Crystal structure of zymonic acid and a redetermination of its precursor, pyruvic acid

Intermolecular interactions in both crystal structures are dominated by hydrogen bonding. The common (8) hydrogen-bonding motif links carboxylic acid groups on adjacent molecules in both structures.

The structure of zymonic acid (systematic name: 4-hydroxy-2-methyl-5-oxo-2,5dihydrofuran-2-carboxylic acid), C 6 H 6 O 5 , which had previously eluded crystallographic determination, is presented here for the first time. It forms by intramolecular condensation of parapyruvic acid, which is the product of aldol condensation of pyruvic acid. A redetermination of the crystal structure of pyruvic acid (systematic name: 2-oxopropanoic acid), C 3 H 4 O 3 , at low temperature (90 K) and with increased precision, is also presented [for the previous structure, see : Harata et al. (1977). Acta Cryst. B33, 210-212]. In zymonic acid, the hydroxylactone ring is close to planar (r.m.s. deviation = 0.0108 Å ) and the dihedral angle between the ring and the plane formed by the bonds of the methyl and carboxylic acid carbon atoms to the ring is 88.68 (7) . The torsion angle of the carboxylic acid group relative to the ring is 12.04 (16) . The pyruvic acid molecule is almost planar, having a dihedral angle between the carboxylic acid and methyl-ketone groups of 3.95 (6) . Intermolecular interactions in both crystal structures are dominated by hydrogen bonding. The common R 2 2 (8) hydrogen-bonding motif links carboxylic acid groups on adjacent molecules in both structures. In zymonic acid, this results in dimers about a crystallographic twofold of space group C2/c, which forces the carboxylic acid group to be disordered exactly 50:50, which scrambles the carbonyl and hydroxyl groups and gives an apparent equalization of the C-O bond lengths [1.2568 (16) and 1.2602 (16) Å ]. The other hydrogen bonds in zymonic acid (O-HÁ Á ÁO and weak C-HÁ Á ÁO), link molecules across a 2 1 -screw axis, and generate an R 2 2 (9) motif. These hydrogen-bonding interactions propagate to form extended pleated sheets in the ab plane. Stacking of these zigzag sheets along c involves only van der Waals contacts. In pyruvic acid, inversion-related molecules are linked into R 2 2 (8) dimers, with van der Waals interactions between dimers as the only other intermolecular contacts.

Chemical context
The Human Metabolome Database (Wishart et al., 2007(Wishart et al., , 2009(Wishart et al., , 2013(Wishart et al., , 2018 lists the compound 4-hydroxy-2-methyl-5-oxofuran-2-carboxylic acid (C 6 H 6 O 5 ), commonly named zymonic acid, with the metabocard HMDB0031210. Zymonic acid is used as a flavor constituent for confectionery and tobacco products (Yannai, 2004). The generation of zymonic acid can proceed by condensation of parapyruvic acid, which itself forms by aldol condensation of pyruvic acid (IUPAC name 2oxopropanoic acid, C 3 H 4 O 3 ; Bloomer et al., 1970). Therefore, zymonic acid is directly derived from pyruvic acid, and is thus related to the compounds present in the tricarboxylic acid (Krebs) cycle (Nelson & Cox, 2004) and its reductive version (Guzman, 2011;Guzman & Martin, 2008;Zhou & Guzman, 2016). As an intermediate in central metabolism, zymonic acid is produced in the cytoplasm at very low concentration, from where it can be excreted to the extracellular region.
The electron-impact mass spectrum (MS) and electrospray ionization fragmentation of zymonic acid following gas and liquid chromatography, respectively, have been reported (Allen et al., 2015(Allen et al., , 2016. The use of 13 C-zymonic acid has enabled mapping of pH changes, independently of concentration, in mammalian organs and tumors via hyperpolarized magnetic resonance . Thus, zymonic acid is a non-invasive extracellular imaging sensor to localize and quantify pH in vivo Hundshammer et al., 2017), with many possible applications in medical diagnosis . As part of the process resulting in the aforementioned invention, the detailed 1 H and 13 C NMR spectra of pure zymonic acid have been reported . Herein, we contribute new information to characterize zymonic acid by reporting for the first time its crystal structure, along with a low-temperature redetermination of pyruvic acid.

Structural commentary
Aside from the effects on the geometry of the carboxylic acid group in zymonic acid that stem from disorder about the twofold axis (see below), there are no unusual bond lengths or angles in either compound.
In zymonic acid ( Fig. 1), the hydroxylactone ring is essentially planar (r.m.s. deviation = 0.0108 Å ), with the largest deviation from planarity [0.0171 (8) Å ] for the ring oxygen atom, O3. The plane defined by the ring carbon atom C4, the methyl carbon atom C6, and the carboxylic acid carbon atom C5, is almost perpendicular to the mean plane of the ring atoms [dihedral angle = 88.68 (7) ]. Lastly, the orientation of the carboxylic acid group relative to the ring, as defined by the torsion angle O4-C5-C4-O3, is 12.04 (16) . For the carboxylic acid group, disorder about the crystallographic twofold axis effectively averages the C O double and C-O single bonds, rendering them equivalent [the C5-O4 and C5-O5 distances are 1.2568 (16) and 1.2602 (16) Å , respectively], and requires modeling of half-occupancy hydrogens (H4O and H5O) on each.
In spite of increased precision resulting from much lower temperature (90 K versus 266 K) and data collection on modern equipment, the redetermined structure of pyruvic acid ( Fig. 2) is largely unchanged from that reported by Harata et al. (1977). For example, the dihedral angle between the planes defined by atoms C1/C2/C3/O3 and C1/C2/O1/O2 is 3.95 (6) at 90.00 (2) K versus 3.5 at 266 (1) K.

Supramolecular features
The main intermolecular interactions in the crystals of both zymonic and pyruvic acids are hydrogen bonds. In zymonic acid, the carboxylic acid groups of adjacent molecules are related by a crystallographic twofold axis to form hydrogen bonds [O4-H4OÁ Á ÁO4 ii and O5-H5OÁ Á ÁO5 ii ; symmetry code: (ii) 1 À x, y, 3 2 À z] giving R 2 2 (8) dimer motifs (Table 1). This common supramolecular construct in carboxylic acids usually occurs between inversion-related or symmetry-independent molecules. Here, the orientation of the dimer relative to the crystallographic twofold axis forces the average struc-

Figure 1
The molecular structure of zymonic acid, with displacement ellipsoids drawn at the 50% probability level.

Database survey
A search of the Cambridge Crystal Structure Database (Version 5.40, Nov. 2018;Groom et al., 2016) for zymonic acid gave no hits for searches on either 'zymonic' or on the structural formula. A search on the structural formula of pyruvic acid gave two hits. CSD entry PRUVAC (Harata et al., 1977) describes the pure compound at 266 K, and is similar to the present pyruvic acid structure (after transformation to a common cell setting). CSD entry FAFGUR (Prohens et al., 2016) describes a co-crystal of pyruvic acid with the drug agomelatine. The CSD does contain structures for derivatives of both zymonic and pyruvic acids, but none of these have features that are especially relevant to the current work.

Synthesis and crystallization
Vacuum distillation of pyruvic acid (Sigma-Aldrich, 98.5%) was used for purification (Eugene & Guzman, 2017a,b). Freshly distilled pyruvic acid was crystallized in a closed vial in a freezer at 253 K. The tail of this distillation, a viscous yellowish residue enriched in parapyruvic and zymonic acids, was isolated in a vial, and the headspace filled with N 2 (g) before sealing it with a cap. Crystals of zymonic acid were produced slowly from this isolated residue kept at 275 K inside a refrigerator. The easily identifiable transparent crystals of zymonic acid appear above the level of the viscous solution within two weeks. Pyruvic acid crystals are deliquescent in air, even at 263 K (Harata et al., 1977), so they had to be kept cold, with minimal exposure to ambient air. Thus, throughout all experimental stages from initial inspection through data collection, special techniques for crystal handling at low temperature (Parkin & Hope, 1998)  The R 2 2 (9) dimer of zymonic acid. Unlabeled atoms are related to their labeled counterparts by a crystallographic 2 1 -screw axis ( 3 2 À x, 1 2 + y, 3 2 À z). Disorder of the carboxylic acid H atoms is omitted to enhance clarity.

Figure 5
A packing plot of zymonic acid viewed down the b axis, showing the stacking along c of zigzag pleated assemblies of molecules. Disorder of the carboxylic acid hydrogen atoms is omitted to enhance clarity.

Refinement
Crystal data, data collection, and structure refinement details are summarized in Table 3. Non-disordered hydrogen atoms were found in difference Fourier maps. For pyruvic acid, the hydroxyl hydrogen-atom coordinates were refined freely, while methyl hydrogen C-H distances used a riding model that allowed the C-H distance to refine. For zymonic acid, riding models were used for all hydrogen atoms apart from those disordered about the twofold axis, which were modeled in accordance with the recommendations of Fá bry (2018). U iso (H) parameters of non-disordered hydrogens were set to either 1.2U eq or 1.5U eq (for the methyl and hydroxyl groups, respectively) of the attached atom. To ensure stable refinement of disordered groups in the zymonic acid structure, constraints (SHELXL command EADP) were used to equalize displacement parameters of superimposed atoms.

Funding information
We are thankful for research funding from the National Science Foundation under NSF CAREER award CHE-1255290 to MIG, and the MRI program, grants CHE-0319176 and CHE-1625732. An International Visiting Scholar grant from the College of Arts and Sciences at UK is gratefully acknowledged.

4-Hydroxy-2-methyl-5-oxo-2,5-dihydrofuran-2-carboxylic acid (zymonic)
Crystal data Extinction correction: SHELXL2018 (Sheldrick, 2015a), Fc * =kFc[1+0.001xFc 2 λ 3 /sin(2θ)] -1/4 Extinction coefficient: 0.0057 (13) Special details Experimental. The crystal was mounted using polyisobutene oil on the tip of a fine glass fibre, which was fastened in a copper mounting pin with electrical solder. It was placed directly into the cold gas stream of a liquid-nitrogen based cryostat (Hope, 1994;Parkin & Hope, 1998). Diffraction data were collected with the crystal at 90K, which is standard practice in this laboratory for the majority of flash-cooled crystals. Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. Refinement progress was checked using Platon (Spek, 2009) and by an R-tensor (Parkin, 2000 where P = (F o 2 + 2F c 2 )/3 (Δ/σ) max < 0.001 Δρ max = 0.40 e Å −3 Δρ min = −0.21 e Å −3 Special details Experimental. The crystal was mounted using polyisobutene oil on the tip of a fine glass fibre, which was fastened in a copper mounting pin with electrical solder. It was placed directly into the cold gas stream of a liquid-nitrogen based cryostat (Parkin & Hope, 1998). Diffraction data were collected with the crystal at 90K, which is standard practice in this laboratory for the majority of flash-cooled crystals. Geometry. All esds (except the esd in the dihedral angle between two l.s. planes) are estimated using the full covariance matrix. The cell esds are taken into account individually in the estimation of esds in distances, angles and torsion angles; correlations between esds in cell parameters are only used when they are defined by crystal symmetry. An approximate (isotropic) treatment of cell esds is used for estimating esds involving l.s. planes. Refinement. Refinement progress was checked using Platon (Spek, 2009) and by an R-tensor (Parkin, 2000). The final model was further checked with the IUCr utility checkCIF.