Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation

Sunithi Gunasekera,Teshome L Aboye,Ulf Göransson,Walid A Madian,Hesham R El-Seedi

doi:10.1007/s10989-012-9331-y

Sunithi Gunasekera, Teshome L Aboye + Show 3 more

Open Access

https://doi.org/10.1007/s10989-012-9331-y

Copy DOI

Abstract

The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.Electronic supplementary materialThe online version of this article (doi:10.1007/s10989-012-9331-y) contains supplementary material, which is available to authorized users.

Highlights

The discovery of proteins whose ends are linked together to produce a circular topology is an unique event in protein biochemistry
We demonstrate the synthesis of disulfide-rich cyclic peptides with accelerated speed of synthesis and with high yields and purity using microwave assisted Fmoc SPPS
The synthesis of the cyclotide kalata B1 was initiated on the Tentagel Rink amide resin with a C-terminal Di-Fmoc-3, 4-diaminobenzoic acid (Di-Fmoc-Dbz) linker and an

Summary

Introduction

The discovery of proteins whose ends are linked together to produce a circular topology is an unique event in protein biochemistry. Even though naturally occurring cyclic peptide products, such as cyclosporine, have been known for years, the presence of genetically encoded circular proteins in nature became known only recently. The cyclotides (Craik et al 1999) form the largest family of circular proteins with more than 200 characterized members. These proteins originate from plants and are commonly found within the plant families Rubiaceae and Violaceae. Cyclotides have captured significant interest because they possess an ultra stable scaffold, the cyclic cystine knot (CCK) motif, derived from the circular backbone and three knotted disulfide bonds.

Methods

Results

Discussion

Conclusion