pubs.acs.org/Langmuir © 2010 American Chemical Society
Complexation between r-Cyclodextrin and PEGylated-PAMAM Dendrimers at Low and High pH Values Salim Khouri and Kam C. Tam* Department of Chemical Engineering, Waterloo Institute for Nanotechnology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada N2L 3G1 Received August 17, 2010. Revised Manuscript Received October 1, 2010 Poly(ethylene glycol)-grafted-poly(amido amine) (PEGylated-PAMAM) dendrimers have attracted increasing amounts of attention because of their improved stability, toxicity, and better particle drug leakage property. The complexation of R-cyclodextrin (R-CD) with grafted PEG segments on the surface of PAMAM dendrimers was elucidated by light scattering and titration calorimetry. At pH 10, complexation between R-CD and PEGylatedPAMAM occurred once R-CD was titrated into the PAMAM solution. We observed for the first time a unique phenomenon at pH 2, where no binding took place until a critical R-CD concentration (C*) of ∼8.0 mM was reached. The size of the nanostructures increased from 6.7 to 57.6 nm when the R-CD concentration was increased from 0.5 to 15 mM at pH 2. The zeta potential of PEGylated-PAMAM at pH 2 was þ6.7 mV. Thus, the dendrimers possessed positive charges attributed to the protonation of primary amine groups on PAMAM chains that impart electrostatic repulsive forces to the system. The morphology of the complex is expected to be different at two different pH values (2 and 10) because the former produces a clear solution and the latter forms a turbid solution with white precipitates.
Introduction The design of polyrotoxanes is one of the most active fields in supramolecular inclusion complexation. To date, a diverse number of polymeric inclusion complexation (PIC) structures via host-guest interactions between cyclodextrins (CDs) and various polymeric systems have been reported.1-8 CDs are cyclic molecules comprising six (R), seven (β), or eight (γ) glucose units linked through R-1-4-glycosidic linkages.9 The average diameters of the cavities of R-, β-, and γ-CD are 4.5, 7.0, and 8.5 A˚, respectively, and the height of each CD’s torus is ca. 7.8 A˚.10-12 They are readily soluble in water, with solubilities of 145 g/L for R-CD, 18.5 g/L for β-CD, and 232 g/L for γ-CD.13-15 The four most important driving forces for the formation of polymer/CD complexes are hydrophobic interactions between host (CD) and guest *To whom correspondence should be addressed. E-mail: mkctam@ uwaterloo.ca. (1) Ceccato, M.; Lo Nostro, P.; Baglioni, P. Langmuir 1997, 13, 2436–2439. (2) Choi, H. S.; Ooya, T.; Lee, S. C.; Sasaki, S.; Kurisawa, M.; Uyama, H.; Yui, N. P. H. Macromolecules 2004, 37, 6705–6710. (3) Lee, S. C.; Choi, H. S.; Ooya, T.; Yui, N. Macromolecules 2004, 37, 7464– 7468. (4) Huh, K. M.; Tomita, H.; Ooya, T.; Lee, W. K.; Sasaki, S.; Yui, N. Macromolecules 2002, 35, 3775–3777. (5) Liu, J.; Sondjaja, H. R.; Tam, K. C. Langmuir 2007, 23, 5106–5109. (6) Okada, M.; Kawaguchi, Y.; Okumura, H.; Kamachi, M.; Harada, A. J. Polym. Sci., Part A: Polym. Chem. 2000, 38, 4839–4849. (7) Ni, X.; Cheng, A.; Li, J. J. Biomed. Mater. Res., Part A 2009, 88A, 1031– 1036. (8) Zheng, P. J.; Wang, C.; Hu, X.; Tam, K. C.; Li, L. Macromolecules 2005, 38, 2859–2864. (9) Challa, R.; Ahuja, A.; Ali, J.; Khar, R. AAPS PharmSciTech 2005, 6, E329–E357. (10) Bender, M. L. In Cyclodextrin Chemistry; Komiyama, M., Ed.; Springer-Verlag: Berlin, 1978. (11) Okumura, H.; Okada, M.; Kawaguchi, Y.; Harada, A. Macromolecules 2000, 33, 4297–4298. (12) Sabadini, E.; Cosgrove, T.; Taweepreda, W. Langmuir 2003, 19, 4812–4816. (13) Harada, A. Acc. Chem. Res. 2001, 34, 456–464. (14) Harada, A. Carbohydr. Polym. 1997, 34, 183–188. (15) Wenz, G. Angew. Chem., Int. Ed. Engl. 1994, 33, 803–822. (16) He, L.; Huang, J.; Chen, Y.; Liu, L. Macromolecules 2005, 38, 3351–3355. (17) Loethen, S.; Ooya, T.; Choi, H. S.; Yui, N.; Thompson, D. H. Biomacromolecules 2006, 7, 2501–2506.
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molecules: van der Waals forces, geometrical compatibility between the host and guest, hydrogen bonding, and dipolar interactions.1,3,16,17 Harada and co-workers were the first to report the formation of a PIC between R-CD and poly(ethylene glycol) (PEG).18-20 When PEG was added to a saturated R-CD solution, white crystalline precipitates in high yield were produced. The complexation was confirmed by NMR with a stoichiometric ratio of 2:1 R-CD/PEG; (i.e., two R-CDs are needed to complex with one ethylene oxide (EO) unit).18,19 The formation of the complexes is thought to be promoted by hydrogen bond formation between neighboring cyclodextrins, while adopting head-to-head or tail-to-tail arrangements.13 Similar complexation was observed for other biodegradable polymers and copolymer systems, which may find interesting applications. Li et al. reported on the formation of vesicular structures when R-CD complexed with poly(ethylene glycol)-b-poly(acrylic acid) (PEO-b-PAA) at a high pH of 11.5 The solution remained clear; however, it became turbid when the pH was reduced to 3. They studied the thermodynamics and morphology of the nanostructures using ITC and light scattering techniques. In another study, Yui et al.3 reported the formation of pH-dependent polyrotoxanes formed by the complexation of R-CD and triblock PEI-b-PEG-b-PEI, where a reduction in pH induced repulsion between the cationic polymer chains and R-CDs that hinder the threading of R-CD. Recently, Tu et al.21 studied the self-assembly of well-defined poly(ethylene oxide-b-N-isopropylacrylamide) (PEO-b-PNIPAM) via inclusion complexation with R-CD. They recorded the selective threading of R-CD onto PEO segments that transforms a random coil conformation into a rod/coil structure. Because of the possible uses of CDs in various applications such as controlled drug delivery,22,23 (18) Harada, A. Coord. Chem. Rev. 1996, 148, 115–133. (19) Harada, A.; Li, J.; Kamachi, M. Nature 1992, 356, 325–327. (20) Harada, A.; Nishiyama, T.; Kawaguchi, Y.; Okada, M.; Kamachi, M. Macromolecules 1997, 30, 7115–7118. (21) Tu, C.; Kuo, S.; Chang, F. Polymer 2009, 50, 2958–2966. (22) Loethen, S.; Kim, J.; Thompson, D. H. Polym. Rev. 2007, 47, 383–418. (23) Luo, D.; Haverstick, K.; Belcheva, N.; Han, E.; Saltzman, W. M. Macromolecules 2002, 35, 3456–3462.
Published on Web 11/01/2010
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PEGylated-PAMAM, resulting in the formation of different microstructures.
Materials and Methods
Figure 1. PEGylated-PAMAM dendrimer under various pH conditions.
food processing,24-27 environmental protection,27 and pharmaceutical formulations,28 the study and formation of these supramolecular structures are significant. Most of the investigations on CD ICs concentrated on welldefined copolymeric systems that are designed and synthesized using techniques, such as ATRP, cationic/anionic polymerization, and so forth. In this study, we examined a PEG-grafted dendritic structure that was prepared by the Michael addition reaction. Modified PAMAM dendrimers can be used as drug carriers,29,30 gene carriers,31 antiviral agents,32 contrast agents,33 and nanoscale catalysts for industrial processes.34 The uniqueness of dendrimers stems from the fact that they are robust, well-defined, and highly branched globular structures, making them flexible systems for various applications. The synthesis of PEGylated-PAMAM dendrimers is generally based on a condensation reaction between PEG and PAMAM as previously reported in several communications.23,35,36 PAMAM dendrimers possess primary, secondary, and tertiary amines, where the pKa value of primary amine groups is about ∼9.2 and that of tertiary amines is ∼6.6. At low pH (e.g., e2), PAMAM would swell and entrap more molecules within the cavity of the core because all of the amine groups are fully protonated. At neutral pH values (7 to 8), studies showed that only the amines on the surface of PAMAM are protonated whereas those in the core are deprotonated because of the fact that their pKa values (∼6.6) are lower than the pH.8,37,38 (Figure 1) In this study, we investigated the complexation of PEGylatedPAMAM dendritic system with R-CD at two extreme pH conditions (2 and 10). The difference in the ionic strength and the pH values has a strong impact on the threading of CD into the (24) Szejtli, J. Chem. Rev. 1998, 98, 1743–1754. (25) Breslow, R. Enzyme Models Related to Inclusion Compounds. In Inclusion Compounds; Atwood, J. L., Davies, J. E., Eds.; Academic Press, Orlando, FL, 1984; Vol. 3, pp 473-501. (26) Szejtli, J. Akad. Kiado, Budapest 1982, 74–87. (27) Ishiwata, S.; Kamiya, M. Chemosphere 2000, 41, 701–704. (28) Duchane, D.; Wouessidjewe, D. Drug Dev. Ind. Pharm. 1990, 16, 2487– 2499. (29) Kojima, C.; Kono, K.; Maruyama, K.; Takagishi, T. Bioconjugate Chem. 2000, 11, 910–917. (30) Kolhe, P.; Misra, E.; Kannan, R. M.; Kannan, S.; Lieh-Lai, M. Int. J. Pharm. 2003, 259, 143–160. (31) Baek, M.; Roy, R. Bioorg. Med. Chem. 2002, 10, 11–17. (32) Wiener, E. C.; Auteri, F. P.; Chen, J. W.; Brechbiel, M. W.; Gansow, O. A.; Schneider, D. S.; Belford, R. L.; Clarkson, R. B.; Lauterbur, P. C. J. Am. Chem. Soc. 1996, 118, 7774–7782. (33) Tomalia, D. A.; Dvornic, P. R. Nature 1994, 372, 617–618. (34) Klajnert, B.; Bryszewska, M. Acta Biochim. Pol. 2001, 48, 199–208. (35) Kim, Y.; Klutz, A. M.; Jacobson, K. A. Bioconjugate Chem. 2008, 19, 1660– 1672. (36) Yang, H.; Morris, J. J.; Lopina, S. T. J. Colloid Interface Sci. 2004, 273, 148–154. (37) Tomalia, D. A. Adv. Mater. 1994, 6, 529–539. (38) Wang, C.; Wyn-Jones, E.; Sidhu, J.; Tam, K. C. Langmuir 2007, 23, 1635– 1639.
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The polymeric materials were used from previously synthesized protocols, where 32 PEG chains were grafted on a generation 3 PAMAM dendrimer (MW of PEG ∼2000 g/mol, MW of generation 3 PAMAM ∼6909 g/mol).39 The amine-terminated generation 3 PAMAM dendrimers were purchased from SigmaAldrich Chemical Co. (>99% purity) and used without further purification. G3-[EDA]-PAMAM-NH2 (Mw = 6909 g/mol) has 32 primary amines on the surface and 30 tertiary amines at the branch points within the core. The synthesized PEG-conjugated PAMAM has an average mole ratio of methylated-poly(ethylene glycol) (MPEG) to PAMAM of 32. It is fully protonated and deprotonated at pH ∼2 and ∼10, respectively, and in the pH range of 7 to 8, only the primary amines on the outermost surface are protonated. R-CD (Mw = 972 g/mol) with 99% purity was purchased from Sigma-Aldrich. All of the solutions were prepared using Millipore water at neutral pH (∼7). Samples were prepared by adding 5, 20, 100, 170, and 300 μL of 150 mM R-CD to 1.5 mL of 0.1 wt % PEGylated-PAMAM to produce 0.5, 2, 9, 15, and 25 mM R-CD solutions. Isothermal Titration Calorimetry (ITC). The calorimetric experiments were carried out using a Microcal isothermal titration calorimeter (VP-ITC, Northampton, MA). This power compensation differential instrument was previously described in detail by Wiseman et al.40 It has a reference cell and a sample cell of 1.35 mL, which are both insulated by an adiabatic shield. Titration was carried out by stepwise injections of different initial R-CD aqueous solutions (40, 70, 80, 90, and 120 mM) from a 250 μL injection syringe into the sample cell filled with 0.1 wt % PEGylated-PAMAM at pH 2 and 10. The syringe is tailor made such that the tip acts as a blade-type stirrer to ensure optimum mixing efficiency at 400 rpm.40 The final concentration of the titrant (R-CD) in the cell was obtained from the expression [Vinjected/(Vinjected þ Vcell)][initial R-CD concentration]. All measurements were carried out at a constant temperature of 25.0 °C, and the calorimetric data were processed with the Origin software supplied with the ITC. Dynamic Light Scattering (DLS). The microstructure of the sample solutions prepared at pH ∼2 was examined using a Brookhaven laser light scattering system that comprises a BI200SM goniometer, a BI-9000AT digital correlator, and analysis software. A 636 nm vertically polarized helium-neon diode laser was used as the light source. The data obtained from DLS measurements in the form of correlation functions were analyzed using the inverse Laplace transform (ILT) technique to obtain the distribution of decay times, τ. The regularized positive exponential sum (REPES) supplied with the GENDIST software package was used, and the reject and grid density probabilities were set to 0.5 and 12, respectively. This decay rate Γ (1/τ) is related to the translational diffusion coefficient D and the wave factor (vector) q according to Γ = Dq2, where q = (4πn/λ) sin(θ/2). The hydrodynamic radius was determined from the Stokes-Einstein relationship given by eq 1 Rh ¼
kT 6πηD
ð1Þ
where k is the Boltzmann constant, T is the absolute temperature, η is the solvent viscosity, and Rh is the hydrodynamic radius. Static Light Scattering (SLS). In SLS, the time-averaged intensity of scattered light was measured at different scattering (39) Lim, A. H. Thesis for Master of Engineering, Nanyang Technology University, 2008; pp 38-48 (40) Wiseman, T.; Williston, S.; Brandts, J. F.; Lin, L. Anal. Biochem. 1989, 179, 131–137.
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Figure 2. Thermograms showing cell feedback (CFB) for (a) pH 2, [R-CD] = 40 mM; (b) pH 10, [R-CD] = 40 mM; (c) pH 2, [R-CD] = 80 mM; and (d) pH 10, [R-CD] = 80 mM. angles. The radius of gyration (Rg) can be determined by plotting I(q) versus q2 according to eq 2. IðqÞ ¼ 1 -
q 2 Rg 2 3
ð2Þ
Results and Discussion The raw cell feedback signals for titrating 40 and 80 mM R-CD into the PEG-PAMAM dendrimers at pH 2 and 10 are shown in Figure 2a-d. Different injection volumes were used, ranging from 2 to 10 μL. Integrating the raw heat under each of the thermograms yielded the enthalpy of interaction between R-CD and dendrimers. Figure 3a,b shows the integrated heat signals with the binding isotherms for the titration of 40 and 80 mM R-CD into 0.1 wt % PEGlyated-PAMAM at pH 2 and 10, respectively. Because the titration of R-CD into water produced a negligible heat change,8 it is apparent that negligible heat was produced for the titration of R-CD into 40 mM PEGylated-PAMAM at pH 2 (filled circles, Figure 3a). Thus, the observed binding enthalpies for 40 and 80 mM PEGylated-PAMAM at pH 10 were attributed to the complexation of R-CD and PEG segments on the PEGylated-PAMAM dendrimer (unfilled circles in Figure 3a,b). As mentioned earlier, the formation of inclusion complexes between a guest and cyclodextrins (CDs) is primarily a result of the hydrophobic interactions between the guest molecule and the relatively hydrophobic cavity of the host; therefore, it is driven by the enthalpic contribution. Langmuir 2010, 26(23), 17969–17974
Figure 3. Differential enthalpy curve vs total R-CD obtained by titrating [R-CD] into a 0.1 wt % solution of PEGylated-PAMAM: (a) [R-CD] = 40 mM and (b) [R-CD] = 80 mM.
The threading of R-CDs onto PEG chains for the 40 and 80 mM PEGylated-PAMAM solutions at pH 10 occurred with the first injection of R-CD solution. At this pH, PAMAM dendrimers DOI: 10.1021/la103287r
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Figure 5. Dependence of the decay rate Γ on the square of the scattering vector, q2, at (O) [R-CD] = 2 mM and (b) [R-CD] = 15 mM.
Figure 4. Calorimetric titration of R-CD into a 0.1 wt % solution
Figure 6. Relaxation distribution functions obtained from DLS measurement at (a) [R-CD] = 2 mM and (b) [R-CD] = 15 mM.
are completely deprotonated and possessed a negligible level of positive charges, thereby permitting the threading of R-CD onto the PEG segments. However, at a low pH of 2, where the PAMAM dendrimers are protonated, R-CD threading occurred only when the R-CD concentration exceeded 8.0 mM, as shown in Figure 3b (filled circles). At pH 2, as more R-CD was injected into the PAMAM solution, the binding progressed to a less-negative ΔH as more R-CD molecules were threaded onto the PEG segments. Similarly, at pH 10, as we increased the concentration of R-CD, the binding between R-CD and PEG segments progressed to completion until it eventually reached the baseline, indicating the completion of the threading process. The results revealed the profound effect of the electrostatic repulsion of positive charges on PAMAM on the threading of R-CD onto PEG chains at pH 2. In both cases, the threading process was exothermic, indicating that the complex formation between PEGylated-PAMAM and R-CD was energetically favorable. The impact of the R-CD concentration on the binding between R-CD and PEG at pH ∼2 can be seen more clearly when the binding thermograms for the titration of different R-CD concentrations into PAMAM dendrimers are compared in Figure 4a,b. The titration of an initial R-CD concentration (40, 70, 80, and 120 mM) into PEGylated-PAMAM at pH 2 showed no interaction until an R-CD concentration of 8.0 mM was reached. At this point, a sufficient number of R-CD molecules are present to induce the threading process, where the cumulative hydrogen bond interactions between the R-CD molecules exceed the electrostatic repulsive forces of the positive charges on the surface and core of the dendritic structure. As shown in Figure 4a, titrating 120 mM requires a lower volume of titrant to initiate binding than
that required for 70 mM because the former has 1.71 times more R-CD molecules, thereby requiring a smaller injected volume and resulting in a shift in the graph to a lower volume. When the data in Figure 4a were replotted with R-CD concentration on the x axis, all of the data collapsed onto a single master curve with a critical transition observable at 8 mM (Figure 4b). Six solutions at pH 2 were prepared with increasing concentrations of R-CD using the method outlined above, and the microstructure was examined using the Brookhaven DLS system. Figures 5 and 6 show the distribution functions for several scattering angles and the decay rate at various angles for low (2 mM) and high (15 mM) R-CD concentrations. From previous studies, the size of PEGylated-PAMAM was found to be around 6.7 nm.23 The decay mode of the PEGylated-dendrimer was contributed by the translational diffusion of the scattering objects in solution. The graph showed a linear relationship between the decay mode, Γ, and wave vector, q2, with R2 = 0.95 and 0.97 for [R-CD] = 2 and 15 mM, respectively. As indicated earlier, q = (4πn) sin(θ/2)/λ, where n is the refractive index of the solvent, θ is the scattering angle, and λ is the wavelength of the incident laser light in a vacuum. By using the Stokes-Einstein equation (eq 1), we can estimate the hydrodynamic radius at both R-CD concentrations. When [R-CD] = 2 mM, the Rh was 6.7 nm (which is identical to the size of an individual PEGylated-PAMAM dendrimer). The size of the PEGylated-PAMAM/CD complex increased to 57.6 nm when [R-CD] was increased from 2 to 15 mM. This increase suggests the impact of R-CD threading on the PEG chains that induces the bridging, association, and aggregation of PEGylated-PAMAM/CD complexes. A plot showing the dependence of the hydrodynamic radius (Rh) and radius of gyration (Rg) on R-CD content for 0.1 wt %
of PEGylated-PAMAM at pH 2. (a) ΔH vs total injected volume. (b) ΔH vs R-CD concentration.
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Figure 7. Particle size of PEGylated-PAMAM at increasing [R-CD] (mM): (a) Rh, (b) Rg, and (c) Rg/Rh.
PEGylated-PAMAM dendrimers at varying R-CD concentrations is shown in Figure 7a,b. The results revealed a significant increase in the particle size with increasing R-CD concentration, which is associated with favorable and stable PEG/R-CD inclusion complexes. As elucidated by previous researchers, the stability of a polypseudorotoxane (PPR) is dependent on the initial concentration of cyclodextrin.41 Therefore, we can reasonably expect that the stability of the assembled aggregates would be greater at higher R-CD concentrations. As mentioned earlier, we believe that at low R-CD concentrations the threading of R-CD is inhibited by the strong electrostatic repulsion from the positive charges on the PEGylated-PAMAM dendrimers. Hence, isolated dendrimers are observed with a size of approximately 6.7 nm. However, when the R-CD concentration was increased from 2 to 25 mM, exceeding the critical concentration of 8.0 mM, R-CD threading onto the PEG chains occurred as evident from the ITC thermogram. Beyond this concentration, the hydrodynamic radius increased by a factor of 8, from 6.7 to 57.6 nm, when the R-CD concentration was increased from 2 to 15 mM. The size of the aggregate increased further to 73.5 nm at [R-CD] = 25 mM because more R-CD molecules are threaded onto the PEG chains, resulting in the bridging of more PEGylated-PAMAM dendrimers. The presence of aggregated cyclodextrin molecules as previously reported by Bonini et al.42 and Rossi et al.43 facilitated the threading of CD onto the PEG chains. The morphological transformation of the dendrimer and the dendrimer/CD complexes can be elucidated from the Rg/Rh data as shown in Figure 7c. The parameter F (Rg/Rh) is commonly used to examine the morphology of the microstructure of aggregates. In the absence (41) Lo Nostro, P.; Giustini, L.; Fratini, E.; Ninham, B. W.; Ridi, F.; Baglioni, P. J. Phys. Chem. B 2008, 112, 1071–1081. (42) Bonini, M.; Rossi, S.; Karlsson, G.; Almgren, M.; Lo Nostro, P.; Baglioni, P. Langmuir 2006, 22, 1478–1484. (43) Rossi, S.; Bonini, M.; Lo Nostro, P.; Baglioni, P. Langmuir 2007, 23, 10959– 10967.
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Figure 8. PEGylated-PAMAM/R-CD complex obtained at (a) pH 2 (clear solution) and (b) pH 10 (turbid solution) at [R-CD] = 20 mM.
of R-CD, Rg/Rh possessed a value of 0.87, which corresponds to a dendritic core with dangling PEG chains. It remained unchanged until the critical R-CD concentration of 8 mM, where it increased sharply to approximately 1.49 at high R-CD concentration. This value is very close to the theoretical values of a Gaussian chain (1.50), confirming that at [R-CD] = 25 mM, interparticle association of R-CD-threaded PEG segments resulted in the formation of a larger aggregate with a Gaussian distribution of polymeric chains. Similar to the results illustrated by other researchers,44 the zeta potential determined for the PEGylated-PAMAM dendrimers at pH 2.2 was þ6.7 mV, which confirmed the presence of positive charges at pH values e2 because most of the amine groups are protonated (Figure 1). With decreasing pH values, the surface of the dendrimer becomes more positive as the core and surface amine groups of PAMAM are increasingly being protonated. Figure 8 illustrates the complete dissolution of the PEGylatedPAMAM/R-CD complex at pH 2 compared to a turbid solution at pH 10. At pH 2, stable PEGylated-PAMAM/R-CD aggregates with a size of less than 100 nm are produced, induced by the balance of electrostatic repulsive forces on the dendrimers and hydrogen bond/hydrophobic forces of the PEG/R-CD (44) Zhu, S.; Hong, M.; Zhang, L.; Tang, G.; Jiang, Y.; Pei, Y. Pharm. Res. 2010, 27, 161–174.
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Scheme 1. Schematic Diagram Showing the Mechanism Nanostructures at pH 2 Induced by the Complexation between PEO Segments and r-CD
chains (Scheme 1). However at pH 10, the dendrimers are neutral37,45 and the hydrogen bonds between the amine and ether groups of the threaded R-CDs result in the formation of larger insoluble aggregates as revealed by the white precipitates. It is important to note that interactions other than hydrogen bonding and the hydrophobic effect, such as spatial dielectric anisotropy, dipolar interactions, and conformational changes, could also play a vital role in the assembly and aggregation of these nanostructures.41 The mechanism that summarizes the threading and association of the PEGylated-PAMAM/R-CD complex at pH 2 is illustrated in Scheme 1. At C
