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Binding of Short-Chain Lecithin by β-Cyclodextrin Noriaki Funasaki,* Seiji Ishikawa, and Saburo Neya Kyoto Pharmaceutical University, Misasagi, Yamashina-ku, Kyoto 607-8414, Japan Received June 12, 2001. In Final Form: October 22, 2001 Complex formation between diheptanoylphosphatidylcholine (DHPC) and β-cyclodextrin (β-CD) in deuterium oxide solution has been investigated by measurements of proton NMR chemical shifts and the ROESY spectrum and molecular mechanics calculations. The vicinal coupling constants for protons of R-, β-, and γ-CDs in their DHPC complexes have been also determined for comparison among these CDs. From the variations in chemical shifts of DHPC and β-CD with the addition of β-CD, the macroscopic equilibrium constant, K1, of the 1:1 complexation of DHPC and β-CD is estimated. The magnitude of K1 is in the order of β-CD > γ-CD > R-CD for DHPC, although it is in the order of R-CD g β-CD > γ-CD for single-chain surfactants. From the vicinal coupling constants of the glycerol C1H2-C2H protons of DHPC, the populations of three rotamers, gauche+ conformer (G+), gauche- conformer (G-), and trans conformer (T), are estimated. The addition of β-CD causes a decrease in the T conformer and an increase in the G+ conformer. These changes are the same as those of γ-CD but opposite to those of R-CD. The microscopic binding constants of CD complexation for the three rotamers are also estimated from the concentration dependence of vicinal coupling constants. The microscopic binding constant for the T conformer is in the order of R-CD > β-CD > γ-CD, as expected. The presence of DHPC causes large changes in vicinal coupling constants of the β-CD protons, suggesting that the β-CD macrocycle is deformed by incorporation of DHPC. A three-dimensional structure of the major complex β-CD-G+ is proposed on the basis of these variations in chemical shift and vicinal coupling constant, the ROESY spectrum, and molecular mechanics calculations: two heptanoyl chains of DHPC are simultaneously incorporated into a β-CD cavity so that the rim of β-CD is deformed to an ellipse. This tight contact between the hydrophobic groups of DHPC and β-CD leads to a large binding constant.
Introduction Cyclodextrins (CDs) have homogeneous toroidal structures of different molecular sizes: most typical are cyclohexaamylose (R-CD), cycloheptaamylose (β-CD), and cyclooctaamylose (γ-CD). The toroidal structure has a hydrophilic surface, making it water soluble, whereas the cavity is composed of the glucoside oxygens and methine hydrogens, giving it a hydrophobic character. Consequently, the CDs can accommodate other hydrophobic molecules of appropriate dimensions and shapes.1,2 The crystal structures of many CD inclusion complexes have been determined by X-ray diffraction techniques.3-5 The CD cavities including large guest molecules are generally deformed. This flexibility of the CD macrocycle has been supported by molecular mechanics calculations and molecular dynamics simulations.6-8 A number of studies have focused on the interactions of CDs with surfactants having a single alkyl chain.9-13 (1) Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344. (2) Szejtli, J. Cyclodextrin Technology; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1988; Chapters 2 and 3. (3) Saenger, W.; Jacob, J.; Gesseler, K.; Steiner, T.; Hoffmann, D.; Sanbe, H.; Koizumi, K.; Smith, S.; Takaha, T. Chem. Rev. 1998, 98, 1787. (4) Harata, K. Chem. Rev. 1998, 98, 1803. (5) Harata, K. In Inclusion Compounds; Atwood, J. L., Davies, J. E. D., MacNicol, D. D., Eds.; Oxford University Press: Oxford, U.K., 1991; Vol. 5, Chapter 9. (6) Sherrod, M. In Spectroscopic and Computational Studies of Supramolecular Systems; Davies, J. E. D., Ed.; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1992; Chapter 9. (7) Lipkowitz, K. B. J. Org. Chem. 1991, 56, 6357. (8) Lipkowitz, K. B. Chem. Rev. 1998, 98, 1829. (9) Park, J. W.; Song, H. J. J. Phys. Chem. 1989, 93, 6454. (10) Funasaki, N.; Yodo, H.; Hada, S.; Neya, S. Bull. Chem. Soc. Jpn. 1992, 65, 1323 and references therein. (11) Wan Yunus, W. M. Z.; Taylor, J.; Bloor, D. M.; Hall, D. G.; WynJones, E. J. Phys. Chem. 1992, 96, 8979. (12) Jobe, D. J.; Verrall, R. E.; Junquera, E.; Aicart, E. J. Phys. Chem. 1994, 98, 10814.
For instance, the 1:1 binding constants of β-CD with single chain surfactants are close to those of R-CD but much larger than those of γ-CD. The small CDs R-CD and β-CD can include one alkyl chain only in their cavities, but the larger γ-CD can include two alkyl chains simultaneously. On the other hand, very few studies have been reported on complex formation between double alkyl chain surfactants and CDs.14-16 For such systems, the stoichiometry, the structure of the complex, and the binding constant remain virtually unexplored. The structure of the complex provides essential information about the magnitude of the binding constant.10,13-15 These studies on complex formation between CDs and surfactants are also interesting from the viewpoint of supramolecular chemistry.17,18 Because CDs are essentially nontoxic, they are added to pharmaceuticals and foods, for example, for stabilization of labile compounds, suppression of bitter tastes and hemolysis,13,19-21 and long-term preservation of color, odor, and flavor.1,2,22 However, because the CDs are hemoly(13) Funasaki, N.; Ohigashi, M.; Hada, S.; Neya, S. Langmuir 2000, 16, 383. (14) Ishikawa, S.; Neya, S.; Funasaki, N. J. Phys. Chem. B 1998, 102, 2502. (15) Funasaki, N.; Neya, S. Langmuir 2000, 16, 5343. (16) Isnin, R.; Yoon, H. R.; Vargas, R.; Quintela, P. A.; Kaifer, A. E. Carbohydr. Res. 1989, 192, 357. (17) Schneider, H.-J.; Hacket, F.; Ru¨diger, V.; Ikeda, H. Chem. Rev. 1998, 98, 1755. (18) Schneider, H.-J.; Yatsimirsky, A. K. Principles and Methods in Supramolecular Chemistry; John Wiley and Sons: New York, 2000. (19) Funasaki, N.; Uemura, Y.; Hada, S.; Neya, S. J. Phys. Chem. 1996, 100, 16298. (20) Funasaki, N.; Ohigashi, M.; Hada, S.; Neya, S. Langmuir 1999, 15, 594. (21) Funasaki, N.; Kawaguchi, R.; Ishikawa, S.; Hada, S.; Neya, S.; Katsu, T. Anal. Chem. 1999, 71, 1733. (22) Fro¨mming, K.-H.; Szejtli, J. Cyclodextrins in Pharmacy; Kluwer Academic Publishers: Dordrecht, The Netherlands, 1994; Chapters 3, 6, and 10.
10.1021/la0108860 CCC: $22.00 © 2002 American Chemical Society Published on Web 02/01/2002
Binding of Short-Chain Lecithin by β-Cyclodextrin
tic,1,20,22-24 parenteral administration of CDs is restricted by health authorities.22 This hemolysis is caused by the extraction of erythrocyte membrane components (cholesterol and phospholipid) by CD.22-24 The hemolytic activity of CDs is in the order of β-CD > R-CD > γ-CD.20,22-24 Therefore, basic studies on complex formation between lecithin and CDs should shed some light on the mechanism of hemolysis by CDs. In this work, we investigated complex formation of 1,2diheptanoyl-3-L-R-phosphatidylcholine (DHPC), a shortchain lecithin, with β-CD by 1H NMR and molecular mechanics. From the variations in chemical shift and vicinal coupling constant for DHPC and β-CD with the addition of β-CD, the equilibrium binding constants (macroscopic and microscopic constants) of DHPC and β-CD were determined. Unexpectedly, this change in vicinal coupling constant of DHPC was similar to that for γ-CD, and the macroscopic 1:1 binding constant for β-CD was larger than that of R-CD and γ-CD. To analyze these unexpected findings, we also determined the vicinal coupling constant for protons of R-, β-, and γ-CDs in the complexes with DHPC. On the basis of the chemical shift, ROESY data, and molecular mechanics calculations, an elliptically deformed structure of β-CD in its DHPC complex is proposed. Experimental Section NMR Measurements. DHPC from Sigma Chemical Co. and deuterium oxide (99.9%) from Aldrich were used as received. Sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) and β-CD were purchased from Nacalai Tesque Co. All 300 MHz proton NMR spectra were recorded with a Varian XL-300 NMR spectrometer at 21.0 ( 0.5 °C. These spectra were deconvoluted with a Nuts NMR data-processing software (Acorn NMR Inc.). This software was also used for spectral simulations. The chemical shift of a sample in deuterium oxide was measured against DSS as external standard. No buffer or salt was added to adjust the pH and the ionic strength, because DHPC is a zwitterion over a wide neutral pH range. The concentration of DHPC, CL, was kept at 1.00 mmol kg-1, which is below the critical micelle concentration of 1.5 mmol kg-1,25 whereas the concentration of CD, CD, was increased up to ca. 14 mmol kg-1. The chemical shifts of all protons of CDs decreased linearly with increasing CD concentration, and this concentration dependence was ascribed to the dimerization of CDs.26 Matsui and Tokunaga argued that the internal reference method using noncomplexing solutes is more reliable than the external one.27 Although we employed the external reference method, our corrected chemical shift data are consistent with those determined by the internal reference method using tetramethylammonium chloride.28 The 500 MHz ROESY (phase-sensitive rotating framework Overhauser enhancement spectroscopy) spectrum of a deuterium oxide solution containing 3 mM DHPC and 3 mM β-CD was recorded with a JEOL Lambda 500 spectrometer using the JEOL standard pulse sequences. The data consisted of 16 transients collected over 2048 complex points. A mixing time of 500 ms, a repetition delay of 1.2 s, and a 90° pulse width of 11.0 ms were used. The ROESY data set was processed by applying an exponential function in both dimensions and zero-filling to 2048 × 2048 real data points prior to the Fourier transformation. Small cross-peaks in these ROESY spectra were neglected, because their magnitude was close to that of noise. (23) Uekama, K.; Hirayama, F.; Irie, T. Chem. Rev. 1998, 98, 2045. (24) Ohtani, Y.; Irie, T.; Uekama, K.; Fukunaga, K.; Petha, J.Eur. J. Biochem. 1989, 186, 17. (25) Ishikawa, S.; Hada, S.; Funasaki, N. J. Phys. Chem. 1995, 99, 11508. (26) Alston, D. R.; Lilley, T. H.; Stoddart, J. F. J. Chem. Soc., Chem. Commun. 1985, 1600. (27) Matsui, Y.; M.; Tokunaga, S. Bull. Chem. Soc. Jpn. 1996, 69, 2477. (28) Funasaki, N.; Nomura, M.; Yamaguchi, H.; Ishikawa, S.; Neya, S. Bull. Chem. Soc. Jpn. 2000, 73, 2727.
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Figure 1. Newman projections of three rotamers of different dihedral angles around the HXC2-C1HAHB axis of the glycerol moiety of DHPC. Molecular Modeling of Complexes. The structure of DHPC was constructed on the basis of the crystal structure of dimyristoylphosphatidylcholine.25,29,30 The DHPC molecule in the crystal was in the gauche+ (G+) form (Figure 1). The 2-heptanoyl chain in the gauche- (G-) and trans (T) forms was assumed as a fully extended conformation, although it may be somewhat folded in the G- form, because of intramolecular hydrophobic interactions between the two heptanoyl chains.31 The structure of β-CD was taken from the crystal structure of its complex with 2-(3phenoxyphenyl)propionic acid.32 The structures of β-CD and DHPC were regarded as rigid bodies unchanged with complexation. Instead of 2-(3-phenoxyphenyl)propionic acid, the heptanoyl groups of a DHPC molecule in the G+ form were inserted into the β-CD cavity, parallel to the symmetry axis of β-CD, and the phosphatidylcholine group was located outside the secondary alcohol side of β-CD. The DHPC molecule was penetrated, regardless of interatomic collision, so deeply that the observed ROESY and chemical shift data were consistent with the structure of the complex. This complex was accommodated by a unit cell (2.5 nm × 2.5 nm × 3.5 nm), where the heptanoyl groups were arranged parallel to the long axis of the cell. This cell was soaked with 627 water molecules. Then, the β-CD molecule was regarded as flexible, whereas the DHPC molecule was kept rigid. This structure of the complex was optimized using the Discover III module of Molecular Simulation Insight II/Discover (98.0) on a Silicon Graphics Octane workstation. The periodic boundary condition was applied to this unit cell. The cutoff distance for van der Waals and electrostatic forces was 1.6 nm. A HyperChem (Hypercube, Inc.) package modeling software and our own software25 were used for molecular modeling and data analysis on a Dell Precision 610 personal computer.
Results Chemical Shifts of DHPC and CD Protons. The assignment of each proton of DHPC in the 300 MHz 1H NMR spectrum has been established.14,31,33 The HX proton of the glycerol C1HAHBC2HX fragment of DHPC (Figure 1) exhibits a complicated multiplet at 5.3 ppm. The HB and HA proton signals appear as quartets around 4.5 and 4.3 ppm, respectively. The proton signals of two hexyl groups of DHPC constitute four main peaks at high field: double triplets of two R-CH2 groups of chains 1 and 2, double multiplets of two β-CH2 groups, a broad tall peak of the middle methylene groups (γ-, δ-, and -CH2 groups), and a single triplet of the ω-methyl groups. Figure 2 depicts the observed and simulated 300 MHz 1H NMR spectra of deuterium oxide solutions containing (a) 1 mmol kg-1 β-CD and (b) 1 mmol kg-1 DHPC and 1 mmol kg-1 β-CD in the region of all β-CD protons except H1. The assignments of the β-CD protons were carried out in comparison with the literature spectra of R-CD and γ-CD.14,34 Computer simulations of these NMR spectra were performed for accurate determinations of chemical (29) Pascher, I.; Pearson, R. H. Nature 1979, 281, 499. (30) Seddon, J. M. In Phosholipids Handbook; Cevc, G., Ed.; Marcel Dekker: New York, 1993; p 909. (31) Hauser, H.; Guyer, W.; Pascher, I.; Skrabal, P.; Sundell, S. Biochemistry 1980, 19, 366. (32) Hamilton, J. A.; Chen, L. J. Am. Chem. Soc. 1988, 110, 5833. (33) Roberts, M. F.; Bothner-By, A. A.; Dennis, E. A. Biochemistry 1978, 17, 935. (34) Wood, D. J.; Hruska, F. E.; Saenger, W. J. Am. Chem. Soc. 1977, 99, 1735.
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Funasaki et al. Table 2. Macroscopic and Microscopic Binding Constants and the Mole Fraction of Each Species for r-, β-, and γ-CDs
Figure 2. Observed (a and b) and simulated (a′ and b′) 300 MHz NMR spectra of 1 mmol kg-1 β-CD (a and a′) and a mixture of 1 mmol kg-1 β-CD and 1 mmol kg-1 DHPC (b and b′) in the region of the CD protons excluding H1.
Figure 3. Chemical-shift variations of the DHPC middle methylene (open circles) and methyl (closed circles) protons and the β-CD H5 (open triangles) and H3 (closed triangles) protons with increasing β-CD concentration in a 1 mmol kg-1 DHPC solution. The solid lines were calculated from eqs 5 and 6 using the parameters shown in Tables 1 and 2. Table 1. Estimated Proton Chemical Shift Variations of CD and DHPC and SS3 Values of CD with Complexation for r-, β-, and γ-CDs params
R-CD
β-CD (SDa)
γ-CD
∆δLD(H1) ∆δLD(H2) ∆δLD(H3) ∆δLD(H4) ∆δLD(H5) ∆δLD(H6) ∆δLD(CH2)γ- ∆δLD(CH3) ∆δLD(1-CH3) ∆δLD(2-CH3) SS3 (Hz2)
0.030 0.014 -0.062 0.098 -0.067 -0.057 0.161b 0.105b
0.001 (0.006) 0.025 (0.006) -0.160 (0.009) 0.022 (0.006) -0.276 (0.014) 0.001 (0.006) 0.059 (0.001) 0.045 (0.001)
-0.019 -0.006 -0.087 0.018 -0.142 -0.030 0.037b
a
1.74
3.26
0.060b 0.074b 0.55
Standard deviation. b Taken from ref 14.
shifts and coupling constants of β-CD as well as R- and γ-CDs. Though two H6 protons, H6a and H6b, are actually nonequivalent, their averaged chemical shift was used for further analysis. Figure 3 shows the variations in chemical shift of the ω-methyl and middle methylene protons of DHPC and the H3 and H5 protons of β-CD with the addition of β-CD in a 1 mmol kg-1 DHPC solution. The negative variation indicates a decrease in chemical shift δ, viz., a shift toward high field, and vice versa. The magnitude of chemical shift variations of the heptanoyl protons is in the order of 2-(CH2)β ) 1-(CH2)β > (CH2)γ- > CH3 > 2-(CH2)R ) 1-(CH2)R. The chemical shift variations of the phosphati-
params
R-CDa
β-CD (SD)b
γ-CDa
K1 (kg mol-1) x1G+ x1Gx1T x2G+ x2Gx2T K1G+ (kg mol-1) K1G- (kg mol-1) K1T (kg mol-1)
550 0.52 0.41 0.07 0.38 0.46 0.16 410 620 1100
1290 (130) 0.52 0.41 0.07 0.65 (0.005) 0.34 (0.005) 0.01 (0.005) 1630 1080 210
750 0.52 0.41 0.07 0.62 0.38 0.00 905 696 1.0 × 10-5
a
Taken from ref 14. b Standard deviation.
dylcholine protons were similar to those of γ-CD already reported.14 The inner proton H5 shifts more largely than the other inner proton H3 (Figures 2 and 3). This finding indicates a rather deep penetration of DHPC into the CD cavity. The chemical shifts of the outer protons H1, H2, and H4 changed little (Table 1). Vicinal Coupling Constants of the C1H2-C2H Bond of DHPC. From the observed vicinal coupling constants of 3JAX and 3JBX for the spin system CHAHBCHX, we can determine the populations of three rotamers of DHPC (Figure 1). If the rotation about the C1-C2 axis is rapid, we can regard the observed coupling constant as the average of the coupling constants for the three rotamers weighted by their populations PG+, PG-, and PT:
JAX ) JAXTPT + JAXG+PG+ + JAXG-PG-
(1)
JBX ) JBXTPT + JBXG+PG+ + JBXG-PG-
(2)
Here we employed literature coupling constant values (Hz) of JAXG+ ) 12, JAXG- ) 0.45, JAXT ) 5.8, JBXG+ ) 2.4, JBXG) 2.4, and JBXT ) 12.7.31 Thus, the major component of JAX is the G+ form and that of JBX is the T form. The populations of the three rotamers of DHPC, calculated from our observed coupling constants, are PG+ ) x1G+ ) 0.52, PG- ) x1G- ) 0.41, and PT ) x1T ) 0.07 (Table 2). The coupling constant JAX increased with increasing β-CD concentration, whereas JBX decreased (data not shown). These changes in JAX and JBX were similar to those of γ-CD but opposite to those of R-CD.14 The increase in JAX and the decrease in JBX for β-CD indicate an increase in the G+ form and decreases in the T and G- forms. Figure 4 shows the populations of three rotamers, calculated from eqs 1 and 2, as a function of the β-CD concentration. The G+ conformer is preferentially bound to β-CD. Estimation of Binding Constants and Population of Each Complex Species. One or two of the heptanoyl groups of DHPC will be incorporated into the hydrophobic β-CD cavity. Because the length of the heptanoyl group is close to the depth of the β-CD cavity, 1:1 and 1:2 complexes of DHPC and β-CD would be formed. We mainly considered the 1:1 complex and did not take into consideration the 2:1 complex, because we used excess β-CD over DHPC. The concentration, [D], of free CD molecules can be obtained from
K1[D]2 + {1 + K1(CL - CD)}[D] - CD ) 0
(3)
Here CL and CD denote the total concentrations of DHPC and CD, respectively, and K1 stands for the macroscopic equilibrium constant of the 1:1 complex (LD) between
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Langmuir, Vol. 18, No. 5, 2002 1789
Figure 4. Populations of the rotational isomers G+ (open squares), G- (open triangles), and T (open circles), calculated from eqs 1 and 2 using the observed coupling constant data as a function of the concentration of β-CD. The solid lines were calculated from the microscopic equilibrium constants and the mole fractions shown in Table 2.
DHPC (L) and CD (D). We can solve eq 3 for a given set of K1, CL, and CD to obtain the concentrations of D, L, and LD. Furthermore, we can calculate the concentrations of the three rotamers (G+, G-, and T) of DHPC in the free form L and the binary complex LD. The concentration of L can be divided into the concentrations of the three rotamers, [G+], [G-], and [T]. Similarly, the concentration of LD is divided into the concentrations of three rotamers, respectively. Using these concentrations, we can define three microscopic equilibrium constants, K1G+, K1G-, and K1T, of CD complexation with specific rotamers. For instance, the microscopic constant of equimolar complexation of CD and G+ is defined as
K1G+ ) [G+D]/[G+][D] ) x2G+[LD]/x1G+[L][D] ) x2G+K1/x1G+ (4) Here x stands for the mole fraction of each species and these mole fractions are connected by the equations of x1G+ + x1G- + x1T ) 1 and x2G+ + x2G- + x2T ) 1. We presumed that the DHPC-CD complexation and the rotational isomerization of DHPC were both rapid on the NMR time scale. Under these conditions, the chemical shift of a DHPC proton can be written as
(or ∆δD-LD ) δD-LD - δD-D) of eq 6 for two CD protons, H3 and H5, as four adjustable parameters. Thus, we obtained the best fit values for these four parameters for β-CD shown in Tables 1 and 2. Using this K1 value, we determined the best fit δL-LD and δD-LD values for protons of DHPC and β-CD excluding these three protons (Table 1). As Figure 3 shows, the theoretical values for the middle methylenes were slightly different from the observed ones. This discrepancy is ascribed to the nonequivalence of the middle methylenes induced by β-CD inclusion. Next, to calculate three microscopic constants K1G+, K1G-, and K1T, we used the observed populations of three rotamers for the free DHPC molecule (x1G-, x1G+, and x1T), the estimated macroscopic constant, and the 12 observed vicinal coupling constant data, JAX and JBX, and then minimized the difference between the observed and calculated coupling constants: 6
SS2 )
} ∑ {(JAXcalc - JAXobsd)2 + (JBXcalc - JBXobsd)2(7)
To calculate 12 theoretical values of the corresponding coupling constants, we regarded x2G+ and x2G- in eqs 1, 2, 5, and 6 as two adjustable parameters. These estimated mole fractions and microscopic binding constants are shown in Table 2. In Table 2 it is notable that the K1 value for β-CD is markedly larger than that of R-CD and γ-CD: β-CD > γ-CD > R-CD. The G+ form has a high affinity to β-CD. For single alkyl chain surfactants, the equilibrium constant of binary complexation is in the order of R-CD g β-CD > γ-CD.9-13 Vicinal Spin-Spin Coupling Constants of CD Protons. We determined the geminal and vicinal coupling constants of CD in the free and bound states. For the equimolar mixture of DHPC and CD (CD ) CL ) 1 mmol kg-1), the vicinal coupling constants for the bound state are shown as deviations ∆3JLD ()3JLD - 3JD) from those for the free state. Under the present conditions, however, not all β-CD molecules were bound to DHPC: [LD]/CD ) 0.507. The ∆3JLD value at full binding was calculated using this degree of binding, and its squared value was summed up over six vicinal spin-spin couples (1-2, 2-3, 3-4, 4-5, 5-6a, and 5-6b) to yield the summation of the squared coupling constant variations: 6
δL ) ([L]δL-L + [LD]δL-LD)/CL
(5)
Here δL-L denotes the average chemical shift of a DHPC proton for the rotational-equilibrium mixture of free DHPC molecules. Similarly, δL-LD denotes the average chemical shift of the rotational-equilibrium mixtures of the binary complex. For the chemical shift of a CD proton, we can write the corresponding equation
δD ) ([D]δD-D + [LD]δD-LD)/CD
(6)
To determine unknown parameters in eqs 3-6, we minimized the difference between the theoretical and observed chemical shifts of DHPC and CD. To estimate the macroscopic equilibrium constant K1, we used the observed chemical shift variations of three kinds of protons, namely, the DHPC terminal methyl proton, the CD H3 proton, and the CD H5 proton, at six CD concentrations (Figure 3). To calculate the theoretical chemical shifts, we regarded K1, δL-LD (or ∆δL-LD ) δL-LD - δL-L) of eq 5 for the terminal methyl proton, and δD-LD
SS3 )
∑(3JLD - 3JD)2
(8)
This SS3 value will reflect the deformation of the CD macrocycle induced by complex formation with DHPC. As Table 1 shows, the SS3 value for β-CD was markedly larger than that of R- and γ-CDs calculated from the published data.14 This suggests that the β-CD macrocycle is deformed more extensively than the R- and γ-CD macrocycles upon inclusion of DHPC. Molecular Structure of the Complex. A partial 500 MHz ROESY spectrum of 3 mM DHPC and 3 mM β-CD is shown in Figure 5. Under these conditions, the concentrations of free and complexed DHPC molecules were estimated to be 1.2 and 1.8 mM, respectively, from the macroscopic binding constant. Because this free DHPC concentration was lower than a cmc value of 1.5 mM, no micelles formed. The middle methylene and terminal methyl groups of DHPC exhibited rather large cross-peaks with the H3, H5, and H6 protons of β-CD (Figure 5) but did not show any cross-peak with the H4 proton (data not shown). The β-methylene group had a small cross-peak
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Discussion
Figure 5. Partial 500 MHz ROESY spectrum of a solution of 3 mM DHPC and 3 mM β-CD.
Figure 6. Top and side views of energetically optimized structure of the β-CD-G+ complex constructed by docking a rigid DHPC(G+ ) molecule with a flexible β-CD molecule.
with the H3 proton, though the R-methylene group had no cross-peaks with any β-CD protons. The cross-peak between the terminal methyl and H6 protons gave an important clue for estimating the structure of DHPC and β-CD. On the basis of the chemical shift and ROESY data, we postulate feasible molecular structures of the major complex β-CD-G+ in Figure 6. The original structure of β-CD shown in Figure 6 was based on the atomic coordinates of β-CD in complex with 2-(3-phenoxyphenyl)propionic acid.32 This structure was searched for as one of the deformed β-CD complexes whose structures are available in the Cambridge Crystallographic Data Center. First, we docked the DHPC(G+) conformer with this β-CD molecule so that the structure of the complex would satisfy the ROESY data. Then, keeping the DHPC(G+) structure rigid, we energy-optimized the β-CD structure in the presence of water by molecular mechanics calculations. As Figure 6 shows, the β-CD rim was deformed from a circle to an ellipse and the difference in radius between the upper and lower rims was much reduced. This large deformation of β-CD was consistent with the largest SS3 value and the largest chemical shift variations of the H3 and H5 protons among the CDs (Table 1). This structure is akin to that of the major complex G+-γ-CD proposed previously.14 This is consistent with the similarity in vicinal spin-spin coupling constants of the CHX-CHAHB group and the chemical shift variations of DHPC protons between β-CD and γ-CD. For example, the chemical shift variation of the middle methylene protons with the formation of the equimolar complex of β-CD was close to that of γ-CD but distant from that of R-CD (Table 1).
For single chain surfactants, two alkyl chains will move too independently to be incorporated in a β-CD cavity. Very recently, we found that the two decyl groups of didecyldimethylammonium bromide are not simultaneously incorporated in the β-CD cavity.15 At first glance, this finding seems to be inconsistent with the present finding for DHPC. Two intramolecular alkyl chains of DHPC can contact tightly,31,35 but those of didecyldimethylammonium bromide would be in loose contact, because of unstable folding of the decyl group. Two unsaturated chains of prostaglandin E2 are assumed to be incorporated in a β-CD cavity.36 To our knowledge, this is the only example for β-CD inclusion of two alkyl chains. The unusually distorted structure of β-CD (Figure 6) is similar to the X-ray structure of β-CD in the β-CD-2,7dihydroxy-naphthalene complex.37 Irrespective of the deformation, the binding constants for β-CD, K1 and K1G+, are larger than those for γ-CD (Table 2). A tighter contact between the hydrophobic groups of DHPC and β-CD gave rise to a larger binding constant than R-CD and γ-CD.38 The binding constant K1G- of the G- form and β-CD is rather large. Two heptanoyl chains of the G- form will be in parallel arrangement, similar to the G+ form.31 Two heptanoyl chains of the T form will be independently bound to CD. Thus, the magnitude of binding constant K1T for the T form is in the order of R-CD > β-CD > γ-CD, as expected. The structure of the β-CD-T complex is close to that of the R-CD-T complex. In this work we neglected the 1:2 and 2:1 ternary complexes of DHPC and β-CD. We attempted to determine the binding constant of the 1:2 complexation, but because this value was very small, we failed to obtain a reliable ternary binding constant. Very recently, the discrepancy between the external and internal standards for the chemical shift determination has been resolved.17,25-28,39 The chemical shift of tetramethylammonium chloride, referred to an external standard, decreases linearly with increasing concentration of various oligosaccharides.27 This linear decrease has been ascribed to the change in volume magnetic susceptibility.39 The ultimate strength of the NMR method is to provide microscopic information on the structure of complexes. The respective peaks of the R- and β-methylene protons of DHPC are distinguishable between chains 1 and 2. The concentration dependence of these proton chemical shifts is close to one another, suggesting that these protons are simultaneously bound to β-CD. The terminal methyl and middle methylenes of DHPC for R-CD exhibited the largest chemical shift variations ∆δL-LD among R-, β-, and γ-CDs (Table 1). This will be due to the largest increase in the T form with 1:1 complexation. Acknowledgment. This work was supported by Grants-in-Aid for the Scientific Research Program (No.11672153) and Frontier Research Program from the Ministry of Education, Science, Culture, Science, and Sports of Japan. LA0108860 (35) Stryer, L. Biochemistry, 2nd ed.; W. H. Freeman and Company: San Francisco, CA, 1975; p 208. (36) Uekama, K.; Otagiri, M. CRC Crit. Rev. Ther. Drug Carrier Syst. 1987, 3, 7. (37) An˜ibarro, M.; Gessler, K.; Uso´n, I.; Sheldrick, G. M.; Saenger, W. Carbohydr. Res. 2001, 333, 251. (38) Ishikawa, S.; Neya, S.; Funasaki, N. J. Phys. Chem. B 1999, 103, 1208. (39) Funasaki, N.; Nomura, M.; Ishikawa, S.; Neya, S. J. Phys. Chem. B 2001, 105, 7361.
