Multicharged Anion

Hydrodynamic injection: 10 mbars, 3 s. Sample: 1.0 g/L G3 + 0.10 g/L pyridine (as a reference) in water. Electrolyte (pH 7.4): TRIS 13.6 mM + boric ac...
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Anal. Chem. 2010, 82, 7362–7368

Neutral Coatings for the Study of Polycation/ Multicharged Anion Interactions by Capillary Electrophoresis: Application to Dendrigraft Poly-L-lysines with Negatively Multicharged Molecules Tao Zou,† Farid Oukacine,†,‡ Thomas Le Saux,§ and Herve´ Cottet*,† Institut des Biomole´cules Max Mousseron (UMR 5247 CNRS-Universite´ de Montpellier 1-Universite´ de Montpellier 2), Place Euge`ne Bataillon CC 1706, 34095 Montpellier Cedex 5, France, COLCOM, Cap Alpha, Avenue de l’Europe, Clapiers, 34940 Montpellier Cedex 9, France, and Ecole Normale Supe´rieure, De´partement de Chimie, (UMR 8640 CNRS-Ecole Normale Supe´rieure-Universite´ Pierre et Marie Curie Paris 6), 24 rue Lhomond, 75231 Paris Cedex 05, France The study of interactions between oppositely multicharged (macro)molecules remains a challenging issue. In frontal analysis capillary electrophoresis (FACE), it is difficult to avoid the adsorption of one of the interacting partners onto the capillary wall. In this work, we demonstrate the possibility to use FACE and affinity capillary electrophoresis (ACE) on a neutrally coated capillary for the study of interactions between a polycationic dendrigraft (or linear) poly-L-lysines, on one hand, and a multicharged anionic biomolecule (adenosine monophosphate, AMP, or adenosine triphosphate, ATP), on the other hand. A systematic comparison of four different neutral coatings (hydroxypropyl cellulose, polydimethylacrylamide, polyacrylamide, polyethylene glycol) has been performed based on the repeatability of the electrophoretic migration of the dendrigraft poly-L-lysines at pH close to neutrality. Both FACE and ACE methodogies were then used to study the interactions and to get the association constants and the stoichiometry of the complex. Multisite interactions, with two classes of independent sites, were determined. The specificity of the dendritic polylysine structure compared with linear polylysine in the interaction with ATP or AMP is also emphasized. Dendritic polymers have been recognized as the fourth major class of synthetic polymer architecture1-3 (after linear, crosslinked, and branched), characterized by a cascade-branching structure typically obtained from polyfunctional monomers under more or less strictly controlled polymerization conditions. Since their discovery, dendritic polymers have found numerous applica* To whom correspondence should be addressed. Phone: +33-4-6714-3427. Fax: +33-4-6763-1046. E-mail: [email protected]. † Institut des Biomole´cules Max Mousseron. ‡ COLCOM. § Ecole Normale Supe´rieure. (1) Tomalia, D. A.; Fre´chet, J. M. Dendrimers and Other Dendritic Polymers; Fre´chet, J. M., Tomalia, D. A., Eds.; John Wiley & Sons: Chichester, U.K., 2001; pp 3-44. (2) Tomalia, D. A. Aldrich Chim. Acta 2004, 37, 39–57. (3) Teertstra, S. J.; Gauthier, M. Prog. Polym. Sci. 2004, 29, 277–327.

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tion domains such as nanomaterials, diagnostics, drug delivery,4 biocides, gene transfer,5 and catalysts.6-9 Dendrigraft polymers are the most recently discovered subset of dendritic polymers. They are usually synthesized by “protect/deprotect” or activation schemes. Compared to dendrimers, the molar mass of dendrigraft polymers increases more rapidly with generation than for dendrimers. Combined with the possibility of large-scale synthesis, dendrigraft polymers offer the possibility to develop applications of dendrimers at a much lower cost.10 Recently, our group described a new synthetic route to dendrigraft poly-L-lysines (DGL) by polymerization of N-carboxyanhydride of amino acids (NCA) in water.11 As a variety of dendritic structures is now available by chemical synthesis, characterization of such objects becomes an important issue. Dendrimers have been analyzed using a wide range of different analytical methods, such as potentiometric titrations to quantify the functional groups,12-14 size-exclusion chromatography (SEC) to determine the molar mass distribution,11,15,16 HPLC to (4) Svenson, S.; Tomalia, D. A. Adv. Drug Delivery Rev. 2005, 57, 2106–2129. (5) Shen, X. C.; Zhou, J.; Liu, X.; Wu, J.; Qu, F.; Zhang, Z. L.; Pang, D. W.; Que´le´ver, G.; Zhang, C. C.; Peng, L. Org. Biomol. Chem. 2007, 5, 3674– 3681. (6) Darbre, T.; Reymond, J. L. Acc. Chem. Res. 2006, 39, 925–934. (7) Caminade, A. M.; Servin, P.; Laurent, R.; Majoral, J. P. Chem. Soc. Rev. 2008, 37, 56–67. (8) Colby, D. E. A.; Mennen, S. M.; Xu, Y.; Miller, S. J. Chem. Rev. 2008, 107, 5759–5812. (9) Helms, B.; Fre´chet, J. M. J. Adv. Synth. Catal. 2006, 348, 1125–1148. (10) Kee, R. A.; Gauthier, M.; Tomalia, D. A. Dendrimers and Other Dendritic Polymers; Fre´chet, J. M. J., Tomalia, D. A., Eds.; Wiley Series in Polymer Science; John Wiley and Sons: West Sussex, U.K., 2001. (11) Collet, H.; Souaid, E.; Cottet, H.; Deratani, A.; Boiteau, L.; Dessalces, G.; Rossi, J. C.; Commeyras, A.; Pascal, R. Chem.sEur. J. 2010, 16, 2309– 2316. (12) Majoros, I. J.; Keszler, B.; Woehler, S.; Bull, T.; Baker, J. R., Jr. Macromolecules 2003, 36, 5526–5529. (13) Shi, X.; Ba´nyai, I.; Islam, M. T.; Lesniak, W.; Davis, D. Z.; Baker, J. R., Jr.; Balogh, L. P. Polymer 2005, 46, 3022–3034. (14) Shi, X.; Lesniak, W.; Islam, M. T.; MuNiz, M. C.; Balogh, L. P.; Baker, J. R., Jr. Colloids Surf. A 2006, 272, 139–150. (15) Majoros, I. J.; Myc, A.; Thomas, T.; Mehta, C. B.; Baker, J. R. Biomacromolecules 2006, 7, 572–579. (16) Majoros, I. J.; Thomas, T. P.; Mehta, C. B.; Baker, J. R. J. Med. Chem. 2005, 48, 5892–5899. 10.1021/ac101473g  2010 American Chemical Society Published on Web 08/04/2010

check the purity,17-,19 mass spectrometric methods (MALDI-MS, ESI-MS) to confirm the molar mass,20,21 and more recently, ultra performance liquid chromatography (UPLC) analysis to monitor surface transformations and product quality22 and Taylor dispersion analysis to determine the hydrodynamic radius.23 Describing the interactions of these polymeric structures with their chemical environment is the next challenge, especially as major applications24 explicitly make use of noncovalent interactions between the dendrimer and a chemical entity. Capillary electrophoresis (CE) is a well-known separation technique for the characterization of (bio)macromolecules and allows to evaluate noncovalent interactions either by frontal analysis capillary electrophoresis (FACE) or by affinity capillary electrophoresis (ACE). CE presents the advantage to require only minute amount of sample. As CE was successfully implemented to separate dendrimers of different generations11,25,26 or to describe functionalized dendrimers,20,23,27 it seems particularly attractive to investigate the interactions developed by dendritic structures. So far, ACE and FACE were mostly used to study interactions between neutral and charged compounds or between charged compounds having the same charge sign.28-34 It has been pointed out in the literature that the main drawback of FACE and ACE is the adsorption of one of the interacting compounds onto the capillary wall in the case of oppositely charged (macro)molecules.35 For instance, protein/ligand interactions were generally studied at a pH well above the pI of the protein to avoid binding of the protein to the negatively charged silica capillary.36,37 Therefore, the study of interactions between oppositely charged (macro)molecules remains a challenging issue, especially when (17) Choi, Y.; Thomas, T.; Kotlyar, A.; Islam, M. T.; Baker, J. R. Chem. Biol. 2005, 12, 35–43. (18) Islam, M. T.; Majoros, I. J.; Baker, J. R. J. Chromatogr., B 2005, 822, 21– 26. (19) Shi, X. Y.; Bi, X. D.; Ganser, T. R.; Hong, S. P.; Myc, L. A.; Desai, A.; Holl, M. M. B.; Baker, J. R. Analyst 2006, 131, 842–848. (20) Kallos, G. J.; Tomalia, D. A.; Hedstrand, D. M.; Lewis, S.; Zhou, J. Rapid Commun. Mass Spectrom. 1991, 5, 383–386. (21) Schwartz, B. L.; Rockwood, A. L.; Smith, R. D.; Tomalia, D. A.; Spindler, R. Rapid Commun. Mass Spectrom. 1995, 9, 1552–1555. (22) Cason, C. A.; Oehrle, S. A.; Fabre, T. A.; Girten, C. D.; Walters, K. A.; Tomalia, D. A.; Haik, K. L.; Bullen, H. A. J. Nanomater. 2008, Article ID 456082, 7 pages. (23) Cottet, H.; Martin, M.; Papillaud, A.; Souaid, E.; Collet, H.; Commeyras, A. Biomacromolecules 2007, 8, 3235–3243. (24) Crampton, H. L.; Simanek, E. E. Polym. Int. 2007, 56, 489–496. (25) Carter, B.; Desai, A.; Sharma, A. Electrophoresis 2007, 28, 335–340. (26) Sedlakova, P.; Svobodova, J.; Miksik, I.; Tomas, H. J. Chromatogr., B 2006, 841, 135–139. (27) Brothers, H. M.; Piehler, L. T.; Tomalia, D. A. J. Chromatogr., A 1998, 814, 233–246. (28) Østergaard, J.; Heegaard, N. H. H. Electrophoresis 2003, 24, 2903–2913. (29) Sun, H. W.; He, P. Electrophoresis 2009, 30, 1991–1997. (30) Jiang, C.; Armstrong, D. Electrophoresis 2010, 31, 17–27. (31) Anderot, M.; Nilsson, M.; Vegvari, A.; Moeller, E. H.; van de Weert, M.; Isaksson, R. J. Chromatogr., B 2009, 877, 892–896. (32) Boija, E.; Lundquist, A.; Nilsson, M.; Edwards, K.; Isaksson, R.; Johansson, G. D. Electrophoresis 2008, 29, 3377–3383. (33) Franzen, U.; Jorgensen, L.; Larsen, C.; Heegaard, N. H. H.; Østergaard, J. Electrophoresis 2009, 30, 2711–2719. (34) Østergaard, J.; Jorgensen, L.; Thomsen, A. E.; Larsen, S. W.; Larsen, C.; Jensen, H. Electrophoresis 2008, 29, 3320–3324. (35) Sperber, B. L. H. M.; Stuart, M. A. C.; Schols, H. A.; Voragen, A. G. J.; Norde, W. Biomacromolecules 2009, 10, 3246–3252. (36) Gao, J. Y.; Dubin, P. L.; Muhoberac, B. B. Anal. Chem. 1997, 69, 2945– 2951. (37) Shen, Y.; Smith, R. D. J. Microcolumn Sep. 2000, 12, 135–141.

the partners are bearing multiple charges. So far, there are very few studies dealing with the interaction of oppositely charged partners.31,33,38 To address this challenging issue, the selection and the use of a robust, neutral coating is highly desirable. In the present study, we investigated and compared the use of four different neutral coatings for the characterization of interactions between highly charged polycationic DGL (or linear poly-L-lysine), on one hand, and negatively multicharged small molecules, on the other hand. Adenosine monophosphate (AMP) and adenosine triphosphate (ATP) were chosen as models of negatively multicharged small biomolecules. The interaction studies were performed at pH 7.4, for which the two interacting partners are oppositely multicharged. In a first part, four different neutral coatings were compared with respect to the repeatability of DGL electrophoretic migration. In a second part, interactions of dendrigraft (or linear) poly-L-lysines with AMP and ATP were studied using FACE and ACE. EXPERIMENTAL SECTION Chemicals. Hydroxypropyl cellulose (HPC) (Mw 105 g/mol), γ-methacryloxypropyltrimethoxysilane (γ-MPS), acrylamide, tetramethylethylenediamine (TEMED), ammonium persulfate (APS), dimethylacrylamide (DMA), boric acid (H3BO3), TRIS ((HOCH2)3CNH2), adenosine 5′-triphosphate disodium salt (ATP), and adenosine 5′-monophosphate monohydrate (AMP) were purchased from Sigma-Aldrich (Steinheim, Germany). All materials were used as received. Deionized water was further purified with a Milli-Q system (Millipore, Molsheim, France). DGL of third generation (G3) was supplied by COLCOM (Montpellier, France). Poly(L-lysine hydrobromide) (PLL) was supplied by Alamanda Polymers (Huntsville). Capillary Coatings. HPC coated capillaries have been coated according to the following procedure:37 the HPC powder was dissolved at room temperature in water to a final concentration of 5% (w/w). The polymer solutions were left overnight to eliminate bubbles. The capillary columns (100 µm i.d.) were filled at 100 µL/h with the polymer solution using a syringe pump for 30 min, and the excess of polymer solution was removed using N2 gas at 3 bar. The HPC polymer layer was immobilized by heating the capillary in a GC oven (GC-14A, Shimadzu, France) from 60 to 140 °C at 5 °C/min and then at 140 °C for 20 min, keeping N2 pressure at 3 bar. Before use, the coated capillaries were rinsed with water for 10 min. Linear polyacrylamide (PAA) coated capillaries have been coated according to the following procedure:39 New bare fusedsilica capillaries (100 µm i.d.) were first cleaned with acetone for 10 min at 100 µL/h using a syringe pump. The capillaries were flushed with 1 M NaOH for 30 min, followed by 0.1 M HCl for 10 min, and finally washed with water for 10 min at the same rate. After these washing steps, the capillaries were silanized at room temperature by filling it with a mixture of 0.6% (v/v) γ-MPS and 0.5% (v/v) acetic acid in dichloromethane. After 50 min, the capillaries were washed with methanol and water for 10 min each and were then filled with a 1.5% acrylamide monomer solution (purged with N2 for 30 min before used) including TEMED (38) Jia, Z. J.; Ramstad, T.; Zhong, M. J. Pharm. Biomed. Anal. 2002, 30, 405– 413. (39) Hjerten, S.; Zhu, M.-D. J. Chromatogr. 1985, 346, 265–270.

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Table 1. Physico-Chemical Characteristics of the Three First Generations of DGL from Reference 22

number average DP polydispersity index hydrodynamic radius (nm)

a

G1

G2

G3

8 1.20 0.77

48 1.38 1.40

123 1.46 2.14

a Measured by Taylor dispersion analysis in 38 mM NaH2PO4 + 38 mM Na2-HPO4 + 154 mM NaCl (pH adjusted to 7.0, 0.306 M ionic strength).

(0.1%, v/v) and APS (0.1%, w/v). Capillaries were kept with this solution at room temperature for 1 h. The capillaries were then washed with water for 10 min and finally dried with a hot N2 flow (100 °C) for 1 h. These coated capillaries can be stored at room temperature. Before use, the coated capillaries were rinsed with water for 10 min. Linear polydimethylacrylamide (PDMA) coated capillaries have been coated according to the following procedure:40 New bare fused-silica capillaries (100 µm i.d.) were first cleaned with acetone for 10 min, then flushed with 1 M NaOH for 30 min, followed by 0.1 M HCl for 10 min, finally washed with water for 10 min. After these washing steps, the capillaries were filled with a solution of 10% (v/v) γ-MPS in acetic acid-acetone (100 µL of γ-MPS + 450 µL of acetic acid + 450 µL of acetone) and the capillary ends were closed with a rubber septum and then kept overnight at room temperature. The capillaries were washed with acetone. A volume of 1 mL of 1% DMA solution was purged with N2 for 30 min. A mixture of 50 µL of 10% (v/v) aqueous TEMED and 50 µL of 10% (w/v) aqueous APS was then introduced in the capillaries. After closing the ends, the capillaries were placed in an oven and mildly heated, 50 °C for 2 h for polymerization. Unreacted monomers were removed by washing with water for 10 min after cooling. Newly coated capillaries were flushed with Milli-Q water for 10 min prior to use. Polyethylene glycol (PEG) coated capillaries (DB-WAX, 0.100 mm i.d., 0.10 µm film thickness) were purchased from Agilent Technologies (Waldbronn, Germany). Capillary Electrophoresis. CE experiments were carried out using a 3D-CE Agilent technologies system (Waldbronn, Germany) equipped with a diode array detector. Separation capillaries prepared from bare silica tubing were purchased from Composite Metal Services (Worcester, U.K.). New fused silica capillaries were conditioned by performing the following washes: 1 M NaOH for 20 min, 0.1 M NaOH for 15 min, and water for 10 min. Neutral polymer coated capillaries were flushed only using buffer solution for 5 min between two runs. The temperature of the capillary cassette was set at 25 °C.

Figure 1. Electropherograms of G3 obtained in a fused silica capillary at pH 2.2 and 7.4. Experimental conditions: Fused silica capillary 33.5 cm (25 cm to detector) × 100 µm i.d. Temperature: 25 °C. Electrolyte (pH 2.2): TRIS 62.5 mM + H3PO4 125 mM. Applied voltage: +5 kV. Hydrodynamic injection: 10 mbars, 3 s. Sample: 1.0 g/L G3 + 0.10 g/L pyridine (as a reference) in water. Electrolyte (pH 7.4): TRIS 13.6 mM + boric acid 150 mM. Applied voltage: +20 kV. Electrokinetic injection: 7 kV, 5 s. Sample: 5.0 g/L G3 + 0.150 g/L imidazole (as a reference) in water.

RESULTS AND DISCUSSION DGL are dendritic structures based on poly-L-lysines. While five successive generations were synthesized,11 we mainly focused in this study on the third generation (G3) whose characteristics are given in Table 1, together with the two first generations (G1 and G2). Briefly, G1 is a linear poly-L-lysines containing a number average of 8 lysine residues (1 R-amine group and 8

ε-amine groups). G2 is a branched poly-L-lysine with a number average of 48 lysine residues (9 R-amine groups and 40 ε-amine groups). G3 is hyperbranched poly-L-lysine having an average of 123 lysine residues (49 R-amine groups and 75 ε-amine groups). For more details on the structure of DGL, the reader can refer to the following papers.11,23 Before studying the interaction between DGL and AMP (or ATP), it was necessary to find a neutral coating that can be used for the analysis of both negatively and positively multicharged molecules at a pH close to neutrality (tris-borate buffer, pH 7.4). Indeed, DGL are highly positively charged at pH 7.4, while AMP (pKa 3.99, 6.73)40 and ATP (pKa 4.68, 7.60),41 respectively, bear -1.83 and -3.39 elementary charges. Four different neutral coatings (HPC, PAA, PDMA, and PEG) were compared with the aim to get repeatable results regarding the electrophoretic migration of DGL. Comparison of Four Neutral Coatings for DGL Analysis. So far in the literature, the analysis of DGL was performed in acidic conditions (phosphate buffer, pH 2.2) using a fused silica capillary.11 In these acidic conditions, the cationic DGL does not interact with the bare silica surface, as shown in Figure 1 for the third generation of DGL (G3). The absence of interaction at pH 2.2 was due to the very weak dissociation of silanol groups at this pH and to the interactions of phosphates onto the silica wall that reduce solute/capillary wall interactions.42 On the contrary, at pH 7.4, strong interactions onto the silica capillary wall lead to tailing peaks without baseline return as shown in Figure 1. Similar results were obtained for the two first generations of DGL (G1 and G2), as presented in the Supporting Information (Figure S1). Electropherograms of the G3 obtained on four different neutral coatings are presented in time-scale and effective mobility-scale in Figure 2 (electropherograms for G1 and G2 are given in the Supporting Information, Figure S2). All the coatings lead to good peak shapes without excessive tailing and without any sign of interaction of the solute onto the capillary wall (i.e., good baseline returns). It is worth noting that 100 µm i.d.

(40) Wan, H.; Ohman, M.; Blomberg, L. G. J. Chromatogr., A 2001, 924, 59– 70.

(41) Alberty, R. A.; Goldberg, R. N. Biochemistry 1992, 31, 10610–10615. (42) McCormick, R. M. Anal. Chem. 1988, 60, 2322–2328.

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Figure 2. Electropherograms of G3 in time-scale (A) and in effective mobility-scale (B) on four different coated capillaries. Experimental conditions: capillary 33.5 cm (25 cm to detector) × 100 µm i.d. Electrolyte: TRIS 13.6 mM + boric acid 150 mM, pH 7.4. Applied voltage: +20 kV. Electrokinetic injection: 7 kV, 5 s. Sample: 5.0 g/L G3 + 0.150 g/L imidazole (as a reference) in water. Temperature: 25 °C. Table 2. Average Migration Time 〈tm〉, Average Effective Mobility (〈µep〉), Average Electroosmotic Mobility (〈µeof〉), Time-Corrected Peak Area (PA), and the Corresponding Relative Standard Deviation (RSD) for the Three Successive Generations of DGL on Four Different Neutral Coating (10 Repetitions)a sample G1

G2

G3

average values

〈tm〉 (min) RSD (%) 〈µep〉a RSD (%) 〈µeof〉a PA b RSD (%) 〈tm〉 (min) RSD (%) 〈µep〉a RSD (%) 〈µeof〉a PAb RSD (%) 〈tm〉 (min) RSD (%) 〈µep〉a RSD (%) 〈µeof〉a PAb RSD (%) 〈RSD〉 on 〈tm〉 (%) 〈RSD〉 on 〈µep〉 (%) 〈µeof〉a 〈RSD〉 on PA (%)a

HPC

PEG

PAA

PDMA

1.75 1.5 40.93 1.3 -0.44 7.26 2.1 1.80 1.1 40.33 0.72 -0.93 13.0 4.5 1.90 0.36 37.64 1.4 -0.41 13.7 4.9 1.0 1.1 -0.59 3.8

1.75 2.8 40.88 3.0 0.30 8.57 3.5 1.80 3.2 40.30 1.2 -1.0 13.3 4.9 2.08 3.1 36.86 4.9 -2.6 13.7 5.1 3.0 3.0 -1.1 4.5

1.80 0.4 40.90 2.2 -1.5 11.6 3.7 1.92 2.7 39.67 1.9 -2.7 12.9 4.6 1.97 2.7 37.43 1.8 -1.4 12.0 4.9 1.9 2.0 -1.9 4.4

1.75 1.3 42.28 2.4 -1.6 8.50 4.2 1.93 1.8 38.68 3.9 -2.0 15.5 4.8 2.04 4.0 37.42 2.5 -3.3 10.1 4.7 2.4 2.9 -2.3 4.6

a In 10-9 V-1 m2 s-1. b Time-corrected peak area ratio relative to imidazole (internal standard). a The last lines (average values) correspond to the average values obtained on the three first generations of DGL.

capillaries were used in this study in order to get lower limits of detection for interaction studies. Indeed, FACE and ACE generally does not require excessive separation performances but may necessitate longer optical path length for better sensitivity. For each DGL generation, 10 repetitions were performed on each capillary coating. Table 2 gathers the average migration time (〈tm〉), the average effective mobility (〈µep〉), the average electroosmotic mobility (〈µeo〉) measured by the Williams and Vigh method43 before each CE separation, and the average time-corrected peak area ratio (PA) relative to imidazole used (43) Williams, B. A.; Vigh, G. Anal. Chem. 1996, 68, 1174–1180.

as an internal standard. Table 2 also provides the RSD on 〈tm〉, 〈µep〉, 〈µeo〉, and PA. For a better comparison, average migration times and average effective mobilities were determined by integration of the entire electrophoretic profile. On the whole, the repeatability results are relatively good for the four neutral coatings and for the three DGL generations, with RSD on migration times and effective mobilities lower than 3% (except for G3 on PDMA and PEG) and with RSD on PA lower than 5%. All the µeo values are lower than 3.0 × 10-9 V-1 m2 s-1 at pH 7.4 (except for G3 on PDMA), demonstrating the success in the preparation of the neutral polymer coatings and their robustness against DGL adsorption. As already observed for dendrimers,25 µep slightly decreases with increasing generation number. To get a better general comparison of the coatings, the average RSD values calculated on the three DGL generations (i.e., 30 runs for each capillary coating) are summarized in the last lines of Table 2 (average values). These summarized results show that the four coated capillaries lead to rather good results in terms of repeatability but the HPC coating seems to give the best results with the lowest RSD on migration times and mobilities (about 1%), the lowest electroosmotic mobility, and the lowest RSD on PA (3.8%). Finally, electrokinetic and hydrodynamic injections were compared in terms of time-corrected peak area repeatability. With the use of an internal standard (imidazole), similar RSD values (between 3 and 4%) were obtained for electrokinetic (+7 kV, 5 s) and hydrodynamic (10 mbars, 3 s) injection modes, and whatever the studied sample matrix (electrolyte or water). Without internal standards, RSD on the time-corrected PA was much lower for electrokinetic injection 6.2% than for hydrodynamic injection 13.3% on the 33.5 cm × 100 µm i.d. capillaries. This is due to the difficulty for the CE apparatus to control the low injection pressure (10 mbar) and small injection times (3 s) required for a “normal” injection volume (lower than 1% of the total capillary volume). Limits of detection (signal-to-noise of 3) of G1 to G3 on HPC coated capillary with hydrodynamic injection mode was, respectively, of 0.12, 0.098, and 0.060 g/L. Study of the Interaction between G3 and Anionic Multicharged Biomolecules (AMP/ATP). FACE of mixtures of ATP (or AMP) and G3 were performed on a HPC coated capillary with negative polarity in a tris-borate buffer at pH 7.4. In these electrophoretic conditions, only the free negatively charged ligand (L ) ATP or AMP) enters in the capillary. The positively Analytical Chemistry, Vol. 82, No. 17, September 1, 2010

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Figure 3. FACE of ATP, or AMP, and G3 mixtures at variable r ratios of negative charge to lysine residue (in moles). Experimental conditions: HPC coated capillary, 33.5 cm (25 cm to detector) × 100 µm i.d. Electrolyte: TRIS 13.6 mM + boric acid 150 mM pH 7.4. Applied voltage: -20 kV. Sample: variable ATP (or AMP) concentrations in a G3 solution prepared at 0.50 g/L (final concentration) in the electrolyte. pH of the sample solution was adjusted to 7.4 by 0.1 M NaOH due to the acid form of ATP (or AMP). Temperature: 25 °C.

Figure 4. Isotherms of adsorption (A) of AMP (b) or ATP (9) onto G3 and the corresponding Scatchard plots (B). n is the number of ligands (ATP or AMP) per substrate (G3) and [L] is the free ligand concentration. Experimental conditions as in Figure 3.

charged G3 and the ligand-G3 complexes do not enter in the capillary using negative polarity since the EOF is close to zero in the HPC coated capillary. It is worth noting that the use of an uncoated capillary would not have been possible since at pH 7-8 the negatively charged ligand would migrate in the counter-electroosmotic flow (with positive polarity). In this mode, cationic and neutral complexes would enter in the capillary and would strongly interact onto the fused silica capillary wall. This selective injection of one of the interacting partners is favorable to study the binding process between the G3 and anionic structures: All of the partners remain in the same solution and only a tiny amount of free anionic AMP or ATP is taken out from a large reservoir of the equilibrated mixture. In particular, the requisite of comigrating complex and free G3 to accurately evaluate the concentration of free ligand is here fulfilled.44 G3 is constituted of a number-average of 123 lysine residues. The maximum positive charge number per G3 is 124, including an average of 49 R amine groups and 75 ε amine groups. At pH 7.4, most of the ε amine groups are protonated, while only a part of the R amine groups are protonated. At pH 7.4, each ATP and AMP molecules, respectively, bear -3.4 and -1.8 negative charge numbers. Because of this difference in charge and in size of the ligand and the substrate, multisite interactions are expected with a relatively high number of ligands per G3.

We can define r as the ratio of negative charge number to the number of lysine residues (in moles) introduced in the mixture. As shown in Figure 3, the front height corresponding to the free ligand concentration present in the mixture at equilibrium increases with increasing r values. After measurement of the calibration curves for ATP and AMP, isotherms of adsorption representing the number of complexed ligands n per G3 were plotted (Figure 4A). Both isotherms display a rapid increase of n with [L] indicating a rather high ligand/substrate interaction constants. Surprinsigly, there is a kind of a discontinuity in the isotherms, especially in the case of ATP, suggesting some changes in the ligand/substrate interactions. This discontinuity is even better observed on the corresponding Scatchard plots (Figure 4B) representing n/[L] as a function of n. As demontrated by eq 1, the Scatchard plot allows the direct determination of the interaction constant k from the slope of the plot, and of the maximum number of ligands at saturation (nmax) from the intercept with the x-axis: n ) nmaxk - nk [L]

Alternatively, the binding isotherm can be directly fitted according to multisite model of independent energies.29 n)

(44) Winzor, D. J. Anal. Biochem. 2008, 383, 1–17.

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(1)

niki[L]

∑ 1 + k [L] i

i

(2)

Figure 5. Isotherms of adsorption (A) of AMP (O) or ATP (0) onto linear poly-L-lysine (PLL) and the corresponding Scatchard plots (B). n is the number of ligands (ATP or AMP) per substrate (PLL) and [L] is the free ligand concentration. Experimental conditions are the same as in Figure 3.

where i designates each class of the ni independent sites having the same constant ki. Clearly, the Scatchard plots evidence two regimes in the interaction G3/ligand interaction. It should be noted that the use of the Scatchard representation for the study of two independent classes of interaction sites has been already reported in the past.45 Up to n1 ) 30 ATP per G3, there is a strong interaction with an association constant k1,G3/ATP of 1.0 × 106 M-1. For higher n values, the interaction seems much weaker (k2,G3/ATP ∼ 1.0 × 104 M-1, n2 ∼ 10). This suggests that two classes of binding sites can be populated in the G3. Similarly with AMP, there is a first strong interaction (k1,G3/AMP ∼ 105 M-1) up to n1 ) 33 AMP per G3 and a much weaker interaction for higher n values (k2,G3/AMP ∼ 1.2 × 104 M-1). Nonlinear curve fitting of the isotherm with two types of binding sites leads to binding constants of k1,G3/AMP ) 1.0 × 105 M-1 (n1 ) 31) and k2,G3/AMP ∼ 1.0 × 104 M-1 (n2 ) 17) for the higher and lower affinities, respectively, in good agreement with the values derived from the Scatchard plot. On the whole, ATP interacts more strongly with G3 than AMP does, as suggested by the difference in charge of the ligands in the case of electrostatic interactions. The number of AMP molecules at saturation of the substrate is higher than ATP because AMP is less charged and it requires a higher number of molecules to neutralize the G3 charge. So far, different possible explanations can be given to explain the existence of two classes of independent sites between G3 and the ligands: (i) one class could be attributed to electrostatic interactions, while the second class could belong to hydrophobic sites of the polymer; (ii) the two classes of independent sites could belong to the two families of amine groups in the dendrimer (R and ε amine groups) that have different pKa and thus different charges. In the first assumption, hydrophobic interactions between poly-L-lysines and ATP (or AMP) could come from the presence of the -(CH2)4- lateral chain in the poly-L-lysine and from the heterocycle in the base. It is worth noting that noncooperative effects due, for example, to a progressive neutralization of the polycationic charge with increasing n values may not be predominant since the linear domain of the Scatchard plot up to ∼30 bounded ligands per G3 suggests that a homogeneous class of binding sites of high (45) Klotzt, I. M.; Hunston, D. L. Biochemistry 1971, 10, 3065–3069.

affinity can be populated. As 30 ATP represents at least 80% of neutralization of the charge borne by the lysine residues in G3, noncooperative effects due to charge neutralization would affect the linearity of the Scatchard plot for n values much lower than 30. To get a better insight on the possible explanations for the two classes of independent sites, G3 was replaced by a linear polylysine (PLL, number average DP ) 100) having a similar number of lysine residues but with almost only ε amine groups (and one R amine group). Comparison between G3 and Linear Poly-L-lysine. Isotherms of adsorption and the corresponding Scatchard plots for PLL are displayed in Figure 5. Similarly to G3, Scatchard plots can be divided into two zones: (i) a strong interaction zone up to n1 ∼ 20 and a much weaker interaction for higher n values. The transition between the two zones was observed at lower n values for PLL than for G3, but this can be explained by the difference in the total number of lysine residues between G3 (123) and PLL (100). PLL/ligand constant values in the strong interaction zone are very close to G3 (k1,PLL/ATP ∼ 7.0 × 105 M-1 (n1 ) 21) and k1,PLL/AMP ∼ 9.7 × 104 M-1 (n1 ∼ 23)). Therefore, the second aforementioned assumption proposed to explain the two different classes of interaction sites based on the difference in the R/ε amine groups can be discarded. To better compare the stoichiometry of the interactions, the number of negative charge per lysine residue was plotted for both G3 and PLL in Figure 6. Higher complexation capacity of G3 compared to the linear PLL can be observed. G3 can complex up to 20% more of ATP and about 10% more of AMP than PLL for a given number of lysine residues. This supports that the cavities inside the DGL may play an important role in the capacity of complexation of the polymer. This specificity is even more impressive if one considers that the DGL (G3) should be less charged than the PLL at pH 7.4 due to the lower pKa values of the R amine groups that are not present in the linear PLL structure. The existence of two classes of interaction sites is also in agreement with a number of ATP/Lys residues of about 1.2 at saturation on the G3. This suggests that electrostatic interactions could not be the sole cause of interactions, otherwise this ratio could not be higher than unity, or even lower, if we consider the partial dissociation of the R amino groups on G3. Analytical Chemistry, Vol. 82, No. 17, September 1, 2010

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data obtained by ACE would be required to have a model for the variation of the mobility of the complex with the number n of ligands linked to the complex. Because of the complexity of this issue in the present case, and despite the good agreement in the trends observed by FACE and ACE, it is not possible to determine the association constants directly from mobility shifts observed in ACE experiments. This supports that the proposed frontal analysis protocol is particularly attractive to estimate binding parameters between oppositely charged entities.

Figure 6. Number of negative charges per lysine residue in the complex versus the free ligand concentration. Experimental conditions as in Figure 3.

Figure 7. Variation of effective mobility of G3 versus the AMP concentration in the electrolyte. Experimental conditions: HPC coated capillary 33.5 cm (25 cm to detector) × 100 µm i.d. Electrolyte: TRIS 13.6 mM + boric acid 150 mM + variable AMP concentration, pH 7.4. Applied voltage: +20 kV. Hydrodynamic injection: 10 mbar, 3 s. Sample: 2.0 g/L G3 in the electrolyte. Temperature: 25 °C. Dashed line corresponds to the occupation of the sites of higher affinity, while the dotted line corresponds to the occupation of the sites of lower affinity.

Study of G3/AMP Interaction by ACE. Finally, ACE has been performed on an HPC coated capillary by injecting a G3 plug in background electrolyte containing different concentrations in AMP. As seen in the insert of Figure 7, the electrophoretic profiles did not show any evidence of undesirable interactions with the capillary wall, demonstrating the interest of using neutral coated capillaries for studying interactions between oppositely charged compounds. The effective mobility of G3 versus the AMP concentration was plotted in Figure 7. According to the constant values determined by frontal analysis, the occupancy of the sites pertaining to the higher affinity interaction increases up to 90% in the 0-60 µM range, when the sites of lower affinity remain below 10% of occupancy (see Figure 7). In relation, the mobility of the G3 is strongly affected in that concentration range. Above 60 µM, the sites of higher affinity are almost saturated and the ones of lower affinity are moderately and linearly populated. Similarly, the variation of the mobility of G3 follows the occupancy of the sites of lower affinity. It is worth noting that in the case of complicated multisite complexations, the determination of the association constants from the effective mobility

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CONCLUSIONS Four different neutral polymer coatings were compared for CE analysis of cationic dendrigraft poly-L-lysines at pH 7.4. HPC coating was found to be the best for CE analysis of DGL at pH 7.4. With the use of this coating, it has been possible to study the interaction of the third generation of DGL (G3) with negatively multicharged biomolecules (AMP and ATP) by FACE and ACE. The neutral coating is a prerequisite to avoid any adsorption of one of the two interacting partners on the capillary surface. Of the two CE techniques investigated, FACE was found to be the most appropriated methodology for determining the association constants of the oppositely charged partners. FACE is straightforward and only requires the determination of the free concentration in ligand for getting the adsorption isotherm and for plotting the Scatchard plot that leads to the determination of the association constants. FACE demonstrated the existence of two different classes of interaction sites that were assigned to electrostatic (high affinity) and hydrophobic interactions (lower affinity). Similar association constants were determined for G3 and linear PLL; however, the dendrigraft poly-L-lysine has a higher capacity of complexation. The number of interaction sites in G3 is 10-20% higher than for the linear PLL. This could be due to the cage effect of the dendrigraft polymer as compared to the linear poly-L-lysine. The use of robust neutral coated capillaries at pH close to neutrality opens up to possibility to study interactions between oppositely charged polyelectrolytes in physiological conditions. To this respect, it would be crucial to evaluate what is the highest value of association constant that could be determined using this approach in regards to the sensitivity of detection of each partner. ACKNOWLEDGMENT H.C. gratefully acknowledges the support from the Re´gion Languedoc-Roussillon for the fellowship “Chercheurs d’Avenir”. We gratefully acknowledge the support from the Scientific Council of the University of Montpellier 2 for the postdoctoral grant to T.Z. We also thank Colcom for the funding of the fellowship for F.O. and for providing the DGL. SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

Received for review June 3, 2010. Accepted July 17, 2010. AC101473G