A Multi-Technique Experimental and Computational Approach To

Article pubs.acs.org/crystal

A Multi-Technique Experimental and Computational Approach To Study the Dehydration Processes in the Crystals of Endomorphin Opioid Peptide Derivative Marta K. Dudek, Tomasz Pawlak, Piotr Paluch, Agata Jeziorna, and Marek J. Potrzebowski* Polish Academy of Sciences, Centre of Molecular and Macromolecular Studies, Sienkiewicza 112, 90-363 Lodz, Poland S Supporting Information *

ABSTRACT: When molecular crystals undergo partial dehydration, determining the crystal contents and precise localization of the remaining water in the crystal lattice becomes challenging, especially when the quality of crystals after dehydration is not suitable for X-ray diffraction studies. In this work, we describe a methodology that allows the determination and refinement of the structures of partially dehydrated crystals employing complementary experimental (advanced solid-state NMR techniques, differential scanning calorimetry, elemental analysis) and computational (gauge-including projector-augmented wave density functional theory) techniques. We present the power of the methodology using an opioid peptide derivative, endomorphin-2-OH (EM2-OH) heptahydrate. The advanced solid state NMR techniques 2D-PASS, inv-HETCOR, and cross polarization variable contact (CP-VC) carried out with very fast magic angle spinning were used as a source of the constraints showing differences and similarities in the structures and local molecular dynamics for crystallized and dehydrated samples. A crystal structure prediction employing the gauge-including projector-augmented wave (GIPAW) method was used for the determination and refinement of dehydrated EM2-OH. After dehydration, three out of the initial seven water molecules remain in the lattice of the EM2-OH crystals, with two water molecules located in the pockets made by the pseudocyclic conformations and one forming a bridge between two independent EM2-OH molecules.

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INTRODUCTION Understanding and accounting for the role of water in the formation and stability of molecular crystals of small organic moieties and natural products is challenging from both the intellectual and experimental points of view.1−3 The challenge is met on each stage of crystal formation starting from preorganization, followed by the growing of regular forms with water content, and finally the dehydration process, which is often related to the collapse of the subtle structure.4 This problem affects a significant population of crystals. A search of the Cambridge Crystallographic Data Center (CDDC) shows that approximately 20 percent of the deposited structures contain water in the crystal lattice.5 Hydrate formation and spontaneous dehydration have many implications in the pharmaceutical industry because they affect the physicochemical properties of materials, such as their density, melting point, and dissolution rate, which in turn can influence their manufacturability and pharmacokinetic properties.6 The bioavailability of a drug is affected through differences in the solubility and dissolution rate between anhydrate, semihydrate, and hydrate forms.7 The unexpected formation of hydrates can thus lead to unpredictable behavior of the drug. Today, modern science offers a number of tools for the analysis of hydrates that allow basic or detailed questions to be answered with different degrees of accuracy and complexity. For this apparent reason, the X-ray diffraction (XRD) of single © XXXX American Chemical Society

crystals is thought to be the most informative. This technique is used for studying the structure of matter at the atomic level. The power of this technique is also due to the possibility of the precise construction of three-dimensional models. The noncovalent intra- and intermolecular interactions (e.g., hydrogen bonding, aromatic−aromatic, van der Waals interactions, etc.) can be unambiguously defined.8,9 Another important technique that provides a rich set of constraints extremely useful in the structural analysis of hydrates and/or semihydrates is solid state NMR spectroscopy (SS NMR).10,11 It is worthwhile to highlight that SS NMR, contrary to XRD, does not require any preconditions for the structural model to the interpretation of data. This technique responds to the short-range environment of relevant atoms and is not directly influenced by the long-range order. Chemical shift, the most important NMR parameter that provides detailed information about the electronic distribution around each individual nucleus, gives information about intermolecular interactions, which are crucial for the analysis of the nature of hydrogen bonding.12,13 Additional insight into an electronic environment of a nucleus can be gained by the determination of the three principal components of the chemical shielding tensor (CST), which describe the distribution of the magnetic Received: June 1, 2016 Revised: July 15, 2016

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DOI: 10.1021/acs.cgd.6b00831 Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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Figure 1. Molecular structure and atom numbering of EM2-OH.

crystal structure of the analogue of endomorphin-2 modified at the C-end by the replacement of a NH2 group with an OH residue.28 This Tyr-Pro-Phe-Phe-OH modification, known in the literature as EM2-OH, crystallizes as a hydrate containing seven water molecules in the unit cell. The molecular structure and numbering system used in this work are shown in Figure 1.

shielding in the three principal directions from the nucleus. Crystallographic disorder related with water migration during the hydration/dehydration process is also detectable, and distinctions between spatial and temporal disorder can be made.14 Finally, measurement of both homo- and heterodipolar coupling constants under favorable circumstances can be a source of information about the local molecular motion of functional residues related to the water content in the crystal lattice.15 Other experimental techniques such as infrared, UV− vis, or THz spectroscopy have also found a number of spectacular applications in the study of water in molecular crystals.16−19 A different approach than experimental methodologies employed in the search of hydrates and semihydrates that can be used is computational chemistry. The basic target of “in silico” routines is to develop algorithms which allow regular crystal forms to be built on the basis of knowledge of the molecular formula.20 Over the past decade, theoretical methods generating crystal structures have progressed considerably, becoming a useful complement to experimental solid form screening.21−23 Crystal structure prediction (CSP) calculations are now being successfully performed on multicomponent systems including on such complex matter as hydrates.24,25 The CSP approach is particularly useful when the quality of crystals after dehydration is not good enough for measurement by Xray diffraction techniques. The presented above procedures have advantages and disadvantages as well as weak and strong points. Hence, it is apparent that a complementary approach joining experimental techniques and theoretical calculations is the most efficient strategy leading to possibility of presenting a clear picture of processes occurring during the crystallization and dehydration of molecular crystals. In the current work, we present the power of such a complementary methodology for the model tetrapeptide belonging to the family of opioid peptides, which is an endomorphin derivative. Endomorphins are relatively new endogenous peptides preferring morphine μ-opioid receptors in many brain regions.26 They exhibit the highest affinity and specificity for the μ-receptors of any compound found thus far in the mammalian nervous system.27 The endomorphin family includes two peptides that differ by one amino acid: endomorphin-1 (Tyr-Pro-Trp-Phe-NH2) and endomorphin-2 (Tyr-Pro-Phe-Phe-NH2) with molecular masses of 665.7 and 626.7 Da, respectively. In 2005 In and co-workers published the

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EXPERIMENTAL SECTION

Materials. EM2-OH was synthesized by the Lipopharm Company (Poland). The purity of the obtained compound was >99%. Crystals of sample 1 were obtained by crystallization from water according to the procedure described in ref 28. Dehydrated crystals of EM2-OH 2 were obtained from a sample of 1 by placing it in a desiccator over phosphorus pentoxide (P2O5) for 12 h. NMR Spectroscopy. A solid-state cross-polarization magic angle spinning (CP/MAS) NMR and one-pulse 1H MAS experiments were performed on a 600 MHz Avance III spectrometer (operating at 600.13, 150.90, and 60.81 MHz for 1H, 13C, and 15N, respectively) equipped with a MAS probe head using 4 mm ZrO2 rotors. A sample of 13C and 15N-labeled histidine hydrochloride was used to set the Hartmann−Hahn conditions for 13C and 15N. The conventional 13C CP/MAS spectra were performed with a proton 90° pulse length of 3.8 μs, a contact time of 2 ms, a repetition delay of 3 s, a spectral width of 40 kHz, and a time domain size of 3.5 k data points. The acquisition data were collected with a Small Phase INcremental ALternation (SPINAL) decoupling sequence.29 The 15N CP/MAS spectra were performed with a proton 90° pulse length of 3.3 μs, a contact time of 4 ms, a repetition delay of 3 s, a spectral width of 73 kHz, and a time domain size of 5.5 k data points. 38000 and 73000 scans were collected for 1 and 2, respectively. In order to determine the principal components of the chemical shielding tensors a 5-π pulse phase-adjusted spinning sidebands (2D PASS) scheme and 1500 as well as 4000 Hz sample spinning speeds were used in the two-dimensional (2D) experiments. The π-pulse length was 8 μs. Sixteen t1 increments using the timings described by Levitt et al. were used in the 2D PASS experiments.30 For each increment, 360 scans were collected. Because the pulse positions in the t1 set returned to their original positions after a full cycle and the t1FID formed a full echo, the 16-point experimental t1 data were replicated to 256 points. After the Fourier transformation in the direct dimension, the 2D spectrum was sheared to align all side bands with the center bands in the indirect dimension of the 2D spectrum. Onedimensional chemical shift anisotropy (CSA) spinning sideband patterns were obtained from t1 slices taken at the isotropic chemical shifts in the t2 dimension of the 2D spectrum. The values of the principal elements of the CSA tensor were obtained from the best-fit simulated spinning sideband pattern. Simulations of the spinning CSA B



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Figure 2. (a) Unit cell of endomorphin-2(OH); (b) alignment of two nonequivalent molecules in the unit cell. Coordinates are taken from ref 28. sideband spectra were performed on a PC using the Bruker TopSpin 3.0 program.31 The 2D cross polarization variable contact (CP-VC) experiments were carried out with a 13C RF field equal to 160 kHz. The 1H RF field was optimized for a given sample to approximately 100 kHz. The spinning speed was set to 60 kHz. Sixty-four t1 points were collected with increments equal to 16.67 μs (1 over rotor period). Data were processed as described in our previous article.32 The 1H−13C HETeronuclear CORrelation (invHETCOR) for indirect detection of 13C experiments was performed using the pulse sequence described by Mao et al.33 The following parameters were used: a 60 kHz spin rate, a proton 90° pulse length of 2.5 μs, a first contact time of 2 ms, a second contact time of 1 ms or 50 μs, and a proton π pulse (5 μs) in the middle of the evolution period (instead of CW 1H decoupling as mentioned by Ishii and Tycko).34 The maximal evolution times were T1max = 4.2 ms and T2max = 20 ms. All data were processed using the Bruker TopSpin 3.0 program. Differential Scanning Calorimetry (DSC). DSC measurements were recorded using a DSC 2920, TA Instruments. The heating rate was 10 K/min for both samples. Theoretical Calculations. The quantum chemical calculations were performed using CASTEP35 and/or the Gaussian0936 code. In Gaussian09, NMR parameters were calculated using the Gauge Invariant Atomic Orbitals (GIAO) approach and Perdew, Burke, and Ernzerhof (PBE0) functional37 with 6-31G** basis set using the CASTEP-optimized structure of 1 as an input. Molecules A and B of 1 were calculated separately but with all of the hydrogen bonds saturated with water molecules. In the case of 2, trihydrates were created from the heptahydrate structure by removing particular water molecules so that 35 possible trihydrates were generated, and the NMR parameters were calculated. Afterward, the differences in the 15N shieldings and 13 C chemical shielding tensors (CSTs) of four carbonyls and the C− OH tyrosine atom between heptahydrate and trihydrate structures were calculated and compared with the differences obtained experimentally. The differences in the computational vs experimental results for 15N and 13C nuclei were expressed in ppm as a ΔΔδ parameter, defined by the following equations:

ΔΔδ =

performed under periodic boundary conditions. For geometric optimization, the X-ray diffraction crystal structure of 1 was used as an input file. Possible structures of 2 were generated by removal of particular water molecules. The generalized density approximation density functional theory (DFT) functional PBE39 was applied. A comparison of the average forces remaining on the atoms after geometric optimization was carried out for proton-only and all-atom optimizations by using a maximum plane wave cutoff energy of 571 eV ultrasoft pseudopotential.40 The P21 unit cell parameters were taken from the X-ray structure of 1 and kept fixed during the optimization of the geometries of structures 1 and 2. A 3 × 1 × 1 Monkhorst−Pack grid41 was used to sample the Brillouin zone. The NMR chemical shifts were computed using the gauge-including projector-augmented wave (GIPAW) method.42 In all cases, the optimization algorithm was Broyden−Fletcher−Goldfarb−Shanno (BFSG)43 with line search. All numerical data are presented in the Supporting Information.

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RESULTS AND DISCUSSION A. Correlation between Crystal Structure of EM2-OH 1 and the 1H, 13C, and 15N Chemical Shifts in the Solid

Figure 3. Comparison of conformers A (blue) and B (red) for structure 1 assuming the same position of atoms for the Pro-Phe bond. To clarify the picture, the hydrogen positions are not shown.

Σii = 11,22,33|(δiihepta − δiitri)exp − (δiihepta − δiitri)theor | 30 13

for C and ΔΔδ =

State. The crystal and molecular structure of EM2-OH 1 is deposited in the Cambridge Crystallographic Data Center (CCDC 271791). EM2-OH 1 crystallizes in the monoclinic system with the P21 space group. The unit cell parameters are a = 19.687(2), b = 6.5058(7), c = 25.869(3), β = 101.370(2), and volume = 3248.27 Å3. The crystal unit contains 4 molecules of peptide and 14 molecules of water, and the asymmetric unit cell contains half of them. The molecular structure and packing are shown in Figure 2. Each of the two independent molecules in the unit cell adopts the cis configuration around the tyrosine−proline amide

hepta tri hepta tri ∑ |(δiso − δiso )exp − (δiso − δiso )theor |

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for 15N. Such a procedure of comparing experimental and theoretical differences between the trihydrate and heptahydrate allows for the elimination of computational systematical errors. The CASTEP code implemented in the Materials Studio software38 was used for the calculations of 1, five best-matching structures of 2 according to the GIAO calculations, and, for comparison purposes, fully dehydrated structure of 1, obtained by removing all water molecules from the crystal lattice. All of these calculations were C



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Figure 4. Conformers A (blue) and B (red) for structure 1, their hydrogen-bonding networks and water molecule locations. Molecules are colored by the symmetry equivalence of the conformer, with each distinct water molecule colored differently.

Figure 5. 13C CP MAS NMR spectrum of 1 recorded at a spinning rate of 12 kHz. The resonances are assigned employing the GIPAW method.

According to the X-ray data, 11 hydrogen bonds per peptide molecule (without counting the water−water attractions) are observed. Comparing the A and B conformers, differences in the hydrogen bonding pattern for the −NH3+ (N1) group and (N4) group are seen. For the former molecule, strong attraction with water molecule w14 is apparent, while the attraction is absent for B. Molecules A and B are bonded via intermolecular hydrogen bonding with the contributions of tyrosine −OH groups and water. The 13C CP/MAS spectrum of freshly crystallized 1 recorded at room temperature with a spinning rate of 12 kHz is shown in Figure 5. Keeping in mind that the structure of 1 consists of two molecules in the asymmetric unit cell, we should observe the splitting of signals. The rough inspection of the spectrum clearly shows that some of the resonances are isochronous and cannot be assigned to molecules A and B, while some are diagnostic. In particular, peaks of the carboxylic residues in the region 175−180 ppm as well as signals of tyrosines and carbons C13 and C15/C17 in region 112−125 ppm are indicative.

Figure 6. 15N CP MAS NMR spectrum of 1 recorded at a spinning rate of 8 kHz. The resonances are assigned employing the GIPAW method.

bond and exists in the zwitterionic form. The pseudocyclic conformation for both peptide molecules, labeled as A and B, is slightly different. The intramolecular distance between the amine (N1) and carboxyl group (C40) for A (blue) is 3.6 Å, while the length for B (red) is 4.3 Å (Figure 3). Such distinction is mostly due to the different nature of hydrogen bonding with the contribution of water. Figure 4 shows the hydrogen bond patterns and locations of water molecules for both conformers A (blue) and B (red). D



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Figure 7. DSC plots of EM2-OH heptahydrate 1 (black line) and trihydrate 2 (red line).

Figure 8. 13C CP MAS NMR spectrum of 2 recorded at a spinning rate of 12 kHz. The crucial resonances used in further discussion are assigned employing the GIPAW method.

NMR shielding parameters are collected in Table 3S attached in the Supporting Information. The theoretical calculations allowed assignment of the carbonyl and amine residues to particular A and B moieties. The most significant difference observed experimentally for the C40 δ22 parameter in molecules A and B (199.1 and 186.4 ppm, respectively) was also visible in the computational results. B. Analysis of the Dehydration Process by Means of Differential Scanning Calorimetry (DSC) and Solid-State NMR. Two basic procedures are usually employed for removing water from the crystal lattice. This process (known as dehydration) can be carried out by the thermal treatment of a sample or by changing the relative humidity of the environment. In the first approach, high stability of the sample is required because in many cases, elimination of water from the crystal lattice causes the destruction of the subtle structure and frequently amorphous material is obtained as a final product. Such a destructive mechanism often operates in the case of small peptides. However, under favorable conditions, the crystallographic structure of the peptide can be preserved. In our recent paper, we revealed that thermal dehydration for tetrapeptide Tyr−D-Ala−Phe−Gly (YAFG) dihydrate heated up to 160 °C leads to a new anhydrous form with a wellorganized crystal lattice.45 It is worth noting that this dehydration takes place below the melting temperature. Unfortunately, this is not the case for sample 1. DSC measurements prove that processes in the crystal lattice of 1 related to the thermal migration of water are very complex (Figure 7). Water molecules usually escape from the crystal lattice of hydrates at approximately 130 °C. The DSC profile of the heptahydrate of EM2-OH 1 (black line in Figure 7) shows an

Figure 9. 15N CP MAS NMR spectrum of 2 recorded at a spinning rate of 8 kHz. The resonances are assigned employing the GIPAW method.

The 15N CP/MAS data for 1 shown in Figure 6 are consistent with the 13C results. The splitting of signals for two nitrogen positions at (N1) and (N4) reflects the different conformations and distinct in hydrogen bonding characters for molecules A and B. The assignment of NMR signals in the solid state and distinguishing between A and B resonances is not a trivial task. In order to solve this problem, we employed a theoretical approach. Recent years have witnessed incredible progress in the advancement of the calculation of NMR parameters in solid matter. State of the art in this field was recently exhaustively discussed in a review article published by Bonhomme et al.44 On the basis of literature reports and our longstanding experience in computing the NMR parameters, for the calculations of 1 we employed the GIPAW approach, which is implemented in the CASTEP code. This technique is based on DFT, pseudopotential plane-wave and periodic system. In the GIPAW approach, the entire crystalline unit cell is included in the calculation. As an input file, we used the X-ray data for 1. The coordinates of hydrogen and heavy atoms were optimized, while the size of the unit cell was preserved. The computed E



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Figure 10. 1H−13C inverse detected HETCOR NMR spectra acquired at a 60 kHz spinning rate and 298 K with a 1 ms contact time for 1 (A) and 2 (B). Expanded part of the overlapping spectra of aromatic and carbonyl/carboxyl residues of 1 and 2 (C).

Figure 11. CPVC VF MAS 2D NMR aromatic part of spectra of EM2-OH 1 (a) and a dehydrated sample of EM2-OH 2 (b). At the top 1D 13C CPMAS spectra are shown.

F



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Figure 12. Differences between experimental and theoretical (GIAO-NMR) changes in the 15N chemical shifts and 13C chemical shielding tensors upon dehydration. Each point marks a distinct trihydrate structure. Blue circles indicate the best structures in terms of the changes in 15N shielding, the green circle indicates the structure with the best agreement in terms of 13C CSTs and reasonably good agreement in terms of 15N shielding. All five marked structures were recalculated using the GIPAW approach. ΔΔδ = ΔΔδ =

Σii = 11,22,33 | (δiihepta − δiitri)exp − (δiihepta − δiitri)theor | 30

for 13C and

hepta tri hepta tri ∑iso |(δiso − δiso )exp − (δiso − δiso )theor |

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.

Table 1. Correlation Coefficients Obtained after Plotting Experimental and Theoretical 15N Chemical Shifts of the Fully Hydrated and Dehydrated EM2-OH Together with the Lattice Energies for the Trihydrates and the C40−N1 Distances CO(40)−N(1) 2

structure trihydrates

dehydrated EM2-OH fully hydrated EM2-OH

1 3 4 6 10

15

2

13

R (δ N)

R (δ C)

energy [kcal/mol]

molecule A

molecule B

0.994 0.995 0.995 0.998 0.998 0.967 0.999

0.982 0.987 0.983 0.982 0.983 0.981 0.995

−929556 −929571 −929544 −929566 −929563

3.56 3.73 3.60 3.46 3.54 3.69 3.60

3.23 3.71 4.16 4.36 4.70 3.97 4.30

can be drawn on the basis of the lack of splitting for carbon signals for the C40 and C13 carbon position. The δ22 parameter for C40, which was different for molecules A and B in the heptahydrate, has a value of 184.4 ppm after dehydration, which is very close to the value for molecule B before dehydration (186.4 ppm). It is worth noting the significant downfield shifting of one of the peptide carbonyl residues, which suggests strengthening of the hydrogen bonding at this position. It further suggests possible interaction between CO and residual water molecules. In fact, the elemental analysis revealed that dehydration over P2O5 does not completely remove the water. According to this analysis, three water molecules from the seven remain in the crystal lattice. The first impression that conformations of the A and B molecules in the crystal lattice are identical or very similar is questioned by the 15N CP/MAS measurement. The splitting of signals is clearly seen for the N3 and N4 positions. On the other hand, signals N1 and N2 are isochronous. The NMR signals of N1, N2, and N3 are upfield shifted, which suggests weakening of the hydrogen bonding for those respective residues. In this structural puzzle, the crucial question involves the localization of water molecules in the crystal lattice after the dehydration process. In the first running hypothesis, we can assume that the resemblance of molecules A and B is due to the

endothermic change already taking place at 88 and next at 114 and 140 °C. Those initial phase transitions at temperatures lower than 140 °C are probably related to the preorganization of the crystal system and migration of water only inside the lattice. At a temperature of 170 °C, a strong exothermic thermal effect was registered, which can be the result of a new state in the structure or the decomposition of EM2-OH. After gentle heating of the sample below the melting point at 160 °C, the collapse of the structure is observed by employing solid state NMR spectroscopy. The alternative pathway of dehydration, keeping the sample in an environment with low humidity, is less invasive. Figure 8 shows the 13C CP/MAS spectrum of sample 2 obtained by keeping sample 1 in a desiccator over P2O5 overnight. The differences between the spectra for freshly crystallized and treated samples are apparent. From initial examination of the spectra, it is clear that crystals undergo changes, but the subtle lattice structure is preserved. Additionally, differences in the curves obtained from the DCS analysis of 2 (red line in Figure 7) are apparent in the region corresponding to the removal of water from the crystal lattice. However, further heating (above 170 °C) does not show any differences in the trihydrate and heptahydrate DSC profiles. More advanced inspection of the NMR spectra suggests that after dehydration, molecules A and B start to structurally resemble each other. Such a conclusion G



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Figure 13. Most probable structures of EM2-OH trihydrate 2: (a) with w14, w18, and w15 water molecules (structure 3) and (b) with w14, w18, and w19 water molecules (structure 10).

similar intermolecular interactions with water. As a consequence, it is further assumed that in both cases one tetrapeptide molecule interacts with one water molecule. The localization of the third water molecule in the crystal lattice is challenging.

Keeping in mind that for the freshly crystallized hydrate of 1, one water molecule is involved in the bridging of the A and B molecules via interaction with tyrosine hydroxyl groups, the monitoring of changes after dehydration in this region is H



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critical. For this purpose, the 1H−13C heteronuclear (HETCOR) correlation with a relatively long contact time for inspection of long-range distances was found to be useful. We employed the HETCOR experiment with inverse detection (via proton) performed under a very fast magic angle spinning (VF MAS) with a sample speed of 60 kHz. By applying such a methodology, we solved two problems: first the resolution of proton spectra, and second the sensitivity of measurements for detecting abundant nuclei. Figure 10A,B shows the 1H−13C invHETCOR spectra of samples 1 and 2 recorded with contact times equal to 1 ms (long contact). In order to visualize differences between both forms, the expanded parts of the spectra showing aromatic and carbonyl/carboxyl residues are displayed in Figure 10C. From analysis of the correlation peaks, it is apparent that 1H and 13C chemical shifts of the C16 tyrosine in the hydrate and dehydrated forms are very similar, indicating that the hydrogen bonding pattern in both cases is similar. Hence, we can assume that the water molecule bridging the A and B moieties remains in the crystal lattice after the dehydration process. Unfortunately, the correlation peaks between water and EM2-OH molecules are not observable, and this is due to (i) too long distances between water and EM2OH, (ii) too small dipolar coupling value, and/or (iii) water dynamics. Therefore, in order to confirm the water location in the EM2-OH trihydrate, we employed theoretical calculations, described in section D. C. Local Molecular Dynamics of Aromatic Residues for the Heptahydrate and Trihydrate Forms of EM2OH. As we highlighted in the previous sections, one of the targets of this project is to work out the methodology, which allows the determination and refinement of the structure of a compound with an unknown composition and/or molecular geometry in the solid phase. Such a goal can be achieved employing a NMR crystallography approach.46 This strategy combines the experimental techniques and advanced GIPAW quantum mechanical calculations. Comparing the experimental chemical shifts δii with theoretical shielding σii parameters and analyzing the consistency between both sets of data, it is possible to determine the quality of a model and validate the reliability of a solution. However, care has to be taken in the interpretation of data. The quantum chemical calculations are typically performed using static structures, i.e., at 0 K where zero-point motion is neglected. The local molecular motion averages NMR tensor parameters such as the CSA, dipolar interactions, and quadrupolar interactions. Knowledge about such processes is crucial in further analysis. It is well-known that side groups in peptides and proteins undergo complex dynamic processes in the crystal lattice. The molecular motion of the phenyl rings of the Phe residues in the solids has been extensively studied for amino acids, polypeptides, and proteins employing different NMR techniques. Hiraoki and co-workers exploring the phenyl ring dynamics of poly(L-phenylalanine) as a function of temperature and echo delay time have revealed that the π-flipping motion is characterized by a fairly broad distribution of correlation times and is strongly dependent on the temperature.47 Kamihira et al. have reported detailed analysis of the molecular dynamics of enkephalin, one of the best-characterized opioid peptides.48 These observations suggest that the degree of motion in the aromatic rings of the phenylalanine and tyrosine residues strongly depends on the environment of the enkephalin molecules, such as the crystalline packing and the state of the bound solvents. In our recent paper employing experimental

and theoretical approaches, we revealed that for the signaling sequence Tyr-D-Ala-Phe of dermorphin and its analogue TyrAla-Phe, the dynamic processes are dramatically different.49 Among commonly used methodologies for the inspection of molecular motion, the analysis of X(13C, 15N)−1H heteronuclear dipolar coupling was found to be extremely useful. A large achievement in the field of the measurement of dipolar couplings was the introduction of the PISEMA technique and its different variants, which allowed the determination of dipolar interactions under MAS.50−52 The applicabilities of other approaches, such as T-MREV and DIPSHIFT, have also been reported in several articles. 53,54 Discussed supra methodological approaches have found a number of spectacular applications for samples under slow (few kHz) and moderate (10−15 kHz) spinning speeds. For such spinning rates, the application of complex and demanding recoupling sequences was found to be crucial. In our recent articles, we have shown that measurement of dipolar coupling can be greatly simplified when the experiment is performed under VF MAS conditions with a sample spinning rate above 40 kHz.55,56 The simple cross-polarization with variable contact (CPVC VF MAS) technique is free from the limitations related with the complexity of recoupling sequences and provides very accurate values of heteronuclear dipolar splittings, D, which reflect the interatomic distances and/or molecular motions. Figure 11 shows the CPVC VF MAS 2D NMR aromatic part of spectra for EM2-OH freshly crystallized 1 (Figure 11a) and after dehydration over P2O5 2 (Figure 11b). The full spectra for both samples and the extracted 1D slices of the aromatic part of the spectra are attached as Supporting Information (see Figures 1S and 2S). From inspection of the contour plot, the assignment of quaternary carbons characterized by small dipolar splitting is unambiguous. There is also no doubt of the assignment of the tyrosine residue. In both cases (1 and 2), for the Tyr group the dipolar splitting was found to be 15.6 kHz, which, according to our recent results, is a typical value for a rigid molecule in the crystal lattice. Similar splitting was established for the para position of phenylalanines Phe3 and Phe4. In contrast, the splitting for the ortho and meta positions is significantly reduced to 9.4 kHz. These results clearly prove that Phe residues undergo rotation around the 1−4 axis (ipsopara); however, the barrier of rotation is higher compared to an unrestricted π-flip. For the latter case, splitting of approximately 7 kHz is usually observed. It is worth noting that dehydration does not dramatically change the dynamics of aromatic residues, meaning that the space in the crystal lattice “reserved” by aromatic phenylalanine rings for rotation around the 1−4 axis for the heptahydrate and trihydrate forms is comparable. D. Crystal Structure Prediction for Dehydrated EM2OH. To confirm the assumptions about the EM2-OH behavior upon dehydration made on the basis of the analysis of solidstate NMR data, theoretical calculations employing the GIPAW methodology are needed. However, crystal structure prediction is a very challenging and computationally demanding task, especially for systems such as dehydrated EM2-OH, which, according to the experimental data, has five distinct molecules in the asymmetric part of a unit cell, i.e., two EM2-OH and three water molecules. To determine which water molecules remain and which are removed from the crystal lattice during the dehydration process, all 35 possible combinations (each 3 out of 7) should be checked. However, such an approach using the GIPAW calculations would consume too much computaI



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and w18 water molecules inside each EM2-OH, but instead of w15, it has the w19 water molecule, which forms only one connection between A and B (Figure 13b).

tional time. Therefore, to rationalize the computational cost, we first performed the NMR-GIAO calculations (without including the crystal lattice but preserving all of the hydrogen bonds) for the 35 different trihydrate structures using Gaussian09 software. The NMR-GIAO calculations revealed that the differences in the changes upon dehydration in the 15N chemical shifts are more distinct than those in the 13C chemical shielding tensors (CSTs, Figure 12) and that the structures that give the best agreement between experimental and computational data in terms of 15N shielding are usually different from those giving the best agreement in terms of 13C chemical shielding tensors. Typically, for the prediction of the crystal structure of a molecule employing GIPAW calculations and solid-state NMR, the latter parameter, i.e., 13C CST values, from the experiment and calculations are compared. However, in the case of EM2OH, the detailed GIPAW-based assignment of the 13C signals is ambiguous. This is due to the following: (i) the signals overlap, which results in the averaging of the experimental 13C CST values and (ii) the dynamics of the phenyl ring, which are not accounted for in the GIPAW calculations (discussed supra). It was shown that in the latter case, the discrepancies between experimental and theoretical CSTs are observed. Then, it is not possible to decide a priori which of the observed differences are the result of dynamic processes in EM2-OH and which indicate the incorrect structural prediction. Therefore, primarily 15N shieldings were considered in further analysis. The 13C CSTs of four carbonyls and the C−OH of tyrosine in both molecules, for which the assignments were more reliable, were used as an additional but not decisive criterion. According to the 15N NMR-GIAO calculations, there are four structures of 2 considered to be the most probable, marked in Figure 12 with the blue circles. The structure marked with a green circle is the one that gives quite good agreement in terms of the 15N shielding and the best agreement in terms of the 13C CSTs. All five structures were optimized with the GIPAW approach, together with fully dehydrated and fully hydrated EM2-OH, and the NMR parameter calculations were performed. The obtained correlation coefficients for the 15N theoretical shieldings vs the experimental chemical shifts are gathered in Table 1. A very good agreement obtained for all five trihydrate structures, only slightly worse than that obtained for the fully hydrated structure and much better than that for the fully dehydrated structure, confirms the validity of the employed computational methodology. Three out of five trihydrate structures (namely, 1, 3, and 4) contain a water molecule that bridges the A and B moieties of EM2-OH, engaging the C−OH group from the tyrosine residue, which is in agreement with the suggestion from the experimental data. In addition, in trihydrate 3 very similar C40−N1 distances for molecules A and B were observed, which confirms the assumption that after dehydration both molecules become more similar. Structure 3 also has the lowest lattice energy. All of these data indicate that 3 is therefore the most probable structure of the EM2-OH trihydrate. It contains one water molecule inside each EM2-OH molecule (w14 and w18), and the third water molecule (w15) constitutes two connections between A and B, namely, one which is involved in the hydrogen bond with the tyrosine hydroxyl group, and the second one between COO− (A) and NH3+ (B) groups (Figure 13a). As for structure 10, which in GIAO calculations performed the best in terms of 13C CSTs, after recalculations with GIPAW, it returned the best result for the 15N shielding but slightly worse results for the 13C CSTs. It also contains w14

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CONCLUSIONS The fully dehydrated crystalline material can be investigated by means of different experimental and theoretical techniques assuming that only one pure component is in the matter being analyzed. The problem becomes more challenging when crystals undergo partial dehydration, and then, more issues need to be addressed. The first issue is related to the crystal contents and the second to the precise localization of the remaining water in the crystal lattice. Addressing these issues is particularly challenging when the quality of crystals is not suitable for X-ray diffraction studies, but the subtle structure is preserved and can be analyzed by other spectroscopic techniques. In this paper, we presented a methodology for solving all the above-mentioned issues. The procedure shown employing EM2-OH as model sample is comprised of advanced solid-state NMR spectroscopy, DSC measurements, and theoretical calculations, which together allowed for the following conclusions to be drawn regarding the dehydration process of EM2-OH: (i) partially dehydrated EM2-OH contains three water molecules and two EM2-OH molecules (A and B) in the asymmetric unit cell, (ii) the hydrogen-bonding patterns of molecules A and B after dehydration become more similar, (iii) two of the remaining water molecules (w14 and w18) are located in the cavities made by the formation of the pseudocyclic conformations of peptide, and the other one (w15) forms a bridge between both EM2-OH molecules, (iv) the dehydration of EM2-OH does not impede molecular motion in the crystal, and finally (v) as was demonstrated previously in the literature 15N shieldings proved to be more sensitive indicators of the changes in the hydrogen bonding network, especially when local dynamics are observed. Finally, it is worthwhile to express that the new three hydrate form is stable and does not undergo reversible hydration.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.cgd.6b00831. Full CP-VC MAS NMR spectra of 1 and 2 together with the expanded aromatic region and extracted respective 1D slices (Figures 1S and 2S), 15N and 13C chemical shift tensor experimental and theoretical data for 1 and 2 (Tables 1S-4S), and GIPAW coordinates along with calculated NMR parameters for 1 as well as the five most probable structures of 2 (PDF)

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AUTHOR INFORMATION

Corresponding Author

*Tel. +48 42 680 3240. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors are grateful to the Polish National Science Centre (NCN) for financial support under Grant No. UMO-2014/13/ B/ST4/03050. The computational resources were partially J



Article

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