PEPTIDES & PROTEINS


the HPLC chromatogram. Mass analysis was performed using an LCQ Advantage ion trap mass spectrometer with electrospray ionisation (Thermo Electron).


Results and discussion The initial experiments outlined in Table 1 highlight the standard microwave peptide synthesis protocol and demonstrate the synthesis of a series of biologically active peptides that contain only proteinogenic amino acids. 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 of exceptions to this general protocol that are sequence-dependent. The coupling temperature for cysteine and histidine residues should be limited to 50°C to limit the amount of racemisation. For aspartic acid containing sequences where the following amino acid is glycine, asparagine, serine, or alanine, the addition of 0.1M HOBt to the deprotection solution and the use the piperazine as the base should be used to


Entry 1 2 3 4 5 6 7 Peptide Octreotide HIV TAT Thymosin VIP Beta-amyloid BAM 3200 HPV


Oncoprotein, E7 (43-77)


8 9 10 11


Melittin, honey bee


significantly reduce the potential for aspartimide formation. Lastly, due to the potential for γ-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 synthesised 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, which generated the peptide in 92% yield (conventional - 93% yield) despite operating at a maximum temperature of 80°C with the more sensitive 2-chlorotrityl resin. The TAT peptide (2) can be difficult to synthesise due to the multiple arginine residues, but the standard microwave method provided the Fmoc-protected peptide in 92% crude purity. Peptide 3 is thymosin α1 and was synthesised in 64% crude purity using microwave irradiation for the entire synthesis process. Using microwave for only the


Sequence fCFwKTCT-ol Fmoc-GRKKRRQRRRPPQ SDAAVDTSSEITTKDLKEKKEVVEEAEN HSDAVFTDNYTRLRKQMAVKKYLNSILN


DAEFRHDSGYEVHHQKLVFFAEDVGS KGAIIGLMVGGVVIA


YGGFMRRVGRPEWWMDYQKRYGGFL


GQAEPDRAHYNIVTFCCKCDSTLRLCV STHVDIR


GIGAVLKVLTTGLPALISWIKRKRQQ Neuropeptide S SFRNGVGTGMKKTSFQRAKS


OVA (241-270) SMLVLLPDEVSGLEQLESIINFEKLT EWTS


Secretin HSDGTFTSELSRLREGARLQRLLQGLV Deprotection 20% piperidine 20% piperidine


5% piperazine, 0.1M HOBt


5% piperazine, 0.1M HOBt


5% piperazine, 0.1M HOBt


20% piperidine, 0.1M HOBt


20% piperidine, 0.1M HOBt


20% piperidine 20% piperidine


20% piperidine, 0.1M HOBt


5% piperazine, 0.1M HOBt


Table 1. Microwave synthesis of biologically active peptides containing standard amino acids using standard microwave peptide synthesis protocols.


coupling reaction resulted in a 10% drop in crude purity, and performing the entire synthesis under conventional synthesis conditions gave only 18% crude purity. VIP (4) is a 28-mer peptide susceptible to aspartimide formation. While the conventional synthesis conditions gave only 45% crude purity in 35 hours, the microwave method provided the peptide in 80% purity in half the time. As a side note to emphasise the importance of 0.1M HOBt in the deprotection solution, the same synthesis without HOBt results in only 27% crude purity with about 50% of the undesirable aspartimide product. β-Amyloid (1-42) is known to be a difficult peptide to synthesise due to the hydrophobic nature of the peptide and its propensity to aggregate. This peptide is easily synthesised with the standard microwave protocol giving 90% crude purity in just 28 hours. The remaining peptides in Table 1 were synthesised using the same standard microwave peptide synthesis conditions. These peptides have been synthesised routinely using conventional techniques, but microwave irradiation provides a fast and


Activation HCTU/DIEA HBTU/DIEA DIC/HOBt HBTU/DIEA DIC/HOBt HBTU/DIEA HCTU/DIEA


Crude purity (%)


85 92 64 80 90 90 72 Time (hrs) 4.5 11 16.5 17 28 16 23


HBTU/DIEA HBTU/DIEA DIC/HOBt HBTU/DIEA


70 87 85 92


16 13 19 18


March/April 2012 sp2 Inter-Active 39


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