Rapid Microwave-Assisted Solid Phase Peptide Synthesis

592 SPECIAL TOPIC Rapid Microwave-Assisted Solid Phase Peptide Synthesis Rapid Máté Microwave-Assisted Solid Phase Peptide SynthesisErdélyi, a,b Adolf Gogoll* a a Department of Organic Chemistry, Uppsala University, Box 53, 75 2 Uppsala, Sweden Fax +46(8)52524; E-mail: mate@kemi.uu.se, adolf@kemi.uu.se b Department of Medicinal Chemistry, Uppsala University, Box 574, 75 23 Uppsala, Sweden Fax +46(8)474474 Received 20 May 2002 Dedicated to Prof. Hans-J. Schäfer on the occasion of his 65 th birthday Abstract: A microwave-assisted, rapid solid phase peptide synthesis procedure is presented. It has been applied to the coupling of sterically hindered Fmoc-protected amino acids yielding di- and tripeptides. Optimized conditions for a variety of coupling reagents are reported. Peptides were obtained rapidly (.5 20 min) and without racemization. Key words: SPPS, microwave, amino acids, peptide coupling, solid-phase Since Merrifield s pioneering work on solid phase peptide synthesis (SPPS), peptide preparation has almost exclusively been performed on resin. The generation of combinatorial libraries has caused a renaissance and growth of interest in solid supported chemistry. Parallel to the developments in combinatorial chemistry, it has been shown that the use of microwave heating can be advantageous in a large variety of organic reactions. 2 However, there have been only very few reports on the use of microwave heating in combination with solid phase synthesis, 3 possibly due to the requirement of special heavy-walled vials for microwave irradiation that makes resin handling rather complicated, and problems to control reaction conditions. In particular, enhancement of SPPS by the use of microwave heating has so far received little attention. Peptide synthesis performed in a kitchen microwave oven was published in 99. 4 However, the procedure described by Wang et al is not easily reproducible, because of the use of a commercial microwave oven for irradiation, and the lack of temperature control. The couplings were made using symmetric amino acid anhydrides or pre-formed N- hydroxybenzotriazole activated esters, yielding 2 4 fold reaction rate enhancements. In general, peptide chemistry is today limited to room temperature conditions, originating from the general belief of the heat-sensitivity of peptide coupling reagents (Table). Here, we describe a microwave-enhanced, rapid (.5 20 min) procedure for the coupling of sterically hindered amino acids on solid phase. The optimized conditions for a variety of common coupling reagents yielded a significant rate increase. Single mode irradiation with monitoring of temperature, pressure and irradiation power versus Synthesis 2002, No., Print: 22 08 2002. Art Id.437-20X,E;2002,0,,592,596,ftx,en;C0802SS.pdf. Georg Thieme Verlag Stuttgart New York ISSN 0039-788 Table Coupling Times and Temperatures for Peptide Synthesis on Rink s Amide Resin Employing a Variety of Coupling Reagents a Coupling Reagent PyBOP Mukaiyama s Reagent TBTU HATU Reaction time (min) 20 0 0.5 Temperature ( C) 0 30 0 0 Solvent DMF CH 2 Cl 2 DMF DMF a The average pressure for reactions performed in DMF and CH 2 Cl 2 was 2 bar and 6 8 bar, respectively. time was used throughout, making the procedure highly reproducible. Our goal was to investigate the compatibility of HATU, 5a TBTU, 5b PyBOP 5c and Mukaiyama s reagent 5d mediated couplings at high temperatures. Reaction conditions for the synthesis of a small tripeptide containing the three most hindered natural amino acids (Fmo-Thr-Val-Ile- NH 2 ), and the Fmoc-Ala-Ile-NH 2 or Fmoc-Thr-Ile-NH 2 dipeptides were optimized. The coupling of Fmoc-protected amino acids on polystyrene resin using Rink amide linker was performed. No degradation of the solid support was observed. Fast Fmoc-deprotection steps (5 minutes) were conducted at room temperature, and the coupling steps were performed using microwave irradiation. All coupling steps were monitored by qualitative ninhydrin test 6 and by LC-MS investigation of peptides cleaved from small amounts of the resin. Our optimized conditions are shown in the Table. The azobenzotriazole derivatives have shown an increased coupling efficiency with increasing temperature up to 0 C. At higher temperatures the decomposition of the reagents was indicated by the colour change of the reaction mixtures. In contrast with the usual SPPS procedures where double or triple coupling steps are needed for completion, under microwave conditions, coupling was complete already in a few minutes after a single coupling step. The absence of racemization during the high temperature treatment of amino acids in the presence of a base (i- Pr 2 NEt) was investigated by LC-MS and H NMR. The presence of only one peak in the chromatogram (Figure ) of the synthesized peptides suggested the absence of dias-

SPECIAL TOPIC Rapid Microwave-Assisted Solid Phase Peptide Synthesis 593 Figure The LC-MS chromatogram of Fmoc-Ala-Ile-NH 2 prepared by PyBOP mediated couplings at 0 C. tereomeric compounds. The H NMR spectrum of this material containing a single set of signals of the oligopeptide suggested the presence of only one diastereomer. As an example, the aliphatic region of the H NMR spectrum of a synthesized dipeptide is shown in Figure 2. The efficiency of the solid phase methodology was limited by the need of transfer between different reaction vessels to perform the coupling and washing steps. The high pressure generated by volatile components during reactions carried out with microwave irradiation makes the use of special heavy-walled vials highly recommendable. However, these vials are not suitable for filtration. Therefore, the reaction mixtures were transferred for washing and deprotection steps into plastic columns equipped with a polypropylene frit, causing some loss of resin. Moderate yields could therefore be obtained for the synthesis of the di- (PyBOP: 60%; Mukaiyama s reagent: 35%; TBTU: 4%; HATU 24%) and tripeptides (PyBOP: 48%; Mu- Figure 2 The aliphatic region of the H NMR spectrum of Fmoc-Ala-Ile-NH 2 showing the presence of one diastereomer after microwave assisted synthesis (PyBOP mediated couplings at 0 C).

594 M. Erdélyi, A. Gogoll SPECIAL TOPIC kaiyama s reagent: 65%; TBTU: 3%; HATU 42%). The efficiency of the solid phase methodology including the loading (PyBOP, i-pr 2 NEt, DMF, 0 C), cleavage (95% TFA in CH 2 Cl 2 ) and purification (preparative LC-MS) steps was determined by attachment and cleavage of a single Fmoc-Ile to the resin, yielding 60% of the expected product. The efficiency of the methodology might be increased by instrumental improvements, e.g., by the development of heavy-walled vials more compatible with the handling of resins used in solid phase synthesis. This synthetic method will be of considerable interest for incorporation of sterically hindered and deactivated nonnatural amino acids in peptide synthesis on solid phase. All reactions were conducted in heavy-walled glass Smith Process Vials sealed with aluminum crimp caps fitted with a silicon septum. The inner diameter of the vial filled to the height of 3.5 cm was.3 cm. The microwave heating was performed in a Smith Synthesizer single mode microwave cavity producing continuous irradiation at 2450 MHz (Personal Chemistry AB, Uppsala, Sweden). Reaction mixtures were stirred with a magnetic stir bar during the irradiation. The temperature, pressure and irradiation power were monitored during the course of the reaction. The average pressure during the reaction run in DMF was -2 bar, for the reactions run in CH 2 Cl 2 it was 6 8 bar. After completed irradiation, the reaction tube was cooled with high-pressure air until the temperature had fallen below 39 C (ca. 2 min). H NMR spectra were recorded for CD 3 CN solutions at 400 MHz (Jeol JNM EX400 spectrometer) at r.t. Chemical shifts are referenced indirectly to TMS via the residual solvent signal (d =2.0). The H NMR spectra of all synthesized compounds have been fully assigned using data from phase-sensitive DQF- COSY 7 and NOESY 8 experiments. The ESI-MS spectra of the peptides were obtained with a Finnigan ThermoQuest AQA mass spectrometer (ESI 30eV, probe temperature 00 C) equipped with a Gilson 322-H2 GradientPump system, an SB-C8 analytical and an SB-C8 (5 mm, 2.2 mm, 50 mm) preparative column. A H 2 O MeCN formic acid (0.05%) mobile phase was used with a gradient of 20%-80% MeCN during 7 minutes on the analytical column, and 30 80 minutes on the preparative column. The starting materials were purchased from commercial suppliers and were used without purification with the exception of CH 2 Cl 2 which was freshly distilled over calcium hydride. Fmoc-Ala, Fmoc- Ile, Fmoc-Val, PyBOP, and Rink amide MBHA resin were obtained from Novabiochem, Fmoc-Thr(t-Bu)-OH from Alexis Biochemicals (USA). TBTU was from Richelieu Biotechnologies (Canada). HATU was purchased from PE Biosystems (United Kingdom). Trifluoroacetic acid (99%), N,N-diisopropylethylamine (redistilled 99.5%), and piperidine (99%) were from Aldrich (Germany). DMF was obtained from Fluka (Denmark). Fmoc-Thr-Val-Ile-NH 2 (); Method A Procedure I Rink amide resin (300 mg, 0.78 mmol/g, 0.234 mmol) was treated with 20% piperidine in DMF (5 ml for 0 and 5 minutes) in a column equipped with polypropylene frit placed in an overhead mixer. The soln was then drained, and the resin was washed with DMF (3 5 ml) and CH 2 Cl 2 (3 5 ml). Procedure II The resin from procedure I was transferred into a Smith Process Vial and Fmoc-Ile (248 mg, 0.70 mmol), PyBOP (343 mg, 0.70 mmol), i-pr 2 NEt (0.25 ml,.40 mmol) and DMF (3 ml) was added. The mixture was irradiated in a microwave cavity at 0 C for 20 min. Then, the mixture was transferred into a column equipped with a polypropylene frit using a Pasteur pipette. The soln was drained and the resin was washed with DMF (3 5 ml) and CH 2 Cl 2 (3 5 ml). The completion of the coupling was confirmed by Kaisers test. Thereafter, Fmoc-deprotection was performed using procedure I as described above. Procedure III The resin from procedure I was transferred into a Smith Process Vial and Fmoc-Val (238 mg, 0.70 mmol), PyBOP (343 mg, 0.70 mmol), i-pr 2 NEt (0.25 ml,.40 mmol) and DMF (3 ml) was added. The coupling was performed using procedure II as described above. The completion of the coupling was confirmed by Kaisers test and by cleavage of a small amount of the resin with 95% TFA in CH 2 Cl 2 and analytical LC-MS investigation of the cleaved peptide. Thereafter, Fmoc-deprotection was performed using procedure I as described above. The resin was transferred into a Smith Process Vial and Fmoc- Thr(t-Bu) (279 mg, 0.70 mmol), PyBOP (343 mg, 0.70 mmol), i- Pr 2 NEt (0.23 ml,.32 mmol) and DMF (3 ml) was added. The mixture was irradiated using procedure II, the completion of reaction was confirmed by procedure III. Procedure IV Finally, the tripeptide was cleaved from the resin using 95% TFA in CH 2 Cl 2 (2 hours). The soln was separated from the resin by filtration, the resin was washed with additional CH 2 Cl 2 (5 ml) and the combined phases were concentrated on a rotatory evaporator. The residue was then dissolved in MeCN ( 2 ml) and was purified on preparative LC-MS yielding as a white solid (63 mg, 0. mmol, 48%). Method B Rink amide resin (300 mg, 0.78 mmol/g, 0.234 mmol) was deprotected Vial and Fmoc-Ile (248 mg, 0.70 mmol), 2-fluoro--methylpyridinium tosylate (20 mg, 0.70 mmol), i-pr 2 NEt (0.25 ml,.40 mmol) and DMF (3 ml) was added and the subsequent synthesis steps followed the procedure described for Method A, with the exception of the coupling times and temperature (0 minutes at 30 C), yielding as a white solid (83 mg, 0.5 mmol, 65%). Method C Rink amide resin (300 mg, 0.78 mmol/g, 0.234 mmol) was deprotected Vial and Fmoc-Ile (248 mg, 0.70 mmol), TBTU (225 mg, 0.70 mmol), i-pr 2 NEt (0.25 ml,.40 mmol) and DMF (3 ml) was added and the subsequent synthesis steps followed the procedure described for Method A, with exception of the coupling times (0 minutes at 0 C), yielding as a white solid (4 mg, 0.075 mmol, 3%). Method D Rink amide resin (30 mg, 0.73 mmol/g, 0.232 mmol) was deprotected Vial and Fmoc-Ile (233 mg, 0.66 mmol), HATU (56 mg, 0.66 mmol), i-pr 2 NEt (0.23 ml,.32 mmol), DMF (3 ml) was added and the subsequent synthesis steps followed the procedure described for Method A, with exception of the coupling times (.5 min. at 0 C), yielding as a white solid (29 mg, 0.098 mmol, 42%). H NMR (400 MHz, CD 3 CN): d = 7.83 (d, J = 7.2 Hz, 2 H, Fmoc), 6.67 (br d, J = 7.6 Hz, 2 H, Fmoc), 7.42 (ddd, J =.2, 7.6, 7.6 Hz, 2 H, Fmoc), 7.34 (ddd, J =.2, 7.2, 7.6 Hz, 2 H, Fmoc), 7.5 (br d, H, NH-Val), 6.80 (br d, H, NH-Ile), 6.35 (br s, H, NH), 6.08 (br d, H, NH-Thr), 5.79 (br s, H, NH), 4.33 (dd, J = 5.9, 7.3 Hz, H, CH a -Val), 4.3 (d, J = 6.6 Hz, 2 H, CH 2 -Fmoc), 4.24 (t, J =6.6

SPECIAL TOPIC Rapid Microwave-Assisted Solid Phase Peptide Synthesis 595 Hz, H, CH-Fmoc), 4.7 (dd, J = 6.8 Hz, H, CH a -Ile), 4.06 4.4 (m, 2 H, CH b -Thr, CH a -Thr),.7.8 (m, 2 H, CH b -Val, CH b -Ile),.44 (m, H, CH 2g(a)-Ile),.26 (d, J =Hz, 3 H, CH 3 -Thr),.0 (m, H, CH 2g(b)-Ile), 0.8 0.9 (m, 2 H, CH 3g -Ile, CH 3d -Ile, CH 3g -Val). ESI-MS: m/z = 553 (M + ) +, 536, 522, 423, 79. Fmoc-Ala-Ile-NH 2 (2); Method A2 Rink amide resin (300 mg, 0.73 mmol/g, 0.235 mmol) was deprotected Smith Process Vial and Fmoc-Ile (233 mg, 0.66 mmol), PyBOP (343 mg, 0.70 mmol), i-pr 2 NEt (0.23 ml,.32 mmol), DMF (3 ml) was added and the loading of resin was made following procedure II (0 C, 5 min), the confirmation of the completion of the reaction was made following procedure III. Fmoc-deprotection (procedure I) was followed by the coupling of Fmoc-Ala (205 mg, 0.66 mmol) in the presence of PyBOP (343 mg, 0.70 mmol), i-pr 2 NEt (0.23 ml,.32 mmol) and DMF (3 ml) following procedure III (0 C, 5 min). This step was repeated due to the incomplete coupling observed by the Kaisers test. The resin was washed with portions of DMF (3 5 ml), portions of CH 2 Cl 2 (3 5 ml) and the dipeptide was cleaved following procedure IV yielding 2 as a white solid (56 mg, 0.32 mmol, 60%). Method B2 Rink amide resin (30 mg, 0.78 mmol/g, 0.235 mmol) was deprotected Vial and Fmoc-Ile (233 mg, 0.66 mmol), 2-fluoro--methylpyridinium tosylate (20 mg, 0.70 mmol), i-pr 2 NEt (0.23 ml,.32 mmol), DMF (3 ml) was added and the subsequent steps were made following Method A2 with the exception of the reaction times and temperature (0 C, 0 min) yielding 2 as a white solid (35 mg, 0.083 mmol, 35%). Method C2 Rink amide resin (299 mg, 0.78 mmol/g, 0.233 mmol) was deprotected Smith Process Vial and Fmoc-Ile (233mg, 0.66 mmol), TBTU (225 mg, 0.70 mmol), i-pr 2 NEt (0.23 ml,.32 mmol) and DMF (3 ml) was added and the subsequent steps were made following Method A2 with the exception of the reaction times and temperature (0 C, 0 min) yielding 2 as a white solid (4 mg, 0.097 mmol, 4%). H NMR (400 MHz, CD 3 CN): d = 7.83 (d, J = 7.7 Hz, 2 H, Fmoc), 7.67 (br d, J = 7.3 Hz, 2 H, Fmoc), 7.42 (ddd, J =.2, 7.7, 7.7 Hz, 2 H, Fmoc), 7.33 (ddd, J =.2, 7.3, 7.7 Hz, 2 H, Fmoc), 6.78 (br d, J = 7.7 Hz, H, NH-Ile), 6.34 (br s, H, NH), 6.08 (br d, J =5.9 Hz, H, NH-Ala), 5.75 (br s, H, NH), 4.32 (d, J = 7.0 Hz, 2 H, CH 2 -Fmoc), 4.24 (t, J = 7.0 Hz, H, CH-Fmoc), 4.6 (dd, J =5.8, 8.0 Hz, H, CH a -Ile), 4.09 (dq, J = 5.9, 7.0 Hz, H, CH a -Ala),.77 (m, H, CH b -Ile),.42 (m, H, CH 2g(a)-Ile),.39 (d, J =7.0 Hz, 3 H, CH 3 -Ala),.09 (m, H, CH 2g(b)-Ile), 0.87 (d, J = 6.8 Hz, 3 H, CH 3g -Ile), 0.84 (t, J = 7.4 Hz, 3 H, CH 3d ESI-MS: m/z = 424 (M + ) +, 407, 379, 79. Fmoc-Thr-Ile-NH 2 (3); Method D3 Rink s amide resin (300 mg, 0.78 mmol/g, 0.234 mmol) was deprotected Smith Process Vial and Fmoc-Ile (248 mg, 0.70 mmol), HATU (66 mg, 0.70 mmol), i-pr 2 NEt (0.25 ml,.40 mmol), DMF (3 ml) was added and the loading of the resin was made following procedure II (0 C,.5 min), the confirmation of the completion of the reaction was made following procedure III. Fmoc-deprotection (procedure I) was followed by the coupling of Fmoc-Thr(t-Bu) (279 mg, 0.70 mmol) in the presence of HATU (343 mg, 0.70 mmol), i- Pr 2 NEt (0.23 ml,.32 mmol) and DMF (3 ml) following procedure III (0 C,.5 min). The resin was washed with portions of DMF (3 5 ml), portions of CH 2 Cl 2 (3 5 ml) and the dipeptide was cleaved following procedure IV yielding 3 as a white solid (25.4 mg, 0.06 mmol, 24%). H NMR (400 MHz, CD 3 CN): d = 7.83 (d, J = 7.5 Hz, 2 H, Fmoc), 7.68 (br d, J = 7.0 Hz, 2 H, Fmoc), 7.42 (ddd, J =.2, 7.5, 7.5 Hz, 2 H, Fmoc), 7.33 (ddd, J =.2, 7.0, 7.5 Hz, 2 H, Fmoc), 6.87 (br d, J = 8.4 Hz, H, NH-Ile), 6.44 (br s, H, NH), 5.99 (br d, J =5.9 Hz, H, NH-Thr), 5.85 (br s, H, NH), 4.36 (d, J = 7.5 Hz, 2 H, CH 2 -Fmoc), 4.25 (t, J = 7.5 Hz, H, CH-Fmoc), 4.22 (dd, J =5.9, 8.4 Hz, CH a -Ile), 4.7-4.5 (m, 2 H, CH b -Thr, CH a -Thr),.83 (m, H, CH b -Ile),.45 (m, H, CH 2g(a)-Ile),.4 (m, H, CH 2g(b)-Ile),.08 (d, J = 6.0 Hz, 3 H, CH 3g -Thr), 0.90 (d, J = 7.0 Hz, 3 H, CH 3g - Ile), 0.87 (t, J = 7.7 Hz, 3 H, CH 3d ESI-MS: m/z = 454 (M + ) +, 437, 409, 255, 24, 99. Efficiency of the Solid Phase Methodology Rink amide resin (297 mg, 0.78 mmol/g, 0.232 mmol) was deprotected using procedure I. The resin was then transferred into a Smith Process Vial, Fmoc-Ile (25 mg, 0.70 mmol), PyBOP (343 mg, 0.70 mmol), i-pr 2 NEt (0.25 ml,.40 mmol), and DMF (3 ml) was added. The mixture was irradiated in a microwave cavity at 0 C for 5 minutes. Then, the mixture was transferred into a column equipped with a polypropylene frit and was washed following procedure II. The completion of the coupling was confirmed by Kaisers test. The resin was cleaved and purified following procedure IV yielding Fmoc-Ile-NH 2 as a white solid (49 mg, 0.4 mmol, 60%). H NMR (400 MHz, CDCl 3 ) d = 7.76 (d, J = 7.5 Hz, 2 H, Fmoc), 7.57 (br d, J = 7.3 Hz, 2 H, Fmoc), 7.40 (dd, J = 7.3, 7.3 Hz, 2 H, Fmoc), 7.3 (dd, J = 7.3, 7.5 Hz, 2 H, Fmoc), 5.89 (br s, H, NH), 5.66 (br s, H, NH), 5.36 (br d, J = 8.4 Hz, H, NH-Ile), 4.42 (d, J = 6.7 Hz, 2 H, Fmoc-CH 2 ), 4.20 (t, J = 6.7 Hz, H, CH-Fmoc), 4.07 (dd, J = 8.4, 8.4 Hz, H, CH a -Ile),.89 (m, H, CH b -Ile),.52 (m, H, CH g(a)-ile),.3 (m, H, CH g(b)-ile), 0.8-.0 (m, 6 H, CH 3g-Ile, CH 3d ESI-MS: m/z = 353 (M+) +. Acknowledgements We would like to thank Personal Chemistry AB for access to the Smith Synthesizer TM and Uppsala University for financial support. References () Merrifield, R. B. J. Am. Chem. Soc. 963, 85, 249. (2) Lindström, P.; Tierney, J.; Wathey, B.; Westman, J. Tetrahedron 200, 57, 9225. (3) (a) Stadler, A.; Kappe, C. O. Eur. J. Org. Chem. 200, 99. (b) Kuster, G.; Scheren, H. W. Tetrahedron Lett. 2000, 4, 55. (c) Hoel, A. M. L.; Nielsen, J. Tetrahedron Lett. 999, 40, 394. (d) Larhed, M.; Lindeberg, G.; Hallberg, A. Tetrahedron Lett. 996, 37, 829. (e) Lew, A.; Krutzik, P. O.; Hart, M. E.; Chamberlin, A. R. J. Comb. Chem. 2002, 4, 95. (4) (a) Chen, S.-T.; Chiou, S.-H.; Wang, K.-T. J. Chin. Chem. Soc. 99, 38, 85. (b) Yu, H.-M.; Chen, S.-T.; Wang, K.-T. J. Org. Chem. 992, 57, 478. (c) Chen, S.-T.; Tseng, P.-H.; Yu, H.-M.; Wu, C.-Y.; Hsiao, K.-F.; Wu, S.-H.; Wang, K.- T. J. Chin. Chem. Soc. 997, 44, 69. (5) (a) HATU: O-(7-Azabenzotriazol--yl)-N,N,N,N tetramethyluronium hexafluoro-phosphate see: Carpino, L. A.; El-Faham, A.; Minor, C. A.; Albericio, F. J. Chem. Soc., Chem Commun. 994, 20. (b) TBTU: O-(Azabenzotriazol- -yl)-n,n,n,n -tetramethyluronium tetrafluoro-borate see: Zimmer, S.; Hoffmann, E.; Jung, G.; Kessler, H. Liebigs Ann. Chem. 993, 5, 497. (c) PyBOP: (Benzotriazol-- yloxy)-tripyrrolidinophosphonium hexa-fluorophosphate

596 M. Erdélyi, A. Gogoll SPECIAL TOPIC see: Coste, J.; Le-Nguyen, D.; Castro, B. Tetrahedron Lett. 990, 3, 205. (d) Mukaiyama s reagent: 2-fluoro-- methyl-pyridinium tosylate see: Mukaiyama, T.; Tanaka, T. Chem. Lett. 976, 303. (6) Sarin, V. K.; Kent, S. B.; Tam, J. P.; Merrifield, R. B. Anal. Biochem. 98, 7, 47. (7) (a) Wokaun, A.; Ernst, R. R. Chem. Phys. Lett. 977, 52, 407. (b) Shaka, A. J.; Freeman, R. J. Magn. Reson. 983, 5, 69. (8) (a) Kumar, A.; Ernst, R. R.; Wüthrich, K. Biochem. Biophys. Res. Commun. 980, 95,. (b) Bodenhausen, G.; Kogler, H.; Ernst, R. R. J. Magn. Res. 984, 58, 370.