Structural Properties, Order–Disorder Phenomena, and Phase Stability of Orotic Acid Crystal Forms

Orotic acid (OTA) is reported to exist in the anhydrous (AH), monohydrate (Hy1), and dimethyl sulfoxide monosolvate (SDMSO) forms. In this study we investigate the (de)hydration/desolvation behavior, aiming at an understanding of the elusive structural features of anhydrous OTA by a combination of experimental and computational techniques, namely, thermal analytical methods, gravimetric moisture (de)sorption studies, water activity measurements, X-ray powder diffraction, spectroscopy (vibrational, solid-state NMR), crystal energy landscape, and chemical shift calculations. The Hy1 is a highly stable hydrate, which dissociates above 135 °C and loses only a small part of the water when stored over desiccants (25 °C) for more than one year. In Hy1, orotic acid and water molecules are linked by strong hydrogen bonds in nearly perfectly planar arranged stacked layers. The layers are spaced by 3.1 Å and not linked via hydrogen bonds. Upon dehydration the X-ray powder diffraction and solid-state NMR peaks become broader, indicating some disorder in the anhydrous form. The Hy1 stacking reflection (122) is maintained, suggesting that the OTA molecules are still arranged in stacked layers in the dehydration product. Desolvation of SDMSO, a nonlayer structure, results in the same AH phase as observed upon dehydrating Hy1. Depending on the desolvation conditions, different levels of order–disorder of layers present in anhydrous OTA are observed, which is also suggested by the computed low energy crystal structures. These structures provide models for stacking faults as intergrowth of different layers is possible. The variability in anhydrate crystals is of practical concern as it affects the moisture dependent stability of AH with respect to hydration.


Conformational Analysis of Orotic Acid
The potential energy surface scan of the OTA diketo tautomer shows two energy minima within 11.1 kJ mol -1 of the global minimum, separated by a significant barrier. The two energy minima are planar and differ by a 180°rotation of the carboxylic acid group ( Figure S1). Both conformations have been observed experimentally, the global minimum in the monohydrate (Hy1) and the local energy minimum in the dimethyl sulfoxide monosolvate (S DMSO ).

DFT-D Calculations: Methodology
The DFT-D calculations were carried out with the CASTEP plane wave code 1 using the Perdew-Burke-Ernzerhof (PBE) generalized gradient approximation (GGA) exchange-correlation density functional 2 and ultrasoft pseudopotentials, 3 with the addition of a semi-empirical dispersion correction, either the Tkatchenko and Scheffler (TS) model, 4 or Grimme06 (G06) 5 . In a first step, the structures were geometry optimized using the TS dispersion correction. Brillouin zone integrations were performed on a symmetrized Monkhorst-Pack k-point grid with the number of k-points chosen to provide a maximum spacing of 0.07 Å −1 and a basis set cut-off of 560 eV. The self-consistent field convergence on total energy was set to 1x10 −5 eV. Energy minimizations were performed using the Broyden-Fletcher-Goldfarb-Shanno optimization scheme within the space group constraints. The optimizations were considered complete when energies were converged to better than 2x10 −5 eV per atom, atomic displacements converged to 1x10 −3 Å, maximum forces to 5x10 −2 eV Å −1 , and maximum stresses were converged to 1x10 −1 GPa. Energy minimizations with variable unit cells (geometry optimizations) were restarted after the first minimization to reduce the effects of changes in unit cell on the basis set. The energies for all anhydrates and monohydrates within 15 kJ mol −1 of the lowest structures were recalculated, without optimization, with the number of k-points chosen to provide a maximum spacing of 0.04 Å −1 and a basis set cutoff of 780 eV, using the G06 dispersion correction, resulting in the final crystal energy landscapes (Figs. 10 & 12). Isolated molecule minimizations to compute the isolated OTA and water energies (Ugas) were performed by placing a single molecule in a fixed cubic 35x35x35 Å 3 unit cell and optimized and recalculated with the same settings used for the crystal calculations.

Computationally Generated Low-Energy Structures (PBE-G06)
All calculated structures are available in .res format from the authors on request. The lowest energy structures derived from DFT-D calculations (PBE-TS optimization and PBE-G06 single point energies) are given in Table S1 (anhydrates) and Table S2 (monohydrates), respectively.

Representation of the Experimental Structures
The computational models were successful in reproducing the experimental Hy1 (OROTAC, 7,8 P-1, Z′=1) and S DMSO (XARBEZ, 9 P21/c, Z′=1) structures (Table S3). The computationally generated structures were compared using the Packing Similarity tool in the Solid Form module of Mercury to determine the root mean square deviation of the non-hydrogen atoms in a cluster of 15 molecules (rmsd15). 10 Table S3. Quality of representation of the PBE-TS structures.   It has to be noted that the non-additivity of the molecule•••molecule polarization energies could not be taken into account for calculating the dimeric energies given in Tables S4-S11. This error was estimated by considering the lattice energies obtained by summing the molecule-molecule pairwise energies. These energies differ from the PIXEL lattice energies when the polarization is calculated from the net field (i.e. accounting for non-additivity of the electrostatic field around a molecule) by approximately a max. ± 6% error in intermolecular energy. The neglect of nonadditivity and distant interactions does not qualitatively affect the interpretation for the structures.

Monohydrate
Selected pair-wise intermolecular interactions for the computed H1 (Table S2) structure are given in Table S11.

DMSO Solvate ( 13 C)
The CASTEP computed shielding constants, σcalc, were converted to chemical shifts, δcalc, according to δcalc = σrefσcalc using a reference value, σref, taken from the zero intercepts of the fits of the calculated shielding vs. experimental chemical shift plot (σCastep = -x•δexp + σref).

Differential Scanning Calorimetry (DSC) and Thermogravimetric Analysis (TGA)
The three OTA AH samples were subjected to DSC and TGA analyses ( Figures S7-S9).
The TGA curves reviled that sublimation starts at temperatures above 280°C upon heating.
The observed mass loss corresponds to sublimation and decomposition of the acid. In DSC experiments the AH decomposition can be observed at temperatures above 355°C.         The 1 H CRAMPS solid-state NMR spectra confirm structural differences between the crystalline forms of OTA. Three different 1 H sites can be distinguished in the anhydrous OTA. They can be assigned to the hydroxyl proton at ca. 13 ppm, two amino protons at ca. 10 ppm and the hydrogen attached to carbon C3 at ca. 5 ppm. The spectrum of Hy1 shows three different sites in which a peak of the hydrogen attached to carbon C3 at ca. 4 ppm shows a significant broadening due to the presence of structural water, which shows a peak in the same region of the spectrum. Two other peaks can be assigned to the two amino protons at ca. 10 ppm and the hydroxyl proton at ca. 14 ppm. The 1 H spectrum of S DMSO shows five different proton sites. The most intense peak at 2.5 ppm can be assigned to the DMSO, the peak at 4.5 ppm is due to the hydrogen attached to carbon C3, while the peak at 14 ppm can be assigned to the hydroxyl group. Interestingly, it is possible to distinguish both amino protons in the 1 H CRAMPS spectrum of S DMSO . Assignment of this two sites can be made based on CASTEP calculations and the 1 H-1 H 2D CRAMPS solid state NMR spectrum. Based on CASTEP calculations peaks at 10 ppm and 11 ppm can be assigned to hydrogens attached to N1 and N2. That is further corroborated by a 1 H-1 H 2D CRAMPS NMR experiment in which cross peaks between hydroxyl proton and N1 proton can be observed, indicating close proximity between both sites, which is agreement with the crystal structure.        The experimental diffraction patterns were indexed using the first twenty peaks with DICVOL04 and the space group was determined based on a statistical assessment of systematic absences 11 as implemented in the DASH structure solution package. 12 The lattice parameters agree with the computated A1 data ignoring temperature effects. Pawley fits 13 were performed with Topas Academic V5. 14 The background was modelled with Chebyshev polynomials and the modified Thompson-Cox-Hastings pseudo-Voigt function was used for peak shape fitting.   56  54  52  50  48  46  44  42  40  38  36  34  32  30  28  26  24  22  20  18  16  14  12  10  8  6  4  2 Counts 9,500 9,000 8,500 8,000 7,500 7,000 6,500 6,000 5,500 5,000 4,500 4,000 3,500 3,000 2,500 2,000 1,500 1,000 500 0 -500 s3 0.00 % -34 -

Determination of the Critical Water Activity (Slurry Method)
Excess OTA AH was stirred (500 r.p.m.) in 2 mL of methanol:water mixtures, each containing a different mole fraction of water corresponding to a defined water activity 15,16 (Figure S26) at 25.0 ± 0.1°C for 40 days. Samples were withdrawn, filtered and the resulting phase was determined using powder X-ray diffraction and thermogravimetric analysis.

Comparison of experimental and calculated densities
The true densities of the hydrate (Hy1) and anhydrous material (AH s1 ) were measured using the helium pycnometer AccuPyc 1330 (Micromeritics Instruments Corp., Norcross, GA, USA). An amount of 5 to 6 g was measured using a 12 cm³ cell with following parameters: number of purges = 10, runs = 5 (triplicate measurements). Table S 18 lists the densities of OTA forms obtained in the present work with those of other sources.