PEPTIDES & PROTEINS Entry
efficient method to generate these peptides using the standard protocols. N-Terminal peptide modifications are prevalent in a variety of sequences, and they serve several different purposes. Fatty acid acylation on the N-terminus of a peptide increases its cell permeability and affinity, and is a common post-translational modification for a wide variety of viral, bacterial and eukaryotic proteins and peptides.14 Biotin-labeled peptides have numerous biochemical and microbiological applications.15 Under conventional conditions, these modifications often require coupling reactions of 24 hours or more due to poor solubility and reactivity of the acid or biotinylating reagents.16 We expected that sluggish reaction kinetics in these couplings could be overcome by the application of microwave irradiation. The acyl carrier protein ACP (65-74) sequence was selected as the platform on which to develop these methods. The backbone ACP (65-74) peptide was synthesised in less than 5 h with a crude purity of more than 95%. Double coupling of n-hexanoic acid to ACP for 5 min at 75°C using HCTU/DIEA activation gave the N-acylated peptide in 80% crude purity (Table 2, entry 1). The benzoic acid coupling was performed under similar conditions using HBTU activation to give the desired peptide in 81% crude purity (Table 2, entry 2). Microwave coupling of biotin-LC to ACP using HATU/DIEA for 5 min at 75°C gave the biotin-labeled peptide in 93% crude purity (Table 2, entry 3). Lastly, a peptide featuring an N-terminal 3-maleimidopropionyl group
1 Sequence
n-Hexanoyl VQAAIDYING
2
Benzoyl VQAAIDYING
3
Biotin-LC VQAAIDYING
4
3-Maleimidopropionyl GGGKKTKPTPPPKP HLKPK
Deprotection Activationa 20%
piperidine, 0.1M HOBt
20%
piperidine, 0.1M HOBt
20%
piperidine, 0.1M HOBt
20% piperidine a: Double coupling for underlined amino acids Table 2. Microwave synthesis of N-terminal modified peptides.
was studied. The maleimide can be used as a Michael acceptor to conjugate the peptide to another peptide or matrix. This peptide features the SH3-binding peptide17, and the synthesis of both the peptide and the incorporation of the 3-maleimidopropionic acid required double coupling as indicated in the table (Table 2, entry 4). With these modifications the peptide was synthesised in 91% crude purity.
Cyclic peptides exhibit improved metabolic stability, and increased potency and bioavailability as compared to their linear counterparts.18 The head-to-tail on-resin cyclisation strategy is an important tool in solid phase peptide synthesis that takes advantage of the resin induced pseudo- dilution effects thereby limiting undesirable
dimerisation. However, such cyclisations often require long reaction times under conventional conditions and result in a low crude purity of the cyclised peptide. Figure 1 outlines the on-resin microwave promoted synthesis of a head-to-tail cyclised peptide. All of the synthesis steps were carried out in a fully automated fashion using the CEM Liberty microwave peptide synthesizer. The peptide backbone (Gly-Val-Tyr-Leu-His-Ile-Glu) was synthesised on Fmoc-Glu(Wang resin)- ODmab with the side-chain γ−carboxyl group anchored to the resin and the α-carboxyl protected by the Dmab orthogonal protecting group in 91% crude purity. Selective on-resin removal of Dmab protection was performed with 5% hydrazine in DMF (2 x 3 min at 75°C) to give the linear precursor quantitatively. Head-to-tail cyclisation of the resin-bound peptide was accomplished using DIC/HOBt (3 x 10 min at 75°C) followed by cleavage of the cyclic peptide from the resin which provided crude product in overall 77% purity.
In conclusion
Fig 1. Microwave synthesis of head-to-tail cyclised peptide. [a] 20% piperidine, 5 min, 75°C. [b] Fmoc-AA-OH/HBTU/DIEA, 5 min, 75°C. [c] 5% hydrazine, 2 x 3 min, 75°C. [d] DIC/HOBt, 3 x 10 min, 75°C. [e] TFA/H2O/TIS/DODT, 40 min, 38°C.
40 sp2 Inter-Active March/April 2012
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 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. Microwave technology can be used routinely to synthesise the most trivial to the most
N-Terminal modification
HBTU/DIEA Double coupling HCTU/DIEA 5 min, 75ºC
HBTU/DIEA Double coupling HCTU/DIEA 5 min, 75ºC
HBTU/DIEA HATU/DIEA 5 min, 75ºC
DIC/HOBt
Double coupling 5 min, 75ºC
Crude purity (%)
80
81
93
91
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