Chemical Synthesis of Circular Proteins

Abstract

Circular proteins and their smaller versions, cyclic peptides, have a characteristic head-to-tail or end-to-end peptide backbone structure. The absence of both N and C termini in these macrocycles confers resistance to exopeptidase and heat degradation, enhances their conformational stability, and maximizes epitope display upon their circular contiguous sequences for interactions with other molecules. These advantages have provided incentives to engineer circular proteins by grafting linear bioactive peptides or epitopes into various structural scaffolds for therapeutic applications (1).For the purpose of this minireview, we refer to a cyclic peptide of Ͼ15 amino acids as a "circular protein" or "miniprotein." This arbitrary cutoff point has two justifications. First, many cyclic peptides of Ͻ15 amino acids are derived from nonribosomal synthesis, and the substrate specificity of a cyclase enzyme such as tyrocidine thioesterase could accommodate precursors of 6 -14 amino acids in length (2). Second, circular proteins are gene-encoded and processed by specific enzymes from their linear precursors containing a signal peptide and one or more prodomains (3). Cyclic peptides and circular proteins are frequently found in bacteria (microcin), fungi (cyclosporin), animals (-defensins), and more commonly, plants (cyclotides) (4 -7).From a synthetic standpoint, chemical synthesis of circular proteins has been a formidable challenge using the traditional enthalpic methods, which require partially or globally protected linear precursors and a strong enthalpic activation of the C-terminal residue for the cyclization reaction (Fig. 1A). Strong activation of the C-terminal moiety is necessary to overcome the entropy barrier in the coupling reactions and often leads to epimerization of the C-terminal amino acid residue and oligomerization to dimers and trimers. Work prior to 1997 showcased the challenges associated with enthalpy-driven cyclization of peptides of Ͻ15 amino acids. In 1997, we reported the total synthesis of circular proteins of 31 amino acids, cyclotides circulin B and cyclopsychotride, and in the following year, two other cyclotides (8,9). These studies represented a breakthrough because they were the first reports on a successful chemical synthesis of naturally occurring circular proteins using an entropy-driven ligation chemistry, which is conceptually different and operationally much simpler than the conventional enthalpy cyclization method.Many review articles have been published over the last few years, with a majority articulating the occurrence, chemistry, and biological functions of cyclic peptides (10 -13). Here, we will focus on contemporary entropy-driven ligation chemistry for the synthesis of circular proteins. Entropy-driven Ligation ChemistryDuring the 1990s, there was a paradigm shift in the synthesis of large peptides and proteins (14 -18). The sea change was driven by the discovery of entropic activation in convergent peptide synthesis using unprotected peptides as building blocks to enable an ...

Sortase-mediated backbone cyclization of proteins and peptides

“…After an acyl shift from the thioester to the thiol side chain of cysteine, an S→N rearrangement takes place, driving the reaction to completion and resulting in an N→C backbone-cyclized peptide with a cysteine insert at the ligation site ( Figure 1D). Cyclization of θ-defensins, containing two anti-parallel motifs of three cysteine residues separated by a single residue (CXCXC), is accelerated due to formation of an intermediate so-called cysteine zipper Tam and Wong, 2012;Borra and Camarero, 2013;Jia et al, 2014). The drawback of the NPL strategy is that it has been incompatible with standard Fmoc-protocols for many years, as C-terminal thioesters are not resistant to the repetitive alkaline conditions during Fmoc removal by piperidine .…”

Defensins: A Novel Approach to Use Plant Antimicrobial Peptides as an Alternative to Antibiotics

“…Nevertheless, bacteria produce polycyclic peptides such as lantibiotics and all major forms of life produce circular peptides which include bacteriocins from bacteria, cyclotides from plants and theta-defensins from animals. [4][5][6] In plants, the majority of AMPs are Cysrich [7] , a feature that enables the formation of multiple disulfide bonds (usually two to six) that contribute to a compact structure and resistance to chemical and proteolytic degradation.…”

Synthesis of Highly-strained Cyclic Tetrapeptides via Peptide Hydrazide Based Ligation

“…具有更大长度和复杂翻译后修饰的多肽/ 蛋白质被合成并应用于相关生物学研究. 与此同时, 通 过分子内天然化学连接反应, 众多环肽与环状蛋白分子 被成功合成 [3] . 作为天然化学连接的底物, 多肽硫酯的 有效制备是该反应成功应用的重要前提, 因此成为蛋白 质合成化学的一个研究热点.…”