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Effect of Neighbors on the Conformational Preferences of Glycosidic Linkages in Glycyrrhizic Acid and Its Mono- and Dideprotonated Forms: X‑ray, NMR, and Computational Studies Ewa Tykarska,*,† Zbigniew Dutkiewicz,† Daniel Baranowski,‡ Zofia Gdaniec,‡ and Maria Gdaniec§ †

Department of Chemical Technology of Drugs, Poznan University of Medical Sciences, Grunwaldzka 6, 60-780 Poznan, Poland Institute of Bioorganic Chemistry, Polish Academy of Sciences, Z. Noskowskiego 12/14, 61-704 Poznan, Poland § Faculty of Chemistry, Adam Mickiewicz University, Umultowska 89b, 61-614 Poznan, Poland ‡

S Supporting Information *

ABSTRACT: Three-dimensional structure of glycyrrhizic acid is determined by the conformation of the disaccharide unit and its relative orientation to a virtually rigid aglycone. X-ray crystallography, NMR spectroscopy, and density functional theory (DFT) calculations were used to study the conformational preferences of the glycosidic linkages in solid state, solution and vacuo, respectively. Experimental data have revealed that conformation of glycosidic bonds, that is adopted in the solid state, is also favored in solution. The molecular geometry optimizations have shown that a strongly twisted orientation of the two glucopyranose units, characteristic of the solid state, is stabilized by the bridging interaction of water molecules and/or cations with disaccharide COOH/COO¯ groups. Our results illustrate the impact of environment on the preferred conformation of disaccharide unit in the studied glycoside and point to a possible reason for the observed rigid conformation of the glycosidic bonds in solution and in the solid state.

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INTRODUCTION Glycosides are a large and ubiquitous family of molecules that play diverse roles in living organisms, and many of them are of pharmacological significance. An important subset of the glycosides are saponins, natural products widely occurring in higher plants and lower marine animals.1 Glycyrrhizic acid (GA), a triterpene saponin, has been shown to be the major biologically active constituent of the liquorice species (Glycyrrhiza), the medicinal plant used in Asia and Europe since prehistoric times for the treatment of respiratory, gastrointestinal, cardiovascular, urogenital, and skin diseases.2−4 In nature, GA occurs as a mixture of magnesium, calcium, and potassium salts (glycyrrhizin), and its amount in liquorice root extracts varies greatly according to the species and growth environment.5 The molecule of GA consists of the hydrophobic aglycone, known as 18β-glycyrrhetinic acid, and a hydrophilic sugar moiety consisting of two β-D-glucuronic acid units (Scheme 1). The amphiphilic structure determines its physicochemical properties. Aqueous solutions of GA show surface activity, and the molecules aggregate forming micelles and gels.6,7 Studies of the last few decades revealed remarkable therapeutic properties of GA and its salts8−19 and its chemically modified derivatives.20,21 Recently, GA has been demonstrated to possess an ability to form water-soluble complexes with © XXXX American Chemical Society

Scheme 1

hydrophobic water-insoluble drugs improving the bioavailability, stability, and therapeutic efficacy of commercially available Received: July 21, 2014 Revised: September 29, 2014

A

dx.doi.org/10.1021/cg5010962 | Cryst. Growth Des. XXXX, XXX, XXX−XXX

Crystal Growth & Design

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medicines.22,23 It has been recognized as a promising compound in the development of a novel drug delivery system.24−26 Since the biological activity of compounds is strictly related to their molecular structure and the ability to form intra- and intermolecular interactions, knowledge of the preferred conformation of GA should lead to a better understanding of its properties. The three-dimensional structure of this molecule depends primarily on the conformation of the diglucuronic unit and its orientation relative to a virtually rigid aglycone. Considering that conformation about glycosidic linkages is generally flexible and dependent on its environment,27 it has been extremely surprising that recently published crystal structures of highly solvated GA and its mono- and dibasic salts (NH4+, K+, Cs+) are practically isomorphic,28−30 indicating nearly identical three-dimensional structures of the neutral GA molecule and its ionic forms. The inner glucuronic acid residue (unit A) is always arranged linearly with the triterpenoid moiety, whereas the glucopyranose units are strongly twisted relative to each other (Figure 1).

Figure 2. Alternating hydrophobic and hydrophilic areas in solvated crystal structures of GA and its salts. The hydrophilic area is formed by hydrogen-bonded diglucuronic chains parallel to the x-axis. Hydrogen atoms and solvent molecules located in the channels are omitted for clarity.

is occupied exclusively by an OH group of a solvent molecule. In dibasic salts, cations are located at sites I and IIa. Site IIb is not occupied because of increasing repulsive forces between similar charges. A solvent molecule is always located at site III. Although the saponins are an ubiquitous class of compounds in nature, X-ray crystal data are rather scarce, and there is no systematic investigation of the factors affecting their structure. The presented in this article new crystal form of a monoammonium salt of GA (AGA) indicates a possible role of solvent molecules and cations incorporated into the sugar bilayer on the conformational stability of glycosidic linkages, suggesting that the structure of investigated saponin can be preorganized in solution and adopts a similar conformation as in the solid state. To determine the preferred conformation of disaccharide unit in solution and its relative orientation with respect to the aglycone, the structure of glycyrrhizinate anion in AGA was established by NMR studies. In addition, information obtained from the crystal structures of GA and its salts allowed us to study by DFT calculations the impact of the nearest environment on the conformational preferences about the sugar−sugar and sugar−aglycone glycosidic linkages.

Figure 1. Molecular structure of GA with water molecules located at sites designated as I, II, and III. Water molecule at II is disordered over two almost equally occupied positions IIa and IIb. C-bound hydrogen atoms are omitted for clarity.

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EXPERIMENTAL SECTION

General Remarks. Ammonium glycyrrhizinate, DMF, DMSO-d6 (99.9% D), and D2O (99.8% D) were purchased from commercial suppliers and used without further purification. X-ray Crystallography. Single plate-like crystals of glycyrrhizic monoammonium salt (AGA) were grown at room temperature by slow evaporation of 50% DMF solution. At start, concentration of AGA was 16 mM. Diffraction intensity data were measured at 130 K with an Oxford Diffraction SuperNova using the microsource and mirror-monochromated Cu Kα radiation and processed with the Agilent Technologies CrysAlis Pro software.31 The structure was solved by direct methods and refined by the full matrix least-squares based on F2 (SHELXS-97 and SHELXL-97).32 All non-hydrogen atoms were refined anisotropically. The C−H hydrogen atoms were introduced in geometrical positions and refined as riding on their pivot atoms with Uiso(H) = 1.2Ueq(C) or 1.5Ueq for methyl groups. Hydrogen atoms of hydroxyl groups and the ammonium ion were located in a difference electron density map with the O−H and N−H distances standardized in the

In the crystals, neutral and ionized forms of GA aggregate to form alternately arranged hydrophobic (aglycone) and hydrophilic (glycone) areas (Figure 2). The hydrophobic part of the crystal consists of partially interpenetrating layers of triterpene moieties, which leads to the formation of intersecting channels filled with solvent and guest molecules. The hydrophilic region, in the form of a sugar bilayer, consists of hydrogen-bonded diglucuronic chains joined by solvent molecules/cations incorporated into the carbohydrate area at sites designated as I−III (Figures 1 and 2). A peculiar feature of this region is the ability to replace some of the water molecules found in the GA crystal structure (Figure 1) by OH groups of organic solvents or monovalent cations (NH4+, K+, Cs+) maintaining practically unchanged the conformation of the glycosidic bonds and the construction of a sugar bilayer. In monobasic glycyrrhizinates, site I is occupied by a monovalent counterion, whereas site IIb B



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final refinement to 0.84 and 0.90 Å, respectively. Some H atoms of water molecules were introduced after a detailed inspection of possible hydrogen bonding. Because of disorder, the hydrogen atom positions for one water molecule with minor occupancy were not included. Summary of the structure determination is given in Table 1, and the hydrogen bond geometry is in Table S1 (Supporting Information).

The atom labeling scheme and the asymmetric unit are shown in Figures S1 and S2 (Supporting Information). Computational Methods. All DFT calculations were performed with the Gaussian 09 suite33 using the long-range corrected CAMB3LYP functional34 and 6-31+G(d,p) basis sets. The geometries of the crystal structures of GA and its mono- and diammonium (AGA, AGA1, A2GA) and mono- and dipotassium (KGA, K2GA) salts were fully optimized with the following options Opt=Tight and Int=UltraFine. Because of SCF convergence problems resulting from the usage of diffuse functions, the triterpenoid unit of saponin was reduced to one ring only (Figure S3 Supporting Information), and such simplified molecular structures were used to study conformations of the diglucuronic moiety. All calculations were carried out in vacuo with the exception of one optimization of the GA fragment, when the default implementation of polarizable continuum method (PCM) was used to simulate water as a solvent (GAscrf Table 2). The crystal structures of GAvac, AGAvac, AGA1vac, KGAvac, A2GAvac, and K2GAvac were optimized with water molecules and/or cations located at sites I and II between two acidic groups of glycone moiety (Table 2). In the case of mono- and diammonium glycyrrhizinates, the structures were recalculated with an additional water molecule at site III (AGAvac, AGA1vac) or DMF2 molecule (A2GAvac) in order to avoid proton transfer from NH4+ to COO− group, and these structures were used for conformational analysis of glycosidic linkages. Because of repulsion forces between two positively charged ions, a DMF1 molecule bridging the cations in the crystal structure was included in calculations of dibasic salts of GA (A2GAvac and K2GAvac). For comparison, the K2GAvac structure was also recalculated without DMF. The fragments of saponin with solvent molecules and different cations used in calculations are presented in Figure S3 (Supporting Information). NMR Experimental. All measurements were done on Bruker Avance III 700 MHz equipped with QCI-P CryoProbe in DMSO-d6/ D2O (7:1) at 298 K. Addition of D2O eliminated overlapping with the residual H2O signal in the 1H spectrum. DMSO-d6 (99.9% D) and D2O (99.8% D) were purchased from Sigma-Aldrich. The concentration of AG was 2.8 mmol. 1H and 13C signals were assigned by means of 1 H−1H COSY, 1H−1H TOSCY, 1H−13C HSQC, and 1H−13C HMBC experiments which are reported as supplementary data in Supporting Information. The 2-D homo- or heteronuclear correlation experiments

Table 1. Crystal Data and Refinement Details for Monoammonium Salt of GA AGA empirical formula

CCDC no. formula weight crystal system temp, K space group unit cell dimensions, Å

volume, Å3 Z calc. density, g/cm3 absorption coefficient, mm−1 refl collected independent refl data/restraints/parameters GOF on F2 final R indices [I > 2σ(I)] R indices (all data) largest peak and hole, e·A−3

C42H61O16·NH4, 3.0 DMF 6.53 H2O 1014908 1176.88 orthorhombic 130 P212121 a = 10.2250(3) b = 11.4024(3) c = 53.281(1) 6212.0(3) 4 1.258 0.844 16375 10103 10103/6/739 1.090 R1 = 0.0947 wR2 = 0.2614 R1 = 0.0963 wR2 = 0.2628 0.81, −0.60

Table 2. Torsion Angles ϕ, ψ, and δ [°] for X-ray Structures and Selected Optimized Structures of Glycyrrhizic Acid and Its Mono- and Dibasic Saltsa sugar B − sugar A X-ray

crystallization solution

GA28 AGA AGA129 AGA228 KGA29 CsGA29 A2GA29 K2GA29 Cs2GA29

propionic acid DMF propionic acid methanol acetic acid propionic acid DMF DMF DMF

calculated GA1scrf GA1vac GAvac AGAvacb AGA1vacb KGAvac A2GAvacb K2GAvac

occupied sites none none WI, WIIb I/NH4+, WIIb, WIV I/NH4+, WIIb I/K+, WIIb I/NH4+, IIa/NH4+ I/K+, IIa/K+

sugar A − triterpenoid

ϕ1

ψ1

δ1

ϕ2

ψ2

δ2

50.0 55.5 56.3 55.8 52.1 53.4 54.9 54.8 46.8

34.8 27.6 34.6 24.6 26.0 36.4 28.0 25.7 36.1

78.3 76.4 83.4 73.9 72.3 83.0 76.5 74.4 76.6

37.5 34.9 41.8 42.1 36.0 39.9 36.2 33.7 38.2

−15.2 −18.1 −18.2 −19.7 −19.2 −15.3 −21.0 −22.1 −15.9

20.5 15.3 21.4 20.1 15.4 22.4 13.7 10.3 20.3

ϕ1 41.1 −16.1 2.6 63.4 30.8 49.6 53.4 48.0

ψ1 30.5 −12.8 −6.4 62.7 18.3 31.3 33.7 38.4

δ1 66.6 −27.3 −3.6 113.9 46.3 74.7 80.6 80.0

ϕ2 44.1 49.6 47.5 41.7 37.6 38.8 44.0 36.9

ψ2 −9.4 −13.7 −12.7 −19.4 −16.0 −16.7 −2.5 2.7

δ2 31.4 31.8 31.3 19.8 19.6 19.9 38.6 37.4

a W - water molecule, GA - glycyrrhizic acid, AGA - monoammonium glycyrrhizinate, KGA − monopotassium glycyrrhizinate, CsGA - monocaesium glycyrrhizinate, A2GA - diammonium glycyrrhizinate, K2GA - dipotassium glycyrrhizinate, Cs2GA - dicaesium glycyrrhizinate bThe structures were calculated with an additional solvent molecule in order to maintain the N−H distances in the NH4+ ion (Figure S3, Supporting Information).

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were acquired using a standard set of parameters. The 1-D NMR measurements of the nuclear Overhauser enhancement (NOE) were performed using the selnogp pulse program.35−37 Data were collected with 64k data points, spectral width of 5144 Hz, 64 scans. The 2-D NOESY experiments (mixing time of 100, 200, 300, 400, 500, 600, and 700 ms) were performed using the noesygpphpp pulse program.38,39 Data were acquired with 2048 and 256 points (F2 and F1, respectively), 72 scans, a spectral width of 5144 Hz. Spectra were processed and prepared with TopSpin 3.0 Bruker Software. Vicinal proton−proton coupling constants were obtained by means of 1D spectrum simulation software (DAISY, BRUKER). A series of 1H NMR spectra in a wide range of concentrations of AGA (0.1, 0.5, 1, 3, 5, 10, 20 mmol/L) and temperatures (298−358 K) were recorded to check whether the micelles are formed in the conditions of the NMR measurement (2.8 mmol/L, DMSO-d6/D2O 7:1, 298 K). Regardless of the concentration and temperature, changes of the chemical shifts were insignificant, 0.01−0.04 ppm and 0.01− 0.06 ppm, respectively. For these concentrations, the diffusion coefficient has been measured using NMR experiments. The obtained values have changed from 8.16 × 10−11 to 9.20 × 10−11 m2 s−1, which strongly suggests that micelles are not formed in these conditions.

conformational analysis of the GA molecule (Figure 3). In contrast to the usual torsion angle, it involves four atoms but the two C atoms are not directly bonded. This angle describes the relative orientation of the C−H bonds across the glycosidic linkages allowing for the estimation of the pyranose−pyranose and pyranose−aglycone twist and easy comparison of the X-ray and NMR data. This torsion angle is approximately the sum of ϕ and ψ (Figure 4, Table 2); however, it does not describe

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RESULTS AND DISCUSSION An important feature of the carbohydrate conformation is the conformational flexibility of glycosidic linkages and its susceptibility to the neighbors interacting with the sugar oxygen atoms. Considering this, the reported conformational stability of GA in its solvated crystal structures and in a variety of salts obtained from different solvents is very unusual.28,29 Although it has been shown that the isomorphism of the investigated solid state structures results from the specific construction of the sugar area, the question concerning the role of the entities incorporated into the hydrophilic region on the conformational behavior of glycosidic linkages remains open. To determine the effect of neighbors on the preferred structure of the diglucuronic unit, the torsion angles ϕ and ψ used in conformational analysis of glycosidic linkages in carbohydrates have been examined. As shown in Figure 3, the relative

Figure 4. Graphs representing the relationship between the δ angle and the sum of ϕ and ψ angles describing glycosidic linkages in the Xray and optimized structures of GA and its mono- and dibasic salts: (a) sugar−sugar glycosidic linkage, (b) sugar−aglycone glycosidic linkage.

Figure 3. Labeling scheme of the atoms from glycosidic linkages (shown as spheres of arbitrary radii).

uniquely the two torsion angles of the glycosidic linkage because for the same value of δ different sets of ϕ and ψ may be adopted. Crystal Structure of AGA. X-ray structural analysis revealed that crystallization of the monoammonium salt of GA (AGA) from DMF resulted in a solvated structure containing in the asymmetric unit one glycyrrhizinate anion, one ammonium cation, three DMF molecules, and seven water molecules of which one was at 0.5 occupancy. The important

orientation of the two glucopyranose units is described by the torsion angles ϕ1 and ψ1 defined by the atoms H1B−C1B− O2A−C2A and C1B−O2A−C2A−H2A, respectively. The ϕ2 and ψ2 torsion angles, defined by the H1A−C1A−O3−C3 and C1A−O3−C3−H31 atoms, respectively, characterize the mutual orientation of the sugar A unit and the aglycone. For the purposes of further discussion, a torsion angle H− C···C−H, denoted as δ, has been introduced to facilitate D



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Figure 6. Basic unit of the supramolecular architecture of AGA consisting of a hydrogen-bonded diglucuronic bilayer covered by sparsely spaced triterpene residues.

Figure 5. Crystals structures of AGA and K2GA showing the bridge between COOH/COO¯ groups of disaccharide unit through a chain consisting of (a) a NH4+ ion occupying site I and two water molecules located at sites IIb and IV, (b) two K+ counterions occupying sites I and IIa bridged by a DMF molecule. Only the first ring of the triterpenoid moiety is presented. C−H hydrogen atoms are omitted for clarity.

Figure 7. Hydrogen-bonded zigzag chains of diglucuronic residues parallel to the unit cell axis a. NH4+ ion at site I and three water molecules at sites IIb-IV incorporated into the sugar area are shown in the ball-and-stick style. The chains belonging to different layers are marked in dark and light gray. R indicates the aglycone moiety.

feature of this structure is an extra water molecule incorporated into the hydrophilic region at site denoted as IV (Figures 5a and 7) that was not previously found in other monodeprotonated forms of GA. This water molecule enables the formation of a hydrogen-bonded chain composed of NH4+ cation and two water molecules that bridges COOH/COO¯ groups of diglucuronic unit. The chain is further stabilized by interactions of NH4+/H2O with oxygen atoms of the same and neighboring host molecules (Figure 5a, Table S1 Supporting Information). Because an ammonium ion at site I replaces a water molecule in the GA crystal structure (Figure 1), it can be assumed that also in a neutral form of GA a similar bridge between the two carboxylic groups of the disaccharide may appear. Until now, such association was achieved only in dibasic salts (NH4+, K+, Cs+) across two cations bridged by DMF molecule (Figure 5b).29 Despite this difference the structure of AGA is isomorphic with previously reported crystal structures of GA and its ionized forms.28,29 The δ1 torsion angle of 76.5° (Table 2) indicates a strongly twisted orientation of the A and B sugar units, while δ2 of 15.3° points to nearly syn arrangement of the C−H bonds across the sugar−aglycone glycosidic linkage. Both rings of the β-D-glucuronic units adopt the 4C1 chair conformation. Similarly as in other crystal structures of GA, the supramolecular architecture is constructed of interdigitated basic units (Figure 6) which form a layered structure of alternating hydrophilic (sugar) and hydrophobic (aglycone) areas presented in Figure 2. The hydrophilic region consists of hydrogen-bonded zigzag disaccharide chains joined by NH4+ counterion and three water

molecules occupying sites IIb−IV (Figure 7). The water at site IV, like the cation located at site IIa in dibasic salts, connects two consecutive diglucuronic units of a sugar chain, enhancing its construction, and restricts the rotation of the carboxylic group of sugar B around the C−C bond observed in GA and its monobasic forms.29 Thus, the new structure of AGA to some extent resembles the structures of dibasic salts of GA. The presented structure for the first time points to the possible dual role of solvent/cations incorporated into the hydrophilic area. These entities join different saccharide chains reinforcing the sugar bilayer, and additionally they can stabilize the twisted conformation of the diglucuronic unit by formation of a bridge between the carboxylic/carboxylate groups, which suggests that the structure of the acid sugar unit in solution may be preorganized and similar to that in solid state. DFT Calculations. DFT calculations were performed for testing the effect of cations and water molecules at sites I−IV in solid state structures on the conformation of glycosidic linkages of GA, its mono- and diammonium (AGA, AGA1, A2GA) and mono- and dipotassium (KGA, K2GA) salts. The CAM-B3LYP functional with 6-31+G(d,p) basis set was applied for DFT calculations of GA in its neutral and ionic forms. In commonly used functionals, long-range correlations are missing, also in the B3LYP hybrid functional, which is very common in various DFT computational studies of carbohydrates.40−44 Lack of these correlations results in describing E



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(X-ray) derived conformations of GA (Figures 1 and 5a). Torsion angles ϕ and ψ are consistent with those obtained in solid state and, similarly to crystal structures, δ correspond to gauche (66.6°) and close to syn (31.4°) conformation for δ1 and δ2, respectively (Table 2). When DFT calculations were carried out in vacuo (GA1vac), the orientation of C−H bonds across both glycosidic linkages exhibited the same close to syn conformation (δ1 = −27.3°, δ2 = 31.8°), and consequently two pyranose rings were arranged in linear fashion stabilized by an intramolecular hydrogen bond between OH groups of sugar units A and B (Table 2, Figure 8b). Comparison of the δ1 and δ2 torsion angles in GA1scrf and GA1vac indicates that sugar− sugar glycosidic linkage is influenced by the solvent, whereas the orientation of the pyranose ring A and the triterpene moiety is independent of the environment (Table 2). To eliminate the impact of implicitly included water as a solvent on the conformation of glycosidic bonds and to study the effect of neighboring cations and water molecules found in the crystal structures at sites I−IV an optimization of a variety of GA solid state structures was carried out in vacuo. As the triterpene moiety is rigid, only the first ring was included in DFT calculations. Starting structures used in the optimization procedures are presented in Figure S3 (Supporting Information). Effect of Neighbors On the Conformation of GAvac and Glycyrrhizinate Ions in Monoammonium (AGAvac) and Monopotassium (KGA vac) Salts. Analysis of optimized structures of GAvac and its monobasic salts (AGAvac, AGA1vac, KGAvac) revealed that the conformation of the sugar−sugar glycosidic linkage is susceptible to the presence of neighboring cations and water molecules, whereas the conformation of the sugar−aglycone glycosidic linkage remains practically unchanged. In GAvac the twisted conformation of the disaccharide unit found in the crystal structure of GA is not maintained. The rotation around ϕ1 and ψ1 results in an extended, linear arrangement of pyranose rings with the δ1 torsion angle of −3.6° indicating the syn orientation of C−H bonds across the sugar−sugar glycosidic linkage (Table 2). Conformation of the glycone is stabilized by a strong intramolecular hydrogen bond between OH groups of sugar A and B (Figure 9a, Table S2 Supporting Information). Replacement of the water molecule at site I with a monovalent cation (NH4+, K+) results in the rearrangement of hydrogen bonds created by H2O at site IIb and formation of a chain that bridges COO¯/COOH groups in the disaccharide unit (Figure 9b−d, Table S2 Supporting Information). The association of disaccharide COO¯/COOH groups through the bridging chain is facilitated by the twisted orientation of the pyranose rings; however, the δ1 torsion angle varies in the optimized monobasic salts of GA (Table 2). Strongly twisted conformation of the disaccharide unit in AGAvac and KGAvac with δ1 of 113.9° and 74.7°, respectively, is stabilized by COO¯--chain--COOH interactions, where the last water molecule from the chain forms a strong hydrogen bond with the carboxylic group of sugar B (Figure 9b,c). Optimization of AGA1 (X-ray) leads to an intermediate structure of AGA1vac with δ1 = 46.3° in which the weak association between carboxylate---carboxylic groups of the diglucuronic unit and the weak intramolecular hydrogen bond between the OH groups of sugar A and B stabilize the sugar−sugar glycosidic linkage conformation (Figure 9d). The cations and water molecules from the chain barely change the conformation of the sugar−aglycone glycosidic

anions as having only a fraction of the additional electron bound, which leads to fractional electron transfer for systems with cationic and anionic sites.45 Thus, long-range corrected functionals are recommended for DFT calculations applied to systems with localized anionic or strongly electron donating sites. Moreover, it has been shown that CAM-B3LYP functional, which belongs to this group was successfully employed in studies of hydrogen-bonded complexes.46,47 It has been also demonstrated by Csonka40 that the applied basis set [6-31+G(d,p)] is sufficient for geometry optimizations and for relative energy calculations in carbohydrates. During optimizations of mono- and diammonium glycyrrhizinates (sites I and II/IIa occupied by H2O/cation) in gasphase a proton transfer from the ammonium cation to the carboxylate group has occurred. A similar effect was observed in computational studies of formic acid complexes with ammonia45,48 and trimethylamine.49 It has been demonstrated that an ion-pair can be stabilized either by inclusion of water molecules or increasing the dielectric constant.49,50 Inclusion of one additional water molecule found at site III in the solid state structures of AGA and AGA1 or DMF2 molecule in A2GA was sufficient to avoid the proton transfer in DFT calculations of ammonium−GA complexes (Figure S3, Table S2 Supporting Information). As shown by ϕ, ψ, and δ torsion angles, these additional solvent molecules have practically no impact on the conformation of glycosidic bonds (Tables 2 and S3, Supporting Information). Optimization of the GA crystal structure with water as solvent included in implicit solvation model (GA1scrf) revealed a strong similarity between theoretically and experimentally

Figure 8. Optimized conformation of the GA fragment (a) in solvent − GA1scrf, (b) in vacuo − GA1vac. C−H hydrogen atoms are omitted for clarity. Water molecules/cations located at sites I−IV in the crystal structures were not included in DFT calculations. F



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Figure 9. (a−f) Conformations of glycosidic linkages in optimized GA and its mono- and dibasic salts. C−H hydrogen atoms are omitted for clarity. W indicates water molecule.

increase of the K+−K+ distance (5.4 Å) and the δ2 torsion angle (50.3°) indicates that the DMF molecule favors close to syn conformation of the sugar−triterpene glycosidic linkage (Table S3 Supporting Information). NMR Conformational Analysis. Conformational analysis of AGA in solution was based on NMR spectroscopy. Chemical shifts were obtained from 1D 1H and 13C spectra and from 2D methods such as 1H−1H COSY, 1H−1H TOCSY, 1H−13C HSQC, 1H−13C HMBC (summarized in Supporting Information) and are in a good agreement with previous results.51,52 Conformations of the six-membered rings were defined by the analysis of the intraring vicinal proton−proton coupling constants (3JH,H) obtained from one-dimensional 1H spectrum and supported by the analysis of the intraring NOE contacts present in the NOESY spectrum. Additionally, several selective 1D-NOESY experiments have been recorded to confirm the observation of weak correlation signals in the NOESY spectra (Supporting Information). The values of 3JH,H determined for both β-D-glucuronic units are listed in Table 3 and are close to those found by Cano53 for model carbohydrates with fixed 4C1 conformation. Values of torsion angles calculated on the basis of coupling constants are collected in Table 3 and compared with the values of corresponding angles found in the crystal structure. Good agreement between the values calculated from the NMR data and those determined from the crystal structure (difference less than 5.5°) suggests that in solution pyranose rings of the β-D-glucuronic units adopt exclusively the 4C1 conformation. Additionally, one bond proton−carbon coupling constant values of 159.5 and 160.2 Hz determined for anomeric carbon atoms of the A and B glucuronic units, respectively, support an axial orientation of C1A−H1A and C1B−H1B bonds that corresponds with the 4C1 conformation in β-Dpyranoses (Table 4).54 Furthermore, the two-dimensional

linkage. Formation of the bridging chain in AGAvac, AGA1vac, and KGAvac causes a slight reduction of the δ2 torsion angle by ca. 10° in comparison to the GAvac and GA1vac structures (Table 2). Nevertheless, δ2 corresponds to close to syn conformation of the glycosidic linkage in the calculated and X-ray structures of monobasic salts. Effect of Neighbors on the Conformation of Glycyrrhizinate Ions in Dibasic Salts of GA − A2GAvac and K2GAvac. Optimization carried out for dibasic salts revealed that the presence of two counterions strongly stabilized a twisted orientation of pyranose rings (Figure 9e,f, Table 2). Thus, similarly to crystal structures, only δ1 values corresponding to gauche conformation are found for the two dibasic salts. On the other hand, repulsive interactions between positively charged cations cause an increase in the distance between the ions, followed by a change mainly in the torsion angle ψ2 (Table 2). This is reflected by changes in the relative orientation of a sugar unit A to the triterpenoid fragment. In the X-ray (A2GA) and optimized (A2GAvac) structures of diammonium salt of GA, the distance between the two NH4+ counterions increases from ca. 4.0 Å to 4.4 Å, respectively, changing δ2 from 10.3° to 38. 6° (Tables 2 and S4, Supporting Information). A similar trend is observed in the dipotassium structure of GA. The distance between the K+ ions is ca. 4.1 Å in K2GA (X-ray) and 4.4 Å in K2GAvac, and consequently δ2 adopts values of 10.3° and 37.4°, respectively (Tables 2, S4 Supporting Information). In calculations, the DMF molecule, which in the crystal structure is located in the channels above the sugar platform bridging the two cations (Figures 5b, S3 Supporting Information), was taken into account. To recognize the impact of DMF on the relative orientation of a sugar unit A to aglycone, an optimization of K2GA structure without DMF has also been performed. Further G


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HLA equation (chemical groups):55 3JHH = 14.34 cos2φ − 0.78 cos φ + 0.58 + ∑λi[0.34 − 2.31 cos2(siφ + 18.4|λi|)]. bDAD equation:55 3JHH = 7.82 − 0.79∑λi − 0.78 cos φ + (6.54 − 0.64∑λi) cos 2φ + 0.70∑siλi2 sin 2φ. cTorsional angles presented in the table were chosen only for those values that correspond with 4C1 conformer.

Table 4. One Bond Proton-Carbon Coupling Constants (1JC,H) of Units A and B 1

Unit A Unit B

JC1′−H1′ (Hz) 159.5 160.2

1

JC2′−H2′ (Hz) 145.6 143.0

1

JC3′−H3′ (Hz) 142.9 142.7

1

JC4′−H4′ (Hz) 145.0 144.0

1

JC5′−H5′ (Hz) 141.7 142.3

NOESY spectrum of AGA revealed a cross-peak intensity pattern characteristic of the 4C1 conformation. A strong NOE was detected between the H1A proton and protons H5A and H3A, and a smaller effect was observed from H1A to H2A and H4A (Figure 10). Similarly, strong intraring NOEs from H1B

Figure 10. Fragment of the 2-D NOESY spectrum (700 MHz, 298 K, DMSO-d6/D2O, mixing time 300 ms) of AGA showing correlations to H1A and H1B protons (A) and corresponding fragments of 1D-NOE spectra after irradiation of H1A (B) and H1B (C). Diglucopyranose region of 1H NMR spectrum (D).

to H5B and H3B were observed (Figure 10), medium to H2B and weak to H4B. Obtained NOE enhancements were used to estimate through−space distances (Table 5). Comparison of these experimentally determined distances is in agreement with corresponding distances in the X-ray structure and confirms that for both residues the 4C1 conformation is predominant. The relative orientation of the two glucopyranose units, A and B, was deduced from the magnitude of NOEs between proton H1B and protons H2A and H3A. Irradiation of H1B produced strong inter-ring NOE to H2A and medium to H3A (Figures 10 and 11), indicating that the pyranose rings adopt strongly twisted orientation, similar to that found in crystal structures (Table 2). The orientation of sugar ring A with respect to the triterpenoid moiety was also derived from the analysis of NOEs. Irradiation of H1A produced a strong NOE to H31 pointing to the syn arrangement of C3−H31 and C1A−H1A bonds (Figures 10 and 11). The observation of weak NOEs

a

H4B−H5B

9.60 172.2 173.2 H4B−C4B−C5B−H5B 176.8

H4A−H5A

9.45 169.6 170.4 H4A−C4A−C5A−H5A 175.9 9.20 (9.15−9.3) 176.1 175.5 H3B−C3B−C4B−H4B 173.6

H3B−H4B H3A−H4A

9.3 (9.10−9.3) 179.7 178.3 H3A−C3A−C4A−H4A 179.1

H2B−H3B

9.00 (8.90−9.10) 190.9 190.1 H2B−C2B−C3B−H3B −173.0 (187.0)

H2A−H3A

8.85 (8.75−9.00) 186.2 186.2 H2A−C2A−C3A−H3A −173.8 (186.2)

H1B−H2B

7.85 (7.60−7.90) 170.2 173.2 H1B−C1B−C2B−H2B 176.1

H1A−H2A

7.75 (7.65−7.85) 170.1 172.3 H1A−C1A−C2A−H2A 172.9 JHH (Hz) HLA cg (deg)a,c DAD (deg)b,c torsion angles X-ray (deg)

coupled protons

3

Table 3. Vicinal Proton−Proton Coupling Constants (3JH,H) for Glucuronate A and B Moieties and Torsion Angles between All Pairs of Coupled Protons Calculated by Means of Haasnoot-de Leeuv-Altona (HLA) or Diez-Altona-Donders (DAD) Equations Compared with Corresponding Torsion Angles Determined from the X-ray Structure


H



Article

neighbors (solvent molecules, cations) on the preferred conformation of glycosidic linkages in the investigated saponin. Optimization of neutral, mono- and dideprotonated GA obtained from the X-ray structure analyses revealed that the major factor responsible for the twisted conformation of the disaccharide unit of GA is the tendency of the COOH/COO¯ groups of the diglucuronic moiety to be bridged by a chain composed of water molecules or water molecules and cations. The relative orientation of the pyranose rings is conserved in the dibasic salts of GA with the δ1 torsion angle of ca. 80° and similar to that in the solid state, whereas the sugar−sugar glycosidic linkage in monobasic salts of GA is more flexible, which is reflected in the δ1 value ranging from 46° to 114° (Table 2). This difference can be related to a different strength of interactions between the bridging chain and COOH/COO− groups of disaccharide. The twisted conformation of diglucuronic unit established by NMR studies for AGA suggests also a strong association of COO¯ and COOH groups in solution. A similar orientation of the sugar unit A relative to the triterpene moiety in the experimental and calculated structures indicates that a close to syn conformation of the sugar− aglycone glycosidic linkage is favored. As shown by DFT calculations, repulsive interaction between positively charged cations in dibasic salts of GA results in a slight increase of the δ2 torsion angle. This effect is not observed in crystal structures in which the construction of a sugar bilayer prevents changes in the disaccharide conformation.

Table 5. Comparison of Interproton Distances [Å] Determined from the X-ray Structure and Calculated from the 300 ms NOESY Spectruma H1B−H5B H1B−H3B H1B−H2A H1B−H3A H1A−H31

X-rayb

NMRc

Δ%d

2.23 2.66 2.60 3.49 2.04

2.34 2.71 2.48 3.73 2.24

4.7 1.8 −4.8 6.4 8.9

a

Distance H181−H121 was taken as reference distance. bDistances taken from the X-ray structure. cDistances calculated from NOESY spectrum, mixing time = 300 ms. dPercentage difference between NMR and X-ray distances.

■

ASSOCIATED CONTENT

S Supporting Information *

Hydrogen bonds tables, atom labeling system, crystallographic information file (CIF), and NMR data are available for compound AGA. This material is available free of charge via the Internet at http://pubs.acs.org.

■

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +48 61 8546639. Tel: +48 61 8546632. Notes

Figure 11. Graphical representation of the NOE for AGA. NOE from irradiation of H1A (a) and H1B (b). Solid line: strong NOEs, dashed line: medium NOEs, dotted line: weak NOEs. For clarity of the figure some parts of the molecule are omitted. Gb-glucopyranose B moiety, Te-terpenoid moiety.

The authors declare no competing financial interest.

■

ACKNOWLEDGMENTS This study was partly supported by UMP Grant No. 502-0103313427-08870 and the European Fund for Regional Development No. UDA-POIG.02.01.00-30-182/09. The calculations were performed at the Poznan Supercomputing and Networking Center affiliated with the Institute of Bioorganic Chemistry of the Polish Academy of Sciences, http://www.man.poznan.pl/online/en/.

between H1A and protons of terpenoid methyl group (C23) and H22 additionally supported the syn orientation of these two bonds. For other conformations across the glycosidic bond, different patterns of NOEs would be expected. An additional argument corroborating similarity of the solution and X-ray structures was obtained from the observation of two long-range NOEs between proton H2A and protons of the two methyl groups C23 and C24. A more quantitative treatment was performed by comparing distances obtained from the X-ray structure with those derived from the NOE intensities. It can be noticed that the agreement between experimental and theoretical values is satisfactory (Table 5).

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REFERENCES

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CONCLUSIONS Comparison of the data obtained from X-ray, NMR, and computational studies allowed an analysis of the impact of I



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