Novel Derivatives of Cyclodextrins, Modified with Poly(ethylene Oxide

676

Bioconjugate Chem. 1998, 9, 676−682

Novel Derivatives of Cyclodextrins, Modified with Poly(ethylene Oxide) and Their Complexation Properties Irina N. Topchieva,* Petra Mischnick,† Gerhard Ku¨hn,‡ Vladimir A. Polyakov,§ Svetlana V. Elezkaya,| Georgy I. Bystryzky,| and Konstantin I. Karezin| Lomonosov Moscow State University, Department of Chemistry, 119899, Lenin Hills, Moscow, Russia, Institut fu¨r Organische Chemie der Universita¨t Hamburg, Martin-Luther-King-Platz 6, D-20146 Hamburg, Berlin, Bundenstalt fu¨r Materialforshung und-pru¨fung, Rudower Chausse 5, Haus 10.1, D-12489 Berlin, Germany, Russian Mendeleev University of Chemical Technology, Miusskaya, 9, 125820, Moscow, Russia, and State Scientific Center “NIOPIK”, B. Sadovaya, 1, bd.4, 113787 Moscow, Russia. Received January 15, 1998; Revised Manuscript Received May 7, 1998

A new family of bouquet-like molecules based on cyclodextrins is described. These compounds were obtained by polymerization of ethylene oxide. This reaction was initiated by primary and secondary hydroxyl groups of cyclodextrins, which constitute an organizing core. Analysis of structure and composition of conjugates based on R-CD and β-CD was performed using the data of MALDI-MS, GC-MS, and 13C-NMR spectra. Glass transition behavior of the conjugates shows that the above compounds are amorphous. Complexation properties of the conjugates are described with respect to sodium 4-nitrophenolate and calcium acetylhomotaurinate, which are used as guest molecules. Binding interaction between cyclodextrins or their conjugates and sodium 4-nitrophenolate was studied using differential absorption spectra. Association constants Ka between CD hosts and calcium acetylhomotaurinate composed of two equal anionic moieties were studied using 1H-NMR spectroscopy. The values of binding constant for β-CD were found to increase by more than 2 orders of magnitude than that of the corresponding system based on R-CD. R-CD was shown to form the inclusion complex with one anionic moiety, whereas β-CD produces a ternary complex with two anionic moieties of CAHT. For PEO derivatives of CDs, Ka decreases as compared with that of parent CD for both guests. These conjugates may be used as potent drug-delivery systems.

INTRODUCTION

Cyclodextrins (CD)1 have been extensively studied because of a unique ability of these cyclic starches to produce dynamic molecular inclusion complexes with various compounds including pharmaceuticals. The ability of CD for molecular recognition is controlled by hydrophobicity of a guest and fitness between the shape of guest species and a CD cavity (1). To change the molecular recognition ability of CDs, many derivatives of CDs containing various groups on both sides of CD have been investigated (1, 2). In this respect CDs, modified with water-soluble polymers, are of a great interest. At the present time, these investigations are * Address correspondence to this author at Moscow State University. Tel: 007-(095)-939-5583. Fax: 7-(095)-939-01-74. † Institut fur Organische Chemie der Universitat Hamburg. ‡ Bundenstalt fur Materialforshung und-prufung. § Russian Mendeleev University of Chemical Technology. | State Scientific Center “NIOPIK”. 1 Abbreviations: CD, cyclodextrin; CI, chemical ionization; EI, electron impact; EO, ethylene oxide; GC-MS, gas chromatography-mass spectrometry; M, molecular mass; Mw,weight average molecular mass; Mn, number average molecular mass; MMD, molecular mass distribution; MALDI-MS, matrix-assisted laser desorption ionization-mass spectrometry; MS, molar degree of substitution; PEO, poly(ethylene oxide); NP, natrium 4-nitrophenolate; CAHT, calcium acetylhomotaurinate; PEO-R-CD, conjugate of PEO and R-CD; PEO-β-CD, conjugate of PEO and β-CD; Tg, temperature of glass transition.

in their infancy. The advantages of polymer derivatives of CD were demonstrated for CDs modified with poly(Nisopropylacrylamide) chains (3). In these systems, polymer chains experience the coil-globule transition upon heating, and the inclusion of a guest molecule is controlled by temperature. Therefore, polymer chains are sensitive to the external action. More interesting polymer derivatives of CD are the conjugates of CD with poly(ethylene oxide), which may be presented as a microcapsule consisting of a CD core with short poly(ethylene oxide) chains attached to primary and secondary hydroxyl groups of CD (bouquet-shaped structures) (4). Even though modification of CD by PEO chains decreases the size of CD cavity and binding constant, one may anticipate that this modification would give rise to new properties such as amphiphilic character of CD derivatives (5) and the adaptation of guest accommodation due to the additional noncovalent fixation of the ligands in polymer surroundings. In this work, the synthesis of the above compounds are described, and effect of polymer on the complexation with two guests, sodium 4-nitrophenolate (SN) and calcium acetylhomotaurinate (CAHT) was examined. EXPERIMENTAL PROCEDURES

Chemicals. In this work, we used R- and β-cyclodextrins (Cyclolab, Hungary) and 2,5-dihydroxybenzoic acid (Aldrich); SN and CAHT2 were analytically grade reagents.

10.1021/bc980005+ CCC: $15.00 © 1998 American Chemical Society Published on Web 09/25/1998

Poly(ethylene Oxide) Derivatives of Cyclodextrins

Methods. Gas chromatography (GC) was performed with a Carlo Erba GC 6000 Vega Series 2 equipped with CPSi18 CB (Chrompack) and retention gap, on-column injector, and a flame ionization detector. Hydrogen was used as a carrier gas. GC-MS. Mass spectra were recorded with a HewlettPackard 5840 A-5985A instrument (CI, ammonia, and EI) and VG/70-250S GC-MS-system (VG analytical). MALDI-mass spectra were recorded using the TOF laser Kratos Compact MALDI III mass spectrometer (Kratos, Shimadzu, U.K.) in a reflection mode. Samples were prepared by casting the matrix compound (2,5dihydroxybenzoic acid, 10 mg/mL in ethanol/water, 1:1, v/v) onto the slide. After evaporation of the solvent, the solution of conjugate (in methanol or for permethylated samples methanol-dichloromethane, 2:1, v/v, 1 mg/mL) was deposited on the matrix. The solvent was evaporated. Ions were produced by laser ionization/desorption at 337 nm (3 ns pulse width). Ionization was accelerated with 20 kV in the positive ion mode. Differential absorption spectra were measured by a Hitachi 557 model UV spectrophotometer. 1H-NMR spectra were recorded on a CXP-200 Bruker spectrometer. 13C-NMR spectra were recorded on AM-300 Bruker spectrometer. Chemical shifts are given in parts per million. Sodium (4,4′-dimethyl-4-)silapentane sulfonate and dimethylsulphoxide are used as internal references. Glass transition temperatures were measured with a Mettler TA 400 thermal analyzer using a DSC-30 unit. Scanning rate was 20 K/min. Synthesis. β-CD (3.5 g, 0.003 mol) in 50 mL of 0.9% NaCl solution (pH 11.2) was placed in the reaction stainless vessel. Ethylene oxide (EO) was added at 80 °C under a pressure of 0.145-0.196 MPa. After stirring for 4-10 h, the reaction mixture was cooled down to 40 °C and unloaded. The product was dissolved in water. The content of insoluble product was estimated to be ∼5%. This product was separated by filtration. The dialysis of the solution was carried out using Seamless tubing (Sigma). The completion of dialysis was controlled using thin layer chromatography on Silufol plates in CHCl3:C2H5OH:H2O (36:12:1). The spots were revealed by iodine. The solution was filtered through a glass fiber filter (Sigma) and lyophilized. A repeated drying of the conjugates was carried out by heating in vacuum in the presence of phosphorous pentoxide at 95-100 °C for 1 h. Water content in the lyophilyzed samples of the conjugates was estimated to be 10-13%. For the PEOβ-CD conjugate, calcd: C, 45.7%; H, 8.7%. Found: C, 45.8%; H, 8.5%. Chemical Modification of PEO-CD Conjugates. PEO-CD conjugates were permethylated using powdered sodium hydroxide and methyl iodide (6). The permethylated products were extracted with dichloromethane. Hydrolysis of the conjugates was carried out in 2 N trifluoracetic acid in a screw cap vial at 120 °C for 2 h. The solvent was evaporated under nitrogen flow. The traces of acid were removed by codistillation with toluene. Remethylation of glucose derivatives of PEO (hydrolysates) was performed using CD3I/NaOH in DMSO according to ref 6. Complexation Properties. Complexation between SN and CD or their conjugates was studied by the measurements of differential absorption spectra in 0.05 M phosphate buffer (pH 9.5). Equilibrium constants of complexation between CD or their conjugates with CAHT 2 CAHT was kindly presented by Dr. R. N. Alyautdin (Moscow Sechenov Medical Academy).

Bioconjugate Chem., Vol. 9, No. 6, 1998 677

Figure 1. Analysis of the PEO-CD conjugates and their degradation products.

were estimated from 1H-NMR spectra. The analysis of binding curves was carried out using computer program Microcal Origin 3.5. Thus, values δ1 (the limiting value of chemical shift of methyl protons of CAHT in the presence of CD derivatives) were calculated. Correlation between chemical shifts of methyl protons of CAHT and concentration of CDs or their derivatives was described by the following equation:

δobs ) δ1 +

δ0 - δ1 [B] 1 + Ka

(1)

where δobs stands for the chemical shift of methyl protons of CAHT; δ1 is the limiting value of chemical shift; δ0 is the value of the chemical shift of the initial CAHT; [B] is the concentration of CDs or their derivatives; Ka is the microscopic binding constant of CDs or their derivatives with one binding center (anionic moiety of CAHT). This equation holds when both anionic moieties of CAHT (centers) are equivalent and do not interact with each other and [B] >> [CAHT]. RESULTS AND DISCUSSION

Synthesis of Conjugates. PEO-CD conjugates were synthesized in one-pot manner by polymerization of EO. The reaction was initiated by hydroxyl groups of multifunctional core of CD. This method was developed for the synthesis of graft copolymers of EO on starch (7). PEO, the main byproduct of this reaction, was produced by the polymerization of ethylene oxide initiated by hydroxide ions (8). Characteristics of Conjugates. Characteristics of the conjugates (molecular mass, molecular mass distribuh n, molar degree of substitution, MS, and mode tion, M h w/M of substitution) were obtained through the procedure of succesive permethylation, hydrolysis, and perdeuteromethylation (6). The consequence of the reactions and the methods of investigation are presented in Figure 1.

678 Bioconjugate Chem., Vol. 9, No. 6, 1998

Topchieva et al.

Figure 2. MALDI-mass spectra of PEO-β-CD (1) and its permethylated derivative (2). Sodium adducts are detected (Minor series of gomologous present sodium adducts).

presented in Table 1 were calculated using the following formula:

Table 1. Characteristics of PEO-CD Conjugates M hw

M h w/M hn

CD

conjugate

hydrolysate

conjugate

hydrolysate

MS-range/ CD

MSav

R β

3025 3540

608 778

1.004 1.005

1.04 1.09

35-60 40-70

8 8

Mass Spectrometry. Three parameters of the conjuh n, and MS were determined gates, molecular mass, M h w/M by MALDI-MS method (Table 1). The MALDI-mass spectrum of the conjugates exhibit the distribution of weight molecular mass Mw. Using graphical method (9) values of average weight and number molecular masses, Mw and Mn, were calculated. Note that the MALDI-mass spectra are characterized by a narrow and symmetrical hn distribution of the components (Figure 2) with M h w/M close to unity. This is characteristic of anionic polymerization. Mass distribution of the permethylated PEGCD conjugates agrees with that of the original samples, ∆M ) n42 (42 is 3 × 14 for threee methyl groups per glucose unit) where n is a number of glucose units per hn CD (Figure 2). To estimate molecular mass and M h w/M of PEO substituents, the permethylated conjugates were hydrolyzed. The MALDI-mass spectra of the resultant PEO-containing glucose derivatives are presented in Figure 3. As it is seen, these products are characterized h n values (1.04-1.09). These by a narrow interval of M h w/M values are slightly higher than those of the initial conjugates but MMD is still narrow. This evidence suggests that the degree of polymerization of PEO substituents attached to different positions of glucose units of CD does not differ substantially. The total number of ethylene oxide units in the conjugates was obtained from molecular mass of the conjugate minus molecular mass of the initial CD divided by 44 (molecular mass ethylene oxide unit) (Table 1). An average numbers of monomer units in each PEO substituent (MSav)

MSav ) (Mw,hyd - 245)/44 where 245 is the total mass of all non-PEO fragments of oligomer (180 for glucose fragment, 42 for 3 methyl groups, and 23 for sodium). Mode of Substitution. Additional information concerning the number of substituents in PEO-CD conjugates was obtained using GLC-MS method. This method allows one to estimate the number of substituents in a given compound (from the molecular mass of separate peaks). For example, using both EI and CI modes, it was shown that methyl glucosides with MS from 0 to 11 present in PEO-β-CD. This implies that, even at these high values of MS, unmodified groups in CD still exist. From the fragmentation patterns of the EI mass spectra, preferred 6-O-hydroxyethylation was demonstrated in the fraction with MS ) 1 (6 >> 2). For the fraction with MS ) 2 the order was 66′ >> 22′ > 26. For methyl glucosides with MS ) 3, the patterns 66′6′′, 266′, 22′2′′, and 22′3 are detected. At higher MS, the formation of oligoether at 2-OH dominates. However, it seems difficult to differentiate between longer PEO chains because the structure of isomers becomes very complex. As known, the relative reactivities of hydroxyl groups of CD or starch during the kinetically controlled etherification are controlled by the concentration of base (10). Polymerization proceeds without any addition of base but due to nucleophilic opening of oxirane by chloride ion of NaCl pH of the reaction media achieves about 11. Under these conditions, the reaction at the most acidic 2-OH, supported by the axial glucoside oxygen, dominates. In general, a poor reactivity of secondary 3-OH in CDs prevents peretherification. Formation of oligoether in 2 and 6 positions is preferable and makes the sterically



Figure 3. MALDI-mass spectra of glucose derivatives of PEO (hydrolysates): (1) From PEO-R-CD; (2) from PEO-β-CD.

hindered 3-OH even less accessible. Therefore, for the various hydroxyl groups in the glucose unit of PEG-CDs, molar degree of substitution changes in the following order: 2 > 6 >> 3. Polymerization of ethylene oxide in the presence of CDs yields randomly distributed PEO chains attached to CD with MSav ) 8-11 per glucose unit. In this case, MMD is symmetrical and narrow, and h n is close to unity. M h w/M NMR Spectra. PEO-CD conjugates were studied using 1H- and 13C-NMR spectra. In the 1H-NMR spectra, the signals of protons of glucose rings were shielded by a strong signal from PEO protons. In the 13C-NMR

spectra, one may distinguish well-defined signals corresponding to carbon atoms of polymer chains which are characterized by the following chemical shifts: 62.173, 71.315, and 73.477 ppm. Furthermore, the spectrum shows broad signals of carbon atoms corresponding to CD moiety (62, 71-75, 80-84, and 100-103 ppm). Our efforts to achieve a better resolution of spectral lines by heating the sample up to 90 °C failed. This is likely to be associated with the statistical character of substitution at different positions of hydroxyl groups and a crowding effect of PEO substituents. Thus, the formula of conjugates may be presented as


Figure 4. Curves of DSC of glass transition of PEG 300 (1) and PEO-β-CD conjugate (preparation 4). (2) The rate of heating is 10 °C/min.

Figure 5. Binding curves for the systems based on SN and R-CD (9), PEO-β-CD (2), PEO-R-CD (preparation 4) (b). Concentration of SN ) 5 × 10-5 M; T ) 297 K.

Additional information concerning the physical state of the conjugates was obtained studying of glass transition behavior and glass transition temperatures Tg (Figure 4). Figure 4 shows the DSC scan for PEG 300. Note that the parent β-CD is a crystalline compound with melting temperature of about -50 °C. As is seen, Tg of PEG 300 is close to Tg of PEO-β-CD conjugate and lies in the range from -75 to -70 °C. At the same time, the temperature interval of transition depending on the degree of cooperativity is much wider for bound polymer chains as compared with that of initial polymer. As follows from Figure 4, the DSC scans of PEG 300 are characterized by both picks of crystallization and melting, whereas the corresponding DSC scans of the conjugates show only the region of glass transition. Complexation Properties of the Conjugates with Sodium 4-Nitrophenolate. The fundamental property of PEO-CD derivatives concerns their complexation ability. In this work, we used two organic compounds SN and CAHT as guest molecules. Upon the addition of R-CD, SN in aqueous solutions exhibits a spectral shift of about 15 mm (11). Figure 5 shows the binding curves for R-CD and for two conjugates with SN. As is seen, an

Topchieva et al.

increase in the concentration of host molecule leads to an abrupt linear increase in the value of optical absorbance, then this value levels off when the guest:host ratio varies in the range from 1:1 to 1:2. One may conclude that (1) the affinity between the ligand and PEO-CD conjugates decreases as compared with that of the system based on parent R-CD; (2) the binding curve for PEOR-CD conjugate shows an S-shaped profile which suggests a far more complex character of binding as compared with that of the native R-CD. As compared to parent R-CD a decrease in the complexation ability of this conjugate is likely to be related to a decrease of the cavity size. The same effect was observed for other types of CD derivatives (12). As was shown in ref 13, the binding constant of β-CD with respect to SN is by 1 order of magnitude lower than that of the system based on R-CD. This is likely to be related to the fact that an increase in the size of β-CD cavity decreases the complex stability. The complexation between β-CD and SN was not identified by differential UV spectroscopy. At the same time, as follows from the binding curve presented in Figure 5, PEO-β-CD forms the complex with SN. For the systems based on PEOβ-CD and R-CD, the comparison between the slopes of linear portions of the corresponding binding curves shows that both hosts are characterized by similar binding constants. Therefore, flexible oligo(ethylene oxide) chains may facilitate the adaptation of guest accommodation because of the additional noncovalent fixation of guests by noncovalent forces. As known, PEO forms inclusion compounds with R-CD (14). It seems important to understand whether selfinclusion of the grafted PEO chains in PEO-R-CD conjugates is possible. Our experiments within the framework of Stuart-Breigleb models show that oligo(oxyethylene) chains grafted to CD core are able to form complexes with one grafted oligo(oxyethylene) chain incorporated in the cavity of the same molecule (autocomplexes). Actually, these structures should show poor complexation properties in respect to the external guest. This conclusion is proved by a low value of the slope of the first portion of the binding curve (Figure 6). Probably, an increase in the affinity of components at a higher concentration of the conjugate is provided by the destruction of autocomplexes and formation of amphiphilic type associates (15). Complexation Properties of the Conjugates with Calcium Acetylhomotaurinate. Taking into account the ability of PEO-CD conjugates for drug targeting (16), it seems interesting to study the complexation between these conjugates and biologically active ligands. As one of these substances, we have choosen CAHT [CH3CONH(CH2)3SO3-)2Ca2+, which is used in the clinic to prevent relapses in weaned alcoholics (17). This ligand is also interesting as a guest molecule containing two anionic moieties in one molecule. Modeling of complexes based on CAHT and R-CD using Stuart-Breigleb models shows that the most stable structure is formed by the incorporation of hydrophobic fragment of CAHT to the cavity of CD, whereas the charged sulfonate groups stay outside of the cycle. For complexes based on β-CD, both of anionic moieties may be incorporated into the cavity. In both cases, the molar stoichiometry of complexes is 1:1. The complexation of CAHT was studied by 1H-NMR spectroscopy. When the guest molecule is accommodated in CD, hydrogen atoms of CAHT located in the channel of CD should be shifted upfield (18). Figure 6 presents 1 H-NMR spectra of free CAHT and its complex with R-CD. As follows from this figure, the character of



Figure 6. 1H-NMR spectra of CAHT (1) and CAHT in the presence of R-CD. (2) Concentration of CAHT ) 1.25 × 10-2 M, concentration of R-CD ) 0.12 M. Table 2. Binding Properties of System Based on CDs and Their PEO Derivatives Toward Calcium Acetylhomotaurinate CDs or their derivatives

Figure 7. Binding curves for the system based on CAHT and β-CD (9), and PEO-β-CD (preparation 4) (b). Concentration of CAHT is 1.25 × 10-2 M, T ) 303 K.

changes in 1H-chemical shifts of methylene and methyl group of CAHT suggests the formation of inclusion complexes. Note that, in the presence of R-CD, the value of 1H-chemical shift of CH3 proton is higher than that of methylene groups. These values were used to estimate of Ka. At the same time, as follows from the spectra of the conjugates, C3-H and C5-H of R-CD are also shifted upfield. However, these shifts cannot be used to estimate Ka of the conjugate because of the complex character of the corresponding 1H-NMR spectrum of CD core. Binding constants Ka were determined from the binding curves (change of 1H-chemical shifts of methyl protons of CAHT, D, as a function of concentration of CDs) by a curve-fitting analysis using eq 1 (Figure 7 and Table 2). For complexes based on R- and β-CD, the comparison between Ka shows that Ka for β-CD-CAHT complex increases by more than 2 orders of magnitude as com-

1

R-CD

2 3

PEO-R-CD β-CD

4

PEO-β-CD

temp K

D ) δ0 - δ1

Ka, 1/mol

313 323 333 313 303 323 343 303 323 343

0.0360 0.0335 0.0320 0.0360 0.0148 0.0150 0.0124 0.0195 0.0148 0.0179

4.6 ( 0.4 3.0 ( 0.2 2.1 (.0.2 2.1 (. 0.1 1800 ( 200 1060 ( 20 860 ( 40 320 ( 20 168 ( 10 63 ( 2

pared this the corresponding constant of R-CD-CAHT. This situation seems to be rather unusual for the complexation of the guests with CDs, which is characterized by a decrease in the Ka with the increase of the size of the cavity (1). This behavior may be explained as follows: β-CD forms a complex with two anionic moieties of CAHT in contrast to R-CD. Both types of PEO-CD conjugates were found to be less effective hosts with respect to CAHT as compared with parent CDs. Upon binding of CAHT with conjugates, no selfinclusion phenomenon was observed. This is likely to be related to the fact that the interval of concentrations of the conjugate was by 2-3 orders of magnitude higher than that of the corresponding systems based on SN. Taking into account the fact that the formation of autocomplexes proceeds at low concentrations, it seems evident that their effect on binding properties of the conjugates is absent at high concentrations of host molecule. As follows from the Table 2, for all systems under study, the values of Ka decrease with increasing of temperature.


Therefore, a facile procedure for synthesis of new derivatives of CD modified with PEO chains was advanced. These compounds present randomly substituted CD molecules with PEO chains predominantly linked at 2 and 6 positions of glucoside units. Note, that PEO chains are characterized by a narrow MMD. Analysis of the binding properties of PEO-modified derivatives of CD reveals some characteristic features of these hosts. In addition, trivial steric effects associated with the decrease in the dimensions of the cavity of CD-PEO chains grafted to the CD were shown to assist the adaptation due to an additional noncovalent fixation of the ligands in polymer surrounding. The application of biologically active compound CAHT composed of two anionic moieties allows one to identify new types of topologic structures of inclusion complexes of CAHT with CD and PEO-CD conjugates. These investigations provide the basis for a new strategy of targeting of this neuroleptic to brain (16). The conjugates may be used as potent drug delivery systems. ACKNOWLEDGMENT

We would like to thank Prof. A. S. Shashkov for his valuable assistance in recording and interpretation of NMR spectra, Dr. T. E. Grokhovskaya, F. A. Kalashnikov, and E. I. Popova for their assistance in experimental work and Prof. V. I. Sokolov and Prof. B. I. Kurganov for their interest and fruitful discussion. This work was supported by the Russian Foundation of Basic Research (Project 93-03-33519a) and International Science Foundation (MPE 300). LITERATURE CITED (1) Bender, M. L., and Komiyama, M. (1978) Cyclodextrin Chemistry, Springer-Verlag, Berlin, Heidelberg, New York. (2) Szejtly, J. Cyclodextrins and their inclusion complexes, Akademiai Kiado, Budapest. (3) Nozaki, T., Maeda, Y., Ito, K., and Kitano, H. (1995) Cyclodextrins modified with polymer chains which are responsive to external stimuli. Macromolecules 28, 522-524. (4) Canceill, J., Jullien, L., Lacombe, L., and Lehn, J.-M. (1992) Channel type molecular structures. Part 2. Synthesis of bouquet-shaped molecules based on b-cyclodextrin core. Helv. Chim. Acta 75, 791-812. (5) Topchieva, I. N., Elezkaya, S. V., Polyakov, V. A., and Karezin, K. I. (1996) Cyclodextrins modified with poly-

Topchieva et al. (ethylene oxide) as complexation and transport agents. Abstracts of the 8th International Symposium on Cyclodextrins, Budapest, Hungary, 2-20. (6) Mischnick, P. (1989) Determination of the patterns of substitution of hydroxyethyl and hydroxypropyl-cyclomaltoheptaoses. Carbohydr. Res. 192, 233-241. (7) Tahan, M., and Zilkha, A. (1969) Anionic graft polymerization of ethylene oxide on starch. J. Polym. Sci. 7, A-1, 18151824. (8) Enyeart, C. R. (1966) Polyoxyethylene alkylphenols. in Nonionic detergents (M. J. Schick, Ed.) p 62, Marcel Dekker, New York. (9) Ke, B. Ed. (1964) Newer methods of polymer characterization p 370, Interscience Publishers, New York. (10) Trinadha Rao, C., Lindberg, B., Lindberg, J., and Pitha, J. (1991) Substitution in β-cyclodextrin directed by basicity: preparation of 2-O- and 6-O-[(R)- and (S)-2-hydroxypropyl] derivatives. J. Org. Chem. 56, 1327-1329. (11) Cramer, F., Saenger, W., and Spatz, H.-Ch. (1967) Inclusion compounds. XIX. The formation of inclusion compounds of a-cyclodextrin in aqueous solutions. Thermodynamics and kinetics. J. Am. Chem. Soc. 89, 14-20. (12) Suzuki, J., Sakurai, Y., Ohkubo, M., Osa, T., and Ueno, A. (1994) Molecular recognition ability of cyclodextrin derivatives modified at secondary hydroxyl side. Proc. 7th Int. Symp. Cyclodextrins Tokyo, Japan, pp 222-225. (13) Tabushi, J., Shimizu, N., Sugimoto, T., Shiozuka, M., and Jamamura, K. (1977) Cyclodextrin flexible capped with metal ion. J. Am. Chem. Soc. 99, 7100-7102. (14) Harada, A., Li, J., Kamachi, M. (1993) Preparation and properties of inclusion complexes of poly(ethylene glycol) with a-cyclodextrin. Macromolecules 26, 5698-5703. (15) Topchieva, I. N., Elezkaya, S. V., Polyakov, V. A., and Say, S.B. (1993) Conjugates of cyclodextrins with poly(ethylene glycol) as complexation agents and emulgators and the method of their synthesis. Russian patent 93043686. (16) Alyautdin, R. N., Petrov, V. E., Elezkaya, S. V., Polyakov, V. A., and Topchieva, I. N. (1993) Transport of neuroleptics to the brain. Russian patent 93043262. (17) Zeise, M. L., Kasparov, S., Capogna, M., Zieglgansberger, W. (1993) Acamprosate (calciumacetylhomotaurinate) decreases postsynaptic potentials in the rat neocortex: possible involvement of excitory amino acid receptors, Eur. J. Pharm. 231, 47-52. (18) Connors, K. A. (1997) Stability of cyclodextrin complexes in solution. Chem. Rev. 97, 1325-1357.

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Novel Derivatives of Cyclodextrins, Modified with Poly(ethylene Oxide

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