Analyte Templating: Enhancing the Enantioselectivity of Chiral

Anal. Chem. 2005, 77, 5009-5018

Analyte Templating: Enhancing the Enantioselectivity of Chiral Selectors upon Incorporation into Organic Polymer Environments Elena Gavioli,† Norbert M. Maier,*,† Karsten Haupt,‡ Klaus Mosbach,§ and Wolfgang Lindner†

Institute of Analytical Chemistry, University of Vienna, Wa¨hringerstrasse 38, A-1090 Vienna, Austria, Department of Bioengineering, Compie` gne University of Technology, UMR CNRS 6022, BP 20529, 60205 Compie` gne Cedex, France, and Swiss Federal Institute of Technology, Biotechnology, ETH Ho¨nggerberg HPT E 77, CH-8093 Zu¨rich, Switzerland

A simple strategy for preserving and enhancing the chiral recognition capacity of polymer-embedded chiral selectors is proposed, capitalizing on a temporary blockage of the receptor binding site with tightly binding analytes during the polymerization process. We demonstrate that the copolymerization of a quinine tert-butylcarbamate selector monomer with chiral (and achiral) 3,5-dichlorobenzoyl amino acids allows one to control to a certain extent the binding characteristics of the resultant polymeric chiral stationary phases. The structural and stereochemical requirements of the templating analytes for maximizing the chiral recognition capacity of the polymer-embedded selectors are probed. The chromatographic chiral recognition characteristics of the analyte-templated polymeric chiral stationary phases are analyzed with respect to binding capacities and affinities and compared to those obtained with a conventional silica-based surface-grafted reference material. Changes in substrate-specific enantioselectivity originating from analyte templating are also addressed. The vast majority of the commercially available chiral stationary phases for direct enantiomer separation comprises chiral selectors grafted or coated onto particulate silica-type supports.1 Silica-based materials offer many desirable features for chromatographic applications, such as mechanical stability, resistance to swelling, and high efficiency. In addition, they can be precisely tailored with respect to particle and pore characteristics, and a well-established repertoire of (silane-based) surface chemistry is available for selector immobilization.2 However, silica-based chiral stationary phases suffer from several inherent drawbacks.3,4 These include pronounced instability in acidic and basic media, restricting their working range to pH 2-8. Generally, even densely ligand-grafted * Corresponding author. E-mail: [email protected]. Phone: ++431-4277-52373. Fax: ++43-1-4277-9523. † University of Vienna. ‡ Compie`gne University of Technology. § Swiss Federal Institute of Technology. (1) Piras, P.; Roussel, C.; Pierrot-Sanders, J. J. Chromatogr., A 2001, 906, 443458. (2) Scott, R. P. W. Silica Gel and Bonded Phases. Their Production, Properties and Use in LC; Wiley: Chichester, 1993. (3) Buchmeiser, M. R. J. Chromatogr., A 2001, 918, 233-266. (4) Claessens, H. A.; van Straten, M. A. J. Chromatogr., A 2004, 1060, 23-41. 10.1021/ac050407s CCC: $30.25 Published on Web 06/09/2005

© 2005 American Chemical Society

materials display a considerable number of residual, noncapped silanol functions, giving rise to nonspecific interactions and rendering the surfaces vulnerable to attack by network-degrading agents. These silica-specific limitations may be circumvented by the use of organic polymeric supports for CSP production.3,5 Not only do organic polymers exhibit stability over the entire pH range, but they also show an enhanced chemical inertness to aggressive media. Polymers are amenable to a variety of surface chemistries, facilitating ligand immobilization and suppression of nonspecific binding.3,5,6 A particularly attractive feature of organic polymers is the ease by which enantiomer separation media of different shapes can be accessed.7 Copolymerization of appropriate selectortype monomers with cross-linking agents and comonomers may be used to shape chiral recognition materials in the form of selfsupporting beads,8-11 membranes,12 and porous monoliths;3,13,14 considerably broadening the scope of applications compared to conventional silica-grafted materials. Unfortunately, the transfer of selectors originally developed for silica-surface grafting into polymeric matrixes is far from being straightforward. Materials produced with polymerizable versions of selectors often exhibit inferior enantiomer separation characteristics as compared to the corresponding silica-grafted binders.15-20 (5) Svec, F. J. Sep. Sci. 2004, 27, 747-766. (6) Jungbauer, A.; Pflegerl, K. In Monolithic Materials: Preparation, Properties, and Applications; Svec, F., Tennikova, T. B., Deyl, Z., Eds.; Journal of Chromatography Library 67; Elsevier: Boston, 2003; pp 725-743. (7) See ref 6. (8) Xu, M.; Brahmachary, E.; Janco, M.; Ling, F. H.; Svec, F.; Fre´chet, J. M. J. J. Chromatogr., A 2001, 928, 25-50. (9) Hahn, R.; Podgornik, A.; Merhar, M.; Schallaun, E.; Jungbauer, A. Anal. Chem. 2001, 73, 5126-5132. (10) Lewandowski, K.; Murer, P.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 1998, 70, 1629-1638. (11) Burguete, M. I.; Fre´chet, J. M. J.; Garcı´a-Verdugo, E.; Janco, M.; Luis, S. V.; Svec, F.; Vincent, M. J.; Xu, M. Polym. Bull. 2002, 48, 9-15. (12) Svec, F.; Tennikova, T. B. J. Bioact. Compat. Polym. 1991, 6, 393-405. (13) La¨mmerhofer, M.; Peters, E. C.; Yu, C.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2000, 72, 4614-4622. (14) La¨mmerhofer, M.; Svec, F.; Fre´chet, J. M. J.; Lindner, W. Anal. Chem. 2000, 72, 4623-4628. (15) Lee, Y.-K.; Yamashita, K.; Eto, M.; Onimura, K.; Tsutsumi, H.; Oishi, T. Polymer 2002, 43, 7539-7547. (16) Lee, Y.-K.; Nakashima, Y.; Onimura, K.; Tsutsumi, H.; Oishi, T. Macromolecules 2003, 36, 4735-4742. (17) Oishi, T.; Lee, Y.-K.; Nakagawa, A.; Onimura, K.; Tsutsumi, H. J. Polym. Sci., Part A 2002, 40, 1726-1741.

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The loss in performance reflects to some extent the problems associated with controlling important macroscopic polymer properties, such as pore size and structure, surface area, different nonspecific binding properties, and unpredictable solvent-induced swelling behavior.11,21,22 To a major part, however, the observed degradation in chiral recognition capacity of polymer-embedded selectors may arise from largely ignored molecular-level insufficiencies. As the incorporation of polymerizable selectors occurs in a random fashion, they will become fixed within the polymeric network in all possible orientations and environments. A certain fraction of the selector molecules may become buried in nonporous polymer regions, rendering them completely inaccessible to target molecules. Alternatively, disposition of the selector in crowded polymer domains may enforce detrimental conformational changes, which will impair the selector’s ability to establish the subtle network of noncovalent contacts essential for efficient and selective target binding. Degeneration of chiral recognition capabilities may also originate from unfavorable influences acting on the selector prior to polymerization. Prepolymer formulations generally containsapart from the selector monomersa variety of other molecular species at considerable concentration levels, such as comonomers, cross-linking agents, and porogenic solvents. Being exposed to these environments, the highly functionalized chiral selector molecules are likely to complex more or less tightly with these nonsubstrate components. In addition, under high concentration conditions, selectors may undergo self-aggregation23-25 to a considerable extent, forming rather stable dimers, oligomers, or both. On polymerization, these undesired hetero-/ homomolecular complexes may also become integrated into the macromolecular network, generating blocked and thus unproductive selector sites. The combination of these deleterious effects may result in materials displaying extremely heterogeneous binding characteristics, becoming macroscopically evident in low levels of enantioselectivity, binding capacity, and lack of peak efficiency.15-20 The availability of procedures that allow an improved level of control over accessibility, conformational constraints, and surface representation of the polymer-embedded selectors certainly would greatly facilitate efforts to develop polymer-type chiral stationary phases. A simple, yet elegant approach toward this goal may consist in a temporary blockage of the selector binding site during polymerization, exploiting its ability to form strong and spatially well-defined, but noncovalent and thus reversible, complexes with favorable analytes. Employing this analyte-templating approach as a protective measure may help to preserve selector functionality in polymeric environments in a number of ways: (i) Specific analyte binding should selectively “saturate” selector sites engaged (18) Lee, K.-P.; Choi, S.-H.; Kim, S.-Y.; Kim, T.-H.; Ryoo, J. J.; Ohta, K.; Jin, J.-Y.; Takeuchi, T.; Fujimoto, C. J. Chromatogr., A 2003, 987, 111-118. (19) Mayr, B.; Schottenberger, H.; Elsner, O.; Buchmeiser, M. R. J. Chromatogr., A 2002, 973, 115-122. (20) Mayr, B.; Sinner, F.; Buchmeiser, M. R. J. Chromatogr., A 2001, 907, 4756. (21) Xu, M.; Peterson, D. S.; Rohr, T.; Svec, F.; Fre´chet, J. M. J. Anal. Chem. 2003, 75, 1011-1021. (22) Buchmeiser, M. R. New J. Chem. 2004, 28, 549-557. (23) Uccello-Barretta, G.; Bari, L. D.; Salvadori, P. Magn. Reson. Chem. 1992, 30, 1054-1063. (24) Duret, P.; Foucault, A.; Margraff, R. J. Liq. Chromatogr., Relat. Technol. 2000, 23, 295-312. (25) Pirkle, W. H.; Pochapsky, T. C. J. Am. Chem. Soc. 1987, 109, 5975-5982.

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in the molecular recognition event, rendering them inaccessible to perturbing interactions with nonsubstrate and other selector molecules. (ii) Tight analyte binding should enforce the selector to adopt and maintain its “productive conformation(s)” throughout the entire polymerization process, enhancing the level of analytespecific preorganization of the resultant polymer-embedded binding site. (iii) Incorporation of intact selector-analyte assemblies should also help to define the specific spatial requirements for efficient selector-analyte interaction within polymeric networks, diminishing the fraction of selector units being nonfunctional due to steric restrictions. (iv) As an additional benefit, embedding selector-analyte complexes into polymers may shape secondary binding sites around the complexes, with the potential to enhance enantioselectivity and substrate specificity. Analyte templating bears resemblance to classical molecular imprinting26-29 in that both exploit template molecules to shape (more or less) target-specific polymer-embedded binding sites. However, there are important conceptual differences concerning the nature of the functional monomers and the specific role of the templates. In molecular imprinting, the template molecules serve to spatially preassemble functional monomers lacking inherent selectivity. Subsequent polymerization is an essential step to stabilize the array of monomers assembled around the template to create selective binding sites. In contrast, analyte templating employs inherently selective functional monomers. These are purposefully complexed with tightly binding analytes prior to polymerization with the intention to preserve (and ideally enhance) existing, rather than create new, binding sites upon incorporation into the polymeric environment. In this contribution, we test the feasibility of this new concept by preparing a series of analyte-templated porous polymers; employing a polymerizable version of a well-established cinchona carbamate-type selector, in the presence of tightly binding analytes. By variation of the structure and chirality of the templating analytes, the molecular requirements for achieving significant levels of binding site preservation of the polymer-embedded selectors are probed. Observed differences in the chromatographic chiral recognition characteristics of the corresponding polymers are analyzed with respect to binding capacities and affinities and compared to those obtained with a conventional silica-grafted reference material. Changes in substrate-specific enantioselectivity originating from analyte templating are also addressed. EXPERIMENTAL SECTION Materials. Ethylene glycol dimethacrylate (EDMA), 2,2′azobisisobutyronitrile (AIBN), thionyl chloride, HPLC-grade methanol, acetonitrile, chloroform, and dimethoxyethane were purchased from Merck (Darmstadt, Germany). N-Hydroxysuccinimide, N-ethyldiisopropylamine, tert-butylamine, methacryloyl chloride, 2-mercaptoethanol, tetrahydrofuran (THF), sodium chloride, sodium hydrogencarbonate, magnesium sulfate, sodium hydroxide, and acetic acid (analytical grade) were supplied from Fluka (Buchs, Switzerland). 3,5-Dichlorobenzoyl chloride, triphenyl(26) Piletsky, S. A.; Andersson, H. S.; Nicholls, I. A. Macromolecules 1999, 32, 633-636. (27) Wulff, G. Chem. Rev. 2002, 102, 1-27. (28) Wulff, G.; Knorr, K. Bioseparation 2002, 10, 257-276. (29) Hosoya, K.; Tanaka, N. In Molecular and Ionic Recognition with Imprinted Polymers; Bartsch, R. A., Maeda, M., Eds.; American Chemical Society: Washington, DC, 1997; pp 143-158.

phosphine, and diethyl azodicarboxylate were from Aldrich (Austria, Vienna). tert-Butylmethacrylamide (tBu-MAA) was prepared following the procedure described in ref 43. General Information. Unless otherwise stated, all reactions were carried out under strictly anhydrous conditions under nitrogen atmosphere. All solvents were dried and distilled by standard procedures prior to use. 1H spectra were acquired on a Bruker DRX 400 MHz spectrometer. Chemical shifts are given in parts per million (δ ppm) with respect to TMS as internal standard. FT-IR spectra were recorded on a Perkin-Elmer Spektrum 2000 spectrometer. Optical rotation values were acquired on a Perkin-Elmer 341 polarimeter at 25 °C. Melting points were determined with a Kofler apparatus, equipped with a Leica microscope. Flash chromatography was performed on Silica 60 (0.040-0.063-mm particle size, Merck). Instruments. The mechanic ball mill (MM 2000) used for polymer grinding was from Retsch (Haan, Germany). A MerckHitachi L-6000 preparative pump was used for column packing of the polymers. Chromatographic data were acquired on a MerckHitachi L-7000 HPLC system consisting of an L-7150 semipreparative pump, a L-7250 autosampler, and a L-7455 diode array detector. A D-7000 software was employed for chromatogram interpretation and data processing. All mobile phases were composed from HPLC-grade solvents. Synthesis of Templating Analytes and Monomers. O-Succinimidyl-3,5-dichlorobenzoate. Dry N-hydroxysuccinimide (15.6 g, 134 mmol) was dissolved in 200 mL of dry THF in the presence of N-ethyldiisopropylamine (23 mL, 134 mmol). To the precooled solution (ice/H2O bath) 3,5-dichlorobenzoyl chloride (23.5 g, 112 mmol) in 50 mL of dry THF was added dropwise with stirring. After 2 h the reaction mixture was filtered to remove precipitated amine hydrochloride. The filtrate was evaporated under reduced pressure at 40 °C. The solid residue was extracted with 2 M HCl and ethyl acetate (100 mL/350 mL). The organic phase was washed with brine (2 × 100 mL) and dried (MgSO4). Evaporation of the solvent under reduced pressure gave 31.2 g (97%) of white crystals: mp 131-134 °C; IR (KBr) 3082, 2946, 1750, 1571, 1427, 1405, 1354, 1304, 1227, 1197, 1118 cm-1; 1H NMR (CDCl3) δ 8.01 (2H, s), 7.67 (1H, s), 2.91 (4H, s). (30) Maier, N. M.; Nicoletti, L.; La¨mmerhofer, M.; Lindner, W. Chirality 1999, 11, 522-528. (31) La¨mmerhofer, M.; Lindner, W. J. Chromatogr., A 1996, 74, 33-48. (32) Krawinkler, K. H.; Maier, N. M.; Sajovic, E.; Lindner, W. J. Chromatogr., A 2004, 1053, 119-131. (33) La¨mmerhofer, M.; Maier, N. M.; Lindner, W. Am. Lab. 1998, 30, 71. (34) Lah, J.; Maier, N. M.; Lindner, W.; Vesnaver, G. J. Phys. Chem., B 2001, 105, 1670-1678. (35) Maier, N. M.; Schefzick, S.; Lombardo, G. M.; Feliz, M.; Rissanen, K.; Lindner, W.; Lipkowitz, K. J. Am. Chem. Soc. 2002, 124, 8611-8629. (36) Czerwenka, C.; Zhang, M. M.; Ka¨hlig, H.; Maier, N. M.; Lipkovitz, K. B.; Lindner, W. J. Org. Chem. 2003, 68, 8315-8327. (37) Wirz, R.; Bu ¨ rgi, T.; Lindner, W.; Baiker, A. Anal. Chem. 2004, 76, 53195330. (38) Lesnik, J.; La¨mmerhofer, M.; Lindner, W. Anal. Chim. Acta 1999, 401, 3-10. (39) Franco, P.; La¨mmerhofer, M.; Klaus, P. M.; Lindner, W. J. Chromatogr., A 2000, 869, 111-127. (40) Franco, P.; Klaus, P. M.; Minguillo´n, C.; Lindner, W. Chirality 2001, 13, 177-186. (41) Sellergren, B. J. Chromatogr., A 2001, 906, 227-252. (42) Haupt, K.; Mosbach, K. Chem. Rev. 2000, 100, 2495-2504. (43) Jodlbauer, J.; Maier, N. M.; Lindner, W. J. Chromatogr., A 2002, 945, 4563.

(R)-N-3,5-Dichlorobenzoylleucine (DCB-(R)-Leu). (R)-Leucine (1.47 g, 11.2 mmol) and NaHCO3 (3.15 g, 56 mmol) were dissolved in 100 mL of water with stirring. O-Succinimmidyl-3,5-dichlorobenzoate (2.16 g, 7.5 mmol) was dissolved in 50 mL of THF and added in single portion to the amino acid solution. The mixture was stirred at ambient temperature for 18 h. The reaction mixture was evaporated under reduced pressure. The solid residue was suspended in 100 mL of water, and concentrated aqueous HCl was added to adjust the pH to ∼2. The solid was isolated by filtration and dried at 60 °C to give 2.22 g (97%) of a white solid: mp after recrystallization from MeOH/H2O (4:3) 158-160 °C; IR (KBr) 3279, 3082, 2961, 1719, 1643, 1567, 1418, 1333, 1274, 1240, 1148 cm-1; 1H NMR (CDCl3) δ 7.65 (2H, s), 7.50 (1H, s), 7.55 (1H, d), 4.83 (1H, m), 1.76 (3H, m), 0.98 (6H, d); optical rotation [R]589 ) +11.2°, [R]546 ) + 12.5°, [R]436 ) + 19.2° (c ) 1.0, MeOH). (S)-N-3,5-Dichlorobenzoylleucine (DCB-(S)-Leu). This compound was prepared from (S)-leucine analogous to the procedure described above for DCB-(R)-Leu: yield 2.12 g (93%) of a white solid; optical rotation [R]589 ) -11.1°, [R]546 ) -12.6°, [R]436 ) 19.3° (c ) 1.0, MeOH). All other physical properties were identical with those of the (R)-enantiomer. (R,S)-N-3,5-Dichlorobenzoylphenylalanine (DCB-(R,S)-Phe). This compound was prepared from (R,S)-phenylalanine analogous to the procedure described above for DCB-(R)-Leu: yield 2.45 g (96%) of a white solid recrystallized from MeOH/H2O (4:3). 3,5-Dichlorobenzoylglycine (DCB-Gly). This compound was prepared from glycine analogous to the procedure described above for DCB-(R)-Leu: yield 1.79 g (96%) of a white solid; mp after recrystallization from MeOH/H2O 1/1, 188-190°C; IR (KBr): 3293, 3079, 1716, 1638, 15552, 1423, 1335, 1239 cm-1; 1H NMR (CDCl3) δ 7.71 (2H, s), 7.52 (1H, s), 3.98 (2H, s). 11-[(2-Hydroxyethyl)thia]-9-tert-butylcarbamoyl dihydroquinine (1). Quinine tert-butylcarbamate (20.00 g, 47.3 mmol) was dissolved in 150 mL of dry CHCl3. 2-Mercaptoethanol (6.60 mL, 94.6 mmol) and AIBN (0.25 g) were added. The mixture was refluxed with stirring for 20 h. The reaction mixture was extracted with 3 M aqueous NaOH (3 × 200 mL). The organic phase was dried (MgSO4) and the solvent evaporated under reduced pressure to give a yellowish oil, which crystallized on addition of ethyl acetate: yield 22.8 g (96%) of white crystals; mp 179-182 °C; IR (KBr) 3339, 2915, 1731, 1619, 1591, 1509, 1457, 1391, 1365, 1267, 1227 cm-1; 1H NMR (CDCl3) δ 8.73 (1H, d), 7.99 (1H, d), 7.48 (1H, s), 7.35 (2H, m), 6.42 (1H, d), 4.73 (1H, s), 3.95 (3H, s), 3.70 (2H, t), 3.28 (1H, d), 3.06 (2H, m), 2.72 (2H, t), 2.62 (1H, m), 2.48 (2H, t), 2.33 (1H, d), 1.28 (9H, s); optical rotation [R]589 ) -24.6°, [R]546 ) - 29.5°, [R]436 ) - 67.6° (c ) 1.0, MeOH). 11-[(2-Aminoethyl)thia]-9-tert-butylcarbamoyl-dihydroquinine (2). 11-[(2-Hydroxyethyl)thia]-9-tert-butylcarbamoyldihydroquinine (10.00 g, 19.9 mmol) was dissolved in 200 mL of dry THF. Triphenylphosphine (6.27 g, 23.9 mmol) and hydrazoic acid (2 M in toluene, 11.90 mL, 23.9 mmol) were added. The mixture was cooled (ice/H2O), and diethylazodicarboxylate (3.77 mL, 23.9 mmol) in 30 mL of dry THF was added dropwise with stirring. After 3 h another portion of triphenylphosphine (5.23 g, 23.92 mmol) was added. The reaction mixture was stirred at 50 °C for 2 h. Then 3 mL of H2O were added, and stirring was continued at the same temperature for 20 h. The solvent was removed under Analytical Chemistry, Vol. 77, No. 15, August 1, 2005

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Table 1. Composition of the Templated (P1-P4) and Control (CP1-CP4) Polymers material

tBuCQN-MAA (mmol)

tBu-MAA (mmol)

template/mmol

EDMA (mmol)

AIBN (mmol)

MeOH (mL)

P1 P2 P3 P4 CP1 CP2 CP3 CP4

1.00 1.00 1.00 1.00 none none none none

3.00 3.00 3.00 3.00 4.00 4.00 4.00 4.00

DCB-(S)-Leu/1.00 DCB-(R)-Leu/1.00 DCB-Gly/1.00 none DCB-(S)-Leu/1.00 DCB-(R)-Leu/1.00 DCB-Gly/1.00 none

20.00 20.00 20.00 20.00 20.00 20.00 20.00 20.00

0.24 0.24 0.24 0.24 0.24 0.24 0.24 0.24

5.60 5.60 5.60 5.60 5.60 5.60 5.60 5.60

reduced pressure to give a yellowish oil. This was taken into 200 mL of CHCl3 and extracted with 2 M HCl (250 mL). The organic phase was discarded. The aqueous phase was washed with 50 mL of CHCl3, which again was discarded. Ice (50 mL) was added to the aqueous phase, and solid sodium hydroxide was added in small portions to adjust to pH 14. The strongly basic mixture was extracted with CHCl3 (1 × 200 mL, 1 × 100 mL). The combined organic phases were dried (MgSO4), and the solvent was removed under reduced pressure to yield 7.85 g (79%) of a yellowish foam. An analytically pure sample was obtained by flash chromatography (aminopropyl-modified silica/ethyl acetate): mp 81-85 °C; IR (KBr) 3351, 2934, 1713, 1621, 1592, 1509, 1474, 1393, 1364, 1267 cm-1; 1H NMR (CDCl3) δ 8.74 (1H, s), 8.00 (1H, d), 7.47 (1H, s), 7.35 (2H, m), 7.26 (1H, s), 6.43 (1H, d), 4.78 (1H, s), 3.95 (3H, s), 3.25 (1H, m), 3.06 (2H, m), 2.84 (2H, t), 2.59 (3H, t), 2.46 (2H, t), 2.33 (1H, d), 1.47 (1H, m), 1.28 (9H, s); optical rotation [R]589 ) -22.0°, [R]546 ) -27.6°, [R]436 ) -64.4° (c ) 1.0, MeOH). 11-[(2-(Methacrylamido)ethyl)thia]-9-tert-butylcarbamoyldihydroquinine (tBuCQN-MAA). The resulting amine (7.80 g, 15.6 mmol) was dissolved in 50 mL of CHCl3 and cooled in ice/H2O bath. Methacryloyl chloride (3.02 mL, 31.15 mmol) was dissolved in 25 mL of CHCl3 and added dropwise to the amine solution with stirring. After 20 h the solvent was removed under reduced pressure at ambient temperature. The resultant white foam was dissolved in 150 mL of CHCl3 and washed with 1 M aqueous NaOH (3 × 100 mL). The organic phase was dried (MgSO4) and evaporated under reduced pressure. The product was purified by flash chromatography (150 g of silica, CHCl3/MeOH, 10:1) to yield 4.87 g (55%) of a white solid. An analytically pure sample was obtained by recrystallization from ethyl acetate: mp 168-171 °C; IR (KBr) 3310, 2932, 2361, 1713, 1660, 1621, 1511, 1456, 1393, 1365, 1267, 1228 cm-1; 1H NMR (CDCl3) δ 8.74 (1H, d), 8.00 (1H, d), 7.49 (1H, s), 7.35 (2H, m), 6.43 (1H, d), 6.18 (1H, s), 5.69 (1H, s), 5.34 (1H, s), 4.77 (1H, s), 3.96 (3H, s), 3.49 (2H, m), 3.29 (1H, m), 3.07 (2H, m), 2.63 (3H, m), 2.50 (2H, t), 2.35 (1H, d), 1.95 (3H, s), 1.28 (9H, s); optical rotation [R]589 ) -22.1°, [R]546 ) -27.6°, [R]436 ) -62.0° (c ) 1.0, MeOH). Preparation and Size Classification of Polymers P1-P4/ CP1-CP4. All of the cinchona receptor-type polymers P1-P4 and corresponding control polymers CP1-CP4 were prepared according to the following general procedure: The respective amounts of monomers, initiator, and porogenic solvent indicated in Table 1 were transferred into in glass screwcap centrifugation tubes (25 mL, 120 × 15 mm i.d.). The solutions were degassed by passing a vigorous stream of dry nitrogen through the viscous mixtures for 5 min. The tubes were tightly closed with Teflon-lined screw caps and additionally sealed with 5012

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several layers of a plastic insulation tape. The tubes were then incubated for thermal polymerization in a water bath at 65 ( 0.1 °C for 24 h. The resultant polymer rods were manually broken into small pieces using a mortar and subsequently subjected to mechanical grinding employing a ball mill. For size classification, the ground polymers were suspended in acetone and wet-sieved through a 25-µm steel sieve by gentle agitation with a brush. To achieve the removal of fines, the fractions of