Binding of Short-Chain Lecithin by â-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 â-cyclodextrin (â-CD) indeuterium oxide solution has been investigated by measurements of proton NMR chemical shifts and theROESY spectrum and molecular mechanics calculations. The vicinal coupling constants for protons of R-,â-, and γ-CDs in their DHPC complexes have been also determined for comparison among these CDs. Fromthe variations in chemical shifts of DHPC and â-CD with the addition of â-CD, the macroscopic equilibriumconstant, K1, of the 1:1 complexation of DHPC and â-CD is estimated. The magnitude of K1 is in the orderof â-CD > γ-CD > R-CD for DHPC, although it is in the order of R-CD g â-CD > γ-CD for single-chainsurfactants. From the vicinal coupling constants of the glycerol C1H2-C2H protons of DHPC, the populationsof three rotamers, gauche+ conformer (G+), gauche- conformer (G-), and trans conformer (T), are estimated.The addition of â-CD causes a decrease in the T conformer and an increase in the G+ conformer. Thesechanges are the same as those of γ-CD but opposite to those of R-CD. The microscopic binding constantsof CD complexation for the three rotamers are also estimated from the concentration dependence of vicinalcoupling constants. The microscopic binding constant for the T conformer is in the order of R-CD > â-CD> γ-CD, as expected. The presence of DHPC causes large changes in vicinal coupling constants of the â-CDprotons, suggesting that the â-CD macrocycle is deformed by incorporation of DHPC. A three-dimensionalstructure of the major complex â-CD-G+ is proposed on the basis of these variations in chemical shift andvicinal coupling constant, the ROESY spectrum, and molecular mechanics calculations: two heptanoylchains of DHPC are simultaneously incorporated into a â-CD cavity so that the rim of â-CD is deformedto an ellipse. This tight contact between the hydrophobic groups of DHPC and â-CD leads to a large bindingconstant.

Introduction

Cyclodextrins (CDs) have homogeneous toroidal struc-tures of different molecular sizes: most typical arecyclohexaamylose (R-CD), cycloheptaamylose (â-CD), andcyclooctaamylose (γ-CD). The toroidal structure has ahydrophilic surface, making it water soluble, whereas thecavity is composed of the glucoside oxygens and methinehydrogens, giving it a hydrophobic character. Conse-quently, the CDs can accommodate other hydrophobicmolecules of appropriate dimensions and shapes.1,2 Thecrystal structures of many CD inclusion complexes havebeen determined by X-ray diffraction techniques.3-5 TheCD cavities including large guest molecules are generallydeformed. This flexibility of the CD macrocycle has beensupported by molecular mechanics calculations and mo-lecular dynamics simulations.6-8

A number of studies have focused on the interactionsof CDs with surfactants having a single alkyl chain.9-13

For instance, the 1:1 binding constants of â-CD with singlechain surfactants are close to those of R-CD but muchlarger than those of γ-CD. The small CDs R-CD and â-CDcan include one alkyl chain only in their cavities, but thelarger γ-CD can include two alkyl chains simultaneously.On the other hand, very few studies have been reportedon complex formation between double alkyl chain sur-factantsandCDs.14-16 Forsuchsystems, thestoichiometry,the structure of the complex, and the binding constantremain virtually unexplored. The structure of the complexprovides essential information about the magnitude ofthe binding constant.10,13-15 These studies on complexformation between CDs and surfactants are also interest-ing from the viewpoint of supramolecular chemistry.17,18

Because CDs are essentially nontoxic, they are addedto pharmaceuticals and foods, for example, for stabiliza-tion of labile compounds, suppression of bitter tastes andhemolysis,13,19-21 and long-term preservation of color, odor,and flavor.1,2,22 However, because the CDs are hemoly-

(1) Saenger, W. Angew. Chem., Int. Ed. Engl. 1980, 19, 344.(2) Szejtli, J. Cyclodextrin Technology; Kluwer Academic Publish-

ers: 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 ofSupramolecular Systems; Davies, J. E. D., Ed.; Kluwer AcademicPublishers: 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.; Wyn-

Jones, E. J. Phys. Chem. 1992, 96, 8979.(12) Jobe, D. J.; Verrall, R. E.; Junquera, E.; Aicart, E. J. Phys. Chem.

1994, 98, 10814.

(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.; Rudiger, 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) Fromming, K.-H.; Szejtli, J. Cyclodextrins in Pharmacy; Kluwer

Academic Publishers: Dordrecht, The Netherlands, 1994; Chapters 3,6, and 10.

1786 Langmuir 2002, 18, 1786-1790

10.1021/la0108860 CCC: $22.00 © 2002 American Chemical SocietyPublished on Web 02/01/2002

tic,1,20,22-24 parenteral administration of CDs is restrictedby health authorities.22 This hemolysis is caused by theextraction of erythrocyte membrane components (choles-terol and phospholipid) by CD.22-24 The hemolytic activityof CDs is in the order of â-CD > R-CD > γ-CD.20,22-24

Therefore, basic studies on complex formation betweenlecithin and CDs should shed some light on the mechanismof hemolysis by CDs.

In this work, we investigated complex formation of 1,2-diheptanoyl-3-L-R-phosphatidylcholine (DHPC), a short-chain lecithin, with â-CD by 1H NMR and molecularmechanics. From the variations in chemical shift andvicinal coupling constant for DHPC and â-CD with theaddition of â-CD, the equilibrium binding constants(macroscopic and microscopic constants) of DHPC andâ-CD were determined. Unexpectedly, this change invicinal coupling constant of DHPC was similar to that forγ-CD, and the macroscopic 1:1 binding constant for â-CDwas larger than that of R-CD and γ-CD. To analyze theseunexpected findings, we also determined the vicinalcoupling constant for protons of R-, â-, and γ-CDs in thecomplexes with DHPC. On the basis of the chemical shift,ROESY data, and molecular mechanics calculations, anelliptically deformed structure of â-CD in its DHPCcomplex is proposed.

Experimental Section

NMR Measurements. DHPC from Sigma Chemical Co. anddeuterium oxide (99.9%) from Aldrich were used as received.Sodium 4,4-dimethyl-4-silapentane-1-sulfonate (DSS) and â-CDwere purchased from Nacalai Tesque Co.

All 300 MHz proton NMR spectra were recorded with a VarianXL-300 NMR spectrometer at 21.0 ( 0.5 °C. These spectra weredeconvoluted with a Nuts NMR data-processing software (AcornNMR Inc.). This software was also used for spectral simulations.The chemical shift of a sample in deuterium oxide was measuredagainst DSS as external standard. No buffer or salt was addedto adjust the pH and the ionic strength, because DHPC is azwitterion over a wide neutral pH range. The concentration ofDHPC, CL, was kept at 1.00 mmol kg-1, which is below the criticalmicelle concentration of 1.5 mmol kg-1,25 whereas the concentra-tion of CD, CD, was increased up to ca. 14 mmol kg-1. The chemicalshifts of all protons of CDs decreased linearly with increasingCD concentration, and this concentration dependence wasascribed to the dimerization of CDs.26 Matsui and Tokunagaargued that the internal reference method using noncomplexingsolutes is more reliable than the external one.27 Although weemployed the external reference method, our corrected chemicalshift data are consistent with those determined by the internalreference method using tetramethylammonium chloride.28

The 500 MHz ROESY (phase-sensitive rotating frameworkOverhauser enhancement spectroscopy) spectrum of a deuteriumoxide solution containing 3 mM DHPC and 3 mM â-CD wasrecorded with a JEOL Lambda 500 spectrometer using the JEOLstandard pulse sequences. The data consisted of 16 transientscollected over 2048 complex points. A mixing time of 500 ms, arepetition delay of 1.2 s, and a 90° pulse width of 11.0 ms wereused. The ROESY data set was processed by applying anexponential 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.

Molecular Modeling of Complexes. The structure of DHPCwas constructed on the basis of the crystal structure of dimyris-toylphosphatidylcholine.25,29,30 The DHPC molecule in the crystalwas in the gauche+ (G+) form (Figure 1). The 2-heptanoyl chainin the gauche- (G-) and trans (T) forms was assumed as a fullyextended conformation, although it may be somewhat folded inthe G- form, because of intramolecular hydrophobic interactionsbetween the two heptanoyl chains.31 The structure of â-CD wastaken from the crystal structure of its complex with 2-(3-phenoxyphenyl)propionic acid.32

The structures of â-CD and DHPC were regarded as rigidbodies unchanged with complexation. Instead of 2-(3-phenox-yphenyl)propionic acid, the heptanoyl groups of a DHPC moleculein the G+ form were inserted into the â-CD cavity, parallel to thesymmetry axis of â-CD, and the phosphatidylcholine group waslocated outside the secondary alcohol side of â-CD. The DHPCmolecule was penetrated, regardless of interatomic collision, sodeeply that the observed ROESY and chemical shift data wereconsistent with the structure of the complex. This complex wasaccommodated by a unit cell (2.5 nm × 2.5 nm × 3.5 nm), wherethe heptanoyl groups were arranged parallel to the long axis ofthe cell. This cell was soaked with 627 water molecules. Then,the â-CD molecule was regarded as flexible, whereas the DHPCmolecule was kept rigid. This structure of the complex wasoptimized using the Discover III module of Molecular SimulationInsight II/Discover (98.0) on a Silicon Graphics Octane worksta-tion. The periodic boundary condition was applied to this unitcell. The cutoff distance for van der Waals and electrostatic forceswas 1.6 nm. A HyperChem (Hypercube, Inc.) package modelingsoftwareandourownsoftware25 wereused formolecularmodelingand data analysis on a Dell Precision 610 personal computer.

Results

Chemical Shifts of DHPC and CD Protons. Theassignment of each proton of DHPC in the 300 MHz 1HNMR spectrum has been established.14,31,33 The HX protonof the glycerol C1HAHBC2HX fragment of DHPC (Figure1) exhibits a complicated multiplet at 5.3 ppm. The HBand HA proton signals appear as quartets around 4.5 and4.3 ppm, respectively. The proton signals of two hexylgroups 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 â-CH2 groups, a broad tall peakof the middle methylene groups (γ-, δ-, and ε-CH2 groups),and a single triplet of the ω-methyl groups.

Figure 2 depicts the observed and simulated 300 MHz1H NMR spectra of deuterium oxide solutions containing(a) 1 mmol kg-1 â-CD and (b) 1 mmol kg-1 DHPC and 1mmol kg-1 â-CD in the region of all â-CD protons exceptH1. The assignments of the â-CD protons were carriedout in comparison with the literature spectra of R-CD andγ-CD.14,34 Computer simulations of these NMR spectrawere performed for accurate determinations of chemical

(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.

(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.

Figure 1. Newman projections of three rotamers of differentdihedral angles around the HXC2-C1HAHB axis of the glycerolmoiety of DHPC.

Binding of Short-Chain Lecithin by â-Cyclodextrin Langmuir, Vol. 18, No. 5, 2002 1787

shifts and coupling constants of â-CD as well as R- andγ-CDs. Though two H6 protons, H6a and H6b, are actuallynonequivalent, their averaged chemical shift was usedfor further analysis.

Figure 3 shows the variations in chemical shift of theω-methyl and middle methylene protons of DHPC andthe H3 and H5 protons of â-CD with the addition of â-CDin a 1 mmol kg-1 DHPC solution. The negative variationindicates a decrease in chemical shift δ, viz., a shift towardhigh field, and vice versa. The magnitude of chemical shiftvariations of the heptanoyl protons is in the order of2-(CH2)â ) 1-(CH2)â > (CH2)γ-ε > CH3 > 2-(CH2)R )1-(CH2)R. The chemical shift variations of the phosphati-

dylcholine protons were similar to those of γ-CD alreadyreported.14 The inner proton H5 shifts more largely thanthe other inner proton H3 (Figures 2 and 3). This findingindicates a rather deep penetration of DHPC into the CDcavity. The chemical shifts of the outer protons H1, H2,and H4 changed little (Table 1).

Vicinal Coupling Constants of the C1H2-C2HBond of DHPC. From the observed vicinal couplingconstants of 3JAX and 3JBX for the spin system CHAHB-CHX, we can determine the populations of three rotamersof DHPC (Figure 1). If the rotation about the C1-C2 axisis rapid, we can regard the observed coupling constant asthe average of the coupling constants for the threerotamers weighted by their populations PG+, PG-, and PT:

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 ofJAX is the G+ form and that of JBX is the T form. Thepopulations of the three rotamers of DHPC, calculatedfrom 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 increasingâ-CD concentration, whereas JBX decreased (data notshown). These changes in JAX and JBX were similar tothose of γ-CD but opposite to those of R-CD.14 The increasein JAX and the decrease in JBX for â-CD indicate an increasein the G+ form and decreases in the T and G- forms. Figure4 shows the populations of three rotamers, calculated fromeqs 1 and 2, as a function of the â-CD concentration. TheG+ conformer is preferentially bound to â-CD.

Estimation of Binding Constants and Populationof Each Complex Species. One or two of the heptanoylgroups of DHPC will be incorporated into the hydrophobicâ-CD cavity. Because the length of the heptanoyl groupis close to the depth of the â-CD cavity, 1:1 and 1:2complexes of DHPC and â-CD would be formed. We mainlyconsidered the 1:1 complex and did not take into consid-eration the 2:1 complex, because we used excess â-CDover DHPC.

The concentration, [D], of free CD molecules can beobtained from

Here CL and CD denote the total concentrations of DHPCand CD, respectively, and K1 stands for the macroscopicequilibrium constant of the 1:1 complex (LD) between

Figure 2. Observed (a and b) and simulated (a′ and b′) 300MHz NMR spectra of 1 mmol kg-1 â-CD (a and a′) and a mixtureof 1 mmol kg-1 â-CD and 1 mmol kg-1 DHPC (b and b′) in theregion of the CD protons excluding H1.

Figure 3. Chemical-shift variations of the DHPC middlemethylene (open circles) and methyl (closed circles) protonsand the â-CD H5 (open triangles) and H3 (closed triangles)protons with increasing â-CD concentration in a 1 mmol kg-1

DHPC solution. The solid lines were calculated from eqs 5 and6 using the parameters shown in Tables 1 and 2.

Table 1. Estimated Proton Chemical Shift Variations ofCD and DHPC and SS3 Values of CD with Complexation

for r-, â-, and γ-CDs

params R-CD â-CD (SDa) γ-CD

∆δLD(H1) 0.030 0.001 (0.006) -0.019∆δLD(H2) 0.014 0.025 (0.006) -0.006∆δLD(H3) -0.062 -0.160 (0.009) -0.087∆δLD(H4) 0.098 0.022 (0.006) 0.018∆δLD(H5) -0.067 -0.276 (0.014) -0.142∆δLD(H6) -0.057 0.001 (0.006) -0.030∆δLD(CH2)γ-ε 0.161b 0.059 (0.001) 0.037b

∆δLD(CH3) 0.105b 0.045 (0.001)∆δLD(1-CH3) 0.060b

∆δLD(2-CH3) 0.074b

SS3 (Hz2) 1.74 3.26 0.55a Standard deviation. b Taken from ref 14.

Table 2. Macroscopic and Microscopic BindingConstants and the Mole Fraction of Each Species for r-,

â-, and γ-CDs

params R-CDa â-CD (SD)b γ-CDa

K1 (kg mol-1) 550 1290 (130) 750x1G+ 0.52 0.52 0.52x1G- 0.41 0.41 0.41x1T 0.07 0.07 0.07x2G+ 0.38 0.65 (0.005) 0.62x2G- 0.46 0.34 (0.005) 0.38x2T 0.16 0.01 (0.005) 0.00K1G+ (kg mol-1) 410 1630 905K1G- (kg mol-1) 620 1080 696K1T (kg mol-1) 1100 210 1.0 × 10-5

a Taken from ref 14. b Standard deviation.

JAX ) JAXTPT + JAXG+PG+ + JAXG-PG- (1)

JBX ) JBXTPT + JBXG+PG+ + JBXG-PG- (2)

K1[D]2 + {1 + K1(CL - CD)}[D] - CD ) 0 (3)

1788 Langmuir, Vol. 18, No. 5, 2002 Funasaki et al.

DHPC (L) and CD (D). We can solve eq 3 for a given setof K1, CL, and CD to obtain the concentrations of D, L, andLD.

Furthermore, we can calculate the concentrations ofthe three rotamers (G+, G-, and T) of DHPC in the freeform L and the binary complex LD. The concentration ofL can be divided into the concentrations of the threerotamers, [G+], [G-], and [T]. Similarly, the concentrationof LD is divided into the concentrations of three rotamers,respectively. Using these concentrations, we can definethree microscopic equilibrium constants, K1G+, K1G-, andK1T, of CD complexation with specific rotamers. Forinstance, the microscopic constant of equimolar complex-ation of CD and G+ is defined as

Here x stands for the mole fraction of each species andthese mole fractions are connected by the equations ofx1G+ + x1G- + x1T ) 1 and x2G+ + x2G- + x2T ) 1.

We presumed that the DHPC-CD complexation andthe rotational isomerization of DHPC were both rapid onthe NMR time scale. Under these conditions, the chemicalshift of a DHPC proton can be written as

Here δL-L denotes the average chemical shift of a DHPCproton for the rotational-equilibrium mixture of free DHPCmolecules. Similarly, δL-LD denotes the average chemicalshift of the rotational-equilibrium mixtures of the binarycomplex. For the chemical shift of a CD proton, we canwrite the corresponding equation

To determine unknown parameters in eqs 3-6, weminimized the difference between the theoretical andobserved chemical shifts of DHPC and CD.

To estimate the macroscopic equilibrium constant K1,we used the observed chemical shift variations of threekinds of protons, namely, the DHPC terminal methylproton, the CD H3 proton, and the CD H5 proton, at sixCD concentrations (Figure 3). To calculate the theoreticalchemical shifts, we regarded K1, δL-LD (or ∆δL-LD ) δL-LD- δL-L) of eq 5 for the terminal methyl proton, and δD-LD

(or ∆δD-LD ) δD-LD - δD-D) of eq 6 for two CD protons, H3and H5, as four adjustable parameters. Thus, we obtainedthe best fit values for these four parameters for â-CDshown in Tables 1 and 2. Using this K1 value, wedetermined the best fit δL-LD and δD-LD values for protonsof DHPC and â-CD excluding these three protons (Table1). As Figure 3 shows, the theoretical values for the middlemethylenes were slightly different from the observed ones.This discrepancy is ascribed to the nonequivalence of themiddle methylenes induced by â-CD inclusion.

Next, to calculate three microscopic constants K1G+, K1G-,and K1T, we used the observed populations of threerotamers for the free DHPC molecule (x1G-, x1G+, and x1T),the estimated macroscopic constant, and the 12 observedvicinal coupling constant data, JAX and JBX, and thenminimized the difference between the observed andcalculated coupling constants:

To calculate 12 theoretical values of the correspondingcoupling constants, we regarded x2G+ and x2G- in eqs 1, 2,5, and 6 as two adjustable parameters. These estimatedmole fractions and microscopic binding constants areshown in Table 2.

In Table 2 it is notable that the K1 value for â-CD ismarkedly larger than that of R-CD and γ-CD: â-CD >γ-CD > R-CD. The G+ form has a high affinity to â-CD.For single alkyl chain surfactants, the equilibrium con-stant of binary complexation is in the order of R-CD gâ-CD > γ-CD.9-13

Vicinal Spin-Spin Coupling Constants of CDProtons. We determined the geminal and vicinal couplingconstants of CD in the free and bound states. For theequimolar mixture of DHPC and CD (CD ) CL ) 1 mmolkg-1), the vicinal coupling constants for the bound stateare shown as deviations ∆3JLD ()3JLD - 3JD) from thosefor the free state. Under the present conditions, however,not all â-CD molecules were bound to DHPC: [LD]/CD )0.507. The ∆3JLD value at full binding was calculated usingthis degree of binding, and its squared value was summedup over six vicinal spin-spin couples (1-2, 2-3, 3-4,4-5, 5-6a, and 5-6b) to yield the summation of thesquared coupling constant variations:

This SS3 value will reflect the deformation of the CDmacrocycle induced by complex formation with DHPC.As Table 1 shows, the SS3 value for â-CD was markedlylarger than that of R- and γ-CDs calculated from thepublished data.14 This suggests that the â-CD macrocycleis deformed more extensively than the R- and γ-CDmacrocycles upon inclusion of DHPC.

Molecular Structure of the Complex. A partial 500MHz ROESY spectrum of 3 mM DHPC and 3 mM â-CDis shown in Figure 5. Under these conditions, theconcentrations of free and complexed DHPC moleculeswere estimated to be 1.2 and 1.8 mM, respectively, fromthe macroscopic binding constant. Because this free DHPCconcentration was lower than a cmc value of 1.5 mM, nomicelles formed. The middle methylene and terminalmethyl groups of DHPC exhibited rather large cross-peakswith the H3, H5, and H6 protons of â-CD (Figure 5) butdid not show any cross-peak with the H4 proton (data notshown). The â-methylene group had a small cross-peak

Figure 4. Populations of the rotational isomers G+ (opensquares), G- (open triangles), and T (open circles), calculatedfrom eqs 1 and 2 using the observed coupling constant data asa function of the concentration of â-CD. The solid lines werecalculated from the microscopic equilibrium constants and themole fractions shown in Table 2.

K1G+ ) [G+D]/[G+][D] ) x2G+[LD]/x1G+[L][D] )x2G+K1/x1G+ (4)

δL ) ([L]δL-L + [LD]δL-LD)/CL (5)

δD ) ([D]δD-D + [LD]δD-LD)/CD (6)

SS2 ) ∑6

{(JAXcalc - JAXobsd)2 + (JBXcalc - JBXobsd)

2}(7)

SS3 ) ∑6

(3JLD -3JD)2 (8)

Binding of Short-Chain Lecithin by â-Cyclodextrin Langmuir, Vol. 18, No. 5, 2002 1789

with the H3 proton, though the R-methylene group hadno cross-peaks with any â-CD protons. The cross-peakbetween the terminal methyl and H6 protons gave animportant clue for estimating the structure of DHPC andâ-CD.

On the basis of the chemical shift and ROESY data, wepostulate feasible molecular structures of the majorcomplex â-CD-G+ in Figure 6. The original structure ofâ-CD shown in Figure 6 was based on the atomiccoordinates of â-CD in complex with 2-(3-phenoxyphenyl)-propionic acid.32 This structure was searched for as oneof the deformed â-CD complexes whose structures areavailable in the Cambridge Crystallographic Data Center.First, we docked the DHPC(G+) conformer with this â-CDmolecule so that the structure of the complex would satisfythe ROESY data. Then, keeping the DHPC(G+) structurerigid, we energy-optimized the â-CD structure in thepresence of water by molecular mechanics calculations.

As Figure 6 shows, the â-CD rim was deformed from acircle to an ellipse and the difference in radius betweenthe upper and lower rims was much reduced. This largedeformation of â-CD was consistent with the largest SS3value and the largest chemical shift variations of the H3and H5 protons among the CDs (Table 1). This structureis akin to that of the major complex G+-γ-CD proposedpreviously.14 This is consistent with the similarity invicinal spin-spin coupling constants of the CHX-CHAHBgroup and the chemical shift variations of DHPC protonsbetween â-CD and γ-CD. For example, the chemical shiftvariation of the middle methylene protons with theformation of the equimolar complex of â-CD was close tothat of γ-CD but distant from that of R-CD (Table 1).

Discussion

For single chain surfactants, two alkyl chains will movetoo independently to be incorporated in a â-CD cavity.Very recently, we found that the two decyl groups ofdidecyldimethylammonium bromide are not simulta-neously incorporated in the â-CD cavity.15 At first glance,this finding seems to be inconsistent with the presentfinding for DHPC. Two intramolecular alkyl chains ofDHPC can contact tightly,31,35 but those of didecyldim-ethylammonium bromide would be in loose contact,because of unstable folding of the decyl group. Twounsaturated chains of prostaglandin E2 are assumed tobe incorporated in a â-CD cavity.36 To our knowledge, thisis the only example for â-CD inclusion of two alkyl chains.

The unusually distorted structure of â-CD (Figure 6) issimilar to the X-ray structure of â-CD in the â-CD-2,7-dihydroxy-naphthalene complex.37 Irrespective of thedeformation, the binding constants for â-CD, K1 and K1G+,are larger than those for γ-CD (Table 2). A tighter contactbetween the hydrophobic groups of DHPC and â-CD gaverise to a larger binding constant than R-CD and γ-CD.38

The binding constant K1G- of the G- form and â-CD is ratherlarge. Two heptanoyl chains of the G- form will be inparallel arrangement, similar to the G+ form.31 Twoheptanoyl chains of the T form will be independently boundto CD. Thus, the magnitude of binding constant K1T forthe T form is in the order of R-CD > â-CD > γ-CD, asexpected. The structure of the â-CD-T complex is closeto that of the R-CD-T complex. In this work we neglectedthe 1:2 and 2:1 ternary complexes of DHPC and â-CD. Weattempted to determine the binding constant of the 1:2complexation, but because this value was very small, wefailed to obtain a reliable ternary binding constant.

Very recently, the discrepancy between the externaland internal standards for the chemical shift determi-nation has been resolved.17,25-28,39 The chemical shift oftetramethylammonium chloride, referred to an externalstandard, decreases linearly with increasing concentrationof various oligosaccharides.27 This linear decrease has beenascribed to the change in volume magnetic susceptibility.39

The ultimate strength of the NMR method is to providemicroscopic information on the structure of complexes.The respective peaks of the R- and â-methylene protonsof DHPC are distinguishable between chains 1 and 2. Theconcentration dependence of these proton chemical shiftsis close to one another, suggesting that these protons aresimultaneously bound to â-CD. The terminal methyl andmiddle methylenes of DHPC for R-CD exhibited the largestchemical shift variations ∆δL-LD among R-, â-, and γ-CDs(Table 1). This will be due to the largest increase in theT form with 1:1 complexation.

Acknowledgment. This work was supported byGrants-in-Aid for the Scientific Research Program(No.11672153) and Frontier Research Program from theMinistry of Education, Science, Culture, Science, andSports 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) Anibarro, M.; Gessler, K.; Uson, 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.

Figure 5. Partial 500 MHz ROESY spectrum of a solution of3 mM DHPC and 3 mM â-CD.

Figure 6. Top and side views of energetically optimizedstructure of the â-CD-G+ complex constructed by docking arigid DHPC(G+ ) molecule with a flexible â-CD molecule.

1790 Langmuir, Vol. 18, No. 5, 2002 Funasaki et al.