I. INTRODUCTION
Folic acid (also known as vitamin M, vitamin B9, or vitamin Bc) is the synthetic form of folate that is normally presented in food supplements. Folate is essential for numerous bodily functions. A lack of folate may lead to glossitis, diarrhea, depression, confusion, anemia, and fetal neural tube and brain defects during pregnancy (Duthie, Reference Duthie1999). Folic acid is commercially available in a dihydrate form, and has the systematic name of (2S)-2-[(4-{[(2-amino-4-hydroxypteridin-6-yl)methyl]amino}phenyl)formamido]pentanedioic acid dihydrate. A two-dimensional (2D) structural diagram is shown in Figure 1.
The molecular structure of folic acid.
The presence of high-quality reference powder patterns in the PDF (Powder Diffraction File; ICDD, 2013) is important for phase identification, particularly by pharmaceutical, forensic, and law enforcement scientists. The crystal structures of a significant fraction of the largest dollar volume pharmaceuticals have not been published, and thus the calculated powder patterns are not present in the PDF-4 Organics database. Sometimes experimental patterns are reported, but they are generally of low quality. Accordingly, a collaboration among ICDD, IIT, Poly Crystallography Inc., and Argonne National Laboratory has been established to measure the high-quality synchrotron powder patterns of commercial pharmaceutical ingredients, include these reference patterns in the PDF, and determine the crystal structures of these active pharmaceutical ingredients (APIs).
Most crystal structures are determined using single-crystal diffraction data, but even in the absence of suitable single crystals improvements in instrumentation and algorithms make it increasingly possible to solve structures using powder diffraction data. The process is made difficult by the collapse of the 3D diffraction information into the one dimension of a powder pattern. The consequences are that fewer structure factors are measured, they are less well determined because of peak overlap, and they rarely extend to atomic resolution. Conventional direct methods developed for single-crystal experiments thus generally do not work very well with intensities extracted from powder data; our experience is that direct methods (or charge flipping) succeed only 10–15% of the time, even when using synchrotron powder data. Methods that incorporate additional information, such as the chemical connectivity (molecular structure), are generally necessary.
Even when the crystal structure of an API is reported, the single-crystal structure was often determined at low temperature. Most powder measurements are performed at ambient conditions. Thermal expansion (often anisotropic) means that the peak positions calculated from a low-temperature single-crystal structure often differ from those measured at ambient conditions. These peak shifts can result in failure of default search/match algorithms to identify a phase, even when it is present in the sample. High-quality reference patterns measured at ambient conditions are thus critical for easy identification of APIs using standard powder diffraction practices.
II. EXPERIMENTAL
The folic acid was commercial material, purchased from The US Pharmacopeial Convention (USP; Lot #Q0G151), and was used as-received. The white powder was packed into a 1.5 mm diameter Kapton capillary, and rotated during the experiment at ~50 cycles s−1. The powder pattern was measured at beam line 11-BM (Lee et al., Reference Lee, Shu, Ramanathan, Preissner, Wang, Beno, Von Dreele, Ribaud, Kurtz, Antao, Jiao and Toby2008; Wang et al., Reference Wang, Toby, Lee, Ribaud, Antao, Kurtz, Ramanathan, Von Dreele and Beno2008) of the Advanced Photon Source at Argonne National Laboratory using a wavelength of 0.413 691 Å at 296 K from 0.5° to 50°2θ with a step size of 0.001° and a counting time of 0.1 s step−1. The pattern was indexed on a primitive orthorhombic unit cell having a = 7.273, b = 8.628, c = 32.409 Å, and V = 2038.0 Å3 using Jade 9.5 (MDI, 2014). A search of this cell in the Cambridge Structural Database (Allen, Reference Allen2002) yielded the Refcode FOLCAH, folic acid dihydrate (Camerman et al., Reference Camerman, Mastropaolo and Camerman1980; Mastropaolo et al., Reference Mastropaolo, Camerman and Camerman1980); this entry contained the unit cell but no atom coordinates. The systematic absences determined the space group to be P212121 (#19) (a common space group for chiral organic compounds), which was confirmed by successful solution and refinement of the structure.
A folic acid molecule was built and its conformation optimized using Spartan ‘14 (Wavefunction, 2013), and saved as a mol2 file. This molecule and two oxygen atoms were used as fragments to solve the structure using Dash 3.3.2 (David et al., Reference David, Shankland, van de Streek, Pidcock, Motherwell and Cole2006). In addition to yielding a good fit to the diffraction data, the solution exhibited a number of potential hydrogen bonds. It would be very surprising if the crystal structure of folic acid dihydrate did not contain many hydrogen bonds, so their presence provided extra confidence in the chemical reasonableness of the solution.
The Rietveld refinement was carried out using GSAS (Larson and Von Dreele, Reference Larson and Von Dreele2004). Only the 1°–25° portion of the pattern was included in the refinement, because the higher-angle portion did not contain visible peaks. The C6H4 phenyl group was refined as a rigid body, and all non-H bond distances and angles were subjected to restraints, based on a Mercury/Mogul Geometry Check (Bruno et al., Reference Bruno, Cole, Kessler, Luo, Motherwell, Purkis, Smith, Taylor, Cooper, Harris and Orpen2004; Sykes et al., Reference Sykes, McCabe, Allen, Battle, Bruno and Wood2011) of the molecule. The Mogul average and standard deviation for each quantity were used as the restraint parameters. The N1–O12 portion of the molecule was subjected to a planar restraint with a standard deviation of 0.01 Å. The restraints contributed 3.00% to the final χ 2. Isotropic displacement coefficients were refined, grouped by chemical similarity. The U iso of each hydrogen atom was constrained to be 1.3× that of the heavy atom to which it is attached. The peak profiles were described using profile function #4, which includes the Stephens (Reference Stephens1999) anisotropic strain broadening model. The background was modeled using a three-term shifted Chebyshev polynomial and an eight-term diffuse scattering function to describe the scattering from the Kapton capillary and any amorphous content of the sample. The final refinement of 115 variables using 24 003 observations yielded the residuals wR p = 0.0703, R p = 0.0564, and χ 2 = 1.388. The largest peak (1.11 Å from C32) and hole (1.45 Å from H45) in the difference Fourier map were 0.18 and −0.15 eÅ−3, respectively. The Rietveld plot is included as Figure 2. The largest errors are in the shapes of the lowest-angle peaks.
(Color online) The Rietveld plot from the refinement of folic acid dihydrate. The red crosses represent the experimental data points, and the green line is the calculated pattern. The magenta curve is the difference pattern, plotted at the same vertical scales as the other patterns. The vertical scale has been multiplied by a factor of 6 for 2θ higher than 8.1°, and by a factor of 20 for angles higher than 13.2°.
(Color online) A density functional geometry optimization (fixed experimental unit cell) was carried out using CRYSTAL09 (Dovesi et al., Reference Dovesi, Orlando, Civalleri, Roetti, Saunders and Zicovich-Wilson2005). The basis sets for the H, C, N, and O atoms were those of Gatti et al. (Reference Gatti, Saunders and Roetti1994). The calculation used eight k-points and the B3LYP functional.
III. RESULTS AND DISCUSSION
The refined atom coordinates of folic acid dihydrate are reported in Table I, and the coordinates from the DFT optimization in Table II. The root-mean-square deviation of the non-hydrogen atoms is 0.21 Å, and the maximum deviation is 0.44 Å, at O30 (Figure 3). The other largest differences are in the dicarboxylic acid side chain. The discussion of the geometry uses the DFT-optimized structure. The folic acid molecule (with atom numbering) is illustrated in Figure 4, and the crystal structure is presented in Figure 5.
(Color online) Comparison of the refined and optimized structures of folic acid dihydrate.
(Color online) The molecular structure of folic acid, with the atom numbering. The non-hydrogen atoms are represented by 50% probability spheroids.
(Color online) The crystal structure of folic acid dihydrate, viewed down the a-axis.
Rietveld refined fractional coordinates of folic acid dihydrate. Space group P212121 with a = 7.275 78(3), b = 8.632 17(4), c = 32.417 19(22) Å, V = 2035.985(18) Å3, and Z = 4.
DFT (CRYSTAL09) optimized fractional coordinates of folic acid dihydrate. Space group P212121 with a = 7.275 776, b = 8.632 17, c = 32.417 19 Å, V = 2035.985(18) Å3, and Z = 4.
Almost all bond distances, angles, and torsion angles fall within the normal ranges indicated by a Mercury Geometry Check. Only the bonds C4–O12 at 1.270 Å (Z-score = 2.1), C8–C9 = 1.421 Å (Z-score = 2.2) and the angles O30–C29–C28 = 122.2° (Z-score = 3.7) and O30–C29–O31 = 118.1° (Z-score = 2.2) lie at the edges of the normal ranges. The torsion angles O30–C29–C28–C32, O35–C34–C33–C32, and O36–C34–C33–C32 lie in the tails of the ranges for these torsion angles. As might be expected, the displacement coefficients for the water molecules O52 and O53 are higher than those of the atoms in the folic acid molecule. Mercury detects no voids in the crystal structure.
The crystal structure is dominated by a 3D network of hydrogen bonds (Table III). The carboxylic acid O30–H50 donates a proton to the water molecule O52, and the carboxylic acid O35–H51 donates a proton to the ketone group O12 = C4. The Mulliken overlap populations indicate that the hydrogen bonds are strong; the correlation developed for O–H···O hydrogen bonds in Rammohan and Kaduk (Reference Rammohan and Kaduk2014) suggests that these hydrogen bonds each account for 15–16 kcal mol−1 of energy. Each water molecule donates two protons and accepts one. There are several N–H···O hydrogen bonds. Some of these are intramolecular, and they (as well as C–H···O hydrogen bonds) seem to contribute to the bent conformation of the dicarboxylic acid side chain. The conformation seems ideal for chelation to metal cations, so this structure should be helpful in determining the structures of folate salts. Most of the hydrogen bonds are discrete; the chains and rings are in general long and hard to visualize. There is one prominent ring with graph set (Etter, Reference Etter1990; Bernstein et al., Reference Bernstein, Davis, Shimoni and Chang1995; Shields et al., Reference Shields, Raithby, Allen and Motherwell2000) R2,2(8), made up of ···H51–O35–C34–O36···H37–N3–C4–O12. This eight-membered ring contains two hydrogen bond donors and two acceptors.
Hydrogen bonds in folic acid dihydrate.
The Bravais–Friedel–Donnay–Harker (Bravais, Reference Bravais1866; Friedel, Reference Friedel1907; Donnay and Harker, Reference Donnay and Harker1937) morphology suggests that we might expect platy morphology for folic acid dihydrate, with {001} as the principal faces. The texture index, modeled using fourth-order spherical harmonics, was 1.001, so preferred orientation was not significant for this rotating capillary specimen.
The anisotropic microstrain is greatest along the a-axis. This is the direction in which the flat portions of the molecules stack, suggesting that occasional mis-stackings are present in the crystallites. The powder pattern of folic acid dihydrate has been submitted to ICDD for inclusion in future releases of the PDF.
ACKNOWLEDGEMENTS
Use of the Advanced Photon Source at Argonne National Laboratory was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02–06CH11357. This work was partially supported by the International Centre for Diffraction Data. We thank Lynn Ribaud for his assistance in data collection, and Silvina Pagola for her participation in the early stages of this project.
SUPPLEMENTARY MATERIALS AND METHODS
The supplementary material refered to in this paper can be found online at http://www.journals.cambridge.org/pdj.
