Research article Received: 24 March 2014
Revised: 8 May 2014
Accepted: 15 May 2014
Published online in Wiley Online Library: 3 June 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4089
The structure-dependent self-association of five phenolic acids in aqueous solution Chaoni Xiao, Man Wu, Yajun Zhang, Xinfeng Zhao, Jie Yu and Xiaohui Zheng* Weak self-interaction plays an important role in interpreting the biomechanisms and modes of drug action. The structuredependent self-association of five phenolic acids with various bioactivities, including danshensu (DSS), caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA), was investigated by 1H NMR. These phenolic acids have similar condensed structures, with a CA moiety and varying numbers of DSS moieties. The strengths of the self-association constants are in the order DSS < CA < RA < LA < SA, which corresponds to the increasing molecular size of these phenolic acids and roughly corresponds to the increasing number of DSS moieties. The binding site for the self-aggregation of these phenolic acids has been identified to be on the CA moiety, rather than on the DSS moiety, as a result of CA’s stronger aromatic π–π interactions, which cause larger chemical shift variations. The thermodynamic parameters for the self-association of these phenolic acids show that the self-association is spontaneous and enthalpically favorable at room temperature in all cases. It was inferred that π–π interactions and intermolecular hydrogen bonding stabilize the stacking structures of the phenolic acids. Knowledge of self-association processes will enable us to quantitatively assess the possible effects of self-aggregation on the interaction between drug and protein. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: NMR; self-association; phenolic acid
Introduction
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Phenolic acids are the largest class of secondary metabolites present in the leaves, bark, and fruit of higher plants. They are important natural, active compounds and are drug candidates with effective antioxidant, anti-inflammatory, anticancer, and diuretic properties.[1,2] However, many studies have demonstrated their harmful effects, including the inhibition of digestive enzymes, decreases in body weight, and growth retardation, especially when applied at high concentrations.[3,4] Phenolic acids with different chemical structures have exhibited different pharmacological effects in humans. Therefore, much attention has been paid to the structure-dependent bioactivities of phenolic acids. The interactions between phenolic acids and various proteins play an important role in the alteration of their biological and pharmacological activities, and these binding processes are affected significantly by the structures of the phenolic acids. The reactions of nine phenolic compounds with several enzymes indicate that inhibition of enzyme activity is related to the number and position of the hydroxyl groups in the structures of the phenolic compounds.[5] The digestion properties and physicochemical characterization of whey proteins were affected differently by reactions with different phenol compounds.[6] The binding processes of several phenolic compounds (with antioxidant qualities) with transfer proteins (e.g., bovine serum albumin/human serum albumin) were compared, and the binding affinity increases with the phenolic molecular weight in the presence of galloyl groups.[7,8] Complete understanding of the interactions of phenolic acids with proteins, however, requires an understanding of the self-associations of these drug ligands, as their aggregations may complicate the interpretation of binding data.[9,10] To determine the amount of a given phenolic acid available for interaction with protein, and to interpret
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the biomechanism and mode of drug action, it is necessary to examine the self-associations of phenolic acids. Nuclear magnetic resonance is a powerful tool for the evaluation of molecular complexation in solution. Self-stacking results in considerable changes in the chemical shifts in the 1H or 13C–NMR spectra, from which the association constant can be calculated. DOSY is typically applied to identify formula weights and estimate the relative size of the organometallic complexes in the self-aggregation phenomena,[11–13] while 2D NOESY or ROESY is known to be very useful to determine the mutual spatial arrangement of the aggregates.[14] The self-associations of a variety of compounds, including caffeine,[15] chloroquine,[16] theophylline,[17] theaflavin,[18] and quinacridone derivatives,[19,20] have been reported. These compounds, containing aromatic rings and unsaturated electrons, tend to self-associate in solution because of strong attractive interactions between π-systems.[21] Nevertheless, the self-associations of phenolic acids with unique structural features have not yet been systematically investigated, and the relationship between the structures and self-association has not been reported. Danshensu (DSS), caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA) are five phenolic acids with similar condensed structures, including a CA moiety and varying numbers of DSS moieties, and are simultaneously present in certain herb, especially those of the Lamiaceae family.
* Correspondence to: Xiaohui Zheng, College of Life Sciences, Northwest University, No. 229 North Taibai Road, Xi’an, 710069, Shaanxi Province, China. E-mail: [email protected] College of Life Sciences, Northwest University, Xi’an 710069, China
Copyright © 2014 John Wiley & Sons, Ltd.
The structure-dependent self-association of five phenolic acids Specifically, the structure of RA consists of one CA moiety and one DSS moiety; LA is composed of one CA moiety and two DSS moieties; and SA consists of one CA moiety and three DSS moieties. In this study, the structure-dependent self-affinities of five phenolic acids, DSS, CA, RA, LA, and SA, were investigated by NMR to evaluate and compare the effects of their different structures on self-association. Knowledge of the self-association process will enable us to quantitatively assess the effects of aggregation on the binding between phenolic acid and protein.
Experimental
Typically, the self-association processes have a relatively fast dynamics, and individual NMR peaks form different aggregates can be not obtained. It was assumed that the chemical shift of the molecules (δobs) is the weighted average between the monomer (δA) and the assembly values (δmax). The progressive association of solute molecules into stacks (dimers, trimers, tetramers, etc.) is expected to occur in accordance with the same affinity constant described by the isodesmic model. Thermodynamic parameters, including the enthalpy change (ΔHΦ), entropy change (ΔSΦ), and the Gibbs free energy change (ΔGΦ), for the self-associations of five phenolic acids were obtained from the van’t Hoff equation and Gibbs free energy equation.
Materials and sample preparation DSS, CA, RA, LA, and SA (all reagent grade) were purchased from Sigma-Aldrich and used as obtained. Analytical grade K2HPO4 and Na2HPO4 · 12H2O were purchased from Guoyao Chemical Co. Ltd. (Shanghai, China). Deuterium oxide (D2O, 99.9% D) and sodium 3-trimethylsilyl [2,2,3,3,-2H4] propionate (TSP) were purchased from Cambridge Isotope Laboratories, Inc (MA, U. S. A.). To eliminate proton peak chemical shift variation resulting from differences in the pH or ionic strength, stock solutions of DSS, CA, RA, LA, and SA (20 mM each) were prepared in a buffer (0.5 M K2HPO4/Na2HPO4, pH 7.4, 100% D2O) containing TSP (0.1%, m/v) and then further diluted with the same buffer to obtain seven different concentrations (0.25, 0.5, 2.5, 5.0, 10, 15, and 20 mM). To ensure accuracy, the pH value for each solution (7.40 ± 0.05) was measured three times using a Mettler Toledo pH meter with a micro glass electrode before 0.5 mL of each solution was transferred into a 5-mm NMR tube for NMR analysis. Nuclear magnetic resonance measurements One-dimensional 1H NMR spectra were measured for each solution at 283, 298, 310, and 323 K on a 600 MHz Varian VNMRS spectrometer with a cold probe. The spectra were acquired using a one-dimensional NOESYPR pulse sequence (RD-90°-t1-90°-tm90°-acquisition; t1 = 4 μs, tm = 100 ms) with water resonance suppression during the recycle delay and the mixing time (tm). For each measurement, 64 transients were collected with 32-k data points and a spectral width of 10 ppm. The pulse length of 90° was adjusted to approximately 10 μs, and recycle delay was set to 2 s. For all spectra, an exponential window function was applied with a line-broadening factor of 0.5 Hz prior to FT, and the phase and baseline were then manually corrected and referenced to TSP (δ 0.00). A total of 35 solutions were subjected to 1H NMR analysis to obtain chemical shift data for investigating the self-interactions. A set of 2D NMR spectra including 1H–1H COSY, 1H–1H TOCSY, 1 H–13C HSQC, and 1H–13C HMBC were acquired and processed on 600-MHz spectrometers to assign the signals. Data fitting The self-association constant Ka is determined by the least-square curve fitting of the experimental proton NMR chemical shift data. n h io2 1 δobs ¼ δA þ ðδmax δA ÞKa ½A 2= 1 þ ð4Ka ½A þ 1Þ 2
(1)
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ΔH∅ 1 ΔS∅ þ R T R
(2)
and ΔG∅ ¼ RT LnKa ;
(3)
where Ka is the self-association constant (M1), R is the gas constant (8.314 J K1 M1) and the temperature T are at 283, 298, 310 and 323 K. The data fitting was performed by Origin software (version 7.5, OriginLab Corporation, MA, USA). All proton chemical shifts in each solution at different temperatures were recorded, and chemical shift dependence on the phenolic concentration was fitted based on Eqn (1), yielding the estimated parameters of Ka, δmax, and δA, together with the error bars of these parameters and correlation coefficients for evaluating the fitting quality. Subsequently, the estimated Ka dependence on temperature was fitted based on Eqns (2) and (3), providing the thermodynamic parameters for the self-association processes of phenolic acids.
Results and Discussion Chemical shift variation of 1H nuclear magnetic resonance signals for five phenolic acids Figure 1 displays the chemical structures, labeled aromatic rings and labeled atoms of five phenolic acids, including DSS, CA, RA, LA, and SA. RA, LA, and SA have similar condensed structures with regard to their common CA moiety (aromatic ring A, with a conjugate double bond) and different numbers of DSS moieties (aromatic rings B, C, and D, with saturated bonds). Figure 2 shows that the 1H NMR spectra of these phenolic acids in D2O at 298 K were stacked with increasing concentration. The 1H resonances were assigned to labeled protons based on previous studies[24–26] and were individually confirmed by the analysis of 2D NMR J-resolved, COSY, TOCSY, HSQC, and HMBC spectra. With increasing concentration from 0.25 to 20 mM, the DSS and CA proton peak positions shifted only slightly, whereas the signals for RA, LA, and SA moved significantly either upfield (lower chemical shift) or downfield (higher chemical shift). The displacements of the phenolic acid proton signals demonstrate that self-aggregation occurs in solution, presumably due to the π–π stacking interactions of aromatic functional groups.[21,27] As the temperature increases from 283 to 310 K, at a fixed concentration, the intensities of 1H signals for all phenolic acids reduced gradually, and the peak positions are shifted downfield to different extents for different phenolic acids (Fig. S1).
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, where δobs is the observed chemical shift and δA and δmax are the proton chemical shift of the monomer and the maximal proton chemical shift of solute A present in a stack, respectively. The value [A] denotes the total molar concentration in solution. [22,23]
lnKa ¼
C. Xiao et al.
Figure 1. The chemical structures, labeled aromatic rings and labeled atoms of five phenolic acids.
1
Figure 2. H NMR spectra for five phenolic acids, danshensu (DSS), caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA), in D2O at 298 K, stacked from bottom to up by increasing concentration. The signals in the stacked spectra were assigned to the labeled protons.
Aggregation of five phenolic acids by self-association
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Isodesmic fitting curves for the phenolic acids, based on Eqn (1), show changes in the chemical shift (Δδ) with increasing concentration (Fig. 3). The value of Δδ is defined as the proton chemical shift of the sample at various concentrations relative to the reference at the smallest concentration of 0.25 mM. Compared with the very small changes in chemical shifts for DSS (nearly constant, not shown), all of the protons in CA displayed a relatively large
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chemical shift variation (Fig. 3A). It is well known that the strong attractive interactions between π systems (i.e., aromatic ring) induce proton chemical shift variations during the process of selfaggregation and are affected by the size and properties of the substituents on the aromatic ring. Here, an electron-withdrawing substituent, such as the double bond in CA (7, 8 CH = CH), tends to have a greater influence on aromatic stacking than an electron-donating substituent, such as the saturated bond in DSS (7′,8′ CH2-CHCOOH). This result is in agreement with electrostatic
Copyright © 2014 John Wiley & Sons, Ltd.
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The structure-dependent self-association of five phenolic acids
Figure 3. Concentration dependences of chemical shift change (Δδ) of the resolved protons of caffeic acid (A), rosmarinic acid (B), lithospermic acid (C), and salvianolic acid B (D) in aqueous solution at 298 K. The value of Δδ is the proton chemical shift of the sample at various concentrations relative to that at the smallest concentration of 0.25 mM. The curves corresponding to each data set have been obtained with the Origin’s the least-square curve fitting based on Eqn (1).
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aromatic D ring protons may be attributable to the predominance of ring-current-induced diamagnetic deshielding effects from the opposing aromatic rings. The protons of aromatic ring A and the olefinic bond (CA moiety) in LA and SA display a greater variation in their chemical shifts, which confirms that CA moieties prefer stacking because of their larger π–π conjugated system. The concentration dependence of the 1H chemical shifts of the phenolic acids at various temperatures was used to estimate the association constants Ka (Table 1). The Ka values are considerably less precise in DSS and CA due to their minimal variations in chemical shift. Moreover, certain aromatic signals for RA, LA, and SA could not be extracted from the 1H spectra because of extensive signal overlap. These Ka values were therefore calculated from the olefinic protons to evaluate the strength of selfassociation in the formation of self-stacked aggregates. The Ka values are in the order CA < RA < LA < SA, which corresponds to the increasing molecular size of the phenolic acids and roughly corresponds to the increasing number of DSS moieties. Therefore, the relative strength of the self-interaction, represented by the Ka values for the olefinic bonds, has an obvious correlation to molecular size. A similar relationship between Ka and molecular size has also been observed for several polyphenols (e.g., tannins).[9] The exact reason for the apparent increase of the Ka value by additional DSS units in the phenolic acid structures remains unclear. However, it can be inferred that the existence of DSS units having aromatic ring might influence the density of π electronic ring-current of caffeic unit, resulting in the enhanced π–π stacking interactions between phenolic acid molecules and the apparent increase of the Ka value. Self-association thermodynamics of five phenolic acids The thermodynamic parameters of the self-association processes for these phenolic acids were calculated from the Ka values for olefinic protons at different temperatures using van’t Hoff plots (Fig. 4) and are presented in Table 2. The negative sign for the Gibbs free energy (ΔG) means that the self-interaction process
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models of the π–π stacking interaction,[21,28] which have found that electron-deficient rings prefer stacking interactions compared to electron-rich rings. Moreover, the perfect planarity of the larger π–π conjugated system of the aromatic ring and double bond in CA further enhance its preference for stacking. All of the signals of RA shift upfield with increasing concentration (Fig. 3B). It is well established that upfield shifts, as observed here, indicate plane to plane or vertical stacking.[17] The magnitudes of the differences in chemical shift varied for the RA signals, with larger chemical shift displacements for the aromatic ring A protons (2,6 ArH) and olefinic protons (7,8 CH = CH) compared to the displacements of the aromatic ring B protons (5′,6′ArH) and saturated protons (7′,8′ CH2-CHCOOH). The larger chemical shift changes correspond to the most favorable binding sites, which are therefore the least subject to conformational averaging of the aggregate with the monomer. This result implies that the π–π stacking occurs primarily with the planar aromatic ring A and its conjugated double bond (CA moiety), confirming that CA favors stacking more than DSS. The different magnitudes of the chemical shift changes within RA most likely reflect different binding geometries rather than differences in binding affinities. The directions of the proton chemical shift displacements for LA follow a different trend (Fig. 3C); the protons of aromatic ring A and aromatic ring C are strongly shifted upfield, while the aromatic ring B protons are shifted slightly downfield. Similarly, an obvious upfield shift in the aromatic A and aromatic C protons was observed in SA, as well as a minimal downfield shift in the aromatic B and aromatic D protons (Fig. 3D). Shifts upfield generally are of larger magnitude than those for shifts downfield, suggesting the upfield-shifted signals have a closer distance than the downfield-shifted signals. Different directions of the chemical shift displacements were also observed for chloroquine and quinacridone.[16,19] The upfield shifts demonstrated by aromatic ring A and aromatic ring C protons in the favored equiplanar geometry are commonly attributed to the strong interactions between the π systems, as in the case of several polyphenolic compounds.[10] The downfield shifts of the aromatic B and
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Copyright © 2014 John Wiley & Sons, Ltd.
SA
LA
RA
CA
DSS
2′,5′,6′ ArH 7′,8′ CH 2 ArH 5 ArH 6 ArH 7 CH = CH 8 CH = CH 2 ArH 5 ArH 6 ArH 7 CH = CH 8 CH = CH 2′ ArH 5′ ArH 6′ ArH 7′ CH 8′ CH 5 ArH 6 ArH 7 CH = CH 8 CH = CH 2′ ArH 5′ ArH 6′ ArH 7′ CH 8′ CH 2″ArH 5″,6″ ArH 7″ CH 8″ CH 5,6 ArH 7 CH = CH 8 CH = CH 2′,5′ ArH 6′ ArH 7′ CH 8′ CH 2″,5″ArH 6″ ArH
Proton
— — 1.02 ± 0.78 — 0.98 ± 0.65 2.31 ± 0.94 1.68 ± 0.65 9.41 ± 0.73 / / 9.83 ± 0.94 9.74 ± 0.97 — / 5.01 ± 0.76 8.24 ± 0.86 / 17.96 ± 3.11 18.42 ± 1.44 18.88 ± 1.36 19.41 ± 1.43 30.57 ± 3.32 30.51 ± 3.40 36.46 ± 3.43 32.25 ± 3.02 29.25 ± 2.94 21.73 ± 1.52 / 21.37 ± 1.57 19.10 ± 1.47 / 31.11 ± 2.47 30.53 ± 2.63 / / 30.18 ± 4.07 28.83 ± 2.80 / 30.10 ± 2.58
Ka(M )
1
— — 7.19 ± 0.00 — 7.08 ± 0.00 6.37 ± 0.00 7.30 ± 0.00 7.23 ± 0.00 / / 6.41 ± 0.00 7.62 ± 0.00 — / 6.81 ± 0.00 3.01 ± 0.00 / 6.95 ± 0.00 7.29 ± 0.00 6.36 ± 0.00 7.70 ± 0.00 6.99 ± 0.00 6.87 ± 0.00 6.79 ± 0.00 3.09 ± 0.00 4.91 ± 0.00 7.00 ± 0.00 / 5.84 ± 0.00 4.40 ± 0.00 / 5.87 ± 0.00 7.04 ± 0.00 / / 2.90 ± 0.00 4.97 ± 0.00 / 6.87 ± 0.00
δa
283 K
— — 6.63 ± 0.40 — 6.61 ± 0.29 6.24 ± 0.05 7.15 ± 0.05 6.31 ± 0.04 / / 5.44 ± 0.05 6.67 ± 0.06 — / 6.62 ± 0.02 2.93 ± 0.00 / 6.52 ± 0.03 6.03 ± 0.04 5.80 ± 0.01 7.08 ± 0.01 7.06 ± 0.00 6.93 ± 0.00 6.86 ± 0.00 3.14 ± 0.00 4.94 ± 0.00 6.67 ± 0.00 / 5.58 ± 0.00 3.78 ± 0.01 / 5.62 ± 0.01 6.52 ± 0.01 / / 3.01 ± 0.00 4.94 ± 0.00 / 6.10 ± 0.00
δmax — — 0.95 ± 0.56 2.16 ± 0.85 2.59 ± 0.77 3.11 ± 0.48 3.09 ± 0.57 7.82 ± 0.75 / 7.17 ± 0.78 8.96 ± 0.50 9.15 ± 0.67 — 9.13 ± 1.58 8.76 ± 0.98 13.46 ± 1.28 14.45 ± 0.83 10.96 ± 2.64 12.61 ± 0.62 13.43 ± 0.64 13.86 ± 0.68 7.33 ± 1.67 15.51 ± 1.52 18.31 ± 1.12 8.73 ± 0.99 9.14 ± 1.60 12.51 ± 0.83 / 16.78 ± 1.08 13.69 ± 0.70 / 23.66 ± 1.48 22.91 ± 1.47 / 28.03 ± 1.83 16.33 ± 2.21 — / 21.45 ± 1.54
Ka(M )
-1
— — 7.19 ± 0.00 6.97 ± 0.00 7.09 ± 0.00 6.37 ± 0.00 7.30 ± 0.00 7.22 ± 0.00 / 7.12 ± 0.00 6.40 ± 0.00 7.61 ± 0.00 — 6.89 ± 0.00 6.82 ± 0.00 3.02 ± 0.00 5.03 ± 0.00 6.90 ± 0.00 7.28 ± 0.00 6.35 ± 0.00 7.70 ± 0.00 6.99 ± 0.00 6.87 ± 0.00 6.79 ± 0.00 2.98 ± 0.00 4.95 ± 0.00 6.99 ± 0.00 / 5.84 ± 0.00 4.38 ± 0.00 / 5.89 ± 0.00 7.07 ± 0.00 / 6.76 ± 0.00 2.91 ± 0.00 — / 6.87 ± 0.00
δa
298 K
— — 6.84 ± 0.19 6.88 ± 0.03 6.97 ± 0.03 6.33 ± 0.01 7.26 ± 0.00 6.47 ± 0.05 / 6.08 ± 0.07 5.66 ± 0.02 6.90 ± 0.03 — 6.85 ± 0.00 6.73 ± 0.00 3.00 ± 0.00 4.95 ± 0.00 6.35 ± 0.07 6.30 ± 0.03 5.93 ± 0.01 7.25 ± 0.01 7.08 ± 0.01 6.91 ± 0.00 6.84 ± 0.00 3.02 ± 0.00 4.96 ± 0.00 6.74 ± 0.00 / 5.67 ± 0.00 3.91 ± 0.01 / 5.68 ± 0.00 6.65 ± 0.01 / 6.80 ± 0.00 3.03 ± 0.00 — / 6.25 ± 0.00
δmax — — 1.15 ± 0.55 — 3.15 ± 0.42 3.28 ± 0.95 6.22 ± 0.53 7.76 ± 0.67 / 3.45 ± 1.46 5.76 ± 0.84 6.71 ± 0.40 — 8.07 ± 1.35 7.60 ± 0.96 14.87 ± 1.56 12.01 ± 1.57 7.94 ± 2.54 8.99 ± 0.34 9.98 ± 0.38 10.37 ± 0.47 1.86 ± 0.44 8.52 ± 1.11 9.86 ± 1.38 5.02 ± 0.79 6.37 ± 1.95 6.96 ± 1.01 / 15.38 ± 1.65 10.04 ± 0.50 / 17.76 ± 0.67 16.11 ± 0.66 / 8.32 ± 1.34 8.67 ± 1.47 — / 14.22 ± 1.78
Ka(M )
1
— — 7.18 ± 0.00 — 7.08 ± 0.00 6.37 ± 0.00 7.30 ± 0.00 7.22 ± 0.00 / 7.12 ± 0.00 6.38 ± 0.00 7.60 ± 0.00 — 6.89 ± 0.00 6.81 ± 0.00 3.11 ± 0.00 5.04 ± 0.00 6.94 ± 0.00 7.28 ± 0.00 6.35 ± 0.00 7.70 ± 0.00 6.99 ± 0.00 6.86 ± 0.00 6.79 ± 0.00 2.98 ± 0.00 4.97 ± 0.00 6.99 ± 0.00 / 5.84 ± 0.00 4.36 ± 0.00 / 5.92 ± 0.00 7.10 ± 0.00 / 6.77 ± 0.00 2.92 ± 0.00 — / 6.86 ± 0.00
δa
310 K
Table 1. The fitting data of five phenolic acids at different temperatures calculated from chemical shift dependent on concentration
— — 7.00 ± 0.08 — 7.02 ± 0.00 6.35 ± 0.00 7.29 ± 0.00 6.63 ± 0.03 / 5.78 ± 0.48 5.65 ± 0.07 6.95 ± 0.03 — 6.86 ± 0.00 6.74 ± 0.00 3.09 ± 0.00 5.00 ± 0.00 6.45 ± 0.10 6.47 ± 0.02 6.03 ± 0.00 7.36 ± 0.01 7.23 ± 0.05 6.90 ± 0.00 6.84 ± 0.00 3.06 ± 0.00 5.02 ± 0.01 6.79 ± 0.02 / 5.74 ± 0.00 4.00 ± 0.01 / 5.75 ± 0.00 6.75 ± 0.00 / 6.83 ± 0.00 3.07 ± 0.01 — / 6.32 ± 0.00
δmax — — / / / — — / / 3.39 ± 0.90 4.59 ± 0.64 4.28 ± 0.67 — 4.04 ± 0.81 6.54 ± 0.82 2.21 ± 1.05 / 7.38 ± 0.76 5.93 ± 0.61 6.97 ± 1.32 6.99 ± 1.29 / 4.59 ± 2.61 / 2.18 ± 0.77 1.30 ± 0.67 / / 9.22 ± 1.56 7.07 ± 1.29 / 13.21 ± 0.53 11.66 ± 0.76 / 5.87 ± 2.94 7.01 ± 1.85 — / 11.06 ± 1.29
Ka(M )
1
— — / / / — — / / 7.11 ± 0.00 6.38 ± 0.00 7.59 ± 0.00 — 6.89 ± 0.00 6.81 ± 0.00 3.02 ± 0.00 / 6.94 ± 0.00 7.27 ± 0.00 6.34 ± 0.00 7.70 ± 0.00 / 6.86 ± 0.00 / 2.99 ± 0.00 4.99 ± 0.00 / / 5.84 ± 0.00 4.35 ± 0.00 / 5.94 ± 0.00 7.12 ± 0.00 / 6.77 ± 0.00 3.09 ± 0.00 — / 6.86 ± 0.00
δa
323 K
(Continues)
— — / / / — — / / 6.09 ± 0.22 5.76 ± 0.06 6.94 ± 0.08 — 6.87 ± 0.00 6.76 ± 0.00 2.99 ± 0.01 / 6.68 ± 0.01 6.60 ± 0.05 6.11 ± 0.00 7.47 ± 0.03 / 6.91 ± 0.02 / 3.16 ± 0.05 5.18 ± 0.14 / / 5.78 ± 0.00 4.09 ± 0.03 / 5.83 ± 0.00 6.84 ± 0.01 / 6.83 ± 0.02 3.15 ± 0.01 — / 6.45 ± 0.02
δmax
C. Xiao et al.
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5.79 ± 0.00 3.54 ± 0.03 6.53 ± 0.04 6.65 ± 0.05 6.19 ± 0.01 / — 5.98 ± 0.00 4.43 ± 0.00 6.32 ± 0.00 6.48 ± 0.00 6.14 ± 0.00 / — 12.53 ± 0.87 9.77 ± 0.55 4.52 ± 1.05 3.20 ± 1.11 3.81 ± 1.90 / — 5.68 ± 0.00 3.32 ± 0.02 6.48 ± 0.01 6.59 ± 0.00 6.19 ± 0.00 2.78 ± 0.04 —
δa Ka(M )
Table 2. Thermodynamic parameters for the self-associations of five phenolic acids
5.99 ± 0.00 4.44 ± 0.00 6.29 ± 0.00 6.46 ± 0.00 6.11 ± 0.00 2.62 ± 0.00 —
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1
ΔH (KJ mol )
Phenolic acids
DSS, danshensu; CA, caffeic acid; RA, rosmarinic acid; LA, lithospermic acid; SA, salvianolic acid B. —, it was not determined because of the minimal chemical shift change; /, it was not determined because of signal overlap.
14.24 ± 0.54 13.97 ± 0.63 7.52 ± 0.92 6.30 ± 0.58 10.50 ± 1.01 3.46 ± 0.94 — 5.58 ± 0.01 3.14 ± 0.04 6.44 ± 0.00 6.55 ± 0.00 6.18 ± 0.00 2.66 ± 0.00 — 5.99 ± 0.00 4.45 ± 0.00 6.26 ± 0.00 6.45 ± 0.00 6.08 ± 0.00 2.57 ± 0.00 — 23.34 ± 1.75 20.90 ± 1.62 13.83 ± 1.46 15.83 ± 1.92 23.38 ± 2.08 11.28 ± 1.88 — 5.99 ± 0.00 4.46 ± 0.01 6.23 ± 0.00 6.43 ± 0.00 6.06 ± 0.00 2.54 ± 0.00 2.90 ± 0.00 30.60 ± 2.54 29.35 ± 2.61 24.61 ± 2.71 29.48 ± 2.89 37.18 ± 2.82 25.52 ± 3.23 30.17 ± 4.08
5.43 ± 0.02 2.82 ± 0.05 6.44 ± 0.00 6.56 ± 0.00 6.23 ± 0.00 2.64 ± 0.00 3.01 ± 0.00
δa δa δa Ka(M )
7″ CH 8″ CH 2‴ ArH 5‴ ArH 6‴ ArH 7‴ CH 8‴ CH
Proton
Figure 4. The van’t Hoff plots for the olefinic protons (7,8 CH = CH) in rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA).
RA LA SA
1
ΔS (J mol
1
K )
1
ΔG (KJ mol )
17.42 ± 5.52 19.18 ± 1.54 16.63 ± 1.14
41.45 ± 18.23 42.80 ± 5.10 28.87 ± 3.75
13.75 ± 4.91 18.03 ± 1.26 15.56 ± 1.12
is spontaneous. The negative enthalpy (ΔH) and negative entropy (ΔS) of the intermolecular interaction indicate that the selfassociation is driven largely by enthalpic contributions at room temperature.[29] These thermodynamic values are characteristic of the π–π stacking interactions of planar aromatic rings. Other compounds with planar aromatic structures have been shown to self-associate according to the same indefinite association model, with similar thermodynamic constants. For example, Sun and coworkers have found values of ΔH = 19.3 ± 2.2 for the aggregation of quinacridone derivatives.[19] Negative enthalpies (ΔH) have also been observed whenever hydrogen bonds are involved in protein-drug binding.[8,30] It is likely that the selfassociation process of phenolic acids involves hydrogen bonds between the hydroxyl groups of one molecule and the carboxyl groups of the partner molecule. Compared to conventional interaction forces such as hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals interactions, the nature of π–π stacking interactions remains less clear because of complications such as multiple points of intermolecular contact, the strong influence of substituent groups and the cooperative effect of various noncovalent interactions. [19] It can be deduced, however, that the combination of π–π interactions and intermolecular hydrogen bonding stabilizes the selfaggregations of condensed structures.
Conclusions Five phenolic acids with similar structural features, DSS, CA, RA, LA, and SA, were identified to self-associate in D2O based on concentration-dependent proton chemical shift variations. The strength of the self-interactions, based on the self-association constants, increases in the following order of DSS < CA < RA < LA SA, which corresponds to the increasing molecular size of these phenolic acids and roughly corresponds to the increasing
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Table 1. (Continued)
1
283 K
δmax
-1
Ka(M )
298 K
δmax
1
Ka(M )
310 K
δmax
1
323 K
δmax
The structure-dependent self-association of five phenolic acids
C. Xiao et al. number of DSS moieties. Obviously, the existences of DSS moieties are very important for stabilizing the self-associations and the stacking structures of these pheonlic acids. The binding sites for the self-association of these phenolic acids were identified to be on the CA moiety rather than the DSS moiety by the larger chemical shift variations that result from the strong aromatic π–π interactions. The self-association processes are spontaneous, as demonstrated by the negative ΔG, and are enthalpically favorable at room temperature as shown by the negative ΔH and ΔS. It was inferred that π–π interactions and packing forces, from hydrogen bonding between the hydroxyl groups of one molecule and the carboxyl groups of the partner molecule, stabilize the stacking structures of the phenolic acids. Acknowledgements We acknowledge financial support from the National Natural Science Foundation of China (21005062), the Ministry of Education of China (20106101120024), the Education Department of Shaanxi province of China (12JK1011), and the Key Program for Science and Technology Innovative Research Team of Shaanxi Province (2013KCT-24).
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[10] K. Wroblewski, R. Muhandiram, A. Chakrabartty, A. Bennick. Eur. J. Biochem. 2001, 268, 4384–4397. [11] P. S. Pregosin, P. G. Anil Kumar, I. Ferna´ndez. Chem. Rev. 2005, 105, 2977–2998. [12] A. Macchioni, G. Ciancaleoni, C. Zuccaccia, D. Zuccaccia. Chem. Soc. Rev. 2008, 37, 479–489. [13] G. Hamdoun, M. Sebban, E. Cossoul, A. Harrison-Marchand, J. Maddaluno, H. Oulyadi. Chem. Commun. 2014, 50, 4073–4075. [14] D. Pavletta, S. L. Luk Van, V. Ingrid, W. Rudolph. J. Phys. Chem. B 2008, 12, 10935–10941. [15] T. H. Lilley, H. Linsdel. J. Chem. Soc. Faraday Trans. 1992, 88, 2865–2870. [16] N. Marchettini, G. Valensin, E. Gaggelli. Biophys. Chem. 1990, 36, 65–70. [17] A. L. Thakkar, L. G. Tensmeyer, W. L. Wilham. J. Pharm. Sci. 1971, 60, 1267–1269. [18] A. J. Charlton, A. L. Davis, D. P. Jones, J. R. Lewis, A. P. Davies, E. Haslam, M. P. Williamson. J. Chem. Soc., Perkin Trans. 2000, 2, 317–322. [19] H. Sun, K. Q. Ye, C. H. Wang, H. Y. Qi, Y. Wang. J. Phys. Chem. A 2006, 110, 10750–10756. [20] H. Sun, Y. F. Z-hao, Z. W. Huang, Y. Wang, F. Li. J. Phys. Chem. A 2008, 112, 11382–11390. [21] M. J. Rashkin, M. L. Waters. J. Am. Chem. Soc. 2002, 124, 1860–1861. [22] N. J. Baxter, M. P. Williamson, T. H. Lilleyb, E. Haslam. J. Chem. Soc., Faraday Trans. 1996, 92, 231–234. [23] M. R. Bruce. Chem. Rev. 1996, 96, 3043–3064. [24] C. N. Xiao, H. Dai, H. B. Liu, Y. L. Wang, H. R. Tang, J. Agric. Food Chem. 2008, 56, 10142–10153. [25] H. Dai, C. N. Xiao, H. B. Liu, F. H. Hao, H. R. Tang. J. Proteome Res. 2010, 9, 1565–1578. [26] H. Dai, C. N. Xiao, H. B. Liu, H. R. Tang. J. Proteome Res. 2010, 9, 1460–1475. [27] C. A. Hunter, J. K. M. Sanders. J. Am. Chem. Soc. 1990, 112, 5525–5534. [28] S. L. Cockroft, C. A. Hunter, K. R. Lawson, J. Perkins, C. J. Urch. J. Am. Chem. Soc. 2005, 127, 8594–8595. [29] J. B. Chaires, N. Dattagupta, D. M. Crothers. Biochemistry 1982, 21, 3927–3932. [30] P. B. Kandagal, S. Ashoka, J. Seetharamappa, S. M. T. Shaikh, Y. Jadegoud, O. B. Ijare. J. Pharm. Biomed. Anal. 2006, 41, 393–399.
Supporting Information Additional supporting information may be found in the online version of this article at the publisher’s website.
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Magn. Reson. Chem. 2014, 52, 460–466
Revised: 8 May 2014
Accepted: 15 May 2014
Published online in Wiley Online Library: 3 June 2014
(wileyonlinelibrary.com) DOI 10.1002/mrc.4089
The structure-dependent self-association of five phenolic acids in aqueous solution Chaoni Xiao, Man Wu, Yajun Zhang, Xinfeng Zhao, Jie Yu and Xiaohui Zheng* Weak self-interaction plays an important role in interpreting the biomechanisms and modes of drug action. The structuredependent self-association of five phenolic acids with various bioactivities, including danshensu (DSS), caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA), was investigated by 1H NMR. These phenolic acids have similar condensed structures, with a CA moiety and varying numbers of DSS moieties. The strengths of the self-association constants are in the order DSS < CA < RA < LA < SA, which corresponds to the increasing molecular size of these phenolic acids and roughly corresponds to the increasing number of DSS moieties. The binding site for the self-aggregation of these phenolic acids has been identified to be on the CA moiety, rather than on the DSS moiety, as a result of CA’s stronger aromatic π–π interactions, which cause larger chemical shift variations. The thermodynamic parameters for the self-association of these phenolic acids show that the self-association is spontaneous and enthalpically favorable at room temperature in all cases. It was inferred that π–π interactions and intermolecular hydrogen bonding stabilize the stacking structures of the phenolic acids. Knowledge of self-association processes will enable us to quantitatively assess the possible effects of self-aggregation on the interaction between drug and protein. Copyright © 2014 John Wiley & Sons, Ltd. Keywords: NMR; self-association; phenolic acid
Introduction
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Phenolic acids are the largest class of secondary metabolites present in the leaves, bark, and fruit of higher plants. They are important natural, active compounds and are drug candidates with effective antioxidant, anti-inflammatory, anticancer, and diuretic properties.[1,2] However, many studies have demonstrated their harmful effects, including the inhibition of digestive enzymes, decreases in body weight, and growth retardation, especially when applied at high concentrations.[3,4] Phenolic acids with different chemical structures have exhibited different pharmacological effects in humans. Therefore, much attention has been paid to the structure-dependent bioactivities of phenolic acids. The interactions between phenolic acids and various proteins play an important role in the alteration of their biological and pharmacological activities, and these binding processes are affected significantly by the structures of the phenolic acids. The reactions of nine phenolic compounds with several enzymes indicate that inhibition of enzyme activity is related to the number and position of the hydroxyl groups in the structures of the phenolic compounds.[5] The digestion properties and physicochemical characterization of whey proteins were affected differently by reactions with different phenol compounds.[6] The binding processes of several phenolic compounds (with antioxidant qualities) with transfer proteins (e.g., bovine serum albumin/human serum albumin) were compared, and the binding affinity increases with the phenolic molecular weight in the presence of galloyl groups.[7,8] Complete understanding of the interactions of phenolic acids with proteins, however, requires an understanding of the self-associations of these drug ligands, as their aggregations may complicate the interpretation of binding data.[9,10] To determine the amount of a given phenolic acid available for interaction with protein, and to interpret
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the biomechanism and mode of drug action, it is necessary to examine the self-associations of phenolic acids. Nuclear magnetic resonance is a powerful tool for the evaluation of molecular complexation in solution. Self-stacking results in considerable changes in the chemical shifts in the 1H or 13C–NMR spectra, from which the association constant can be calculated. DOSY is typically applied to identify formula weights and estimate the relative size of the organometallic complexes in the self-aggregation phenomena,[11–13] while 2D NOESY or ROESY is known to be very useful to determine the mutual spatial arrangement of the aggregates.[14] The self-associations of a variety of compounds, including caffeine,[15] chloroquine,[16] theophylline,[17] theaflavin,[18] and quinacridone derivatives,[19,20] have been reported. These compounds, containing aromatic rings and unsaturated electrons, tend to self-associate in solution because of strong attractive interactions between π-systems.[21] Nevertheless, the self-associations of phenolic acids with unique structural features have not yet been systematically investigated, and the relationship between the structures and self-association has not been reported. Danshensu (DSS), caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA) are five phenolic acids with similar condensed structures, including a CA moiety and varying numbers of DSS moieties, and are simultaneously present in certain herb, especially those of the Lamiaceae family.
* Correspondence to: Xiaohui Zheng, College of Life Sciences, Northwest University, No. 229 North Taibai Road, Xi’an, 710069, Shaanxi Province, China. E-mail: [email protected] College of Life Sciences, Northwest University, Xi’an 710069, China
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The structure-dependent self-association of five phenolic acids Specifically, the structure of RA consists of one CA moiety and one DSS moiety; LA is composed of one CA moiety and two DSS moieties; and SA consists of one CA moiety and three DSS moieties. In this study, the structure-dependent self-affinities of five phenolic acids, DSS, CA, RA, LA, and SA, were investigated by NMR to evaluate and compare the effects of their different structures on self-association. Knowledge of the self-association process will enable us to quantitatively assess the effects of aggregation on the binding between phenolic acid and protein.
Experimental
Typically, the self-association processes have a relatively fast dynamics, and individual NMR peaks form different aggregates can be not obtained. It was assumed that the chemical shift of the molecules (δobs) is the weighted average between the monomer (δA) and the assembly values (δmax). The progressive association of solute molecules into stacks (dimers, trimers, tetramers, etc.) is expected to occur in accordance with the same affinity constant described by the isodesmic model. Thermodynamic parameters, including the enthalpy change (ΔHΦ), entropy change (ΔSΦ), and the Gibbs free energy change (ΔGΦ), for the self-associations of five phenolic acids were obtained from the van’t Hoff equation and Gibbs free energy equation.
Materials and sample preparation DSS, CA, RA, LA, and SA (all reagent grade) were purchased from Sigma-Aldrich and used as obtained. Analytical grade K2HPO4 and Na2HPO4 · 12H2O were purchased from Guoyao Chemical Co. Ltd. (Shanghai, China). Deuterium oxide (D2O, 99.9% D) and sodium 3-trimethylsilyl [2,2,3,3,-2H4] propionate (TSP) were purchased from Cambridge Isotope Laboratories, Inc (MA, U. S. A.). To eliminate proton peak chemical shift variation resulting from differences in the pH or ionic strength, stock solutions of DSS, CA, RA, LA, and SA (20 mM each) were prepared in a buffer (0.5 M K2HPO4/Na2HPO4, pH 7.4, 100% D2O) containing TSP (0.1%, m/v) and then further diluted with the same buffer to obtain seven different concentrations (0.25, 0.5, 2.5, 5.0, 10, 15, and 20 mM). To ensure accuracy, the pH value for each solution (7.40 ± 0.05) was measured three times using a Mettler Toledo pH meter with a micro glass electrode before 0.5 mL of each solution was transferred into a 5-mm NMR tube for NMR analysis. Nuclear magnetic resonance measurements One-dimensional 1H NMR spectra were measured for each solution at 283, 298, 310, and 323 K on a 600 MHz Varian VNMRS spectrometer with a cold probe. The spectra were acquired using a one-dimensional NOESYPR pulse sequence (RD-90°-t1-90°-tm90°-acquisition; t1 = 4 μs, tm = 100 ms) with water resonance suppression during the recycle delay and the mixing time (tm). For each measurement, 64 transients were collected with 32-k data points and a spectral width of 10 ppm. The pulse length of 90° was adjusted to approximately 10 μs, and recycle delay was set to 2 s. For all spectra, an exponential window function was applied with a line-broadening factor of 0.5 Hz prior to FT, and the phase and baseline were then manually corrected and referenced to TSP (δ 0.00). A total of 35 solutions were subjected to 1H NMR analysis to obtain chemical shift data for investigating the self-interactions. A set of 2D NMR spectra including 1H–1H COSY, 1H–1H TOCSY, 1 H–13C HSQC, and 1H–13C HMBC were acquired and processed on 600-MHz spectrometers to assign the signals. Data fitting The self-association constant Ka is determined by the least-square curve fitting of the experimental proton NMR chemical shift data. n h io2 1 δobs ¼ δA þ ðδmax δA ÞKa ½A 2= 1 þ ð4Ka ½A þ 1Þ 2
(1)
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ΔH∅ 1 ΔS∅ þ R T R
(2)
and ΔG∅ ¼ RT LnKa ;
(3)
where Ka is the self-association constant (M1), R is the gas constant (8.314 J K1 M1) and the temperature T are at 283, 298, 310 and 323 K. The data fitting was performed by Origin software (version 7.5, OriginLab Corporation, MA, USA). All proton chemical shifts in each solution at different temperatures were recorded, and chemical shift dependence on the phenolic concentration was fitted based on Eqn (1), yielding the estimated parameters of Ka, δmax, and δA, together with the error bars of these parameters and correlation coefficients for evaluating the fitting quality. Subsequently, the estimated Ka dependence on temperature was fitted based on Eqns (2) and (3), providing the thermodynamic parameters for the self-association processes of phenolic acids.
Results and Discussion Chemical shift variation of 1H nuclear magnetic resonance signals for five phenolic acids Figure 1 displays the chemical structures, labeled aromatic rings and labeled atoms of five phenolic acids, including DSS, CA, RA, LA, and SA. RA, LA, and SA have similar condensed structures with regard to their common CA moiety (aromatic ring A, with a conjugate double bond) and different numbers of DSS moieties (aromatic rings B, C, and D, with saturated bonds). Figure 2 shows that the 1H NMR spectra of these phenolic acids in D2O at 298 K were stacked with increasing concentration. The 1H resonances were assigned to labeled protons based on previous studies[24–26] and were individually confirmed by the analysis of 2D NMR J-resolved, COSY, TOCSY, HSQC, and HMBC spectra. With increasing concentration from 0.25 to 20 mM, the DSS and CA proton peak positions shifted only slightly, whereas the signals for RA, LA, and SA moved significantly either upfield (lower chemical shift) or downfield (higher chemical shift). The displacements of the phenolic acid proton signals demonstrate that self-aggregation occurs in solution, presumably due to the π–π stacking interactions of aromatic functional groups.[21,27] As the temperature increases from 283 to 310 K, at a fixed concentration, the intensities of 1H signals for all phenolic acids reduced gradually, and the peak positions are shifted downfield to different extents for different phenolic acids (Fig. S1).
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, where δobs is the observed chemical shift and δA and δmax are the proton chemical shift of the monomer and the maximal proton chemical shift of solute A present in a stack, respectively. The value [A] denotes the total molar concentration in solution. [22,23]
lnKa ¼
C. Xiao et al.
Figure 1. The chemical structures, labeled aromatic rings and labeled atoms of five phenolic acids.
1
Figure 2. H NMR spectra for five phenolic acids, danshensu (DSS), caffeic acid (CA), rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA), in D2O at 298 K, stacked from bottom to up by increasing concentration. The signals in the stacked spectra were assigned to the labeled protons.
Aggregation of five phenolic acids by self-association
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Isodesmic fitting curves for the phenolic acids, based on Eqn (1), show changes in the chemical shift (Δδ) with increasing concentration (Fig. 3). The value of Δδ is defined as the proton chemical shift of the sample at various concentrations relative to the reference at the smallest concentration of 0.25 mM. Compared with the very small changes in chemical shifts for DSS (nearly constant, not shown), all of the protons in CA displayed a relatively large
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chemical shift variation (Fig. 3A). It is well known that the strong attractive interactions between π systems (i.e., aromatic ring) induce proton chemical shift variations during the process of selfaggregation and are affected by the size and properties of the substituents on the aromatic ring. Here, an electron-withdrawing substituent, such as the double bond in CA (7, 8 CH = CH), tends to have a greater influence on aromatic stacking than an electron-donating substituent, such as the saturated bond in DSS (7′,8′ CH2-CHCOOH). This result is in agreement with electrostatic
Copyright © 2014 John Wiley & Sons, Ltd.
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The structure-dependent self-association of five phenolic acids
Figure 3. Concentration dependences of chemical shift change (Δδ) of the resolved protons of caffeic acid (A), rosmarinic acid (B), lithospermic acid (C), and salvianolic acid B (D) in aqueous solution at 298 K. The value of Δδ is the proton chemical shift of the sample at various concentrations relative to that at the smallest concentration of 0.25 mM. The curves corresponding to each data set have been obtained with the Origin’s the least-square curve fitting based on Eqn (1).
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aromatic D ring protons may be attributable to the predominance of ring-current-induced diamagnetic deshielding effects from the opposing aromatic rings. The protons of aromatic ring A and the olefinic bond (CA moiety) in LA and SA display a greater variation in their chemical shifts, which confirms that CA moieties prefer stacking because of their larger π–π conjugated system. The concentration dependence of the 1H chemical shifts of the phenolic acids at various temperatures was used to estimate the association constants Ka (Table 1). The Ka values are considerably less precise in DSS and CA due to their minimal variations in chemical shift. Moreover, certain aromatic signals for RA, LA, and SA could not be extracted from the 1H spectra because of extensive signal overlap. These Ka values were therefore calculated from the olefinic protons to evaluate the strength of selfassociation in the formation of self-stacked aggregates. The Ka values are in the order CA < RA < LA < SA, which corresponds to the increasing molecular size of the phenolic acids and roughly corresponds to the increasing number of DSS moieties. Therefore, the relative strength of the self-interaction, represented by the Ka values for the olefinic bonds, has an obvious correlation to molecular size. A similar relationship between Ka and molecular size has also been observed for several polyphenols (e.g., tannins).[9] The exact reason for the apparent increase of the Ka value by additional DSS units in the phenolic acid structures remains unclear. However, it can be inferred that the existence of DSS units having aromatic ring might influence the density of π electronic ring-current of caffeic unit, resulting in the enhanced π–π stacking interactions between phenolic acid molecules and the apparent increase of the Ka value. Self-association thermodynamics of five phenolic acids The thermodynamic parameters of the self-association processes for these phenolic acids were calculated from the Ka values for olefinic protons at different temperatures using van’t Hoff plots (Fig. 4) and are presented in Table 2. The negative sign for the Gibbs free energy (ΔG) means that the self-interaction process
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models of the π–π stacking interaction,[21,28] which have found that electron-deficient rings prefer stacking interactions compared to electron-rich rings. Moreover, the perfect planarity of the larger π–π conjugated system of the aromatic ring and double bond in CA further enhance its preference for stacking. All of the signals of RA shift upfield with increasing concentration (Fig. 3B). It is well established that upfield shifts, as observed here, indicate plane to plane or vertical stacking.[17] The magnitudes of the differences in chemical shift varied for the RA signals, with larger chemical shift displacements for the aromatic ring A protons (2,6 ArH) and olefinic protons (7,8 CH = CH) compared to the displacements of the aromatic ring B protons (5′,6′ArH) and saturated protons (7′,8′ CH2-CHCOOH). The larger chemical shift changes correspond to the most favorable binding sites, which are therefore the least subject to conformational averaging of the aggregate with the monomer. This result implies that the π–π stacking occurs primarily with the planar aromatic ring A and its conjugated double bond (CA moiety), confirming that CA favors stacking more than DSS. The different magnitudes of the chemical shift changes within RA most likely reflect different binding geometries rather than differences in binding affinities. The directions of the proton chemical shift displacements for LA follow a different trend (Fig. 3C); the protons of aromatic ring A and aromatic ring C are strongly shifted upfield, while the aromatic ring B protons are shifted slightly downfield. Similarly, an obvious upfield shift in the aromatic A and aromatic C protons was observed in SA, as well as a minimal downfield shift in the aromatic B and aromatic D protons (Fig. 3D). Shifts upfield generally are of larger magnitude than those for shifts downfield, suggesting the upfield-shifted signals have a closer distance than the downfield-shifted signals. Different directions of the chemical shift displacements were also observed for chloroquine and quinacridone.[16,19] The upfield shifts demonstrated by aromatic ring A and aromatic ring C protons in the favored equiplanar geometry are commonly attributed to the strong interactions between the π systems, as in the case of several polyphenolic compounds.[10] The downfield shifts of the aromatic B and
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SA
LA
RA
CA
DSS
2′,5′,6′ ArH 7′,8′ CH 2 ArH 5 ArH 6 ArH 7 CH = CH 8 CH = CH 2 ArH 5 ArH 6 ArH 7 CH = CH 8 CH = CH 2′ ArH 5′ ArH 6′ ArH 7′ CH 8′ CH 5 ArH 6 ArH 7 CH = CH 8 CH = CH 2′ ArH 5′ ArH 6′ ArH 7′ CH 8′ CH 2″ArH 5″,6″ ArH 7″ CH 8″ CH 5,6 ArH 7 CH = CH 8 CH = CH 2′,5′ ArH 6′ ArH 7′ CH 8′ CH 2″,5″ArH 6″ ArH
Proton
— — 1.02 ± 0.78 — 0.98 ± 0.65 2.31 ± 0.94 1.68 ± 0.65 9.41 ± 0.73 / / 9.83 ± 0.94 9.74 ± 0.97 — / 5.01 ± 0.76 8.24 ± 0.86 / 17.96 ± 3.11 18.42 ± 1.44 18.88 ± 1.36 19.41 ± 1.43 30.57 ± 3.32 30.51 ± 3.40 36.46 ± 3.43 32.25 ± 3.02 29.25 ± 2.94 21.73 ± 1.52 / 21.37 ± 1.57 19.10 ± 1.47 / 31.11 ± 2.47 30.53 ± 2.63 / / 30.18 ± 4.07 28.83 ± 2.80 / 30.10 ± 2.58
Ka(M )
1
— — 7.19 ± 0.00 — 7.08 ± 0.00 6.37 ± 0.00 7.30 ± 0.00 7.23 ± 0.00 / / 6.41 ± 0.00 7.62 ± 0.00 — / 6.81 ± 0.00 3.01 ± 0.00 / 6.95 ± 0.00 7.29 ± 0.00 6.36 ± 0.00 7.70 ± 0.00 6.99 ± 0.00 6.87 ± 0.00 6.79 ± 0.00 3.09 ± 0.00 4.91 ± 0.00 7.00 ± 0.00 / 5.84 ± 0.00 4.40 ± 0.00 / 5.87 ± 0.00 7.04 ± 0.00 / / 2.90 ± 0.00 4.97 ± 0.00 / 6.87 ± 0.00
δa
283 K
— — 6.63 ± 0.40 — 6.61 ± 0.29 6.24 ± 0.05 7.15 ± 0.05 6.31 ± 0.04 / / 5.44 ± 0.05 6.67 ± 0.06 — / 6.62 ± 0.02 2.93 ± 0.00 / 6.52 ± 0.03 6.03 ± 0.04 5.80 ± 0.01 7.08 ± 0.01 7.06 ± 0.00 6.93 ± 0.00 6.86 ± 0.00 3.14 ± 0.00 4.94 ± 0.00 6.67 ± 0.00 / 5.58 ± 0.00 3.78 ± 0.01 / 5.62 ± 0.01 6.52 ± 0.01 / / 3.01 ± 0.00 4.94 ± 0.00 / 6.10 ± 0.00
δmax — — 0.95 ± 0.56 2.16 ± 0.85 2.59 ± 0.77 3.11 ± 0.48 3.09 ± 0.57 7.82 ± 0.75 / 7.17 ± 0.78 8.96 ± 0.50 9.15 ± 0.67 — 9.13 ± 1.58 8.76 ± 0.98 13.46 ± 1.28 14.45 ± 0.83 10.96 ± 2.64 12.61 ± 0.62 13.43 ± 0.64 13.86 ± 0.68 7.33 ± 1.67 15.51 ± 1.52 18.31 ± 1.12 8.73 ± 0.99 9.14 ± 1.60 12.51 ± 0.83 / 16.78 ± 1.08 13.69 ± 0.70 / 23.66 ± 1.48 22.91 ± 1.47 / 28.03 ± 1.83 16.33 ± 2.21 — / 21.45 ± 1.54
Ka(M )
-1
— — 7.19 ± 0.00 6.97 ± 0.00 7.09 ± 0.00 6.37 ± 0.00 7.30 ± 0.00 7.22 ± 0.00 / 7.12 ± 0.00 6.40 ± 0.00 7.61 ± 0.00 — 6.89 ± 0.00 6.82 ± 0.00 3.02 ± 0.00 5.03 ± 0.00 6.90 ± 0.00 7.28 ± 0.00 6.35 ± 0.00 7.70 ± 0.00 6.99 ± 0.00 6.87 ± 0.00 6.79 ± 0.00 2.98 ± 0.00 4.95 ± 0.00 6.99 ± 0.00 / 5.84 ± 0.00 4.38 ± 0.00 / 5.89 ± 0.00 7.07 ± 0.00 / 6.76 ± 0.00 2.91 ± 0.00 — / 6.87 ± 0.00
δa
298 K
— — 6.84 ± 0.19 6.88 ± 0.03 6.97 ± 0.03 6.33 ± 0.01 7.26 ± 0.00 6.47 ± 0.05 / 6.08 ± 0.07 5.66 ± 0.02 6.90 ± 0.03 — 6.85 ± 0.00 6.73 ± 0.00 3.00 ± 0.00 4.95 ± 0.00 6.35 ± 0.07 6.30 ± 0.03 5.93 ± 0.01 7.25 ± 0.01 7.08 ± 0.01 6.91 ± 0.00 6.84 ± 0.00 3.02 ± 0.00 4.96 ± 0.00 6.74 ± 0.00 / 5.67 ± 0.00 3.91 ± 0.01 / 5.68 ± 0.00 6.65 ± 0.01 / 6.80 ± 0.00 3.03 ± 0.00 — / 6.25 ± 0.00
δmax — — 1.15 ± 0.55 — 3.15 ± 0.42 3.28 ± 0.95 6.22 ± 0.53 7.76 ± 0.67 / 3.45 ± 1.46 5.76 ± 0.84 6.71 ± 0.40 — 8.07 ± 1.35 7.60 ± 0.96 14.87 ± 1.56 12.01 ± 1.57 7.94 ± 2.54 8.99 ± 0.34 9.98 ± 0.38 10.37 ± 0.47 1.86 ± 0.44 8.52 ± 1.11 9.86 ± 1.38 5.02 ± 0.79 6.37 ± 1.95 6.96 ± 1.01 / 15.38 ± 1.65 10.04 ± 0.50 / 17.76 ± 0.67 16.11 ± 0.66 / 8.32 ± 1.34 8.67 ± 1.47 — / 14.22 ± 1.78
Ka(M )
1
— — 7.18 ± 0.00 — 7.08 ± 0.00 6.37 ± 0.00 7.30 ± 0.00 7.22 ± 0.00 / 7.12 ± 0.00 6.38 ± 0.00 7.60 ± 0.00 — 6.89 ± 0.00 6.81 ± 0.00 3.11 ± 0.00 5.04 ± 0.00 6.94 ± 0.00 7.28 ± 0.00 6.35 ± 0.00 7.70 ± 0.00 6.99 ± 0.00 6.86 ± 0.00 6.79 ± 0.00 2.98 ± 0.00 4.97 ± 0.00 6.99 ± 0.00 / 5.84 ± 0.00 4.36 ± 0.00 / 5.92 ± 0.00 7.10 ± 0.00 / 6.77 ± 0.00 2.92 ± 0.00 — / 6.86 ± 0.00
δa
310 K
Table 1. The fitting data of five phenolic acids at different temperatures calculated from chemical shift dependent on concentration
— — 7.00 ± 0.08 — 7.02 ± 0.00 6.35 ± 0.00 7.29 ± 0.00 6.63 ± 0.03 / 5.78 ± 0.48 5.65 ± 0.07 6.95 ± 0.03 — 6.86 ± 0.00 6.74 ± 0.00 3.09 ± 0.00 5.00 ± 0.00 6.45 ± 0.10 6.47 ± 0.02 6.03 ± 0.00 7.36 ± 0.01 7.23 ± 0.05 6.90 ± 0.00 6.84 ± 0.00 3.06 ± 0.00 5.02 ± 0.01 6.79 ± 0.02 / 5.74 ± 0.00 4.00 ± 0.01 / 5.75 ± 0.00 6.75 ± 0.00 / 6.83 ± 0.00 3.07 ± 0.01 — / 6.32 ± 0.00
δmax — — / / / — — / / 3.39 ± 0.90 4.59 ± 0.64 4.28 ± 0.67 — 4.04 ± 0.81 6.54 ± 0.82 2.21 ± 1.05 / 7.38 ± 0.76 5.93 ± 0.61 6.97 ± 1.32 6.99 ± 1.29 / 4.59 ± 2.61 / 2.18 ± 0.77 1.30 ± 0.67 / / 9.22 ± 1.56 7.07 ± 1.29 / 13.21 ± 0.53 11.66 ± 0.76 / 5.87 ± 2.94 7.01 ± 1.85 — / 11.06 ± 1.29
Ka(M )
1
— — / / / — — / / 7.11 ± 0.00 6.38 ± 0.00 7.59 ± 0.00 — 6.89 ± 0.00 6.81 ± 0.00 3.02 ± 0.00 / 6.94 ± 0.00 7.27 ± 0.00 6.34 ± 0.00 7.70 ± 0.00 / 6.86 ± 0.00 / 2.99 ± 0.00 4.99 ± 0.00 / / 5.84 ± 0.00 4.35 ± 0.00 / 5.94 ± 0.00 7.12 ± 0.00 / 6.77 ± 0.00 3.09 ± 0.00 — / 6.86 ± 0.00
δa
323 K
(Continues)
— — / / / — — / / 6.09 ± 0.22 5.76 ± 0.06 6.94 ± 0.08 — 6.87 ± 0.00 6.76 ± 0.00 2.99 ± 0.01 / 6.68 ± 0.01 6.60 ± 0.05 6.11 ± 0.00 7.47 ± 0.03 / 6.91 ± 0.02 / 3.16 ± 0.05 5.18 ± 0.14 / / 5.78 ± 0.00 4.09 ± 0.03 / 5.83 ± 0.00 6.84 ± 0.01 / 6.83 ± 0.02 3.15 ± 0.01 — / 6.45 ± 0.02
δmax
C. Xiao et al.
Magn. Reson. Chem. 2014, 52, 460–466
5.79 ± 0.00 3.54 ± 0.03 6.53 ± 0.04 6.65 ± 0.05 6.19 ± 0.01 / — 5.98 ± 0.00 4.43 ± 0.00 6.32 ± 0.00 6.48 ± 0.00 6.14 ± 0.00 / — 12.53 ± 0.87 9.77 ± 0.55 4.52 ± 1.05 3.20 ± 1.11 3.81 ± 1.90 / — 5.68 ± 0.00 3.32 ± 0.02 6.48 ± 0.01 6.59 ± 0.00 6.19 ± 0.00 2.78 ± 0.04 —
δa Ka(M )
Table 2. Thermodynamic parameters for the self-associations of five phenolic acids
5.99 ± 0.00 4.44 ± 0.00 6.29 ± 0.00 6.46 ± 0.00 6.11 ± 0.00 2.62 ± 0.00 —
Magn. Reson. Chem. 2014, 52, 460–466
1
ΔH (KJ mol )
Phenolic acids
DSS, danshensu; CA, caffeic acid; RA, rosmarinic acid; LA, lithospermic acid; SA, salvianolic acid B. —, it was not determined because of the minimal chemical shift change; /, it was not determined because of signal overlap.
14.24 ± 0.54 13.97 ± 0.63 7.52 ± 0.92 6.30 ± 0.58 10.50 ± 1.01 3.46 ± 0.94 — 5.58 ± 0.01 3.14 ± 0.04 6.44 ± 0.00 6.55 ± 0.00 6.18 ± 0.00 2.66 ± 0.00 — 5.99 ± 0.00 4.45 ± 0.00 6.26 ± 0.00 6.45 ± 0.00 6.08 ± 0.00 2.57 ± 0.00 — 23.34 ± 1.75 20.90 ± 1.62 13.83 ± 1.46 15.83 ± 1.92 23.38 ± 2.08 11.28 ± 1.88 — 5.99 ± 0.00 4.46 ± 0.01 6.23 ± 0.00 6.43 ± 0.00 6.06 ± 0.00 2.54 ± 0.00 2.90 ± 0.00 30.60 ± 2.54 29.35 ± 2.61 24.61 ± 2.71 29.48 ± 2.89 37.18 ± 2.82 25.52 ± 3.23 30.17 ± 4.08
5.43 ± 0.02 2.82 ± 0.05 6.44 ± 0.00 6.56 ± 0.00 6.23 ± 0.00 2.64 ± 0.00 3.01 ± 0.00
δa δa δa Ka(M )
7″ CH 8″ CH 2‴ ArH 5‴ ArH 6‴ ArH 7‴ CH 8‴ CH
Proton
Figure 4. The van’t Hoff plots for the olefinic protons (7,8 CH = CH) in rosmarinic acid (RA), lithospermic acid (LA), and salvianolic acid B (SA).
RA LA SA
1
ΔS (J mol
1
K )
1
ΔG (KJ mol )
17.42 ± 5.52 19.18 ± 1.54 16.63 ± 1.14
41.45 ± 18.23 42.80 ± 5.10 28.87 ± 3.75
13.75 ± 4.91 18.03 ± 1.26 15.56 ± 1.12
is spontaneous. The negative enthalpy (ΔH) and negative entropy (ΔS) of the intermolecular interaction indicate that the selfassociation is driven largely by enthalpic contributions at room temperature.[29] These thermodynamic values are characteristic of the π–π stacking interactions of planar aromatic rings. Other compounds with planar aromatic structures have been shown to self-associate according to the same indefinite association model, with similar thermodynamic constants. For example, Sun and coworkers have found values of ΔH = 19.3 ± 2.2 for the aggregation of quinacridone derivatives.[19] Negative enthalpies (ΔH) have also been observed whenever hydrogen bonds are involved in protein-drug binding.[8,30] It is likely that the selfassociation process of phenolic acids involves hydrogen bonds between the hydroxyl groups of one molecule and the carboxyl groups of the partner molecule. Compared to conventional interaction forces such as hydrogen bonds, hydrophobic interactions, electrostatic interactions, and van der Waals interactions, the nature of π–π stacking interactions remains less clear because of complications such as multiple points of intermolecular contact, the strong influence of substituent groups and the cooperative effect of various noncovalent interactions. [19] It can be deduced, however, that the combination of π–π interactions and intermolecular hydrogen bonding stabilizes the selfaggregations of condensed structures.
Conclusions Five phenolic acids with similar structural features, DSS, CA, RA, LA, and SA, were identified to self-associate in D2O based on concentration-dependent proton chemical shift variations. The strength of the self-interactions, based on the self-association constants, increases in the following order of DSS < CA < RA < LA SA, which corresponds to the increasing molecular size of these phenolic acids and roughly corresponds to the increasing
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Table 1. (Continued)
1
283 K
δmax
-1
Ka(M )
298 K
δmax
1
Ka(M )
310 K
δmax
1
323 K
δmax
The structure-dependent self-association of five phenolic acids
C. Xiao et al. number of DSS moieties. Obviously, the existences of DSS moieties are very important for stabilizing the self-associations and the stacking structures of these pheonlic acids. The binding sites for the self-association of these phenolic acids were identified to be on the CA moiety rather than the DSS moiety by the larger chemical shift variations that result from the strong aromatic π–π interactions. The self-association processes are spontaneous, as demonstrated by the negative ΔG, and are enthalpically favorable at room temperature as shown by the negative ΔH and ΔS. It was inferred that π–π interactions and packing forces, from hydrogen bonding between the hydroxyl groups of one molecule and the carboxyl groups of the partner molecule, stabilize the stacking structures of the phenolic acids. Acknowledgements We acknowledge financial support from the National Natural Science Foundation of China (21005062), the Ministry of Education of China (20106101120024), the Education Department of Shaanxi province of China (12JK1011), and the Key Program for Science and Technology Innovative Research Team of Shaanxi Province (2013KCT-24).
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