8992
J. Phys. Chem. B 1999, 103, 8992-8997
NMR Study of the Solution Structures of the Inclusion Complexes of β-Cyclodextrin with (+)-Catechin and (-)-Epicatechin Takashi Ishizu,*,† Kanako Kintsu,‡ and Hideji Yamamoto‡ Faculty of Pharmacy and Pharmaceutical Sciences, and Department of Applied Biological Science, Faculty of Engineering, Fukuyama UniVersity, Sanzo Gakuen-cho 1, Fukuyama, Hiroshima 729-0292, Japan ReceiVed: April 8, 1999
The probable structures of the inclusion complexes of β-cyclodextrin (β-CD) with (+)-catechin (CA) and (-)-epicatechin (EC) in D2O at 35 °C were investigated using NMR. In the β-CD‚CA inclusion complex, a large portion of the flavonoid skeleton was included in the β-CD cavity and the axis of C1′-C4′ of the B ring inclined to the molecular axis of β-CD, and the CA molecule fitted tightly with β-CD. In the β-CD‚EC inclusion complex, the B ring was included deeply in the β-CD cavity from the secondary hydroxyl group side and the A ring was left outside the cavity near the secondary hydroxyl group side and EC molecule fitted loosely.
Introduction
CHART 1
Cyclodextrins (CDs) are R-1,4-linked cyclic oligomers of which possess the remarkable property of forming inclusion complexes with a variety of small molecules of the appropriate size through the influences of noncovalent interactions. The resultant inclusion complexes can increase the stability and improve the solubility and bioavailability of molecules of pharmaceutical interest.1 Therefore, it is important to clarify the structures of the inclusion complexes from a viewpoint of substrates within the hydrophobic cavities of CD in aqueous solution. Recently, the catechins contained in green tea have attracted attention as a natural food additive, since they have been reported to have many biological functions, including antioxidation, deodorization, antivirus, and cancer inhibiting properties.2 However, catechin powders are bitter, brown, and easily oxidized, and hence difficult to use as a natural food additive or medicine. In order to solve these problems, we have investigated the fundamental property of the inclusion complex of cyclodextrin (CD) with catechins. We reported previously the isolation of crystal form of the inclusion complex of β-CD with (+)-catechin (CA) and studied its structure in the solid state.3 In the present study, the solution structures of the inclusion complexes of β-CD with (+)-catechin (CA) and (-)-epicatechin (EC) (Chart 1), which consist of three rings (the A, B, and C rings) and four phenolic hydroxyl groups and 3-OH group, were investigated in D2O at 35 °C using NMR. The detailed differences between the inclusion modes of β-CD with CA and EC, which have equatorial and axial 3-OH groups, respectively, are discussed. D-glucopyranose
monitoring the chemical shift of 8-H in the 1H-NMR measurements. Figure 1 shows plots of the shift values of the 8-H signal of CA or EC by adding β-CD versus the mole concentration ratio of β-CD to CA or EC. The stoichiometries of both the inclusion complexes were determined by the molar method to be 1:1. The stoichiometries in the range of 35-75 °C were the same (1:1). The dimerizations of CA and EC were not observed at these temperatures and concentration in the experiments. The stability constant of the inclusion complex Kc (M-1) is represented in eq 1, where [CA or EC], [β-CD], and [CX] represent the mole concentration of CA or EC, β-CD, and the β-CD‚CA or EC complex (M), respectively, and [ ]0 represents the initial concentration (M). In eq 3, δCA or EC, δCX, and δobs represent the chemical shift (ppm) of 8-H of CA or EC, the inclusion complex of β-CD with CA or EC, and the mixture of β-CD with CA or EC in 1H NMR spectra, respectively.4,5 Kc
CA or EC + β-CD y\z CX
Results
Kc )
The stoichiometries and the stability constants of the inclusion complexes of β-CD with CA and EC were determined by
) * Corresponding author. E-mail:
[email protected]. Tel: 0849-36-2111 (Ex 5253). Fax: 0849-36-2024. † Faculty of Pharmacy and Pharmaceutical Sciences. ‡ Faculty of Engineering.
[CX] [CA or EC][β-CD]
[CX] ([CA or EC]0 - [CX])([β-CD]0 - [CX])
which on rearrangement transforms to
10.1021/jp991178e CCC: $18.00 © 1999 American Chemical Society Published on Web 10/04/1999
(1)
Inclusion Complexes of β-Cyclodextrin
J. Phys. Chem. B, Vol. 103, No. 42, 1999 8993 TABLE 1: Kc, ∆G, ∆H, and ∆S of the Inclusion Complex Formation of β-CD with CA and EC
Figure 1. Plots of the shift values of the 8-H signal of CA (O) and EC (b) by adding β-CD versus the molar concentration ratio of β-CD to CA or EC at 35 °C.
(
)
[CA or EC]0 1 - 1 ([β-CD]0 - [CX]) ) Kc [CX] 1 ) [CA]0Kc [CA or EC]0
(
[CX]
)(
-1
[β-CD]0
[CA or EC]0
-
)
[CX] (2) [CA or EC]0
θ is expressed as
θ)
[CA or EC]0 - [CA or EC] [CA or EC]0
)
[CX] [CA or EC]0
The observed chemical shift (ppm) of 8-H of catechins will be given by
δobs ) δCA or EC (1 - θ) + δCXθ δCA or EC - δobs ∆δobs ) δCA or EC - δCX ∆δCX
θ)
(3)
M is expressed as
M)
[β-CD]0
(4)
[CA or EC]0
Combining eqs 2, 3, and 4 gives
1 1 ) -1 (M - θ) θ [CA or EC]0Kc
( )
which on rearrangement transforms to
M)θ
[
]
1 +1 (1 - θ)[CA or EC]0Kc
Combining eqs 5, 3, and 4 and transforming gives
(5)
[β-CD]0 )
[(
∆δobs ∆δCX
]
1 + [CA or EC]0 ∆δobx 1K ∆δCX c
)
(6)
∆G (kJ mol-1)
∆H (kJ mol-1)
∆S (J K-1 mol-1)
T (°C)
Kc (M-1)
35 45 55 65 75
2209 1558 1078 952 643
β-CD + CA -19.7 -26.1 -19.4 -26.1 -19.0 -26.1 -19.3 -26.1 -18.7 -26.1
-20.8 -21.0 -21.6 -20.2 -21.3
35 45 55 65 75
1212 1023 847 764 686
β-CD + EC -18.2 -12.8 -18.3 -12.8 -18.4 -12.8 -18.7 -12.8 -18.9 -12.8
17.5 17.4 17.0 17.3 17.5
The Kc values at 35-75 °C were estimated from the change in ∆δobs ()δCA or EC - δCX) versus increasing [β-CD]0 at constant [CA or EC]0 using eq 6, incorporated into the Marquardt nonlinear fitting program. These Kc values for the β-CD‚CA and EC inclusion complex at 35 °C were 2209 and 1212 M-1, respectively. These values are little different from Kc values obtained by Cai et al. using NMR (which were 2908 ( 87 and 464 ( 14 M-1 at 45 °C, respectively ).4 This gap is due to the difference of the remarked signals for chemical shifts which are 8-H of CA in this study and 5-H of β-CD in the literature.4 It was difficult to observe the 5-H signal of β-CD because of broadening and separation by the complexation. Then the singlet-like 8-H signal of CA was chosen. The Kc values of the β-CD‚CA complex at 20 and 10 °C estimated by the temperature dependence of Kc are 3638 and 5313 M-1, respectively. Smith has estimated the Kc value as 8700 M-1 by using circular dichroism at lower temperature.6 Considering the difference of the methods, the Kc values obtained in this study were thought to be adequate. The Kc value for the β-CD‚CA inclusion complex was about 1.8 times (6.3 times the Kc values obtained by Cai et al.4) than that for β-CD‚ EC inclusion complex. From the dependency of Kc on temperature, the changes of free energy ∆G, enthalpy ∆H, and entropy ∆S, of the complex formations were estimated as shown in Table 1. The values of the thermodynamic function ∆G, ∆H, and ∆S are considered to be the same order as those for complexes of phenol and 4-nitrophenol with β-CD reported previously.7 The entropy (∆S) at 35 °C for the inclusion complex formation of β-CD‚CA takes a negative value (-20.8 J K-1 mol-1); on the other hand, ∆S for β-CD‚EC takes a positive value (+17.5 J K-1 mol-1) (Table 1). This suggests that upon complexation EC is more flexible than CA in the cavity of β-CD. Figure 2, a and c, shows NMR spectra of 3 × 10-3 M CA and EC, respectively. The proton signals of CA and EC were assigned on the basis of 1H-1H and 13C-1H chemical shift correlation spectroscopy (COSY), and the literature.8 When β-CD (3 × 10-3 M) was added to CA, the proton signals of CA, except for that of 6-H, became remarkably broader (Figure 2b), and as the temperature rose from 35 to 85 °C, these broadening proton signals of CA sharpened (Figure 3), indicating that the motions of these protons were restricted. On the other hand, in the presence of β-CD the proton signals of EC did not broaden (Figure 2d). In addition to the viewpoint of ∆S and Kc for the inclusion complex formation of β-CD‚CA or EC, these findings indicate that CA fits tightly with β-CD, and EC fits loosely.
8994 J. Phys. Chem. B, Vol. 103, No. 42, 1999
Ishizu et al.
Figure 2. 1H NMR spectra of CA and EC in the presence of β-CD in D2O at 35 °C: (a) CA alone (3 × 10-3 M), (b) CA (3 × 10-3 M) + β-CD (3 × 10-3 M), (c) EC alone (3 × 10-3 M), and (d) EC (3 × 10-3 M) + β-CD (3 × 10-3 M).
Structure of the β-CD‚CA Inclusion Complex: The rotating frame nuclear Overhauser effect spectroscopy (ROESY) spectrum was measured in a solution containing β-CD (28.7 × 10-3 M) and CA (28.7 × 10-3 M) (Figure 4). Strong rotating frame nuclear Overhauser effect (ROE) correlations between 2′-H, 5′-H, 6′-H of CA and 5-H of β-CD were observed,
indicating that the B ring, the catechol moiety, of CA is included in the β-CD cavity from the wide secondary hydroxyl group side and close to 5-H on the inner surface of the cavity at the primary hydroxyl group side of β-CD. A 1H NMR spectrum of 3.0 × 10-3 M β-CD solution is shown in Figure 5a. The spectrum is different from those (Figure
Inclusion Complexes of β-Cyclodextrin
J. Phys. Chem. B, Vol. 103, No. 42, 1999 8995
Figure 5. 1H NMR spectra of β-CD in the presence of CA or EC in D2O at 35 °C: (a) β-CD alone (3 × 10-3 M), (b) β-CD (3 × 10-3 M) + CA (3 × 10-3 M), and (c) 5-CD (3 × 10-3 M) + EC (3 × 10-3 M).
TABLE 2: Shift Values (ppm) of Chemical Shifts of β-CD in the Presence of CA and EC β-CD in the presence of proton no.
CAa
ECb
1 2 3 4 5 6
0.0079 0.0177 -0.0226 0.0348 -0.1134 -0.0634
0.0030 0.0043 -0.0274 0.0293 -0.0359 -0.0165
a β-CD (3.0 × 10-3 M) + CA (3.0 × 10-3 M) in D O (550 µL). 2 β-CD (3.0 × 10-3 M) + EC (3.0 × 10-3 M) in D2O (550 µL), a -3 control sample β-CD (3.0 × 10 M) in D2O (550 µL).
b
Figure 3. 1H NMR spectra of CA (3 × 10-3 M) in the presence of β-CD (3 × 10-3 M) in D2O at 35-85 °C: (a) CA alone (3 × 10-3 M) at 35 °C, (b) CA + β-CD at 35 °C, (c) CA + β-CD at 45 °C, (d) CA + β-CD at 55 °C, (e) CA + β-CD at 65 °C, (f) CA + β-CD at 75 °C, and (g) CA + β-CD at 85 °C.
Figure 6. Possible structure of the β-CD‚CA inclusion complex in D2O at 35 °C.
Figure 4. ROESY spectrum of solution containing CA (28.7 × 10-3 M) and β-CD (28.7 × 10-3 M) in D2O at 35 °C.
5b,c) in the presence of 3.0 × 10-3 M of CA or EC. Comparison of these spectra reveals that all proton signals in the β-CD
molecule shifted upfield or downfield (Table 2) in the presence of CA or EC. Upon β-CD‚CA inclusion complex formation the upfield shift of 5-H was most prominent, followed by the 6-H lying on the inner surface of the primary hydroxyl group side. Though 3-H lying on the inner surface of the secondary hydroxyl group side shifted upfield, the upfield shift was not as prominent as that of 6-H of β-CD. Upfield shifts of signals of 1-H, 2-H, and 4-H which lie on the outer surface of the cavity were not observed. Upfield shifts of protons lying on the inner surface of β-CD may result mainly from anisotropic shielding by ring current from the aromatic rings of CA, suggesting that upshield shifts of 5-H and 6-H were due to the B ring of CA and that of 3-H was due to the A ring.
8996 J. Phys. Chem. B, Vol. 103, No. 42, 1999
Figure 7. ROESY spectrum of solution containing EC (28.7 × 10-3 M) and β-CD (28.7 × 10-3 M) in D2O at 35 °C.
Upon inclusion complex formation with CA, the multiplets of the protons of 5-H and 6-H of β-CD became remarkably broader (Figure 5b). This fact suggests that the axis of the C1′C4′ of the B ring of CA inclines to the molecular axis of β-CD in the cavity as shown in Figure 6, and that a gap in the strength of the shielding effect to each 5-H and 6-H among the 7 glucose residues produces, as a result, loss of magnetic equivalents. In Figure 2b, the broadening of the protons signals of CA, except for that of 6-H, in the presence of β-CD suggests that the B ring, the C ring, and a portion of the A ring are included in the cavity and hence the motions of these protons are restricted, and that the 6-H of the A ring is left outside of the cavity. These results suggest that Figure 6 is a possible structure of the β-CD‚CA inclusion complex. Smith et al. reported previously that CA penetrated partially into the β-CD cavity upon complexation and one of the hydroxyl groups of the A ring came into closer contact with the hydroxyl groups on the rim of the β-CD.6 In this study, the structure of the β-CD‚CA inclusion complex was made more clear by using ROESY. Structure of the β-CD‚EC Inclusion Complex. As listed in Table 2, upon β-CD‚EC inclusion complex formation the upfield shift of 5-H was most prominent, followed by 3-H and 6-H. These upfield shifts may result mainly from anisotropic shielding by ring current from the aromatic rings (the A and B rings) of EC. In the ROESY spectrum of a solution containing β-CD (28.7 × 10-3 M) and EC (28.7 × 10-3 M) (Figure 7), the ROE
Figure 8. Two possible β-CD‚EC inclusion modes in D2O at 35 °C.
Ishizu et al. correlations between 4-H, 2-H, 3-H of EC and 2-H of β-CD and between 8-H of EC and 3-H, 2-H, 4-H of β-CD were observed. The broadening of the proton signals of EC in the presence of β-CD was not observed (Figure 2d), indicating that in the β-CD‚EC inclusion complex the motions of the protons of EC are not restricted. Therefore, this fact suggests that EC molecule is included deeply in the β-CD cavity from the wide secondary hydroxyl group side without close contact with the atoms on the inner surface of β-CD, and hence positioned at the center of the cavity along the molecular axis of β-CD. As the possible β-CD‚EC inclusion modes, judging from the investigation using the ROE correlations, the data obtained from 1H NMR spectra mentioned above, and Corey-Pauling-Koltun (CPK) atomic models, two modes, a and b, as shown in Figure 8 are considered. In mode a the upfield shift of 5-H is due to the B ring of EC and in the mode b that is due to the A ring. In Figure 2c the two proton signals of 5′-H and 6′-H of EC appeared as a singlet-like signal because of the fast rotation rate of the B ring relative to the NMR time scale. It is considered that the rotation rate of the B ring is slowed by the β-CD‚EC inclusion complex formation, and as a result, those of 5′-H, and 6′-H signals appear as a normal NMR pattern of the catechol moiety in Figure 2d. From the investigation with CPK atomic models, in mode a the B ring cannot rotate freely around the molecular axis of β-CD, while in mode b it can rotate. It is therefore considered that the mode a is more plausible than mode b (Figure 8). Discussion We will discuss the inclusion modes of β-CD with CA and EC from the standpoint of the stereochemical configuration of the 3-OH group. While CA has an equatorial 3-OH group which is buried in the flavonoid skeleton, EC has an axial 3-OH group which juts out from that. Our results show that upon β-CD‚CA inclusion complex formation, a large portion of the flavonoid skeleton of CA is included in the β-CD cavity from the wide secondary hydroxyl group, and the B ring of CA is close to 5-H of β-CD, and that the CA molecule is inclined to the molecular axis of β-CD. We assume that CA fits tightly with β-CD because of smaller steric hindrance from the equatorial 3-OH group of CA than that from the axial 3-OH group of EC, and that the four phenolic hydroxyl groups of the A and B rings of CA are left out of the hydrophobic cavity of β-CD because of their hydrophilic characters, as shown in Figure 6.
Inclusion Complexes of β-Cyclodextrin Furthermore, our results show that upon the β-CD‚EC inclusion complex formation, the B ring of EC is included deeply in the β-CD cavity from the wide secondary hydroxyl group to form the inclusion mode a (Figure 8). We presume that the B and C rings of EC did not incline to the molecular axis of β-CD such as CA molecule in the β-CD‚CA inclusion complex, but positioned at the center of the cavity along the molecular axis of β-CD in order to avoid the steric hindrance and the hydrophilic character due to the axial 3-OH group of EC. Then it is considered that the anisotropic shielding by the aromatic rings of EC is weak, because the A ring of EC is positioned at the center of the β-CD cavity and away from the inner surface of the cavity. Thus the magnitudes of upfield shifts of 5-H and 6-H of β-CD in the presence of EC are not as prominent as those in the presence of CA (Table 2). Experimental Section NMR spectra were obtained on a JEOL JMN-LA500 spectrometer operating at 500.00 MHz for 1H and 125.65 MHz for 13C in a 5 mm i.d. sample tube at 35-85 °C. Chemical shift values are expressed in ppm downfield using sodium 2,2dimethyl-2-silapentane-5-sulfonate (DSS) as an internal stan-
J. Phys. Chem. B, Vol. 103, No. 42, 1999 8997 dard. Samples were dissolved in D2O (99.9 atom % D, Aldrich Chemical Co., Inc.). ROESY experiments were carried out using a mixing time of 250 ms in the phase-sensitive mode. Materials. β-CD was supplied by ENSUIKO Sugar Refining Co., Ltd. and CA and EC by Kurita Water Industries Ltd. References and Notes (1) (a) Szejtli, J. Cyclodextrins and Their Inclusion Complexes; Akademiai Kiado: Budapest, Hungary, 1982. (b) Szejtli, J. Cyclodextrins Technology; Kluwer Academic Publishers: Dordrecht, 1988. (2) (a) Horita, H. Bull. Natl. Res. Inst. Veg., Ornam. Plants Tea Japan, Ser. B 1989, No. 3, 65. (b) Fujiki, H. Pharmacia 1998, 34, 223-225. (c) Yamada, M. Chem. Chem. Ind. 1998, 51, 582-584. (3) Hosono, T.; Miyamoto, S.; Kobayashi, U.; Yamamoto, H.; Harano, Y. Kagaku Kogaku Symp. Ser. 1995, 47, 120-125. (4) Cai, Y.; Gaffney, S. H.; Lilley, T. H.; Magnolato, D.; Martin, R.; Spencer, C. M.; Halam, E. J. Chem. Soc., Perkin Trans. 2 1990, 21972209. (5) Bergeron, R. J.; Channing, M. A.; Gibeily, G. J.; Pillor, D. M. J. Am. Chem. Soc. 1977, 99, 5146-5151. (6) Smith, V. K.; Ndou, T. T.; Warner, I. M. J. Phys. Chem. 1994, 98, 8627-8631. (7) Ueno, A., Ed. Cyclodextrin-Fundamentals and Application; Sangyou Tosho: Tokyo, 1995. (8) Agrawal, P. K., Ed. Carbon-13 NMR of FlaVonoids; Elsevier: Amsterdam, 1989.
