. Bartolomeo Civalleri for advice concerning CRYSTAL computations. This research was financed by the Ministry of Education and Science of Poland from the budget funds for science in the years 2008−2011 within the research project NN401 015035. M.M. acknowledges the financial support within the Polish National Science Centre (NCN) Project Number UMO-2011/01/N/ST4/ 03616. The authors gratefully acknowledge the Wrocław Networking and Supercomputing Centre for providing computer facilities.

■

ASSOCIATED CONTENT

S Supporting Information *

REFERENCES

(1) (a) Omura, S. Macrolide Antibiotics, Chemistry, Biology and Practice, 2nd ed.; Omura, S., Ed.; Academic Press Inc.: New York, 2002; p 623. (b) Omura, S.; Tanaka, H. Production, structure and

Supporting materials contain experimental details regarding synthesis of AmB-I, single crystal X-ray diffraction studies, a 2344



Article

antifungal activity of polyene macrolides. In Macrolide Antibiotics; Omura, S, Ed.; Academic Press, Inc.: Orlando, 1984; pp 351−404; (c) Hay, R. J. Recent Advances in the Chemistry of Antifungal Agents; Bentley, P. H.; Ponsdorf, R., Eds.; Royal Society of Chemistry: Cambridge, U.K., 1993; pp 163−181. (2) Woodward, R. B. Angew. Chem., Int. Ed. 1957, 69, 50−58. (3) Ellis, D. J. Antimicrob. Chemother. 2002, 49, 7−10. (4) (a) Dutcher, J. D.; Gold, W.; Pagano, J. F.; Vandepatte, J. Amphotericin B, its production and its salts. Patent U.S. 2,908,611, 1959; (b) Gold, W.; Stout, H. A.; Pagano, J. F.; i Donovick, R. Antibiot. Ann. 1956, 579−585. (c) Lemke, A.; Kiderlen, A. F.; Kayser, O. Appl. Microbiol. Biotechnol. 2005, 68, 151−62. (d) Sternberg, T. M.; Wright, E. T.; Oura, M. Antibiot. Ann. 1956, 3, 566−573. (5) (a) Trejo, W. H.; Bennett, R. E. J. Bacteriol. 1963, 85, 436−439. (b) Challis, G. L.; Hopwood, D. A. Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 14555−14561. (6) (a) Hartsel, S.; Bolard, J. Trends Pharmacol. Sci. 1996, 17, 445−9. (b) Cereghetti, D. M.; Carreira, E. M. Synthesis-Stuttgart 2006, 6, 914− 942. (c) Ng, A. W. K.; Wasan, K. M.; Lopez-Berestein, G. J. Pharm. Pharm. Sci. 2003, 6, 67−83. (7) Gold, W.; Stout, H. A.; Pagano, J. F.; i Donovick, R. Antibiot. Ann. 1956, 579−585. (8) Gallis, H. A.; Drew, R. H.; Pickard, W. W. Amphotericin Rev. Infect. Dis. 1990, 12, 308−329. (9) (a) Gagoś, M.; Arczewska, M. Biochim. Biophys. Acta 2010, 1798 (11), 2124−30; 2010, 1798, 2124−2130. (b) Gagoś, M.; Arczewska, M. J. Phys. Chem. B 2011, 115, 3185−3192. (c) Gagoś, M.; Arczewska, M.; Gruszecki, W. I. J. Phys. Chem. B 2011, 115, 5032−5036. (d) Gagoś, M.; Hereć, M.; Arczewska, M.; Czernel, G.; Dalla Serra, M.; Gruszecki, W. I. Biophys. Chem. 2008, 136, 44−49. (e) Gagoś, M.; Koper, R.; Gruszecki, W. I. Biochim. Biophys. Acta 2001, 1511, 90−98. (10) De Kruijff, B.; Gerritsen, W. J.; Oerlemans, A.; Demel, R. A.; van Deenen, L. L. Biochim. Biophys. Acta 1974, 339, 30−43. (11) Andreoli, T. E. Ann. N. Y. Acad. Sci. 1974, 235, 448−468. (12) (a) Czub, J.; Borowski, E.; Baginski, M. Biochim. Biophys. ActaBiomembr. 2007, 1768, 2616−2626. (b) Cohen, B. E. Biochim. Biophys. Acta 1986, 857, 117−122. (c) Cohen, B. E. Biochim. Biophys. Acta 1992, 1108, 49−58. (13) Nicolaou, K. C.; Daines, R. A.; Chakraborty, T. K.; Ogawa, Y. J. Am. Chem. Soc. 1987, 109, 2821−2822. (14) Ganis, P.; Avitabile, G.; Mechlinski, W.; Schaffner, C. P. J. Am. Chem. Soc. 1971, 93, 4560−4564. (15) Sowinski, P.; Pawlak, J.; Borowski, E.; i Gariboldi, P. Magn. Reson. Chem. 1992, 30, 275−279. (16) Petersen, N. O.; Henshaw, P. F. Can. J. Chem.-Rev. Can. Chim. 1981, 59, 3376−3378. (17) Wright, J. J. Derivatives of polyene macrolide antibiotics containing an amino sugar moiety, process for the preparation thereof, and pharmaceutical compositions containing them. Patent U.S. 4272525, 1980. (18) (a) Sheldrick, G. M. Acta Crystallogr. 2008, A64, 112−122. (b) APEX2, 2010.3-0; Bruker AXS Inc.: Madison, WI, USA: 2010; (c) SAINT, 7.68A; Bruker AXS Inc.: Madison, WI, USA, 2010. (19) (a) Hirshfeld, F. L. Theor. Chim. Acta 1977, 44, 129−138. (b) Spackman, M. A.; Jayatilaka, D. CrystEngComm 2009, 11, 19−32. (20) Dovesi, R.; Saunders, V. R.; Roetti, R.; Orlando, R.; ZicovichWilson, C. M.; Pascale, F.; Civalleri, B.; Doll, K.; Harrison, N. M.; Bush, I. J.; D’Arco, P.; Llunell, M. CRYSTAL09 (CRYSTAL09 User’s Manual); University of Torino: Torino, 2009. (21) (a) Becke, A. D. Phys. Rev. A 1988, 38, 3098−3100. (b) Lee, C.; Yang, W.; Parr, R. G. Phys. Rev. B 1988, 37, 785−789. (c) Perdew, J. P. Phys. Rev. B 1986, 33, 8822−8824. (22) Krishnan, R.; Binkley, J. S.; Seeger, R.; Pople, J. A. J. Chem. Phys. 1980, 72, 650−654. (23) (a) Civalleri, B.; Zicovich-Wilson, C. M.; Valenzano, L.; Ugliengo, P. CrystEngComm 2008, 10, 405−410. (b) Grimme, S. J. Comput. Chem. 2006, 27, 1787−1799. (24) Allen, F. H.; Bruno, I. J. Acta Crystallogr. 2010, B66, 380−386.

(25) Weissbuch, I.; Addadi, L.; Berkovitchyellin, Z.; Gati, E.; Weinstein, S.; Lahav, M.; Leiserowitz, L. J. Am. Chem. Soc. 1983, 105, 6615−6621. (26) Punin, Y. O.; Franke, V. D. Crystallogr. Rep. 2004, 49, 256−260. (27) Hollander, F. F. A.; Kaminski, D.; Duret, D.; van Enckevort, W. J. P.; Meekes, H.; Bennema, P. Food Res. Int. 2002, 35, 909−918. (28) Baginski, M.; Gariboldi, P.; Bruni, P.; Borowski, E. Biophys. Chem. 1997, 65, 91−100. (29) (a) Mazerski, J.; Borowski, E. Biophys. Chem. 1995, 54, 49−60. (b) Sowinski, P.; Pawlak, J.; Borowski, E.; Gariboldi, P. Magn. Reson. Chem. 1992, 30, 275−279. (30) Baginski, M.; Resat, H.; Borowski, E. Biophys. J. 1999, 76, A244−A244. (31) (a) Hirshfeld, F. L. Acta Crystallogr. 1976, A32, 239−244. (b) McKinnon, J. J.; Mitchell, A. S.; Spackman, M. A. Chem.Eur. J. 1998, 4, 2136−2141. (32) Wolff, S. K.; Grimwood, D. J.; McKinnon, J. J.; Jayatilaka, D.; Spackman, M. A. CrystalExplorer, 2.1 (381); University of Western Australia: Crawley, Western Australia, 2005−2007. (33) (a) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; J. A. Montgomery, J.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, C.02; Gaussian, Inc.: Wallingford, CT, 2004; (b) Jarzembska, K. N.; Dominiak, P. M. Acta Crystallogr. 2012, A68, 139−147. (c) Volkov, A.; Macchi, P.; Farrugia, L. J.; Gatti, C.; Mallinson, P.; Richter, T.; Koritsanszky, T. XD2006 - a computer program package for multipole refinement, topological analysis of charge densities and evaluation of intermolecular energies from experimental and theoretical structure factors, 2006. (34) Mulliken, R. S. J. Chem. Phys. 1955, 23, 1833−1840. (35) Gagoś, M.; Gabrielska, J.; Dalla Serra, M.; Gruszecki, W. I. Mol. Membr. Biol. 2005, 22, 433−42. (36) Hubschle, C. B.; Dittrich, B. J. Appl. Crystallogr. 2011, 44, 238− 240. (37) Dominiak, P. M.; Volkov, A.; Li, X.; Messerschmidt, M.; Coppens, P. JTCT 2007, 3, 232−247.

2345


Controlled Crystallization, Structure, and Molecular Properties of

Recommend Documents