4254
J. Phys. Chem. 1990, 94, 4254-4259
Stability and Structure of the Inclusion Complexes of Alkyl-Substituted Hydroxyphenylazo Derivatives of Sulfanilic Acid with CY- and @-Cyclodextrlns Noboru Yoshida,* Akitoshi Seiyama, and Masatoshi Fujimoto* Laboratory of Coordination Chemistry, Department of Chemistry 11, Faculty of Science, Hokkaido Unioersity, Sapporo 060, Japan (Received: August 30, 1989; In Final Form: Nocember 28, 1989)
The dissociation constants for inclusion reactions, Kd and K,,', of alkyl-substituted hydroxyphenylazo derivatives of sulfanilic acid with a- and 0-cyclodextrinsare determined spectrophotometrically. In most cases, stabilities of the inclusion complexes with P-cyclodextrin are found to be lower than those of the corresponding inclusion complexes with a-cyclodextrin. The values of Kd and Kd for P-cyclodextrin complexes to some extent depend on the shape and the size of the alkyl substituents in the guest azo molecules, whereas those for a-cyclodextrin are almost the same except for the guests having bulky alkyl substituents. On the basis of 'H NMR measurements, we propose a possible structure of the final inclusion complexes and the intermediate species in the two-step inclusion between the guest azo molecules and cyclodextrins.
Introduction In the preceding paper we reported the kinetics of the inclusion reactions between the alkyl-substituted guest azo molecules, 1-8, and a-cyclodextrin. R3R,(O H ) Ph-N=N-PhS03-Na+, Three types of reaction mechanisms depending on the shape and the size of the alkyl substituents (R, and R,) were established. One-step inclusion was observed for the guest systems 1 (R3 = R, = H), 2 (R3 = Me), and 8 (R3 = R5= i-Pr), whereas in the guest systems, 4 (R, = Pr), 5 (R, = i-Pr), and 6 (R, = s-Bu), two-step inclusion was found. In the latter two-step inclusion, the first step was ascribed to the fast binding of the guest with a - C D and the subsequent slower process to the intramolecular structural interconversion for the intermediate species to attain a more stable final inclusion complex. The guest molecules, 3 (R, = Et) and 7 (R, = t-Bu), lie on the border line between the one-step and the two-step inclusion mechanisms. In the present paper we report a static feature characteristic of the inclusion processes with a-cyclodextrin and @-cyclodextrin. Directional multistep inclusions reported in the preceding paper are of special interest with respect to the multistep binding mechanisms of the enzyme substrate. The structural information on the intermediates and the final inclusion complex obtained by ' H N M R measurements are shown to provide additional information about the detailed mechanism of the directional inclusion process. Experimental Section Materials. Guest azo molecules (abbreviated as x-alkylHABSO,-Na+) were synthesized by azo coupling and purified by use of cellulose column chromatography.' The structural formulas of the guest azo molecules are shown in the preceding paper. The steric hindrance in the inclusion process and the hydrophobicity of the guest molecule depend strongly on the type and the position of the alkyl substituents (Rid)in the phenol ring. A change in the alkyl substituents (Rid) ortho to the hydroxyl group causes a significant change in the size and the shape of the phenol moiety. The values of a and b depicted in Figure 1 of the preceding paper vary from 6.5 to 10.5 A. a- and @-Cyclodextrins were purchased from Tokyo Kasei. Measurements. ' H Fourier transformed N M R spectra of the inclusion complexes were taken on a JEOL J N M GX-270 spectrometer (270 MHz) in a 5-mm spinning tube at 25 "C. Deuterium oxide (99.9%), sodium hydroxide-d (NaOD), and 20% hydrochloric-d acid (DCI) were purchased from E. Merck. Values of the chemical shift were referred to an external tetramethylsilane ( I 5% T M S in CDCI,) with an accuracy of 0.001 ppm. pH was measured with Hitachi-Horiba F-7ss and Hanna microcomputerized HI8424 pH meters. The absorption spectra of the inclusion ( I ) ,Yoshida, N.;Seiyama, A.; Fujimoto, M. J . Phys. Chem.. preceding paper i n this issue.
0022-3654/90/2094-4254$02.50/0
SCHEME I HA-
+ a-CD
e
HA--a-CD
complex in acidic (pH 4.6-4.8; phosphate) and in alkaline (pH 12.0, NaOH) conditions were recorded on a Hitachi 808 spectrometer. Ionic strength was adjusted at I = 0.1 mol dm-3 (NaCI). The temperature was controlled at 25.0 f 0.1 "C with a Lauda Type K2R thermostat. The acid form (HA-) and the base form (A2-) of the guest molecules (pK, = 8-9) were found to form 1:1 inclusion complexes with a - C D (Scheme I), judging from the occurrence of one set of isosbestic points and the nature of the Hildebrand-Benesi plot.2
Results and Discussion Absorption and CD Spectra and Dissociation Constants. Guest molecules (1-8) are classified into three groups, A, B, and C. These molecules are amphiphilic in nature; that is, they contain a hydrophobic (-Ph(OH)R,R,) and a hydrophilic (-PhSO H-6 (-0.196) >> H-1 (A6/ppm = -0.288) (-0.089) H-4 (-0.083) H-2 (-0.079). The shifts in the protons located at the exterior of the torus (H-I, H-2, and H-4) are relatively small. Since the magnitude of upfield shifts of both inner methine H-3 and H-5 protons is almost the same, the possible orientation of the /3-CD ring remains uncertain.22 The ' H N M R spectrum of the a-CD inclusion complex of guest 4 is more complicated than that of the corresponding @-CDinclusion complex (Figure 4a). At least two different kinds of inclusion species are present in equilibrium, judging from the ' H N M R data in Figure 4a. Each signal splits into two different types of peaks ( 0 and e). One is assigned to the signals of the final inclusion complex (0)and the other the intermediate inclusion species (e); the splitting is large particularly for the protons, H-8, H-9 and H-1 1, H-12, of the benzenesulfonate ring. This 'H N M R evidence supports the two-step inclusion process which was kinetically confirmed previously.' Large downfield shifts of the benzenesulfonate ring protons in the final inclusion complex indicates that the time-averaged interacting site of guest 4 is the
-
- -
(19) As to the inclusion of the hydroxyl group of 1,6-naphthalenediol, a very interesting fluorescence study was reported: Agbaria, R. A.; Uzan, B.; Gill, D. J . Phys. Chem. 1989, 93, 3855. (20) Berjeron, R. J.; Channing, M. A. Bioorg. Chem. 1976, 5, 437. (21) Berjeron, R . J.; Rowan, R. Bioorg. Chem. 1976, 5, 425. (22) For example, from the comparison of the relative magnitudes of Ab(H-5) and Ab(H-3). it appears that association takes place by the approach of barbiturate from the primary hydroxyl side of p-CD. Thakkar, A . L.; Demarco. P. V . J . Pharm Sci. 1971, 60. 652.
l ~ " " ' ' " ' ' ' " ' ' ' / " ' '
9
B
7
6 lppn
Figure 5 . Dependence of the 'H NMR spectra of the guest molecule, 1, on the concentration of a-cyclodextrin. The a-CD concentration varied between 6.43 X lo-, and 6.61 X lo-* mol dm-, and the concentration of the guest 1 between 2 X and 4 X IO-* mol dm-,. The bound (%): (A) 0; (B) 16; (C) 34; and (D) 99 at pD = 3.02. The rate constant of association with a-CD may be in the order of lo'-* mol-' dm3 s-' (immeasurably fast by SF method); therefore no separate complexed and uncomplexed atom signals can be observed. Increasing complexing, for example with increasing a-CD concentration, results in a continuous shift of the signals of included atom, approximating to a saturation limit. The asterisk (*) denotes the signal due to the reference solvent, CHCI,.
benzenesulfonate ring. In order to clarify the structure of the intermediate species and the final inclusion complex, we further examined the dependence of the chemical shift of each proton of the guest on the concentration of a - C D in detail. Three types of the concentration-dependence of the chemical shifts (6) were found (Figures 5-7). The 'H N M R observation is in accord with the kinetic interpretation that the inclusion reactions can be classified into three modes, guests A, B, and C. In group A, the inclusion of guest 1 with a-CD is too rapid to follow by the stopped-flow method (k+' > lo7 mol-' dm3 s-' and k-, > lo3 s-'). Therefore, the chemical shift (6&,d) at an appropriate concentration resonates in the region between that of the guest (6,) and that of the inclusion complex (6G-a-CD), according to eq 2,6d,23where [G-a-CD] = [ Q - 4[G],[a-
[e2
CD]T]]1/2/2,Q = [GIT + [a-CD],
+ Kd. The position of bobsd
(23) Berjeron, R. J.; Channing, M. A,; Gilbeily, G. J.; Pillor, D. M. J . Am. Chem. SOC.1977, 99, 5146.
Inclusion Complexes of Sulfanilic Acid Derivatives
The Journal of Physical Chemistry, Vol. 94, No. 10, 1990 4259
---,I ' , ' ' " ' , '
8
8
5/ PPm
7
Figure 6. Dependence of the IH NMR spectra of the guest molecule, 4, on the a-CD concentration. The bound (%): (A) 0; (B)25; (C) 50; (D) 70; and (E) 90 at pD = 3.20. No saturation curve is obtained, but the separate complexed atom signals (0and e) are obtained. The signals ( 0 )shifting to the downfield due to the fast equilibrium between the free guest and the intermediate diminish completely at higher a-CD concentration.
shifts gradually from bG to aGda.CD upon increasing the complexation as shown in Figure 5, indicating that only one type of complex occurs and the chemical exchange proceeds rapidly compared with the N M R time scale. This type of binding dependence of dobsd can be observed in a number of other a- and @-CDsystems. On the other hand, hOM of each proton of the bulky guest 7 (group C) shows intriguing dependence on the percentage bound. Upon increasing the percentage bound, the peak due to the free guest is gradually diminished, while the new peaks ascribed to an inclusion complex appear at a fixed position and its relative intensities increase with the increase of percentage bound. These concentration dependencies demonstrate that the chemical exchange is slow compared with the N M R time scale (ktl = 460 mol-' dm3 s-' and = 0.55 s-I from the stopped-flow measurements) and that only one type of inclusion complex is formed. As was reported in the previous kinetic study, the inclusion reaction of guest 4 (group B) with a-CD was found to proceed via two-step mechanism involving an intermediate species. Therefore, one can expect that the IH N M R spectra would be
" '
___p_
1
6 /PPm Figure 7. Dependence of the 'HNMR spectra of the guest molecule, 7, on the a-CD concentration. The bound (%): (A) 0; (B) 19; (C)45; (D) 98 at pD = 3.04. Unexpected concentration dependencies different from Figures 5 and 6 are observed. The separate free atom signal ( 0 ) is obtained.
complicated as compared with the guest systems 1 and 7 showing the one-step mechanism. Actually, a very complicated 'H N M R spectral pattern is obtained in Figure 6. This spectral pattern seems to be the mixture of Figures 5 and 7. The intermediate species can be detected in appreciable quantities upon increasing the a - C D concentration (% bound: 0, 25, 50, 70, 90). Several peaks (0) due to the fast equilibrium, G + a-CD intermediate, shift gradually to the lower field and the intensity becomes lower. At higher bound percentages where the ratio of the free guest to the complexed guest is very small, the peaks (0)rapidly diminished and only two peaks (e and 0)ascribable to the intermediate and the final inclusion complex are observed.24 Interestingly, for the protons (H-2, H-5, and H-6) of the phenol ring the difference in the chemical shift is small between the intermediate species and the final inclusion complex. However, for the protons (H-8, H-12 and H-9, H-11) of the benzenesulfonate the corresponding difference is markedly large. This indicates that the structures of both species in solution are quite different from each other particularly in the sulfanilate side. (24) A splitting of peaks in the NMR could have several interpretations. More extensive NMR studies are now in progress. Yoshida, N.; Fujimoto, M., unpublished data.
