PEPTIDES & PROTEINS
Microwave technology: a versatile tool for solid phase peptide synthesis
The use of microwave technology for solid phase peptide synthesis is one of the most significant breakthroughs of the past decade in the field of peptide chemistry. This article describes an extensive study of the standard microwave synthesis protocols for the synthesis of a series of peptides that span a range of complexities including N-terminal modifications and peptide cyclisation.
U
sing microwave technology for solid phase peptide synthesis (SPPS) is one of the most significant breakthroughs of the past decade in
the field of peptide chemistry. While much of the research on microwave-assisted peptide synthesis has focused on difficult-to- synthesise peptides, this technology is a useful tool that can be applied to a variety of different types of peptides. This article describes an extensive study of the standard microwave synthesis protocols for the synthesis of a series of peptides that span a range of complexities including N-terminal modifications and peptide cyclisation. Each peptide was rapidly prepared in excellent purity, and often the microwave method far outperformed conventional synthesis techniques.
Microwave technology is quickly becoming the preferred tool for performing solid phase peptide synthesis, especially for the synthesis of ‘difficult’ peptides.1-4 Microwave irradiation significantly reduces the synthesis time while also improving the quality of the peptides produced. Routine methods have been developed that minimise the potential for side reactions including the racemisation of the cysteine and histidine residues during coupling and aspartimide formation in aspartic acid containing sequences during Fmoc removal.5
While nearly 100 papers are published annually that highlight the use of microwave technology for performing peptide synthesis, many of these studies focus on the synthesis of ‘difficult’ peptides.6-12 The use of microwave energy to promote peptide synthesis provides two advantages over conventional room temperature synthesis conditions: significantly faster reaction times
38 sp2 Inter-Active March/April 2012
and, in many cases, higher purity peptide product. During the peptide synthesis process there are many polar and ionic species present that can be rapidly heated by microwave energy. The resulting temperature increase can help break up chain aggregation due to intra- and interchain association and allow for easier access to the growing end of the peptide chain. Thus microwave irradiation can provide access to peptides previously inaccessible by conventional techniques.13 The goal of this study is to demonstrate that microwave irradiation is a versatile tool for the synthesis of a range of peptides using routine methods without the need for extensive method optimisation. First, a series of eleven biologically important peptides were synthesised using commonly employed activation strategies. Then a series of peptides containing a variety of different N-terminal modifications were synthesised. Lastly, microwave technology was used to synthesise a head-to-tail cyclised peptide in a fully automated process. In all cases, the peptides were prepared in moderate to excellent crude purity in a fraction of the time it would take to synthesise these sequences conventionally. Comparative experiments for several peptides in this study demonstrated that the higher purities obtained in microwave SPPS are the result of enhancements in both the deprotection and coupling reactions.
Materials and methods Reagents: All Fmoc amino acids, N-[(1H- benzotriazol-1-yl)(dimethylamino)methylene]- N-methylmethanaminium hexafluorophosphate N-oxide (HBTU), N-hydroxybenzotriazole (HOBt), and N-[(1H-6- chlorobenzotriazol-1-yl- (dimethylamino)methylene]-
N-methylmethanaminium hexafluorophosphate N-oxide (HCTU) were obtained from CEM Corporation. N-[(dimethylamino)-1H-1,2,3-triazolo[4,5- b]pyridino-1ylmethylene]- N-methylmethanaminium hexafluorophosphate N-oxide (HATU) was obtained from Anaspec.
Diisopropylethylamine (DIEA), piperidine, N,N’-diisopropylcarbodiimide (DIC), trifluoroacetic acid (TFA), triisopropylsilane (TIS), and 3,6-dioxa-1,8-octanedithiol (DODT) were obtained from Sigma-Aldrich. Dichloromethane (DCM), N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), anhydrous diethyl ether, acetic acid, HPLC grade water and acetonitrile were obtained from VWR.
Standard microwave synthesis protocol: The peptides were prepared using the CEM Liberty automated microwave peptide synthesizer. Deprotection was performed in two stages with an initial deprotection of 30 sec followed by 3 min at 75°C. Coupling reactions were performed with fivefold excess Fmoc-AA-OH, with the activation strategy indicated in each table for 5 min at 75°C. Special coupling conditions were 10 min with DIC/HOBt, 50°C for Cys and His residues, and double coupling for Arg. Cleavage was performed using 92.5:2.5:2.5:2.5 TFA/H2O/TIS/DODT for 30 min at 38°C. Following cleavage, the peptide was precipitated and washed with diethyl ether.
Peptide analysis: The peptides were analysed on a Waters Atlantis C18 column (5 µm, 2.1 ×150 mm) at 214 nm with a gradient of 5-70% MeCN (0.1% formic acid), 0 to 20 min. The crude purity is based on integration of
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