Multicomponent Reaction Toolbox for Peptide Macrocyclization and

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

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Multicomponent Reaction Toolbox for Peptide Macrocyclization and Stapling Leslie Reguera and Daniel G. Rivera*

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Center for Natural Product Research, Faculty of Chemistry, University of Havana, Zapata y G, Havana 10400, Cuba ABSTRACT: In the past decade, multicomponent reactions have experienced a renaissance as powerful peptide macrocyclization tools enabling the rapid creation of skeletal complexity and diversity with low synthetic cost. This review provides both a historical and modern overview of the development of the peptide multicomponent macrocyclization as a strategy capable to compete with the classic peptide cyclization methods in terms of chemical efficiency and synthetic scope. We prove that the utilization of multicomponent reactions for cyclizing peptides by either their termini or side chains provides a key advantage over those more established methods; that is, the possibility to explore the cyclic peptide chemotype space not only at the amino acid sequence but also at the ring-forming moiety. Owing to its multicomponent nature, this type of peptide cyclization process is well-suited to generate diversity at both the endo- and exo-cyclic fragments formed during the ring-closing step, which stands as a distinctive and useful characteristic for the creation and screening of cyclic peptide libraries. Examples of the novel multicomponent peptide stapling approach and heterocycle ring-forming macrocyclizations are included, along with multicomponent methods incorporating macrocyclization handles and the one-pot syntheses of macromulticyclic peptide cages. Interesting applications of this strategy in the field of drug discovery and chemical biology are provided.

CONTENTS 1. Introduction 2. Multicomponent Reactions in Macrocyclization Chemistry 2.1. Dawn and Development of Multicomponent Macrocyclizations 2.2. Lessons from the Multicomponent Macrocyclization with Nonpeptide Substrates 3. Peptide Macrocyclization by Multicomponent Reactions 3.1. Head-to-Tail Cyclization by IsocyanideBased MCRs 3.2. Synthetic Scope of Peptide Macrocyclization by Yudin and Ugi Reactions 3.3. Head-to-Tail Multicomponent Macrocyclization on Solid Phase 3.4. Peptides Macrocyclization by Passerini and Ugi−Smiles Reactions 3.5. Peptide Macrocyclization by Strecker Reaction 3.6. Peptide Macrocyclization by Heterocycle Ring-Forming MCRs 4. Multicomponent Peptide Stapling 4.1. Stapling by Ugi Reaction 4.2. Stapling by Ugi−Smiles Reaction 4.3. Stapling by A3-Coupling Reaction 4.4. Stapling by Petasis Reaction 5. Sequential MCR/Macrocyclization Approach to Cyclic Peptides © XXXX American Chemical Society

5.1. MCR-Mediated Peptide Functionalization with Ring-Closing Group 5.2. MCR-Mediated Peptide Backbone Stapling 5.3. MCR-Mediated Activation of the Peptide CTerminus 6. Multicomponent Synthesis of Multicyclic Peptide Cages 7. Summary and Outlook Author Information Corresponding Author ORCID Notes Biographies Acknowledgments Dedication Abbreviations References

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1. INTRODUCTION Cyclic peptides are important biologically active compounds which combine the intrinsic properties of their amino acid components with the conformational bias of macrocycles,1,2 thus targeting protein surfaces not easily accessed by traditional small-molecule drugs.3,4 Naturally occurring cyclic

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Special Issue: Macrocycles Received: December 4, 2018

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DOI: 10.1021/acs.chemrev.8b00744 Chem. Rev. XXXX, XXX, XXX−XXX

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Figure 1. Natural cyclic peptides with diverse macrocyclization variants including the peptide termini and the side chains.

closing metathesis (RCM).33,34 However, the stabilization of helical peptide sequences by the introduction of side-chain lactam bridges had been proven even earlier.22,23 Both stapling techniques have provided a variety of helical peptides with important biological and medicinal applications,35,36 some of them even acting at intracellular targets.37 Since then, the stapling chemistry has greatly diversified,38 including the click CuI-catalyzed alkyne−azide cycloaddition (CuAAC),39 Cys Sarylation40,41 and alkylation,42 and Pd-catalyzed C−H activation,43−45 among others. Over the past decade, the field of synthetic chemistry has witnessed the renaissance of multicomponent reactions (MCRs) as powerful peptide macrocyclization tools. The effective implementation of multicomponent macrocyclization approaches46 enables the generation of high levels of molecular diversity during the ring closure of the peptide skeleton, something that is not easy to achieve with the nowadays standard lactamization,28,32 RCM,36 and click39 approaches. The synthetic benefits of using MCRs in peptide macrocyclization are not only derived from the diversity-oriented character of these processes but also include the possibility of locking specific conformations and fine-tuning of physicochemical properties, both through the installation of nonproteinogenic endo- and exo-cyclic fragments. This Review focuses on synthetic strategies that employ MCRs for the macrocyclization and stapling of peptides. We also include examples where the MCR itself is responsible for the incorporation of an activating group or reactive handle, which next allows for the peptide cyclization. Selected examples of the synthesis of macrocyclic peptide cages are also covered. However, the multicomponent syntheses of nonand pseudo-peptide macrocycles (e.g., cyclopeptoids) are not included, as various excellent reviews have been published in this field.46−48

peptides have found medicinal applications as antimicrobial and anticancer agents, immunosuppressants, and inhibitors of enzymes and protein−protein interactions.4−6 Alternatively, synthetic macrocyclic peptides and peptidomimetics have proven equally useful in these areas4,6,7 as well as in other fields such as vaccinology,8 molecular and ion recognition, and catalysis.9 The cyclic peptide class of compounds also includes N-alkylated peptides and depsipeptides10 (e.g., RAVII11 and surfactin A12 shown in Figure 1), the latter ones containing one or more hydroxylated amino acids as part of their backbone, thus adding one or various lactone moieties to the cyclic scaffold. Besides the common peptide head-to-tail connectivity, nature provides numerous linkages involving the amino acid side chains such as disulfide bond13 (e.g., somatostatin), lactam bridge between a side chain and a peptide end (e.g., bacitracin), and biaryl and biaryl-ether bridges14 (e.g., biphenomycin B and RAVII, respectively). Many of these peptide backbone and side chain connectivities are equally considered when designing novel synthetic macrocyclization strategies for medicinal chemistry applications.15,16 Today, it is widely accepted that macrocyclizationin its dissimilar variantsstands as the most effective way of introducing conformational constraints in short- and medium-size peptides designed either as protein ligands1−6,15 or mimetics of protein secondary structures17−19 (i.e., β-hairpins,20 β-strands,21 and α-helix22−24). Peptide cyclization may also help improve membrane permeability25,26 and peptide resistance to proteases,27 while it usually enhances the binding affinity to biological targets compared to their acyclic analogues.2,4 In addition to macrolactamization and macrolactonization,28 synthetic chemists have added a wide repertoire of methods to macrocyclize peptides either by their termini or the side chains.29−32 A special class of peptide cyclization is the so-called peptide stapling; that is, the side chain-to-side chain tethering of two amino acid residues separated along the sequence. Originally, the term peptide stapling was coined for the synthesis of allhydrocarbon bridged α-helical peptides by means of ring-

2. MULTICOMPONENT REACTIONS IN MACROCYCLIZATION CHEMISTRY MCRs49 comprise an important class of chemical transformations that have been applied to almost all fields of organic B


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Another functional group of increasing incidence in the field of peptide chemistry is the isocyanide.64−66 The first known isocyanide-based MCR is the Passerini-3CR,67 discovered as far back as in 192168 but just applied in recent years to cyclize oxo-peptides, leading to depsipeptide skeletons.69 The other face of the coin is the MCR recently developed by Yudin and coworkers,70 which has been broadly employed for the synthesis of cyclic peptides and their post-MCR derivatization. The Yudin’s aziridine−aldehyde reaction comprises the diastereoselective condensation of this latter amphoteric substrate with an amino acid and an isocyanide to produce a hybrid piperazin-2-one/aziridine scaffold (Scheme 1B), while oligopeptides equally react to form aziridine-containing cyclopeptides.16 The Ugi four-component reaction (4CR)71 is possibly the MCR most widely used in all branches of synthetic chemistry, including in the synthesis of cyclic peptidomimetics.10,48,72 As shown in Scheme 1C, this reaction comprises the condensation of an amine, a carbonyl component (i.e., aldehyde or ketone), a carboxylic acid, and an isocyanide to produce a N-substituted peptide skeleton. The Ugi reaction proceeds through the formation of the corresponding Schiff base, followed by activation via protonation by the carboxylic acid and subsequent addition of the iminium and the carboxylate to the isocyanide to form the so-called α-adduct.64 The final step of the Ugi-4CR is the Mumm rearrangement, i.e., an intramolecular acylation of the amine nitrogen atom followed by the subsequent hydroxylimine → amide tautomeric conversion. This MCR was not only the first one to be used in peptide cyclization73 but also the first report of any type of multicomponent macrocyclization approach. An interesting variant of the classic Ugi-4CR is the Ugi−Smiles reaction developed by El Kaı̈m et al.74 In this latter process, the carboxylic acid component is replaced by electron-poor phenols such as 2- or 4-nitrophenol which, upon condensation with a primary amine, an aldehyde, and an isocyanide, give rise to tertiary nitroanilines of pseudo-peptidic nature. The substrate scope of the acidic component of this reaction is notable, including not only electron-poor phenols but also hydroxyl-heterocycles and conjugated enols.75

and medicinal chemistry.50 These convergent procedures incorporate three or more reactants into a final product in one pot, thus combining high levels of complexity and diversity generation with low synthetic cost and high atom economy.49−52 MCRs are currently well-established methods for the synthesis of heterocycles,49,51,53 natural products,54 and polymers,55 while the multicomponent glycoconjugation56 and labeling56,57 of proteins is a growing field of research.58 Most applications of MCRs employed in the covalent modification of peptides include an amino or carboxylic acid group as one of the components. Because these functionalities are present in natural peptides, no pre-MCR derivatization is required. The carbonyl component is also of great occurrence in the realm of MCRs, as many multicomponent sequences are initiated with condensation steps leading to imine species which, eventually upon protonation, react with external nucleophiles. As depicted in Scheme 1, the first reaction Scheme 1. Three and Four-Component Reactions Employed in Peptide Macrocyclization

2.1. Dawn and Development of Multicomponent Macrocyclizations

The first report of a macrocyclization using an MCR was described by Failli et al. in 1979 with the head-to-tail cyclization of hexapeptides by means of the Ugi reaction.73 Initial attempts to cyclize tripeptide Gly-Gly-Gly (1) in the presence of isobutyraldehyde and cyclohexyl isocyanide did not afford the 9-membered cyclopeptide 2, but furnished only the doubly Ugi reaction-cyclized cyclic hexapeptide 3 as a mixture of diastereomers (Scheme 2). The authors realized the difficulty for obtaining such a strained cyclic tripeptide 2 and turned to a more direct Ugi macrocyclization of a linear hexapeptide. Thus, both the cyclization of hexaglycine and of peptide 4 proved successful in producing the corresponding cyclic hexapeptides. In the case of 5, a mixture of diastereomers was formed, which could be separated.73 Perhaps the most interesting feature of this multicomponent strategy, and what makes it different from the classic macrolactamization, is the generation of an exo-cyclic functionality as part of the resulting N-substituted amide bond. This Review will show how such an exo-cyclic functionalization can be used for labeling purposes

known by proceeding through this pathway is the Strecker three-component reaction59 (3CR), which intriguingly only recently was applied for the macrocyclization of peptides.60 Other MCRs also based on imine formation are the metalcatalyzed coupling of an aldehyde, alkyne, and amine61 (i.e., A3-coupling) and the Petasis-3CR,62,63 again only recently utilized in peptide stapling approaches. C


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netics46,72 (not related to the dilution effect) compared to the standards of other common ring closing reactions such as CuAAC and macrolactamization. Second, most Ugi-type MCRs proceed with very poor stereocontrol,76,77 thus producing a mixture (almost 1:1) of epimers that complicates chromatographic purification, even by HPLC, as well as spectroscopic characterization. Both aspects might have been seen as impractical by peptide chemists, who are used to working with fast and reproducible peptide coupling protocols that have been improved over the years to minimize epimerization.

Scheme 2. First Report of a Multicomponent Macrocyclization Using the Ugi Reaction for the Head-toTail Cyclization of Linear Peptides

2.2. Lessons from the Multicomponent Macrocyclization with Nonpeptide Substrates

In contrast to the few applications of MCRs in peptide macrocyclization described over three decades after the first report,73 multicomponent macrocyclizations have been intensively used for the synthesis of complex steroid-based supramolecular receptors,78−80 topologically defined macromulticycles,81−83 macrocyclic peptoids,84,85 and peptidomimetics.86−89 In this sense, Wessjohann10,46,78−83 and Zhu90 can be considered as pioneers of multicomponent macrocyclization strategies, while the groups of Dömling,86−88 Andrade,84,85 and Rivera91,92 have also increased the repertoire of multicomponent ring-closing procedures directed to artificial and pseudo-peptidic macrocycles. Among the interesting examples of MCR-based macrocyclizations is the development of the multiple multicomponent macrocyclization including bifunctional building blocks (MiBs) strategy.46 In a series of relevant reports between 2005 and 2009,78−82 Wessjohann and collaborators described the utilization of rigid bifunctional steroids (e.g., bile

and to enhance the lipophilic or ionic character of a peptide without affecting the amino acid side chains. However, one might wonder why MCRs of Ugi or any other type were not employed in peptide macrocyclization for about 30 years,46 a scenario that changed in 2010 when Yudin and coworkers applied their new reaction to cyclize oligopeptides.70 In our opinion, the reason for this may be due to practical rather than strategic causes. First, Ugi multicomponent macrocyclizations have an intrinsic slow ki-

Scheme 3. Successful Examples of Multicomponent Macrocyclizations with Bifunctional Building Blocks

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Scheme 4. Schematic Comparison of (A) Macrolactamization with the (B) Ugi and (C) Yudin Macrocyclizations of Peptidesa

a

Concept partially adapted from ref 70.

substrates and/or the rigidifying effect of heterocyclic or aryl moieties formed in the first step may be helpful to overcome the entropically disfavored ring closure. For example, good results have been obtained with the utilization of phenyl and biaryl ether diisocyanides and diamines when combined with very flexible bifunctional counterparts.82,94 As shown in Scheme 3B, Lys methyl ester was used as diamine component in the double Ugi reaction-based macrocyclization with mxylylene diisocyanide and different Boc-amino acids (carboxylic acid components), thus leading to various pseudo-peptidic macrocycles (9).94 The rigid structure of the 1,3-disubtitited phenyl substrate proved to be important for the macrocyclization success, as the same combination with very flexible diisocyanides and diamines resulted in very low yields. Alternatively, Zhu and coworkers described the utilization of a heterocycle-forming reaction (Zhu-3CR) in a double MCRbased macrocyclization.90 Scheme 3C depicts the macrocyclization of dimeric Cys and a diisocyanoacetamide as flexible of bifunctional building blocks, which upon the first Zhu-3CR in the presence of an aldehyde led to an acyclic intermediate bearing a 1,3-disubstituted oxazole ring at the middle of the intermediate main chain. The authors considered the rigidifying effect introduced by the newly formed heterocycle rings as crucial for the macrocyclization efficiency.90

acids) as substrates of the MiBs strategy based on the Ugi reaction. The synthetic setup of this macrocyclization method includes the simultaneous slow addition of two bifunctional building blocks (i.e., diamine, diacid, dialdehyde, and diisocyanide) with syringe pumps to a stirring solution of the two remaining monofunctional components (2 equiv). Under such pseudo-dilution conditions set to reduce the formation of higher oligomers,93 the bifunctional components undertake an initial Ugi reaction to assemble a linear intermediate which may next undergo a final multicomponent ring closure to afford a hybrid macrocycle (Scheme 3). As only two of the four components are initially employed in a bifunctional form, paraformaldehyde has been traditionally employed as the oxocomponent to avoid formation of multiple stereoisomers. Scheme 3A depicts a typical example of MiBs featuring the diacid/diisocyanide combination and leading to an amino acid−steroid−biaryl ether macrocyclic hybrid (7),79 which were designed to serve as supramolecular receptors. Wessjohann’s group showed that the efficiency of MiBs is greatly improved when bifunctional components with favorable structural preorganization were employed, e.g., curved steroidal skeletons and 1,3-disubstituted phenyl rings,78−82 as compared with either too flexible or too rigid and linear bifunctional building blocks. Thus, despite the dilution conditions used in the MiBs protocols, (cyclo)oligomerization processes did take place when very rigid and extended steroidal substrates were used instead of the concave-shaped cholic acid derivatives.79 On the other hand, the utilization of building blocks featuring long aliphatic chains was not very effective either,89 as lower macrocyclization yields were obtained due to the entropically disfavored end-to-end cyclization of such substrates lacking any rigidifying effect. The fine-tuning between conformational rigidity and flexibility turned out to be crucial to understand the success or failure of the peptide macrocyclizations further developed using this technology. Other reports from Wessjohann’s94 and Zhu’s90 groups further illustrated how the structural preorganization of

3. PEPTIDE MACROCYCLIZATION BY MULTICOMPONENT REACTIONS 3.1. Head-to-Tail Cyclization by Isocyanide-Based MCRs

The head-to-tail cyclization of short peptides stands as one of the most challenging procedures in synthetic chemistry.16,28−31 Most difficulties in peptide macrocyclizations using MCRs are, however, not due to the multicomponent nature of the process, but to the intrinsic structural properties of peptides. Both the sequence and the ring size determine the synthetic restrictions such as cyclodimerization, C-terminal epimerization, and E


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frequent low yields; therefore, such limitations are observed for most cyclization methods. In a comprehensive review of Yudin and coworkers,16 the main structural and conformational problems that make peptide cyclization so difficult are discussed as well as some answers to these problems. In short, typical solutions are (i) to conduct the macrocyclization at extreme dilution and (ii) to incorporate temporary turninducing elements or metal templates capable to facilitate the macrocyclic ring closure by bringing both termini closer. The development of highly specialized coupling reagents has become also necessary to diminish epimerization upon activation of the C-terminus.32,95 In this sense, a key advantage of the head-to-tail cyclization of peptides by MCRs like the Ugi and Yudin reactions is the lack of epimerization of the terminal carboxylic group (Scheme 4). Whereas the Ugi macrocyclization does produce two epimers if nonsymmetric carbonyl components are employed, this problem can be easily solved by using paraformaldehyde or acetone, of course at the expense of losing one site of diversification. Scheme 4 illustrates a comparison of Ugi and Yudin macrocyclizations with the macrolactamization of a linear peptide. In their seminal publication on the multicomponent peptide macrocyclization using the amphoteric aziridine− aldehyde,70 Yudin and coworkers commented on the benefits of ion pairing interactions for bringing the two peptide termini closer during the multicomponent cyclization. This feature remains after formation and further protonation of the imine by the carboxylic acid, thus leading to an iminium−carboxylate ion pairing that favors a folded peptide conformation upon addition of the isocyanide component (Scheme 4B and C). Of course, other structural features such as interchain hydrogenbonding found in β-turn and β-hairpin structures as well as the presence of Pro, N-methyl, or D-amino acids as turn-inducing elements when embedded midway along the sequence all favor folded conformational states in acyclic peptides. However, a characteristic of the macrolactamization is that the ion pairing between the two termini disappears upon activation of the Cterminal carboxylate, thus erasing such an enthalpic contribution96 (Scheme 4A). Consequently, when no additional turninducing elements are present to reduce the entropic cost of bringing the two ends together, it is likely that the macrolactamization is thermodynamically less favored than an MCR transiting via iminium-carboxylate ion pairing. A proof of this was found by Yudin et al. in the peptide macrocyclization studies with the aziridine−aldehyde component and isocyanide,70 in which even short peptides of two, three, and four amino acid residues could be readily cyclized with high yields in remarkably short time and in a high concentration of 0.2 M without noticeable cyclodimerization. As is known,16,28,30 the macrolactamization of peptides often requires submilimolar concentration, and even so, it is impossible to achieve the head-to-tail cyclization of such short peptides without significant dimerization.

which otherwise would render a complex mixture of acyclic and cyclic oligomers by standard macrolactamization protocols. In addition, whereas amino acids react well with the aziridine−aldehyde dimer and isocyanide with any combination of stereocenters, the reaction of oligopeptides featuring a mismatch between the N-terminal Pro and the aziridine− aldehyde component typically gave intractable mixtures.70,97 This mandatory matching between the aziridine−aldehyde dimer and the Pro residue means that the use of L-Pro at the N-terminus needs to be combined with the (S)-aziridine aldehyde dimer and the D-Pro with the (R)-aziridine aldehyde component. This matching stereochemistry issue should not be seen as a synthetic limitation, as both enantiomerically pure aziridine−aldehydes are readily available from chiral pool reagents like α-amino acids. Key for the high diastereoselection and fast kinetics of Yudin macrocyclization is that the aziridine group ends up as an exo-cyclic functionality in the α-adduct intermediate (see Scheme 4C). Because of this, the nucleophilic attack of the aziridine nitrogen atom to the mixed anhydride fragment is expeditious and proceeds with stereocontrol, while it provides a final cyclic scaffold having the same ring size as the α-adduct intermediate. On the other hand, peptide macrocyclization by the Ugi reaction has mechanistic characteristics that make it a slow process, which frequently leads to mixtures of monomeric and dimeric cyclopeptides. As depicted in Scheme 4B, the main reason for the slow kinetics of the Ugi macrocyclization in short peptides is the slow transannular attack of the amine to the mixed anhydride. The migratory capacity of this endo-cyclic amine depends on both the ring strain and the bulky character of such amino acid residue. In addition, the cyclomonomer/ cyclodimer ratio markedly depends on the ring size, as the transit from the α-adduct to the final product reduces ring size by three atoms. Such an intramolecular acylation, known as Mumm rearrangement, comprises a ring contraction that is detrimental for the macrocyclization efficiency of short peptides (especially tri, tetra, and pentapeptides) due to the increase in ring strain. To get deeper insights into the scope of the Ugi macrocyclization, Rivera and Wessjohann conducted a synthetic study within the frame of a program toward cyclic lipopeptides.69 The focus was both to enable the introduction a lipidic tail during the peptide ring closure and address the scope of this multicomponent macrocyclization with short and medium size peptides. Previously, Failli et al. 73 had demonstrated that tripeptides fully dimerize to render hexapeptides derived from two Ugi reactions, while hexapeptides cyclize without noticeable dimerization (Scheme 3). In addition, Kim and coworkers reported in 2003 the Ugi cyclization of dipeptides to form 2,5-diketopiperazines,98 although the success and particularities of this process could not be translated to a macrocyclization. For a long time, no information about the scope of the head-to-tail Ugi cyclization of tetra- and pentapeptides was available. The experience of our group with the Ugi macrocyclization of higher peptides (6−10 residues) encouraged us to conduct the process at 10 mM concentration using MeOH or MeOH/THF as solvent, conditions upon which the cyclization process is completed in 48−72 h. As depicted in Scheme 5, these conditions were implemented for the cyclization of various tetrapeptides aimed at determining the influence of the sequence on the macrocyclization outcome. Thus, the all-L-peptide 17 led to the desired cyclic lipotetrapeptide 18 in 62% conversion (42%

3.2. Synthetic Scope of Peptide Macrocyclization by Yudin and Ugi Reactions

Scheme 4D depicts some of the cyclic peptides produced with high diastereoselectivity by Yudin’s method, all containing an aziridine ring embedded in the cyclopeptide skeleton. Peptides successfully cyclized by this method usually have Pro as Nterminal residue, which might be seen as a limitation of the procedure. Interestingly, this approach has been effectively employed for the macrocyclization of oligoproline peptides,97 F


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Scheme 5. Study on the Ugi Multicomponent Macrocyclization of Tetrapeptides for the Synthesis of Cyclic Lipopeptides

Scheme 6. Ugi Multicomponent Macrocyclization of Tetraand Pentapeptides in the Side Chain-to-Terminus and Head-to-Tail Variants

isolated yield) but also rendering the corresponding cyclodimer (derived from two Ugi reactions) in 24% conversion. On the other hand, conducting the Ugi cyclization of peptide 17 at 2 mM concentration afforded only the cyclomonomer 18, albeit 96 h of reaction was required to reach at least 80% conversion. Interestingly, when tetrapeptide 19 including three Gly in the sequence was subjected to the macrocyclization procedure at 10 mM, only the cyclic tetrapeptide 20 was obtained in 83% conversion and 41% isolated yield. These results corroborate the key effect of the amino acid sequence on the outcome of the multicomponent cyclization. For tetrapeptides, the intermediate α-adduct is endowed with a 15membered ring, while the final product has a 12-membered macrocyclic ring, which has an intrinsic strain and might be forced to accommodate at least one amide bond in the S-cis configuration. In this sense, a positive structural factor is the presence of an amide N-alkylation in the final product, which facilitates access to the cis-amide bond and partially releases the strain. The incorporation of a D-amino acid into an all-Lpeptide has been also employed to improve the cyclization yield of short peptides,29,99 mainly due to the turn-inducing capability of this modification. This was further proven in the Ugi macrocyclization of tetrapeptide 21 containing a D-amino acid, which led to the formation of the cyclic lipopeptide 22 in 81% conversion and 51% isolated yield.69 The absence of cyclodimer in the cyclization of the Gly- and D-amino-acidcontaining peptides indicates that the presence of these residues along with the resulting N-alkylated amide makes the Ugi-derived cyclic scaffolds strain-free enough to favor their formation over the intermolecular event leading to the dimeric products, even at 10 mM concentration. For comparison purpose, the side chain-to-terminus Ugi macrocyclization of tetrapeptide skeletons was also carried out in our laboratory. As shown in Scheme 6, tetrapeptide 23 was cyclized by the Glu side chain and the N-terminal residue to furnish cyclopeptide 24 in 42% yield, which is equivalent to

that of the head−tail version but without leading to dimerization product (as proven by HPLC/ESI-MS analysis). An even better result was obtained in the side chain-to-Cterminus Ugi cyclization of peptide 25, which bears Lys separated two residues from the C-terminal amino acid. It must be noticed that this Ugi macrocyclization including the Lys side chain resulted more efficient than the variants comprising the N-terminal amino group. We believe that this is not only due to the larger ring size of 26 compared with 24 and 28 but also to the greater migration capacity of the flexible Lys side chain during the transannular acylation step (Mumm rearrangement). In the experience of our group, the most efficient Ugi macrocyclizations of peptides are those involving a Lys side chain as amino component. Scheme 6 also exemplifies the head-to-tail Ugi cyclization of the all-Lpentapeptide 27, furnishing the cyclic peptide 28 in moderate yield after 72 h of reaction. It is worth mentioning that a systematic HPLC/ESI-MS analysis performed in our group for Ugi macrocyclizations at 2 mM concentration of this class of short peptides revealed that 72 h of reaction is not always enough to achieve a conversion of at least 80%. As the option of increasing the concentration is not always optimal, we recommend 96 h of reaction for the Ugi cyclization of tetraand pentapeptides, especially when they involve the Nterminus as amino component. So far, we can summarize that the Ugi reaction is a suitable method for the head-to-tail and side chain-to-terminus cyclization of short peptides, thus enabling a straightforward access to N-substituted macrocyclic peptides. In comparison with Yudin peptide cyclization, the Ugi macrocyclization is much slower, mainly due to the slow transannular acylation of the endo amine and the ring contraction that takes place during such amine migration. A key feature of both multicomponent macrocyclizations is that they can introduce endo- and/or exo-cyclic functionalities derived from the aldehyde and isocyanide components, which may not be present in natural cyclopeptides. Thus, the Ugi reaction introduces an exo-cyclic peptidic fragment as amide Nalkylation, while Yudin’s method produces both an endo-cyclic G


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aziridine and an exo-cyclic amide, this latter one having the capability of controlling the turn conformation100 and the membrane permeability101 of such macrocyclic peptides. The possibility of incorporating exo-cyclic functionalities of biological relevance during the macrocyclic ring closure was exploited by Rivera and Wessjohann69 in the synthesis of analogues of antimicrobial cyclic lipopeptides such a surfactin A and mycosubtilin. As shown in Scheme 7, the mycosubtilin

the Ugi macrocyclization of the linear octapeptide with paraformaldehyde and n-dodecyl isocyanide. In compound 29, the lipidic fragment appears shifted one position compared to the natural lipopeptide, as it is part of the N-alkylated amide exo-cyclic fragment instead of the β-substituent of the Nterminal β-amino-fatty acid. A very important result that demonstrates the power of the Ugi macrocyclization was the direct incorporation of two lipids into a cyclopeptide skeleton bearing the amino acid sequence of surfactin A (see Figure 1). Thus, bilipidated cyclopeptide 30 derives from the Ugi reaction of a peptide aldehyde with both n-dodecyl amine and isocyanide, proving the readily double functionalization of a peptide during this multicomponent macrocyclization. An interesting feature of these latter macrocyclizations is that they could be performed with full conversion at 25 mM concentration of the peptide component in only 48 h, thus providing very good isolated yields of cyclic lipopeptides 29 and 30. The key factor for this success is the presence of various D-amino acids along the peptide sequence, which may favor folded, turn-like conformations and aid engagement of the two reactive ends. Together with the D-amino acid residues, the large ring size of both the α-adduct intermediates and the final macrocycles facilitates the Ugi reaction sequence as compared with shorter peptides. Besides the direct incorporation of exo-cyclic fragments derived from one of the component taking part in the multicomponent macrocyclizations, these approaches enable the installation of reactive functionalities or handles suitable for the late-stage, eventually site-specific derivatization of cyclopeptides. As illustrated in Scheme 8, the presence of the aziridine ring in peptide macrocycles derived from the Yudin reaction allows for the derivatization via ring opening with nucleophiles.70 Thus, cyclopeptide 31 derived from the macrocyclization of a pentapeptide with the aziridine− aldehyde and t-butyl isocyanide could be fluorescently labeled in a site-specific manner with the 7-mercapto-4-methyl-

Scheme 7. Simultaneous Ugi Macrocyclization and Lipidation of Peptides for the Synthesis of Analogues of Naturally Occurring Mycosubtilin and Surfactin A

analogue 29 was produced in good yield through the simultaneous incorporation of an exo-cyclic lipid tail during

Scheme 8. Post-MCR Derivatization of Cyclopeptides Using the endo- and exo-Cyclic Reactive Moieties Introduced during Yudin and Ugi Macrocyclization

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Scheme 9. Solid-Phase Synthesis of Cyclic Peptides Using On-Resin Multicomponent Macrocyclization in the Head-to-Tail Variant

coumarin (7 mmc).70 The nucleophilic opening of the activated aziridine ring by the thiol functionality of the 7 mmc tag efficiently provided the labeled cyclopeptide 32, proving the versatility of such endo-cyclic moiety. As a continuation of this strategy, Yudin’s group also reported a site-specific integration of amino acid fragment into cyclic peptide scaffolds derived from this MCR, with a sequence initiated with the chemoselective hydrolysis of the activated aziridine ring.102 In addition, the isocyanide component can be also used for the introduction of a reactive handle suitable for conjugation. This was exemplified in the synthesis of cyclopeptide 33 using a thioester-isocyanide, which enabled the subsequent native chemical ligation procedure with a linear peptide bearing Cys as N-terminal amino acid to produce peptide 34.103 Finally, in the Wessjohann and Rivera group, we also exploited the possibility of incorporating into cyclopeptides reactive handles derived from the isocyanide component.104 As shown in Scheme 8, cyclopeptide 35, synthesized by means of the Ugi peptide macrocyclization protocol using the 4-isocyanopermethylbutane-1,1,3-triol (IPB) as convertible isocyanide105 bears a N-peptidoacyl pyrrole moiety derived from the acidic treatment of the exocyclic IPB-amide.104 Such an activated acyl group could be employed for the chemoselective labeling of cyclopeptide 35 with the 7-nitrobenz-2-oxa-1,3-diazol-4-yl-derived amine in the

presence of another carboxylic group (i.e., Glu side chain) not affected during this amidation protocol. These examples demonstrated the utility of isocyanide-based MCRs not only for accessing novel cyclopeptide skeletons but also for their late-stage conjugation and labeling without modifying other amino acid side chains. 3.3. Head-to-Tail Multicomponent Macrocyclization on Solid Phase

In 2015, Yudin, Marsault, and coworkers reported106 a solidphase approach for the synthesis of cyclic peptides derived from the aziridine−aldehyde mediated MCR. In the realm of peptide chemistry, the translation of a novel synthetic procedure from solution to solid phase is a key step for the generalization of the method and its application in the rapid construction and screening of peptide libraries. On-resin methodologies such as high-throughput parallel synthesis and split-and-pool combinatorial chemistry have previously validated the success of SPS in peptide drug discovery.107 In this regard, it was clear the importance of proving the versatility of isocyanide-based MCRs in the on-resin peptide macrocyclization. As depicted in Scheme 9A, the protocol employed by the authors followed a tactic typically used in solid-phase peptide synthesis (SPPS), i.e., the attachment of an amino acid side to the resin, followed by peptide elongation, cyclization, and cleavage from the resin.106 Thus, the solid-phase protocol I


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Passerini reaction-based macrocyclization. As depicted in Scheme 10A, the Passerini macrocyclization approach

was initiated by attaching the Glu side chain either to the Wang or to the Rink amide resin, thus leading to final peptides having the Glu side chain either with the free carboxyclic group or in the form of Gln after cleavage from resin. Similarly, the Lys side chain was attached to the Wang resin through a carbamate group, while Thr and Ser could be attached to the Merrifield resin bearing a tetrahydropyranyl linker. The whole protocol included not only the on-resin Yudin macrocyclization of oligopeptides but also the aziridine ring opening by nucleophilic attack of phenyl thiols prior to the cyclopeptide cleavage from the resin by acidic treatment. The parallel solidphase procedure rendered a small library of macrocyclic peptides 37−41 of variable ring size and amino acid sequence.106 In our laboratory, a novel strategy has been developed for the synthesis of gramicidin S analogues, which comprises the introduction of a turn-inducing N-alkylated dipeptide moiety derived from the Ugi multicomponent reaction.108 As depicted in Scheme 9B, the solid-phase approach began with the attachment of a termini-protected tripeptide fragment to the 2chlorotrityl (2CT) resin by the ornithine (Orn) side chain. Elongation of the peptide chain by a standard SPPS using three degrees of orthogonality led to resin-bound acyclic decapeptide 42, which was further subjected to the Ugi macrocyclization to furnish a novel class of gramicidin S analogue 43 bearing an additional phenyl ring as part of the exo-cyclic N-functionality. A key step in Ugi-type reactions is the imine formation, which is typically accomplished prior to addition of the isocyanide component to avoid the competing Passerini reaction. However, on-resin imine formation with paraformaldehyde results as inefficient at both the reactive amino group of a Lys side chain and the N-terminus. Our group devised a solution for this problem encompassing an initial aminocatalysismediated transimination protocol that fully converts the aminopeptide into an imine.109 The procedure comprises the addition to the resin-bound peptide of a piperidinium or pyrrolidinium ion arising from the previous condensation of paraformaldehyde with piperidine or pyrrolidine. Of note, this approach toward gramicidin S analogues focuses on mimicking the β-hairpin structure of the natural product by installing an Ugi reaction-derived N-alkylated dipeptide capable to replicate the β-turn conformation fixed by the central L-Pro-D-Phe fragment. The design of 43 included the incorporation of 2-amino-isobutyric acid (Aib) as the Nterminal residue to be cyclized, thus leading to the N-alkylatedAib-D-Phe fragment opposed to the L-Pro-D-Phe one found at the other corner. Varied combinations of amino acids (e.g., Ala and Gly) with dissimilar exo-cyclic fragments of cationic (e.g., 44) and anionic (e.g., 45) nature were performed during the construction of a parallel library of analogues, which also includes the installation of two Ugi-derived fragments with the consequent deletion of Pro from the sequence (unpublished results). Again, it is worth mentioning that this strategy allowed for modulating the polarity (i.e., hydrophobic or charged groups) of a natural cyclopeptide skeleton without affecting the canonical amino acids side chains.

Scheme 10. Macrocyclization of Nonpeptidic Substrates and an Oxo-Peptide by the Passerini Multicomponent Reaction

employed a dicarboxyclic acid and a diisocyanide in the presence of isobutyraldehyde as carbonyl component to assemble the macrocyclic compound 46 with formation of eight new covalent bonds in one pot. More recently, Dömling et al. also employed the Passerini reaction as ring closing procedure for the synthesis of a small library of tetrazole-based macrolactones (Scheme 10B).87 Despite that neither of these approaches involves peptides, they paved the way for the use of the Passerini protocol in peptide head-to-tail cyclization. Scheme 10C shows the synthesis of surfactin analogue 49 by the Passerini macrocyclization of a peptide aldehyde precursor with a lipidic isocyanide.69 Cyclic lipopeptide 49 actually bears a depsipeptide skeleton as it does the naturally occurring compound but differs in the presence of an exo-cyclic amide in the lipid tail. The Passerini ring-closing procedure of this class of peptide could be conducted at 25 mM concentration in very good yield of isolated product 49, likely as a result of the preorganization of the peptide−aldehyde precursor bearing Damino acids. The diastereoselectivity of this process was also satisfactory compared to acyclic versions of this MCR. Following the success of the Ugi and Passerini macrocyclizations of peptides, Rivera, Wessjohann, and coworkers expanded the repertoire of this class of multicomponent ring closure with the development of the Ugi−Smiles macrocyclization.111 As mentioned before, the Ugi−Smiles reaction is a modern variant of the Ugi reaction in which the acid component is a nitro-phenol. As this reaction had never been employed in a macrocyclization approach, the experience with the Ugi peptide macrocyclization was taken as starting point in terms of reaction time and dilution conditions. The approach comprised the preparation of 3-nitrotyrosine (NTy)-containing peptides followed by the head-to-side chain macrocyclization by means of the Ugi−Smiles reaction. Parallel studies were carried out in MeOH with the peptides at 2, 10, and 25 mM concentration in MeOH, and the conversion was monitored by HPLC at 24, 48, and 96 h.111 This study showed

3.4. Peptides Macrocyclization by Passerini and Ugi−Smiles Reactions

The first example of the utilization of the Passerini reaction in a macrocyclization procedure was reported by Wessjohann and coworkers in 2008.110 These authors applied the principles of the MiBs strategy to the synthesis of macrolactones via double J


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that the peptidic substrates were fully consumed after 48 h of reaction at both 10 and 25 mM with no formation of oligomeric products. As shown in Scheme 11, this multi-

ism with a high rotational energy barrier at the N−C(sp2) single bond. Thus, this novel linkage shows prospect for introducing conformational constraints in peptides and also when installed by stapling procedures.

Scheme 11. Head-to-Side Chain Macrocyclization of Peptides by the Ugi−Smiles Multicomponent Reaction

3.5. Peptide Macrocyclization by Strecker Reaction

Despite being the oldest MCR, the Strecker reaction remained unexplored as a macrocyclization procedure until very recently, when Baran and coworkers60 developed the thermodynamic macrocyclization of peptide aldehydes followed by trapping with either an internal or external nucleophile, including cyanide. To devise such a simple and at the same time powerful multicomponent procedure, the authors were inspired by nonribosomal imino cyclopeptides,112,113 which derived from the reductive cleavage of the C-terminus in the nonribosomal peptide synthetase machinery. Scheme 12 illustrates the Strecker macrocyclization procedure in which a linear peptide aldehyde undertakes an intramolecular condensation to form a cyclic imine or iminium ion. As imine formation is a reversible step, the macrocyclic imine are subsequently trapped by addition of the cyanide. The propensity for the spontaneous cyclization of the linear peptide aldehydes depends of course on both the amino acid sequence and the ring size, albeit the imine trapping by the nucleophile shifts the equilibrium toward the final cyclopeptide. The macrocyclization protocol required high dilution (1 mM of the peptide in aqueous buffer), upon which dimerization products were not generally observed. Whereas the process worked well with peptides containing all proteinogenic amino acids, best results were obtained with those including Pro and Gly in the sequence (Scheme 12). The SPPS methodologies employed to grow the linear peptides enabled the installation of the carbonyl group at both the C-terminus and a glutamine-like side chain, thus leading to head-to-tail (54 and 55) and headto-side chain (56−60) variants. Cyclic cyanopeptides were obtained in good isolated yields but generally as a 1:1 mixture of diastereomers that could be separated by HPLC. Importantly, the Strecker macrocyclization tolerated most side chain functional groups in deprotected form, including carboxylic acids, the imidazole group (His), phenol (Tyr), and

component cyclization incorporated paraformaldehyde and ndodecylisocyanide by connecting the N-terminal residue with the nitro-phenol side chain placed one to four amino acids away. The process delivered in good yields a variety of cyclic peptides bearing a unique lipidated N-aryl-bridge, a class of covalent linkage not found in natural or even in synthetic cyclopeptides. Due to the nitro group present in ortho-position to the tertiary aniline, these compounds showed atropisomer-

Scheme 12. Head-to-Tail and Head-to-Side Chain Macrocyclization of Peptides by the Strecker Multicomponent Reaction

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Scheme 13. Peptide Macrocyclization by a Heterocycle-Forming MCR with a Bifunctional Isocyanide

isomer of cyclopeptide 61 revealed the (S)-configuration for the newly formed stereocenter arising from the aldehyde component. The substrate scope of the oxadiazole-forming macrocyclization was broad regarding the sequence and ring size of the macrocyclic peptides (i.e., 15- to 24-membered rings), which were combined with three different aliphatic aldehydes (Scheme 13). In this method, the amino acid side chains needed to be protected during the multicomponent ring closure, and as before, the method generally worked better with Pro as N-terminal residue. This may be due not only to the enhanced reactivity of the iminium species, but also to the fact that with Pro there is no competition of the classic Ugi reaction because of the impossible acylation of the tertiary endo-amine. Among the most relevant features of this macrocyclization is the control over the cyclopeptide conformation through the installation of the noncanonical reduced amide/heterocycle moiety (highlighted in red in Scheme 13).114 Thus, the network of intramolecular hydrogen bonds formed between the reduced amide/heterocycle moiety and other backbone amides serves as endo-cyclic element of conformational control that stabilizes turn motifs and facilitates passive membrane permeability. A very detailed conformational analysis of this novel class of heterocycle-grafted peptides was recently conducted by Yudin’s group, including the comparison with the canonical cyclopeptide congeners.118 Considering the recent interest in macrocyclic peptides bearing heterocyclic grafts,119 the utilization of heterocycle-forming MCRs in the ring closure of peptides may receive an important boost. For example, other heterocycle-forming MCRs such as the Ugi-azide-4CR120 and the Staudinger-3CR110 have been previously employed in macrocyclization approaches, leading to tetrazole- and β-lactam-containing macrocycles, respectively.

even the primary amine of Lys side chain; albeit the thiol group of Cys needs to be in the form of disulfide due its good nucleophilic character. Of note, peptides bearing the Nmethylated amino acids provided better results than those with a terminal primary amine, likely due to the enhanced reactivity of the iminium macrocycle compared to the imine species. Besides trapping with cyanide, the macrocycle imines could be also efficiently reduced with NaBH3CN, thus representing a powerful reductive amination−macrocyclization protocol.60 3.6. Peptide Macrocyclization by Heterocycle Ring-Forming MCRs

In an endeavor to provide new methods leading to noncanonical cyclopeptides, Yudin and coworkers described the multicomponent synthesis of oxadiazole-grafted macrocyclic peptides.114 The approach comprises the reaction of a special isocyanide component, namely (N-isocyanimino)triphenylphosphorane (Pinc), with an aldehyde and a linear peptide, thus generating a cyclic architecture in which the heterocycle ring is embedded within the cyclopeptide backbone. As depicted in Scheme 13, the synthetic design shares a successful setup previously implemented by this group. This encompasses the use of an isocyanide or an aldehyde component that (a) favors a curved peptide conformation by the zwitterionic engagement of the two ends upon condensation with the N-terminal amine and (b) proves capable to deviate the classic Ugi mechanism using an external nucleophilic center that intercepts the mixed anhydride fragment formed during the Ugi reaction mechanism. In this sense, Pinc is well-suited for such a purpose, as it incorporates a nucleophilic nitrogen atom and the isocyano group in the same reagent.115 Previously, Ramanazi and coworkers had developed the synthesis of substituted oxadiazole by the reaction of Pinc with the other three independent components, i.e., carboxylic acid, amine, and aldehyde.116,117 Thus, the vision of Yudin et al. rested on exploiting the Ugi-type/azaWittig mechanism to leverage the macrocyclization reaction. By avoiding the difficult transannular migration of the endoamino group, this novel multicomponent macrocyclization resembles more the aziridine−aldehyde mediated peptide cyclization than the classic Ugi macrocyclization. As a result, the oxadiazole-forming cyclization can be undertaken at higher concentration and in shorter reaction time than the Ugi peptide macrocyclization, although it also proceeds with poor stereoselectivity, furnishing a mixture of two diastereomers.114 HPLC purification and X-ray crystal analysis of the major

4. MULTICOMPONENT PEPTIDE STAPLING Peptide stapling has been traditionally used to lock peptide conformations into α-helical structures using a variety of macrocyclization methods. Despite the recognized value of MCRs in macrocyclization approaches, successful inputs of these processes in peptide stapling strategies have emerged only recently. An advantage of utilizing an MCR for crosslinking two amino acid side chains is that the ring closure procedure may lead to the stabilization of helical peptides at the same time that enables the diversity-oriented derivatization of the biomolecule. The traditional amino acid side-chain L


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tion,42,134 and bis-arylation40,41 of two Cys thiols, among others. Interestingly, there is a special class of two-component stapling in which the side chain linker bears either an additional functionality or handle enabling the subsequent conjugation or diversification.38,135 In this regard, Spring and coworkers136−139 successfully employed their double-click, two-component stapling with functionalized dialkyne linkers for modulating cellular activity, labeling, etc. Similarly, Dawson and coworkers described an acetone-linked side-chain bridge capable of stabilizing a helical structure and providing the carbonyl functional group for further labeling and conjugation through oxime ligation.140 Depending on the length of the side chain tethers and the linker employed, the one- and twocomponent stapling processes may expand either one or two helical turns, which also influences the stability of the α-helical peptide. In the Rivera and Wessjohann groups, the concept of multicomponent stapling was introduced for those processes relying on MCRs for both the direct side-chain bridging and for cross-linking two side chains with a bifunctional linker. As shown in Scheme 14C and D, the multicomponent stapling approaches may be based either on a single or double MCR and can be adjusted to staple peptides either at i, i + 4 or i, i + 7 positions. In contrast to the two-component variants in which the linker is either previously functionalized for a specific biological purpose or for a subsequent derivatization, the multicomponent stapling incorporates one or various exocyclic functionalities during the ring closure. Indeed, one of the advantages of the latter approach is the greater diversitygenerating capacity compared to those not employing MCRs. It might be noticed that the double multicomponent stapling (Scheme 14D) is actually an implementation of the MiBs strategy46 with peptides, in which up to six components react with formation of eight covalent bonds and incorporation of various exocyclic moieties, all in one pot. This section describes macrocyclization approaches based on MCRs capable to staple peptides by either bridging or cross-linking a pair of side chains. Whereas the main focus is on the synthetic methods, reference to the conformational analysis of the constrained cyclic peptides is also included.

tethering (Scheme 14A) can be referred to as one-component stapling38 because the peptide per se is the only component, Scheme 14. Stapling Strategies Comprising One-, Two-, and Multicomponent (Represented with a 4CR) Approaches Targeting Helical Peptides

and its side-chain functional groups are the responsible ones for the bridging process. Most one-component stapling methods are based on RCM,33,34,121 lactamization22,23,122 and click CuAAC,39,123−125 whereas disulfide bond formation,13,126 oxime ligation,127 thioether formation,128 and Pd-catalyzed C− H activation43−45,129 have also proven success, among others. As proposed by Spring et al.,38 there is also a two-component stapling approach in which a bifunctional linker macrocyclizes with a peptide bearing a pair of counter-reactive functional groups (Scheme 14B). This alternative peptide stapling comprises the execution of two reactions, one forming the acyclic intermediate and another conducting the ring closing step. The diversity of chemical processes applied to this technique is also very high,38 including the double-click approach,130−132 double thiol−ene coupling,133 bis-alkyla-

4.1. Stapling by Ugi Reaction

Aiming at providing diversity-generating tools to the repertoire of peptide stapling approaches, Rivera, Wessjohann, and workers introduced two alternative multicomponent stapling approaches based on the powerful Ugi reaction.141,142 As depicted in Scheme 15, the initial method comprises the

Scheme 15. Multicomponent Peptide Stapling by Solution-Phase Ugi Macrocyclization of Lys and Glu/Asp Side Chains

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Scheme 16. On-Resin Ugi Multicomponent Peptide Stapling Enabling the Stabilization of α-Helical Structures and the Simultaneous Functionalization of the Lactam Bridge

carbocyclic acid groups that take part in the Ugi macrocyclization. Scheme 16 illustrates the on-resin multicomponent stapling procedure developed by Rivera, Wessjonann, and coworkers for the preparation of helical peptides bearing diverse exocyclic functionalities.140 This variation of the isocyanide component enabled the incorporation of lactam N-substituents of lipidic (69), anionic (70), saccharide (71), and polyethylene glycol (PEG, 72) nature, among many other cationic and fluorescent tags that are not shown herein. Importantly, this unique type of lactam-bridge functionalization is achieved with the parallel introduction of conformational constraints leading to α-helical structures, as proven by the circular dichroism and NMR studies.140 This work encompassed the first example of a multicomponent peptide stapling that combines the stabilization of α-helical secondary structure with the simultaneous (and not subsequent) incorporation of functionalized bridges of biologic (lipid), chemical (label), or pharmaceutical (PEG) relevance. In parallel to the development of the side chain cross-linking approach using a single Ugi reaction-based macrocyclization, our laboratory extended the multicomponent stapling concept to the synthesis of stapled peptides using a double Ugi reaction-based macrocyclization.142 As shown in Scheme 17, in this case, the multicomponent macrocyclization was conducted in solution because, according to our experience with on-resin macrocyclizations, the use of bifunctional linkers frequently leads to complex mixtures of cross-linked products, unless a very low resin loading is employed. As previously proven by Wessjohann and coworkers,78−82 pseudo-dilution conditions work very well in MiBs approaches, in this case comprising the simultaneous slow addition with syringe pumps of the peptide dicarboxylic acid and the diisocyanide linker to a stirring solution of the preformed imine. This stapling protocol required from 72 to 96 h of reaction, including the addition process via syringe pumps, but remarkably it provided very high macrocyclization yields while avoiding the use of large solvent volumes. Scheme 17 highlights stapled peptides 73 and 74 cross-linked with biarylether and p-xylylene spacers,

multicomponent ring closure of Lys and Asp/Glu side chains by reaction with paraformaldehyde and an isocyanide.141 As it is traditionally done in stapling procedures based on lactamization, the cross-linked residues were separated three or four amino acids from each other which, depending of the amino acid combination, may favor either 310 or α-helices.22,23 Such Ugi macrocyclizations were performed in solutionphase in 20 mM concentration of the peptide, thus producing stapled peptides 66−68 in very good isolated yields as compared with the head-to-tail Ugi cyclization of short peptides. As these were the first examples of stapled peptides derived from the Ugi reaction, our group conducted a conformational analysis based on NMR and molecular dynamics simulation aiming at studying the effect of the Ugi cyclization on three-dimensional solution structure of the three cyclic peptides. As shown in Scheme 15, the NMR-derived structures of 66 and 67, bridged at i, i + 3 residues, show their occurrence as reversed turns, with cyclopeptide 66 displaying a partial helical content. Alternatively, conformational analysis of peptide 68 proved the presence of a α-turn motif in this i, i + 4 lactam bridged cyclopeptide. Whereas the lactamization of Lys and Asp residues placed at i, i + 4 positions is a very effective way to stabilize an α-helix in peptides,23,122 the presence of the turn-inducing Gly-Pro sequence favored a pseudo-planar turn instead of an α-helical one. Recently, the Ugi stapling approach was extended to the actual stabilization of helical secondary structures and the simultaneous installation of very diverse exocyclic Nfunctionalities at the lactam bridge.143 Instead of using solution-phase macrocyclization, a solid-phase methodology was developed in which both the peptide growth and the Ugimacrocyclization were conducted on resin. The choice of the on-resin method was considered due to the lack of bioorthogonality of the Ugi reaction, i.e., the impossibility of the peptide to bear other free amino and carboxylic acid groups. In this sense, a solid-phase protocol based on three dimensions of orthogonality could enable the on-resin preparation of peptide, followed by the selective deprotection of the amino and N


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according to personal information provided by authors working in this field.

Scheme 17. Multicomponent Peptide Stapling by a Double Ugi Reaction Macrocyclization

4.2. Stapling by Ugi−Smiles Reaction

On the basis of the early success of the Ugi−Smiles macrocyclization involving the N-terminal amino group with the 3-nitrotyrosine (NTy) side chain, a translation to peptide stapling procedures by bridging two amino acid side chains was feasible. Considering the good results achieved with the onresin multicomponent macrocyclization approach, Rivera, Wessjohann, and coworkers adapted the solution-phase Ugi− Smiles macrocyclization to a solid-phase procedure conducting all synthetic operations on-resin.111 As shown in Scheme 18, the protocol comprised the initial on-resin transimination step109 employing the iminium ion derived from the reaction of piperidine with paraformaldehyde, followed by addition of the isocyanide to allow the Ugi−Smiles ring closure leading to the N-aryl-bridged peptide. Again, a lipidic isocyanide was employed aiming at installing such a biologically relevant moiety as exo-cyclic tail, but the on-resin protocol also proved suitable with the variety of isocyanides used in the Ugi stapling method (see Scheme 16). Besides the uniqueness of this type of lipidated N-aryl-bridged covalent bridge, which is not accessible in one step by any other macrocyclization process, it is promising that the Ugi−Smiles stapling provides very good HPLC yields of cyclic peptides cross-linked by the Lys and NTy side chains at positions ranging from i, i + 3 to i, i + 7. Indeed, the flexibility and high reactivity of the Lys side chain plays a key role in this success, besides the intrinsic mechanism of the Ugi−Smiles reaction that favors the engagement of the two reactive ends by ion pairing even after formation of the imine intermediate (see explanation in Section 3.1.).

respectively, derived from the diisocyanide components employed. The linker length and flexibility could be tuned depending on the distance expanded between the two reacting amino acid residues, whose position could vary from i, i + 4 to i, i + 7. This method allows for the ready generation of diversity at the cross-linker fragment, not only through the variation of the diisocyanide building block (e.g., aromatic, aliphatic, and heterocyclic linkers), but also of the amine component, which can include all proteinogenic amino acid in a C-protected form. In addition, other combinations of bifunctional building blocks can be considered, e.g., the diacid/diamine and diamine/diisocyanide, which are both suitable for peptide stapling. Considering the rapid scanning of the stapled peptide chemical space enabled by this macrocyclization technology−proven with varied substrates and dissimilar MCRs46 − it is expected that several applications in the field of drug discovery will emerge during the next years.144 Of note, some applications are already under development

4.3. Stapling by A3-Coupling Reaction

One of the relevant applications of the multicomponent peptide stapling strategy in drug discovery is the development of A3-coupling-based macrocyclization of azapeptide side chains. Azapeptides comprise an important class of peptidomimetic in which the α-carbons of one or more amino acids have been substituted by nitrogen. Lubells’s laboratory has specialized in the diversification of these peptide mimics,145 including the on-resin derivatization of alkyne-functionalized azapeptides by the multicomponent A3-coupling reaction with secondary amines and aldehydes.146 In an endeavor to exploit

Scheme 18. On-Resin Peptide Stapling by Ugi−Smiles Multicomponent Macrocyclization

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Scheme 19. On-Resin Stapling of Azapeptides by the A3-Coupling Macrocyclization

Scheme 20. Multicomponent Peptide Stapling by Petasis Macrocyclization

the diversity-generating capacity of the A3-coupling reaction to the synthesis of stapled azapeptides, Ong, Lubell, and coworkers described the Cu(I)-catalyzed A3-macrocyclization of peptides by cross-linking propargyl aza-residues and Nεalkyl-Lys side chains.147 As depicted in Scheme 19, the authors also chose to implement a solid-phase protocol enabling both the peptide elongation and A3-macrocyclization to be carried out on resin. The focus was on the creation of a compound library of cyclic azapeptide analogues of GHRP-6 (His-D-TrpAla-Trp-D-Phe-Lys-NH2). As for other MCRs, a key advantage of the A3-macrocyclization is the possibility of incorporating an additional component which could help to modulate the biological activity.148 As stated by the authors, the intrinsic features of both the azapeptide skeleton and the cyclization method facilitate the macrocyclic ring closure, i.e., (i) the insertion of aza-residues favors reverse turn conformations and (ii) the metal center brings together the alkyne and imine groups during the coupling step. Scheme 19 also depicts some macrocyclic azapeptides (79− 82) out of the 15 aza-GHRP-6 macrocycles produced by the on-resin A3-macrocyclization, which exemplifies the variety of stapling positions (from i, i + 2 to i, i + 5) and exo-cyclic

substituents inserted at the tertiary amine-bridging moiety. Remarkably, this diversity-oriented multicomponent approach allowed for the identification of compound 81, which displays the highest affinity for CD36 ever reported for a GHRP-6 analogue.147 4.4. Stapling by Petasis Reaction

A very recent addition to the repertoire of multicomponent macrocyclization methods is the development of a peptide stapling approach based on the Petasis reaction. Rivera, Wessjohann, and coworkers recently envisioned the utilization of this remarkable three-component process for the late-stage diversification of peptides at the side chains and the Nterminus.149 Since the discovery of this borono-Mannich reaction by Petasis et al.,62 it has been widely exploited in the synthesis of nonproteinogenic amino acids and natural products,63 but applications in the realm of peptide chemistry have been limited to the on-resin modification of amino acids and secondary amides. As a result, the implementation of this MCR with larger peptides required a detailed optimization process, including the screening of the carbonyl and boronic acid components suitable for the efficient condensation with a P


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peptide A154 using the convertible isocyanide IPB in the Ugi reaction step. The remarkable turn-inducing effect of the peptide backbone N-alkylation derived from the Ugi reaction has also been exploited to assist the cyclization of short peptides.155 Thus, the use of acid or photolabile amines as well as the Rink amide resin in the solid-phase version led to the development of Ugi reaction-derived traceless turn-inducers, which are subsequently removed after peptide macrocyclization. Several other groups have exploited the complexitygenerating character of the Ugi reaction for the assembly of natural product-like peptide architectures that are macrocyclized by non-MCR methods. In 2006, two independent reports156,157 described the preparation of analogues of cyclopeptide alkaloid-like macrocycles utilizing an initial Ugi reaction and followed by dissimilar ring closing steps. Thus, Zhu and coworkers employed an intramolecular SNAr156 for the macrocyclization of Ugi reaction-derived peptide skeleton, while Wessjohann and collaborators used a macro-etherification156 for the same purpose. More recently, Kazmaier’s158 and Sello’s159 groups implemented similar approaches based on Ugi-type reactions, albeit using other macrocyclization methods for the synthesis of analogues of the antibiotic cyclic peptides bottromycin and enopetin, respectively.

peptide amino group. In our laboratory, the original experiments found that the free Lys ε-NH2 group was significantly dibenzylated and diallylated when reacted with formaldehyde and p-OMe-phenyl boronic and (E)-styrylboronic acid, respectively, due to subsequent carbonylation of the resulting secondary amine forming an equally reactive iminium intermediate. Fortunately, it was well-known that the utilization of an enantiomerically pure α-hydroxy-aldehyde results in a diastereoselective Petasis reaction,63 so the diversity of carbonyl components could be expanded to aldehydes such as small glyceraldehyde and small sugars. Nonetheless, such carbonyls reagents also led (although only partially) to double Petasis reaction products. In consequence, the solution was to employ peptides bearing a secondary amine as side chain, e.g., Nε-MeLys, which worked well for the multicomponent incorporation of dissimilar aldoses and ketoses (as carbonyl component) and boronic acids endowed with fluorescent labels, steroid skeletons, PEG chains, etc. In addition, it was possible to develop an efficient multicomponent stapling protocol based on the Petasis reaction with Nε-MeLyscontaining peptides. As depicted in Scheme 20, dihydroxyacetone was initially chosen as carbonyl component to be combined with p- and m-phenylene and biphenylene diboronic acids during the multicomponent stapling of a peptide bearing two Nε-MeLys at i, i + 7 positions to furnish macrocycles 83, 84, and 85, respectively. Another example included the stapling of a peptide bearing two Nε-Me-ornithines placed at i, i + 4 positions employing glyceraldehyde and p-phenylene diboronic acid to give macrocyle 86. These stapled peptides were produced in good isolated yields by a macrocyclization protocol performed under pseudo-dilution conditions,149 thus paving the way also for the incorporation of aldoses and ketoses of biological relevance.

5.1. MCR-Mediated Peptide Functionalization with Ring-Closing Group

There are approaches in which the MCR is actually responsible for the installation of the functional groups taking part in the peptide macrocyclization. Kazmaier et al.160 pioneered the endeavor of accessing conformationally constrained Ugiderived cyclopeptides by RCM macrocyclization, a strategy also employed by other groups161,162 to produce nonpeptidic artificial macrocycles, including potent p53-MDM2 inhibitors. 163 As shown in Scheme 21, Kazmaier’s tactic

5. SEQUENTIAL MCR/MACROCYCLIZATION APPROACH TO CYCLIC PEPTIDES Over the years, isocyanide-based MCRs such as the Passerini and Ugi reactions have been employed for the assembly of pseudo-peptide fragments that are subsequently incorporated into natural cyclic peptides. However, for some of these reports, we only include a brief description of the inputs provided by the MCRs but not a schematic representation because such works have been previously reviewed46,54,144 and, more importantly, the MCR is utilized very early in the synthetic route and is not related with the ring-closing step. The first report of this strategy was described Joullié and coworkers,150 which employed a notable variant of the Ugi reaction using a pyrroline derivative as imine component, nowadays known as Ugi-Joullié-3CR, to access a prolylisoleucine fragment of the 14-membered cyclopeptide alkaloid nummularine F. Follow-up reports of Schmidt’s151 and Semple’s152 groups on the synthesis of the prolyl endopeptidase inhibitor eurystatin A further supported the feasibility of using an isocyanide-based MCR for the straightforward construction of a natural cyclopeptide fragment. Both groups relied on the Passerini reaction for the preparation of the key α-ketoamide core during the assembly of the cyclic peptide. In parallel, Bauer and Armstrong used a convertible isocyanide and methylamine for the Ugi reaction-based synthesis of a Nmethylated peptide fragment, subsequently employed in the assembly of the cyclic peptide motuporin.153 This concept was recently applied by Rivera, Wessjohann, and coworkers in the synthetic of the poly-N-methylated cyclopeptide cordyhepta-

Scheme 21. Synthesis of Olefin-Bridged Cyclic Peptides by an Ugi Reaction/RCM Sequence

encompassed the utilization of an isocyanoacetate allyl ester and an N-Alloc-amino acid in the Ugi reaction along with a chiral amine intended to enhance the diastereoselectivity and isoburyaldehyde to mimic a Val side chain in the newly created amino acid residue. Due to this synthetic setup, acyclic intermediates 87 were obtained in excellent yield and good diastereoselectivity, with the major diastereomer featuring the (S)-configuration at the newly formed stereocenter. Thus, the presence of the alkene functionalities derived from the allyl (All) and allyloxycarbonyl (Alloc) protecting groups enabled the utilization of the RCM using the Grubbs catalyst to achieve the cyclic peptides in moderate yields and bearing the olefinbridge with the E-configuration. A very recent example in which the MCR is responsible for the installation of the functional groups directly participating in the subsequent peptide macrocyclization is the report of Rivera, Paixão, and coworkers describing the synthesis of Q


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without significant epimerization of terminal residues.164 This work represents an alternative approach to the use of heterocycle-forming MCRs in peptide cyclization strategies, as here the MCR is employed to build the heterocycle core at the same time that introduces the functionalities suitable for the subsequent macrocyclization step.

tetrahydropyridine-grafted peptide macrocycles using a highly stereoselective multicomponent approach for the creation of the heterocycle core.164 As depicted in Scheme 22, the authors Scheme 22. Synthesis of a Hybrid TetrahydropyridinePeptide Macrocycle by an MCR/Macrolactamization Sequence

5.2. MCR-Mediated Peptide Backbone Stapling

A recent cooperation between the groups of Rivera and Wessjohann enabled the development of a very general and versatile strategy for stapling peptides at the backbone instead of the side chains.166 As shown in Scheme 23, a solid-phase protocol including on-resin Ugi reactions was employed to introduce functionalized amide N-substituents at different positions of the peptide backbone. This multicomponent backbone N-modification method is efficient and diversity oriented, as it allows the incorporation of very different functionalities suitable for ring closure approaches. As a result, diverse macrocyclization chemistries were implemented with such backbone N-substituents, including the ring-closing metathesis, macrolactamization, and thiol bis-alkylation. Scheme 23A exemplifies the execution of the whole sequence on-resin, including the two Ugi reactions installing the backbone N-modifications, the peptide elongation and the final RCM involving the two olefin functionalities placed at i, i + 6 amides. Of note, backbone stapled peptide 91 was obtained in good crude yield and purity considering the complex solid-phase sequence employed. A different approach is shown in Scheme 23B, in which the N-substituted peptide is previously built on solid phase and then released from the resin to carry out the thiol bis-alkylation in solution. As mentioned before, the reason for not conducting the two-component macro-thioetherification on resin is the likely formation of cross-linker peptides instead of cyclic ones, unless a very low and unpractical resin load is employed. Thus, backbone stapled peptide 92 was also obtained with very good conversion and isolate yield by the two-component macrocyclization of the released peptide.

employed an initial asymmetric conjugate addition of 3-phenyl3-oxo-propanenitrile to cinnamaldehyde catalyzed by diarylprolinol silyl ether to furnish a chiral hemiacetal bearing an aldehyde and an enol functionality. This enantiomerically enriched bifunctional intermediate was employed in an isocyanide-based MCR recently developed in cooperation between the two laboratories165 which encompasses the diastereoselective condensation of the bifunctional substrate with an amine and an isocyanide, in this case both of peptidic nature. Thus, the use of a isocyano-tripeptide and a methyl ester amino acid led to the tetrahydropyridine-grafted peptide 89 in good yield and excellent diastereoselectivity, which was subsequently deprotected and subjected to a two-component macrolactamization with the propane-1,3-diamine counterpart to afford the heterocycle-peptide hybrid macrocycle 90

Scheme 23. Peptide Backbone Stapling Strategy Enabled by the Incorporation of Amide N-Substituents Derived from OnResin Ugi Reactions

R


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Scheme 24. Peptide Macrocyclization Enabled by an MCR-Mediated Activation of the C-Terminal Carboxylic Acid

carboxylate to furnish a putative spirolactone, and final nucleophilic attack of the primary hydroxyl group to render the depsipeptide macrocycle. This mechanism conveys that the MCR-derived 5-aminooxazole moiety acts as a traceless activator of the C-terminal carboxylic acid, thus enabling the ring closure in the presence of an internal nucleophile. The use of an initial MCR guarantees the installation of molecular diversity at the final peptide macrocycle, including the creation of sugar−depsipeptide hybrid scaffolds.168 In an extension of the synthetic program dealing with MCRs using convertible isonitriles, Rivera and Wessjohann reported an interesting peptide macrocyclization approaches also relying on the MCR-mediated activation of the peptide C-terminus.104 As illustrated in Scheme 24B, the method relies on the use of a convertible isocyanide which, upon reaction with a peptide carboxylic acid, methylamine, and paraformaldehyde, affords an N-methylated linear peptide functionalized at terminal carboxamide. The acidic treatment implemented to remove the N-terminal Boc group also permitted the concomitant conversion of the C-terminal amide into an N-peptidoacyl indole, which is well-suited for an amidation reaction. As before, the MCR is the responsible for the incorporation of the activated carboxyl group, which in this case derives from the convertible isocyanide 1-isocyano-2-(2,2-dimethoxyethyl)benzene. Thus, the C-terminal activated peptide can be subjected to macrolactamization without previous isolation via basification and stirring under diluted conditions using either standard heating or microwave irradiation. This approach was employed for the synthesis of N-methylated head-to-tail cyclized penta- (96), hexa- (97), and heptapeptides (98) in good yield over three steps. An alternative protocol was utilized for the installation of a N-methylated lactam bridged in a side chain-to-side chain cyclized peptide 99, although using the IPB convertible isocyanide introduced in Scheme 8. This report improves the repertoire of peptide macrocyclization methods by combining the synthetic

A crucial step for the success of the backbone stapling approach is the aminocatalytic transimination that enables the subsequent on-resin Ugi reaction, which is accomplished by treatment of the resin-linked peptide with paraformaldehyde and a secondary amine (i.e., piperidine or pyrrolidine) prior to addition of the carboxylic acid and the isocyanide. This onresin Ugi protocol has proven success in the incorporation of all amino acids (in protected form) and very complex isocyanides (as shown in Schemes 16 and 23). In our opinion,166 this approach provides a better alternative than the classic methods used to produce complex N-substituted peptides (i.e., having N-alkyl amino acids other than N-methyl ones), which require a difficult acylation step of the Nalkylated terminal amino acid. As demonstrated herein, the onresin Ugi protocol skips this difficulty by incorporating the next Fmoc-amino acid and the N-substitution simultaneously, thus paving the way for the subsequent tethering of the amide Nsubstituents by diverse ring closure procedures. 5.3. MCR-Mediated Activation of the Peptide C-Terminus

Besides the easy incorporation of reactive handles suitable for peptide macrocyclization (e.g., olefins, thiols, etc.), it has been reported that the MCR step can also provide an activated Cterminal carboxylic group prompted for ring closure by intramolecular attack of a proper nucleophile. Zhu and collaborators were the first to implement this concept for the development of a novel macrolactonization approach toward cyclodepsipeptides. 167 As shown in Scheme 24A, the procedure comprises a three-step sequence starting with the Zhu-3CR of an isocyanoacetamide, a secondary amine, and an aldehyde to furnish a 5-aminooxazole-containing peptidomimetic. Considering that the 5-aminooxazole moiety is a masked dipeptide equivalent and can be hydrolyzed back to the diamide fragment, the authors implemented a sequential process involving the ester saponification followed by an acidmediated domino sequence that comprises the oxazole-ring protonation, trapping of the resulting iminium species by the S


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Scheme 25. Synthesis of Multicyclic Peptide Cages by Double Ugi Reaction Macrocyclization

In the same report,169 the authors increased the complexity of the system to the highest limit by implementing a templatedriven macrocyclization using four different bifunctional building blocks, one of peptidic nature. As depicted in Scheme 25B, the macrocyclization setup comprised the template formation of a macrocyclic diimine by reaction of pyridine2,6-dicarbaldehyde with a PEG-diamine biased by the addition of Mg2+. The second step consisted of the slow addition of the diacid and diisocyanide building blocks to the macrocyclic diimine, which allowed the formation of hybrid, peptidecontaining, macromulticycle 101 in acceptable yield considering the complexity of this approach. The metal template proved crucial for obtaining the final macromulticycle because no macrocyclic products were formed in the absence of such metal template.

potential of MCRs with the utility of convertible isocyanides to install masked activated carboxylic groups.

6. MULTICOMPONENT SYNTHESIS OF MULTICYCLIC PEPTIDE CAGES An important input from the field of multicomponent macrocyclization has been the development by Wessjohann’s group of Ugi reaction-derived molecular cages. In this laboratory, several synthetic approaches have been devised for the synthesis of amino-acid-based cryptands, cryptophanes, and cages with high chemical efficiency and atom economy.81,82 In this regard, a very straightforward method toward amino-acid-bridged macrocycles is the recent report of Wessjohann and Rivera169 describing the utilization of a double Ugi reaction-based macrocyclization for the assembly of macromulticycles having up to four different tethers, i.e., hybrid cages. Differently from any other previous approach, this multicomponent procedure focuses on setting up the macromulticycle connectivities by the bridgeheads instead of by the tethers, thus bringing together at least three dissimilar bifunctional building blocks in one pot. As shown in Scheme 25, this method permits the rapid construction of hybrid macromulticycles in which one the tethers is a peptide chain, and using both dilution and metaltemplate driven macrocyclization conditions. Scheme 25A depicts the assembly of the peptide macrobicycle 100 by reaction of a peptide dicarboxylic acid (Glu side chains) with a heterocycle-containing diamine and a biaryl-ether diisocyanide in the presence of paraformaldehyde. It is worth mentioning that when aryl-based diisocyanides are employed, flexible diamines or dicarboxylic acids are required because the employment of three rigid bifunctional building blocks usually leads to complex mixture of acyclic oligomers due to the difficult macrobicycle ring closure. To reduce this latter possibility, pseudo-dilution conditions need to be implemented by the simultaneous slow addition of the bifunctional components. Nonetheless, it was proved by ESI-MS studies169 that the macrocyclization takes places by an initial Ugi reaction that engages the three bifunctional components, followed by the second Ugi reaction that is responsible for the macrobicycle ring closure.

7. SUMMARY AND OUTLOOK We illustrated the versatility of MCRs for the synthesis of peptide macrocycles featuring varied types of chemical topologies. Perhaps the most important feature of this strategy is the rapid generation of both endo- and exo-cyclic diversity during the ring-closing step. In terms of chemical efficiency, the peptide multicomponent macrocyclization has proven as effective as other classic peptide cyclization methods such as lactamization, click chemistry, and RCM but with the former one creating much higher molecular complexity at lower synthetic cost. Of special interest for this chemistry is the field of inhibitors of protein−protein interactions, as MCR-derived cyclic peptides can be designed, at will, for interacting with the protein surfaces not only by the peptide skeletons but also by the external moiety incorporated during the multicomponent ring closure. In the future development of cyclic peptide drugs, MCR may also play a key role due to the possibility of adding exo-cyclic fragments that help improve the pharmacological properties or serve for labeling or conjugation purposes. For example, methods based on the multicomponent stapling concept have been shown in which side-chain bridges can be readily functionalized to modulate properties such as membrane permeability (lipidation) and metabolic stability (PEGylation). The potential of these processes also rests on their simplicity to produce complex macromulticyclic architectures, a key issue for future applications in the T


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ABBREVIATIONS Alloc allyloxycarbonyl All allyl CuAAC CuI-catalyzed alkyne−azide cycloaddition 3CR three-component reaction 2CT 2-chlorotrityl 4CR four-component reaction IPB 4-isocyanopermethylbutane-1,1,3-triol MCRs multicomponent reactions MiBs multiple multicomponent macrocyclization including bifunctional building blocks NTy 3-nitrotyrosine PEG polyethylene glycol Pinc (N-isocyanimino)triphenylphosphorane RCM ring-closing metathesis SPPS solid-phase peptide synthesis

combinatorial production and screening of, e.g., protein ligands or macrocyclic peptide hosts. We certainly believe that the multicomponent macrocyclization approach offers one of the fastest ways to explore the chemical and topological space of macrocyclic peptide scaffolds, thus showing promise not only in drug discovery but also in molecular/ion pair recognition and biomimetic catalysis.

AUTHOR INFORMATION Corresponding Author

*Tel. +53 78792331; E-mail: [email protected]. ORCID

Daniel G. Rivera: 0000-0002-5538-1555 Notes

The authors declare no competing financial interest.

REFERENCES

Biographies

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Leslie Reguera graduated in Chemistry with honors at the University of Havana in 2002. She conducted her doctoral work at both the Universidad Nacional Autónoma de México and the University of Havana, where she did the Ph.D defense in 2009 with the award of the “Best Ph.D Thesis of the Year”. She has been a Visiting Scientist at the National Polytechnic Institute of Mexico and at Leipzig University, Germany. Currently, she is Associate Professor at the University of Havana. Her research interests focus on synthetic methods and the development of coordination polymers for catalysis and gas adsorption. Daniel G. Rivera graduated in Chemistry with honors at the University of Havana in 2002 and completed his Master Thesis in Organic Chemistry in the same year. He earned a Ph.D (summa cum laude) in 2007 working on the development of multicomponent macrocyclization strategies in the group of Prof. L. A. Wessjohann, Leibniz Institute of Plant Biochemistry (IPB), Germany. After return to his Alma Mater in 2008, he was appointed Research Professor, Head of Bioorganic Chemistry and, in 2015, Director of the Center for Natural Products Research. His research interests focus on the synthesis of natural products, multicomponent and organocatalytic reactions, and the development of novel macrocyclization and bioconjugation strategies. He is a Fellow of the Alexander von Humboldt Foundation and a Member of the Cuban Academy of Science. He served as President of the Cuban Society of Chemistry from 2016−2018 and currently is the President of the Latin American Federation of Chemical Associations (FLAQ).

ACKNOWLEDGMENTS We are sincerely grateful to all coworkers contributing to this field, especially to Aldrin V. Vasco, Manuel G. Ricardo, Micjel C. Morejón, and Alfredo R. Puentes, who shaped the results our group herein presented. We also thank Professors Ludger A. Wessjohann, Bernhard Westermann, and Marcio Weber Paixão for their continuous and invaluable support to our group. D.G.R. acknowledges support from the Alexander von Humboldt Foundation for an Experienced Researcher fellowship. DEDICATION Dedicated to Prof. Ludger A. Wessjohann, a pioneer of multicomponent macrocyclizations U


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