J. Phys. Chem. B 1999, 103, 2607-2613
2607
Preparation and Characterization of Inclusion Complexes of Poly(propylene glycol) with Methylated Cyclodextrins Miyuko Okada, Mikiharu Kamachi, and Akira Harada* Department of Macromolecular Science, Graduate School of Science, Osaka UniVersity, Toyonaka, Osaka 560-0043, Japan ReceiVed: May 27, 1998; In Final Form: February 5, 1999
The complex formation between methylated cyclodextrins (CDs) and poly(propylene glycol) (PPG) in water is described. PPG diamine (Mw ) 4000) (PPGN4000) was solubilized in the water phase in the presence of 2,6-O-dimethyl-β-CD (DM-β-CD). 2,3,6-Trimethyl-O-R-CD (TM-R-CD), DM-R-CD, and TM-β-CD did not solubilize PPGN and did not give any precipitated complexes. The effects of DM-β-CD on the solubility of PPG were dependent on the Mw of PPG. The interactions between DM-β-CD and PPG were studied by NMR relaxation spectroscopy in D2O. The 1H resonance of C(3)H at the inner wall of DM-β-CD shifted upfield on addition of PPG. The 1H spin-lattice relaxation times of C(3)H and C(5)H of DM-β-CD markedly increased on addition of PPG. These results show that a PPG chain is included in the cavities of DM-β-CD in an aqueous solution. When PPGN4000 was vigorously stirred with a saturated aqueous solution over 2 days, the mixture became turbid to give precipitated complexes. The DM-β-CD-PPGN4000 complexes were characterized by 1H NMR, solid-state 13C NMR, powder X-ray diffraction, atomic force microscopy, and FT-IR techniques. The complex formation between DM-β-CD and PPG was found to consist of two processes: the solubilization of PPG and the precipitation of complexes. The complexation phenomena between poly(ethylene glycol) (PEG) and methylated CDs were also studied in a similar manner. The interaction of PEG with DM-β-CD is much weaker than that of PPG.
1. Introduction
TABLE 1: Complex Formation between CDs and Polymers yield (%)
Cyclodextrins(CDs)1-5
are a series of cyclic oligosaccharides consisting of 6-8 glucose units linked by R-1,4 linkages. They are called R-, β-, and γ-CD, respectively. The remarkable property of CDs is the formation of inclusion complexes with a variety of small molecules or ions of appropriate size. These complexes of simple structure in the experimental system are significantly different from the huge and complex aggregates in biological system. Studies of the macromolecular recognition are expected to bridge the gap between chemistry and biology. We have found that CDs form inclusion complexes with a variety of polymers.6-11 When a polymer was mixed with an aqueous solution of CD, the mixture became turbid to give crystalline complexes. Table 1 lists the yields of crystalline complexes between CDs and polymers. There is a good correlation between cavity sizes of CDs and cross-sectional areas of polymers. Previously, we reported that poly(propylene glycol) (PPG) formed crystalline inclusion complexes with β- and γ-CD, although R-CD, with a smaller cavity, did not form crystalline complexes with PPG of any weight-average molecular weight (Mw).8,9 The complex formation was Mw-dependent and stoichiometric. On the basis of the X-ray studies, the structures of β- and γ-CD-PPG complexes were found to be “channeltype”. It was shown that the networks of intermolecular hydrogen bondings between OH groups of CDs are effective for complex formation. Recently, the complexation of modified CDs has been investigated by many chemists. CD derivatives show different properties of the complexation in comparison with nonsubsti* To whom correspondence should be addressed. E-mail: harada@ chem.sci.osaka-u.ac.jp. Fax: 06-850-5446.
polymer
Mw
R-CDa
β-CDb
γ-CDc
PEG
1000
92
0
trace
PPG
1000
0
96
80
PMVE
2000
0
0
82
a R-CD saturated aqueous solution, 1.5 mL (R-CD, 0.22 g ) 2.2 × 10-4 mol); polymer, 15 mg. b β-CD saturated aqueous solution, 7.0 mL (β-CD, 0.13 g ) 1.1 × 10-4 mol); polymer, 15 mg. c γ-CD saturated aqueous solution, 2.0 mL (γ-CD, 0.46 g ) 3.6 × 10-4 mol); polymer, 15 mg.
tuted CDs. Some examples of CD derivatives are methylated CDs (Chart 1).1-5 Compared with native CDs, methylated CDs are more hydrophilic at room temperature (water solubility in g/100 mL: 1.85 for β-CD, 60 for DM-β-CD, and 30 for TMβ-CD) and prefer intramolecular hydrogen bondings. However, the hydrophobic cavities of methylated CDs interact strongly with guest compounds through hydrophobic interaction. To our knowledge, there are only some papers12-16 about complex formation between modified CDs and polymers. Topchieva et al.12-15 found that pluronics (triblock copolymers consisting of PPG and poly(ethylene glycol) (PEG) sequences) form complexes with β-CD and 2,6-O-dimethyl-β-CD (DM-β-CD). They studied the interaction between DM-β-CD and pluronics both in solution using X-ray diffraction or NMR spectroscopy and at the water-air interface using the Langmuir-Blodgett technique. Gaitano et al.16 reported that pluronics form hydrophilic
10.1021/jp9823852 CCC: $18.00 © 1999 American Chemical Society Published on Web 03/20/1999
2608 J. Phys. Chem. B, Vol. 103, No. 14, 1999 CHART 1: Methylated CDs
inclusion complexes with DM-β-CD in the water phase. They used dynamic or static light scattering techniques to investigate the complexation phenomena. Although both groups proposed the structures of the inclusion complexes between DM-β-CD and pluronics, the complexation between a triblock copolymer and a methylated CD is not a simple system. It is necessary to investigate the interaction between homopolymers and methylated CDs in detail using various methods. Now we reported here the complex formation of PPG with methylated CDs, 2,3,6-O-trimethyl-R-CD (TM-R-CD), DM-RCD, TM-β-CD, and DM-β-CD, in aqueous solutions. We found that PPG selectively forms inclusion complexes with DM-βCD. Compared with β-CD-PPG complexation, the complex formation of DM-β-CD and PPG shows great differences. When PPG is mixed with a β-CD aqueous solution, the mixture immediately becomes turbid to give precipitates. On the other hand, when PPG is mixed with a DM-β-CD solution, PPG is solubilized in the water phase to give a clear solution. The interaction between DM-β-CD and PPG in an aqueous solution was investigated by turbidity titration, 1H NMR, and 13C NMR analysis. NMR relaxation spectroscopy is a powerful technique for investigating the dynamics and structure of molecules and aggregates.17 The CD substrate coupling can be directly determined by the spin-lattice relaxation time of 1H and 13C (T1(1H) and T1(13C), respectively) or spin-spin relaxation time (T2) measurements.18-23 We used here 1H and 13C NMR relaxation techniques to gain insight into the structure and dynamics of the DM-β-CD-PPG complex in D2O. But it should be noted that the experiments in the liquid phase set a limit in the research of the complex formation because of the equilibrium of the complexation reaction. It is necessary to isolate the complexes themselves from solution and to carry out experiments using the solid samples. We could succeed in obtaining of DM-β-CD-PPG complexes and directly investigate the structures by 1H NMR, solid-state 13C NMR, powder X-ray diffraction, tapping-mode atomic force microscopy (AFM), and FT-IR measurements. In addition, we investigated the complexation phenomena between methylated CD and the other polymer, poly(ethylene glycol) (PEG). Now we extend the investigation to a variety of polymers. We already found that methylated CDs selectively form inclusion complexes with some hydrophobic polymers such as poly(tetrahydrofuran) (PTHF), poly(propylene), and poly(ethylene). Studies of the dynamics and structures are now in progress. 2. Experimental Section Material. R-, β-, and γ-CD were obtained from Nacalai Tesque Ltd. 2,6-Di-O-methyl-β-CD (DM-β-CD) and 2,3,6-triO-methy-β-CD (TM-β-CD) were purchased from Nacalai
Okada et al. Tesque Ltd. DM-R-CD was kindly supplied by Nippon Food Chemical Ltd. TM-R-CD was purchased from Cyclolab R&D Lab. PPG diamines (PPGN) (Mw ) 4000 and 2000) were purchased from Aldrich. PPG hydroxide (PPGOH) (Mw ) 1000) was obtained from Wako Pure Chemical Ltd. PPGOH (Mw ) 725 and 425) were purchased from Aldrich, and PPGOH (Mw ) 400) was from Ishidzu Chemical. Di(propylene glycol) and tri(propylene glycol) were purchased from Nacalai Tesque Ltd. PEG (Mw ) 2000 and 20 000) were purchased from Nacalai Tesque Ltd. D2O and DMSO-d6 were obtained from Aldrich. Measurement. Absorption spectra were recorded on a Shimadzu UV-2001 spectrometer at room temperature. Samples for turbidity analysis were prepared by sonicating the mixture of PPG (20 mg) and DM-β-CD aqueous solution consisting of 2.00 mL of H2O for 20 min. NMR experiments were carried out on a JEOL EX-270 NMR spectrometer operating at 270 MHz for 1H measurements and at 67.8 MHz for 13C. D2O solutions for NMR analysis were prepared by stirring a mixture of 1.00 mL of D2O solutions of DM-β-CD (3.4 × 10-1 M) and polymers (10 mg, 1.7 × 10-1 M (PG unit)) at room-temperature overnight, then bubbling N2 gas. Chemical shifts were referenced to external DSS (δ ) 0.00 ppm). All relaxation measurements were performed at 25 °C. Spin-lattice relaxation times (T1s) were determined by inversion-recovery pulse sequences and delay times greater than 3T1. 1H spin-spin relaxation times (T2(1H)s) were determined by the Carr-Purcell-Meilboom-Gill (CPMG) method. Chemical shifts of NMR spectra in DMSOd6 at 30 °C were referenced to the solvent value (δ ) 2.50 for 1H resonance and 39.50 for 13C). Powder X-ray diffraction patterns were taken using Cu KR irradiation with a Rigaku RAD-ROC X-ray diffractometer (voltage, 40kV; current, 100 mA; scanning speed, 3°/min). FT-IR measurements were performed on a Jusco FT/IR-410 spectrometer by KBr method. 13C cross-polarization magic angle spinning (CP/MAS) spectra were recorded at 75.6 MHz on a Chemagnetics JMNCMX300W spectrometer at room temperature. Chemical shifts were referenced to external hexamethylbenzene (δ ) 17.36 ppm). Optical rotatory measurements were carried out on a JASCO Dip-370 digital polarimeter at 589 nm with a cell length of 10 cm. Tapping-mode atomic force microscopy (AFM) measurements were performed with a Digital Instruments Nanoscope III microscope. Preparation of Solid DM-β-CD-PPGN4000 Complex. PPGN4000 (20 mg, 3.4 × 10-4 mol (PG unit)) was added to 2.8 mL of DM-β-CD saturated aqueous solution (0.32 M), and then the mixture were vigorously stirred for 3 days. The turbid solution was allowed to stand for 2 days. The products precipitated were collected by centrifugation, dried under vacuum, washed with distilled water, and dried under vacuum to give the DM-β-CD-PPGN4000 complxes.24 3. Result and Discussion Effects of Methylated CD on Solubility of PPG. Previously, we reported that PPG gave crystalline complexes with β-CD when a β-CD aqueous solution was mixed with PPG.8,9 First, we mixed PPGs of various Mws with aqueous solutions of methylated CD, and then the mixture was allowed to stand overnight. Although no precipitate could be obtained, we found that DM-β-CD solubilizes PPG in the water phase. The effects of DM-β-CD were measured using the turbidity method. PPG diamine (Mw ) 4000) (PPGN4000) is only sparingly soluble in an aqueous solution. However, upon addition of DM-β-CD, PPGN4000 was solubilized into water and the suspension gave
Complexes of Poly(propylene glycol)
Figure 1. Effects of CDs on the turbidity of PPGN4000 system.
J. Phys. Chem. B, Vol. 103, No. 14, 1999 2609
Figure 3. Mw dependency of turbidity of PPG on concentration DMβ-CD.
Figure 2. Effects of DM-β-CD on the turbidity of PPGTrt1 system. PPGTrt1 (10 mg) was mixed with a DM-β-CD solution (2.00 mL of distilled water).
CHART 2: End-Labeled PPG
Figure 4. 1H NMR spectra of DM-β-CD without (a) and with (b) PPGN4000 in D2O at 25 °C.
a clear solution (Figure 1). DM-R-CD, TM-R-CD, TM-β-CD, and R-CD did not solubilize PPGN4000, and the mixtures did not give precipitates. These results show that the effect of PPGN4000 is characteristic for DM-β-CD. We supposed that a hydrophobic PPG chain is included in hydrophilic DM-β-CDs to give a hydrophilic complex. Studies of molecular modeling showed that the cross section of a PPG chain fits the cavity of DM-β-CD. The cavity sizes of other CDs, such as methylated R-CD, R-CD, and TM-β-CD, are too small to thread PPG. Probably these CDs cannot include PPG because of the steric hindrance. To study the effects of DM-β-CD on the PPG chain, we synthesized hydrophobic PPG derivatives that have bulky end groups (Chart 2). All end-labeled PPGs were prepared from PPGN2000 (the average number n in Chart 2 is 34). PPGTrt1, which has a bulky triphenyl group at one end, could be solubilized on addition of DM-β-CD (Figure 2). On the other hand, PPGTrt, which has bulk triphenyl moieties at both ends, was not solubilized in water at any concentration of DM-βCD. PPG3NB is not soluble in DM-β-CD aqueous solutions. These phenomena indicate that DM-β-CDs thread a PPG chain through small end groups of polymers, such as amino groups, to cause an increase in the solubilities of PPG.
Figure 3 shows the turbidity changes of PPGs of a variety of Mw mixed with DM-β-CD aqueous solutions. Solubility effects were observed in PPGN4000, -2000, and PPG diol (Mw ) 1000) (PPGOH1000) systems. In Figure 3, the transmittance suddenly increased at a ca. 0.2 ratio ([DM-β-CD]/[PG unit]) in the PPG2000 system and 0.4 in PPGN4000. These significant increases indicate cooperative processes of complex formation between PPGN and DM-β-CD. PPGOH725, -425, -400, tri(propylene glycol), and di(propylene glycol) are so hydrophilic that solubility changes with and without DM-β-CD could not be observed by the turbidity experiments. These results show that the solubilization of PPG by DM-β-CD is Mw-dependent and stoichiometric. CD, consisting of D-(+)-R-1,4 glycopyranoyl moieties, can be detected by optical rotation measurement at 25 °C. The optical rotation (R) of DM-β-CD (0.45 g ) 3.4 × 10-4 mol/ 1.00 mL of water) increased about 1° on addition of PPGN4000 (10 mg, 1.7 × 10-4 mol (PG unit)). The change did not result from PPGN4000 of atactic structure. When a clear DM-β-CDPPGN4000 solution was freeze-dried, the powder X-ray diffraction patterns showed a typical broad structure at about 10°. The freeze-dried sample prepared from a DM-β-CD aqueous solution did not give a diffraction peak at about 10°. These results suggested that changes of DM-β-CD structure in the water phase derive from interactions with PPGN4000. 1H NMR Studies of Methylated CD-PPG System. We investigated the mode of binding between DM-β-CD and PPG by 1H NMR spectroscopy. PPGN4000 and a D2O solution of DM-β-CD in a molar ratio of 1:2 (PG unit: DM-β-CD) were stirred for several hours to give a clear solution. 1H NMR experiments of the D2O solution were carried out after N2 bubbling. Figure 4 shows the 1H NMR spectra of DM-β-CD
2610 J. Phys. Chem. B, Vol. 103, No. 14, 1999
Okada et al.
Figure 5. Plots of water solubility of PPGN4000 at various concentrations of DM-β-CD.
TABLE 2: T1(1H)s of DM-β-CD-PPG System T1(1H) [s] DM-β-CD- DM-β-CD- DM-β-CDDM-β-CD PPGN4000 PPGN2000 PPGOH1000 DM-β-CD C(1)H C(3)H C(5)H O(2)CH3 O(6)CH3 PPG methyl H
0.70 0.48 0.65 0.87 0.79
1.19 1.18 1.08 1.24 1.15 0.20
1.13 1.08 1.05 1.17 1.06 0.40
1.17 1.13 1.06 1.17 1.09 0.38
Figure 6. 13C NMR spectra of DM-β-CD without (a) and with (b) PPGN4000 in D2O at 25 °C.
TABLE 3: T1(13C)s of DM-β-CD-PPG System T1(13C) (s) DM-β-CD
with and without PPGN4000. The signals of DM-β-CD in the presence of PPGN4000 were broadened and shifted in comparison to those of free DM-β-CD. The broad signal of methyl protons of PPG chain could be observed. The 1H resonance of C(3) protons of DM-β-CD was markedly broadened and shifted upfield from 3.95 to 3.88 ppm. C(3)protons are located at the inner portion of a CD cavity. These spectral changes indicate that a PPG chain is included in DM-β-CD cavities. Signals of the DM-β-CD-PPGN2000 and PPGOH1000 system did not show significant changes. To get a picture of the interaction between DM-β-CD and PPG, 1H relaxation measurements were carried out at 25 °C in D2O using the inversion-recovery method. T1(1H)s are given in Table 2. It is shown that T1(1H)s of DM-β-CD in the presence of PPGN4000 are larger than those of DM-β-CD in the absence of PPG. The T1(1H)s of C(3)H and C(5)H of DM-β-CD markedly increased on addition of PPG. For example, the T1(1H) of C(3)H increased to be more than twice as long as that before addition of PPG. Protons at C(3) and C(5), inside the CD cavity, are probably located closest to the PPG chain. The changes of C(1)H, O(2)CH3, and O(6)CH3, outside the cavity, are so much smaller that these protons are located further from PPG. T1(1H)s show greater changes in the PPGN4000 system than those in PPGN2000 and PPGOH1000. The Mw dependency of T1(1H) indicates that the interaction between DM-βCD and PPGN4000 is the strongest. It is likely that PPGN4000 with many binding sites interacts with more DM-β-CDs than the other PPGs in water. 1H relaxation studies suggest that a PPG chain is located in DM-β-CD cavities. We tried to evaluate the binding constant between DM-βCD and PPGN4000 by solubility titration measurements. Sample preparation was carried out in a manner similar to that of the turbidity analysis. Figure 5 shows changes of water solubility of PPGN4000 on addition of DM-β-CD. The water solubilities were determined by 1H NMR spectra in D2O at 25 °C. If the soichiometries of DM-β-CD-PPGN4000 complexes are only 1:1 and 1:2 PPG/DM-β-CD, we would not be able to determine the binding constants from a fit of the solubility curve in Figure 5 because solubility changes of PPGN4000 were observed at
DM-β-CD C(1) C(4) C(2) C(3) C(6) C(5) O(2)CH3 O(6)CH3 PPG methyl C
0.16 0.15 0.17 0.17 0.09 0.15 0.57 0.58
DM-β-CDPPGN4000
DM-β-CDPPGOH1000
0.29 0.27 0.24 0.30 0.13 0.23 0.82 0.72 0.32
0.26 0.26 0.26 0.25 0.13 0.24 1.09 1.04 0.68
[DM-β-CD] > 1.5 × 10-2 M. It is probable that a PPG chain interacts with a number of DM-β-CDs. We failed to determine the binding constants of multiple complexation processes. We investigate the interactions PPG with DM-R-CD, TMR-CD, and TM-β-CD by 1H NMR spectroscopy. All signals of methylated CDs did not change in the presence of PPGs of any Mw. These results show that DM-R-CD, TM-R-CD, and TMβ-CD do not form inclusion complexes with PPG. 13C NMR Studies of DM-β-CD-PPG System. We also performed 13C NMR measurements on the DM-β-CD-PPG system. Figure 6 shows the 13C NMR spectra of DM-β-CD without (a) and with (b) PPGN4000 in D2O at 298 K. In the presence of PPGN4000, the 13C resonances of DM-β-CD are broadened and shifted. In particular, the 13C signals of C(1), C(3), and C(4) of DM-β-CD are broadened. T1(13C)s of DM-β-CD with PPG are shown in Table 3. The T1(13C)s of C(1), C(3), and C(4) significantly increased with added PPG. The T1(13C) ratio with and without PPGN4000 follows the sequence 1.81 (C(1)) > 1.80 (C(4)) > 1.76 (C(3)) > 1.53 (C(5)) > 1.44 (C(6) and O(2)CH3) > 1.41 (C(2)) > 1.23 (O(6)CH3). Carbons C(1) and C(4) are located at R-1,4glycopyranosyl linkages of CD. Carbon C(3) points to the interior of a CD cavity. Carbons of C(2), C(6), O(2)CH3, and O(6)CH3 are located outside the CD pocket. The 13C NMR results show that a PPG chain is included in the DM-β-CD cavity to cause changes of the ring structure of DM-β-CD. Precipitation of DM-β-CD-PPG Complexes. As described above, hydrophobic PPGN4000 is solubilized in a DM-β-CD aqueous solution to form the hydrophilic complex. When a
Complexes of Poly(propylene glycol)
J. Phys. Chem. B, Vol. 103, No. 14, 1999 2611
Figure 7. Time dependency of turbidity of DM-β-CD-PPG system.
Figure 9. Complex formation between DM-β-CD and PPGN4000 in water phase.
Figure 8. 1H NMR spectra of precipitated DM-β-CD-PPG complex in DMSO-d6 at 30 °C.
mixture of a saturated aqueous solution of DM-β-CD (distilled water, 4.0 mL; DM-β-CD, 2.4 g) and PPGN4000 (40 mg) was stirred at room temperature, the turbidity change of the mixture was detected (Figure 7). After being stirred for 1 h, the initial heterogeneous solution became clear. After 2 days the mixture became turbid again and then gave precipitates. Although the clear solution consisting of DM-β-CD and PPGN2000 also became turbid to give products precipitated, the yield of precipitated complex was too low (
