On the Polymorphism of l-Citrulline: Crystal Structure and

Article pubs.acs.org/crystal

On the Polymorphism of L‑Citrulline: Crystal Structure and Characterization of the Orthorhombic δ Form Hassan Allouchi,† Béatrice Nicolaï,*,‡ Maria Barrio,§ René Céolin,§,⊥ Nathalie Mahé,‡ Josep-Lluís Tamarit,§ Bernard Do,# and Ivo B. Rietveld*,‡ †

Recherche et Innovation en Chimie Médicinale (RICM, ISP-UMR 1282), Faculté de Pharmacie, Université François Rabelais, 31 avenue Monge, 37200 Tours, France ‡ Laboratoire de Chimie Physique (EA4066), Faculté de Pharmacie, Université Paris Descartes, 4, Avenue de l’Observatoire, 75006 Paris, France § Grup de Caracterització de Materials (GCM), Departament de Física i Enginyeria Nuclear, Universitat Politècnica de Catalunya, ETSEIB, Diagonal 647, 08028 Barcelona, Spain # Etablissement Pharmaceutique de l′Assistance Publique-Hôpitaux de Paris, Agence Générale des Equipements et Produits de Santé, 7, rue du Fer à Moulin, 75005 Paris, France S Supporting Information *

ABSTRACT: Solid-state properties of active pharmaceutical compounds are closely related to their dissolution behavior and bioavailability, and understanding their phase behavior leads to better control in the drug formulation process. LCitrulline is an amino acid, which is known to exhibit polymorphism; however, only the structure of the α form is reported in the literature. The structure of the δ form has been elusive due to lack of good quality single crystals. It has been found to be orthorhombic P212121 with the cell parameters a, b, and c respectively 14.895(5) Å, 9.852(2) Å, and 5.353(2) Å. The unit cell contains four molecules, and its volume is 785.5(4) Å3. Both the α form and the δ form possess uniaxial negative thermal expansion along the direction of the charge exchange of the L-citrulline zwitterions. In mixtures containing solid and saturated aqueous solution, the α form quickly transforms in a dihydrate, whereas the δ phase persists for over three weeks. Although the evidence is not conclusive, the δ phase is most likely the more stable form and most suitable for storage.

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INTRODUCTION L-Citrulline, of which the chemical structure is presented in Figure 1, is an amino acid that does not naturally occur in

solubility and dissolution rate. They in turn depend on the solid-state properties of L-citrulline, the main subject of this paper. In the review by Curis et al., several crystal structures of Lcitrulline, a zwitterion, have been listed comprising one anhydrous crystal structure,2 one dihydrate,3,4 and three salts with L-citrulline: a hydrochloride,5,6 a perchlorate,7 and a cocrystal with L-malic acid.8 Another salt, L-citrulline oxalate monohydrate, has been published recently.9 Despite L-citrulline being a flexible molecule with six torsion angles, Curis et al. recognized only two main conformations of L-citrulline in the various crystalline forms, which they confirmed by molecular dynamics simulations.1 The flexibility of L-citrulline suggests a capacity to crystallize in different crystalline forms or polymorphs. Although the existence of polymorphs is not mentioned by Curis et al.,

Figure 1. Chemical structure of L-citrulline.

proteins. Its properties and metabolism have been described exhaustively in a fairly recent review by Curis et al.1 In mammals, it is a precursor of L-arginine and nitrogen monoxide in the urea cycle. Examples of uses of L-citrulline as a therapeutic agent include treatment of L-arginine deficiency and use as a marker of bowel function.1 For the use of L-citrulline as an active pharmaceutical ingredient (API), its absorption behavior in the body is of interest, which is related to its © XXXX American Chemical Society

Received: December 2, 2013 Revised: January 22, 2014

A

dx.doi.org/10.1021/cg401801u | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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further scrutiny of the scientific literature did result in a Japanese patent by Nagata, who reported the existence of at least three polymorphs, α, γ, and δ, besides the aforementioned dihydrate.10 The “whereabouts” of a possible polymorph β, as suggested by the classification in the patent, is not mentioned.10 The three polymorphs occur naturally, can be recognized by their appearance, and are therefore easily separated. X-ray diffraction patterns of the three forms have been provided in the patent. Form δ can be obtained from aqueous solutions in a temperature range of 50−60 °C by seeding. It is mentioned that form δ does not decompose under storage conditions unlike the initial anhydrate (the form is not specified). Neither does form δ take up water to form the dihydrate, according to the patent.10 A preceding patent describes that the dihydrate can be obtained from aqueous solutions below 45 °C by seeding.11 Uncontrolled crystallization from an aqueous solution results in a powdery sample of an undefined anhydrate that decomposes quickly with time. The solubilities of the dihydrate and the undefined anhydrate can be calculated from the examples given in the patent: 60 g of L-citrulline dissolved in 150 mL (resulting in a 200 mL solution at 60 °C) results in 47 g of dihydrate crystal (39 g of crystallized L-citrulline) at 30 °C, whereas spontaneous crystallization of an identical aqueous solution without seeding results in 21 g of L-citrulline crystals at 30 °C.11 This implies that the solubility of L-citrulline at 30 °C is about 21 g of L-citrulline in 150 mL for the dihydrate and 39 g of Lcitrulline in 150 mL for the undefined anhydrous form, almost double the concentration. Taking into account the volume increase due to L-citrulline in the solutions (60 g of L-citrulline leads to an increase of 50 mL of solution), the solubilities become roughly 125 g L−1 for the dihydrate and 215 g L−1 for the undefined anhydrous form. Thermal analysis of form δ resulted in a peak at 477 K, which was interpreted as a fusion/decomposition.10 Other reported melting points of L-citrulline range from 477 to 508 K as found in a search in the Chemical Abstracts database (Scifinder, June 11, 2013). Crystals obtained from water−ethanol mixtures appear to melt or decompose around 493 K.12,13 However, the crystal forms have not been specified in these references. A binary phase diagram of the system L-citrulline−water has been reported by Fournival et al.14 The diagram was obtained by two methods. The first method consisted of direct mixing of water with commercial L-citrulline in different concentrations, and the second method consisted of mixing L-citrulline dihydrate with either commercial L-citrulline or water to sample the L-citrulline-rich side and the water-rich side respectively. In both cases, the melting point related to the liquidus of pure L-citrulline was found to be around 461 K.14 The L-citrulline anhydrate was the same as the one for which the crystal structure had been determined exhibiting the monoclinic space group P21.2 Its calculated diffraction pattern matches the diffraction pattern of the α form in the patent of Nagata, contrary to the statement in the paper of Fournival et al. that it is the δ form.10,14 Furthermore, Nagata states that the δ form does not change into the dihydrate, when in contact with water.10 In the present paper, the long-overdue crystal structure of form δ is reported in combination with the thermal expansion of forms α and δ. Infrared spectra and solubility studies are also reported.

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MATERIALS AND METHODS

Materials. L-Citrulline (L-2-amino-5-ureido-pentanoic acid, 175.19 g mol−1), α and δ forms of medicinal grade, were kindly provided by Biocodex laboratories (Gentilly, France). All attempts to grow δ form crystals from solution suitable for X-ray diffraction failed; only powder samples were obtained. Although most crystals in the commercial batch were either twinned or crystal clusters (Figure 2), serendipitously, a single δ form crystal of reasonable quality, suitable for X-ray diffraction studies, was found among them.

Figure 2. SEM photograph of crystals from the commercial batch provided by Biocodex.

Single Crystal X-ray Diffraction. A single crystal directly obtained from the commercial product was mounted on a glass rod. Intensities were collected by an Enraf-Nonius CAD4 diffractometer using Cu Kα radiation (λ = 1.54178 Å) at 293 K. Data collection and reduction were carried out with the CAD4 Express Enraf-Nonius programs package and XCAD4.15,16 All data were corrected for Lorentz polarization effects. The structure was solved by direct methods and refined by a least-squares method on F2 with the SHELX program.17 Non-hydrogen atoms were refined with anisotropic thermal parameters. H-atoms were positioned geometrically. High Resolution X-ray Powder Diffraction. X-ray powder diffraction was carried out on a transmission mode diffractometer using Debye−Scherrer geometry equipped with cylindrical positionsensitive detectors (CPS120) from INEL (France) containing 4096 channels (0.029° 2θ angular step) with monochromatic Cu Kα1 (λ = 1.5406 Å) radiation. For the measurements as a function of temperature, a liquid nitrogen 700 series Cryostream Cooler from Oxford Cryosystems (United Kingdom) was used. Ground specimens were introduced in a Lindemann capillary (0.5 mm diameter) rotating perpendicularly to the X-ray beam during the experiments to improve the average over the crystallite orientations. Samples have been measured as a function of temperature from 100 K up to 450 K. The sample temperature was equilibrated for about 10 min followed by an acquisition time of ca. 1 h per measurement isotherm. The heating rate in between data collection was 1.33 K min−1. The powder data were analyzed with FullProf v2.05.18 Scanning Electron Microscopy. Electron microscopy pictures have been obtained by a Hitachi TM-1000 (Japan) tabletop scanning electron microscope. Differential Scanning Calorimetry. Differential scanning calorimetry (DSC) experiments were carried out with a Q100 analyzer from TA-Instruments at 5−10 K min−1. Indium was used as a standard for the calibration of temperature and enthalpy change. Samples were weighed with a microbalance sensitive to 0.01 mg and sealed in aluminum pans of 30 μL inner volume. B



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Table 1. Crystal Data and Structure Refinement of δ LCitrulline

Infrared Spectrometry. Infrared spectra were obtained by a Fourier transform Perkin-Elmer Septum spectrometer 1000 using the Spectrum 10000 analysis software. Spectra were recorded at room temperature from 400 to 5000 cm−1. Dissolution and Solubility Studies. Two solutions of 2 g of Lcitrulline in 7 mL of water were prepared, one for form α and one for form δ. Both solutions were kept at 298 K and stirred with an excess of solid remaining. Samples were taken at 5, 10, 20, 40, and 60 min and centrifuged, and the concentration of L-citrulline was determined by colorimetry following the method described in ref 19. Fingerprint Plots and Hirshfeld Surfaces. Hirshfeld surface calculations were performed with CrystalExplorer v3.020 and experimental crystal geometries as input. A Hirshfeld surface encloses the molecular volume based on the electron density difference with the average value of the crystal. Distance di is defined as the distance from the Hirshfeld surface to the nearest interior atom, and de is the distance from the surface to the nearest exterior atom. Plots of de against di, called fingerprint plots, offer the possibility to classify crystals by the nature of their intermolecular interactions and to rapidly identify similarities.21 Isobaric Thermal Expansion Tensor. Intermolecular interactions can be studied with the isobaric thermal expansion tensor, which is a measure for how intermolecular distances change with temperature. A small value for a tensor eigenvalue is commonly referred to as a “hard” direction and a large value as a “soft” direction. The tensor has been calculated by the Deform software.22 The anisotropy of the thermal expansion can be expressed by the aspherism coefficient,23 which is a single value comprising the three coefficients of the tensor and is also provided by the Deform software.

chemical formula molecular mass, g mol−1 crystal system space group Z a, Å b, Å c, Å V, Å3 dc, g cm−3 F(000) absorption μ(Cu Kα), cm−1 crystal dimensions, mm hmin/hmax kmin/kmax lmin/lmax θmax measured reflections independent reflections observed reflections (I > 2σ(I)) parameters Rint/Rσ refinement R (I > 2σ(I)) wR (I > 2σ(I)) S (goodness of fit) Δρmin /Δρmax, e Å−3

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RESULTS Crystal Structure. The single crystal of the δ form found in the commercial batch led to a crystal structure possessing the chiral, orthorhombic space group P212121 with one molecule in the asymmetric unit. Crystal and structure-refinement data have been compiled in Table 1. Supplementary crystallographic data can be found in the CCDC deposit number 973632 and obtained free of charge from the Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif/. The molecular structure with atomic numbering is presented in Figure 3. Diffraction patterns for forms α and δ are presented in Figure 4. No changes in the diffraction patterns have been observed for both polymorphs from 100 K up to 450 K. Unit cell parameters and cell volumes have been determined as a function of temperature by Le Bail fits of the diffraction patterns (Tables S1 and S2 in the Supporting Information for forms α and δ, respectively). The eigenvalues of the thermal expansion tensors of forms α and δ, calculated with the Deform program,22 have been compiled in Table 2, and the 3D-tensors representing the thermal expansion at 300 K are presented in the inset of Figure 4. The principal axes of the tensor of the δ form correspond directly to the cell parameters a, b, and c, as the δ form is orthorhombic. For the monoclinic form α, the principal axis e2 is parallel to the b axis of the unit cell, and the axes e1 and e3 can be found in the ac plane; the latter two move slightly with temperature (Table S3 in the Supporting Information). Thermal Analysis. DSC measurements of the α and δ form did not lead to any detectable transition between the two solid phases. The observed endothermic peaks were always combined with decomposition phenomena. An endothermic peak was observed for δ L-citrulline with an onset at about 480 K at a scanning rate of 5 K min−1. After the measurement, a grainy, brown residue was found in the DSC pan. For α Lcitrulline, an endothermic event reflecting decomposition and

C6H13N3O3 175.19 orthorhombic P212121 4 14.895(5) 9.852(2) 5.353(2) 785.5(4) 1.481 376 1.008 0.175 × 0.113 × 0.075 −14/14 −9/9 0/5 49.94 1820 812 658 133 0.0420/0.0429 F2 0.047 0.133 1.089 −0.21/0.19

Figure 3. Ortep representation of the structure of δ L-citrulline with atomic numbering and thermal ellipsoids (20% probability level).24

starting at 483 K was accompanied by the formation of brown foam leaving the DSC pan through a hole pierced in its cover. Infrared Spectra. The infrared spectra of the α and δ forms are presented in Figure 5. They are clearly different. The spectrum of the δ form resembles the infrared spectrum provided in the Japanese patent.10 Although less well-defined, the infrared spectrum in the Aldrich library resembles the one of the δ form too.26 The infrared spectra of the α and δ forms contain several broad peaks between 2400 and 3200 cm−1 characteristic for the ammonium group. The peak around 1600 cm−1 can be attributed to a carboxylate group. There is no band around 1700 cm−1 characteristic of carboxylic acid. These observations indicate that citrulline is a zwitterion in both solid forms α and δ. Dissolution and Solubility Measurements. The dissolution profiles of α and δ L-citrulline in saturated solutions are presented in Figure 6. It can be seen that the dissolution of α is faster than that of δ. The final concentration of the δ form is C



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Figure 5. Measured infrared spectra of L-citrulline α and δ at room temperature. Vertical line indicates change of scale.

Figure 4. X-ray powder diffraction patterns of the (a) α and (b) δ forms at room temperature (Cu Kα1 radiation). Inset. Thermal expansion tensor obtained with DEFORM 22 and drawn by Wintensor.25

Table 2. Isobaric Thermal Expansion Tensor Eigenvalues of α and δ L-Citrulline in the Orthogonal (e1 e2 e3) Basis T/K

α11/10−5 K−1

100 150 200 250 300 350 400 450

7.4957 7.4705 7.4454 7.4204 7.3954 7.3706 7.3459 7.3213

150 200 250 300 350 400 450

7.1767 7.1510 7.1256 7.1003 7.0751 7.0502 7.0254

α22/10−5 K−1

α33/10−5 K−1

α form 1.7693 −1.8258 1.7678 −1.8535 1.7662 −1.8813 1.7648 −1.9091 1.7631 −1.9370 1.7615 −1.9651 1.7600 −1.9932 1.7584 −2.0215 δ form −1.0349 4.8983 −1.0355 4.8863 −1.0360 4.8744 −1.0366 4.8626 −1.0371 4.8508 −1.0376 4.8391 −1.0152 4.8274

Figure 6. Concentration of L-citrulline form α (solid circles) and form δ (solid squares) in water as a function of time. Broken lines are guides to the eye.

aspherism coefficient 0.7297 0.7350 0.7404 0.7459 0.7514 0.7571 0.7629 0.7688

peaks in the X-ray diffraction pattern, which could be ascribed to the δ form. This indicates that the α form transforms into the δ form under ambient conditions by providing additional energy by grinding.

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DISCUSSION Crystal Structure of the δ Form, Interactions, and Comparisons. The crystal structure of L-citrulline form δ is orthorhombic P212121 with Z = 4, whereas form α has a monoclinic P21 crystal structure with Z = 2.2 Form δ is more compact with a smaller V/Z of 197 Å3 versus 211 Å3 for α at 300 K. In form α, columns of antiparallel L-citrulline molecules are present related by the 21 screw axis (Figure 7) parallel to the c axis. In form δ, only pairs of antiparallel L-citrulline molecules exist. The dihydrate possesses similar pairs of antiparallel molecules separated by water molecules.3 In the crystal structure of the δ form, hydrogen bonds form a complex three-dimensional network. Each L-citrulline molecule is involved in 10 fairly strong hydrogen bonds and 4 weaker ones with 10 different molecules (Table 3). The nitrogen atoms are the donors and the oxygen atoms are the acceptors; all hydrogen bonds are therefore of the N−H···O type. Some donors and acceptors form single hydrogen bonds; others are involved in multiple bonds. The oxygen atom O1 in the carboxylate group participates in two hydrogen bonds with NH3+ groups on two separate molecules. Atom O2 in the carboxylate group is involved in a single hydrogen bond with an NH3+ group on a third molecule. The oxygen atom O3 of the ureido group is involved in two hydrogen bonds, one with an

0.4434 0.4437 0.4439 0.4442 0.4445 0.4448 0.4451

almost equivalent to the solubility of the dihydrate at 125 g L−1 as calculated from the patent by Nagata.10 The dissolution behavior of form α coincides quite well with the value found for the solubility of the unidentified anhydrous form at 215 g L−1, in particular in the early stages of solubilization. Additional experiments by X-ray diffraction demonstrated that α L-citrulline in water became L-citrulline·2H2O in one hour. With ground δ crystals however, the X-ray profile remained that of δ for at least three weeks even with a large excess of H2O. This indicates that the δ form reaches equilibrium only very slowly. Grinding Experiments. Grinding a sample of α L-citrulline with pestle and mortar led to the appearance of additional D



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Figure 7. Comparison of the lattices of α and δ L-citrulline.

Table 3. Hydrogen Bonds in δ L-Citrullinea DH···A N1H1NA···O1i N1H1NB···O3ii N1H1NC···O2iii N2H2N···O1iv N3H3NA···O3v N3H3NB···O2iv N3H3NB···O1iv

DH 0.90 1.03 1.03 0.84 0.90 0.90 0.90

(8) (7) (6) (6) (6) (6) (6)

H···A 1.90 1.84 1.91 2.28 2.00 2.27 2.43

(8) (7) (6) (6) (6) (6) (6)

D···A 2.767 2.767 2.938 3.030 2.897 3.156 3.106

(6) (6) (7) (6) (7) (7) (7)

C5). Comparing the conformations of L-citrulline in its hydrochloride and perchlorate salts with the one of form α, the following can be observed: a rotation of about 10−20° around C2−C1, a rotation of 130° around C2−C3 and a rotation of 120° around C4−C5. The conformation in the perchlorate salt exhibits two additional rotational changes around N2−C5 and around C6−N2. Hence, at least four different conformers are observed in the published structures (see Table 4). In form δ, L-citrulline exhibits yet another conformation rendering forms α and δ conformational polymorphs; only the torsion angle N3−C6−N2−C5 is comparable for the two structures. The existence of conformational polymorphism becomes evident too from the infrared spectra of the two forms demonstrating clear differences between the vibrational frequencies (see Figure 5). The conformations of the dihydrate and form δ are also very different. The various conformations can be seen in Figure S1 in the Supporting Information. Thermal Expansion of the α and δ Forms. The thermal expansion of the δ form can be expressed in terms of the specific volume, v (cm3 g−1):

DH···A 163 147 172 149 178 169 132

(7) (5) (4) (5) (5) (6) (5)

a Symmetry codes: (i) −x + 1, y + 1/2, −z + 1/2. (ii) −x + 1, y − 1/2, −z + 3/2. (iii) x, y, z − 1. (iv) −x + 1/2, −y + 2, z + 1/2. (v) −x + 1/ 2, −y + 3, z + 1/2.

NH3+ group and another with the proton attached to the atom N3. Four weaker hydrogen bonds between the carboxylate group and H3NB also exist (Table 3). In Figure 8, 2D fingerprint plots are presented for form α, form δ, and the dihydrate. These plots visualize the characteristics of intermolecular interactions in the crystals. Their similarity indicates that overall the interactions are comparable in the three crystal structures. The two longest spikes in each diagram of Figure 8 correspond to the N−H···O hydrogen bonds between L-citrulline molecules (or between Lcitrulline and water molecules in the dihydrate). For form α, these spikes are sharp due to an almost perfect geometry for the hydrogen bonds; they have angles ranging from 161.29° to 177.56°.2 For the δ form, there are additional winglike appearances attached to the hydrogen spikes due to a variation between 142.9(4)° and 174.0(4)° for the angles of the five different hydrogen bonds. For the dihydrate, wings can be observed as well, because of hydrogen bond angles varying between 161° and 171°.3 The Conformations of L-Citrulline. As stated in the introduction, L-citrulline is a flexible molecule possessing six torsion angles (Table 4). Curis et al. observed two main conformations based on a single torsion angle. If all six torsion angles are considered, the picture becomes more complicated (Table 4). In form α and in the cocrystal with L-malic acid, Lcitrulline exhibits almost the same conformation; only the position of the carboxylate group (torsion defined by C3−C2− C1−O1) differs by about 10°. This torsion angle is also the same in the dihydrate. The main difference between the conformations of the dihydrate and of form α is a rotation around the bond N2−C5 of about 140° and a small rotation in the ureido group (defined by the torsion angle N3−C6−N2−

vδ = 0.6621 + 2.955 × 10−5T + 8.098 × 10−8T 2

(1)

with T the temperature in kelvin. And for the α form the specific volume can be given by vα = 0.7141 + 2.169 × 10−5T + 5.582 × 10−8T 2

(2)

It can be seen in Figure 9 and eq 1 that the volume of the unit cell of δ and hence the specific volume increases with temperature, as expected. On a molecular scale, the behavior is more complicated, however. In Table 2, it can be seen that the δ form exhibits uniaxial negative thermal expansion along the b axis. The eigenvalues in Table 2 remain fairly constant, indicating that the thermal expansion over the entire temperature range of 150−450 K does not change significantly. The same can be concluded from the aspherism coefficient, whose value remains constant at 0.44. The latter value indicates that the expansion is not isotropic, and this is obviously due to the uniaxial contraction along the b axis. The cause of the uniaxial contraction can be found in the proton exchange between the carboxylate and the ammonium groups, as L-citrulline is a zwitterion. The vector of this charge exchange runs almost parallel to the b axis. The hydrogen bond network mainly controls the thermal expansion in the two other directions (see also Figure S2 and S3 in the Supporting Information). E



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Figure 8. Fingerprint plots obtained by the program CrystalExplorer20 for the crystal structures of L-citrulline form α,2 form δ, and its dihydrate.3 On the left-hand side, all di versus de distances are plotted, the figures marked by O present the relative contributions of the contacts of O atoms with all other atoms, the figures marked by N provide those of the N atoms with all other atoms, and the figures marked by C those of the C atoms. The percentages relative to the entire Hirshfeld surface are given too.

Table 4. Values for the Six Torsion Angles (°) of L-Citrulline in Different Crystal Structures C3−C2−C1−O1 C4−C3−C2−C1 C5−C4−C3−C2 N2−C5−C4−C3 C4−C5−N2−C6 N3−C6−N2−C5 conformation a

form αa

co-crystal with L-malic acidb

dihydratec

hydrochlorided

perchloratee

form δf

109.5 174.3 176.9 56.7 104.3 170.2 1

98.8 174.8 179.1 57.6 98.3 176.0 1

98.4 174.7 176.8 68.8 −115.2 177.9 2

121.7 −55.2 172.1 179.4 91.2 169.9 3

131.7 −53.7 177.4 179.4 162.7 −4.7 4

80.0(6) 59.9(7) 80.6(6) 175.6(5) 173.4(5) 176.5(5) 5

Ref 2. bRef 8. cRef 3. dRef 6. eRef 7. fThis work.

The thermal expansion of the α form exhibits a similar behavior as δ. However, it is more complicated to analyze due to the monoclinic P21 unit cell, and therefore the orthogonal axes describing the thermal expansion do not coincide with

those of the unit cell. From the aspherism coefficient of around 0.75, it can be concluded that the expansion is not isotropic over the considered temperature range. The eigenvalues for the e1 vector are relatively large indicating a strong expansion, F



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decomposition occurring simultaneously with fusion. The fact that melting of the α form is observed, even if in combination with decomposition, and no melting of the δ form is observed, suggests that the δ form melts at a higher temperature than the α form. Considering the solubility data, it is clear that the α form exhibits the so-called parachute effect indicative for dissolution of metastable forms (Figure 6), well-known in the pharmaceutical solid state.28 The δ form exhibits a more gradual increase in dissolved concentration until it levels off. Interestingly, the obtained solubility value is very close to the one found by Nagata for the hydrate form of L-citrulline10 even though δ does not readily form the dihydrate, whereas the α form is replaced within the hour by the dihydrate. This indicates that the δ form takes much longer than the α form to reach equilibrium in solution, which suggests again that the δ form is more stable. It remains unclear however whether δ turns into the dihydrate eventually in the presence of water. The density of form δ is higher, which indicates that it would become more stable under increasing pressure; however, this information alone does not establish whether form δ would be the stable form under ambient conditions. A third element pointing toward the δ form as the more stable form is the behavior of α L-citrulline under grinding, which turns into δ. However, because grinding adds energy to the mixture and can be considered as pressurizing the sample, the formation of the δ form is not entirely unexpected, as it is the denser form of the two polymorphs. Thus, although there are indications that the δ form may be the more stable form under ambient conditions and also close to the melting point, it cannot be concluded with certainty.

Figure 9. Volume per molecule (V/Z) in Å3 for the α (triangles) and δ (circles) polymorphs of L-citrulline as a function of temperature.

whereas in the direction of e2 the expansion is very limited (Table 2). The eigenvalues for the e3 vector are negative, indicating contraction. Again this contraction is in the direction of the charge displacement approximately parallel to the axis connecting the carboxylate and the ammonium groups. Perpendicular to this negative expansion, the thermal expansion is relatively large; it can be observed that there are only few hydrogen bonds in this direction. Along the b axis, the expansion is fairly small, which is probably due to a number of strong hydrogen bonds that all share at least in part this direction (see also Figure S3 in the Supporting Information). The average value of the strongest expansion tensor values in forms α and δ (+7.3 × 10−5 K−1) is smaller than in tienoxolol (+12.3 × 10−5 K−1).27 Along b (e2) in form δ and e3 in form α, the crystals contract on average −1.5 × 10−5 K−1, this negative coefficient is larger than the one observed in tienoxolol (−0.5 × 10−5 K−1).27 From the two expressions for the specific volume (eqs 1 and 2 and also Figure 9), it can be seen that form δ is denser, but it has a larger overall thermal expansion. So although the δ form has a better packing with a higher density, the overall strength of the interactions appears to be somewhat less. Phase Behavior of L-Citrulline. No phase transition between form α and form δ has been observed by DSC or by X-ray diffraction as a function of temperature (Tables S1 and S2 in the Supporting Information). Apparently, the hydrogen networks in the two solid forms are quite strong and prevent one transforming into the other, in particular because the two conformations are quite different. This fact alone may prevent a solid−solid transition. Above 480 K, decomposition can be observed for the δ form. It does not appear to be connected to a melting transition, because only brown grainy material is observed after the experiment, without any trace of liquid formation. Although the value coincides with the temperature mentioned in the Japanese patent of 477 K, the present result refutes the observation of a melting/decomposition by Nagata.10 In the paper by Fournival et al., the melting point of the α form appears to be 461 K.14 In the present study, no melting point was observed below 483 K, after which the sample started to decompose under the appearance of foam. This indicates that melting and decomposition occur simultaneously. In the literature, a melting point around 493 K is often quoted (Scifinder search, June 11, 2013). Enthalpy changes associated with these events cannot be relied upon, because of the

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CONCLUSION

Despite L-citrulline being a difficult system to investigate, a number of conclusions about the crystal structure and the stability behavior of the δ form can be reached. The crystal structure of the δ phase has been determined by single crystal X-ray diffraction at room temperature using a single crystal serendipitously found in the commercial batch. Both forms α and δ possess an extended hydrogen bond network. For the thermal expansion in both solid forms, a uniaxial contraction is observed along the direction of charge exchange between the Lcitrulline zwitterions. L-Citrulline possesses at least five different conformations, almost one for each crystal structure studied. The conformations between the α and the δ phases are very different. Together with the strong hydrogen bond networks in both polymorphs, this most likely explains why no solid−solid transition has been observed between the two phases. It complicates the analysis of the stability hierarchy between the two solid forms. Although the findings are not conclusive, the δ form is most likely more stable than the α form for three observations. (1) No melting transition for the δ form has been observed by DSC, whereas α exhibits a melting decomposition. (2) Grinding helps transformation of α into δ. (3) The dissolution behavior of α reflects that of a metastable phase, whereas δ persists in a saturated solution for at least three weeks. From the point of view of drug formulation, it may therefore be concluded that the δ phase is the best form for storage. G



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ASSOCIATED CONTENT

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AUTHOR INFORMATION

Article

(18) Rodriguez-Carvajal, J. Physica B 1993, 192 (1−2), 55−69. (19) Rahmatullah, M.; Boyde, T. R. C. Clin. Chim. Acta 1980, 107 (1−2), 3−9. (20) Spackman, M. A.; Jayatilaka, D. CrystEngComm 2009, 11 (1), 19−32. (21) Spackman, M. A.; McKinnon, J. J. CrystEngComm 2002, 4, 378− 392. (22) Filhol, A.; Lajzerowicz, J.; Thomas, M. DEFORM, 1987. (23) Parat, B.; Pardo, L. C.; Barrio, M.; Tamarit, J. L.; Negrier, P.; Salud, J.; Lopez, D. O.; Mondieig, D. Chem. Mater. 2005, 17 (13), 3359−3365. (24) Burnett, M. N.; Johnson, C. K. ORTEP-III: Oak Ridge Thermal Ellipsoid Plot Program for Crystal Structure Illustrations; Oak Ridge National Laboratory: Oak Ridge, 1996. (25) Kaminski, W. WinTensor, 1.1; University of Washington, Department of Chemistry: Seattle, WA, 2004; http://cad4.cpac. washington.edu/wintensorhome/wintensor.htm. (26) Pouchert, C. J. The Aldrich Library of Infrared Spectra, 2nd ed.; Aldrich Chemical Co.: Milwaukee, Wisconsin, USA, 1975. (27) Nicolai, B.; Rietveld, I. B.; Barrio, M.; Mahe, N.; Tamarit, J. L.; Ceolin, R.; Guechot, C.; Teulon, J. M. Struct. Chem. 2013, 24 (1), 279−283. (28) Caira, M. R. Top. Curr. Chem. 1998, 198, 163−208.

S Supporting Information *

Table S1: lattice parameters of form α as a function of temperature. Table S2: lattice parameters of form δ as a function of temperature. Table S3: coordinates of the e1 e2 and e3 vectors within the unit cell as a function of temperature for form α. Figure S1: conformations of L-citrulline in different structures. Figure S2: projection of the tensor of form δ in the bc plane. Figure S3: projection of the tensor of form α in the ac plane. Figure S4: curvedness of L-citrulline within the structures of form α and form δ. This information is available free of charge via the Internet at http://pubs.acs.org/.

Corresponding Authors

*(I.B.R.) Tel. +33 1 73539675, e-mail: ivo.rietveld@ parisdescartes.fr. *(B.N.) E-mail: [email protected]. Present Address ⊥

Faculté de Pharmacie, Université Paris Descartes, 4, Avenue de l’Observatoire, 75006 Paris, France. Notes

The authors declare no competing financial interest

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ACKNOWLEDGMENTS Biocodex Laboratories, Gentilly, France, is kindly acknowledged for supplying L-citrulline. M.B. and J.L.T. were supported by the Spanish Ministry of Science and Innovation (Grant FIS2011-24439) and the Catalan Government (Grant 2009SGR-1251).

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REFERENCES

(1) Curis, E.; Nicolis, I.; Moinard, C.; Osowska, S.; Zerrouk, N.; Benazeth, S.; Cynober, L. Amino Acids 2005, 29 (3), 177−205. (2) Toffoli, P.; Khodadad, P.; Rodier, N.; Astoin, J. Acta Crystallogr. C 1987, 43, 945−947. (3) Toffoli, P.; Rodier, N.; Astoin, J. Bull. Soc. Chim. Fr. 1986, 1, 119−123. (4) Xianglin, J.; Zuohua, P.; Ronghan, G.; Qichen, H. Acta Chim. Sinica 1985, 43, 5−9. (5) Naganathan, P. S.; Venkatesan, K. Acta Crystallogr. B 1971, 27, 1079−1085. (6) Ashida, T.; Funakoshi, K.; Tsukihara, T.; Ueki, T.; Kakudo, M. Acta Crystallogr. B 1972, B 28, 1367−1374. (7) Sridhar, B.; Srinivasan, N.; Dalhus, B.; Rajaram, R. K. Acta Crystallogr. E 2002, 58, O1177−O1179. (8) Toffoli, P.; Rodier, N.; Ceolin, R.; Lepage, F.; Astoin, J. Acta Crystallogr. C 1988, 44, 2128−2131. (9) Sreevalsa, V. G.; Jayalekshmi, S. J. Cryst. Growth 2011, 324 (1), 172−176. (10) Nagata, K. Method of crystallization of citrulline. JP 56-99453, 1981. (11) Matsuda, T.; Ohta, H.; Takeyama, T.; Sato, H.; Hayashi, H. Crystallization method of photo-active citrulline dihydrate. JP 46-174, 1971. (12) Rivard, D. E.; Carter, H. E. J. Am. Chem. Soc. 1955, 77 (5), 1260−1261. (13) Kurtz, A. C. J. Biol. Chem. 1949, 180 (3), 1253−1267. (14) Fournival, J. L.; Ceolin, R.; Rouland, J. C.; Toffoli, P.; Khodadad, P.; Astoin, J. J. Therm. Anal. 1987, 32 (1), 213−225. (15) CAD4 Express Software; Enraf-Nonius, Delft, The Netherlands, 1994. (16) Harms, K.; Wocadlo, S. XCAD-CAD4 Data reduction; University of Marburg: Marburg, 1995. (17) Sheldrick, G. M. Acta Crystallogr A 2008, 64, 112−122. H


On the Polymorphism of l-Citrulline: Crystal Structure and

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