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A disappearing metastable hydrate form of L-citrulline: Variable conformations in polymorphs and hydrates
Journal Pre-proof A disappearing metastable hydrate form of L-citrulline: Variable conformations in polymorphs and hydrates Palash Sanphui, Renjith S. Pillai PII:
S0022-2860(19)31288-8
DOI:
https://doi.org/10.1016/j.molstruc.2019.127179
Reference:
MOLSTR 127179
To appear in:
Journal of Molecular Structure
Received Date: 11 July 2019 Revised Date:
19 September 2019
Accepted Date: 3 October 2019
Please cite this article as: P. Sanphui, R.S. Pillai, A disappearing metastable hydrate form of L-citrulline: Variable conformations in polymorphs and hydrates, Journal of Molecular Structure (2019), doi: https:// doi.org/10.1016/j.molstruc.2019.127179. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
A disappearing metastable hydrate form of L-Citrulline: Variable conformations in polymorphs and hydrates Palash Sanphui* and Renjith S. Pillai Department of Chemistry, SRM Institute of Science and Technology, Kattankulathur-603203, India
Abstract An unstable monohydrate crystalline form of L-citrulline was harvested during high throughput solid form screening and its crystal structure and hydrogen bonding patterns are elaborated. The crystal structures of the previously reported anhydrous polymorphs and dihydrate are analysed and compared their conformational flexibility. Periodic Density Functional Theory (DFT) approach was employed to unveil the relative stability of citrulline upon hydration and its microscopic interactions with water molecules. The relative binding energy suggests the order of stability as dihydrate > citrulline (anhydrous) > monohydrate. Metastable nature of the monohydrate is reasoned based on the presence of water molecules in the channel structure, less interactions with the citrulline molecules and lower crystal density compared to the other solid forms. Unlike the reported dihydrate form, monohydrate transformed to the anhydrous citrulline (Form II) on spontaneous dehydration at ambient conditions because of their similar conformational flexibility. First time, we report reproducible crystallization process of polymorphs, dihydrate and a novel metastable monohydrate of L-Citrulline.
Keywords: Citrulline, polymorphs, hydrates, hydrogen bond, binding energy
1. Introduction The crystallization of hydrates cannot be speculated during solid form screening involving moist solvents or water, which often favour better packing than the corresponding anhydrous form. Generally hydrates/solvates are crystallized when there are imbalance in the number of donorsacceptors in a crystal structure [1-4]. The crystalline hydrates are useful solid form because of their ability to tune physical properties of a drug [5-6]. Approximately, 33% of active pharmaceutical ingredients (APIs) form hydrates due to the small size of water molecule, which can fit nicely into the crystal lattice and also its donors (2)–acceptor (1) balance capability to make multiple hydrogen bonds. Hydrates are predominantly less soluble than their anhydrous counterparts [7-9]. In the hydrate form, the API interacts closely with water and free energy released during the crystal dissolution. Hence, further interactions with water is less probable for the hydrate than the anhydrous phase. Pharmaceutical solids may come in contact with water during crystallizations, lyophilisation, wet granulation etc. Hence thorough investigation on the probable hydrate form of a drug is necessary to avoid late stage discovery, which may cost a huge loss to the innovator. 1
L-Citrulline (chemical name: L-2-Amino-5-ureidovaleric acid, Scheme 1) is an active ingredient of watermelon and other dietary sources like cucumber, squash, pumpkin etc. It is found to relax blood vessels and hence improve cardiovascular health [10]. Greco et al. claimed that citrulline can also be used to treat erectile dysfunction [11]. It is marketed in the form of citrulline malate (brand name Stimol®) as a performance enhancing athletic health drinks. L-citrulline catabolises to Larginine after absorption and goes to Kidneys. Hence, it is actually a better method to obtain supplemental L-arginine into the blood [12]. Although, L-citrulline is not among the 18 essential amino acids, the conformation and size of citrulline is identical with the basic amino acids like lysine, arginine and ornithine, see Scheme 1. Citrulline is more readily absorbed and more bioavailable than L-arginine. Hence, L-citrulline has commercial importance as nutraceuticals and its solid forms need to be screened for its pharmaceutical interest. Being an amino acid (zwitterionic form), citrulline shows high aqueous solubility of 200 g/L at 20 °C. The crystal structures of L-citrulline polymorphs (Form I and II), dihydrate, hydrochloride, perchlorate, oxalate salt monohydrate and cocrystal with L-malic acid are reported in the literature [13-18]. According to a Japanese patent JP 56-99453, L-citrulline crystallizes in three polymorphs [19]. There is no clear specification about the polymorphs crystallizations and also their packing differences are not discussed in the prior art. We have revisited citrulline polymorphs via high throughput solid form screening and reproduced the two reported polymorphs (3D coordinates), dihydrate and a new metastable monohydrate. Crystal structure of citrulline (Form I) was reported more than three decades ago [13]. Allouchi et al. reported 2nd form, which was difficult to harvest the single crystals of Form II from solvent crystallizations [18]. Recently, Rossi and co-workers also reported single crystal X-ray data of Form II and proved spontaneous hydrolysis of L-arginine to Lcitrulline as energetically preferred reaction using Density functional theory [20]. Here we provide the reproducible crystallization methods of two anhydrous forms and dihydrate for the first time. In addition, we report the crystal structure of citrulline monohydrate, which appeared only once during high throughput crystallization. Citrulline monohydrate is a kind of disappearing solid form [21], which we could not able to reproduce irrespective of our best crystallization efforts. The subtle hydrogen bonding differences in the conformational polymorphs of citrulline are discussed in details. The relative stability of citrulline upon hydration and its microscopic interactions with water molecules are explained based on relative binding energy calculations using periodic Density Functional Theory (DFT) approach. All the citrulline solid forms are further characterized with FTIR, PXRD and DSC and elaborated. 2. Materials and methods L-Citrulline was obtained from Sigma Aldrich, Bangalore and used directly for the crystallization experiments. The solvents were purchased from commercial sources and used without further purification. Water filtered through a double distilled water purification system (Siemens, Ultra Clear, 2
Germany) was used in all the crystallization experiments. Powder X-ray diffraction (PXRD) data was recorded using a Philips X’pert Pro X-ray powder diffractometer (Cu-Kα radiation, λ = 1.54056 Å) equipped with an X’cellerator detector at room temperature with the scan range 2θ= 5 to 40º and step size 0.017º. X'Pert HighScore Plus was used to compare the experimental XRD pattern of Citrulline polymorphs and dihydrate with the simulated X-ray lines from their crystal structures (Fig. S2, supporting information). Crystal Explorer (version 17.5) was employed to compare the intermolecular interactions. Fourier transform IR (FT-IR) spectra were recorded using KBr pellets with a Perkin– Elmer (UK) spectrophotometer (4000–400 cm–1). Differential Scanning Calorimetry (DSC) was performed to obtain exact melting onset/peak on a Mettler Toledo DSC 822e module at the heating rate of 10 ºC/min in the temperature range of 30-250 ºC under nitrogen atmosphere. 2.1 Single Crystal X-ray Diffraction Single crystal X-ray diffraction data of the L-Citrulline polymorphs and monohydrate were collected on a Rigaku Mercury 375/M CCD (XtaLAB mini) diffractometer using graphite monochromated MoKα radiation at 150 K. The data were processed with the Rigaku Crystal clear software [22]. Structure solution and refinements were executed using SHELX-97 [23] and WinGX [24] suite of programs. Refinement of 3D coordinates and anisotropic thermal parameters of non-hydrogen atoms were performed with the full-matrix least-squares method. The hydrogen atoms attached to N/O atoms were located from difference Fourier map or calculated using riding model and further refined isotropically. PLATON software [25] was used to confirm the correctness of the space group. Xseed [26] and Mercury 3.9 softwares [27] were utilized for molecular representations and packing diagrams.
Crystallographic
cif
files
(CCDC
Nos.
1006847-1006849)
are
available
at
www.ccdc.cam.ac.uk/data_request/cif or as part of the Supporting Information.
2.2 Computational Section The experimentally elucidated structure of the L-Citrulline polymorphs (anhydrous form) was initially geometry optimized at the Density Functional Theory (DFT) level using the CP2K package [28-31]. In these simulations, both the positions of atoms of the framework and the unit cell parameters were relaxed. All the structural optimizations were carried out using Perdew-Burke-Ernzerhof (PBE) functional [32] along with a combined Gaussian basis set and pseudopotential. For all the atoms of Lcitrulline, a triple zeta (TZVP-MOLOPT) basis set was considered. The pseudopotentials used for all the atoms were derived by Goedecker et al. [33]. The van der Waals interactions were taken into account via the use of semi-empirical dispersion corrections as implemented in the DFT-D3 method [34]. Hydrated forms of L-Citrulline structural models, i.e. 0.5, 1, 1.5 and 2 water molecules per LCitrulline molecule were generated by incorporating the water molecules into the initially optimized L-Citrulline structure in a progressive manner. These structure models (labeled as L-Citrulline3
0.5H2O,
L-Citrulline-1H2O,
L-Citrulline-1.5H2O
and
L-Citrulline-2H2O
for
hemihydrate,
monohydrate, sesquihydrate and dihydrate, respectively) were further geometry optimized by DFT allowing the relaxation of the atomic positions and the unit cell parameters while maintaining similar to the monoclinic symmetry. All these basis sets had been optimized already to reduce the basis set superposition error (BSSE) to the total system energy, however BSSE have been taken into account when comparing energies for hydrated L-Citrulline structural models. The BSSE corrected energies were obtained from the counterpoise correction method of Boys and Bernardi [35], where the energies are modified to take into account the lowering of the energy caused by the overlap of basis functions between water molecules and L-Citrulline. The optimized unit cell parameters for these structures are reported in Table S3, supporting information. Additionally, the binding energy for water molecules and relative stability energy of L-Citrulline during hydration were calculated from the following equation Ebinding =EL-citrulline+nH2O ‒ EL-citrulline + nEH2 O
Erelative stability =EL-Citrulline+nH2O ‒nEH2 O Where EL-citrulline+nH2O , E
L-citrulline
(1) (2)
, and EH2O represent the total energies of L-citrulline with water
at each hydration stage (n = 0.5, 1, 1.5 and 2), the empty L-citrulline (n= 0) and gas phase H2O, respectively. Equation 1 defines negative values as exothermic and positive values as endothermic processes. The relative stability of L-citrulline, E , upon hydration could be extracted from Equation 2.
3. Results and discussion Crystal structures of L-citrulline polymorphs (Refcode-FIFGOQ/01) were reported earlier by two different research groups [13, 18], we define as Form I and II. Interestingly, Form I was crystallized from polar aprotic solvent such as dioxane, THF, acetone, EtOAc with the addition of dropswise water; whereas stable polymorph II harvested from polar protic solvents (MeOH to butanol) with water, see Table 1 for the details of the crystallization methods. Both the single crystal X-ray diffraction data of the reported Forms are recollected at 150 K to compare their structural difference and packing motifs, see Table 2. Being chiral molecule, both the L-citrulline polymorphs crystallize in non-centrosymmetric space groups (P212121 and P21). Unlike polymorphs or dihydrate form (P212121), monohydrate crystallizes in the monoclinic c-centered (C2) lattice, as the reported hydrochloride salt (Refcode-LCITHC01) [15]. We reproduced the dihydrate phase from the high pressure crystallization method (under vacuum) of saturated aqueous solution and also from pH 4 acetate buffer medium. Lack of crystallography details of citrulline polymorphs and their reproducibility problems in the prior art prompted us to revisit the commercially important compound. 4
We discuss the structural details of the polymorphs and hydrates and then compared their conformational flexibility. 3.1 Crystal structure analysis Form I: In the crystal structure of Form I (P21), citrulline molecules extend as a ribbon motif using alternate N–H···O (between carbonyl group with the terminal ureido NH) and N+–H···O– (between tertiary amine NH+ and carboxylate) hydrogen bonds (Fig. 1). Citrulline molecules form inversion related N+–H···O– hydrogen bonded dimer of R22(18) ring motif of graph set [36-37] through hydrogen bonding between ureido NH and carboxylate. In addition, four L-citrulline molecules are assembled as a tetramer via R42(8), R44(14) and R44(28) ring motifs. Unlike its close analogue Larginine [37], citrulline molecules arrange in a head (carboxylate) to head (ammonium ion) fashion via N+–H···O– hydrogen bonds along the crystallographic b-axis (Fig. S1a, ESI). Carbamoyl moiety of citrulline forms N–H···O hydrogen bonded catemer chains along the crystallography b-axis. 3D packing (Fig. S1b, ESI) along the crystallographic a axis indicates the clear separation of hydrophobic (intermediate aliphatic chains) and hydrophilic moiety (carbamoyl amino and amino acid), which may favour its higher solubility than other forms [18]. Form II: The citrulline conformation in Form II (P212121) is quite distinct from Form I. Similar to Form I, here also citrulline molecules assemble as tetramer ring motif of R43(12), but in a different molecular orientation via N–H···O and N+–H···O– hydrogen bonds (Fig. 2). The tetramer units interact to another via ammonium ion (one side) and ureido motif (other side). Ureido NH's form bifurcated hydrogen bond of R21(6) ring motif with the carboxylate moiety. Like Form I, citrulline molecules arrange in a head to head fashion via N+–H···O– ionic interactions and extended by N– H···O hydrogen bonded catemer chain along the a-axis (Fig. S1c, ESI). Because of their difference in molecular orientation, two forms are termed as conformational polymorphs [39-40]. 3D packing view of Form II indicates zigzag kind of packing arrangement in which two parallel molecules are arranged in a perpendicular fashion to the next ones along the diagonal of bc plane (Fig. S1d, ESI). Two polymorphs differ by conformation in the side alkyl chain moiety including the carbamoylamino group (Fig. 3). Allouchi et al. mentioned, there are minimum five conformations of L-citrulline exists based on the six torsion angles in the crystal structures reported till now [18]. The PXRD pattern of Form I and II in bulk quantity matches with the calculated X-ray lines indicates the bulk phase purity (Fig. S2, ESI). Commercial form (Sigma Aldrich) is identified as Form II by XRD comparison. Calculated density (1.40 vs 1.49 g/cc) and packing fraction (70.6 vs 75.2%) indicates better stability of Form II than Form I. Even more frequent crystallization of Form II than Form I suggests more stability of the former. In addition, grinding of Form I transformed to Form II, which further supports more stability of Form II. Of course, the N···O bond distances (Table S1, ESI) in 5
Form II is slightly longer than in Form I, which may favor little more stability of Form I than Form II. To conclude, both the polymorphs are close to each other in the lattice energy landscape [41-42] and concomitant crystallization occurs when appropriate conditions like slurry experiment or crystallization in water are followed, see Table 1. L-citrulline monohydrate: During high throughput crystallization, L-citrulline monohydrate (Form M) as plate like crystals were harvested from saturated aqueous solution followed by drop wise addition of EtOAc (anti-solvent) after a day. The crystals were not good quality and unstable (opequed) in the air and the corresponding single crystal X-ray diffraction data was collected at 150 K. The crystal structure was solved in C-centered monoclinic C2 space group (Z=4). Form M has similar torsional flexibility as the reported dihydrate (Refcode-DILFAF01) conformation [14], but differ in carbamoyl moiety. Both the hydrates follow the conventional urea tape motif [R21(6)] via bifurcated hydrogen bonds between two NH’s with oxygen atom. Hydrogen bonding parameters of Form M are summarized in Table 3. Similar to polymorphs, citrulline molecules arrange in a head to head fashion, but interrupted in presence of water molecules. One water molecule forms hydrogen bonds with two more water and carbonyl moiety of one citrulline molecule via O–H···O hydrogen bonds (O4–H4···O3: 2.18, 2.81 Å, 158°), see Fig. 4a. Two fold axis related L-citrulline molecules form left handed helix [43] via N+–H···O– ionic hydrogen bonds between ammonium cation and carboxylate anion (Fig. 4b). Water molecules are present in a channel keeping the fixed distance (O···O: 2.79 Å) between them that’s suggests the probability of phase transformation from Form M to anhydrous form. Strong hydrogen bonded water molecules interacting with carboxylate, ammonium ions and ureido carbonyls in L-citrulline dihydrate makes macrocylic complex of six membered ring, R65(12) and is the main reason behind its high stability at ambient conditions. One water molecule interacts with two ammoniam ions and one amide carbonyl involving three citrulline molecules and also with another water molecule in the dihydrate form. Second water molecule forms hydrogen bonds with two carboxylates of citrulline molecules and one water molecule (Fig. S3, ESI). Unlike Form M, water···water hydrogen bonds are limited to two only, which have shorter contacts (O···O: 2.68 Å) in dihydrate form. All these interactions contribute higher crystal density (1.385 vs 1.370 g/cc) and packing efficiency (70.9 vs 68.5%) of dihydrate form compared to monohydrate. L-citrulline in Form M maintains extended chain motif, whereas in the reported dihydrate form or Form II, it is much angular especially in the carbamoyl moiety (Fig. 5 and Fig. S4, ESI). 3.2 Computational Studies A microscopic picture of the hydration process can be obtained from the analysis of the geometry optimized structures of L-Citrulline upon sequential addition of H2O per single crystal (Fig. S5, ESI). Fig. S6c shows that the simulated arrangement of water in the dihydrate form, which agrees with the single crystal X-ray data (Fig. S3, ESI), i.e. a macrocylic complex of six membered ring is formed by 6
strong hydrogen bonded water molecules with carboxylate, ammonium ions and ureido carbonyls in L-citrulline. During the hydration process, the first H2O molecule (Fig. S6b) interacts with carboxylate, ammonium ions and ureido carbonyls in L-citrulline by strong hydrogen bond. In this step, one water molecule interacts with one ammonium ions and two amide carbonyl involving three citrulline molecules only. The possibility of making hydrogen bond with another water molecule is not there in the monohydrate. Therefore, the stability of this structure is very weak as compared to the dihydrate form, which is clearly observed from the large difference in their binding energy of water molecules in sequential addition of H2O to the single crystal of L-Citrulline, i.e. 34.6 kcal/mol and ‒ 50.4 kcal/mol for mono and dihydrate, respectively. The binding energy calculation further supports the kinetic nature of the monohydrate crystals.
3.3 Phase transformations Crystallization is a very complex phenomenon, which initiates with the appearance of the high energy metastable form (more soluble) and ends with the most stable form (less soluble) via several intermediate phase transformation [44-46]. The nucleation of the stable form can be initiated by the dissolution of the metastable form and growth of the stable form continues until the supersaturation achieve [47]. The real challenge is to trap the metastable phases to understand the crystallization phenomena. Several molecular conformations of L-citrulline in its polymorphs and hydrates constitute a crystal structure landscape [41-42], in which all the high energy conformations can simultaneously compete with each other during the crystallization events and ultimately try to pack more efficient way. Sometimes it may crystallize as an anhydrous form or hydrate form, when water molecule satisfies donor/acceptor interactions in the crystal system depending upon the need of the molecular conformation. Subtle differences in the molecular conformations of citrulline resulting from the difference in hydrogen bonding interactions provide two anhydrous forms and mono/dihydrates. Phase transformations between the four crystalline forms of L-citrulline are summarized in Fig. 6. Form M was only stable in the mother liquor and started getting opaque when exposed in the air within 5-10 minutes. This seems like crystallization process is endothermic in nature, i.e. binding energy of 34.6 kcal/mol, so it requires extra energy to be stable without the mother liquor. The stabilization energies of anhydrous and hydrated structures of L-citrulline are in the order of dihydrate > sesquihydrate > anhydrous> hemihydrate> monohydrate according to the relative stabilization energy profile (Fig. 7) obtained from the computational studies on hydration of L-citrulline, which further support the experimental observation. Experimentally, we have not found hemi/sesquihydrate during high throughput screening and the probability of sesquihydrate is more due to its close stabilization energy as dihydrate. In the crystal structure, water molecules present in the channel along the crystallographic b axis, which is easier to escape from the crystal system due to highly unstable conformation when compared to the strongly hydrogen bonded dihydrate form. Even in Form M, only one hydrogen bonded contact exists between water molecule and amide carbonyl of Citrulline, which 7
may be the reason behind its poor stability. Hydrate solid forms after dehydration, generally transform to a metastable form or stable anhydrous form [48]. Form M spontaneously transformed to Form II, which was further confirmed by DSC and FT-IR spectroscopy (discussed next). Citrulline molecules in Form M rearrange themselves to adopt Form II conformation in the supramolecular level followed by spontaneous dehydration due their conformational similarity (Fig. S4, ESI). The DFT optimized monohydrate single crystal structure further confirm the different molecular conformation of Lcitrulline. Dihydrate form showed better stability at ambient conditions with exothermic binding energy of -50.0 kcal/mol. Only on heating above 100°C, it dehydrated and transformed to the mixture of Forms I and II, which was confirmed from its XRD pattern (Fig. S7, ESI). 3.3 Hirshfeld surface analysis Hirshfeld surface (using Crystal Explorer, version 17.5 software) serves as an effective tool to obtain additional information of short or long range contacts in a crystal structures [49-50]. The 2D fingerprint plot provides the nature and type of intermolecular interactions quantitatively between the molecules in the crystal surface. Here we would like to find out the reason behind metastable nature of Form M and why it transformed to the anhydrous Form II, not Form I by examining their polar/non-polar interactions on their crystal surfaces. Due to the presence of water molecules in the hydrates, polar interactions (O···H) contribute more towards hydration stability compared to the anhydrous Forms (Fig. 8). However, both Form I and II have comparable interactions, which make difficult task for a crystallographer about the stability preference. If we consider only van der Walls interactions (H···H), there is a close relation between Form II and Form M (43.8 vs 43.7%). Of course, there is an important role of both polar and nonpolar interactions in stabilizing crystal lattice. Hirshfeld surface analysis suggests the stability order may be as dihydrate (93.5%)>Form M (90.8%)>Form I (90.5%)>Form II (88.6%) considering both polar and non-polar interactions. However, Form M is the least stable form at the ambient conditions and dihydrate is the most stable hydrate form, observed from experimental and DFT calculation. 3.4 Vibrational spectroscopy and thermal stability Vibrational spectroscopy is a particularly useful tool to differentiate between the polymorphs of a compound depending upon the functional groups involved in different synthon formation and also conformational change in the molecular level. Although both the L-citrulline polymorphs have similar tetramer synthons, they differ in molecular orientations and packing arrangement which may affect the vibrational pattern in the solid state. L-citrulline exhibits N+–H band above 3300 cm–1, N–H band within 3100-3220 cm–1, C=O band at 1670-1690 cm–1 (Table S2, supporting information). IR bands of Form I and II exhibited quite different vibrational frequencies in the C=O (1684 vs 1671 cm–1) and NH (3374 vs 3357 cm–1) stretching regions, see Fig. 9. Inspite of close (carbamoyl) C=O bond
8
distances (1.25 and 1.26 Å) and carbonyl group is hydrogen bonded with tertiary ammonium ion and NH2 of ureido group, Form I and II exhibit distinct vibrational frequencies may be because of differences in conformations and overall packing. Form M was not stable in air (got opaque) and showed similar absorption bands as commercial form II and this suggests there was a quick phase transition to anhydrous form II, when exposed to air. The dihydrate phase showed C=O band (1669 cm–1) close to Form II and also exhibits characteristic O–H bands of two water molecules at 3436 cm– 1
. Differential scanning Calorimetry (DSC) measures the exact melting point, phase transition
and suggests the relationship between the polymorphs [51]. Both the citrulline polymorphs melted without any phase transition (Fig. 10). Enthalpy of fusion of Form I and II are 30.4 and 22.6 Kcal/mol indicates slightly higher thermal stability of Form I than Form II. Even Form I exhibited slightly higher melting endotherms compared to Form II (214 vs 209°C). According to the “Enthalpy of fusion rule” [52], both the polymorphs are monotropically related. Thermodynamic stability of Form II was explained based on the slurry experiments as mixture of Forms I and II resulted Form II only. Opequed Form M (blue trace) did not exhibit any endotherm corresponds to water loss and directly melts at 209 °C, similar to Form II. This indicates Form M undergoes phase transformation to Form II after dehydration. Stable citrulline dihydrate (green trace) releases two water molecules in two successive steps at 65.8 and 78.5 °C, followed by phase transform to Form II and finally melts at 233.5 °C, which is quite higher than its polymorphs and suggests the possibility of a new polymorph.
4. Conclusions L-Citrulline crystallized as two anhydrous polymorphs and two hydrates; metastable monohydrate and a stable dihydrate during solid form screening. Being a chiral L-amino acid, both the anhydrous forms and hydrates crystallize in chiral space groups. Here we report the reproducible crystallization processes for Form I, II and dihydrate, along with a new metastable monohydrate. Both the polymorphs differ by conformation in the side alkyl chain moiety including the carbamoylamino group, which reflected in distinct vibrational frequencies. Slightly higher melting point and polar/nonpolar interactions indicated better stability of Form I compared to Form II. However, frequent crystallizations, better crystal packing (or calculated density), mechanical/slurry grinding and lower solubility of Form II indicated more stability than Form I. Dihydrate is highly stable at ambient conditions that is reflected in higher crystal density (or packing fractions) than the monohydrate. Monohydrate (channel hydrate) spontaneously transformed to Form II and confirmed by DSC and IR of the corresponding opaque solid form. Binding energy calculation suggests that dihydrate is the most stable solid form, followed by anhydrous form and the monohydrate is the least stable form. Crystallizing monohydrate and stabilizing the kinetic phase may be challenging one.
9
Supporting Information Appendix A. Neutron normalized hydrogen bonds, vibrational frequencies of solid Forms, DFT optimized cell parameters, packing diagrams, PXRD comparison, hydrogen bonding network in geometry optimized crystal structures and crystallographic information files (.cif) are available in the electronic supporting information.
Appendix B. CIF file CCDC no. 1006847-1006849 Author information Corresponding author *Email: [email protected] ORCiD ID: https://orcid.org/0000-0001-7854-1964 Tel.: +91 91 68280808 Acknowledgements P.S. and R. S. P. thank SRM Institute of Science and Technology for giving basic research facilities and acknowledge DST˗FIST fund (No. SR/FST/CST-266/2015(c)) for improvement of S&T infrastructures of Department of Chemistry. We thank Prof. Gautam R. Desiraju for the single crystal X-ray data.
Conflicts of interest There are no conflicts to declare. References 1) R. K. Khankari, D. J. W. Grant, Pharmaceutical Hydrates, Thermochim. Acta 248 (1995) 6179. 2) S. R. Vipagunta, H. G. Brittain and D. J. W. Grant, Crystalline solids, Adv. Drug Delivery Rev. 48 (2001), 3-26. 3) D. Braga, F. Grepioni, L. Chelazzi, M. Campana, D. Confortini, G. C. Viscomi, The structure–property relationship of four crystal forms, CrystEngcomm 14 (2012) 6404-6411. 4) D. E. Braun, R. M. Bhardwaj, J. B. Arlin, A. J. Florence, V. Kahlenberg, U. J. Griesser, D. A. Tocher, S. L. Price, Absorbing a Little Water: The Structural, Thermodynamic, and Kinetic Relationship between Pyrogallol and Its Tetarto-Hydrate, Cryst Growth Des. 13 (2013) 40714083.
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22) Rigaku Mercury375R/M CCD. 2009 CrystalClear-SM Expert 2.0 rc14; Rigaku Corporation: Tokyo, Japan. 23) G. M. Sheldrick, SHELXT - Integrated space-group and crystal-structure determination, Acta Cryst. A71 (2015) 3-8. 24) L. J. Farrugia, WinGX and ORTEP for Windows: an update J. Appl. Cryst. 45 (2012) 849854. 25) A. L. Spek, Single crystal structure validation with the program PLATON, J. Appl. Crystallogr. 36 (2003) 7-13. 26) L. J. Barbour, X-Seed - A Software Tool for Supramolecular Crystallography, J. Supramol. Chem. 1 (2001) 189-191. 27) C. F. Macrae, P. R. Edgington, P. McCabe, E. Pidcock, G. P. Shields, R. Taylor, M. Towler, J. van de Streek, Mercury Visualization and Analysis of Crystal Structures, J. Appl. Cryst. 39 (2006) 453-457. 28) J. VandeVondele, M. Krack, F. Mohamed, M. Parrinello, T. Chassaing, J. Hutter, Quickstep: Fast and accurate density functional calculations using a mixed Gaussian and plane waves approach, Comput. Phys Commun, 167 (2005) 103-128. 29) J. VandeVondele, J. Hutter, An efficient orbital transformation method for electronic structure calculations, J. Chem. Phys. 118 (2003) 4365-4369. 30) G. Lippert, J. Hutter, M. Parrinello, The Gaussian and augmented-plane-wave density functional method for ab initio molecular dynamics simulations, Theor Chem Acc, 103 (1999) 124-140. 31) G. Lippert, J. Hutter, M. Parrinello, A hybrid Gaussian and plane wave density functional scheme, Mol. Phys. 92 (1997) 477-488. 32) J. P. Perdew, W. Yue, Accurate and simple density functional for the electronic exchange energy: Generalized gradient approximation, Phys. Rev. B33 (1986) 8800-8802. 33) S. Goedecker, M. Teter, J. Hutter, Separable dual-space Gaussian pseudopotentials, Phys. Rev. B. 54 (1996) 1703-1710. 34) S. Grimme, J. Antony, S. Ehrlich, H. Krieng, A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements, HPu J. Chem. Phys. 132 (2010) 154104-154119. 35) S. F. Boys, F. Bernardi, The calculation of small molecular interactions by the differences of separate total energies. Some procedures with reduced errors, Mol. Phys. 19 (1970) 553-566. 36) M. C. Etter, J. C. Macdonald, J. Bernstein, Graph-set analysis of hydrogen-bond patterns in organic crystals, Acta Cryst. B46 (1990) 256-262. 37) J. Bernstein, R. E. Davis, L. Shimoni, N. L. Chang, Patterns in Hydrogen Bonding: Functionality and Graph Set Analysis in Crystals Angew. Chem. Int. Ed. Eng. 34 (1995) 15551573. 12
38) E. Courvoisier, P. A. Williams, G. K. Lim, C. E. Hughesa, K. D. M. Harris, The crystal structure of L-arginine, Chem. Commun. 48 (2012) 2761-2763. 39) A. Nangia, Conformational polymorphism in organic crystals, Acc Chem Res 41(2008) 595604. 40) A. Cruz-Cabeza, J. Bernstein, Conformational Polymorphism Chem. Rev. 114 (2014), 21702191. 41) T. S. Thakur, R. Dubey, G. R. Desiraju, Crystal Structure and Prediction, Annual Review of Physical Chemistry 66 (2015) 21-42. 42) R. Dubey, M. S. Pavan, G. R. Desiraju, Structural landscape of benzoic acid: using experimental crystal structures of fluorobenzoic acids as a probe, Chem. Commun. 48 (2012) 9020-9022. 43) M. Novotny, G. J. Kleywegt, A survey of left-handed helices in protein structures, J. Mol. Biol. 347 (2005) 231-241. 44) Y. Lian, Nucleation of One Polymorph by Another, J. Am. Chem. Soc. 125 (2003) 6380-6381. 45) L. Yu, Survival of the fittest polymorph: how fast nucleater can lose to fast grower, CrystEngComm 9 (2007) 847-851. 46) G. R. Desiraju, Crystal engineering: from molecule to crystal, J. Am. Chem. Soc. 135 (2013) 9952-9967. 47) W. F. Ostwald, Studies on formation and transformation of solid materials, Z Phys Chem 22 (1897) 289-310. 48) J. Mahieux, M. Sanselme, G. Coquerel, Cryst. Growth Des. 16 (2016) 396-405. 49) J. J. McKinnon, M. A. Spackman, A. S. Mitchell, Novel tools for visualizing and exploring intermolecular interactions in molecular crystals, Acta Crystallogr. B60 (2004) 627-668. 50) M. A. Spackman, D. Jayatilaka, Hirshfeld surface analysis, CrystEngComm 11 (2009) 19-32. 51) W. Y. Zhang, Y. Y. Tang, P. F. Li, P. P. Shi, W. Q. Liao, D. W. Fu, H. Y. Ye, Y. Zhang, R. G. Xiong, Precise Molecular Design of High‐Tc 3D Organic−Inorganic Perovskite Ferroelectric: [MeHdabco]RbI3 (MeHdabco = N‐Methyl-1,4-diazoniabicyclo[2.2.2]octane), J. Am. Chem. Soc. 139 (2017) 10897−10902. 52) A. Burger, R. Ramberger, On the polymorphism of pharmaceuticals and other molecular crystals, Microchimica Acta. II 72 (1979) 259-271.
13
Table 1 Crystallizations and outcome of citrulline polymorphs (forms) Solvents of crystallization Water MeOH-water EtOH-water 2-PrOH-water n-butanol-water Aceteone-water THF-water EtOAc-water CH3NO2-water (beaker) CH3NO2-water (conical)
Forms
Solvent of crystallization
Forms
Form I+Form II Form II Form II Form II Form II Form I Form I Form I Form I Form II
Form II Form II Form II Monohydrate Form II Form II Form II Dihydrate Form II Form II
Slurry in water (24h)
Form I+Form II
pH 4 buffer (conical) pH 7 buffer pH 9 buffer Water-EtOAc (beaker) Water-EtOAc (conical) Ethylacetoacetate-water DMF-water pH 4 buffer (beaker) Citrullline+glycine (water) Citrullline+asparagine (water) Aqueous solution under vaccuam
CHCl3-water
Form II
Dihydrate
Table 2 Crystallographic parameters of L-Citrulline polymorphs and monohydrate L-Citrulline
Form I
Form II
Monohydrate
(recollected)
(recollected)
(Form M, new)
C6Hl3N3O3
C6Hl3N3O3
C6Hl3N3O3, H2O
175.19
175.19
193.21
Monoclinic
Orthorhombic
Monoclinic
P21
P212121
C2
150(2)
150(2)
150(2)
a/Å
8.940(4)
5.330(2)
10.284(11)
b/Å
5.135(2)
9.895(5)
4.661(5)
c/Å
9.082(5)
14.741(6)
20.01(2)
α/°
90
90
90
β/°
95.169(16)
90
102.356(10)
γ/°
90
90
90
Chemical formula Formula weight Crystal system Space group T (K)
14
V/Å3
415.3(3)
777.4(6)
936.9(18)
1.401
1.497
1.370
0.112
0.120
0.114
2
4
4
R1 [I > 2 σ(I)]
0.0417
0.0464
0.0661
wR2 (all)
0.1314
0.1239
0.2244
GOF
1.205
1.095
1.096
Ck (%)
70.6
75.2
69.3
Rigaku-CCD
Rigaku-CCD
Rigaku-CCD
1006847
1006848
1006849
calc/g
cm−3
µ/mm−1 Z
Diffractometer CCDC Nos.
Table 3 Neutron normalized hydrogen bond (Å/ º) Crystal Form
Interactions
H···A /Å
D···A /Å
∠D–H···A /º
Symmetry code
Form M
N1–H1A···O1
1.84
2.784(5)
164
-1/2+x,1/2+y,z
N1–H1A···O2
2.43
3.115(5)
128
-1/2+x,1/2+y,z
N1–H1B···O2
1.85
2.781(4)
164
1/2-x,1/2+y,-z
N1–H1C···O1
2.08
2.982(5)
168
-1/2+x,-1/2+y,z
N2–H2A···O3
2.16
2.836(6)
163
x,1+y,z
N3–H3A···O4
2.50
3.153(6)
143
-x,y,1-z
N3–H3B···O3
2.43
3.078(6)
145
x,1+y,z
O4–H4···O3
2.18
2.808(5)
158
½+x,-1/2+y,z
Scheme 1 Chemical structures of zwitterionic L-citrulline and its close analogues L-arginine, Lornithine and L-lysine. O H 2N
NH COO
N H
+ NH3
H 2N
L-Citrulline
H 2N
COO
N H
+ NH3
L-Arginine
COO
COO
H 2N
+ NH3
+ NH3
L-Ornithine
L-Lysine
15
Fig. 1 Hydrogen bonded ribbon motif in Form I.
Fig. 2 Hydrogen bonding in Form II.
Fig. 3 (a) L-citrulline molecules show flexible torsions along the intermediate alkyl chains. (b) Molecular overlay of L-Citrulline polymorphs: Form I-red and Form II-blue. Notice ammonium ion and carbamoylamino attached to aliphatic carbons are anti to each other in Form II, but they are close to syn orientation in Form I.
16
O H 2N
COO
N H
+ NH3
(a)
(b)
Fig. 4 (a) Hydrogen bonding in Form M. (b) Packing diagram of Form M, viewed down the a-axis. Left handed α-helix is highlighted in green lines. Water molecules present in a channel parallel to the b-axis.
(a)
(b) Fig. 5 Molecular overlay of L-citrulline in Form II (blue trace), Form M (green trace) and dihydrate (magenta trace) form indicates close similarity in conformation except in the side chain ureido moiety.
17
Fig. 6 Phase transformations between anhydrous and hydrate forms of L-citrulline.
Fig. 7. The relative energy profile for the hydration of L-citrulline obtained from the periodic DFT calculations (provided 1 eV=23.061 Kcal/mol) that indicates monohydrate is the least stable form.
18
Fig. 8 Polar and non-polar interactions of L-Citrulline forms. Percentages are given on the histogram only for the major atom-type/atom-type contacts.
Fig. 9 Vibrational frequency comparisons of Form I, II, M (opaque) and dihydrate.
19
Fig. 10 DSC endotherms of L-citrulline polymorphs (black & red trace) indicate monotropic relationship between the two forms. Monohydrate transformed to Form II after dehydration.
20
Graphical abstract
A metastable monohydrate of L-Citrulline was crystallized, which spontaneously transformed to anhydrous Form II because of its channel hydrate structure. The relative binding energy profile for the hydration of L-citrulline obtained from the periodic DFT calculations that indicates the monohydrate is the less stable form compared to other possible hydrates.
21
Highlights of “A disappearing metastable hydrate form of L-Citrulline: Variable conformations in polymorphs and hydrates” are mentioned as below. • • • •
First time, we have reported a metastable L-Citrulline monohydrate crystal structure. Metastable nature of the monohydrate was explained based on the binding energy calculations using Periodic Density Functional Theory (DFT) approach. Highlighted the structural differences between monohydrate, dihydrate and metastable nature of the former. The conformational similarity between monohydrate and anhydrous (Form II) prompted rapid phase transformation between them.
S0022-2860(19)31288-8
DOI:
https://doi.org/10.1016/j.molstruc.2019.127179
Reference:
MOLSTR 127179
To appear in:
Journal of Molecular Structure
Received Date: 11 July 2019 Revised Date:
19 September 2019
Accepted Date: 3 October 2019
Please cite this article as: P. Sanphui, R.S. Pillai, A disappearing metastable hydrate form of L-citrulline: Variable conformations in polymorphs and hydrates, Journal of Molecular Structure (2019), doi: https:// doi.org/10.1016/j.molstruc.2019.127179. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier B.V.
A disappearing metastable hydrate form of L-Citrulline: Variable conformations in polymorphs and hydrates Palash Sanphui* and Renjith S. Pillai Department of Chemistry, SRM Institute of Science and Technology, Kattankulathur-603203, India
Abstract An unstable monohydrate crystalline form of L-citrulline was harvested during high throughput solid form screening and its crystal structure and hydrogen bonding patterns are elaborated. The crystal structures of the previously reported anhydrous polymorphs and dihydrate are analysed and compared their conformational flexibility. Periodic Density Functional Theory (DFT) approach was employed to unveil the relative stability of citrulline upon hydration and its microscopic interactions with water molecules. The relative binding energy suggests the order of stability as dihydrate > citrulline (anhydrous) > monohydrate. Metastable nature of the monohydrate is reasoned based on the presence of water molecules in the channel structure, less interactions with the citrulline molecules and lower crystal density compared to the other solid forms. Unlike the reported dihydrate form, monohydrate transformed to the anhydrous citrulline (Form II) on spontaneous dehydration at ambient conditions because of their similar conformational flexibility. First time, we report reproducible crystallization process of polymorphs, dihydrate and a novel metastable monohydrate of L-Citrulline.
Keywords: Citrulline, polymorphs, hydrates, hydrogen bond, binding energy
1. Introduction The crystallization of hydrates cannot be speculated during solid form screening involving moist solvents or water, which often favour better packing than the corresponding anhydrous form. Generally hydrates/solvates are crystallized when there are imbalance in the number of donorsacceptors in a crystal structure [1-4]. The crystalline hydrates are useful solid form because of their ability to tune physical properties of a drug [5-6]. Approximately, 33% of active pharmaceutical ingredients (APIs) form hydrates due to the small size of water molecule, which can fit nicely into the crystal lattice and also its donors (2)–acceptor (1) balance capability to make multiple hydrogen bonds. Hydrates are predominantly less soluble than their anhydrous counterparts [7-9]. In the hydrate form, the API interacts closely with water and free energy released during the crystal dissolution. Hence, further interactions with water is less probable for the hydrate than the anhydrous phase. Pharmaceutical solids may come in contact with water during crystallizations, lyophilisation, wet granulation etc. Hence thorough investigation on the probable hydrate form of a drug is necessary to avoid late stage discovery, which may cost a huge loss to the innovator. 1
L-Citrulline (chemical name: L-2-Amino-5-ureidovaleric acid, Scheme 1) is an active ingredient of watermelon and other dietary sources like cucumber, squash, pumpkin etc. It is found to relax blood vessels and hence improve cardiovascular health [10]. Greco et al. claimed that citrulline can also be used to treat erectile dysfunction [11]. It is marketed in the form of citrulline malate (brand name Stimol®) as a performance enhancing athletic health drinks. L-citrulline catabolises to Larginine after absorption and goes to Kidneys. Hence, it is actually a better method to obtain supplemental L-arginine into the blood [12]. Although, L-citrulline is not among the 18 essential amino acids, the conformation and size of citrulline is identical with the basic amino acids like lysine, arginine and ornithine, see Scheme 1. Citrulline is more readily absorbed and more bioavailable than L-arginine. Hence, L-citrulline has commercial importance as nutraceuticals and its solid forms need to be screened for its pharmaceutical interest. Being an amino acid (zwitterionic form), citrulline shows high aqueous solubility of 200 g/L at 20 °C. The crystal structures of L-citrulline polymorphs (Form I and II), dihydrate, hydrochloride, perchlorate, oxalate salt monohydrate and cocrystal with L-malic acid are reported in the literature [13-18]. According to a Japanese patent JP 56-99453, L-citrulline crystallizes in three polymorphs [19]. There is no clear specification about the polymorphs crystallizations and also their packing differences are not discussed in the prior art. We have revisited citrulline polymorphs via high throughput solid form screening and reproduced the two reported polymorphs (3D coordinates), dihydrate and a new metastable monohydrate. Crystal structure of citrulline (Form I) was reported more than three decades ago [13]. Allouchi et al. reported 2nd form, which was difficult to harvest the single crystals of Form II from solvent crystallizations [18]. Recently, Rossi and co-workers also reported single crystal X-ray data of Form II and proved spontaneous hydrolysis of L-arginine to Lcitrulline as energetically preferred reaction using Density functional theory [20]. Here we provide the reproducible crystallization methods of two anhydrous forms and dihydrate for the first time. In addition, we report the crystal structure of citrulline monohydrate, which appeared only once during high throughput crystallization. Citrulline monohydrate is a kind of disappearing solid form [21], which we could not able to reproduce irrespective of our best crystallization efforts. The subtle hydrogen bonding differences in the conformational polymorphs of citrulline are discussed in details. The relative stability of citrulline upon hydration and its microscopic interactions with water molecules are explained based on relative binding energy calculations using periodic Density Functional Theory (DFT) approach. All the citrulline solid forms are further characterized with FTIR, PXRD and DSC and elaborated. 2. Materials and methods L-Citrulline was obtained from Sigma Aldrich, Bangalore and used directly for the crystallization experiments. The solvents were purchased from commercial sources and used without further purification. Water filtered through a double distilled water purification system (Siemens, Ultra Clear, 2
Germany) was used in all the crystallization experiments. Powder X-ray diffraction (PXRD) data was recorded using a Philips X’pert Pro X-ray powder diffractometer (Cu-Kα radiation, λ = 1.54056 Å) equipped with an X’cellerator detector at room temperature with the scan range 2θ= 5 to 40º and step size 0.017º. X'Pert HighScore Plus was used to compare the experimental XRD pattern of Citrulline polymorphs and dihydrate with the simulated X-ray lines from their crystal structures (Fig. S2, supporting information). Crystal Explorer (version 17.5) was employed to compare the intermolecular interactions. Fourier transform IR (FT-IR) spectra were recorded using KBr pellets with a Perkin– Elmer (UK) spectrophotometer (4000–400 cm–1). Differential Scanning Calorimetry (DSC) was performed to obtain exact melting onset/peak on a Mettler Toledo DSC 822e module at the heating rate of 10 ºC/min in the temperature range of 30-250 ºC under nitrogen atmosphere. 2.1 Single Crystal X-ray Diffraction Single crystal X-ray diffraction data of the L-Citrulline polymorphs and monohydrate were collected on a Rigaku Mercury 375/M CCD (XtaLAB mini) diffractometer using graphite monochromated MoKα radiation at 150 K. The data were processed with the Rigaku Crystal clear software [22]. Structure solution and refinements were executed using SHELX-97 [23] and WinGX [24] suite of programs. Refinement of 3D coordinates and anisotropic thermal parameters of non-hydrogen atoms were performed with the full-matrix least-squares method. The hydrogen atoms attached to N/O atoms were located from difference Fourier map or calculated using riding model and further refined isotropically. PLATON software [25] was used to confirm the correctness of the space group. Xseed [26] and Mercury 3.9 softwares [27] were utilized for molecular representations and packing diagrams.
Crystallographic
cif
files
(CCDC
Nos.
1006847-1006849)
are
available
at
www.ccdc.cam.ac.uk/data_request/cif or as part of the Supporting Information.
2.2 Computational Section The experimentally elucidated structure of the L-Citrulline polymorphs (anhydrous form) was initially geometry optimized at the Density Functional Theory (DFT) level using the CP2K package [28-31]. In these simulations, both the positions of atoms of the framework and the unit cell parameters were relaxed. All the structural optimizations were carried out using Perdew-Burke-Ernzerhof (PBE) functional [32] along with a combined Gaussian basis set and pseudopotential. For all the atoms of Lcitrulline, a triple zeta (TZVP-MOLOPT) basis set was considered. The pseudopotentials used for all the atoms were derived by Goedecker et al. [33]. The van der Waals interactions were taken into account via the use of semi-empirical dispersion corrections as implemented in the DFT-D3 method [34]. Hydrated forms of L-Citrulline structural models, i.e. 0.5, 1, 1.5 and 2 water molecules per LCitrulline molecule were generated by incorporating the water molecules into the initially optimized L-Citrulline structure in a progressive manner. These structure models (labeled as L-Citrulline3
0.5H2O,
L-Citrulline-1H2O,
L-Citrulline-1.5H2O
and
L-Citrulline-2H2O
for
hemihydrate,
monohydrate, sesquihydrate and dihydrate, respectively) were further geometry optimized by DFT allowing the relaxation of the atomic positions and the unit cell parameters while maintaining similar to the monoclinic symmetry. All these basis sets had been optimized already to reduce the basis set superposition error (BSSE) to the total system energy, however BSSE have been taken into account when comparing energies for hydrated L-Citrulline structural models. The BSSE corrected energies were obtained from the counterpoise correction method of Boys and Bernardi [35], where the energies are modified to take into account the lowering of the energy caused by the overlap of basis functions between water molecules and L-Citrulline. The optimized unit cell parameters for these structures are reported in Table S3, supporting information. Additionally, the binding energy for water molecules and relative stability energy of L-Citrulline during hydration were calculated from the following equation Ebinding =EL-citrulline+nH2O ‒ EL-citrulline + nEH2 O
Erelative stability =EL-Citrulline+nH2O ‒nEH2 O Where EL-citrulline+nH2O , E
L-citrulline
(1) (2)
, and EH2O represent the total energies of L-citrulline with water
at each hydration stage (n = 0.5, 1, 1.5 and 2), the empty L-citrulline (n= 0) and gas phase H2O, respectively. Equation 1 defines negative values as exothermic and positive values as endothermic processes. The relative stability of L-citrulline, E , upon hydration could be extracted from Equation 2.
3. Results and discussion Crystal structures of L-citrulline polymorphs (Refcode-FIFGOQ/01) were reported earlier by two different research groups [13, 18], we define as Form I and II. Interestingly, Form I was crystallized from polar aprotic solvent such as dioxane, THF, acetone, EtOAc with the addition of dropswise water; whereas stable polymorph II harvested from polar protic solvents (MeOH to butanol) with water, see Table 1 for the details of the crystallization methods. Both the single crystal X-ray diffraction data of the reported Forms are recollected at 150 K to compare their structural difference and packing motifs, see Table 2. Being chiral molecule, both the L-citrulline polymorphs crystallize in non-centrosymmetric space groups (P212121 and P21). Unlike polymorphs or dihydrate form (P212121), monohydrate crystallizes in the monoclinic c-centered (C2) lattice, as the reported hydrochloride salt (Refcode-LCITHC01) [15]. We reproduced the dihydrate phase from the high pressure crystallization method (under vacuum) of saturated aqueous solution and also from pH 4 acetate buffer medium. Lack of crystallography details of citrulline polymorphs and their reproducibility problems in the prior art prompted us to revisit the commercially important compound. 4
We discuss the structural details of the polymorphs and hydrates and then compared their conformational flexibility. 3.1 Crystal structure analysis Form I: In the crystal structure of Form I (P21), citrulline molecules extend as a ribbon motif using alternate N–H···O (between carbonyl group with the terminal ureido NH) and N+–H···O– (between tertiary amine NH+ and carboxylate) hydrogen bonds (Fig. 1). Citrulline molecules form inversion related N+–H···O– hydrogen bonded dimer of R22(18) ring motif of graph set [36-37] through hydrogen bonding between ureido NH and carboxylate. In addition, four L-citrulline molecules are assembled as a tetramer via R42(8), R44(14) and R44(28) ring motifs. Unlike its close analogue Larginine [37], citrulline molecules arrange in a head (carboxylate) to head (ammonium ion) fashion via N+–H···O– hydrogen bonds along the crystallographic b-axis (Fig. S1a, ESI). Carbamoyl moiety of citrulline forms N–H···O hydrogen bonded catemer chains along the crystallography b-axis. 3D packing (Fig. S1b, ESI) along the crystallographic a axis indicates the clear separation of hydrophobic (intermediate aliphatic chains) and hydrophilic moiety (carbamoyl amino and amino acid), which may favour its higher solubility than other forms [18]. Form II: The citrulline conformation in Form II (P212121) is quite distinct from Form I. Similar to Form I, here also citrulline molecules assemble as tetramer ring motif of R43(12), but in a different molecular orientation via N–H···O and N+–H···O– hydrogen bonds (Fig. 2). The tetramer units interact to another via ammonium ion (one side) and ureido motif (other side). Ureido NH's form bifurcated hydrogen bond of R21(6) ring motif with the carboxylate moiety. Like Form I, citrulline molecules arrange in a head to head fashion via N+–H···O– ionic interactions and extended by N– H···O hydrogen bonded catemer chain along the a-axis (Fig. S1c, ESI). Because of their difference in molecular orientation, two forms are termed as conformational polymorphs [39-40]. 3D packing view of Form II indicates zigzag kind of packing arrangement in which two parallel molecules are arranged in a perpendicular fashion to the next ones along the diagonal of bc plane (Fig. S1d, ESI). Two polymorphs differ by conformation in the side alkyl chain moiety including the carbamoylamino group (Fig. 3). Allouchi et al. mentioned, there are minimum five conformations of L-citrulline exists based on the six torsion angles in the crystal structures reported till now [18]. The PXRD pattern of Form I and II in bulk quantity matches with the calculated X-ray lines indicates the bulk phase purity (Fig. S2, ESI). Commercial form (Sigma Aldrich) is identified as Form II by XRD comparison. Calculated density (1.40 vs 1.49 g/cc) and packing fraction (70.6 vs 75.2%) indicates better stability of Form II than Form I. Even more frequent crystallization of Form II than Form I suggests more stability of the former. In addition, grinding of Form I transformed to Form II, which further supports more stability of Form II. Of course, the N···O bond distances (Table S1, ESI) in 5
Form II is slightly longer than in Form I, which may favor little more stability of Form I than Form II. To conclude, both the polymorphs are close to each other in the lattice energy landscape [41-42] and concomitant crystallization occurs when appropriate conditions like slurry experiment or crystallization in water are followed, see Table 1. L-citrulline monohydrate: During high throughput crystallization, L-citrulline monohydrate (Form M) as plate like crystals were harvested from saturated aqueous solution followed by drop wise addition of EtOAc (anti-solvent) after a day. The crystals were not good quality and unstable (opequed) in the air and the corresponding single crystal X-ray diffraction data was collected at 150 K. The crystal structure was solved in C-centered monoclinic C2 space group (Z=4). Form M has similar torsional flexibility as the reported dihydrate (Refcode-DILFAF01) conformation [14], but differ in carbamoyl moiety. Both the hydrates follow the conventional urea tape motif [R21(6)] via bifurcated hydrogen bonds between two NH’s with oxygen atom. Hydrogen bonding parameters of Form M are summarized in Table 3. Similar to polymorphs, citrulline molecules arrange in a head to head fashion, but interrupted in presence of water molecules. One water molecule forms hydrogen bonds with two more water and carbonyl moiety of one citrulline molecule via O–H···O hydrogen bonds (O4–H4···O3: 2.18, 2.81 Å, 158°), see Fig. 4a. Two fold axis related L-citrulline molecules form left handed helix [43] via N+–H···O– ionic hydrogen bonds between ammonium cation and carboxylate anion (Fig. 4b). Water molecules are present in a channel keeping the fixed distance (O···O: 2.79 Å) between them that’s suggests the probability of phase transformation from Form M to anhydrous form. Strong hydrogen bonded water molecules interacting with carboxylate, ammonium ions and ureido carbonyls in L-citrulline dihydrate makes macrocylic complex of six membered ring, R65(12) and is the main reason behind its high stability at ambient conditions. One water molecule interacts with two ammoniam ions and one amide carbonyl involving three citrulline molecules and also with another water molecule in the dihydrate form. Second water molecule forms hydrogen bonds with two carboxylates of citrulline molecules and one water molecule (Fig. S3, ESI). Unlike Form M, water···water hydrogen bonds are limited to two only, which have shorter contacts (O···O: 2.68 Å) in dihydrate form. All these interactions contribute higher crystal density (1.385 vs 1.370 g/cc) and packing efficiency (70.9 vs 68.5%) of dihydrate form compared to monohydrate. L-citrulline in Form M maintains extended chain motif, whereas in the reported dihydrate form or Form II, it is much angular especially in the carbamoyl moiety (Fig. 5 and Fig. S4, ESI). 3.2 Computational Studies A microscopic picture of the hydration process can be obtained from the analysis of the geometry optimized structures of L-Citrulline upon sequential addition of H2O per single crystal (Fig. S5, ESI). Fig. S6c shows that the simulated arrangement of water in the dihydrate form, which agrees with the single crystal X-ray data (Fig. S3, ESI), i.e. a macrocylic complex of six membered ring is formed by 6
strong hydrogen bonded water molecules with carboxylate, ammonium ions and ureido carbonyls in L-citrulline. During the hydration process, the first H2O molecule (Fig. S6b) interacts with carboxylate, ammonium ions and ureido carbonyls in L-citrulline by strong hydrogen bond. In this step, one water molecule interacts with one ammonium ions and two amide carbonyl involving three citrulline molecules only. The possibility of making hydrogen bond with another water molecule is not there in the monohydrate. Therefore, the stability of this structure is very weak as compared to the dihydrate form, which is clearly observed from the large difference in their binding energy of water molecules in sequential addition of H2O to the single crystal of L-Citrulline, i.e. 34.6 kcal/mol and ‒ 50.4 kcal/mol for mono and dihydrate, respectively. The binding energy calculation further supports the kinetic nature of the monohydrate crystals.
3.3 Phase transformations Crystallization is a very complex phenomenon, which initiates with the appearance of the high energy metastable form (more soluble) and ends with the most stable form (less soluble) via several intermediate phase transformation [44-46]. The nucleation of the stable form can be initiated by the dissolution of the metastable form and growth of the stable form continues until the supersaturation achieve [47]. The real challenge is to trap the metastable phases to understand the crystallization phenomena. Several molecular conformations of L-citrulline in its polymorphs and hydrates constitute a crystal structure landscape [41-42], in which all the high energy conformations can simultaneously compete with each other during the crystallization events and ultimately try to pack more efficient way. Sometimes it may crystallize as an anhydrous form or hydrate form, when water molecule satisfies donor/acceptor interactions in the crystal system depending upon the need of the molecular conformation. Subtle differences in the molecular conformations of citrulline resulting from the difference in hydrogen bonding interactions provide two anhydrous forms and mono/dihydrates. Phase transformations between the four crystalline forms of L-citrulline are summarized in Fig. 6. Form M was only stable in the mother liquor and started getting opaque when exposed in the air within 5-10 minutes. This seems like crystallization process is endothermic in nature, i.e. binding energy of 34.6 kcal/mol, so it requires extra energy to be stable without the mother liquor. The stabilization energies of anhydrous and hydrated structures of L-citrulline are in the order of dihydrate > sesquihydrate > anhydrous> hemihydrate> monohydrate according to the relative stabilization energy profile (Fig. 7) obtained from the computational studies on hydration of L-citrulline, which further support the experimental observation. Experimentally, we have not found hemi/sesquihydrate during high throughput screening and the probability of sesquihydrate is more due to its close stabilization energy as dihydrate. In the crystal structure, water molecules present in the channel along the crystallographic b axis, which is easier to escape from the crystal system due to highly unstable conformation when compared to the strongly hydrogen bonded dihydrate form. Even in Form M, only one hydrogen bonded contact exists between water molecule and amide carbonyl of Citrulline, which 7
may be the reason behind its poor stability. Hydrate solid forms after dehydration, generally transform to a metastable form or stable anhydrous form [48]. Form M spontaneously transformed to Form II, which was further confirmed by DSC and FT-IR spectroscopy (discussed next). Citrulline molecules in Form M rearrange themselves to adopt Form II conformation in the supramolecular level followed by spontaneous dehydration due their conformational similarity (Fig. S4, ESI). The DFT optimized monohydrate single crystal structure further confirm the different molecular conformation of Lcitrulline. Dihydrate form showed better stability at ambient conditions with exothermic binding energy of -50.0 kcal/mol. Only on heating above 100°C, it dehydrated and transformed to the mixture of Forms I and II, which was confirmed from its XRD pattern (Fig. S7, ESI). 3.3 Hirshfeld surface analysis Hirshfeld surface (using Crystal Explorer, version 17.5 software) serves as an effective tool to obtain additional information of short or long range contacts in a crystal structures [49-50]. The 2D fingerprint plot provides the nature and type of intermolecular interactions quantitatively between the molecules in the crystal surface. Here we would like to find out the reason behind metastable nature of Form M and why it transformed to the anhydrous Form II, not Form I by examining their polar/non-polar interactions on their crystal surfaces. Due to the presence of water molecules in the hydrates, polar interactions (O···H) contribute more towards hydration stability compared to the anhydrous Forms (Fig. 8). However, both Form I and II have comparable interactions, which make difficult task for a crystallographer about the stability preference. If we consider only van der Walls interactions (H···H), there is a close relation between Form II and Form M (43.8 vs 43.7%). Of course, there is an important role of both polar and nonpolar interactions in stabilizing crystal lattice. Hirshfeld surface analysis suggests the stability order may be as dihydrate (93.5%)>Form M (90.8%)>Form I (90.5%)>Form II (88.6%) considering both polar and non-polar interactions. However, Form M is the least stable form at the ambient conditions and dihydrate is the most stable hydrate form, observed from experimental and DFT calculation. 3.4 Vibrational spectroscopy and thermal stability Vibrational spectroscopy is a particularly useful tool to differentiate between the polymorphs of a compound depending upon the functional groups involved in different synthon formation and also conformational change in the molecular level. Although both the L-citrulline polymorphs have similar tetramer synthons, they differ in molecular orientations and packing arrangement which may affect the vibrational pattern in the solid state. L-citrulline exhibits N+–H band above 3300 cm–1, N–H band within 3100-3220 cm–1, C=O band at 1670-1690 cm–1 (Table S2, supporting information). IR bands of Form I and II exhibited quite different vibrational frequencies in the C=O (1684 vs 1671 cm–1) and NH (3374 vs 3357 cm–1) stretching regions, see Fig. 9. Inspite of close (carbamoyl) C=O bond
8
distances (1.25 and 1.26 Å) and carbonyl group is hydrogen bonded with tertiary ammonium ion and NH2 of ureido group, Form I and II exhibit distinct vibrational frequencies may be because of differences in conformations and overall packing. Form M was not stable in air (got opaque) and showed similar absorption bands as commercial form II and this suggests there was a quick phase transition to anhydrous form II, when exposed to air. The dihydrate phase showed C=O band (1669 cm–1) close to Form II and also exhibits characteristic O–H bands of two water molecules at 3436 cm– 1
. Differential scanning Calorimetry (DSC) measures the exact melting point, phase transition
and suggests the relationship between the polymorphs [51]. Both the citrulline polymorphs melted without any phase transition (Fig. 10). Enthalpy of fusion of Form I and II are 30.4 and 22.6 Kcal/mol indicates slightly higher thermal stability of Form I than Form II. Even Form I exhibited slightly higher melting endotherms compared to Form II (214 vs 209°C). According to the “Enthalpy of fusion rule” [52], both the polymorphs are monotropically related. Thermodynamic stability of Form II was explained based on the slurry experiments as mixture of Forms I and II resulted Form II only. Opequed Form M (blue trace) did not exhibit any endotherm corresponds to water loss and directly melts at 209 °C, similar to Form II. This indicates Form M undergoes phase transformation to Form II after dehydration. Stable citrulline dihydrate (green trace) releases two water molecules in two successive steps at 65.8 and 78.5 °C, followed by phase transform to Form II and finally melts at 233.5 °C, which is quite higher than its polymorphs and suggests the possibility of a new polymorph.
4. Conclusions L-Citrulline crystallized as two anhydrous polymorphs and two hydrates; metastable monohydrate and a stable dihydrate during solid form screening. Being a chiral L-amino acid, both the anhydrous forms and hydrates crystallize in chiral space groups. Here we report the reproducible crystallization processes for Form I, II and dihydrate, along with a new metastable monohydrate. Both the polymorphs differ by conformation in the side alkyl chain moiety including the carbamoylamino group, which reflected in distinct vibrational frequencies. Slightly higher melting point and polar/nonpolar interactions indicated better stability of Form I compared to Form II. However, frequent crystallizations, better crystal packing (or calculated density), mechanical/slurry grinding and lower solubility of Form II indicated more stability than Form I. Dihydrate is highly stable at ambient conditions that is reflected in higher crystal density (or packing fractions) than the monohydrate. Monohydrate (channel hydrate) spontaneously transformed to Form II and confirmed by DSC and IR of the corresponding opaque solid form. Binding energy calculation suggests that dihydrate is the most stable solid form, followed by anhydrous form and the monohydrate is the least stable form. Crystallizing monohydrate and stabilizing the kinetic phase may be challenging one.
9
Supporting Information Appendix A. Neutron normalized hydrogen bonds, vibrational frequencies of solid Forms, DFT optimized cell parameters, packing diagrams, PXRD comparison, hydrogen bonding network in geometry optimized crystal structures and crystallographic information files (.cif) are available in the electronic supporting information.
Appendix B. CIF file CCDC no. 1006847-1006849 Author information Corresponding author *Email: [email protected] ORCiD ID: https://orcid.org/0000-0001-7854-1964 Tel.: +91 91 68280808 Acknowledgements P.S. and R. S. P. thank SRM Institute of Science and Technology for giving basic research facilities and acknowledge DST˗FIST fund (No. SR/FST/CST-266/2015(c)) for improvement of S&T infrastructures of Department of Chemistry. We thank Prof. Gautam R. Desiraju for the single crystal X-ray data.
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Table 1 Crystallizations and outcome of citrulline polymorphs (forms) Solvents of crystallization Water MeOH-water EtOH-water 2-PrOH-water n-butanol-water Aceteone-water THF-water EtOAc-water CH3NO2-water (beaker) CH3NO2-water (conical)
Forms
Solvent of crystallization
Forms
Form I+Form II Form II Form II Form II Form II Form I Form I Form I Form I Form II
Form II Form II Form II Monohydrate Form II Form II Form II Dihydrate Form II Form II
Slurry in water (24h)
Form I+Form II
pH 4 buffer (conical) pH 7 buffer pH 9 buffer Water-EtOAc (beaker) Water-EtOAc (conical) Ethylacetoacetate-water DMF-water pH 4 buffer (beaker) Citrullline+glycine (water) Citrullline+asparagine (water) Aqueous solution under vaccuam
CHCl3-water
Form II
Dihydrate
Table 2 Crystallographic parameters of L-Citrulline polymorphs and monohydrate L-Citrulline
Form I
Form II
Monohydrate
(recollected)
(recollected)
(Form M, new)
C6Hl3N3O3
C6Hl3N3O3
C6Hl3N3O3, H2O
175.19
175.19
193.21
Monoclinic
Orthorhombic
Monoclinic
P21
P212121
C2
150(2)
150(2)
150(2)
a/Å
8.940(4)
5.330(2)
10.284(11)
b/Å
5.135(2)
9.895(5)
4.661(5)
c/Å
9.082(5)
14.741(6)
20.01(2)
α/°
90
90
90
β/°
95.169(16)
90
102.356(10)
γ/°
90
90
90
Chemical formula Formula weight Crystal system Space group T (K)
14
V/Å3
415.3(3)
777.4(6)
936.9(18)
1.401
1.497
1.370
0.112
0.120
0.114
2
4
4
R1 [I > 2 σ(I)]
0.0417
0.0464
0.0661
wR2 (all)
0.1314
0.1239
0.2244
GOF
1.205
1.095
1.096
Ck (%)
70.6
75.2
69.3
Rigaku-CCD
Rigaku-CCD
Rigaku-CCD
1006847
1006848
1006849
calc/g
cm−3
µ/mm−1 Z
Diffractometer CCDC Nos.
Table 3 Neutron normalized hydrogen bond (Å/ º) Crystal Form
Interactions
H···A /Å
D···A /Å
∠D–H···A /º
Symmetry code
Form M
N1–H1A···O1
1.84
2.784(5)
164
-1/2+x,1/2+y,z
N1–H1A···O2
2.43
3.115(5)
128
-1/2+x,1/2+y,z
N1–H1B···O2
1.85
2.781(4)
164
1/2-x,1/2+y,-z
N1–H1C···O1
2.08
2.982(5)
168
-1/2+x,-1/2+y,z
N2–H2A···O3
2.16
2.836(6)
163
x,1+y,z
N3–H3A···O4
2.50
3.153(6)
143
-x,y,1-z
N3–H3B···O3
2.43
3.078(6)
145
x,1+y,z
O4–H4···O3
2.18
2.808(5)
158
½+x,-1/2+y,z
Scheme 1 Chemical structures of zwitterionic L-citrulline and its close analogues L-arginine, Lornithine and L-lysine. O H 2N
NH COO
N H
+ NH3
H 2N
L-Citrulline
H 2N
COO
N H
+ NH3
L-Arginine
COO
COO
H 2N
+ NH3
+ NH3
L-Ornithine
L-Lysine
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Fig. 1 Hydrogen bonded ribbon motif in Form I.
Fig. 2 Hydrogen bonding in Form II.
Fig. 3 (a) L-citrulline molecules show flexible torsions along the intermediate alkyl chains. (b) Molecular overlay of L-Citrulline polymorphs: Form I-red and Form II-blue. Notice ammonium ion and carbamoylamino attached to aliphatic carbons are anti to each other in Form II, but they are close to syn orientation in Form I.
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O H 2N
COO
N H
+ NH3
(a)
(b)
Fig. 4 (a) Hydrogen bonding in Form M. (b) Packing diagram of Form M, viewed down the a-axis. Left handed α-helix is highlighted in green lines. Water molecules present in a channel parallel to the b-axis.
(a)
(b) Fig. 5 Molecular overlay of L-citrulline in Form II (blue trace), Form M (green trace) and dihydrate (magenta trace) form indicates close similarity in conformation except in the side chain ureido moiety.
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Fig. 6 Phase transformations between anhydrous and hydrate forms of L-citrulline.
Fig. 7. The relative energy profile for the hydration of L-citrulline obtained from the periodic DFT calculations (provided 1 eV=23.061 Kcal/mol) that indicates monohydrate is the least stable form.
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Fig. 8 Polar and non-polar interactions of L-Citrulline forms. Percentages are given on the histogram only for the major atom-type/atom-type contacts.
Fig. 9 Vibrational frequency comparisons of Form I, II, M (opaque) and dihydrate.
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Fig. 10 DSC endotherms of L-citrulline polymorphs (black & red trace) indicate monotropic relationship between the two forms. Monohydrate transformed to Form II after dehydration.
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Graphical abstract
A metastable monohydrate of L-Citrulline was crystallized, which spontaneously transformed to anhydrous Form II because of its channel hydrate structure. The relative binding energy profile for the hydration of L-citrulline obtained from the periodic DFT calculations that indicates the monohydrate is the less stable form compared to other possible hydrates.
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Highlights of “A disappearing metastable hydrate form of L-Citrulline: Variable conformations in polymorphs and hydrates” are mentioned as below. • • • •
First time, we have reported a metastable L-Citrulline monohydrate crystal structure. Metastable nature of the monohydrate was explained based on the binding energy calculations using Periodic Density Functional Theory (DFT) approach. Highlighted the structural differences between monohydrate, dihydrate and metastable nature of the former. The conformational similarity between monohydrate and anhydrous (Form II) prompted rapid phase transformation between them.