pubs.acs.org/Langmuir © 2009 American Chemical Society
Rapid Magnetic Catch-and-Release Purification by Hydrophobic Interactions Motoyuki Iijima,† Yuzuru Mikami,‡ Tomohiko Yoshioka,‡ Shokaku Kim,‡ Hidehiro Kamiya,† and Kazuhiro Chiba*,‡ † Institute of Symbiotic Science and Technology, Tokyo University of Agriculture and Technology, 2-24-16 Nakacho, Koganei, Tokyo 184-8588, Japan, and ‡Laboratory of Bio-organic Chemistry, Tokyo University of Agriculture and Technology, 3-5-8 Saiwaicho, Fuchu, Tokyo 183-8509, Japan
Received April 16, 2009. Revised Manuscript Received June 15, 2009 A reversible, conventional, and rapid purification method of hydrophobically tagged products using hydrophobic magnetic nanoparticles was developed. The reversible purification system entails simply controlling the polarity of solvents. First, for the catching procedure, poor solvents were added into a well-dispersed system of magnetic nanoparticles and tagged products. Once the poor solvents were added to the system, the products were recrystallized among the nanoparticles and the aggregation of magnetic nanoparticles occurred due to hydrophobic interactions. These aggregates with the products contained within them were able to be collected rapidly by magnets. Then, the releasing procedure can be easily performed by redispersing the collected aggregates into good solvents. The availability of this purification protocol was confirmed by using a hydrophobically tagged fluorescent model product. Furthermore, this rapid purification method was successfully applied to a peptide elongation reaction system which enabled the synthesis of peptides such as Leu-Enkephalin in high purity, in high yield, and in a short time.
1. Introduction Ever since Merrifield introduced the concept of solid-phase peptide synthesis,1 it has gained widespread use as a platform for the automated organic synthesis of polypeptides,2-5 oligonucleotides,6-9 and oligosaccharides.10-13 The great advantage of solidphase synthesis is the ease of handling during the purification process. However, there are still some issues to be addressed in the process, such as low reagent accessibility, solvent limitations due to swollen properties of resins, and difficulties in monitoring the reaction. Liquid-phase organic synthesis, on the other hand, with affinity tags and chromatographic separation, is also widely *Corresponding author. E-mail:
[email protected]. Telephone: 81-42367-5667. Fax: 81-42-360-7167.
(1) Merrifield, R. B. J. Am. Chem. Soc. 1963, 85, 2149. (2) Houghten, R. A.; Pinilla, C.; Blondelle, S. E.; Appel, J. R.; Dooley, C. T.; Cuervo, J. H. Nature 1991, 354, 84. (3) Dawson, P. E.; Muir, T. W.; Clark-Lewis, I; Kent, S. B. H. Science 1994, 266, 776. (4) Broyer, R. M.; Quaker, G. M.; Maynard, H. D. J. Am. Chem. Soc. 2008, 130, 1041. (5) Mosse, W. K. J.; Koppens, M. L.; Gengenbach, T. R.; Scanlon, D. B.; Gras, S. L.; Ducker, W. A. Langmuir 2009, 25, 1488. (6) Gait, M. J.; Sheppard, R. C. J. Am. Chem. Soc. 1976, 98, 8514. (7) Stein, C.; Cheng, Y. Science 1993, 261, 1004. (8) Varghese, O. P.; Barman, J.; Pathmasiri, W.; Plashkevych, O.; Honcharenko, D.; Chattopadhyaya, J. J. Am. Chem. Soc. 2006, 128, 15173. (9) Bouillon, C.; Meyer, A.; Vidal, S.; Jochum, A.; Chevolot, Y.; Cloarec, J.-P.; Praly, J.-P; Vasseur, J.-J.; Morvan, F. J. Org. Chem. 2006, 71, 4700. (10) Douglass, S. P.; Whitfield, D. M.; Kerpinsky, J. J. J. Am. Chem. Soc. 1991, 113, 5095. (11) Ban, L.; Mrksich, M. Angew. Chem., Int. Ed. 2008, 47, 3396. (12) Love, K. R.; Seeberger, P. H. Angew. Chem., Int. Ed. 2004, 43, 602. (13) Burt, J.; Dean, T.; Warriner, S. Chem. Commun. 2004, 4, 454. (14) Han, H.; Wolfe, M. M.; Brenner, S.; Janda, K. D. Proc. Natl. Acad. Sci. U. S.A. 1995, 92, 6419. (15) Ge, X.; Yang, D. S. C.; Trabbic-Carlson, K.; Kim, B.; Chilkoti, A.; Filipe, C. D. M. J. Am. Chem. Soc. 2005, 127, 11228. (16) Johnson, E. C. B.; Malito, E.; Shen, Y.; Rich, D.; Tang, W.-J.; Kent, S. B. H. J. Am. Chem. Soc. 2007, 129, 11480. (17) Manzoni, L.; Castelli, R. Org. Lett. 2006, 8, 955. (18) Fustero, S.; Garcia, S. A.; Chiva, G.; Sanz-Cervera, J. F.; del Pozo, C.; Acena, J. L. J. Org. Chem. 2006, 71, 3299.
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accepted because of the benefits of high reaction rate and the possibility of reaction monitoring.14-18 However, contrary to the solid-phase concept, there are some problems in the separation process such as yield loss, longer time, and higher cost of chromatographic separation. The introduction of a new process that combines the benefits of organic reactions in the liquid phase and the ease of separation in the solid phase should play an important role in the development of automated organic synthesis. Recently, magnetic nanoparticles (MNPs) have attracted much attention for separation and purification techniques because of their high surface area, reversible flocculation, and site selective collection by magnets.19-23 In most cases, the tagged product directly interacts with the nanoparticle itself or with a specific functional group introduced on the nanoparticle surface and can then be separated by magnets. Though the reported separation ratio is quite high, their release ratio is relatively low. If we can control both the separation and recovery processes of tagged products on MNPs repeatedly with ease, this technique should introduce a new concept for solid-phase and/or liquid-phase organic synthesis. In order to obtain a site selective reversible collection and recovery process of tagged products with ease, the use of hydrophobic/hydrophilic interactions between the MNPs and the products will be one of the simplest methods. For example, hydrophobic MNPs were used to control the reversible switching of electrochemical reactions at electrode surfaces. When hydrophobic MNPs were site selectively attracted on the electrodes by magnets, the electrochemical reactions were inhibited due (19) Xu, C.; Xu, K.; Gu, H.; Zhong, X.; Guo, Z.; Zheng, R.; Zhang, X.; Xu, B. J. Am. Chem. Soc. 2004, 126, 3392. (20) Lee, I. S.; Lee, N.; Park, J.; Kim, B. H.; Yi, Y.-W.; Kim, T.; Kim, T. K.; Lee, I. H.; Paik, S. R.; Hyeon, T. J. Am. Chem. Soc. 2006, 128, 10658. (21) Willner, I.; Katz, E. Langmuir 2006, 22, 1409. (22) Stevens, P. D.; Fan, J.; Gardimalla, H. M. R; Yen, M.; Gao, Y. Org. Lett. 2005, 7, 2085. (23) Bao, J.; Chen, W.; Liu, T.; Zhu, Y.; Jin, P.; Wang, L.; Liu, J.; Wei, Y.; Li, Y. ACS Nano 2007, 1, 293.
Published on Web 07/02/2009
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Figure 1. Concept of magnetic catch-and-release purification with hydrophobic interactions. (a) MNPs, tagged products, and byproducts are well dispersed/dissolved in a good solvent. (b) Aggregation of hydrophobic MNPs and recrystallization of hydrophobically tagged products among MNPs are induced by the addition of poor solvents. These aggregates can be easily collected by magnets. (c) Washing out the byproduct contained solvents by magnetic decantation. (d) Redispersion of MNPs and hydrophobically tagged products in good solvents.
to retraction of redox species from the electrodes by the formation of a hydrophobic thin film on the conducted support. On the contrary, electrochemical reactions switched on when MNPs were retracted from the electrodes and redox species were attracted to the electrodes.24,25 In this paper, we report the easy, rapid, and repeatable purification process of hydrophobically tagged products by hydrophobic Fe3O4 MNPs with hydrophobic interactions toward the conventional peptide elongation reaction. Figure 1 shows the concept of magnetic purification using hydrophobic interactions. First, by adding an adequate amount of poor solvent into a well dissolved and/or dispersed solution of tagged products, byproducts, and hydrophobic MNPs, the MNPs form aggregates and the dissolved hydrophobically tagged products selectively recrystallize among those aggregated MNPs due to hydrophobic interactions. Next, by the magnetic decantation procedure, the MNPs and the hydrophobically tagged products are attracted to the magnet while the byproducts remain in the solvent which is then washed out. Finally, by adding a good solvent into the collected aggregates, the MNPs and the tagged products easily redisperse and redissolve into these solvents by sonication. The resulting solution can then be applied to the next reaction between the tagged products and newly added reagents and purified by the same procedure.
2. Experimental Section 2.1. Materials. FeSO4 3 7H2O, FeCl3 3 6H2O, and 25 wt % NH4OH aqueous solution were purchased from Wako Pure Chemical Industry Ltd., Japan. Anhydrous tetrahydrofuran (THF, >99.5%) and anhydrous acetonitrile (>99.5%) were from Kanto Chemical Co., Inc. Diisopropylcarbodiimide (DIPCI, >97%), N,N-diisopropylethylamine (DIPEA, >98%), trifluoroacetic acid (TFA, >99.0%), and triisopropylsilane (TIS, >98.0%) were from Tokyo Chemical Industry, Ltd., Japan. 4Dimethylaminopyridine (DMAP, 99%) and 1,8-diazabicyclo[5.4.0]-undec-7-ene (DBU, 98%) were from Sigma-Aldrich, Inc. (24) Katz, E.; Sheeney-Haj-Ichia, L.; Basnar, B.; Felner, I.; Willner, I. Langmuir 2004, 20, 9714. (25) Katz, E.; Weizmann, Y.; Willner, I. J. Am. Chem. Soc. 2005, 127, 9191.
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N,N,N0 ,N0 -Tetramethyl-O-(benzotriazol-1-yl)uronium hexafluorophosphate (HBTU) and all Fmoc-amino acids were supplied from Watanabe Chemical Industries, Ltd. 1-Hydroxybenzotriazole (HOBt) and standard Leu-Enkephalin were purchased from Peptide Institute, Inc. All materials were used without further purifications. 2.2. Synthesis of Hydrophobic Fe3O4 MNPs. The hydrophobic Fe3O4 nanoparticles were prepared by a simple and conventional coprecipitation method.26 First, 0.765 g of FeSO4 3 7H2O and 1.23 g of FeCl3 3 6H2O were dissolved into 30 mL of deionized water. Then, with vigorous stirring, 3.75 mL of 25 wt % NH4OH aqueous solution was rapidly added. During raising the solution temperature up to 80 °C, 0.30 mL of oleic acid was carefully injected. After mixing for 1 h, 30 mL of toluene and an excess amount of NaCl was added into the solution with vigorous stirring, which obtained oleic acid capped Fe3O4 MNPs dispersed into the toluene phase. The toluene phase was separated, and the hydrophobic Fe3O4 MNPs were collected by magnetic decantation after adding 30 mL of acetone. The collected Fe3O4 MNPs were dried under vacuum overnight at 40 °C before further use.
2.3. Synthesis of Hydrophobically Tagged Pyrene Butyric Acid (1). The detail synthesis procedure of the hydrophobic
tag is reported elsewhere.27 For the synthesis of hydrophobically tagged pyrene butyric acid (1), 200 mg of hydrophobic tag and 140 mg of Fmoc-leucine was dissolved into 5 mL of THF. Then, 0.06 mL of DIPCI and a catalytic amount of DMAP were added into the THF solution. After mixing for 1 h, the white precipitate which appeared just after adding 30 mL of acetonitrile was collected by filtration. The collected white precipitate was dried under vacuum for 12 h. Next, 150 mg of the collected hydrophobically tagged Fmoc-Leucine was dissolved into 5 mL of THF with an addition of 0.07 mL of DBU. After stirring for 5 min, 30 mL of acetonitrole was added into the synthesis solution and the white precipitate was collected and dried in a way similar to the method described above which obtained hydrophibically tagged leucine. Finally, 120 mg of hydrophibically tagged leucine was redispersed into 5 mL of THF with an addition of 70 mg of 1pyrenebutyric acid, 30 mg of HOBt, and 60 mg of HBTU. After stirring for 1 h, 30 mL of acetonitrile was added into the synthesis solution and the white precipitate was collected and dried in a way similar to the method described above which obtained hydrophobically tagged pyrene butyric acid (1) in a yield of 93%.
2.4. Catch-and-Release Properties of Hydrophobically Tagged Pyrene Butyric Acid (1) by Using MNPs. The catch-and-release properties of hydrophobically tagged products on/from hydrophobic MNPs were tested by using fluorescent labeled products (1). First, 60 mg of hydrophobically tagged pyrene butyric acid (1) was dissolved into 3 mL of THF. Then 60 mg of hydrophobic NMPs was dispersed into this solution with sonication. As soon as 30 mL of acetonitrile was added into this solution, the homogeneously dispersed MNPs rapidly aggregated. These aggregated MNPs were collected by magnetic decantation and redispesed into THF. The catch-and-release properties of hydrophobically tagged pyrene butyric acid (1) to/ from MNPs were investigated by visible UV luminescent and Fourier transform infrared (FTIR) measurements (Nexus 470 (Thermo Fisher Scientific Co.)).
2.5. Peptide Elongation Reactions by Using Magnetic Catch-and-Release Purification Techniques. Two types of tripeptides (H-Phe-Phe-Phe-hydrophobic tag (5) and Fmoc-ProPro-Ile-hydrophobic tag (6)) and a pentapeptide (Leu-Enkephalin) were synthesized in order to ensure the availability of the catch-and-release purification to peptide elongation reactions. The synthesized peptides were characterized by TOF-MS (JEOL
(26) Sun, Y.; Ding, X.; Zheng, Z.; Cheng, X.; Hu, X.; Peng, Y. Chem. Commun. 2006, 26, 2765. (27) Tamaki, H.; Obata, T.; Azefu, Y.; Toma, K. Bull. Chem. Soc. Jpn. 2001, 74, 733.
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Figure 2. Procedure for synthesis of hydrophobically tagged triphenylalanine (5). JMS-T100LP), HPLC (JASCO CO 2067 Plus, JASCO PU 2080 Plus), and NMR (JEOL JNM-R600).
2.5.1. Synthesis of H-Phe-Phe-Phe-Hydrophobic Tag (5). For the synthesis of hydrophobically tagged triphenylalanine, hydrophobically tagged Fmoc-phenylalanine (2) was synthesized in a way similar to the synthesis of hydrophobically tagged Fmoc-leucine, where Fmoc-phenylalanine was used instead of Fmoc-leucine. For the peptide elongation of 2, 90 mg of 2 and 60 mg of hydrophobic Fe3O4 nanoparticles were dissolved into 3 mL of THF. After addition of 0.05 mL of DBU, the synthesis solution was stirred for 5 min. As soon as 30 mL of acetonitrile was added, the homogeneously dispersed Fe3O4 nanoparticles aggregated with hydrophobically tagged products and were separated by magnet. The separated Fe3O4 nanoparticles and hydrophobically tagged products were washed by redispersing into 20 mL of acetonitrile and separating by magnets for three times. After the washing procedure, the acetonirile was completely evaporated off from the magnetically collected nanoparticles and products (hydrophobically tagged phenylalanine (3)) under vacuum. These procedures occurred in one pot: a round-bottom flask. Next, 3 mL of THF, 60 mg of Fmoc-phenylalanine, 20 mg of HOBt, and 60 mg of HBTU was added into the flask where the dried Fe3O4 nanoparticles and hydrophobically tagged phenylalanine (3) were obtained. After stabilizing the synthesis solution by sonication, the solution was stirred for 1.5 h and 30 mL of acetonitrile was added. As soon as acetonitrile was added, the Fe3O4 nanoparticles and the product (hydrophobically tagged Fmoc-diphenylalanine (4)) aggregated and the resulting mixture was purified in a way similar to the method mentioned above. By repeating the deprotection of 4 and further peptide elongation by the method mentioned above, hydrophobically tagged triphenylalanine (5) was obtained. The final product was separated from Fe3O4 nanoparticles by redispersing the dried Fe3O4 nanoparticles/product mixture into acetone/THF solution and subjecting the it to further magnetic decantation. Since the final product and the Fe3O4 nanoparticles can dissolve and form weak flocculates in acetone/THF solvent, respectively, the final product can be separated. The overall yield of this product was 81% (Figure 2).
2.5.2. Synthesis of Fmoc-Pro-Pro-Ile-Hydrophobic Tag (6). Fmoc-Pro-Pro-Ile-hydrophobic tag (6) was prepared similarly to product 5. Hydrophobilally tagged Fmoc-isoleucine (90 mg) was deprotected by DBU and then further elongated with Fmocproline (55 mg) by using HBTU, HOBt, and DIPEA (25 μL) in THF. The magnetic catch-and-release purification was also performed in a similar way as that for product 5. Product 6 was obtained in a yield of 99%.
2.5.3. Synthesis of Fmoc-Tyr(tBu)-Gly-Gly-Phe-Leu-Hydrophobic Tag (7). Fmoc-Tyr(tBu)-Gly-Gly-Phe-Leu-hydrophobic tag (7) was prepared in a similar way as product 6, where 0.07 mmol of hydrophobilally tagged leucine was elongated with 0.15 mmol of Fmoc-phenylalanine, Fmoc-glycine, and Fmoctyrosine(tBu). This product was prepared in a yield of 86%. Furthermore, in order to enable the characterization of this Langmuir 2009, 25(18), 11043–11047
Figure 3. Magnetic catch-and-release purification of hydrophobically tagged pyrenebutyric acid (1). Images of (a) Fe3O4 nanoparticles dispersed in THF, (b) 1 dissolved in THF, (c) solution after addition of acetonitrile where Fe3O4 nanoparticles and 1 were dispersed into THF, and (d) solution after redispersion of magnetically collected Fe3O4 nanoparticles with 1 into THF. Panels (e) and (f) are the fluorescent microscope images from the solutions in (c) and (d), respectively. (Bar = 500 μm.) product by TOF-MS, Leu-Enkephalin (8) was prepared by the following manner. First, 7 (90 mg) was dissolved into 10 mL of THF with 25 μL of DBU and stirred for 10 min. Then the white flocculates that appeared after addition of excess acetonitrile were collected by filtration and dried. The dried product was dissolved into dichloromethane with TFA (19 mL), TIS (0.5 mL), and water (0.5 mL). After stirring for 2 h, an excess amount of acetonitrile was added and the white flocculates were separated. The residual solution was condensed, and the final product 8 was recrystallized using diethylether in a yield of 78%.
3. Results and Discussion 3.1. Magnetic Catch-and-Release Properties of Hydrophobically Tagged Products. Figure 3 shows an example of the magnetic catch-and-release purification of hydrophobically tagged pyrenebutyric acid (1). In Figure 3a, the oleic acid capped Fe3O4 nanoparticles were well dispersed into their primary particle sizes in THF and the Fe3O4 nanoparticles acted like a magnetic fluid. Thus, the nanoparticles could not be collected by magnets individually. In Figure 3b, the product 1 was well dissolved into THF and showed strong fluorescence in the solution. Figure 3c shows a photograph just after addition of acetonitrile into the THF solution of product 1 and Fe3O4 nanoparticles. As soon as the acetonitrile was added, the Fe3O4 nanoparticles rapidly formed aggregates, and the aggregates could be separated by magnets within 30 s. As shown in Figure 3c, fluorescence was observed only from the aggregated Fe3O4 nanoparticles while no fluorescence was observed from the bulk solution. Figure 3e shows the fluorescent microscope image of aggregated Fe3O4 nanoparticles in the mixed solvent of THF and acetonitrile. As in Figure 3e, a strong fluorescence was observed only from the largely aggregated Fe3O4 nanoparticles while no fluorescence was detected from the solvent, even in the dark field image, indicating that product 1 was successfully collected on the Fe3O4 nanoparticles. Figure 3d shows the THF solution DOI: 10.1021/la901351s
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Figure 4. FTIR spectrum of (a) product 1, (b) as prepared Fe3O4 nanoparticles, (c) Fe3O4 nanoparticles aggregated with 1, and (d) Fe3O4 after release of 1.
in which the separated Fe3O4 nanoparticles and product 1 were redispersed. The fluorescence from the solution was recovered. We can also see that there is no fluorescence from the Fe3O4 nanoparticles while that can be detected in the interparticle area, where the solvent is, as seen in the dark field image of Figure 3f. They show that product 1 was successfully released from the Fe3O4 nanoparticles. The Fe3O4 nanoparticles collected by magnets can be easily redispersed in THF with sonication and can be used again for multiple purification processes. Figure 4 shows the FTIR spectrum of product 1, as-synthesized Fe3O4 nanoparticles, Fe3O4 nanoparticles aggregated with product 1, and Fe3O4 nanoparticles after releasing product 1. From the as-synthesized Fe3O4 nanoparticles, strong peaks at 3006, 2962, 2824, 2853, 1716, and 1429 cm-1 were observed which were attributed to vinyl groups, νasCH3, νasCH2, νsCH2, νCdO, and δCH2, respectively, from oleic acid on Fe3O4 nanoparticles.28,29 From the spectrum of product 1, strong peaks at 3040, 2958, 2921, 2849, 1748, 1639, 1601, and 1512 cm-1 were detected which were attributed to νAr-H, νasCH3, νasCH2, νsCH2, νCdO (ester), νCdO (amide), aromatic ring breathing, and δNH (amide), respectively.30,31 In the case of Fe3O4 nanoparticles aggregated with product 1, typical peaks which correspond to νCdO (ester), νCdO (amide), δNH (amide), and aromatic ring breathing modes were detected at 1748, 1639, 1512, and 1589 cm-1, respectively. On the contrary, from the Fe3O4 nanoparticles after releasing product 1, peaks which are attributed to oleic acid (strong peaks at 3006, 2962, 2824, 2853, 1716, and 1429 cm-1 which were related to vinyl groups, νasCH3, νasCH2, νsCH2, νCdO, and δCH2, respectively) were only detected while no peaks which contribute to product 1 (νCdO (ester), νCdO (amide), δNH (amide), and aromatic ring breathing modes at 1748, 1639, 1512, and 1589 cm-1, respectively) remained. These results strongly indicate the successful collection and complete release of product 1. 3.2. Peptide Elongation Reaction with Magnetic Catchand-Release Purifications. The above-mentioned rapid catchand-release purification process has been applied to peptide elongation reactions. First, hydrophobically tagged Fmoc-phenylalanine (28) Baruwati, B.; Nadagouda, M. N.; Varma, R. S. J. Phys. Chem. C 2008, 112, 18399. (29) Wu, X.; Zou, L.; Yang, S.; Wang, D. J. Colloid Interface Sci. 2001, 239, 369. (30) Yamada, N.; Matsubara, K.; Narumi, K.; Sato, Y.; Koyama, E.; Ariga, K. Colloids Surf., A 2000, 169, 271. (31) Podstawka, E.; Kafarski, P.; Proniewicz, L. M. J. Phys. Chem. A 2008, 112, 11744.
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Figure 5. HPLC chromatogram of (a) H-Phe-Phe-Phe-hydrophobic tag (5), (b) Fmoc-Pro-Pro-Ile-hydrophobic tag (6), and FmocLeu-Enkephalin-hydrophobic tag (7).
(2) was dissolved into THF in the presence of oleic acid capped Fe3O4 nanoparticles. The deprotection reaction was then induced by adding DBU. The reaction was performed in the liquid phase, so that hydrophobically tagged phenylalanine (3) could be produced within 10 min. In order to purify the product 3, acetonitrile was added to the system. Fe3O4 nanoparticles started to form aggregates, product 3 was embedded among the aggregated Fe3O4 nanoparticles, and both were able to be rapidly separated by magnets. The collected Fe3O4 nanoparticles and product 3 were then redispersed into THF with the addition of another Fmoc-phenylalanine activated by DIPCI and DMAP. Hydrophobically tagged Fmoc-dipeptide 4 was produced within 1.5 h and rapidly purified by the catch-and-release method. By repeating this deprotection and condensation reaction scheme with the aid of purification with Fe3O4 nanoparticles, hydrophobically tagged triphenylalanine 5 was synthesized in one pot without any complicated techniques. Furthermore, various peptides such as hydrophobically tagged Fmoc-Ile-Pro-Pro (6) and hydrophobically tagged Fmoc-Leu-Enkepahaline (7) were also able to be prepared by similar methods. Figures 5 and 6 show the HPLC chromatogram and TOFMS spectrum of the prepared tetra/pentapeptides, respectively. The detection of a strong single peak in each sample from the HPLC analysis verifies that the purity of products 5-7 synthesized by our newly developed process is high. (Note: the strong peak at 4 min in Figure 5b and c are due to the solvent injection.) Furthermore, since the largest mass numbers measured in Figure 6 were the same as those of the final products, we can state that the expected products (6 and 7) were prepared by our newly developed process. It is expected that the small mass numbers detected in Figure 6 were due to the fragmentation of final products during the ionization procedure of TOF-MS analysis, because only a single peak was observed in HPLC chromatograms (Figure 5). In order to confirm the possibility of deprotection and tag detachment of hydrophobically tagged peptides, Leu-Enkephalin was synthesized from product 7. Figure 6c shows the TOF-MS spectrum of as-synthesized Leu-Enkephalin (8). The detected Langmuir 2009, 25(18), 11043–11047
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Figure 6. TOF-MS spectra of (a) H-Phe-Phe-Phe-hydrophobic tag (5) (calculated for C88H143N3O7 [M þ H]þ, 1355; found, 1355), (b) FmocPro-Pro-Ile-hydrophobic tag (6) (calculated for C92H151N3O9 [M þ Na]þ, 1465; found, 1465), and Leu-Enkephalin (8) (calculated for C28H37N5O7 [M þ H]þ, 556; found, 556).
and standard samples. (Note: the strong peak at 4 min is due to the solvent injection.) This verifies the successful synthesis of LeuEnkephalin by using our developed methods. A pentapeptide such as Leu-Enkephalin was able to be synthesized rapidly in high purity by applying the magnetic catch-and-release purification process to the Fmoc peptide elongation chemistry.
4. Conclusion
Figure 7. HPLC chromatogram of Leu-Enkephalin: (a) standard sample and (b) as-synthesized sample.
largest mass number was 556.27 which contributes to 8. Figure 7 shows the HPLC chromatograms of the synthesized (8) and standard samples of Leu-Enkephalin. A single strong peak was detected at a retention time of 13 min from both the as-synthesized
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We have developed a conventional and rapid purification technique of hydrophobically tagged products by using hydrophobic nanoparticles. This process comprises a homogeneous chemical reaction in the liquid phase and a rapid separation in the solid phase. We have successfully applied this technique to synthesize tri/pentapeptides in high purity, in high yield, and in a short time. This concept promises to be a useful process for automated organic synthesis. Supporting Information Available: 1H and 13C NMR spectra, HPLC methods, and DLS and TEM analysis of Fe3O4 nanoparticles. This material is available free of charge via the Internet at http://pubs.acs.org.
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