pubs.acs.org/Langmuir © 2010 American Chemical Society
Adsorbents with High Selectivity for Uremic Middle Molecular Peptides Containing the Asp-Phe-Leu-Ala-Glu Sequence Yitao Qiao, Jianxin Zhao, Pinglin Li, Jun Wang, Jing Feng, Wei Wang, Hongwei Sun, Yi Ma, and Zhi Yuan* Key Laboratory of Functional Polymer Materials, Ministry of Education, College of Chemistry, Nankai University, Tianjin 300071, PR China Received November 11, 2009. Revised Manuscript Received February 1, 2010 Asp-Phe-Leu-Ala-Glu (DE5) is a frequent sequence of many toxic middle molecular peptides that accumulate in uremic patients. To eliminate these peptides by hemoperfusion, three adsorbents (CP1-Zn2þ, CP2-Zn2þ, and CP3-Zn2þ) were designed on the basis of coordination and hydrophobic interactions. Adsorption experiments indicated that CP2Zn2þ had the highest affinity for DE5 among these three adsorbents. Also, the adsorption capacity of CP2-Zn2þ in DE5 and DE5-containing peptides was about 2-6 times higher than that of peptides without the DE5 sequence. Linear polymers bearing the same functional groups of the adsorbents were used as models to study the adsorption mechanism via isothermal titration calorimetry (ITC) and computer-aided analyses. The results indicated that coordination and hydrophobic interactions played the most important roles in their affinity. When two carboxyl moieties on Asp and Glu residues coordinated to CP2-Zn2þ, the hydrophobic interaction took place by the aggregation of the hydrophobic amino acid residues with phenyl group on CP2-Zn2þ. The optimal collaboration of these interactions led to the tight binding and selective adsorption of DE5-containing peptides onto CP2-Zn2þ. These results may provide new insight into the design of affinity adsorbents for peptides containing DE5-like sequences.
1. Introduction Adsorption technology has become increasingly important owing to its wide range of applications in the separation of toxic compounds from environmental and industrial waste. This technology has also been used for the elimination of toxins that accumulate in the human body.1-3 Affinity adsorbents, with specific adsorption and high elimination efficiency, have attracted much attention in this field,4-6 and researchers are now focused on the design of affinity adsorbents for the specific adsorption of target toxins. There are two main types of affinity adsorbent ligands: biomolecules and synthetic ligands. Bioactive ligand adsorbents have good selectivity and high capacity, but they are usually inconvenient because of the low structural stability and high cost of biomolecules. The advantages of synthetic ligands are stability and low cost, and they could be promising for the preparation of affinity adsorbents. Traditionally, to identify a proper affinity adsorbent, different synthetic ligands are immobilized onto matrices and the more efficient adsorbents are screened out by adsorption experiments; however, this approach is time-consuming and labor-intensive. Recently, the design of affinity adsorbents for specific targets has attracted much interest and has been extensively developed. Computer simulation has been used to find
a more efficient ligand as reported by Labrou and Liu.7,8 Molecular recognition theory has also been used to direct the design of affinity adsorbents to improve adsorption selectivity.9 The accumulation of metabolites in the human body is thought to be toxic and contributes to many pathological changes in patients with organ failure.10,11 These toxins could be eliminated by hemodialysis or hemoperfusion; for example, low-density lipoprotein could be removed by adsorbents with sulfonic and cholesterol groups.12 According to the middle molecule hypothesis,13 middle-molecular-weight toxins ranging from 500 to 5000 Da are the main toxic substances that accumulate in uremic patients, and middle molecular peptides have been assumed to be one type of major uremic toxin. Kaplan et al. reported that of the 51 middle molecular peptides isolated from the serums of uremic patients 38 contain DE5 sequences.14 It seems that DE5 is related to the uremia, so if there are efficient adsorbents that can eliminate DE5-containing peptides by hemoperfusion, this will be very helpful in the treatment of uremia. In this work, the frequent DE5 sequence was used as the target peptide. On the basis of the structural features of DE5, three adsorbents, CP1-Zn2þ, CP2-Zn2þ, and CP3-Zn2þ, were designed and synthesized on the basis of the coordination and hydrophobic interactions whereas CP4-Zn2þ and CP5-Zn2þ, bearing only coordination groups or hydrophobic groups, were used as the controls. Static adsorption experiments were performed to
*Corresponding author. Tel: þ86-22-23501164. Fax: þ86-22-23503510. E-mail:
[email protected]. (1) Davankov, V.; Tsyurupa, M.; Ilyin, M.; Pavlova, L. J. Chromatogr., A 2002, 956, 65–73. (2) Hughes, R. D. Int. J. Artif. Organs 2002, 25, 911–917. (3) Rozga, J. Xenotransplantation 2006, 13, 380–389. (4) Duarte, I. S.; Zollner, R. L.; Bueno, S. M. A. Artif. Organs 2006, 30, 606–614. (5) Tani, N. Artif. Organs 1996, 20, 922–929. (6) Zhong, W.; Huang, W.; Li, J. H.; Hou, G. H.; Fang, J.; Yuan, Z. J. Chromatogr., B 2007, 852, 288–292. (7) Labrou, N. E.; Eliopoulos, E.; Clonis, Y. D. Biotechnol. Bioeng. 1999, 63, 322–332. (8) Liu, F. F.; Wang, T.; Dong, X. Y.; Sun, Y. J. Chromatogr., A 2007, 1146, 41–50.
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(9) Platis, D.; Sotriffer, C. A .; Clonis, Y.; Labrou, N. E. J. Chromatogr., A 2006, 1128, 138–151. (10) Vanholder, R.; De Smet, R. J. Am. Soc. Nephrol. 1999, 10, 1815–1823. (11) Mitzner, S. R.; Stange, J.; Klammt, S.; Peszynski, P.; Schmidt, R.; NoldgeSchomburg, G. J. Am. Soc. Nephrol. 2001, 12, S75–S82. (12) Wang, S. Q.; Yu, Y. T.; Cui, T.; Cheng, Y. Biomaterials 2003, 24, 2799– 2802. (13) Babb, A. L.; Farrell, P. C.; Uvelli, D. A.; Scribner, B. H. ASAIO Trans. 1972, 18, 98–105. (14) Kaplan, B.; Cojocaru, M.; Unsworth, E.; Knecht, A.; Martin, B. M. J. Chromatogr., B 2003, 796, 141–153.
Published on Web 03/04/2010
DOI: 10.1021/la904272e
7181
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examine their adsorption ability with respect to DE5 and DE5containing peptides. Soluble linear polymers bearing the same functional groups were used as models of the adsorbents to determine the adsorption mechanism using isothermal titration calorimetry (ITC) and computer simulation.
2. Experimental Section 2.1. Materials. Tetrahydrofuran (THF) was dried over sodium/ benzophenone and distilled immediately before use. Acrylamide (AM) was recrystallized from acetone, 2,20 -azobisisobutyronitrile (AIBN) was recrystallized from ethanol, and 2,6-diaminopyridine was recrystallized from toluene. Phenylacetyl chloride and 3-phenylpropanoyl chloride were prepared by the reaction of thionyl chloride with the corresponding carboxylic acid. Acryloyl chloride was prepared by the reaction of benzoyl chloride with acrylic acid. Peptides were purchased from GL Biochem (Shanghai) Ltd. 2-Aminopyridine was purchased from Shanghai Chemical Co. and recrystallized in petroleum ether. The other reagents and solvents were commercially available and used without purification.
2.2. General Procedure for the Synthesis of Functional Monomers. 2.2.1. Synthesis of 2-Acryloylamido-6-benzoylamindo-pyridine (M1). M1, M2, and M3 were synthesized from the corresponding acylchloride by reaction with 2,6-diaminopyridine in tetrahydrofuran (THF) at -10 °C ((1 °C) under a nitrogen atmosphere.15 Typical synthesis procedures are as follows. Benzoyl chloride (3.05 mL, 26.25 mmol) in tetrahydrofuran (100 mL) was added dropwise (finished within 5 h) to a solution of 2,6-diaminopyridine (2.73 g, 25 mmol) and triethylamine (3.85 mL, 26.25 mmol) in tetrahydrofuran (150 mL) at -10 °C ((1 °C) under a nitrogen atmosphere. After the mixture was stirred overnight, triethylamine (3.49 mL, 23.75 mmol) was added to the mixture and acryloyl chloride (1.93 mL, 23.75 mmol) was then added dropwise; the mixture was again stirred overnight. After being filtered to remove the deposited material, the mixture was decolored by acticarbon for 6 h at room temperature. The solution was concentrated to give a reddish-brown oil. The residue was purified by column chromatography, eluting with 8:1 dichloromethane/acetic ether to give 2-acryloylamido-6-benzoylamindo-pyridine (M1, 3.55 g, 53%) as a white solid. 1H NMR (400 MHz, CDCl3) δ: 5.81 (d, 1H), 6.25 (m, 1H), 6.46 (d, 1H), 7.49 (t, 2H), 7.57 (t, 1H), 7.78 (t, 1H), 7.90 (d, 2H), 7.99 (d, 1H), 8.08 (d, 1H). ESI-MS: [M þ H]þ m/z = 268.34.
2.2.2. Synthesis of 2-Acryloylamido-6-phenylacetamidopyridine (M2). Compound M2 was prepared from phenylacetyl chloride according to the preparation procedure of M1. Yield: 41%. The mobile phase of column chromatography was 10:1 dichloromethane/acetone. 1H NMR (400 MHz, CDCl3) δ: 3.74 (s, 2H), 5.76 (d, 1H), 6.21 (d, 1H), 6.44 (d, 1H), 7.33-7.92 (m, 8H). ESI-MS: [M þ H]þ m/z = 282.11.
2.2.3. Synthesis of 2-Acryloylamido-6-hydrocinnamamidopyridine (M3). Compound M3 was prepared from 3-phenylpropanoyl chloride according to the preparation procedure of M1. Yield: 48%. The mobile phase of column chromatography was 12:1 dichloromethane/acetic ether. 1H NMR (400 MHz, CDCl3) δ: 2.67 (t, 2H), 3.04 (t, 2H), 5.77 (d, 1H), 6.23 (d, 1H), 6.41 (d, 1H), 7.18-7.31 (m, 5H), 7.70 (t, 1H), 7.89-7.97 (m, 2H). ESI-MS: [M þ H]þ m/z = 296.20.
2.2.4. Synthesis of N-(6-Phenylacetylaminobenzen-2-yl)acrylamide (M4). Phenylacetyl chloride (3.48 mL, 26.25 mmol)
Qiao et al. (3.49 mL, 23.75 mmol) was added. After that, acryloyl chloride (1.93 mL, 23.75 mmol) was added dropwise. The mixture was stirred at -10 °C for another 12 h. After being filtered to remove the deposited material, the solution was decolorized with activated charcoal for 6 h at room temperature. The solution was concentrated under reduced pressure and purified by column chromatography (5:1 CH2Cl2/EtOAc) to give M4 as a white solid (5.26 g, 79%). 1H NMR (400 MHz, CDCl3) δ: 7.31-7.48 (m, 5H), 6.53-6.56 (m, 1H), 6.39 (d, 1H), 5.84 (d, 1H), 3.75 (s, 2H). ESIMS: [M þ H]þ m/z = 281.08. 2.2.5. Syntheses of 2-Acryloylamidopyridine (M5). Compound M5 was prepared from acryloyl chloride and amidopyridine according to the literature.16 Yield: 4.38 g, 61%. 1H NMR (400 MHz, CDCl3) δ: 8.56 (s, 1H), 8.29 (d, 1H), 7.72 (t, 1H), 7.05(d, 1H), 6.46 (d, 1H), 6.25 (m, 1H), 5.81 (d, 1H). ESI-MS: [M þ H]þ m/z = 149.28.
2.3. Preparation of Adsorbents (CP1, CP2, CP3, CP4, and CP5). The functional monomer (80% molar fraction),
acrylamide (10% molar fraction), cross-linking agent N,N0 -methylenebisacrylamide (10% molar fraction), AIBN (w = 3%), and THF (concentration of monomer, 0.1 g/mL) were placed in a round-bottomed flask under a nitrogen atmosphere with a magnetic stir bar. The reaction was carried out at 60 °C in an oil bath for 24 h. The filtered precipitate was extracted in a Soxhlet extractor successively by acetone and distilled water for 48 h, respectively. The final adsorbents were ground and treated with standard sieves (80-100 meshes).
2.4. Preparation of Linear Polymers (P1, P2, P3, P4, and P5). The linear polymers were synthesized and purified accord-
ing to our previous work.15 Briefly, the functional monomer (10% molar fraction), acrylamide (90% molar fraction), AIBN (w = 3%), and 1:1 v/v THF/H2O (concentration of monomer, 0.1 g/mL) were placed in a round-bottomed flask under a nitrogen atmosphere with a magnetic stir bar. The reaction was carried out at 60 °C in an oil bath for 24 h. The crude linear polymer was obtained by precipitation in acetone. Then, the crude product was purified by reprecipitation in acetone three times until no monomer remained as analyzed by 1H NMR. The mole fractions of the functional monomer in P1, P2, P3, P4, and P5 were 4.2, 2.7, 3.0, 3.6, and 7.7%, respectively, as determined by 1H NMR. 2.5. ITC Experiments. ITC titration was performed on a VP-ITC microcalorimeter (MicoCal Inc., Northampton, MA). In general, a solution of the peptide (1 mM) was injected in portions (7 μL 39 times) into a solution (cell volume = 1.4685 mL) of the polymer (0.05 mM, calculated by the number of functional monomer repeat units) or polymer (0.05 mM)-Zn2þ (0.165 mM) under different conditions. The heat flow was recorded, plotted against time, and converted into enthalpy (ΔH) by integration of the appropriate reaction peaks. Dilution effects were eliminated by the subtraction of the data from a blank experiment. All of the data were calculated with Origin ITC data-analysis software using one set of site models. 2.6. Adsorption Experiments. Dry adsorbent (0.1 g) was incubated with ZnCl2 solution (0.66 mM, 10 mL) and shaken for 24 h at 30 °C. The peptide (0.2 mM) was then added with further shaking. The peptide absorption was determined by a Unico UV4802 spectrometer at 220 nm. The control experiments were carried out under the same conditions. The adsorption capacity was calculated from the equation Ac ¼
ðc1 - c2 ÞMV m
in THF (100 mL) was added dropwise to a solution of 2,6diaminobenzene (2.70 g, 25 mmol) and triethylamine (3.85 mL, 26.25 mmol) in THF (150 mL) within 4.5 h at -10 °C. The mixture was stirred at -10 °C for 12 h, and then triethylamine
where Ac stands for the adsorption capacity, c1 and c2 stand for the peptide concentrations before and after adsorption, respectively, V stands for the volume of solution used in adsorption,
(15) Zhao, J. X.; Qiao, Y. T.; Feng, J.; Luo, Z. F.; Yuan, Z. Chem. J. Chin. Univ. 2008, 29, 658–660.
(16) Qiao, Y. T.; Wei, Z.; Feng, J.; Chen, Y. C.; Li, P. L.; Wang, W.; Ma, Y.; Yuan, Z. J. Sep. Sci. 2009, 32, 2462–2468.
7182 DOI: 10.1021/la904272e
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M stands for the peptide molar mass, and m stands for the mass of the dry adsorbent. All of the adsorption experiments were carried out in triplicate, and data are expressed as the mean ( SD. 2.7. Molecular Modeling Studies. Hartree-Fock (HF) and density functional theory (DFT) calculations were performed with the Gaussian 03 suite of programs.17 N-(6-Formylaminopyridin-2-yl)-2-phenylacetamide (FPP) was selected as the modeling complexes for the adsorbent. The complex structure of FPP-Zn2þ was built and optimized at the HF/6-31G (D) level. A Silicon Graphics SGI Indigo 2 workstation was used for the simulation of the adsorption. Molecular building, geometry optimization, and conformational searching were carried out using molecular modeling package Sybyl, version 6.6. In the calculations, the molecular mechanics and molecular dynamics methods were used.18
3. Results and Discussion 3.1. Design of Functional Monomers. To our knowledge, the adsorption of proteins and peptides onto adsorbents is usually based on various noncovalent interactions, including electrostatic forces, coordination interactions, hydrophobic interactions, and hydrogen bonding. Hence, the introduction of functional groups bearing these noncovalent interaction binding sites to adsorbents may improve their adsorption ability. To achieve high affinity, the collaboration of multiple interactins is also a promising strategy.19 In this work, target peptide DE5 has a special structure with hydrophobic amino acid residues in the middle (Phe-Leu) and an acidic amino acid residue (Asp and Glu) at each terminus. Considering the structural feature of DE5, the collaboration of coordination and hydrophobic interactions was expected to have a substantial effect on peptide adsorption. Metal-coordination interactions show a high affinity for electron-enriched groups such as phosphate and carbonate on peptides20-22 whereas hydrophobic interactions are also an important driving force in the adsorption of hydrophobic peptides in aqueous solution. Moreover, these interactions could be efficiently achieved in aqueous solution. It may therefore be beneficial to utilize these interactions in improving the adsorption of DE5 in aqueous solution. Until now, the coordination interaction between pyridyl group and divalent transition-metal ions has been widely applied in binding studies,23 and the phenyl group has been involved in many hydrophobic interactions owing to its hydrophobicity. Thus, functional monomers bearing the coordination interaction (17) Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision C.02; Gaussian, Inc.: Wallingford, CT, 2004.Frisch, M. J.; Trucks, G. W.; Schlegel, H. B. et al. . Gaussian 03, Revision C.02, Gaussian, Inc., Wallingford CT, 2004. (18) Vaidyanathan, J.; Vaidyanathan, T. K.; Yadav, P.; Linaras, C. E. Biomaterials 2001, 22, 2911–2920. (19) Peczuh, M. W.; Hamilton, A. D. Chem. Rev. 2000, 100, 2479–2494. (20) Yamaguchi, S.; Yoshimura, I.; Kohira, T.; Tamaru, S.; Hamachi, I. J. Am. Chem. Soc. 2005, 127, 11835–11841. (21) Ojida, A.; Mito-oka, Y.; Inoue, M.; Hamachi, I. J. Am. Chem. Soc. 2002, 124, 6256–6260. (22) Imai, H.; Munakata, H.; Uemori, Y.; Sakura, N. Inorg. Chem. 2004, 43, 1211–1213. (23) Kruppa, M.; Konig, B. Chem. Rev. 2006, 106, 3520–3560.
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Figure 1. Adsorption capacities of DE5 onto adsorbents at 30 °C (n = 3). Scheme 1. Chemical Structures of the Functional Monomers
group (M5), hydrophobic group (M4), and both coordination and hydrophobic groups (M1, M2, and M3) were designed and synthesized (Scheme 1). To facilitate geometrical matching, M1, M2, and M3 differed only in the distance between the terminal phenyl group and the pyridyl groups. 3.2. Adsorption Capacity of DE5. Polyacrylamide (PAM) has been extensively used in recent years for protein separation owing to its good biocompatibility and environmental stability. Moreover, hydrophilic PAM on the adsorbent can decrease the nonspecific adsorption of peptides.24 Previous work has confirmed that no obvious interaction occurs between PAM and DE5.15 Therefore, the corresponding adsorbents were synthesized by the copolymerization of functional monomers, acrylamide, and cross linker. We evaluated the adsorption capacities of CP1Zn2þ, CP2-Zn2þ, CP3-Zn2þ, CP4-Zn2þ, and CP5-Zn2þ for DE5 at 30 °C by static adsorption experiments. As shown in Figure 1, all static adsorption experiments reached an equilibrium state within 1.75 h. As expected, CP1-Zn2þ, CP2-Zn2þ, and CP3-Zn2þ bearing both coordination and hydrophobic groups exhibited better adsorption to DE5. In particular, CP2-Zn2þ had an optimal adsorption capacity for DE5, which was almost 8 times greater than that of CP4-Zn2þ or CP5-Zn2þ. It has been found that cross-linked polyacylamide showed neglected adsorption to DE5 (
