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First Example of Supramolecular Assemblies in Water of New Amphiphilic Glucose-persubstituted Poly(amidoamine) Dendrimers A. Schmitzer,† E. Perez,† I. Rico-Lattes,*,† A. Lattes,† and S. Rosca‡ Laboratoire des IMRCP, UMR CNRS 5623, Universite´ Paul Sabatier, 118 Route de Narbonne, 31062 Toulouse Cedex, France, and Universitatea “Politehnica” Bucuresti, DSI-FF 313 Splaiul Independentei, 77206 Bucuresti, Romania Received October 7, 1998. In Final Form: March 22, 1999
We described in this work new glucose-persubstituted poly(amidoamine) dendrimers. The physicochemical properties of these new amphiphilic dendrimers were investigated in water, showing that these new amphiphilic dendrimers were able to solubilizate increasing quantities of hydrophobic compounds (pyrene or aromatic ketones) in water, depending on the number of microcavities in each dendrimer. These dendrimers were observed to behave like unimolecular micelles. For the first time the formation of aggregates of these carbohydrate dendrimers in superstructures could be observed by electron microscopy and light scattering. Moreover, it is the first example of asymmetric induction in the “pseudomicellar” solution of a chiral amphiphilic dendrimer: therefore, asymmetric reduction of the prochiral aromatic ketone (benzoylcyclohexane) was achieved by NaBH4 with a chemical yield of 95% and an enantiomeric excess of 50%.
Introduction Dendrimers,1 the most highly branched functionalized molecules in existence, are attracting considerable attention in chemistry. Dendrimers are macromolecules with morphological architectures differing from the classical micelles which are formed by intermolecular aggregation. The architectural features of dendrimers include their precise constitutions with high overall symmetries, their well-defined internal cavities, and their nanometer dimensions. One function of biological systems arousing substantial current interest is self-assembly.2 The first reports of selfassembled dendritic structures generally focused on the association of hydrophobic subunits in aqueous solutions.3 Zimmerman and co-workers, however, described a discrete hexameric structural assembly of dendritic wedges which was held together by directional hydrogenbonding interactions.4 One application of assembled structures is the exploitation of their liquid-crystalline properties. Recently, saccharides have been used to functionalize these dendrimers, leading to amphiphilic dendrimers with polar sugar heads. The synthesis of several saccharide residues attached at the periphery of preformed poly(amidoamine) (PAMAM) dendritic cores5 has been reported by Aoi et al.;6 in their work, PAMAM dendrimers conjugated with 48 carbohydrate residues were prepared from 4-O-(β-D-galactopyranosyl)-D-gluconic acid. Recently, * To whom correspondence should be addressed. Fax: (33) 5 61 25 17 33. E-mail:
[email protected]. † Universite ´ Paul Sabatier. ‡ Universitatea “Politehnica” Bucuresti. (1) Flory, P. J. J. Am. Chem. Soc. 1941, 63, 3083, 3091, and 3096. (2) Lindsey, J. S. New J. Chem. 1991, 15, 153-180. (3) Newkome, G. R.; Lin, X.; Yaxiong, G. J. Org. Chem. 1993, 58, 3123-3129. (4) Zimmermann, S. C.; Zeng, F.; Reichert, D. E. C.; Kolotuchin, S. V. Science 1996, 271, 1095-1098. (5) (a) Tomalia, D. A.; Naylor, A. M.; Goddart, W. A. Angew. Chem., Int. Ed. Engl. 1990, 29, 138. (b) Fre´chet, J. M. J. Science 1994, 263, 1710. (6) Aoi, K.; Itoh, K.; Okada, M. Macromolecules 1995, 28, 5391.
Lindhorst and Kieburg7 have described a number of cluster glycosides, also starting from PAMAM dendrimers. It is certain that dendrimers may incorporate functional groups (like sugars, for example) suitable for molecular recognition and catalysis. As in biology, supramolecular glycodendrimer assemblies will display distinct functions and properties that are absent in the individual molecular building block. Moreover, a series of papers on the chemical modification of dendrimers,8 aimed at the preparation of biologically active glycodendrimers, has been published by Roy and co-workers.9a-c We have used the hydrophobic character of the poly(amidoamine) dendrimers in the preparation of a new class of amphiphilic glycodendrimers. We now present our results on the modification of PAMAM dendrimers involving the multiple attachment of glucose units to the peripheral primary amine groups, with amide bonds as glucose-dendrimer linkages. As our previous work on the synthesis of new glycopolymers10 indicated, the approach combines commercially available dendrimers, simple glucose derivatization (lactone), and standard coupling techniques to yield well-defined structures (Figure 1). The isolation and characterization of glucose-containing dendrimers (Chart 1) are described in this paper. The glucose-persubstituted dendrimers were studied by the point of view of the structure of the macromolecule. Moreover, the physicochemical and chemical properties of these new amphiphilic dendrimers were evaluated in water. (7) Lindhorst, T. K.; Kieburg, C. Tetrahedron Lett. 1997, 38 (22), 3885. (8) Jayaraman, N.; Nepogodiev, S. A.; Stoddart, J. F. Chem. Eur. J. 1997, 3 (8), 1193. (9) (a) Zanini, D.; Park, W. K. C.; Roy, R. Tetrahedron Lett. 1995, 36 (41), 7383. (b) Roy, R.; Park. W. K. C.; Wu, Q.; Wang, S. Tetrahedron Lett. 1995, 36 (25), 4377. (c) Roy, R.; Zanini, D. J. Chem. Soc., Chem. Commun. 1993, 1869. (10) Puech, L.; Perez, E.; Rico-Lattes, I.; Bon, M.; Lattes, A. New J. Chem. 1997, 21, 1229.
10.1021/la981402b CCC: $18.00 © 1999 American Chemical Society Published on Web 05/27/1999
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Figure 1. Attachment of glucose units to the peripheral primary amine groups on the G(3)G PAMAM dendrimers.
Experimental Section 1. General Methods. 1H NMR spectra were recorded on either a Bruker AC200 (200 MHz) spectrometer or a Bruker AC400WB (400 MHz) spectrometer with either the solvent reference or tetramethylsilane (TMS) as the internal standard. 13C NMR spectra were recorded on a Bruker AC200 (50 MHz) spectrometer or a Bruker AC400WB (100.6 MHz) spectrometer.
Thin-layer chromatography (TLC) was carried out on aluminum sheets coated with Kieselgel 60 F254 (Merck). The plates were inspected under UV light and developed with mixture “MoCe” (a mixture of 900 mL of water, 100 mL of H2SO4, 25 g of MoNH4, and 10 g of CsSO4NH4) at 120 °C. Molecular Simulation. Simulations were carried out using the COSMIC force-field as implemented in PIMMS (Oxford Molecular) running on a Silicon Graphics Indigo 2 workstation. The
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Chart 1
Figure 2. Computer-assisted molecular simulations for G(2)G.
Table 1. Structural Data Calculated for the Glucose-Persubstituted PAMAM Dendrimers generation of glucose-persubstituted PAMAM dendrimers no. of glucose units approx. diameter (nm)a
0
1
2
3
4 2.4
8 3.2
16 4.8
32 8.0
a Measured from the centroid of the ethylenedimaine core to the furthest point on the periphery of the dendrimer surface.
whole assembly was minimized using the conjugated gradients method. Microanalyses were carried out at the microanalysis center of the Ecole Nationale Superieure de Chimie de Toulouse (Toulouse, France). Size-exclusion chromatography (SEC) was taken with a highperformance liquid-chromatograph apparatus (three columns, Waters-Shodex SB802HQ, SB802.5HQ, SB804HQ; solvent, water at room temperature, and poly(oxyethylene) as the standard). 2. General Procedure for Coupling the Poly(amidoamine) Dendrimers (PAMAM Dendrimers) with D-Glucono1.5-lactone. Chemicals, including D-glucono-1.5-lactone and PAMAM dendrimers (generations 0-3), were purchased from Aldrich. The PAMAM dendrimer samples (all generations) used in this paper were stored in a methanol solution. All solvents were obtained from Aldrich and used without further purification. The solvents were preserved with molecular sieves (4 Å). In a two-necked, round-bottomed flask equipped with a thermometer and a magnetic stir bar was introduced in 5 mL of dry DMSO under argon 7.25 × 10-4 mol of PAMAM dendrimers (generation n, n ) 1, 2, 3). A 10% molar excess of 2n+2 × 7.25 × 10-4 mol of D-glucono-1.5-lactone in 3 mL of DMSO was added to the solution by a syringe with stirring. The mixture was stirred
Figure 3. SEC chart of G(3)G (refractive index detector, eluent water, 25 °C). at 40 °C for 24 h under an argon atmosphere. The reaction was followed by TLC using CHCl3/CH3OH (80/20, v/v) as the eluent. The solution was poured into an amount of propan-2-ol (IPA). The azeotrope formed by DMSO with IPA was removed under vacuum. Glucose-persubstitued poly(amidoamine) was isolated after vigorous washing of the reaction mixtures with methanol (these compounds showed hydrophilic properties and were soluble in water and DMSO; they were insoluble in methanol and chloroform, which dissolved d-glucono-1.5-lactone). The precipitate was lyophilized to yield a white powder. The linking of the sugars is achieved by amide bond formation, provided that the reaction proceeds quantitatively or nearly so (G(0)G yield 94%, G(1)G yield 95%, G(2)G yield 93%, G(3)G yield 96%). The compounds exhibit versatile high hygroscopic behavior shown by elemental analyses: Anal. Calcd for G(1)G, C110H208N26O60‚30H2O: C, 42.41; H, 7.96; N, 10.73; O, 38.91. Found: C, 42.91; H, 7.92; N,10.16; O, 39.01. Anal. Calcd for G(2)G, C238H448N58O124‚27H2O: C, 43.36; H, 7.67; N, 12.32; O, 36.64. Found: C, 43.32; H, 7.57; N, 10.96; O, 38.15. Anal. Calcd for G(3)G, C494H928N122O252‚55H2O: C, 43.8; H, 7.74; N, 12.4; O, 36.8. Found: C, 43.64; H, 7.91; N, 11.6; O, 36.8. 3. Physicochemical Methods. (i) Fluorescence Methods. Pyrene fluorescence spectra were recorded with a PTI QM1 spectrofluorimeter (Photon Technology International Quantum Master 1). All measurements were performed at 25 °C. For pyrene the excitation wavelength was set at 335 nm. All solutions were made with pyrene-saturated water (pyrene concentration ca. 6 × 10-7M)
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Figure 4. Variation of the pyrene solubility with dendrimer concentration at 25 °C. unless otherwise specified. It should be mentioned that aqueous solutions of all sugar-dendrimers produce a weak fluorescence around 430 nm. We have always subtracted from the fluorescence of pyrene the fluorescence of appropiate blanks containing the same sugar-dendrimer concentration as the samples. All of the solutions, either in pure water or in sugar-dendrimer aqueous solutions, were prepared by allowing the solvent to stay overnight undisturbed and in contact with the solute at 40 °C. The maximum concentration of pyrene in water was found to be equal to 6 × 10-7 M. (ii) UV spectra were recorded with a Hewlett-Packard HP 8452A diode-array UV-visible spectrophotometer, which is a single beam microprocessor controlled with collimating optics (wavelength range, i.e., 190-820 nm). All of the solutions, either in pure water or in sugar-dendrimer aqueous solutions, were prepared by allowing the solvent to stay overnight undisturbed and in contact with the solute at 40 °C. (iii) Transmission Electron Microscopy. Aqueous solutions of glucose-persubstituted dendrimers (2, 3, and 5 wt %) were applied on carbon-coated Formvar grids, negatively stained with a 2% solution of uranyl acetate and examined in a JEOL JEM 200CX electron microscope operating at 200 kV. (iv) Dynamic Light Scattering. Measurements were performed on a Coulter N4MD, suitable for samples containing particles from 1 to 10 000 nm. 4. General Procedure for Reduction of Prochiral Ketones. A glucose-dendrimer aqueous solution with cyclohexylphenyl ketone in excess was allowed to stay for 12 h undisturbed at 40 °C. The maximum concentration of ketone in a 10-2 M aqueous solution of glucose-persubstituted PAMAM dendrimers (generation 3) was equal to 10-4 M. Sodium borohydride (22 mg) was added to 10 mL of this saturated solution. The mixture was stirred for 2 h, followed by two successive extractions with CCl4. After evaporation of the solvent, the cyclohexylphenylcarbinol was obtained at a 95% chemical yield and analyzed. The enantiomeric excess (ee 50%) of the product was determined by optical rotation (the optical rotation and configuration of the optically pure alcohol was taken as to the literature [R]20D(max) value: -68.1 (c ) 0.118, C6H6) for (S)-(-)-cyclohexylphenylmethanol) and by gas chromatography with a chiral separation column (SUPELCO R DEX 120 fused silica capillary column of 30 m × 0.25 mm × 0.25 µm film thickness).
Results and Discussion These new amphiphilic dendrimers possess numerous advantages over conventional micelles. First, the glucosepersubstituted dendrimers which are formed by covalent bonds have greater structural stability than conventional micelles. Second, depending on the synthetic strategy, a high degree of control of both molecular weight and shape is achieved, leading to precise compositions and constitutions of these unimolecular entities. I. Structural Characterization. NMR Spectroscopy. Evidence for the introduction of glucose moieties at the surface of the dendrimers was forthcoming from inspection of the NMR spectra of the coupled products. In all cases, the 13C NMR spectra are more diagnostic of the products than are the 1H NMR spectra. In the 13C NMR spectrum of the PAMAM dendrimers in D2O, signals due to R- and β-methylene carbons of terminal amino groups appeared at 41.9 and 41.2 ppm, respectively. No signal was observed in this region in the spectra of the glucose-persubstituted PAMAM. Glucosamide formation was shown by the appearance of a new carbonyl carbon signal at 179 ppm as shown below:
1 H NMR (D2O, 400 MHz) for G(n)G: 4.25 (d); 4.02-3.67 (m); 3.3 (m); 2.84-2.7 (m); 2.65 (m); 2.4 (m). 13 C NMR (D2O, 100.6 MHz) for G(n)G: δ 179 (carbonyl carbon of glucosamide); 175.4 and 174.9 (carbonyl carbons of the PAMAM dendrimer); 74.4, 73.7, 72.5, 71.4, 70.7, and 62.9 (other carbons derived from the sugar); 51.5 (b); 49.3 (c); 39 (f); 38.4 (e); 37.1 (a); 32.9 (d). Molecular Modeling Studies. In an attempt to gain some insight into the nature of the three-dimensional structures
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Figure 5. Variation of the benzoylcyclohexane solubility with dendrimer concentration at 25 °C.
for these amphiphilic glucose-persubstituted dendrimers, we modeled these compounds. The structural data obtained from these preliminary studies are provided in Table 1. Molecular simulations give some information concerning the key role of the degree of generation on molecular shape, morphology, surface, and cavities structure (Figure 2). Specifically, according to these molecular simulations, “low” generations of glucose-persubstituted PAMAM (generations 0-2) possess a highly asymmetric, open “starfish-like” shape, while the “high” generations have a nearly spherical shape and present a more closed and densely packed structure from the third generation. Size-Exclusion Chromatography. Mw/Mn values were determined by size-exclusion chromatography. Figure 3 shows SEC profiles of glucose-persubstituted PAMAM generation 3. The sharp unimodal peak indicates the absence of unsubstituted PAMAM dendrimers and shows an unexpected high molecular mass peak. The results suggested that aggregation of all generations of glucosepersubstituted PAMAM was occurring in water even at low working concentration (10-4 mol/L). These results have been confirmed by the following physicochemical studies. II. Physicochemical Studies of the Amphiphilic Glucose-Persubstituted Dendrimers. We have focused our studies on the glucose-persubstituted PAMAM generations 0-3 in order to elucidate the ability of these dendrimers to interact with guest organic molecules,11,12 their binding capabilities, and the nature of their interior cavities.13
Figure 6. Variation of the pyrene I1/I3 ratio with dendrimer generation and concentration.
UV-Vis Absorption Spectroscopy. Glucose-persubstitued dendrimers, which look like unimolecular micelles, were expected to increase the solubility of water insoluble or weakly soluble organic compounds. In the present study, we have examined, by UV-vis absorption spectroscopy, aqueous glucose-substituted dendrimer solutions of generations 1-3 saturated with pyrene and benzoylcyclohexane. The concentration of the maximum solubilized organic compounds was found to increase linearly with the generation and the concentration of dendrimer, expressed (11) Pistolis, G.; Malliaris, A.; Paleos, C. M.; Tsiourvas, D. Langmuir 1997, 13, 5870. (12) Naylor, A. M.; Goddard, W. A.; Kiefer, G. E.; Tomalia, D. A. J. Am. Chem. Soc. 1989, 111, 2339. (13) Watkins, D. M.; Sayed-Sweet, Y.; Klimash, J. W.; Turro, N. J.; Tomalia, D. A. Langmuir 1997, 13, 3136.
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Figure 7. Aggregation of G(3)G observed by transmission electron microscopy.
in terms of the number of microcavities in each dendrimer, according to the recent work of Pistolis and co-workers11 (Figure 4 for pyrene and Figure 5 for benzoylcyclohexane). Dendrimers in this family are of particular interest because they mimic unimolecular micelles with sugar heads, leading to a large polar and chiral interface that can be used in asymmetric synthesis. Fluorescence. To investigate the micropolarities of the solubilization sites of these dendrimers, we used the I1/I3 ratio of the fluorescence intensity of the first to the third vibrational peak of pyrene. Thus, G(0)G has only four repeat units and consequently possesses the smallest radius and a very open structure which appears unable to protect pyrene molecules from the aqueous phase. The results are rationalized in terms of water penetration into the dendrimer microcavities (Figure 6), according to the work of Pistolis and co-workers,11 in the absence of sugars in the periphery of the dendrimer. The glucose-persubstituted PAMAM generations 0 and 1 possess an open structure, whereas the generations 2 and 3 provide better water protection to the solubilized pyrene molecules. These results agree with the theory given by computer-assisted molecular simulations. The trend across the generation series exhibits a sharp increase at generation sizes beyond G(3)G. This breaking point indicates that this structure allows the amount of pyrene to access the hydrophobic core. The structure becomes more tightly packed. Note that the values of I1/I3 in G(0)G and G(1)G are close to those observed in pure water (1.5-1.6), whereas in G(2)G and G(3)G I1/I3 has a value close to that in aqueous micelles (1-1.4). Transmission Electron Microscopy. Moreover, we expected that the glucose-persubstituted dendrimers had potential aggregation properties in aqueous media arising from their distinctive dual-natured structure, i.e., an external periphery bearing multiple functional groups for solubility in aqueous media, combined with an internal hydrophobic core. Therefore, we investigated these aggregation properties in water by transmission electron microscopy. To our knowledge it is the first time that a self-organization of dendrimers to form aggregates in water has been observed (Figure 7). Size distributions of all glucose-persubstituted PAMAM generations were confirmed according to dynamic light
Table 2. Size Distribution of Assemblies of Glucose-Persubstituted PAMAM Dendrimers by DLS and Electron Microscopy Generation of glucose-persubstituted PAMAM dendrimers D (nm) by DLS D (nm) by electron microscopy
0
1
2
3
46-264 80-340
100-464 130-600
215-1000 300-600
464-2150 800-2000
scattering carried out on samples in water at room temperature. The aggregates showed a polydisperse size distribution with diameters ranging from about 100 to 2000 nm; therefore, there is no doubt that these objects fall into the size of assemblies consisting of several molecules (Table 2). We propose that this aggregation phenomenon is due to the intermolecular hydrogen bonds between the sugar moieties which persist in aqueous solutions. This phenomenon is well-known in biology with glycoconjugates, for example.14 III. Study of the Amphiphilic Glucose-Persubstituted Dendrimers in Chemical Reactivity. To investigate the subtle effects related to the conformational rigidity of glucose end groups and hydrophobic cavities of these dendrimers, we have studied the asymmetric reduction of aromatic prochiral ketones in water solutions of glucose-persubstituted dendrimers by sodium borohydride. For the first time, we have chosen the third generation dendrimer G(3)G, which is the first generation G(n)G leading to a nearly spherical shape similar to that of a micelle and presenting a more closed and densely packed structure than previous generations G(0)G to G(2)G. It is anticipated that although the reaction takes place at the chiral interface of the aggregates, the stereoselectivity of the reduction might be effected. The concentration of maximum solubilized benzoylcyclohexane in a 10-2 M aqueous solution of G(3)G dendrimer was found to be equal to 10-4 M. Optically active alcohol obtained in high chemical yield (95%) showed satisfactory NMR characteristics, and the enriched enantiomer of product has the S configuration (ee 50%). (14) Ricoul, F.; Dubois, M.; Belloni, L.; Zemb, T.; Andre-Barres, C.; Rico-Lattes, I. Langmuir 1998, 14 (10), 2645 and references herein.
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Conclusion In this work, we presented a new series of amphiphilic dendrimers involving the multiple attachment of glucose units to the peripheral amine groups by amide linkage. These preliminary results presented above showed that the glucose-persubstituted dendrimers were able to selectively “catalyze” the reduction of prochiral ketones. Moreover, these results are better than all of the results previously observed in asymmetric induction in direct or reverse micelles (the ee varied from 1.7% to 27% for reduction of aromatic ketones,15,16 with enantioselectivity no more than 10% for oxidation of sulfides.17 One important reason for all of these failures is the dynamic of the micelles leading to a chiral interface that is not rigid enough. These new amphiphilic dendrimers could therefore be considered rigid unimolecular micelles, able to provide stable asymmetric complexes. (15) Goldberg, S. I.; Baba, N.; Green, R. L.; Pandiar, R.; Stowers, J. J. Am. Chem. Soc. 1978, 100, 6768. (16) Zhang, Y.; Sun, P. Tetrahedron: Asymmetry 1996, 7 (1a), 3055 and references herein. (17) Fan, W.; Zhou, Q.; Zhang, Y.; Shen, J.; Lu, P. Acta Chim. Sin. 1987, 45, 287.
These new amphiphilic dendrimers are able to solubilizate in water increasing quantities of pyrene or aromatic ketones (hydrophobic compounds), with the generation and the concentration of dendrimer depending on the number of microcavities in each entity. Therefore, these results show that these amphiphilic molecules act as unimolecular micelles. Moreover, we have been able to show, for the first time, that these glycodendrimers aggregated in water to form superstructures, probably by intermolecular hydrogen bonds between the sugar moieties which persist in aqueous solution, as is observed in biological systems. These superstructures were used for studies in the reduction of benzoylcyclohexane by sodium borohydride in an aqueous solution of glucose-persubstituted dendrimers (G(3)G). Work is now in progress to develop this new concept in asymmetric synthesis. LA981402B
