Ligation Technologies for the Synthesis of Cyclic Peptides - American

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Cite This: Chem. Rev. XXXX, XXX, XXX−XXX

Ligation Technologies for the Synthesis of Cyclic Peptides Hoi Yee Chow,† Yue Zhang,† Eilidh Matheson,‡ and Xuechen Li*,†,§ †

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Department of Chemistry, State Key Laboratory of Synthetic Chemistry, The University of Hong Kong, Pokfulam Road, Hong Kong SAR, P. R. China ‡ School of Chemistry, University of Edinburgh, Edinburgh EH8 9LE, United Kingdom § Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, P. R. China ABSTRACT: Cyclic peptides have been attracting a lot of attention in recent decades, especially in the area of drug discovery, as more and more naturally occurring cyclic peptides with diverse biological activities have been discovered. Chemical synthesis of cyclic peptides is essential when studying their structure−activity relationships. Conventional peptide cyclization methods via direct coupling have inherent limitations, like the susceptibility to epimerization at the C-terminus, poor solubility of fully protected peptide precursors, and low yield caused by oligomerization. In this regard, chemoselective ligation-mediated cyclization methods have emerged as effective strategies for cyclic peptide synthesis. The toolbox for cyclic peptide synthesis has been expanded substantially in the past two decades, allowing more efficient synthesis of cyclic peptides with various scaffolds and modifications. This Review will explore different chemoselective ligation technologies used for cyclic peptide synthesis that generate both native and unnatural peptide linkages. The practical issues and limitations of different methods will be discussed. The advance in cyclic peptide synthesis will benefit the biological and medicinal study of cyclic peptides, an important class of macrocycles with potentials in numerous fields, notably in therapeutics.

CONTENTS 1. Introduction 1.1. Cyclic Peptides As Promising Therapeutic Agents 1.2. Strategies for Cyclic Peptide Synthesis 1.2.1. Peptide Cyclization via Direct Coupling with Side Chain Fully Protected Peptides 1.2.2. Peptide Cyclization via Intramolecular Aminolysis with Side Chain Partially Protected Peptides 1.2.3. Chemoselective Ligation-Mediated Peptide Cyclization 2. Chemoselective Ligation-Mediated Peptide Cyclization Creating Nonpeptide Linkages 2.1. Oxime and Hydrazone Ligation-Mediated Peptide Cyclization 2.2. Azide−Alkyne Cycloaddition-Mediated Peptide Cyclization 2.3. Aziridine Aldehyde-Based Multicomponent Macrocyclization 2.4. Imine-Mediated Macrocyclization 3. Chemoselective Ligation-Mediated Peptide Cyclization Creating Peptide Linkages 3.1. N-Terminal Cysteine (and Variants)-Mediated Chemoselective Peptide Cyclization 3.1.1. Thiazolidine-Generated Cyclization

© XXXX American Chemical Society

3.1.2. Native Chemical Ligation-Mediated Cyclization 3.1.3. Thia-zip Peptide Cyclization Derived from NCL 3.1.4. On-Resin NCL for Peptide Cyclization 3.1.5. Cyclization at Non-Cys Site 3.1.6. Selenocysteine-Based NCL 3.1.7. Summary 3.2. Traceless Staudinger Ligation-Mediated Peptide Cyclization 3.3. α-Ketoacid Hydroxylamine Ligation-Mediated Peptide Cyclization 3.3.1. Synthesis of Cyclic Peptides with Type I KAHA Ligation 3.3.2. Synthesis of Cyclic Peptide with Type II KAHA Ligation 3.3.3. Summary 3.4. Serine/Threonine Ligation-Mediated Peptide Cyclization 3.4.1. Synthesis of Cyclic Peptides by STL 3.4.2. Synthesis of Cyclic Peptides by Aspartic Acid Ligation Method Based on STL 3.4.3. Summary

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DOI: 10.1021/acs.chemrev.8b00657 Chem. Rev. XXXX, XXX, XXX−XXX

Chemical Reviews 4. General Considerations for Suppressing Oligomerizations in Chemical Ligation-Mediated Peptide Cyclization 4.1. Preorganization by Internal Elements of the Linear Precursor 4.2. Preorganization Driven by Mechanism of the Ligation Strategy Employed 5. Enzyme-Mediated Peptide Cyclization 5.1. Sortase A 5.2. Butelase 1 5.3. GmPOPB 5.4. Peptiligase 5.5. Summary 6. Conclusions Author Information Corresponding Author ORCID Notes Biographies Acknowledgments Abbreviations References

Review

In addition to their therapeutic applications, cyclic peptides have also been applied in different fields like material science to build nanomaterials23 and biochemistry as imaging agents.6,24 The wide applications of cyclic peptides make them a popular class of macrocycles for extensive studies, in both academia and industry. The access to cyclic peptides and their analogues is the first step to cultivate the full potential of cyclic peptides for various applications. With the growing field in cyclicpeptide-based drug development, finding the most efficient and practical synthetic route for cyclic peptides becomes a new task for synthetic chemists. To achieve this goal, different methods for generating cyclic peptides have been developed and have evolved ever since. In addition to the conventional peptide macrolactamization, ligations by chemoselective reactions to stitch the ends of a linear peptide chain to form cyclic peptides are now the prevailing strategy for chemical or semisynthesis of cyclic peptides.

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1.2. Strategies for Cyclic Peptide Synthesis

The formation of cyclic peptides found in nature usually involves reactions of the two reactive groups via lactamization, lactonization, thiolactonization, or disulfide bridge formation.25,26 On the basis of the site of the two reactive groups within a peptide, peptide cyclization can be generally categorized into four types: head-to-tail, head-to-side chain, side chain-to-tail, and side chain-to-side chain cyclization (Figure 1). For head-to-tail cyclic peptides, the macro-

1. INTRODUCTION 1.1. Cyclic Peptides As Promising Therapeutic Agents

Cyclic peptides have been attracting increasing attention in recent decades, especially in the area of drug discovery. Having a more rigid conformation compared to linear peptides, cyclic peptides possess favorable pharmacological properties and have potentials to be developed into therapeutic agents.1−4 Naturally occurring cyclic peptides exist in all kingdoms of life and exhibit diverse biological activities, including but not limited to antimicrobial, anti-inflammatory, antitumor, antiviral, and antifungal activities.5,6 Notable examples include the antibacterial agents gramicidin S7 and daptomycin8 and immunosuppressant cyclosporin.9 Teixobactin10 and malacidin11 are recently discovered members of cyclic peptide antibiotics with new antibacterial mechanisms. Cyclic peptides are more resistant toward enzymatic proteolysis12 compared to their linear peptide counterparts and thus are metabolically more stable. The ring architecture preorganizes and restricts the conformation of cyclic peptides, which lowers the entropic cost during receptor-binding processes.2 This feature increases their binding affinity and specificity toward receptors and protein targets. As a result, they are suitable for probing and perturbing protein−protein interactions (PPIs), which are “undruggable” targets in conventional drug discovery using small molecules.13−18 By suitable designs, cyclic peptides can mimic protein secondary structures (e.g., α-helices and β-hairpins) that are key motifs in receptor recognitions.19,20 All these favorable pharmacological features render cyclic peptides promising potential drug candidates. From 2006 to 2015, nine new cyclic peptide drugs were given full approval from the Food and Drug Administration (FDA) and European Medicines Agency (EMA).21 Among them, peginesatide,22 approved in 2012, is the first de novo cyclic peptide drug developed by rational design. It is projected that more de novo cyclic peptide drugs will enter the market in the near future, as many candidates are in the late stages of clinical trials.1,21

Figure 1. Four types of cyclic peptides.

cyclization involves the amino group and the carboxylate group at two termini of the linear precursor through lactamization. Besides the two termini, the side chain amino group of Lys, the side chain carboxylic acid group of Asp or Glu, the hydroxy group of Ser and Thr, and the sulfhydryl group of Cys are also common functional groups that are involved in the macrocyclization to yield cyclic peptides of the other three types. Other than utilizing the functional moieties that exist at the termini and the side chain of a peptide for peptide cyclization, functional groups installed at the backbone N-α atoms could also be involved.27,28 Such nonclassical Nbackbone peptide cyclization relies on the use of functionalized N-alkylated amino acids.27,28 Many naturally occurring cyclic peptides are cyclized in the head-to-tail fashion. The absence of N- and C-termini renders B


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peptide precursor. The macrocyclization efficiency via the direct-coupling approach is highly sequence-dependent. With all these issues, one has to carefully optimize the reaction conditions on a case-by-case basis to improve the efficiency for obtaining cyclic peptides in this way. To circumvent the undesired intermolecular reaction during the cyclization process and the use of large excess solvent for highly diluted conditions, solid-phase on-resin cyclization26 has been developed and employed. By immobilizing the linear precursor on a solid support, a pseudodilution is achieved,42 and thus oligomerization of linear peptides can be minimized. There are two methods to achieve head-to-tail cyclization on resin by direct coupling, depending on the linkage strategy employed. By anchoring the side chain of a trifunctional amino acid43−46 or the N-α atom of the C-terminal residues (except Pro)47 to the resin, the C-terminal carboxylate could be revealed for cyclization after orthogonal deprotection. The onresin cyclization by the direct coupling method, followed by global deprotection of side chain protecting groups and cleavage from the solid support, afforded the targeted cyclic peptides (Figure 2a and b). Nevertheless, potential epimeriza-

these types of cyclic peptides resistant to hydrolysis by exopeptidases, which further enhances their metabolic stability compared to other types of cyclic peptides. In this Review, we focus on the head-to-tail macrocyclization via chemoselective peptide ligation methods. The methods summarized in this Review are, however, generally applicable for synthesizing the other three types of cyclic peptides illustrated in Figure 1, if the reactive functional groups are introduced at the side chain of internal amino acid residues instead. Readers could refer to recent reviews about strategies for synthesizing stapled peptides29 and bicyclic peptides.30 They are two other very promising classes of cyclic peptides of which the macrocyclization involves functional moieties at the side chains. The challenges of cyclic peptide synthesis are present in various aspects. Besides the potential epimerization at the cyclization site with nonglycine/proline C-terminus,31 the tendency to form oligomers arising from intermolecular reactions is another major concern. Linear precursors without any turn-inducing element tend to adopt extended conformation due to a more stable all-trans configuration of amide bonds. As a result, the N- and C-termini of the linear peptide are far away from each other, and thus, they are less likely to react intramolecularly. The solubility of the macromolecule in the reaction solvent is another issue that needs to be addressed.32 The discovery of biologically active cyclic peptides and extensive studies to reveal their potentials in drug development have stimulated chemists to develop efficient synthetic methods to prepare cyclic peptides. The conventional method using amide coupling reagents to cyclize peptides requires side chain globally protected peptides.33 Peptide aminolysis allows cyclization using side chain partially protected peptides.34−39 The toolbox for peptide cyclization has been expanded rapidly over the past few decades. Cyclization using unprotected peptides as precursors is also feasible by employing chemoselective ligation strategies for which the unprotected side chains are tolerant.25,40 1.2.1. Peptide Cyclization via Direct Coupling with Side Chain Fully Protected Peptides. Direct coupling in solution phase using fully protected linear precursors (except the two cyclization ends) with coupling reagents is the conventional way for synthesizing cyclic peptides. However, this strategy suffers from several drawbacks that lead to low synthetic efficiency. First, the cyclization has to be performed in high dilution to prevent/reduce oligomerization arising from intermolecular reactions because peptide cyclization is an entropy-disfavored process. Second, the activated C-terminal carboxylic acid (except glycine and proline) is prone to epimerization, generating a pair of cyclic epimers as products. The origin of epimerization of the activated C-terminal carboxylic acid is mainly due to formation of oxazolones that causes the loss of the α-carbon chirality and generates the epimer upon ring-opening. Another less likely pathway for epimerization is the enolization of the activated C-terminal carboxylic acid via deprotonation of the α-proton under basic conditions.31 To tackle the epimerization problem, many different types of coupling reagents have been developed41 to minimize the epimerization during the activation of amino acids. Lastly, the solubility of some fully protected linear peptide precursors in organic solvent is poor,32 prohibiting the precursor to undergo reactions. The use of additives or a mixture of solvents may help solubilize the fully protected peptides, but there is no “magic solvent” that can dissolve every

Figure 2. Methods for synthesizing head-to-tail cyclic peptide via onresin methods: (a) peptides anchored to the resin via the side chain of a trifunctional amino acid; (b) peptides anchored to the resin via the N-α atom of the C-terminal residue; and (c) cyclization inducing the cleavage of the cyclic peptide from the resin through nucleophilic attack by the N-terminal amine.

tion at nonglycine/proline cyclization site remains a problem. Alternatively, head-to-tail cyclic peptides could be generated via on-resin aminolysis, which will be discussed in section 1.2.2 (Figure 2c). 1.2.2. Peptide Cyclization via Intramolecular Aminolysis with Side Chain Partially Protected Peptides. Aminolysis-based reactions using partially protected linear peptide with a mildly activated C-terminus could be used to C


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orthogonally to other side chains and deprotected after the cyclization. The major drawback of aminolysis-mediated cyclization is that epimerization often occurs during cyclization at nonglycine/proline sites. A recent report by Ohara et al.39 used the o-aminoanilide (Dbz) group48 as a masked active ester for aminolysis-mediated peptide cyclization, and their studies indicated that the degree of epimerization was dependent on the cyclization site. They also showed that addition of HOBt or HOAt could suppress but not eliminate the epimerization.39 Li and co-workers36 demonstrated that aminolysis-mediated peptide cyclization proceeded much faster with peptide 2formylthiophenol esters than with other thioesters. Five natural cyclic peptides with a ring size of 7−11 amino acids were synthesized in DMSO at 1 mM concentration. The resultant cyclic peptides were obtained in moderate yields (40−50%) and a high monomer-to-dimer ratio. The epimerization problem was also observed by the authors. Therefore, the five cyclic peptide examples they reported were all designed to be cyclized at C-terminal glycine or proline sites. Arora and coworkers37 found that aminolysis could proceed even faster with peptide selenobenzylaldehyde esters, although applications on cyclic peptide synthesis using peptide selenoesters38 or selenobenzylaldehyde esters37 have yet to be reported. Aminolysis-mediated peptide cyclization could also be conducted on-resin to prepare head-to-tail cyclic peptides. The peptide was linked to the resin through an amine-labile linker/safety-catch linker as a mildly activated ester attached to the solid support, such as oxime linkers.49 The cyclization was initiated by the nucleophilic attack of the N-terminal amine, which induced the cleavage of the peptide from the solid support simultaneously (Figure 2c).50−57 1.2.3. Chemoselective Ligation-Mediated Peptide Cyclization. Due to the limitations of the direct coupling and intramolecular peptide aminolysis in cyclic peptide synthesis discussed in previous sections, highly chemoselective, epimerization-free, and efficient methods have been developed to overcome the limitations for cyclic peptide synthesis. Conceptually, novel ligation technologies that have been developed for synthesis of longer peptides (>50 amino acid) and proteins58,59 can be adopted intramolecularly for cyclic peptide synthesis60 of different ring sizes after minor refinement of reaction conditions. Reactions between bioorthogonal functional groups61−64 as well as cutting-edge strategies such as multicomponent peptide cyclization65 and enzymatic cyclization66 are also promising chemoselective methods for macrocyclization on peptides. Some ligations create peptide linkages at the cyclization site, while others create a variety of nonpeptide backbone linkages upon ringclosure (Figure 5). Herein, we define chemoselective peptide ligation as reactions between the reactive groups in unprotected peptide fragments forming a new linkage, i.e., a condensation reaction tolerant of the side chain functional groups of all amino acids. Applications of such chemoselective ligations on head-to-tail cyclic peptide synthesis from late 1990s to present will be discussed, with an emphasis on the practical considerations in contrast to segment ligations. Enzymatic ligation for cyclic peptide synthesis will also be covered.

generate head-to-tail cyclic peptides (Figure 3). Houghten and co-workers35 synthesized a series of cyclic peptides containing

Figure 3. Aminolysis-mediated peptide cyclization. Different mildly activated esters that could undergo aminolysis are shown.

5−11 amino acids and a natural cyclic peptide tyrocidine A through direct intramolecular aminolysis between the Nterminal amino group and the C-terminal thioesters. Using the fully unprotected peptide thioester in a mixture of acetonitrile and imidazole aqueous solution, they obtained the target cyclic peptides with yields ranging from 15% to 46%. However, this method cannot distinguish the N-terminal amino group from internal amines present on the unprotected linear peptide. Both the side chain amino group of internal Lys and the Nterminal amine can react with the thioester to yield head-toside chain and head-to-tail cyclic peptides, respectively, at the same time (Figure 4). Hence, amino groups that are not intended to participate in cyclization should be protected

Figure 4. Regioselectivity studies by Houghten and co-workers35 showed that aminolysis-mediated cyclization with a C-terminal thioester reacted with both the N-terminal amine and the side chain amine of Lys. D


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Figure 6. (a) Hydrazone-based cyclization. (b) Oxime-based cyclization. The two reactive groups for both types of cyclization can be put at either N- or C-terminus, and the reaction generates a set of E/Z isomers upon cyclization. Ligations are typically performed in acidic aqueous buffers.

Figure 5. Chemoselective ligations for cyclic peptide synthesis. (Upper) Ligations generating a nonpeptide bond at the cyclization site. (Lower) Ligations generating a peptide bond at the cyclization site.

2. CHEMOSELECTIVE LIGATION-MEDIATED PEPTIDE CYCLIZATION CREATING NONPEPTIDE LINKAGES 2.1. Oxime and Hydrazone Ligation-Mediated Peptide Cyclization

Figure 7. Synthetic route of the 34-mer cyclic hybrid peptide. The Met cleavage site is labeled in red. Cyclization proceeded spontaneously with the oxidation of N-terminal Ser such that the intermediate was not observed. The Z-isomer of the cyclized peptide is shown.

Oxime and hydrazone ligation methods are based on the chemistries between an aminooxy moiety or hydrazide with an aldehyde or ketone. These two ligations have been widely used in bioconjugation.63 They have been adopted for cyclic peptide synthesis when the two reactive moieties are incorporated on the same peptide fragment (Figure 6). These two ligations have been more frequently employed in peptide cyclization involving side chains,67−69 while their applications for head-totail cyclization have rarely been reported.70 The Kolmar group70 utilized the hydrazone ligation to cyclize a peptide fragment of 34 residues, which is a hybrid miniprotein containing domains from two proteins (Figure 7). The hybrid peptide was found to inhibit human β-II tryptase activity. The head-to-tail cyclization was carried out between the N-terminal ketoaldehyde and C-terminal hydrazide. The 34-mer hybrid miniprotein was obtained by cyanogen bromide digestion at the methionine site of the recombinant protein construct, generating a N-terminal Ser and a C-terminal γlactone71 in the acyclic precursor. These two distinctive functional groups served as handles for site-specific incorporation of the prerequisite hydrazide at the C-terminus and

ketoaldehyde at the N-terminus in the presence of unprotected side chain functionalities. The cyclization was initiated as soon as the N-terminal serine was oxidized to the ketoaldehyde by NaIO472 in aqueous medium to yield the cyclic peptide with hydrozone linkage within minutes. The extremely fast cyclization was likely due to preorganization of both termini in close proximity by the folded conformation of the hybrid peptide.70 Hydrazone linkage was reversible but was found to be stable at the physiological pH conditions. A stable rigid bond could be generated by reduction with NaCNBH373 to form the hydrazide. The hydrazone linkage was a good peptidomimetic of amide bonds. The cyclized hybrid protein was found to be a more efficient inhibitor than the cyclic form with native amide bonds, illustrating that the unnatural peptide linkage was well-tolerated in the biological system.70 E


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The application of oxime and hydrazone ligation for the preparation of head-to-tail cyclic peptides relies on the accessibility of the peptide precursors with reactive groups on N- and C-termini, regardless of the absolute position of the two reactive groups (Figure 6). Various methods have been developed for functionalizing the termini for oxime or hydrozone forming cyclization such as the use of the functionalized solid support74,75 or incorporations by chemical72,73,76 and enzymatic77,78 strategies. The oxime and hydrozone ligations are highly chemoselective because the reactive moieties (aminooxy, hydrazide, and aldehyde) are bioorthogonal with the side chain functionalities of the 20 natural amino acids. Side reactions from unprotected nucleophilic side chains could be suppressed by conducting the ligation at acidic pH buffers, taking advantage of protonation of the basic side chain nucleophiles.76 Like most peptide cyclization methods, high dilution is needed to prevent oligomerization arising from intermolecular ligation. The low usage of these ligations for head-to-tail cyclic peptide synthesis is most probably due to the possible hydrolysis of the hydrazine and oxime linkage.77 The formation of a mixture of products with E/Z isomers68 and the potential side reactions that may occur arising from the instability of the aldehyde or aminooxy-containing precursor62 cause these ligations to be a less attractive strategy when it comes to cyclic peptide synthesis. Nevertheless, oxime and hydrozone ligation provide means for generating rigid backbone macrocycles with unnatural peptide linkages as peptidomimetics.

Figure 9. (a) trans-Amide bond and (b) cis-amide bond in a dipeptide.

Angell and Burgess investigated the use of 1,2,3-triazole in a ring as β-turn peptidomimetics.79 They synthesized a group of 14-membered ring compounds with a dipeptide scaffold via CuAAC-mediated ring-closure. The linear precursors were prepared by installing an aryl azide and an alkyne on the N-and C-termini of the dipeptide, respectively, via solution-phase synthesis. However, the ring-closure by CuAAC yielded cyclodimer as the major product while the target cyclic monomer was the minor product, possibly due to the high ring strain of the cyclic monomer.79 Besides being able to generate a trans-amide bond mimic, CuAAC could generate a 1,5-disubstituted 1,2,3-triazole when the catalyst was changed from copper(I) to ruthenium(II)86 (Figure 8), which was found to be good peptidomimetics of the cis-amide bond (Figure 9b).87 Because CuAAC is operationally simple, highly efficient, and able to generate regioselective 1,2,3-triazole at the cyclization site as good peptidomimetics, it is very suitable for synthesis of cyclic peptides. However, the use of CuAAC in preparation of head-to-tail cyclic peptides is limited. Synthesis of cyclic peptides by CuAAC mostly involves the N-terminus and side chains but not the C-terminus. This is because the introduction of the two reactive moieties to the N-terminus and side chains of the peptide during SPPS is convenient by incorporating derivatized building blocks with an azide (e.g., N-α-azido amino acids) or an alkyne (e.g., propargyl glycine).88−91 On the contrary, due to the lack of an efficient and convenient method to incorporate an azide or alkyne at the C-terminus by SPPS, an extra step of solution-phase reaction is usually needed to install the alkyne/azide at the Cterminus after peptide cleavage from the solid support.79,85,92,93 Only recently, a method of introducing C-terminal propargyl amide solely by Fmoc-SPPS became available by modification of the aminomethyl resin using a backbone amide linker.94 Applications of CuAAC in peptide cyclization have been thoroughly discussed in other reviews61,95 that readers can refer to for more details.

2.2. Azide−Alkyne Cycloaddition-Mediated Peptide Cyclization

The first head-to-tail cyclic peptide synthesized via copper(I)catalyzed azide−alkyne cycloaddition (CuAAC) was reported by Angell and Burgess in 2005.79 Copper(I)-catalyzed 1,3dipolar cycloaddition80 between an alkyne and azide under mild conditions in either organic solvents or water to form a 1,4-disubstituted 1,2,3-triazole (Figure 8) (a type of click

2.3. Aziridine Aldehyde-Based Multicomponent Macrocyclization

Figure 8. 1,3-Dipolar cycloaddition between the terminal azide and alkyne using Cu(I) and Ru(II) catalyst, yielding 1,4-substituted and 1,5-substituted 1,2,3-triazole at the cyclization site, respectively.

Yudin and co-workers65 have developed an Ugi-typed96 fourcomponent-based macrocyclization. This method involved an aziridine aldehyde, an isocyanide, the N-terminal amino group, and the C-terminal carboxylic acid of a peptide segment (Figure 10). The cyclization went through an imidoanhydride intermediate formed by the concerted addition among the aziridine aldehyde, isocyanide, and the N-terminal amine with the adjacent amide. The concerted addition mechanism accounted for the exceptional stereoselectivity of this cyclization. Attack on the imidoanhydride intermediate by the C-terminal carboxylate afterward, followed by an O-to-Nacyl shift to the aziridine, yielded the cyclic peptide. As seen, the cyclization resulted in an unnatural peptide backbone linkage with an acyl aziridine at the cyclization site, which could further react with different nucleophilic moieties via the

chemistry81) regioselectively was developed by the Meldal group82 and the Sharpless group83 independently in 2002. In Meldal’s work, the application of CuAAC for both linear82 and side chain-to-side chain cyclic peptide84 syntheses in solid phase had been demonstrated. The reaction conditions were fully compatible with peptide chemistries, and the reaction could be performed on side chain unprotected peptides,84 displaying the high chemoselectivity of the reaction between alkynes and azides. The resultant 1,4-disubstituted 1,2,3triazole was found to effectively mimic the native trans-amide bond (Figure 9a),85 which provided peptide chemists with another option of amide bond isosteres. F


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Figure 11. Synthesis of aziridine aldehydes from (a) serine esters, (b) N-Boc-protected amino acids, and (c) ester diols with aziridine esters as the common intermediate.

cyclization site served as a handle for late-stage, site-specific modification.65 The modification could proceed via highly regioselective nucleophilic ring-opening99 by thiols100 or other nucleophiles99 for rapid generation of derivatives from one single compound. This feature is particularly useful for generating an array of cyclic peptides with the same scaffold but diverse modifications at specific sites for the highthroughput drug screening. The cyclization was conducted in an unusually high concentration (0.2 M in TFE or HFIP) without observable formation of cyclodimers or oligomers.65 This is probably because the amide bond adjacent to the Nterminal residue was involved in the reaction, which created a highly effective concentration around the N-terminus, therefore preventing intermolecular reactions from happening. Interestingly, the absolute configuration of the N-terminal amino acid and the aziridine determined the reaction pathway, with an S-aziridine needed for reacting with the N-terminal Lamino acid to generate the cyclic peptides. Mismatch of configurations yielded linear products (e.g., S-aziridine with Damino acid), which upon extended reaction time did not transform into the cyclic form. This result further verified the absence of epimerization at the N-terminus during the multicomponent cyclization, distinct from the Ugi-type macrocyclization that epimerization at the N-terminal residue was usually observed when a monofunctional aldehyde was used instead.65 The absence of epimerization at the C-terminus was supported by the NMR analysis.65 This high-yielding multicomponent peptide macrocyclization will benefit the growing field of cyclic peptide synthesis and cyclic-peptidebased drug discovery.

Figure 10. Multicomponent peptide macrocyclization. The four components involved are the N-terminal amino group (proline in the figure), the C-terminal carboxylate, an alkyl or aryl isocyanide, and the aziridine aldehyde dimer. The acyl aziridine ring is formed in the backbone of the cyclic peptide after the cyclization and O-to-N-acyl transfer. Ring-opening of the aziridine by nucleophiles generates 1,2substituted ethylenediamine-like linkage.

ring-opening of the aziridine (Figure 10).97 The peptide fragment can be obtained via the standard Fmoc-SPPS, while isocyanides are either commercially available or can be readily synthesized. The aziridine aldehyde used for cyclization is in dimeric form and can be prepared from serine ester to form the aziridine ester via the Mitsunobu reaction followed by DIBALH reduction. Alternatively, N-Boc protected amino acid or ester diol can be used as starting material if substitutions on the aziridine are needed. The aziridine ester is the common intermediate98 in different synthetic routes for aziridine aldehydes preparation (Figure 11).65 Aziridine aldehydes are stable for storage for months as 5 M aqueous stock solutions at room temperature.99 Using this multicomponent peptide cyclization, Yudin and co-workers demonstrated that linear peptide precursors, with 2−5 residues, could readily form a cyclic structure with the exogenous aziridine aldehyde and isocyanide within hours. It is worth noting that the N-terminal residue of the linear precursors was proline in all the examples illustrated.65 The cyclization was also feasible using a primary amine but with a much slower rate.99 This multicomponent peptide cyclization was found to be highly chemoselective in that the nucleophilic and acidic side chains were not competing with the termini for reaction, due to the lower nucleophilicity and acidity of the side chains under the reaction conditions.65,99 Although such a head-to-tail peptide cyclization method generated a non-native peptide linkage, the acyl aziridine moiety formed at the

2.4. Imine-Mediated Macrocyclization

Recently, Baran and co-workers developed a peptide macrocyclization method utilizing the N-terminal amino group and the C-terminal aldehyde of an unprotected linear peptide.64 The inspiration of their work was from cyclic peptides produced by the nonribosomal peptide synthetase (NRPS) enzymatic machinery via peptide aldehydes. Like many other cyclization strategies, this cyclization had to be performed at highly diluted conditions (1 mM). This peptide cyclization started with spontaneous intramolecular imine formation in aqueous media, followed by the nucleophilic attack of the imine to “trap” the cyclic peptide (Figure 12a). Depending on the choice of nucleophiles, different moieties at the cyclization site were formed. External nucleophiles such as KCN and NaBH3CN generated α-aminonitriles or secondary amines at the ring-closure site, respectively (Figure 12b), while internal nucleophiles adjacent to the N-terminal amino group, G


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Figure 13. Naturally occurring cyclic peptide containing nonamide backbones.

products.64 Also, minor products with cyclization at the Lys side chain have been observed even though the cyclization method well-tolerated functional groups on unprotected peptides and selectively reacted with the α-amino group at the N-terminus over the internal Lys side chain amino group.64 Formation of this minor product arising from internal Lys lowered the yield and the purity of the cyclic peptide when the mixture of products was not well-separated by HPLC purification. The amount of byproduct from the macrocyclization with internal amino groups and side products from other pathways were found to be pH-dependent and therefore could be minimized after condition optimizations.64 The relatively slow cyclization rate (a reaction time of 24−48 h in general)64 may also be a consideration when employing this method for synthesizing cyclic peptides.

Figure 12. (a) Cyclization initiated by imine formation between the N-terminal amine and the C-terminal aldehyde. The imine linkage is transformed to a more stable bond by (b) inter- or (c) intramolecular nucleophilic attack.

including indole, imidazole, thiol, and selenol, generated heterocycles at the cyclization site (Figure 12c).64 The linear precursor of the peptide C-terminal aldehyde could be readily prepared by Fmoc-SPPS where an Fmocamino aldehyde101 was reacted with the 1,2-hydroxyamine moiety of Thr anchored onto the Rink-amide resin as the ThrGly dipeptide and formed an oxazolidine.74 After protecting the oxazolidine nitrogen with the Boc group, the peptide was elongated via standard Fmoc-SPPS conditions. The unprotected linear peptide bearing a C-terminal aldehyde was revealed after global deprotection of all protecting groups and the hydrolysis of oxazolidine in one attempt by the TFA cleavage cocktail. The scope of this high-yielding macrocyclization method has been illustrated with 28 examples with ring size of 5−10 amino acids. Three natural cyclic peptide analogues whose native form consists of either imine linkage (scytonemide A and koranimine) or thiazolidine linkage (lugdunin), as well as the natural cyclic peptide sanguinamide A, containing a thiazole motif within its structure, were included to demonstrate the scope of this macrocyclization strategy (Figure 13).64 The versatility of this cyclization technique lies in its ability to introduce diverse modifications after cyclization, which enables one to generate an array of analogues derived from the same linear precursors. This strategy makes the construction of a library for a set of cyclic peptide lead compounds very convenient. The absence of a C-terminal activated ester eliminates the competing hydrolysis reactions that are commonly found in other cyclization strategies during cyclization. However, although rarely observed by the Baran group, α-carbon epimerization at the C-terminal aldehyde during both Fmoc-SPPS and the cyclization step may be problematic and produce a mixture of diastereomeric

3. CHEMOSELECTIVE LIGATION-MEDIATED PEPTIDE CYCLIZATION CREATING PEPTIDE LINKAGES 3.1. N-Terminal Cysteine (and Variants)-Mediated Chemoselective Peptide Cyclization

The “soft base” nucleophilicity of the thiol group and the 1,2aminothiol bifunctionality of the N-terminal cysteine can distinguish themselves from other reactive functionalities present on an unprotected peptide, which contributes to the highly chemoselective nature of the N-terminal cysteinemediated chemoselective peptide cyclization. Selenium and selenocysteine, being chemically similar to sulfur and cysteine, respectively, could also mediate peptide cyclization in a similar way. 3.1.1. Thiazolidine-Generated Cyclization. Tam and co-workers were among the first to prepare cyclic peptides using side chain fully unprotected peptide segments as precursors.102 After the group showed that a peptide with a C-terminal glycoaldehyde ester could undergo chemoselective ligation with another peptide segment with a N-terminal Cys (or Ser/Thr) to generate an amide bond with a thiazolidine at the ligation site,103 they moved to put the two reactive groups on the same peptide for producing head-to-tail and side chainto-tail cyclic peptides (Figure 14).102 For head-to-tail cyclic H


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feasibility of native chemical ligation (NCL), another ligation method utilizing the N-terminal Cys, for synthesizing cyclic peptides.105 Native chemical ligation was developed by Kent and co-workers in 1994 as a groundbreaking and revolutionary development in the field of peptide and protein chemical synthesis.106 NCL involves the reaction between the Nterminal Cys and the C-terminal thioester of unprotected peptides in an aqueous solution of pH 7. The ligation is initiated by the addition of a thiol catalyst to facilitate the equilibrium-driven transthioesterification between the two termini. The chemoselectivity of NCL originates from the reversible transthioesterification step. The driving force of the ligation is the irreversible S-to-N-acyl transfer by the Nterminal amino group, and the ligated product acts as the thermodynamic sink to push the reaction to completion. The reversible nature of the transthioesterification step ensures the chemoselectivity of NCL. Internal Cys lacks the unique 1,2aminothiol group that is only present in N-terminal Cys. Without the amino group adjacent to the sulfhydryl group, Sto-N-acyl transfer cannot occur. Therefore, internal Cys will not interfere with the ligation. Other nucleophilic side chains present in the 20 natural amino acids have poor nucleophilicity at pH 7 and thus will not compete with thiols for the ligation reaction.107 Since its first introduction in 1994, enormous efforts have been paid to advance NCL for protein chemical synthesis. For example, various methods have been developed to generate the requisite peptide thioesters via SPPS108,109 and through biosynthesis using intein-based systems.110 To expand the scope of NCL beyond requiring cysteine as the N-terminal residue, the use of cysteine surrogates,58,111−113 thiolcontaining auxiliary group,114−116 and desulfurization methods117,118 have been explored. These improvements that have been made for the linear peptide/protein synthesis will certainly be translatable for cyclic peptides synthesis, which will be illustrated with examples in this section. In the intramolecular version of NCL, a thiolactone is first formed. Subsequent irreversible S-to-N-acyl transfer provides the cyclic peptide with a natural peptide bond (Xaa-Cys) at the site of cyclization. Only a trace amount of epimerization at the C-terminal residue of the linear peptide precursor was observed in the cyclized product (Figure 15). In the first examples of intramolecular NCL, Tam and co-workers successfully cyclized analogues of enkephalins sized from 5 to 8 residues. Two larger cyclic peptides whose sequences were

Figure 14. Thiazolidine-forming cyclization. A stereocenter is generated on the thiazolidine, resulting in a mixture of two diastereomeric cyclic peptide products.

peptide synthesis, the C-terminal glycolaldehyde esters were generated via oxidation of the C-terminal glyceric esters.102 The preformed Fmoc-amino acid glyceric ester was first immobilized on the benzaldehyde resin,104 followed by standard Fmoc-SPPS procedures. After the TFA cleavage, the peptide C-terminal glyceric ester was obtained, and oxidation by NaIO4 at pH 5 generated the required peptide glycoaldehyde ester. The intramolecular macrocyclization was initiated in a pH 5.5 aqueous solution. The rate-determining O-to-N-acyl transfer was triggered by adjusting the pH to 5.9. This reaction afforded a pair of diastereomeric products bearing a thiazolidine with an amide bond at the cyclization site. The resultant thiazolidine moiety could serve as a proline mimic. The reaction concentration for this cyclization could be as high as 20 mM with no oligomer formation observed. This could be attributed to the ring-chain tautomerization of the aldehydeamine system, but the authors did not rule out the possibility of the preorganization of the two reactive termini in their model peptides that minimized oligomerizations at high concentrations. Cyclic peptides with the ring sized from 5 to 26 residues were successfully synthesized in good yields in ∼100 h at room temperature. However, for the cyclopentapeptide, only 25% yield (determined by HPLC analysis) was observed after reacting at 52 °C for 100 h. Although Tam and co-workers did not mention any limitation on the C-terminal amino acid, all model peptides had Gly-Cys at the cyclization sites. Thus, it is plausible to deduce that the reaction rate would be rather slow for other Cterminal amino acids. The slow rate of the cyclization is because a highly strained tricyclic intermediate was involved in the step of the O-to-N-acyl transfer. Heating could speed up the O-to-N-acyl transfer, but side reactions such as hydrolysis and decomposition occurred at elevated temperatures. The rate of the cyclization was dependent on the ring size, such that the larger the ring, the faster was the reaction.102 Therefore, this method is likely suitable to prepare cyclic peptides with a larger ring size (>12 amino acids). Besides the slow reaction rate, the generation of a pair of diastereomers due to the introduction of a stereocenter on the C-5 carbon of the thiazolidine ring with a hydroxyl methyl group102,103 also restricts its wide applications in cyclic peptide synthesis. 3.1.2. Native Chemical Ligation-Mediated Cyclization. Tam and co-workers were also the first to show the

Figure 15. Intramolecular native chemical ligation (NCL). A thiolactone is formed as the intermediate, which after S-to-N-acyl transfer generates the cyclic peptide with a native peptide bond at the cyclization site. I


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taken from the N-terminal of human parathyroid hormone (14-mer) and salmon calcitonin (16-mer) were also synthesized.105 They showed that no oligomers were formed even when the cyclizations were conducted in relatively high concentration (in mM range), in contrast to the case that high dilution was required for the conventional macrocyclization using fully protected peptide. Zhang and Tam105 proposed that the absence of oligomerization at higher concentrations resulted from the ring-chain tautomeric equilibrium of the transthioesterification step between the thioester and the thiol on N-terminal Cys. This observation was consistent with a recent study by Liu and co-workers on the synthesis of highly strained cyclic tetrapeptides by NCL.119 In Liu’s work, they found that cyclic monomers were formed exclusively at a concentration up to 1 mM and that significant cyclic dimer formation occurred at a concentration of 3 mM. Camarero and Muir120 also showed that a 15-residue peptide containing the Arg-Gly-Asp (RGD) motif was cyclized under standard NCL conditions. The linear precursor of this cyclization only required a simple purification of trituration with cold diethyl ether upon peptide cleavage from resins. In their later work,121 Muir and co-workers found that, although cyclization between the two flexible termini under denatured conditions went smoothly at diluted conditions (50 μM), the head-to-tail cyclization was facilitated by the preorganization of the two reactive termini in close proximity. They chose a 44mer sequence from WW domain of human Yes kinaseassociated protein, in which the N- and C-termini are held in close proximity by the three-stranded antiparallel β sheet structure present in the domain. The 44-mer linear precursor was cyclized, both with and without the denaturant (6 M guanidine hydrochloride). Despite the successful cyclization in both cases, the rate of cyclization was 1 order of magnitude slower under the denatured conditions. This study provided direct evidence that the rate of cyclization could be sped up by the preorganization of the two termini.121 This observation was supported by Tam and co-workers,122 on the synthesis of a 40mer cyclic peptide with the sequence taken from the second extracellular loop of chemokine receptor CCR5. They found that the rate of cyclization was much faster in the absence of guanidine hydrochloride than in the presence of guanidine. During the synthesis of this CCR5 cyclic peptide, the Nterminal Cys was temporarily protected by ninhydrin to avoid formylation during the HF cleavage step caused by the Bom protecting of the His side chain. The ninhydrin protecting group spontaneously fell off under NCL conditions (Figure 16). Camarero and Muir were the first to demonstrate that cyclic peptides synthesis by NCL could be aided by the recombinant technology.123 They made use of the intein system to produce linear peptides consisting of both the N-terminal Cys and the C-terminal thioester biosynthetically. Using the intein system, they successfully obtained the cyclic form of the Src homology 3 (SH3) domain. This biosynthetic approach is attractive because it overcomes the size limitation of peptide thioester preparation by SPPS124 and is cost-effective for large-scale production. This evoked further development in intein-based technologies for cyclic peptide synthesis. Examples of such technologies include expressed protein ligation (EPL),125 twointein (TWIN) system,126 and split intein-mediated circular ligation of peptides and proteins (SICLOPPS).127 Intein-based cyclic peptide synthesis is not without limitations. Peptides with post-translational modification and unnatural amino acids

Figure 16. Synthesis of cyclic peptides using a ninhydrin-protected Nterminal cysteine. The ninhydrin group was stable under HF treatment and protected the N-terminal cysteine from formylation during deprotection of the Bom group on His residues by HF. Ninhydrin deprotection and NCL-mediated cyclization were conducted in one-pot fashion to yield the cyclic peptide.

cannot be incorporated in the sequence as easily as when using chemical synthesis. Over the past two decades, NCL-mediated peptide cyclization has been applied to synthesize many cyclic peptides, mainly naturally occurring cyclic peptides with different biological functions from different classes of organisms, as listed in Table 1.117,128−153 Herein, practical considerations during cyclic peptide synthesis by NCL will be discussed in detail with examples of head-to-tail cyclic peptides and with the advancement in NCL showcased. 3.1.3. Thia-zip Peptide Cyclization Derived from NCL. The notable extension of NCL-mediated peptide cyclization was demonstrated with the synthesis of cysteine-rich cyclic peptides, especially cyclotides. Cyclotides are a family of headto-tail cyclic peptides found in plants, typically consisting of 28−31 amino acids and three disulfide bonds arranged into knots.154 Their Cys-rich feature facilitated the NCL-mediated ring-closure via a series of thiolactone exchanges followed by the S-to-N-acyl transfer to reside at the N-terminal Cys and form a cyclic peptide (Figure 17). This thiolactone-exchangeassisted cyclization was termed the thia-zip reaction by the Tam group.130 Using the thia-zip reaction, followed by the oxidative folding, they have synthesized cyclopsychotride (31mer, 6 Cys, 3 disulfide bonds),128−130 an analogue of rabbit αdefesin NP-1 (33-mer, 4 Cys, 2 disulfide bonds),129 circulin A (30-mer, 6 Cys, 3 disulfide bonds),131 circulin B (31-mer, 6 Cys, 3 disulfide bonds),129 kalata B1 (29-mer, 6 Cys, 3 disulfide bonds),131 cyclized analogues of protegrins (18-mer, 2−6 Cys, 1−3 disulfide bonds),138 and cyclic tachyplesins analogues (18-mer, 6 Cys, 3 disulfide bonds).139 The Craik132,142,143,145,148,155,156 and Tate140,141 groups also implemented this strategy to synthesize a number of cyclic peptides and their analogues to examine the effects of cyclization on their structure, stability, and activities. Their study targets included MCoTI-I and II (trypsin inhibitor cyclotides),140,141 kalata B1,132 sunflower trypsin inhibitor-1 J


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Table 1. Naturally Occurring Cyclic Peptides Synthesized by NCL cyclic peptide

no. of residues

origin

biological activity

cyclopsychotride α-defesin NP-1 circulin A circulin B kalata B1 protegrins tachyplesins MCoTI-I MCoTI-II conotoxin

31 33 30 31 29 18 18 34 34 16−22

Psychotria longipes (plant) rabbit Chassalia parvifolia (plant) Chassalia parvifolia (plant) Oldenlandia affinis (plant) pig horseshoe crab Momordica cochinchinensis (plant) Momordica cochinchinensis (plant) marine cone snail

sunflower trypsin inhibitor-1 (SFTI-1) cycloviolacin O2 (cVO2) rhesus θ-defensin-1 (RTD-1) destoamide B

14

sunflower

30 18 6

stellarin G stylostatin 1 cyclonellin crotogossamide tyrocidin A somatostatin microcin J25 Cter M dichotomin C dichotomin E

5 7 8 9 10 11 21 29 6 5

Viola odorata (plant) monkey Streptomyces scopuliridis (marine microbe) Stellaria yunnanensis (plant) Stylotella aurantium (marine sponge) Axinella carteri (marine sponge) Croton gossypifolius (plant) Bacillus brevis (soil bacteria) human E. coli Clitoria ternatea (plant) Stellaria dichotoma (plant) Stellaria dichotoma (plant)

ref

neurotensin (NT) antagonist antiviral antiviral, antimicrobial antiviral, antimicrobial uteroactive, antimicrobial antimicrobial antimicrobial protease inhibitor protease inhibitor targets on neuroreceptors and ion channels trypsin inhibitor

128−130 130 131 129 131−137 138 139 140 134, 136, 140, 141 142−144

antitumor, antibacterial antimicrobial, antiviral, antifungal antibacterial

134, 137, 148 137, 149 150

not determined anticancer not determined not determined antibacterial inhibiting growth hormone antibacterial insecticidal inhibiting cell growth inhibiting cell growth

150 150 150 150, 151 150 152 117 137 153 153

137, 144−147

(SFTI-1),145 conotoxins from the venom of marine cone snails,143 and cyclotide cycloviolacin O2.148 NCL enables easy synthesis of Cys-rich cyclic peptides in a high-yielding and epimerization-free manner. The subsequent oxidation for the formation of disulfide bonds was found to be straightforward in most cases. In some cases where misfolding was a problem, a two-step oxidation sequence with the use of orthogonal protecting groups on different Cys proved to be effective to afford the desired product.129 The in-depth study of the structure−activity relationship of these Cys-rich cyclic peptides depends heavily on NCL.142 These peptides have also been a popular choice as model cyclic peptides for demonstrating the feasibility of novel methods and techniques developed for improving NCL (Table 1). 3.1.4. On-Resin NCL for Peptide Cyclization. On-resin cyclization is preferred to in-solution cyclization due to the beneficial pseudodilution effect42 when immobilizing the linear precursor on solid supports. This minimizes the oligomerization between the peptide chains and favors the intramolecular cyclization. Muir and co-workers were the first to perform onresin NCL for cyclic peptide synthesis (Figure 18a).157 They functionalized the aqueous buffer-compatible aminomethylated PEGA resin with thiol groups by anchoring a suitable carboxylic acid carrying a terminal mercapto group through an amide bond. The first amino acid was loaded to create a thioester on resin. Upon the completion of Boc-SPPS with the N-terminal cysteine assembled, global deprotection of the peptide was followed. Then, the resin-bound peptide was immersed into the NCL buffer for cyclization, releasing the peptide from the resin simultaneously. They demonstrated that this on-resin NCL cyclization was effective in preparing a 15mer cyclic peptide containing the RGD motif of fibronectin

Figure 17. Thia-zip peptide cyclizations. The two ends of the linear peptide were “zipped” by a series of transthioesterifications. An irreversible S-to-N-acyl transfer at the N-terminal Cys afforded the cyclic peptide.

K


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alkoxybenzyl ester as the linker, and the α-carboxyl group was protected by the allyl group. The linear peptide precursor was then assembled through standard Fmoc-SPPS conditions. After the incorporation of the N-terminal Cys using Trt-Cys(Xan)OH, deprotection of the allyl group was followed. The use of Trt-Cys(Xan)-OH was crucial. It allowed the removal of Trt and Xan on Cys under very mild acidic conditions to reveal the 1,2-aminothiol bifunctional group for NCL. The mild condition left the acid-labile benzyl ester bond between the peptide and the resin intact so that the peptide remained attached to the solid support. The base-sensitive thioester moiety was introduced in the last step of the peptide assembly to prevent thioester hydrolysis by base treatment during Fmoc deprotection. The thioester was installed via the coupling between the α-carboxylic acid of Asp and the amino acid containing a preformed thioester. After the deprotection of Trt and Xan on the N-terminal Cys by 1% TFA in DCM, the resin was immersed in an aqueous buffer of pH 7.5 with 6 M guanidine hydrochloride and thiophenol as the thiol additive to initiate the intramolecular NCL. The cyclized product was cleaved from the resin using TFA cleavage cocktail typical for cleavage with Cys-containing peptide, containing thioanisole, 1,2-ethanedithiol, and anisole as scavengers (reagent R). Note that in this case the linear precursor was partially protected instead of fully deprotected because the global deprotection condition and cleavage condition are not orthogonal. The use of partially protected peptide possibly impeded the NCL cyclization. The slow cyclization rate observed (∼75% cyclized after 7.5 h in the fastest case) was likely due to the limited solubility of the partially protected peptide in the NCLaqueous buffer. Another major drawback is the potential epimerization problem at Asp when coupling with the preformed thioester building block. Because the peptide is anchored to the resin via the side chain of Asp, this method for preparing cyclic peptide is only suitable for peptides consisting of Asp or Glu in their sequence or Asn/Gln if Rink-amide resin is used. This aspect greatly limits the scope of this method. Nevertheless, the Fmoc-SPPS compatible on resin cyclization is more favorable than the Boc-SPPS because the usage of toxic HF is eliminated. Very recently, Gless and Olsen developed another FmocSPPS-compatible on-resin NCL protocol for the preparation of cyclic peptides (Figure 19).150 Their design was analogous to the Boc-SPPS version developed by Muir and co-workers.157 A

Figure 18. Two types of on-resin cyclizations. (a) The intramolecular NCL induced the cleavage of peptide from the resin as soon as the thiolactone intermediate was formed. Spontaneous S-to-N-acyl shift yielded the cyclic peptide. (b) The thioester was installed via the C-α carboxy group of Asp with its side chain anchored to the resin. The cyclized peptide on the solid support was released by TFA treatment.

and the 44-mer WW domain of Yes kinase-associated protein, the two cyclic peptides that they had prepared via solutionphase intramolecular NCL in previous studies.120,121 Although the cyclization conducted on-resin was slower (1−2 h compared to 10 min in solution phase), it is operationally simple. The simple workup by filtration after global deprotection of the peptide and clean conversion of the cyclized product made the last purification step much easier. Tulla-Puche and Barany adopted a different design for onresin NCL cyclization (Figure 18b).158 They developed the first Fmoc-SPPS compatible NCL cyclization. Instead of having thioesters anchored onto the resin like in the work of Muir and co-workers,157 they anchored the side chain of Asp to water-compatible resins (PEGA and CLEAR resin) using p-

Figure 19. On-resin NCL peptide cyclization realized by utilizing MeDbz as the linker. The unprotected linear peptide precursor was still attached to the resin after TFA global deprotection. Immersing the resin into NCL buffer initiated cleavage and cyclization simultaneously. L


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fully deprotected linear precursor remained anchored to the solid support after the global deprotection step, and the peptide cyclization induced the cleavage of the peptide from the resin. Their success lay in the use of o-amino(methyl)aniline (MeDbz) as the linker.136 MeDbz is stable under Fmoc-SPPS conditions and at the same time can withstand the global deprotection conditions by TFA cocktail. A peptide thioester equivalent for NCL will be generated after the activation of MeDbz. The SPPS started with the installation of MeDbz onto the aqueous-phase-compatible aminomethyl resin, followed by routine Fmoc-SPPS. After all amino acids of the linear peptide were incorporated, the peptide MeDbz was activated by 4-nitrophenyl chloroformate, followed by global deprotection. The peptide cyclization and cleavage were initiated by treatment of the resin with an aqueous buffer with pH 6.8, followed by the addition of TCEP to yield the cyclic peptide. In their study, they discovered that cyclodimerization was severe when the resin loading was high, but such undesired side reaction could be minimized by using resins with lower loading. By using this method, followed by one-pot desulfurization151 to change the Cys to Ala, they successfully synthesized several natural cyclic peptides containing Ala or Cys with 5−10 residues in their sequence. The examples included stellarin G, stylostatin 1, cyclonellin, crotogossamide, and SFTI-1. An analogue of the antibiotic tyrocidine A with its valine mutated to alanine and two analogues of destoamide B with their valine mutated to cysteine or alanine were also obtained. This one-pot cyclization−desulfurization method resulted in moderate yield (11−31% based on the resin loading). However, when using penicillamine as the N-terminal Cys surrogate for the generation of Val after desulfurization, the major product observed was the hydrolyzed linear peptide in the cases of both destoamide B and tyrocidine A. They proposed that the bulky side chain of penicillamine diminished the rate of S-to-N-acyl transfer, and therefore, the hydrolysis of the thiolactone intermediate became significant. Moreover, they found that highly strained cyclic tetrapeptides could not be formed using their method. Although with limitations, this method is suitable for preparation of cyclic peptides of medium ring sizes. Moreover, this method requires only common reagents and simple operation to produce cyclic peptide in moderate yield. 3.1.5. Cyclization at Non-Cys Site. NCL requires a Nterminal cysteine to mediate chemoselective ligation.106 Tam and co-workers used a homocysteine in place of a cysteine for ligation. Through methylation of the thiol group after the ligation, a Met was generated at the ligation site. This study is an early example to achieve peptide ligation at non-Cys sites (Figure 20a).159 Alternatively, to expand the scope of NCL beyond cysteine containing peptide/protein, two general methods have been developed, namely, the use of thiolcontaining auxiliary as the N-terminal cysteine surrogate113,115 (Figure 20b) and NCL desulfurization118 (Figure 20c). 3.1.5.1. Thiol Auxiliaries at N-Terminal Amino Group. Kent and co-workers extended the NCL to Xaa-Gly sites by using N-α-(oxyethanethiol)glycine as the cysteine surrogate.114 The N-α-oxyethanethiol group could be removed by zinc dust reduction under mild acidic conditions after the intramolecular NCL to obtain the cyclic peptides. Using this method, cyclization at the Gly-Gly site was achieved and the binding loop region of Eglin c (a serine protease inhibitor containing no Cys) was successfully synthesized. Cyclizations at Ala-Gly or Phe-Gly sites were also possible, but the efficiency was low,

Figure 20. Methods for extending NCL to noncysteine sites. (a) The use of homocysteine as the N-terminal cysteine surrogate followed by methylation produced a methionine at the ligation site. (b) The use of a thiol-containing auxiliary followed by its removal could expand ligation to any amino acid residue. (c) NCL followed by desulfurization produced an alanine at the ligation site.

requiring 48 h to finish.160 Botti and co-workers showed that, by using a N-α-(1-(4-methoxyphenyl)mercaptoethyl) auxiliary, cyclization at Ala-Gly sites was also practical, with an example of an 11-residue cyclic peptide.161 Installation of auxiliaries at the N-α-amino group generated a secondary amine, which reduced the ligation rate when reacting with sterically hindered C-terminal thioesters. The slow ligation rate caused the epimerization of C-terminal residues other than Gly, and significant hydrolysis of the peptide thioesters was observed. As a result, the thiol auxiliaries attached at the N-α-amine as cysteine surrogate are only practical and limited to the Gly-Gly and Gly-Ala sites.58 Other types of thiol auxiliary-based methods developed for peptide ligation115,116 could theoretically be adopted for peptide cyclization. 3.1.5.2. NCL-Desulfurization Method for Peptide Cyclization. The desulfurization of Cys to Ala after NCL was developed by Dawson and co-workers in 2001,117 which opened up the field of chemical peptide synthesis by expanding the scope of NCL from Xaa-Cys sites to Xaa-Ala sites. The desulfurization could be achieved in near-quantitative yields by using Pd or Raney nickel as the metal catalyst in acidic medium. Using this approach, they obtained an analogue of a cyclic 21-mer peptide antibiotic, Microcin J25, by NCLdesulfurization method. The synthetic Microcin J25 analogue was highly bactericidal toward E. coli.117 The limitations of this Raney nickel desulfurization include the potential overreduction on the Met side chain and the low peptide recovery. These problems were overcome by a milder and metal-free radical-based desulfurization method introduced by the Danishefsky group151 in 2007, using a water-soluble radical initiator 2,2′-azobis[2-(2-imidazolin-2-yl)propane]dihydrochloride (VA-044), tris(2-carboxyethyl) phosphine hydrochloride (TCEP), and tert-butylthiol (tBuSH). This radical-mediated desulfurization only removes free thiol, with M


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Cys surrogate because both compounds do not contain Cys or Ala in their sequences. However, they failed to obtain the corresponding compound due to the steric hindrance of the side chain of β-thiol Val. Using Cys instead of β-thiol Val, the two peptides with Ala at the cyclization sites were obtained after on-resin NCL followed by desulfurization (Figure 22). The steric-hindrance problem associated with β-thiol Val during NCL was expected, so the Danishefsky group developed a γ-thiol Val165 (Figure 21) with a less sterically hindered thiol group. They showed that the use of a γ-thiol Val was effective to solve the problem. 3.1.6. Selenocysteine-Based NCL. Hilvert and coworkers prepared cyclic peptides containing selenocysteine (Sec) by NCL methodology through replacing the N-terminal Cys with Sec and with adaptations on reaction conditions (Figure 23).166 Recently, Iwaoka and co-workers obtained four selenium-containing cyclic octapeptides with sequences mimicking the catalytic tetrad of selenium-dependent glutathione peroxidase. They utilized linear peptide precursors having a N-terminal Sec and a C-terminal latent thioester for the macrocyclization.167 This extension of NCL by the Nterminal Sec was first illustrated by three groups independently in 2001.168−170 Two optimizations from the NCL conditions were needed to allow Sec-based NCL. First, a reducing agent is needed in the ligation buffer to generate the active form of the selenol from its dominant dimeric form of diselenide. Second, the pH of the buffer has to be lowered because Sec has a lower pKa than Cys. Otherwise, extensive hydrolysis of the thioester may result due to the slow ligation rate at neutral pH for Secbased NCL. Hilvert and co-workers showed that the selenol on the cyclic peptide could act as a handle for postcyclization derivations including alkylation, oxidative elimination, and reduction (Figure 24),166 which was useful for generation of analogues having the same scaffold. Strategies that are used to improve NCL can be translated to Sec-based NCL. For example, deselenization,166 analogous to desulfurization in NCL, allows one to transform Sec to Ala or to Ser when an oxidizing agent is added171,172 during the deselenization step (Figure 24). Using selenylated amino acid as Sec surrogates173,174 followed by deselenization allows Sec-based NCL beyond the Sec site. When using Sec/Sec surrogates in place of Cys and selenoesters in place of thioesters as the N- and C-termini, respectively, a significant enhancement in the ligation rate was observed. This ligation was termed diselenide-selenoester ligation (DSL)175 by Payne and co-workers (Figure 25). Although examples of cyclic peptide synthesis using DSL have yet to be reported, it is conceivable that the required linear precursor with a N-terminal Sec and a C-terminal selenoester on the same peptide should be readily accessible chemically. 3.1.7. Summary. The advancement in NCL and its related methodologies have greatly improved the efficiency and expanded the scope for peptide synthesis cyclization. The thia-zip reaction and the on-resin NCL cyclization were particularly useful for the preparation of cyclic peptides. Various examples of cyclic peptide synthesis by NCL (Table 1) showcased that NCL was suitable for the synthesis of cyclic peptides of various sizes, from the highly strained cyclic tetrapeptide to a large and relatively flexible ring consisting of ∼45 amino acids. The development of peptide thioester surrogates for efficient thioester synthesis has streamlined the procedures for cyclic peptide synthesis by NCL. NCL is a robust and well-established ligation strategy that has already

Thz and Acm protected thiols being untouched. The integrity of this desulfurization method has been verified on the synthesis of a non-Cys containing cyclic nonapeptide crotogossamide151 and proteins. Recently, a highly efficient radical-free desulfurization method using TCEP and sodium borohydride (P−B desulfurization) in aqueous buffer was developed by Li and co-workers.153 Two cell-growth-inhibiting cyclic peptides, dichotomin C (6 residues) and dichotomin E (5 residues), have been synthesized with NCL cyclization followed by P−B desulfurization strategy. Readers can refer to the recent review118 about desulfurization methodology in NCL for details. 3.1.5.3. Mercapto Amino Acids as Cys-Surrogates. The scope of the NCL-desulfurization strategy has been extended to other amino acids, by using unnatural mercapto amino acids as Cys surrogates. As seen in Figure 21, different amino acids

Figure 21. Mercapto amino acids developed as Cys surrogates for NCL-desulfurization strategy to achieve ligation at non-Cys sites.

with β- or γ-mercapto group at their side chain have been developed by various groups.58,111−113,162,163 These Cys surrogates have been used in various peptide/protein chemical synthesis by NCL and could be readily adopted for cyclic peptide synthesis. This clever design, however, is limited by the tedious preparation of the mercapto amino acids. A wider use of this group of Cys surrogates could be anticipated if simpler and more practical synthetic routes were developed or the amino acids were made commercially available at an affordable price. Olsen and co-workers150 attempted syntheses of two natural cyclic peptides, tyrocidine A and destoamide B, using the commercially available β-thiol Val (penicillamine)164 as the N


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Figure 22. Tyrocidin A analogue synthesis using cysteine surrogates and the NCL-desulfurization approach. The use of β-thiol valine to produce valine at cyclization after desulfurization was unsuccessful due to the steric hindrance. On the contrary, NCL and desulfurization went smoothly using a N-terminal cysteine.

Figure 25. Diselenide-selenoester ligation (DSL).

success in transitioning to continuous-flow and microfluidic technologies.176 3.2. Traceless Staudinger Ligation-Mediated Peptide Cyclization

Figure 23. Selenocysteine-based NCL. Selenocysteine exists as dimeric form under atmosphere. Thiophenol has a dual function in the ligation reaction: reducing the diselenide to the selenol and serving as the thiol additive for the generation of the more active thioester. The product will dimerize once in contact with oxygen.

Kleineweischede and Hackenberger first demonstrated that the traceless Staudinger ligation could be utilized for cyclic peptide synthesis using side chain unprotected linear peptides as precursors.177 Staudinger ligation was first developed by Bertozzi and co-workers in 2000 as a chemoselective ligation between azides and triaryl phosphines or N-acylimidazole phosphines to generate an amide containing a triaryl phosphine oxide, inspired by the Staudinger reaction.178 The traceless version of such ligation was independently developed by the Raines group179 and the Bertozzi group;180 in it, an amide bond was generated at the ligation site and the phosphine oxide was released as the byproduct. Raines and coworkers utilized a phosphinothioester while Bertozzi and coworkers employed phenol ester phosphines and N-acylimidazole phosphines to achieve traceless ligation (Figure 26). To obtain the peptide precursor for intramolecular traceless Staudinger ligation with both the N-terminal azido group and the C-terminal phosphinothioester on the same linear peptide, the azido group was installed at the N-terminus via Fmoc-SPPS using N-α-azido containing amino acid building blocks. Subsequent thioesterification between the C-terminus of the side chain protected N-α-azido peptide and a borane-protected phosphinothiol generated a C-terminal phosphinothioester protected by borane. After the global deprotection by TFA to remove both peptide side chain protecting groups and the borane, the requisite linear peptide precursor was obtained (Figure 27).177,181 The use of the borane-protected phosphinothiol was crucial here. If an unprotected phosphinothiol was used for the thioesterification, the premature Staudinger reaction between the N-α-azido group and the external phosphinothiol would have occurred.182 This will not be an

Figure 24. Postcyclization derivations of Sec-based NCL.

found its way for potential large-scale cyclic peptide synthesis in the pharmaceutical industry, encouraged by the preliminary O


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Figure 28. Intramolecular traceless Staudinger ligation. The cyclization was initiated by the addition of base in DMF followed by S-to-N-acyl transfer. The cyclic peptide was obtained after hydrolysis, releasing a phosphine oxide.

azido peptide thioester. The generation of the cyclic peptide was achieved by microwave heating at 50 °C for 5 min in THF with an external phosphine added, with the yield ranging from 48% to 75%.186 Although the traceless Staudinger ligation is highly chemoselective187,188 and free of epimerization at the ligation site,184,189 the problems of low solubility of the phosphinothiols190 and the corresponding phosphinothioesters in aqueous solution191 and low yield at the nonglycyl ligation site due to side reactions192 have hampered its widespread use in peptide synthesis. It is worth mentioning that, in Hackenberger’s works, the cyclic peptide examples were all cyclized at the Gly-Gly site,177 except one that was cyclized at the Gly-Ala site.181 The limitations mentioned earlier have been addressed by fine-tuning the structure of phosphinothiol used,191,192 but tedious preparation of the water-soluble phosphinothiol was inevitable.191 Optimizations of the preparation of the water-soluble phosphinothiol have been reported,193 but perhaps a 5% increase in the overall yield to 23% is not significant enough to promote the usage. The applications of such water-soluble phosphinothiol on cyclic peptide synthesis have not been reported either.

Figure 26. Types of phosphines used for traceless Staudinger ligation.

Figure 27. Preparation of linear peptide precursor for traceless Staudinger ligation-mediated cyclization.

issue in the case of intermolecular traceless Staudinger ligation, as the azide and phosphinothioester will be installed on two separate peptide fragments. Apart from TFA treatment,177,181 the borane group could be selectively deprotected under basic conditions by DABCO at elevated temperatures,181−184 but this harsh condition is less desirable for peptide/protein synthesis.177,181 Moreover, an extra step to remove all the side chain protecting groups by TFA will be needed.181 After obtaining the linear peptide precursor, the cyclization was initiated by the addition of excess DIEA to the linear peptide precursor in DMF with a concentration of millimolar range. The addition of DIEA was to neutralize residual TFA present after the global deprotection step.177,181 In the first step of the ligation-mediated cyclization, a cyclic iminophosphorane was generated by the intramolecular reaction between the azide and phosphinothioester at the N- and C-termini, respectively. Then, an S-to-N-acyl transfer followed by hydrolysis generated an amide bond at the ring-closure site, and the phosphine oxide was released.185 This method was shown to be effective in preparing a medium-sized cyclic peptide containing 11 amino acid residues with moderate yields (∼30%) after HPLC purification (Figure 28).177 Katritzky and co-workers showed that the traceless Staudinger ligation was also effective in preparing cyclic diand tripeptide. In their version, their linear precursor was an

3.3. α-Ketoacid Hydroxylamine Ligation-Mediated Peptide Cyclization

The α-ketoacid hydroxylamine (KAHA) ligation has been used to prepare head-to-tail cyclic peptides. KAHA ligation was developed by Bode and co-workers in 2006.194 It is a chemoselective peptide ligation reaction between the Nterminal hydroxylamine (HA) and the C-terminal α-ketoacid (KA) that form an amide bond at the ligation site.195 As the Nterminal peptide hydroxylamine is prone to oxidation to form oxime, O-substituted hydroxylamine variants have been investigated. While O-acyl and O-alkyl hydroxylamine were found to be unsuitable for peptide ligation,196 5-oxaproline197 (5-OPr), a stable cyclic alkoxymine, has been developed and used for synthesis of peptide and proteins. The use of Osubstituted hydroxylamines (type II KAHA ligation) for KAHA ligation led to a different mechanistic pathway compared to using free hydroxylamines (type I KAHA ligation).198 The major primary product formed in the 5oxaproline-KAHA ligation was a homoserine depsipeptide, which was rearranged to an amide product by O-to-N-acyl shift under basic conditions. 5-OPr-KAHA ligation has been used to synthesize proteins with a homoserine mutation at the ligation P


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Figure 29. KAHA ligation. (a) General strategies for preparation of peptides with the C-terminal α-ketoacid. (b) N-terminal hydroxylamines and derivatives. (c) Amide linkage generated after KAHA ligation. Different amino acids at the ligation site were generated by using different N-terminal hydroxylamines.

site. More recently, oxazetidine199 (Ozt), a cyclic alkoxime with a four-membered ring, has been utilized in the KAHA ligation. This led to the formation of a serine, instead of a homoserine, at the ligation site (Figure 29). Furthermore, the Ozt-KAHA ligation proceeded much faster than the 5-OPrKAHA ligation method, even under low concentration and mild temperatures. However, the difficult preparation of Ozt building block (7 steps with 5.5% overall yield) and its instability may limit its application.199 3.3.1. Synthesis of Cyclic Peptides with Type I KAHA Ligation. KAHA ligation was first applied in cyclization for the synthesis of an aza-analogue of surfactin by Bode and coworkers in 2011, using the free hydroxylamines as the precursor.200 The cyclization was chosen at the Leu-β3amino acid site (Figure 30a). The linear peptide was prepared by using a sulfur ylide linker as the precursor of the C-terminal α-ketoacid, followed by Fmoc-SPPS with the isoxazolidinone as the precursor of β3-N-hydroxyamino acid moiety coupled at the N-terminus. The coupling of the highly hydrophobic isoxazolidinone was found to be difficult, such that harsh coupling conditions were unavoidable. After the cleavage from the resin, temporary protection of the N-terminal hydroxyamino acid as N-benzylidene nitrone201 was required. The protection was essential to prevent oxidation of the hydroxylamine to oxime during the oxidative treatment with dimethyldioxirane (DMDO) on the sulfur ylide to reveal the C-terminal α-ketoacid in the subsequent step.200 The resultant linear peptide was dissolved in a DMF/water (50:1) mixture with excess oxalic acid at 45 °C for one-pot deprotection and KAHA cyclization. The N-benzylidene nitrone slowly fell off under KAHA-ligation conditions via hydrolysis by the trace amount of water in the reaction mixture. This reaction regenerated the N-terminal hydroxylamine needed for the cyclization. The cyclization was rather slow, such that it took 2 days to complete. (Figure 30c). In addition to the synthesis of epi-aza-surfactin with the βamino acid linkage, Bode and co-workers have explored KAHA ligation to prepare cyclic peptides with various ring sizes and side chain functional groups.202 During the synthesis of cyclic peptides by KAHA ligation, a diluted linear peptide solution of 1 mM was considered as the standard cyclization concentration. Dimerization was observed at concentrations higher

Figure 30. (a) Structure of the synthesized analogue of surfactin. The KAHA-ligation site was labeled. The NH colored in blue was an oxygen in the native surfactin. (b) Scaffold of β3-amino acid. (c) Scheme of 3(S)-epi-aza-surfactin by type I KAHA ligation-mediated cyclization.

than 1 mM. The N-terminal residue stereocenters were found to be preserved during the KAHA ligation. However, the Q


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oxidation step to form the α-ketoacids caused a small but detectable amount of epimerization (ca. 5%). In their cases, the desired cyclic products had entirely different retention times compared to the epimerized peptides, which allowed the separation of the desired product from the epimer after HPLC and obtained the target cyclic peptide in pure form. Five natural cyclic peptides were synthesized via type I KAHAcyclization strategy: gramicidin S, tyrocidine A (Figure 31),

Figure 31. Structures of gramicidin S and tyrocidine A. KAHA ligation was performed at Leu-D-Phe site for both cases.

hymenamide B, semi-gramicidin S, and stylostatin A, with yields ranging from 7% to 22%.202 Hydroxylamine was normally installed to the peptide by coupling with the preformed hydroxylamine building blocks. To simplify the process of cyclic peptide synthesis, direct oxidation of the Nterminal amine to nitrones on the solid support by an oxaziridine reagent was employed.203 This oxaziridine reagent was used in the synthesis of the precursor of cyclic tyrocidine A via Fmoc-SPPS. The peptide nitrone was obtained in 17% yield by this method.202 3.3.2. Synthesis of Cyclic Peptide with Type II KAHA Ligation. In 2012, a stable hydroxylamine derivative, 5oxaproline, was introduced for the KAHA ligation.197 Bocprotected 5-oxaproline could be installed via standard FmocSPPS conditions204,205 to the N-terminal of the growing linear peptide chain, while the C-terminal α-ketoacid was introduced using a protected α-ketoacid resin, which upon resin cleavage generated an entirely unprotected C-terminal α-ketoacid peptide.206,207 The protected α-ketoacid resins can be prepared in large scale and stored at room temperature. Thus, this method becomes the preferred method for the preparation of C-terminal α-ketoacid peptides in protein and cyclic peptide synthesis using KAHA ligation. The linear peptide with the Nterminal 5-oxaproline and the C-terminal α-ketoacid would cyclize in the aqueous buffer directly after the general resincleavage protocol, yielding a homoserine depsipeptide as the major intermediate and the desired macrolactam as the minor product.208 The depsipeptides were found to be stable under the KAHA-ligation conditions. Under basic conditions, the depsipeptides underwent O-to-N-acyl shift and transformed to the desired cyclic peptide with the homoserine amide bond linkage (Figure 32).209 After several case investigations, aqueous ammonia was considered as the best choice for Oto-N-acyl shift in one-pot KAHA-cyclization reactions. The fully converted amide product was obtained after treating depsipeptides with aqueous ammonium for several hours. No noticeable side products were observed during this step. By using this approach, Bode and co-worker synthesized 24 cyclic peptides, ranging from 8 to 20 residues.209 The KAHA

Figure 32. KAHA ligation-mediated cyclization using 5-oxaproline as the N-terminal hydroxylamine. The C-terminal α-ketoacid was generated upon cleavage from resins. The homoserine lactone formed after the ligation was transformed to the cyclic peptide with an amide linkage and a homoserine at the ligation site under basic conditions.

cyclization gave a higher crude purity and required less operation time when compared to the traditional HATU cyclization. Furthermore, Bode and co-workers synthesized a lasso peptide Microcin J25, by combining the KAHA ligation (using O-diethylcarbamoyl hydroxylamine) and NCL to form a peptide rotaxane.210 KAHA ligation is also suitable for head-to-tail cyclic protein synthesis. In 2017, Bode and co-workers synthesized the amphiphilic antibacterial cyclic protein, AS-48. AS-48 is a challenging synthetic target composed of 70 amino acids and a highly hydrophobic domain. The synthesis was achieved by using acetal-protected leucine α-ketoacid resins and the Nterminal 5-oxaproline via two KAHA ligations (Figure 33).211 To control the site for sequential ligation as well as to increase the solubility of the peptide fragments, a photolabile protecting group212 was used to protect the N-terminal 5-oxaproline of one segment and the C-terminal α-ketoacid of a second segment. After the first ligation, the resultant depsipeptide was not transformed into the corresponding amide. Instead, it was left as the ester on purpose to increase the solubility of the ligated segment when the two dual-function photolabile groups R


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peptides,196 such mutations may have profound effects on small- and medium-sized cyclic peptides in terms of structures and functions. Although this limitation could be overcome by using Ozt in place of 5-Opr to produce serine at the ligation site, the sluggish and low-efficiency preparation of the unstable Ozt building block hinders its wide applications in both linear and cyclic peptide/protein synthesis. An improved synthetic method for obtaining Ozt building block would certainly increase the usage of KAHA ligation for cyclic peptide synthesis. 3.4. Serine/Threonine Ligation-Mediated Peptide Cyclization

In 2010, Li and co-workers developed an alternative chemoselective peptide ligation, termed serine/threonine ligation (STL).213,214 The 1,2-hydroxy amine bifunctional groups of the N-terminal serine or threonine residue of an unprotected peptide undergo chemoselective oxazolidine formation with the aldehyde group of the C-terminal peptide salicylaldehyde (SAL) ester via the reversible imine capture. Subsequent O-toN-acyl transfer affords N,O-benzylidine acetal-linked peptide as the stable intermediate. Followed by acidolysis, the natural peptide Xaa-Ser/Thr linkage at the ligation site is generated (Figure 34).213 STL is fully compatible with side chain

Figure 33. Synthesis of AS-48 via two KAHA ligations with the use of photolabile protecting group to achieve segment ligation followed by ligation-mediated cyclization. Protein folding and O-to-N-acyl transfer in one pot after two KAHA ligations yielded AS-48 with two sites bearing homoserine mutations.

were being removed. After the second KAHA ligation to cyclize the protein, followed by two O-to-N-acyl transfers and protein folding in one pot, the synthetic protein was obtained. The synthetic AS-48 had similar activities as the native AS-48. Highly hydrophobic domains pose a general synthetic challenge in peptide and protein synthesis due to the low solubility in aqueous buffer system. In the synthesis of AS-48, the solubility problem of the highly hydrophobic segment was solved by the optimization of solvent conditions (HFIP and acetic acid), the incorporation of the photolabile protecting groups on both fragments, and retaining of the ester bond formed after ligation.211 3.3.3. Summary. Although type I KAHA ligation-mediated cyclization in principle can cyclize peptide at any sites and generate the native amide bond linkage, it has been rarely used. The low usage is mostly due to the instability of free hydroxylamines and the need of temporary protection of the N-terminus during precursor synthesis. The development of the stable 5-oxaproline as the N-terminal hydroxylamine and the protected α-ketoacid resins simplify the synthesis of precursors required for KAHA ligation, making synthesis of cyclic peptides operational, simple, and less laborious. The major drawback would be the epimerization that occurred at the C-terminal residues, with a noticeable amount (8%) of epimerization observed in the examples where 5-oxprolineKAHA ligation was used. The generation of a nonproteinogenic homoserine at the cyclization site is less desirable, especially in the case of total synthesis. While mutations of amino acids to homoserine may not be detrimental to the structures and functions of proteins or medium- to large-sized

Figure 34. Proposed Ser/Thr ligation mechanism. After the imine capture and the 5-endo-trig cyclization, O-to-N-acyl transfer produced the stable intermediate N,O-benzylidene acetal. After acidolysis, a peptide with a native peptide bond at the ligation site was generated.

unprotected peptides. Except C-terminal Asp, Glu, and Lys, salicylaldehyde ester with the remaining 17 natural amino acids as the C-terminal residue is suitable for STL.215 The requisite peptide SAL esters can be prepared by several methods, including safety-catch-based phenolysis of the peptide Nbz, direct coupling esterification, ozonolysis-based ester generation, and the “n + 1” coupling approach (Figure 35). 3.4.1. Synthesis of Cyclic Peptides by STL. In addition to peptide/protein synthesis,216−223 STL-mediated peptide cyclization (Figure 36) has been used to prepare a variety of cyclic peptides.224−231 Cyclic peptides with a small ring size (20 h) are necessary.241

The reaction pathway of the ligation techniques can also play a role in the cyclization efficiency. In the aziridine aldehydebased multicomponent macrocyclization (section 2.3), cyclization was conducted in an unusually high concentration (0.2 M in TFE or HFIP) and yielded only cyclic monomers. This outcome is due to the increased effective molarity of the Cterminal carboxylate at the N-terminus of the same peptide, promoted by the formation of the imidoanhydride intermediate via concerted addition among the N-terminus, the amide bond next to the N-terminus, the aziridine, and the isocyanide. As a result, intermolecular reactions with Cterminal carboxylate from other linear peptides were less likely to occur.97 For NCL discussed in section 3.1.2, the C-terminal thioester and the N-terminal Cys are brought together by the formation of the thiolactone from the transthioesterification. Then a ring-contraction step by S-to-N-acyl transfer afforded the cyclic product. The preorganization of the two ends in a larger ring followed by a ring-contraction step overcome the disfavored formation of the highly strained cyclic tetrapeptide at a highly diluted solution of 1 mM.119 A similar phenomenon is also observed in other ligation strategies involving a ringcontraction step. In STL, the imine-capture step preorganizes the linear precursor for cyclization by accommodating the Nand C-termini in a ring. The ring contraction by O-to-N-acyl transfer allowed the formation of cyclic tetrapeptides through lowering the activation energy of the cyclization.226 Ligation strategies that involve ring-contraction steps by first forming a larger ring are effective alternatives for constructing highly strained cyclic peptides compared to the the traditional directcoupling method.236

5.2. Butelase 1

5. ENZYME-MEDIATED PEPTIDE CYCLIZATION Enzyme-mediated ligation is providing an alternative to chemical synthesis for the cyclization step. Enzyme activities are highly chemoselective. Their nontoxic and catalytic properties are of great value to the pharmaceutical industry in the preparation of cyclic peptide drugs considering purity and cost-effectiveness. Although, there is a surprising scarcity of enzymes that mediate protein ligation in nature,66 the potential to obtain ligases for cyclization with modified substrate-recognition sites by genetic engineering, as well as genome-mining for discovering more new ligases, will aid the development in this field. In this section, both naturally occurring and modified, unnatural enzymes that mediate headto-tail cyclization will be discussed.

Butelase 1 is another naturally occurring peptide ligase. The enzyme was found in the plant Clitoria Ternatea, where it acts as a transpeptidase.242 Butelase 1 recognizes peptides with Cterminal Asp/Asn-His-Val tripeptide motifs. Butelase 1 cleaves the Asp/Asn-His bond, removing the His-Val dipeptide to form a catalyst-peptide intermediate. The attack from the nucleophilic N-terminus leads to the dissociation from the catalyst and the formation of the cyclic peptide. The first amino acid at the N-terminus can be of any amino acid, except Asp, Glu, and Pro. The adjacent amino acid is more restricted; only Cys, Ile, Leu, and Val are allowed for successful ligations.243,244 Butelase 1 has a high catalytic molar efficiency (0.005 molar equivalency).238 This feature, combined with the more flexible recognition motifs, makes butelase 1 more appealing and practical than sortase A. When used in the cyclization step of growth hormone production, yields of >95% were achieved in a reaction time of only 15 min.243 Despite the requirement for the N- and C-termini to be preorganized in close proximity because denatured proteins failed to be cyclized, butelase 1 does have the potential to be used for ligation and cyclization for a wide range of proteins due to its broad scope of substrate recognition. However, the current limitation of using butelase 1 is its failure in the production by recombinant techniques. The traditional isolation from the plant is required, and this can be a long and laborious process.238 Increasing the obtainability of this protein ligase would allow its full potential to be studied and utilized in industrial-scale production of cyclic peptides and proteins.

5.1. Sortase A

5.3. GmPOPB

Sortase A is an enzyme found in Staphylococcus aureus that mediates the attachment of proteins to the cell wall.237 This enzyme has found its application in protein and peptide cyclization. The substrate peptide must possess an LPXTG Cterminal combination (where X can be of any amino acid) and a N-terminal glycine. In the presence of Ca2+ ions, sortase A cleaves the glycine-threonine bond to create a peptide-enzyme intermediate, bonded through the threonine. This then allows the nucleophilic N-terminal glycine to attack the acyl enzymeprotein bond. This process results in the cleavage of the protein from the enzyme and formation of the desired cyclic peptide with a TG cyclization site.238 The required amino acid sequence on both the C- and Ntermini severely restricts the possible cyclic peptide products.

Another naturally occurring peptide cyclase extracted from plants is GmPOPB. It is a prolyl oligopeptidase found in the mushroom Galerina marginata. Its main role is to mediate the cyclization of α-amanitin, a fatal toxin that inhibits RNA polymerase II and III. GmPOPB works by first hydrolyzing the 35 amino-acid-long propeptide, GmAMA1, at Pro. This process removes the N-terminal 10-mer sequence MFDTNATRLP. The second role of GmPOPB is to promote the transpeptidation between Ile and a second Pro. This step causes the cleavage of the remaining 17 amino acids and the cyclization of the octapeptide product, cyclo-(IWGIGCNP). Monitoring of the reaction progression for different alterations of the C- and N-termini showed that the C-terminal residue cannot be altered. It must stay as PWTAEHVDQTLASGNV


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of peptides. Peptiligase has a much higher catalytic molar efficiency than that of sortase A or butelase 1 (

Ligation Technologies for the Synthesis of Cyclic Peptides - American

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