Biomacromolecules 2002, 3, 280-283
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Amphiphilic Block Copolymer with a Molecular Recognition Site: Induction of a Novel Binding Characteristic of Amylose by Self-Assembly of Poly(ethylene oxide)-block-amylose in Chloroform Kazunari Akiyoshi,*,†,‡ Naoyuki Maruichi,† Masaharu Kohara,† and Shinichi Kitamura§,| Department of Synthetic Chemistry and Biological Chemistry, Graduate School of Engineering, Kyoto University, Yoshida-Hommachi, Sakyo-ku, Kyoto 606-8501, Japan; PRESTO, Japan Science and Technology Corporation, JST, Kyoto, Japan; and Department of Biological Resource Chemistry, Kyoto Prefectural University, Shimogamo, Sakyo-ku, Kyoto 606-8522, Japan Received September 10, 2001; Revised Manuscript Received November 13, 2001
Solution properties of amphiphilic methoxy poly(ethylene oxide)-block-amyloses (MPEO-amyloses) in chloroform were investigated by SLS and DLS. The results indicated that MPEO-amyloses dissolved in chloroform containing 2 wt % DMSO by their self-associations. The complexation of MPEO-amylose with methyl orange (MO) was significantly enhanced in the amylose domain of the associate in chloroform. The blue shift of the maximum absorption and strong induced circular dichroism with exciton coupling were observed in the MPEO-amylose MO complex in chloroform. The self-assembly of MPEO-amylose in chloroform shows a unique feature for binding with MO. MPEO-block-amylose is a novel amphiphilic polymer with amylose as a molecular recognition site. Introduction Amphiphilic polymers have attracted growing interest with respect to biotechnological and pharmaceutical applications.1 Although many amphiphilic block copolymers have been synthesized and their properties in solution have been extensively studied, few studies have addressed molecular recognition by polymer micelle of such copolymers. We report here self-assembly of an amphiphilic block copolymer containing poly(ethylene oxide) (PEO) and an amylose chain as a molecular recognition site. Amylose, a linear polysaccharide consisting of R-1,4-linked glucose units, is an interesting macromolecule that complexes various hydrophobic guest molecules in a helical cavity. The diameter of the helix can vary depending on the guest size. However, amylose and amylose complexes are not colloidally stable in aqueous solution. Therefore, study of the host-guest chemistry of amylose in solution has been limited2 despite a potential importance in supramolecular science and also in biomedical application. We synthesized amphiphilic amylose derivatives bearing methoxy poly(ethylene oxide), MPEO-block-amyloses (Figure 1).3 PEO is a unique amphiphilic polymer that dissolves in water and even in organic solvents such as chloroform and toluene.4 This should enable the investigation of the host-guest chemistry of amylose in * To whom correspondence should be addressed at Kyoto University. Fax: +81-75-753-5912. E-mail:
[email protected]. † Kyoto University. ‡ PRESTO, Japan Science and Technology Corporation. § Kyoto Prefectural University. | Present address: Laboratory of Biophysical Chemistry, Graduate School of Agriculture and Biological Sicences, Osaka Prefecture University, Sakai, Osaka 599-8531, Japan.
Figure 1. Chemical structure of MPEO-block-amyloses.
various microenvironments. The precipitation (aging) of amylose in water is drastically prevented by conjugation with PEO. We describe here solubilization of amylose into chloroform containing 2 wt % DMSO by the formation of associates of MPEO-amylose. The self-assembly of MPEOamylose in chloroform induced a novel binding characteristic of amylose for methyl orange (MO). Experimental Section Materials. MPEO-p-nitrophenyl carbonate (Mw ) 5000, DP ) 114 ethylene oxide units) was purchased from Sigma Chemical Co., St. Louis, MO. Other organic and inorganic reagents were commercially available and used without further purification. MPEO-amyloses (degree of polymerization of amylose, DP ) 36, 73, and 112 glucosyl residues) were synthesized by enzymatic reaction (potato phosphorylase) from a maltopentaosylamine derivative of MPEO as a primer and R-D-glucose-1-phosphate as a substrate.3 The polydispersities of the polymers (Mw/Mn) were less than 1.2 by size-exclusion chromatography in water using pullulans as standards as previously reported.3 All other reagents were commercially available and used without any further treatment.
10.1021/bm010144l CCC: $22.00 © 2002 American Chemical Society Published on Web 12/21/2001
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Amphiphilic Block Copolymer
Light-Scattering Measurements. Light-scattering measurements were carried out using a DLS-700 instrument (Otsuka Electronics Co, Ltd., Kyoto, Japan). Light of 633 nm wavelength from a He-Ne laser was used as the incident beam. All measurement were performed at 20.0 ( 0.2 °C. Chloroform solutions of MPEO-amylose were prepared by injecting a DMSO solution (100 µL) of MPEO-amylose into chloroform (5 mL). Caution! Skin contact of DMSO results in primary irritation with redness, and chloroform is found to be carcinogenic. Please take care with their handling. The resulting solution was then placed into a cylindrical cell through filtration with a membrane filter (nominal pore size, 0.2 µm, ADVANTEC DISMIC-25JP). In the static light-scattering (SLS) measurements, the light scattered by a dilute polymer solution may be expressed as
Table 1. Static Light Scatering Data of MPEO-Amylose in Chloroforma sample
Mw,app
association no.
Rg (nm)
A2 (mol‚mL/g2)
MPEO-amylose-36 MPEO-amylose-73 MPEO-amylose-112
35 300 49 600 91 100
3.3 3.0 4.0
19.7 25.8 28.8
-8.13 × 10-4 1.06 × 10-3 1.89 × 10-4
a The association number was calculated from M w,app and average molecular weight of MPEO-amylose.
Kc 1 1 1 + Rg2q2 + 2A2c ) M 3 ∆R(θ) w
(
)
where q is the magnitude of the scattering vector, q ) 4πn sin(θ/2)/λ, ∆R(θ) is the difference between the Rayleigh ratio of the solution and that of the solvent, Mw is the weight average molecular mass, Rg2 is the mean square radius of gyration, A2 is the second virial coefficient, and K ) (4π2n2(dn/dc)2/(NAλ4) (NA is Avogadro’s number). The refractive index increments, dn/dc, of PEO-amyloses were measured using differential refractmeter RM-102 (Otsuka Electronics Co., Ltd.). The dn/dc values for poly(ethylene oxide) homopolymer and PEO-amyloses were measured. The values are -0.0464 for PEO, -0.0603 for PEO-amylose-36, -0.0645 for PEO-amylose-73, -0.0665 for PEO-amylose-112, respectively. The weight averaged molecular weight (Mw), the radius of gyration (Rg2) and second virial coefficient (A2) were obtained by the Zimm plot. In the dynamic light-scattering (DLS) measurements, measured autocorrelation function was analyzed by cumulant method. The hydrodynamic radius, Rh, can be calculated using Stokes-Einstein equation Rh )
kBT 6πηD
where kB is the Boltzman constant, T is the absolute temperature, and η is the solvent viscosity. Interaction with Methyl Orange. The chloroform solution (5.0 mL) of MPEO-amylose-73 (0.71 mg/mL) was added to water or phosphate buffer (0.1 M phosphate buffer) (1.0 mL) or containing methyl orange (MO) (2.57 mg/mL, 7.85 × 10-3) and the two phases were shaken for 60 s. The water content was measured using a Karl Fischer moisture automatic titration apparatus (Kyoto Electronics, model MKA). The absorption spectra of the chloroform layer were taken on HITACHI 220A spectrophotometer. CD spectra were obtained on a JASCO J-720 spectrometer. Result and Discussion Self-Association of MPEO-Amyloses in Chloroform. Phannenmuler et al. first demonstrated the synthesis of
Figure 2. Zimm plots for PEO-amylose-73 in chloroform. (temperature, 20.0 ( 0.2 °C; concentration, 0.508, 0.858, 1.07, and 1.43 mg/ mL.)
amylose derivatives by an enzymatic reaction using potato phosphorylase.5 Recently, enzymatic synthesis has been used to obtain various amylose-based materials.6-8 MPEOamyloses (degrees of polymerizations of amylose: DP ) 36, 73, and 112 glucosyl residues) were synthesized by enzymatic reaction from a maltopentaosylamine derivative of MPEO (Mw ) 5000, DP ) 114 ethylene oxide units) as previously reported.3 We can prepare transparent samples of MPEO-amylose in DMSO and chloroform. MPEOamylose dissolves well in DMSO. Chloroform solutions of MPEO-amylose were prepared by injecting a DMSO solution (100 µL) of MPEO-amylose (3.56 mg/mL) into chloroform (5.0 mL). A chloroform solution of MPEOamylose containing 2.0% (v/v) DMSO and 0.04 wt % water was obtained. Molecular weights of MPEO-amyloses in chloroform were determined by static light scattering (Table 1). Figure 2 shows Zimm plots for MPEO-amylose-73. The molecular weights data indicate that three or four MPEOamyloses self-associate in chloroform. The associates were colloidally stable for at least a month without precipitation at room temperature. Amyloses are completely insoluble in chloroform, while chloroform is a good solvent for MPEO. Therefore, MPEOamylose should form a structure with an inner core of amylose chain and an outer shell of MPEO chain in chloroform. However, the large sizes and low aggregation numbers of associates of MPEO-amyloses do not seem to be consistent with the usual picture of a core-shell-type polymer micelle. Water molecules play an important role in the formation of a reverse micelle of low molecular weight surfactant in organic solvent.9 The aggregation numbers of
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Figure 3. Absorption spectra of methyl orange (A) in water (broken line) ([methyl orange] ) 4.06 × 10-5 M) and in the presence of MPEO-amylose-73 (solid line) ([MPEO-amylose-73] ) 3.18 × 10-5 M; [methyl orange] ) 4.06 × 10-5 M) in chloroform. (B) Corresponding induced CD spectra of methyl orange in the presence of MPEOamylose in chloroform.
the reverse micelle in the presence of small amount of water are very small compared to that of aqueous micelles. For example, the aggregation number of the reverse micelle of dipalmitoyl phosphatidylcholine in chloroform is approximately three.10 In our experimental condition, chloroform contains 0.04 wt % of H2O and also 2.0% (v/v) of DMSO. DMSO is also good solvent for amylose. In the absence of DMSO, MPEO-amylose did not dissolve in chloroform. DMSO should play an important role in the solvation and self-assembly of MPEO-amylose. The amylose chain of MPEO-amylose should be selectively solvated by water and DMSO. The plausible structure of the associates of MPEOamylose could be a reverse polymer micelle with DMSO and water as selective solvent. To check this point, the chloroform solution (5.0 mL) of MPEO-amylose-73 (0.71 mg/mL) was added to water (1.0 mL) and the two phases were shaken for 30 s. The mixture separated into two phases within 5 min after shaking. More than 90% of the MPEOamylose remained in the chloroform layer. The water content in the chloroform layer increased from 0.04 to 0.13-0.14 wt % after mixing. In a control experiment without MPEOamylose, the water content increased from 0.02 to 0.080.10 wt %. The hydrodynamic radius of the associate of MPEO-amylose-73 in chloroform was 23 nm. After the treatment of water, the radius a little increased to 26 nm. The associates incorporated water molecules in the domain of amylose of MPEO-amylose. The root-mean-square radius of gyration for MPEO of DP ) 114 in unperturbed condition is calculated to be about 3 nm. 11 This value is reasonable in chloroform. However, the conformation of amylose chain may change by accompanying with association of MPEO-amylose in chloroform. To explain the results of SLS in terms of the structure of a reverse polymer micelle, Rg of amylose should be 7 nm for
Akiyoshi et al.
MPEO-amylose-36, 10 nm for MPEO-amylose-73, and 11.5 nm for MPEO-amylose-112. These values are quite large if amyloses form random coils because those of amyloses of DP ) 36, 73, and 112 are calculated to be about 2, 4, and 6 nm, respectively.12 If amylose chain forms a rigid helix, the size should be larger. On the basis of X-ray diffraction data, the size of six single helical turns ()36 glucose units) for VH-amylose is calculated to be 5.4 nm.13 In such helical structures, the values of the radii of amyloses of DP ) 36, 73, and 112 are calculated to be about 2.7, 5.4, and 17 nm, respectively. If amylose chains were expanded to almost linear structures (one glucose unit is estimated to be 0.5 nm), the maximum radii are 9 nm for MPEOamylose-36, 18 nm for MPEO-amylose-73 and 28 nm for MPEO-amylose-112. Judging from these considerations, the amylose chain of PEO-amylose may be quite expanded or may form helical structure in the special microenvironment of the aggregate in chloroform. Interaction of the Self-Associate of MPEO-Amyloses with Methyl Orange in Chloroform. The chloroform solution (5.0 mL) of MPEO-amylose-73 (0.71 mg/mL) was added to water (1.0 mL) containing methyl orange (MO, 1) (2.57 mg/mL, 7.85 × 10-3 M) and the two phases were shaken for 60 s.
The absorption spectrum of the chloroform layer is shown in Figure 3A. MO molecules were extracted to the chloroform layer, although MO did not extract at all to chloroform without MPEO-amylose. A large blue shift of the maximum absorption (428 nm) of MO was observed in the chloroform layer compared with that in water (464 nm). When phosphate buffer (0.1 M, pH 7.0) was used instead of water, the same spectral change was observed. Therefore, the spectral change is not due to a pH change in the microenvironment. The spectral changes were not observed in an aqueous solution of amylose and MPEO-amylose under the same conditions. The complexation of MPEO-amylose with MO was significantly enhanced in the amylose domain of the associate in chloroform. The circular dichroism (CD) spectrum of the MPEO-amylose MO complex in chloroform is shown in Figure 3B. The pattern of the CD bands is typical for a cotton effect separated by Davydov splitting.14 Few studies of induced CD in the amylose inclusion complex in water have been conducted because of low solubility.2b,15 In water, no induced CD of MO was observed in the presence of the same concentration of MPEO-amylose or amylose. The interaction of MO with the chiral helix of amylose could induce CD. The induced CD of MO is reported by complexation with various cyclodextrins in water.16 We checked the interaction of MO with various cyclodextrins in water. The spectra as shown in Figure 4 are much different from that of MPEO-amylose in chloroform. The absorption spectrum of MO did not change at all in the presence of cyclodextrins.
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Such side by side associated helices due to a folding of the chain involving intermolecular association was proposed by an amylose-iodine-iodide complex in aqueous solution.19 It may be possible to use such a self-associate to solubilize various biomaterials such as enzymes or DNA/RNA in organic solvent. The associates in organic solvent are able to be cross-linked by appropriate cross-linking reagents and nanoparticles should be obtained. The nanoparticles are soluble in water as well as organic solvent and will be applicable to novel nanobiomaterials. Such applications are under investigation in our laboratory.
Figure 4. Induced CD spectra of methyl orange (3.93 × 10-3 M) in water in the presence of (1) R-cyclodextrin (R -CD) (5.15 × 10-4 M), (2) β-cyclodextrin (β -CD) (4.41 × 10-4 M), and (3) γ-cyclodextrin (γ -CD) (3.86 × 10-4 M).
Acknowledgment. The authors are grateful to Prof. Junzo Sunamoto, Emeritus Professor of Kyoto University, for their helpful discussion. This work was supported by a Grant-inAid for Scientific Research in Priority Areas from the Ministry of Education, Science, Culture, and Sports, Japan. References and Notes
The intensity of the CD spectra was approximately 100-fold lower than that in the MPEO-amylose associate. These results indicate that the interactions of MO with MPEOamylose in chloroform are significantly strong. All cyclodextrins mainly form a 1:1 complex with MO because of no observation of the exciton coupling though only γ-cyclodextrin slightly complexes MO as a dimer.16 MPEOamylose shows a unique feature for binding with MO in chloroform. The exciton coupling in the CD spectrum and the blue shift of the maximum absorption of the MPEOamylose MO complex suggest that at least a dimer of MO formed in the chiral helix of amylose. In general, the blue shift of the absorption in the dimer of azobenzenes is expected when the transition dipole moment of the compound arranges in parallel (H aggregate).17 Therefore, a parallel arrangement of MO is plausible for the structure of the dimer. The dimerization of MO would be facilitated by complexation with amylose in the core of the associate. Amylose forms a left-handed 6/1-helical (s-helix) structure in crystal.18 The sign of the CD band of the complex shows that MO molecules bound to the amylose chain have s-chirality. MO molecules may bind to the groove of a s-helical structure of the amylose chain. The amylose domain in the associate of MPEO-amylose should provide a unique microenvironment for the complexation of MO. This complexation may be promoted by the side-by-side association of helices of amylose due to their intra- or intermolecular aggregation in the restricted domain.
(1) Dubin, P.; Bock, J.; Davis, R.; Schults, D. N.; Thies, C. Macromolecular Complexes in Chemistry and Biology; Splinger-Verlag: Berlin, 1994. (2) (a) Wulff, G.; Kubik, S. Makromol. Chem. 1992, 193(5), 1071. (b) Kubik, S.; Wulff, G. Starch/Sta¨ rke, 1993, 45, 220. (c) Wulff, G.; Steinert, A.; Holler, O. Carbohydr. Res. 1998, 307, 19. (3) Akiyoshi, K.; Kohara, M.; Ito, K.; Kitamura, S.; Sunamoto, J. Macromol. Rapid Commun. 1999, 20, 112. (4) Bailey, F. E., Jr.; Koleske, J. V. Poly(Ethylene Oxide); Academic Press: New York, 1976. (5) Ziegast, G.; Pfa¨nnemuler, B. Carbohydr. Res. 1987, 160, 185. (6) (a) Kobayashi, K.; Kamiya, S. Macromolecules 1996, 29, 8670. (b) Kobayashi, K.; Kamiya, S. Macromol. Symp. 1997, 120, 139. (7) Loos, K.; Stadler, R. Macromolecules 1997, 30, 7641. (8) Enomoto, N.; Furukawa, S.; Ogasawara, Y.; Akano, H.; Kawamura, Y.; Yashima, E.; Okamoto, Y. Anal. Chem. 1996, 68, 2798. (9) ReVerse Micelles: Biological and Technological ReleVance of Amphiphilic Structures in Apolar Media; Luisi, P. L., Straub, B. E., Eds.; Plenum Press: New York, 1982. (10) Haque, R.; Tinsley, I. J.; Schmedding, D. J. Biol. Chem. 1972, 247, 157. (11) Smith, G. D.; Yoon, D. Y.; Jaffe, R. L. Macromolecules 1993, 26, 5213. (12) Nakanishi, Y.; Norisuye, T.; Teramoto, A.; Kitamura, S. Macromolecules 1993, 26, 4220. (13) Immel, S.; Lichtenthaler, F. W. Starch/Sta¨ rke 2000, 52, 1. (14) Nakanishi, K.; Verova, N.; Woody, R. W. Circular Dichroism; VCH Publisher: New York, 1994. (15) Kim, O.-K.; Choi, L.-S, Langmuir 1994, 10, 2842. (16) Clarke, R. J.; Coates, J. H.; Lincoln, S. F. Carbohydr. Res. 1984, 127, 181. (17) Shimomura, M.; Ando, R.; Kunitake, T. Ber. Bunsen-Ges. Phys. Chem. 1983, 87, 1134. (18) Sarko, A.; Biloski, A. Carbohydr. Res. 1980, 79, 11. (19) Handa, T.; Yajima, H. Biopolymers, 1981, 20, 2051.
BM010144L
