Novel Method for Solid Phase Peptide Synthesis Using Microwave Energy Jonathan M. Collins, Michael J. Collins, Rebecca C. Steorts CEM Corporation, Matthews, NC 28106-0200, U.S.A. Presented at American Peptide Symposium Boston, MA 22 July 2003 The application of microwave energy has proved to be a major enabling tool for many chemical applications requiring energy input. A new technique for microwave assisted solid phase peptide synthesis (SPPS) has been developed that allows for enhanced deprotection and coupling reactions. A single mode cavity is used to allow for a high microwave power density and a uniform field distribution. Inside of the cavity, a vessel has been placed to allow for filtration of the solution and easy addition of reagents. Also, enhanced cleavage of the final peptide product from the resin takes place in the microwave. The procedure allows for complete cycle times of well under ten minutes for the synthesis of even some of the most difficult peptide sequences. The well-known Acyl Carrier Peptide, 65-74 Acp was synthesized to completion with a 60 second deprotection and a 120 second coupling reactions for each cycle. Substitution of the non-controlled deprotection reagent, Piperazine was used successfully to synthesize the 65-74 Acp sequence with a 90 second deprotection time. Furthermore, the microwave procedure was used to prepare the notoriously difficult deca-alanine peptide, with 120 second deprotection and 300 second coupling reaction times for each cycle. Microwave energy allows for higher resin substitution and less excess reagents to be used that increases scale up potential when compared to traditional methods. Aggregation is overcome by microwave energy allowing for the possibility of purer and longer peptide sequences. Microwave Peptide Synthesis Since its inception in 1963, by Bruce Merrifield, Solid Phase Peptide Synthesis (SPPS) has become the standard for peptide synthesis 1. However, SPPS is still plagued with inherent difficulties due to intermolecular aggregation, B-sheet formation, steric hindrance from protecting groups and premature termination of the sequence. As a result, standard amino acid cycle times range from 30 minutes to 2 hours and incomplete deprotection and coupling reactions are still common. Traditionally, solid phase peptide synthesis (SPPS) has been performed at room temperature and been a very time consuming process. In an effort to speed this process, nitrogen bubbling and vortex mixing have been applied to increase deprotection and coupling reaction rates. Elevated temperature up to 60ºC has been used to enhance coupling rates and efficiency in difficult sequences due to thermal disruption of peptide aggregation. 2a,b In the last ten years, microwave synthesis has become widely accepted to increase reaction rates in organic synthesis up to 1000 fold. Unlike conventional heating, microwave energy directly activates any molecule with a dipole moment and allows for rapid heating at the molecular level. 3,4 Microwave energy has also been successfully used to increase the rate of peptide coupling
reactions and not generate appreciable racemization. 5a,b Intermolecular aggregation, B-sheet formation, and steric hindrance can be overcome with microwave energy. Figure 1. Disruption of Aggregation due to Microwave Absorption The speed of microwave synthesis brings the potential for the use of less expensive and reactive reagents. The combination of microwave energy with SPPS allows for more rapid cycle times with purer products. In this paper, we present a complete process for peptide synthesis in a single vessel with enhanced deprotection and coupling reactions utilizing microwave energy. Method and Materials A 25mL pear-shaped glass vessel was used for the reaction vessel. Inside of the vessel a tube was placed to allow for gas pressure, while a second filtered tube was placed to allow for solution delivery and removal. The reaction vessel was placed inside of a Discover TM single mode microwave with irradiation at 2450MHz (CEM Corporation, Matthews, NC). The vessel was held at a constant position inside of the cavity throughout all reactions. The main solvent used in all washings, DMF was connected to the reaction vessel through the filtered tube line. The resin was manually inserted into the reaction vessel at the beginning of the synthesis. Deprotection and coupling solutions were also manually added to the reaction vessel. 25mL glass vessel CEM Discover TM Microwave System 2
Analysis was performed on an LCQ ion trap mass spectrometer (ThermoFinnigan, San Jose, CA) with electrospray ionization coupled to a Surveyor MS (ThermoFinnigan, San Jose, CA). A Zorbax C-18 column (3.5µm, 2.1x 150mm) was used for all separations. The preloaded Fmoc-Wang resin and all amino acids were purchased from Peptides International (Louisville, KY). The Fmoc amino acids contained the following side chain protecting groups, Asn (Trt), Asp (OtBu), Gln (Trt), Tyr (tbu). Dimethylformamide (DMF), Dichloromethane(DCM), Trifluoroacetic acid (TFA), N-hydroxybenzotriazole (HOBt), Piperidine, Piperazine, Diisopropylethylamine (DIPEA), and Triisopropylsilane (TIS) were purchased from Sigma- Aldrich (St. Louis, MO). Ethyl Ether Anhydrous contained 1ppm 2,6-Di-tert-butyl-p-cresol as a peroxide inhibitor and was purchased from EM Science (Gibbstown, NJ). Procedure I. 65-74 ACP Peptide: VQAAIDYING The well-known acyl carrier peptide, 65-74 ACP was synthesized on 0.417g of a 0.6 mmol/g Glyfunctionalized Wang Resin purchased from Peptides International. Deprotection was performed with a 15% Piperidine in DMF solution. All coupling reactions were performed with 0.9mmol/1mmol PyBOP/HOBt and 2mmol DIPEA dissolved in 15mL of DMF. Each deprotection and coupling reaction was performed with microwave energy and nitrogen bubbling. All deprotection reactions were for 60 seconds, and all coupling reactions were for 120 seconds. The cleavage reaction was performed with a 95% TFA, 2.5% TIS, and 2.5% H 2 O solution for 90 minutes at room temperature. The product was filtered from the resin and precipitated in cold ethyl ether. The ether solution was centrifuged for 5 minutes at medium speed and the solid product dried under vacuum. Procedure II. 65-74 ACP Peptide: VQAAIDYING The acyl carrier peptide, 65-74 ACP was synthesized exactly as in Procedure I., except with a 5% Piperazine in DMF solution as the deprotection solution and a deprotection time of 90 seconds. Procedure III. Deca-Alanine: AAAAAAAAAA The deca-alanine sequence was synthesized on 0.500g of a 0.5mmol/g Ala-functionalized CLEAR Resin purchased from Peptides International. Deprotection was performed with a 20% Piperidine in DMF solution. All coupling reactions were performed with 0.9mmol/1mmol HBTU/HOBt and 2mmol DIPEA dissolved in 15mL of DMF. Each deprotection and coupling reaction was performed with microwave energy and nitrogen bubbling. All deprotection reactions were for 120 seconds, and all coupling reactions were for 240 seconds. The cleavage reaction was performed with a 95% TFA, 2.5% TIS, and 2.5% H 2 O solution for 90 minutes at room temperature. The product was filtered from the resin and precipitated in cold ethyl ether. The ether solution was centrifuged for 5 minutes at medium speed and the solid product dried under vacuum. 3
Results: The crude product from Procedure I was directly infused into the mass spectrometer with an electrospray ionization source. The spectra for this analysis yielded a single peak at 1063.3. Figure 3. ESI mass Spectra of VQAAIDYING from Procedure I. 4
The crude product was further analyzed by reverse phase HPLC and mass spectrometry. A 30 min gradient of 0-60% solvent B(0.1% TFA in acetonitrile) was used. Solvent A was 0.1% TFA in H 2 O. Absorbance was monitored at 214nm. The desired product eluted after 11.9 min and was confirmed by mass spectrometry. Figure 4. LC-MS Data for VQAAIDYING from Procedure I. 5
The crude product from Procedure II was analyzed by mass spectrometry using an electrospray source. The spectrum yielded a major peak at 1063.4, as well as two small peaks at 935.4 corresponding to a loss of valine, and 1285.2 corresponding to an incomplete final deprotection. Figure 5. ESI mass Spectra of VQAAIDYING from Procedure II. 6
The crude product from Procedure III was then analyzed by mass spectrometry using an electrospray source. The spectrum showed a major peak at 729.1 corresponding to deca-alanine. Figure 6. ESI mass Spectra of AAAAAAAAAA from Procedure III. 7
Conclusion: The application of microwave energy to SPPS allows for much faster cycle times while driving deprotection and coupling reactions to completion. With this protocol, complete cycle times of under 10 minutes are attainable for difficult hydrophobic peptides. Thus it is possible to synthesize a hydrophobic 10mer peptide in under two hours. Also, the speed of microwave synthesis allows the use of alternative reagents for coupling and deprotection reactions that have historically not been practical. Future work will be performed to further optimize microwave SPPS methods as well as resin cleavage. The applicability of this new microwave method will be enhanced by the recent creation of the first microwave peptide synthesizer, Odyssey(CEM Corporation, Matthews,NC). This system allows chemists to use this new microwave method to create up to 12 different peptides automatically. References: 1. Merrifield, R.B. J. Am. Chem. Soc. 1963, 85, 2149. 2. (a) Lloyd, D.H., Petrie, G.M., Noble, R.L., and Tam, J.P. Increased coupling efficiency in solid phase peptide synthesis using elevated temperature, Peptides: Chemistry, Structure and Biology, Proceedings of the 11 th American Peptide Symposium, Escom, Leiden, Netherlands, 909-910, 1990. (b) Kates, S.A.; Solé, N.A.;Beyermann, M.;Barany, G.; Albericio, F.; Peptide Research, 1996, 9, 106. 3. Hayes, B.L. Microwave Synthesis: Chemistry at the Speed of Light; CEM Publishing: Matthews, NC, 2002. 4. Loupy, A. Microwaves in Organic Synthesis; Wiley-VCH, Weinheim, 2002. 5. (a) Yu, H.-M.; Chen, S.-T.; Wang, K.-T. J. Org. Chem. 1992, 57, 4781. (b) Erdélyi, M.; Gogoll, A. Synthesis 2002, 11, 1592. Figure 7. Automated Microwave Peptide Synthesizer, Odyssey 8