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Monographic supplement series: Oligos & Peptides - chimica oggi/Chemistry Today - vol. 30 n. 2 March/April 2012

Scientific article ● Peer reviewed

KEYWORDS

ABSTRACT

INTRODUCTION

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 minimize the potential for side reactions including the racemization of the cysteine and histidine residues during coupling and aspartimide formation in aspartic acid containing sequences during Fmoc deprotection (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 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 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 can be used for the synthesis of range of standard peptides using routine methods without the need for extensive method

optimization. Also a series of peptides containing unusual and difficult to couple amino acid derivatives were synthesized. In all cases the peptides were prepared in moderate to excellent crude purity in a fraction of the time it would take to synthesize these sequences conventionally. Comparative experiments for several peptides demonstrated that the higher purities obtained in microwave SPPS are the result of enhancements in both the deprotection and coupling reactions.

MATERIALS AND METHODS

ReagentsAll 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)-1 H - 1 , 2 , 3 - t r i a z o l o [ 4 , 5 - b ] p y r i d i n o - 1 y l m e t h y l e n e ] - 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.

Peptide synthesisStandard 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 seconds followed by 3 minutes at 75°C. Coupling reactions were performed with 5 fold excess Fmoc-AA-OH with the activation strategy indicated in each table for 5 minutes at 75°C. Special coupling conditions: 10 minutes with DIC/HOBt, 50°C for C and H, and double coupling for R. 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.Conventional synthesis protocol: The conventional syntheses were also performed on the CEM Liberty peptide synthesizer with no microwave irradiation. Deprotection was performed in two stages with an initial deprotection of 5 minutes followed by 10 minutes at 21°C. Coupling reactions were performed with 5 fold excess Fmoc-AA-OH with the activation strategy indicated in each table for 30 minutes at 21°C.

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. Much of the research on microwave-assisted peptide synthesis has focused on difficult to synthesize peptides. Described herein is an extensive study of the standard microwave synthesis protocols for the synthesis of a series of peptides that span a range of complexities. Each peptide was rapidly prepared in excellent purity, and often the microwave method far outperformed conventional synthesis techniques.

Microwave-assisted peptide synthesis; microwave irradiation; solid-phase peptide synthesis; difficult peptides.

JONATHAN M. COLLINS, SANDEEP K. SINGH, GRACE S. VANIER**Corresponding author CEM Corporation Life Science Division, P.O. Box 200 Matthews, NC 28106, USA

Microwave technology for solid phase peptide synthesisIt’s not just for difficult peptides

Grace Vanier

Monographic supplement series

Peptide analysisThe peptides were analysed on a Waters Atlantis C18 column (3 mm, 2.1 ×150 mm) at 214 nm with a gradient of 5-70 percent MeCN (0.1 percent formic acid), 0-20 min. The crude purity is based on integration of the HPLC chromatogram. Mass analysis was performed using an LCQ Advantage ion trap mass spectrometer with electrospray ionization (Thermo Electron).

RESULTS AND DISCUSSION

The initial experiments outlined in Table 1 highlight the standard microwave peptide synthesis protocol and also demonstrate the difference in synthesis time and quality between microwave and conventional peptide synthesis. The standard microwave conditions for Fmoc-based solid phase peptide synthesis are 30 seconds followed by three minutes at 75°C for deprotection and five minutes at 75°C for coupling. There are a couple exceptions to this general protocol that are sequence dependent. The coupling temperature of cysteine and histidine residues should be limited to 50°C to limit the amount of racemization. For aspartic acid containing sequences where the following amino acid is glycine, asparagine, serine, or alanine, the addition of 0.1 M HOBt to the deprotection solution and the use the piperazine as the base should be used to significantly reduce the potential for aspartimide formation. Lastly, due to the potential for g-lactam formation during difficult arginine couplings, a double coupling is used for each arginine residue in both the conventional and microwave approaches to ensure complete coupling.

The linear sequence of octreotide (1) was synthesized on O-t-butylthreoninol 2-chlorotrityl resin. While the microwave method provided comparable purity results to the conventional synthesis, the peptide was prepared in less than half the time. Also of note is the microwave method generated the peptide in 92 percent yield (conventional - 93 percent yield) despite operating with the more sensitive 2-chlorotrityl resin. The TAT peptide (2) can be difficult to synthesize due to the multiple arginine residues, but the standard microwave method provided the Fmoc protected peptide in 92 percent crude purity.

Table 1. Comparison of microwave and conventional peptide synthesis conditions.

Figure 1. Crude HPLC chromatogram for the microwave synthesis of peptide 3 in Table 1. The major peak eluting at 12.60 minutes comprises a mass of 1945.4, the [M + H]+ of peptide 3 in Table 1.

Monographic supplement series: Oligos & Peptides - chimica oggi/Chemistry Today - vol. 30 n. 2 March/April 2012

used interchangeably. If guanidine capping occurs when coupling with the aminium activators, DIC or a phosphonium activator should be utilized. In each case, the peptides were produced in good to excellent crude purity in a fraction of the time it would take to synthesize these peptides conventionally.Non-standard amino acid derivatives and other peptide modifications can often present additional synthetic challenges due to increased steric hindrance, decreased reactivity, and/or poor solubility. Microwave irradiation can overcome many of these challenges, and Table 3 highlights several different non-standard amino acids including N-methylated amino acids, methylated arginine, aminoisobutyric acid (Aib), and γ-carboxyglutamic acid (Gla). Included is also one example of an N-terminal modification with 3-maleimidopropionic acid. The coupling of the hindered N-Me amino acid derivatives are difficult and even with special reagents the synthesis quality is often low. Peptide 1 contains two N-Me amino acids, and using microwave irradiation in conjunction with HATU activation the peptide was prepared in 85 percent crude purity in just 5 hours (Figure 2). The conventional synthesis of peptide 1 resulted in significant deletion products with only 14 percent purity of the desired peptide. The incorporation of two asymmetric methylated arginine residues into peptide 2 was easily accomplished with microwave irradiation by only a slight modification to the standard conditions. The coupling time for the methylated arginine residues were increased to 10 minutes to give the peptide in 74 percent crude purity. Conventional conditions gave only 39 percent crude purity with significant deletions observed. The Aib amino acid derivative is a convenient way to introduce a helix inducer into the peptide sequence, but the added steric hindrance from the additional methyl can make coupling difficult especially with multiple Aib residues in the sequence. Peptide 3 contains two adjacent Aib residues, but microwave irradiation can easily overcome the challenge of synthesizing such a difficult sequence. The Aib-7 residue required the use of HATU while

activation for all other cycles was by DIC/HOBt. Under these microwave conditions the synthesis was performed in 93 percent purity in just 11 hours. The rising popularity and synthetic utility of the copper catalysed click chemistry has increased the need for reliable methods for the incorporation of the coupling partners including derivatives containing the propargyl functional group. The coupling of propargylglycine into peptide 4 was performed using standard microwave synthesis conditions to give the peptide in 93 percent crude purity. Syntheses

of peptides containing the γ-carboxyglutamic acid (Gla) residue can suffer from poor coupling efficiency due to the increased sterics from the two t-butyl protecting groups on the side chain. Peptide 5 includes three Gla derivatives early

Peptide 3 was prepared in 88 percent crude purity using microwave irradiation for the entire synthesis process (Figure 1). The use of microwave energy for just the coupling reaction produced the peptide in just 33 percent purity, and the synthesis under conventional synthesis conditions gave only 19 percent crude purity. Peptide 4 is thymosin a1 and was synthesized in 64 percent crude purity using microwave irradiation for the entire synthesis process. Using microwave for only the coupling reaction resulted in a 10 percent drop in crude purity, and performing the entire synthesis under conventional conditions gave only 18 percent crude purity. VIP (5) is a 28-mer peptide susceptible to aspartimide formation. While the conventional synthesis conditions give only 45 percent crude purity in 35 hours, the microwave method provides the peptide in 80 percent purity in half the time. As a side note to emphasize the importance of the 0.1 M HOBt in the deprotection solution, the same synthesis without the HOBt results in only 27 percent crude purity with about 50 percent of the aspartimide product.

Table 2 features a series of biologically active peptides that contain only proteinogenic amino acids. These peptides have been synthesized routinely using conventional techniques, but microwave irradiation provides a fast and efficient method to generate these peptides using the standard protocols. As general rule, 0.1 M HOBt is added to any aspartic acid containing peptides regardless of the propensity for aspartimide formation (Table 2, entry 1, 2, and 5). Peptides containing an “Asp-Gly” segment that are particularly susceptible to base catalyzed side reactions, such as secretin (6) can be easily synthesized in microwave SPPS with use of the weaker base piperazine and HOBt resulting in high purity with negligible aspartimide formation. HBTU and DIC typically perform well for the majority of sequences and can usually be

Table 3. Microwave synthesis of peptides containing unusual amino acids.aDouble coupling for underlined amino acids.

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Table 2. Microwave synthesis of biologically active peptides containing standard amino acids.

in the sequence making it necessary to use double coupling for the latter half of the synthesis. Using this modified method the peptide was prepared in 82 percent crude purity. Peptide 6 features an N-terminal 3-maleimidopropionyl group. The maleimide can be used as a Michael acceptor to conjugate the peptide to another peptide or matrix. Peptide 6 features the SH3-binding peptide (14), and the synthesis of both the peptide and the incorporation of the 3-maleimidopropionic acid required double coupling as indicated in the table. With these modifications the peptide was synthesized in 91 percent crude purity.

CONCLUSIONS

The standard microwave peptide synthesis protocol is a generally applicable method for the synthesis of a variety of peptide sequences. In all cases the microwave method generated the peptide in at least comparable crude purity to

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Figure 2. Crude HPLC chromatogram for the microwave synthesis of N-Me containing peptide 1 in Table 3. The major peak eluting at 8.99 minutes comprises a mass of 683.7, the [M + Na] of peptide 1 in Table 3.

conventional methods and importantly in a fraction of the time. Interestingly, in many cases the microwave method proved superior to the conventional synthesis technique giving the peptide in significantly increased purity. Both the deprotection and coupling reaction can benefit from microwave irradiation to give peptides in excellent purity. Microwave technology can be used routinely to synthesize the most trivial to the most difficult peptide sequences, and the general protocol includes the means by which to minimize side reactions such as racemization and aspartimide formation.

REFERENCES AND NOTES

1. J.M. Collins, M.J. Collins, Microwaves in Organic Synthesis, Wiley-VCH, pp. 898-930 (2006).

2. J.M. Collins, N.E. Leadbeater, Org. Biomol. Chem., 5, pp. 1141-1150 (2007).

3. G.S. Vanier, Microwave heating as a tool for sustainable chemistry, CRC Press, pp. 231-269 (2010).

4. S.L. Pedersen, A.P. Tofteng et al., Chem. Soc. Rev., 41, pp. 1826 - 1844 (2012).

5. S.A. Palasek, Z.J. Cox et al., J. Pept. Sci., 13, pp. 143-148 (2007).6. V. Santagada, F. Fiorino et al., Tetrahedron Lett., 42, pp. 5171-5173

(2001).7. S.J. Tantry, R.V.R. Rao et al., Arkivoc, pp. 21-30 (2006).8. S.A. Rahman, A. El-Kafrawy et al., Amino Acids, 33, pp. 531-536 (2007).9. F. Rizzolo, G. Sabatino et al., Int. J. Pept. Res. Ther., 13, pp. 203-208

(2007).10. B. Bacsa, K. Horvati et al., J. Org. Chem., 73, pp. 7532-7542 (2008).11. S. Northfield, K. Roberts et al., Int. J. Pept. Res. Ther., 16, pp. 159-165

(2010).12. S.L. Pedersen, K.K. Sørensen et al., Peptide Science, 94, pp. 206-212

(2010).13. H. Rodríguez, M. Suarez et al., Journal of Peptide Science, 16, pp. 136-

140 (2010).14. T. Yamaguchi, M. Asay et al., Journal of the American Chemical

Society, 134, pp. 886-889 (2011).