5359
J. Phys. Chem. 1991,95, 5359-5361
An ESR Study of Pressure Effects on the Inclusion-Complex Formation of Cyclodextrins with Di-tert-butyl Nltroxide Yoshimi Sueishi,* Norio Nishimura, Department of Chemistry, Faculty of Science, Okayama University, Tsushima, Okayama, 700 Japan
Koichi Hirata, and Keiji Kuwata Department of Chemistry, Faculty of Science, Osaka University, Toyonaka, Osaka, 560 Japan (Received: October 11, 1990; In Final Form: January 2, 1991)
The volume change upon the inclusion-complex formation of cyclodextrins (j3,yCDs) with di-tert-butyl nitroxide (DTBN) has been studied by a high-pressure ESR technique. Anisotropic motion of DTBN in y-CD suggested a loose inclusion of DTBN in the y C D cavity. The equilibrium constants (Ks)for the inclusion-complexformation of CDs with DTBN decreased with increasing external pressure. From the pressure dependence of K,the reaction volumes were estimated to be 5 f 1 cm3 mol-' for the DTBNIB-CD system and 20 f 2 cm3 mol-' for the DTBN/yCD system, respectively. The difference has been ascribed to the difference in the number of water molecules excluded from the CD cavity upon inclusion.
8- and ycyclodextrins are cyclic polysaccharides consisting of seven and eight glucuse monomer units, respectively, of different cavity size. Due to their large cavity and hydrophobic nature, CD's are known to form inclusion complexes with various kinds of molecules.' They have been recognized as good model compounds of enzymes? and many investigations have been focused on their characterization. Atherton and Strach' have observed a complex formation of 8-CD with nitroxide radicals in aqueous solution by ESR spectroscopy. Later, Okazaki and Kuwata4*'developed a new technique for analyzing ESR spectra of inclusion complexes using the line-width theory of Freed et al. They have discussed the dynamical properties of the inclusion complex of j3-CD with DTBN, and deduced how DTBN is included in 8-CD on the basis of the analysis of ESR spectra. It is of interest to know how volume changes upon inclusion-complex formation; however, little work concerning inclusion complexes at high pressure has been done so far. Recently, we have examined the pressure dependence of inclusion-complex formation of j3-CD with DTBN and found that the equilibrium constant decreases with external pressure.6 In the present study, the anisotropic motion of DTBN in 8-CD and y-CD and the pressure dependence of inclusion-complex formation of y-CD with DTBN are examined. The factors affecting the volume change upon inclusion of DTBN in CD's (&r)are discussed. Experimental Section Cyclodextrins (8.7) and DTBN were commercially purchased and used as received. High-pressure techniques and procedures for ESR measurements were the same as those described elsewhere.' A sample solution was deoxygenated by bubbling nitrogen for 20 min. ESR spectra were recorded on a home-made X-band spectrometer with a 100-kHz field-modulation unit. For density measurements, a digital precision densitometer (Kyoto Denshi, DA-1OlB) was used. Specific volumes at various concentrations of DTBN were measured, and the partial molar (1) Saenger, W. Angew. Chem., Int. Ed. Engl. 1980. 19. 344. (2) For example: Seanger, W.; Noltemeyer, M.; Manor, P. C.; Hingerty, E.;Klar, E. Bioorg. Chem. 1976,5, 187. Thoma, J. A.; Stewart, L. Starch: Chemistry and Technology;Academic Press: New York, 1965; Vol. 1. (3) Atherton, N. M.; Strach, S. J. J . Chem. Soc., Faraday Trans. 1,1975, 71, 357. (4) Okazaki. M.; Kuwata, K. J . Phys. Chem. 1984,88, 3163. ( 5 ) Okazaki, M.; Kuwata, K. J . Phys. Chem. 1984, 88, 4181. (6) Sueishi, Y.; Niahimura, N.; Hirata, K.; Kuwata, K. Chem. Express 1989, 9, 567. (7) Sueishi. Y.; Nishimura, N.; Hirata, K.; Kuwata. K. Bull. Chem. SOC. Jpn. 1988,61, 4253.
0022-3654/91/2095-5359$02.50/0
TABLE I: Reciprocal of Rotational Correlation Times (lO%/s) of DTBN Included in CD's at 1 Bar
0-CD 7-CD
l / T l
1/Tr
l/Tco
2.3 6.5
39 21
2.5 1.8
l/Tlm'
-0
1/Tin'
36
5
25
volume of DTBN at infinite dilution was estimated in the usual manner. Results and Discussion Inclusion Complex of CD with DTBN. The formation of the inclusion complex can be expressed as follows: DTBN + CD = IC (1)
where IC denotes the inclusion complex. The equilibrium constant Kappin concentration terms is given by Kapp
= [IC1/ W B N I [CDI
(2)
ESR spectra of DTBN in the presence of excess CD's (&y) are shown in Figure lA, B. The three-line spectra are due to a I4N nucleus in DTBN with hyperfine coupling constants (hfc) of 1.662 mT for DTBN in 0-CD and 1.675 mT for DTBN in y C D , respectively. The ESR parameters did not change appreciably upon addition of a-CD, which suggests incomplete, if any, inclusion of DTBN in a-CD. Figure lA, B show that the order of peak heights of the three spectral lines for DTBN included in y-CD is the same as that for @-CD,which indicates that the molecular disposition of DTBN in y C D is the same as that for &CD.5 The order did not change with pressure up to 588 bar. By the simulation of ESR spectra,' the rotational correlation time T,,and r1 of DTBN about the symmetry axis of the C D molecule and the axis perpendicular to the symmetry axis, respectively, have been estimated, and are given in Table I. The correlation times of 7Hand T* include the rotational diffusion of the inclusion c o m p l e ~ .Thus ~ it is essential to determine the rotational correlation time T~~~ of DTBN relative to the C D molecule by using the following relationship:* 1/Ti = 1/TCD + l/Tim' (3) where "i" refers to "11" or "I", and TCD denotes the rotational correlation time of the CD molecule. The correlation time T~~ for the isotropic rotational diffusion of CD may be approximated by the Debye equationg TCD
= 4rsa3/3kT
(8) Stejskal, E. 0.;Gutowsky, H. S.J. Chem. Phys. 1958. 28, 388.
0 1991 American Chemical Society
(4)
5360 The Journal of Physical Chemistry, Vol. 95, No. 13, 1991
TABLE 11: J~@MuRI
Sueishi et al.
C~artutr. 8t V U i ~ l gPIWSWU ud R ~ c t i O aVdvmcr for ~ c M o o - C ~ @Forpatioll8t ~X 298 K Klmo1-I dm3
Pb=
196
294
392
490
588
@-CD 939 910 840 7-CD 272 264 249 #Precision within 3% error. bIndicated pressures in bars.
1
98
817 215
822 223
792 197
821 156
A
AV/cm3 mol-’ 5+1 20 k 2
- } 6.0
I
B
I
I
I
I 0
2
6
4
[Y-CDlx103 /mol dm-3
lnclus i o n
I
I
1-1
0.5 mT
D
at 588 bar
al 1 bar
LL
0.1 mT
Figure 1. ESR spcctra of DTBN (4.4 X l P mol din-’) in the presence of (A) 8-CD (1.5 X IO-’ mol dm-)); (B)y-CD (2.8 X IO-’ mol dm-9; (C) y-CD (3.9 X IO-’ mol dm-)); (D) (a) high-field line of HFS, (b)
computer simulated spectrum. where TJ is the viscosity coefficient of the solution and a is the hydrodynamic radius of CD. The relative correlation times qnl have been estimated from eqs 3 and 4 by using available viscosity datal0 and the hydrodynamic radii” of C D s (r = 0.74 nm for &CD, 0.80 nm for y-CD), and they are given in Table I. The value of 1/ T of~ DTBN included in j3-CD is almost q u a l to 1/T,-,, which indicates that the rotation of DTBN about the axis perpendicular to the symmetry axis is determined by rotational diffusion of the inclusion complex. For DTBN included in the ~ is ~ obviously large y C D cavity, on the other hand, the 1 / value compared with I/T,-- This indicates that DTBN is included less tightly in y C D than in 0-CD. This could be attributed to the differin the cavity size between & and y-CD’s. The rotational rate I / T ~ inI the loose y C D complex about the symmetry axis is smaller than that in the tight 0-CD complex. The reason for this will be given later. Pressure Effect on Complex Formation. The ESR spectrum of DTBN (4.4 X IO-‘ mol dm-9 in the presence of y-CD (3.9 X lo-’ mol dm-3) is shown in Figure 1C. The high-field signal (MI = -1) splits into two, one corresponding to free DTBN and (9)
Abragam, A. The Principles of Nuclear Magwrism; Oxford Univ.
Press: New York, 1961. (IO) Eastman, M. P.;Freiha, B.; Hsu, C.C.; Change, C.A. J . fhys. Chem. 19811,92, 1682. Flohr, K.; Paton, R. M.; Kaiser, E. T. J . Am. Chem. Soc.
1975,97, 1209. (1 I) &CD is a doughnut-shaped molecule.’ However, a hydrodynamically equivalent shape of CD in solution is regarded as a sphere.’
Figure 2. Concentration dependence of equilibriumanstant for complex formation of y-CD with DTBN: ( 0 )1 bar; (A)294 bar; (m) 588 bar.
the other to included DTBN. The high-field signal was simulated as shown in Figure lD, and the relative concentraions of free and included DTBN were calculated by methods described elsehere.^^^.^ The equilibrium constants were estimated as a function of y-CD concentrations at various external pressures. This is shown in Figure 2. It is notable that the K value depends on the y-CD concentration. Atherton and S t r a a have pointed out that the variation in K, could be attributed to the change in the activity coefficient of%= DTBN. Thus, the extrapolated values to zero CD concentration should give true thermodynamic equilibrium constants. The K values obtained in this manner at various pressures are given in Table 11. The value of K at 1 bar in the DTBNIP-CD system is by about 3 times larger than that in the DTBN/y-CD system. The stability of an inclusion complex may be determined by the fitness of a guest in the CD cavity.l2 Therefore, the present finding indicates that the fit of DTBN to the cavity space of 8-CD is better than that of y-CD. The equilibrium constants given in Table I1 decrease as the external pressure increases. The reaction volumes AYextrapolated to 1 bar were estimated from the In K vs p plot according to the equation A V = -RT(8 In K/ap)TPII AnKTRT (5)
Fapp
+
where K T is the isothermal compressibility of solvent and An (= -1 in this case) is the difference between the numbers of species in the final and initial states. The value of K ~ RatT 1 bar was calculated from available data for water.I3 The estimated reaction volumes are 5 cm3 mol-’ for the DTBNIB-CD system and 20 an3 mol-’ for the DTBN/y-CD system. The reaction volumes for inclusion-complex formation are unexpectedly positive, and the AVvalue for y-CD is obviously larger than that for @-CD. These values can not be straightforwardly explained in terms of a primitive idea of the decrease in volume accompanied by the inclusion. The following volumetric contributions upon formation of an inclusion complex are likely: (1) inclusion of DTBN into the CD cavity (AVi,,, C 0). (2) desolvation of DTBN upon inclusion (AV,, > 0), (3) pushing included water molecules out of the CD cavity (AVN > O), (4) conformational change of CD (AV, > 0). Thus it follows AVm
AVinclu
+ AVdwl, + AI‘,,
+ AV,
(6)
The conformational change of a-CD upon inclusion-complex (12) Cramer, F.; Henglein, F. M. Chem. Ber. 1957, 90,2563. ( 1 3) International Crfrical Tables: McGraw-Hill: New York,1928; Vol. 111.
5361
J . Phys. Chem. 1991, 95, 5361-5366
formation has been observed by circular dichroism and X-ray studies.'J' The a-CD molecule takes an unsymmetrically collapsed coqformation to fit the cavity to two water molecules contained in the cavity. When the water molecules are replaced by other guest molecules in the cavity, the collapsed conformation changes into a circular unstrained conformation. 8- and y-CD's, however, are not strained'J5 and therefore, conformational volume changes of 8- and y-CD's upon inclusion-complex formation should be small. In the j3-CD cavity, an average of 6.5 water molecules are involved.16 The vacancy of the 8-CD cavity is almost fully occupied by DTBN;' hence, all water molecules in &CD may be excluded from the CD cavity upon inclusioncomplex formation. The 6.5 water molecules excluded give rise to the increase in volume by AVpuh= 117 cm3 mol-'. Accordingly, the sum of AVAV* becomes -1 12 cm3mol-' by eq 6. DTBN in CD rotates about the symmetry axis of CD as mentioned above, thus DTBN in CD is regarded as a cylinder in shape whose height and diameter were tentatively estimated to be 0.88 and 0.62 nm, respectively, with the aid of the Corey-Pauling-Koltun (CPK) model. Since the height of 8- and y-CDs is 0.80 nm,' DTBN should stick out of CD by 0.08 nm. The van der Waals volume of this part is then calculated to be 14.5 cm3 mol-'. It is generally known that the partial molar volume of a species in solution is about twice as large as its van der Waals volume. Thus,the partial molar volume of the excluded part is estimated to be 29 cm3mol-'. If we subtract this from the partial molar volume of DTBN in water, 159 cm3mol-', obtained experimentallyin this work, AV&,, becomes -130 cm3 mol-'. Thus, the volume change due to the desolvation of DTBN upon inclusion AVwv is estimated to be 18 cm3mol-' by using eq 6. In the cation/crown-ether ~omplex,'~
+
(14) Recs, D.A. J. Chem. Soc. B 1980,877. (15) Holand, H.; Hald, L. H.; Kvammer, 0. R.J. Solution Chem. 1981, 10, 775. (16) Lingner, K.; Saenger, W.Angew. Chem. 1978, 90,738. (17) Heiland, H.; Ringscth, J. A,; Vikingstad, E.J. Solution Chem. 1978, 7, 515. Heiland, H.; Ringseth, J. A.; Brun, T. S.J. Solution Chem. 1979, 8, 779.
the reaction volumes AVin water have been estimated to be 8-25 cm3 mol-', and Harada et a1.'* have pointed out that the main contribution to AVis the mnoval of water molecules from around guest ions. In our case, DTBN interacts with surrounding water molecules through dipoltdipole interaction as well as hydrogen bonding. Hence, the above value of AV- seems to be reasonable, though the above estimationswere carried out on the basis of some assumptions. In case of y-CD, 12 water molecules are originally situated in its c a ~ i t y ,and ' ~ all or a part of the water molecules are excluded according to the size of the guest molecule upon complex formation. In the present case, AV = 20 cm3 mol-' and Ayiadu AV= -1 12 cm3mol-'. Therefore, on the average 7.3 out of 12 water molecules are replaced by DTBN upon complex formation. In conclusion, DTBN is shown to be included more ti htly in &CD than in y-CD on the basis of the values of l / r L J a n d K for the inclusion complexes. The water molecules remaining in the y-CD cavity upon complex formation interact with the >N-O group of DTBN as well as the inner wall of CD through hydrogen bonding. This will cause a restriction of free rotation about the symmetry axis, resulting in the reduced l/qd in the y-CD cavity. The presence of polar water molecules in the CD cavity will favor the ionic form (b) in the following canonical resonance structures, which causes an increase in the nitrogen hyperfine coupling constant of DTBN in y-CD."
+
> N e a
-
>N*g-O-
R a m NO. a-CD, 10016-20-3; 8-CD, 7585-39-9; Y-CD, 1746586-0;
DTBN, 2406-25-9; water, 7732-18-5.
(18) Harada, S.; Sahara, H.; Nakapwa, T. Bull. Chem. Soc. Jpn. 1983,
56, 3833.
(19) Maclennan, J. M.; Stemwski, J. J. Biochem. Biophys. Res. Commun. 1980,92,926. (20) Kawamura, T.; Matsunami, S.;Yonezawa, T. Bull. Chem. Sa.Jpn. 1966,10, 11 11. Zger, S. A.; Frecd, J. H. J. Chem. Phys. 1982,77,3344.
Bilayer Structure and Stability in Dihexadecyl Phosphate Dlsperslons A. M. Carmona-Ribeiro,**tC. E. Cashma,$ A. Sess0,t and S. Schreiert Departamento de Bioquimica, Instituto de Quimica, Universidade de Sao Paulo, CP 20780 Sao Paulo, Brazil, Instituto de Investigaciones Bioquimicas, UNLP, CONICET, Facultad de Ciencias Medicas, 60 y 120, 1900. La Plata, Argentina, and Departmento de Patologia. Faculdade de Medicina. Universidade de Sao Paulo, Sao Paulo, Brazil (Received: December 13, 1990) The pHdependent structure and stability of dihexadecyl phosphate (DHP) bilayer dispersionsare determined by using fluorescent and spin probes. The hydrocarbon core region is probed by diphenylhexatriene (DPH) while the polar head/water interface is probed by either rrans-parinaric acid (TPA) or an iminoxyl derivative of stearamide spin-label (SSL). At 5 mM NaCI, the fluorescence depolarization (FP) of DPH or TPA goes through a maximum as pH increases for vesicles prepared and maintained at a given pH value. On the other hand, if the pH is changed after vesicle preparation, as during titration, FP against pH is a typical sigmoidal curve. SSL in large vesicles (LV) exhibits a higher degree of spectral anisotropy than in small vesicles (SV) which is interpreted as a deeper penetration of SSL in the LV bilayer due to its higher fluidity. The SV bilayer fluidity increases as a function of time after sonication. From electron microscopy, the number of bilayer fragments in the SV dispersion decreases with increasing pH. For LV up to pH 6, however, the bilayer structure remained unchanged as a function of time. The degree of ionization of the headgroups is suggested to be important in determining the nonstationary bilayer structures occurring during titration. Nevertheless, for stationary DHP bilayers, headgroup hydration and hydrogen-bonding ability are important factors in determining the bilayer structure and vesicle size.
Introduction Bilayers b r i n g ionizable polar heads have been a matter of some controversy in the literature.'-' The effect of increasing To whom correswndenoe should be addresstd. 'Instituto de Quimica, Universidade de Sao Paulo. Instituto de lnvestigaciones Bioquimicas. 1 Faculdade de
Medicina, Universidade de Sao Paulo.
0022-36S4/91/2095-S361$02.50/0
charge on the bilayer structure has been proposed to be a decrease in the bilayer packing density in earlier work'*2and an increase (1) Traueble, H.; Teubner, M.; Woolley, P.; Eibl, H. Biophys. Chem. 1976, 4. 319. (2) Traueble, H.; Eibl, H. Proc. Nail. Acad. Scf. U.S.A. 1974, 71, 214. (3) Eibl, H.; Blume, A. Biochim. Biophys. Acra 1979, 553, 476. (4) Blume, A.; Eibl, H. Biochim. Biophys. Acra 1979, 558, 13.
0 1991 American Chemical Society
