Rapid Solid-Phase Synthesis of a Calmodulin-Binding Nonapeptide using Thermal and Controlled Microwave Irradiation

Bernadett Bacsa1, Bimbisar Desai2, Gbor Dib1* and Christian Oliver Kappe2*
1Institute

of Chemistry, Etvs Lornd University, PO Box 32, H-1518 Budapest 112, Hungary Laboratory for Microwave Chemistry and Institute of Chemistry, Karl-Franzens University, Heinrichstrasse 28, A-8010 Graz, Austria E-mail: oliver.kappe@uni-graz.at, dibo@chem.elte.hu

2Christian-Doppler

Introduction
Recently, there are a growing number of publications that report the use of microwave heating for solid-phase organic synthesis, either by using a variety of polymeric supports or applying polymer-supported reagents and catalysts. Although solid phase synthesis was originally introduced for the preparation of oligopeptides in 1963 by R.B. Merrifield [1], surprisingly, there are only a few reports so far on the use of microwave irradiation for the preparation of peptides and related species [2,3]. In fact, it is widely accepted that microwave-assisted chemical reactions can be completed in minutes, providing less by-products and higher yields [4]. Moreover, due to the recent progress in microwave technology, a new generation of the microwave ovens reached the market. By using the latest developments in microwave technology, we developed various protocols for the synthesis test peptides under microwave irradiation. The syntheses were performed without temperature control or under temperature monitoring with a fiber optic sensor under pulsed microwave irradiation with intermittent cooling to room temperature or below. Thus, all synthetic steps were performed under very mild conditions, similar to that is used in traditional solid phase peptide synthesis. After the final cleavage step, the crude peptide products were subjected to HPLC-MS analysis. As it was expected, the reaction time of each step decreased to minutes (instead of hours), and the peptides were obtained in better yields and significantly higher purity by the MAPS strategy, as compared to peptides synthesized by classical SPPS protocols.

Instrumentation
CEM Discover Single Mode Reactor
NH H N O O N H CO2 H OH H N O

HN HN O N H

NH 2

NH2 OH H N O

Fiber-optic probe Stirring bar The temperature was monitored by an internal fiber optic probe.

H 2N

H N O

O N H

O N H CO2 H

Results
We herewith report the microwave-assisted solid-phase synthesis of a nonapeptide (calmodulin-binding peptide, H-Trp-Asp-Thr-Val-Arg-Ile-Ser-Phe-Lys-OH) containing amino acid residues which require extensive side chain protection. The syntheses were performed under different conditions including a novel strategy based on the use of pre-cooled reaction vessels to sub-ambient temperatures in combination with a pulsed microwave irradiation sequence. For temperature monitoring an internal fiber-optic sensor was used. The peptide syntheses were performed by using standard Fmoc/tBu orthogonal protection [H-Trp(Boc)-Asp(OtBu)-Thr(tBu)-Val-Arg(Pbf)-Ile-Ser(tBu)-Phe-Lys(Boc)-OH] on polystyrene Wang resin. Starting from Fmoc-Lys(Boc)Wang-resin (100-200 mesh, loading 0,66 mmol/ g), the model nonapeptide was synthesized under five different reaction conditions. Method A: conventional SPPS protocol at room temperature; Method B: under (pulsed) microwave irradiation with intermittent cooling of the reaction to ambient temperature (~20 C); Method C: under (pulsed) microwave irradiation with intermittent cooling of the reaction mixture to sub-ambient temperature (~0 C); and Method D: under (pulsed) microwave irradiation in MicroKan reactors with intermittent cooling of the reaction mixture to sub-ambient temperature (~0 C). Method E: conventional SPPS protocol at elevated temperature (~65 C). The peptides were cleaved from the solid support with a cleavage cocktail of TFA/ethanedithiol/thioanisole/water/phenol using vigorous shaking for 3 hr at ambient temperature.

Method A

Method B

Method C

Method D MicroKan

Method E

Deprotection: 20 min 20% piperidine/DMF, RT Coupling: 1 hr, RT 3x molar excess AA 3x molar excess DIC 3x molar excess HOBt

Deprotection: 3x30sec, 40 W (power contolled) Tmax.100 C, 20% piperidine/DMF Coupling: 4x30sec 30 W (power contolled) Tmax.100 C 3x molar excess AA 3x molar excess DIC 3x molar excess HOBt

Deprotection: 3x30sec 40 W (power contolled) Tmax.65 C, 20% piperidine/DMF Coupling: 4x30sec 30 W (power contolled) Tmax.65 C 3x molar excess AA 3x molar excess DIC 3x molar excess HOBt

Deprotection: 3x30sec 40 W (power contolled) Tmax.65 C, 20% piperidine/DMF Coupling: 4x30sec 30 W (power contolled) Tmax.65 C 9x molar excess AA 9x molar excess DIC 9x molar excess HOBt

Deprotection: 7.5 min 20% piperidine/DMF, 65 C Coupling: 10 min, 65 C 3x molar excess AA 3x molar excess DIC 3x molar excess HOBt

Temperature profiles for the coupling steps

100 90 80 70 60 50 40 30 20 10 0 0 20 40 60 t / sec no cooling cooling power 80 100 100 90 80 70 60 50 40 30 20 10 0

Summary
Preparation of H-Trp-Asp-Thr-Val-Arg-Ile-Ser-Phe-Lys-OH by five different solid-phase peptide synthesis (SPPS) protocols: Method Conditionsb Reaction Yieldc Purityd time (h) (%) (%) MS-dataa [M+2H]2+ 576.50 576.52 576.47 576.63 576.59

[M+H]+

Power / W

T/oC

A B C D E
aPeptide

standard SPPS MW SPPS

20 C

11.0 2.5 2.5 2.5 2.5

82 64 71 95 80

81 63 86 83 78

1151.83 1151.87 1151.83 1152.02 1152.00

MW SPPS 2096 C 065 C

MW SPPS 065 C in MicroKans standard SPPS 65 C

b See text for details. identified by ESI-MS. Calcd Mw for C54H82N14 O14 1150.61. cYield of crude peptides after cleavage from resin. d Purity of crude peptides (peak area percent from analytical RP-HPLC/MS monitored via UV at 220 nm).

Conclusion
We have applied microwave irradiation for the rapid and efficient synthesis of peptides. A test peptide was synthesized under five different conditions (Methods AE) on solid-phase by using an internal fiber optic temperature sensor. The best results were obtained by alternating short pulses of microwave irradiation of constant power with intermittent cooling of the reaction vessel to sub-ambient temperature. The application of MicroKans as microcontainers for the resin beads provided higher purity and better yields in significantly shorter time as compared with that of the conventional SPPS approach or with a standard microwave method not involving exhaustive pre-cooling of the reaction mixture. We are currently investigating the underlying effects of the pre-cooling technique in more detail and plan to exploit this method for the synthesis of longer peptide sequences on a larger scale in the future.

References
[1] Kappe, C. O. Angew. Chem. Int. Ed. 2004, 43, 62506284; Kappe, C. O.; Stadler, A. Microwaves in Organic and Medicinal Chemistry, Wiley-VCH, Weinheim, 2005. [2] Yu, H.-M.; Chen, S.-T.; Wang, K.-T. J. Org. Chem. 1992, 57, 47814784. [3] Erdlyi, M.; Gogoll, A. Synthesis 2002, 11, 15921596. [4] Murray, J. K.; Gellman, S.H. Org. Lett. 2005, 7, 15171520.

Acknowledgments
Financial support from the European Union Ceepus H-076 project and the Austrian Science Fund (Project P15582) are gratefully acknowledged. The authors thank Mr. Blint Hegymegi-Barakonyi for performing the HPLC/MS experiments.