Native Chemical Ligation and Extended Methods: Mechanisms

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

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Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations Vangelis Agouridas,† Ouafâa El Mahdi,§ Vincent Diemer,† Marine Cargoeẗ ,† Jean-Christophe M. Monbaliu,*,‡ and Oleg Melnyk*,† †

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UMR CNRS 8204, Centre d’Immunité et d’Infection de Lille, University of Lille, CNRS, Institut Pasteur de Lille, F-59000 Lille, France ‡ Center for Integrated Technology and Organic Synthesis, Department of Chemistry, University of Liège, Building B6a, Room 3/16a, Sart-Tilman, B-4000 Liège, Belgium § Faculté Polydisciplinaire de Taza, University Sidi Mohamed Ben Abdellah, BP 1223 Taza Gare, Morocco ABSTRACT: The native chemical ligation reaction (NCL) involves reacting a C-terminal peptide thioester with an N-terminal cysteinyl peptide to produce a native peptide bond between the two fragments. This reaction has considerably extended the size of polypeptides and proteins that can be produced by total synthesis and has also numerous applications in bioconjugation, polymer synthesis, material science, and micro- and nanotechnology research. The aim of the present review is to provide a thorough mechanistic overview of NCL and extended methods. The most relevant properties of peptide thioesters, Cys peptides, and common solvents, reagents, additives, and catalysts used for these ligations are presented. Mechanisms, selectivity and reactivity are, whenever possible, discussed through the insights of computational and physical chemistry studies. The inherent limitations of NCL are discussed with insights from the mechanistic standpoint. This review also presents a palette of O,S-, N,S-, or N,Se-acyl shift systems as thioester or selenoester surrogates and discusses the special molecular features that govern reactivity in each case. Finally, the various thiol-based auxiliaries and thiol or selenol amino acid surrogates that have been developed so far are discussed with a special focus on the mechanism of long-range N,S-acyl migrations and selective dechalcogenation reactions.

CONTENTS 1. Introduction 1.1. Chemoselective Amide Bond Forming Reactions and Protein Chemical Synthesis 1.2. The NCL Reaction: From Discovery to Modern Protein Chemical Synthesis and Chemical Biology 1.3. Scope and Organization of the Review 2. Peptide Thioesters and Peptide Selenoesters: General Properties and Synthesis 2.1. General Properties of Thioesters 2.1.1. General Structural Aspects of Thioesters 2.1.2. Reactivity of Thioesters toward Nucleophile Addition: General Trends 2.2. Chemical Synthesis of Peptide Thioesters 2.2.1. Classification of Peptide Thioester Production Methods 2.2.2. Synthesis of Peptide Thioesters: Category I 2.2.3. Synthesis of Peptide Thioesters: Category II 2.3. Latent Thioester Surrogates 2.3.1. Latent Thioesters of Type I 2.3.2. Latent Thioesters of Type II

© XXXX American Chemical Society

2.4. Peptide Selenoesters: General Properties 2.4.1. Reactivity toward Oxygen and Amine Nucleophiles 2.5. Chemical Synthesis of Peptide Selenoesters 2.5.1. Synthesis of Peptide Selenoesters. Category I 2.5.2. Synthesis of Peptide Selenoesters: Category II 3. Properties of Cysteine or Selenocysteine Residues 3.1. Cysteine 3.1.1. Nucleophilicity and Acid−Base Properties of the Cysteine Residue 3.1.2. Controlling the Redox State of Cysteine during NCL 3.1.3. Controlling the Reactivity of Cysteine by the Use of Protecting Groups 3.2. Selenocysteine 3.2.1. Acid−Base Properties and Nucleophilicity of Selenocysteine 3.2.2. Controlling the Redox State of Selenocysteine during NCL

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Received: November 23, 2018

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DOI: 10.1021/acs.chemrev.8b00712 Chem. Rev. XXXX, XXX, XXX−XXX

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3.2.3. Controlling the Reactivity of Selenocysteine by the Use of Protecting Groups Dissecting the Individual Chemical Steps in NCL 4.1. Origin of the Regio- and Chemoselectivity of NCL 4.2. The Thiol−Thioester Exchange Reaction 4.3. Nucleophilic Catalysis 4.3.1. Catalysis by Aryl Thiols 4.3.2. Catalysis by Alkyl Thiols 4.3.3. Catalysis by Internal Cysteine Thiols 4.3.4. Non-Thiol-Based Catalysts 4.3.5. Nucleophilic Catalysis of Peptide Selenoester-Based NCL 4.4. Formation of the Thioester-Linked Intermediate 4.4.1. The Capture of Peptide Aryl Thioesters 4.4.2. The Capture of Peptide Aryl Thioesters: The Special Case of Kinetically Controlled Ligations 4.4.3. Folding or Template-Assisted Ligations 4.4.4. Intramolecular Ligations 4.5. Rearrangement of the Thioester-Linked Intermediate 4.5.1. Experimental Studies 4.5.2. Computational Studies 4.6. NCL at Selenocysteine 4.6.1. Reversibility of the N,Se-Acyl Shift Ligations Utilizing N,S-, N,Se-, and O,S-Acyl Shift Systems 5.1. General Presentation of N,S-, O,S-, and N,SeAcyl Shift Systems 5.2. N,S-Acyl Shift Systems 5.2.1. Mechanisms 5.2.2. N,S-Acyl Shift Systems: Application to Peptide Transamidation and Metathesis 5.3. N,Se-Acyl Shift Systems 5.4. O,S-Acyl Shift Systems 5.4.1. Mechanistic Aspects of the O,S-Acyl Migration Reactions 5.5. Latent Thio- or Selenoesters Based on N,S(Se)-Acyl Shift Systems 5.5.1. Latent Thioester Surrogates Based on N,S-Acyl Shift Systems 5.5.2. Latent Selenoester Surrogates Based on N,Se-Acyl Shift Systems Role of the C-Terminal Residue on the Reactivity of C-Terminal Peptide Thioesters and the Occurrence of Side Reactions 6.1. C-Terminal Residues Leading to Poor Reaction Rates 6.1.1. β-Branched Amino Acids 6.1.2. Peptidyl Prolyl Thioesters 6.2. C-Terminal Residues Leading to Side Reactions 6.2.1. Peptidyl Asparaginyl Thioesters 6.2.2. Peptidyl Aspartyl or Glutamyl Thioesters 6.2.3. Peptidyl Prolyl Thioesters 6.2.4. Peptidyl Lysyl Thioesters NCL beyond Cys and Sec Junctions 7.1. Short Range Acyl Transfers: Auxiliary-Mediated NCL 7.1.1. Thiol Auxiliaries Based on the 2-Mercaptobenzyl Scaffold

Review

7.1.2. Thiol Auxiliaries Based on the 2-Mercaptoethyl Scaffold 7.2. Short Range Acyl Transfers: Chalcogeno Amino Acid Surrogates Combined with Dechalcogenation Techniques 7.2.1. Metal-Free Desulfurization 7.2.2. Metal-Free Deselenization 7.2.3. Ligation/Dechalcogenation Approaches 7.2.4. Chemical Treatments Other than Dechalcogenation 7.3. Long-Range Acyl Transfers 7.3.1. Long Range S,N-Acyl Transfer Assisted by Internal Cysteines 7.3.2. Side-Chain Assisted Ligations 8. The Role of Phosphate Buffer, Denaturants, Detergents, and Organic Solvents in the NCL Reaction 8.1. Role of Phosphate Buffer 8.2. Role of Denaturants 8.2.1. Guanidine Hydrochloride 8.2.2. Urea 8.3. Detergents for Improving the Solubility of Hydrophobic Peptide Segments and Proteins 8.4. Organic Solvents 9. NCL Mechanistic Overview and Perspectives 10. Conclusion Author Information Corresponding Authors ORCID Notes Biographies Acknowledgments Abbreviations References

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1. INTRODUCTION BF 1.1. Chemoselective Amide Bond Forming Reactions and Protein Chemical Synthesis

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The amide bond has been stimulating the creativity of organic chemists since the inception of modern organic chemistry, particularly due to the high occurrence of the amide group in naturally occurring organic molecules. The simplest amides, i.e., formamide and acetamide, were recently detected by mass spectrometry on comet 67P/Churyumov−Gerasimenko by Rosetta’s Philae lander.1 Present in low molecular weight natural compounds such as insect sex pheromones, e.g., N-(2methylbutyl)acetamide, lipids, e.g., N-acyl ethanolamines, and oligo- or polysaccharides, e.g., chitin, the amide group also connects the amino acids in proteins. Since the chemical synthesis of the first dipeptides by Curtius2 and by Fischer,3 the development of amide bond forming reactions has attracted considerable attention. In his seminal paper published in 1901, Fischer3 reported the synthesis of glycylglycine (GlyGly) from glycine ethyl ester as well as the preparation of some protected derivatives of this dipeptide, thereby paving the way toward the synthesis of larger peptides. The need to efficiently concatenate amino acids or peptide fragments has logically prompted peptide chemists to design a large variety of efficient amide coupling methods and orthogonal protecting group strategies, some of which are now considered as gold standards in

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Figure 1. (A) Principle of peptide bond formation based on tandem chemoselective capture and intramolecular rearrangement. The methods are classified according to the position of the functional group(s) involved in the chemoselective capture step within the amine component: type I, side chain; type II, α-nitrogen; type III, α-nitrogen and side-chain. Unless otherwise noted, side-chain functional groups will no longer be represented on peptide blocks throughout the document. (B) Type I: NCL,8 DSL (not shown).9 (C) Type II: KAHA,10 traceless Staudinger ligation.11,12 (D) Type III: pseudoproline ligation (not shown),13 serine/threonine ligation (STL).14

methods in three distinct types depending on the position of the functional group(s) involved in the capture step within the amine component. The chemical ligation methods included in type I involve the participation of the side chain in the capture step (Figure 1B), while the methods belonging to type II involve the reactivity of the α-nitrogen (Figure 1C). Type III methods involve both the α-nitrogen and the side chain in the capture step (Figure 1D). The subject of this review, i.e., the native chemical ligation (NCL), is a type I ligation (Figure 1B).8,15 Introduced by Kent and co-workers in 1994, this reaction involves the coupling of a C-terminal peptide thioester with an N-terminal cysteinyl peptide. For NCL, the chemoselective capture step is a thiol− thioester exchange reaction involving the thioester and the cysteine (Cys) thiol functionalities. The transient thioester-

synthetic organic chemistry. The solid-phase peptide synthesis method4,5 and the coupling of protected peptide fragments and combinations thereof6,7 have considerably extended the size of polypeptides accessible through chemical synthesis. However, the need to overcome the handling of protected peptides, which are notoriously difficult to solubilize in organic solvents, as well as the quest for ever-increasing protein complexity amenable to chemical synthesis have led to the emergence of new synthetic strategies based on a unique chemistry carried out in water with unprotected peptide segments. The key to these new water-compatible chemistries is the idea that a peptide bond can be formed by an intramolecular rearrangement subsequent to a chemoselective capture step (Figure 1A). Such strategies are named “peptide ligation methods”, and we propose here a classification of these C


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Scheme 1. Principle of the Diselenide Selenoester Peptide Ligation (DSL)

Figure 2. (A) Number of proteins prepared by chemical synthesis per year over the period 1994−2018 (moving average of years n, n − 1, and n − 2). (B) Frequency of use of the different chemoselective peptide ligation methods for protein synthesis (% of junctions formed between 2011 and 2018). The data were extracted from the Protein Chemical Synthesis (PCS) database (http://pcs-db.fr).18 Note that the PCS database includes synthetic peptides and proteins of biological relevance only and does not consider ligation reactions used for producing model peptides, polymers, or hybrid materials.

Serine/threonine ligation (STL) is a type III peptide ligation method which was introduced in 2013 (Figure 1D).14 STL is reminiscent of the pseudoproline ligation method developed by the group of Tam in the 1990s.13,16,17 It proceeds through the chemoselective formation of an oxazolidine intermediate by the reaction of an aldehyde with the β-amino alcohol functionality of an N-terminal serine or threonine. Acyl migration and hydrolysis from the oxazolidine intermediate yields the target peptide with a native peptide bond to serine or threonine. A recent survey of the literature has brought to light the net increase in the number of proteins prepared by chemical synthesis since 2010 and therefore the significance of the field (Figure 2A).18 It is noteworthy that a large majority of the junctions considered for assembling these proteins in the 2011−2018 period were produced using NCL or extended methods, which are the main focus of this review (Figure 2B).

linked intermediate generated rearranges spontaneously by intramolecular S,N-acyl transfer to give a peptide with a native peptide bond to cysteine. The diselenide selenoester ligation (DSL,9 Scheme 1) that was introduced in 2015 by Mitchell et al. is classified as a type I ligation, although the exact mechanism of this reaction has not yet been elucidated. In this reaction, peptide selenophenyl esters react with peptide diselenides in the absence of thiol or selenol additives to produce a peptide bond to selenocysteine (Sec). The initial report proposed a redox associative mechanism allowing selenoesters to rapidly combine with diselenides.9 However, this mechanism has been challenged by recent experimental studies from the group of Payne that should shed light on the intimate processes occurring in this important reaction. In addition to NCL, a few mechanistically unrelated peptide ligation methods have also been developed. The traceless Staudinger ligation was reported in 2000 and is a ligation of type II (Figure 1C).11,12 In this case, the capture step relies on the chemoselective reaction of the phosphine component with the azide segment to produce an iminophosphorane intermediate. The later evolves spontaneously into a native peptide by acyl migration followed by hydrolysis. The ketoacid−hydroxylamine ligation (KAHA), also type II, was introduced in 2006.10 The C-terminal ketoacid group and the N-terminal hydroxylamine functionalities combine chemoselectively to produce a nitrone intermediate. The latter undergoes a rearrangement/decarboxylation process which results in the formation of a native peptide bond.

1.2. The NCL Reaction: From Discovery to Modern Protein Chemical Synthesis and Chemical Biology

The discovery of NCL followed more than 50 years of intense efforts worldwide to investigate the role of thioesters in biosynthesis and their interest in synthetic organic chemistry. One important step that shed light on the key role of thioester chemistry in living organisms was the discovery in 1951 of the thioester structure of S-acetyl coenzyme A by Lynen and Reichert,19 who were involved in the study of lipid biosynthesis.20 The fact that a thioester was used by cells as an energy-rich intermediate challenged conventions and stimulated considerable efforts to understand the structural and chemical properties of thioesters and by extension of selenoesters. In particular, the fact that cells use S-acetyl D


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coenzyme A as an acyl donor prompted Wieland to investigate the formation of peptide bonds in biomimetic conditions using the unique reactivity of α-amino acid thiophenyl esters.21−23 Such amino acid thioesters allow peptide couplings to be carried out using very mild conditions. But most importantly, Wieland realized that the reaction in aqueous solution at pH > 7 was particularly efficient with cysteine (Cys, C) in comparison to other amino acid residues such as glycine or alanine (Scheme 2).24 He attributed the excellent reactivity of

Although NCL was initially developed for accessing large polypeptides without resorting to protected and often poorly soluble peptide segments, it is also attractive for producing amides with a higher atom economy37 compared to classical methods relying on the enthalpic activation of carboxylic acids.38 Today, the conception of chemoselective, watercompatible amide bond forming reactions still constitutes one of the most challenging goals in modern synthetic organic chemistry.39−41 Since 1994, NCL has found wide synthetic applications in chemical biology, medicinal chemistry, and material science. More specifically, the synergistic combination of NCL with SPPS has largely contributed to orienting its utilization toward the chemical synthesis of peptides and proteins (Figure 3).42,43 According to the PCS database, over 700 proteins of biological relevance were synthesized using NCL and extended methods.18 Continuous and significant advances in the field have recently culminated with the report of synthetic objects of exceptional size such as fully functional synthetic analogues of bacterial polymerases (∼350 amino acids).44,45 Beyond their size, the diversity of synthetic targets also reflects the versatility of chemical approaches based on NCL for accessing proteins that can be produced by living systems only with great difficulty. Among the latter, cyclic peptides have been the subject of numerous synthetic studies.46−56 For example, Li et al. conducted the full sequence amino acid scanning of θdefensin RTD-1 by building a library of more than 100 variants with the routine application of cyclative NCL.57 In the same way, the synthesis of branched architectures such as (poly)ubiquitinated58−64 and sumoylated65−67 chains can be facilitated by ligation approaches that provide full control over the number of units connected and their attachment site. Of course, other post-translational modifications are also accessible and well documented in the literature. Just to mention a few representative examples, glycosylated,68−71 phosphorylated,72,73 acetylated,72,74 methylated,75 or sulfated76,77 proteins have been regularly synthesized using NCL over the past two decades. More recent work has resulted in the emergence of applications

Scheme 2. Wieland et al.26 Synthesis of Val-Cys Dipeptide by Reaction of Valine Phenyl Thioester with Cysteine in Water

Cys to the rapid formation of a thioester intermediate by thiol− thioester exchange, followed by an intramolecular S-to-N transfer of the aminoacyl group. This discovery followed previous findings by the same laboratory on the synthesis and rearrangement of S-acetyl cysteamine to N-acetyl cysteamine.25 The major steps that punctuated this seminal work were discussed by Wieland in a review published in 1995.26 Following the pioneering works of Wieland 24 and Brenner27,28 on the formation of a peptide bond by proximity-induced intramolecular acyl transfer, groundbreaking works by the groups of Kemp,29−31 Kent,32 and Tam13,33 has further developed the selective capture/acyl transfer principle and established the core principles that are involved in NCL8 and all the chemoselective peptide ligation methods that emerged afterward.9−14,34−36

Figure 3. Synthetic applications of NCL and extended methods. E


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Figure 4. Scope of the review.

protein thioesters with cysteine-containing phospholipids.91 In the same way, peptide−oligonucleotide (DNA92 or tRNA mimics93) conjugates have been obtained. Thioester-based ligation reactions have also proved to be a valuable tool for the incorporation of fluorescent probes in proteins.94,95 Recently, Reinhardt et al. even designed a method for the rapid and selective labeling of membrane receptors in living cells, making use of the effectiveness of templated ligations.96 In another application, NCL enabled the selective modification of complex samples for the identification of N-terminally homocysteinylated peptides by proteomic approaches.97 It is also worth mentioning that NCL has been used to produce complex lipids and phospholipid bilayers.98−100 A recent application of this concept to the reconstitution of G protein-coupled receptors (GPCR) by in situ production of the proteoliposomes was described by Brea et al.101 Finally, the NCL reaction has been extensively applied in macromolecular chemistry for the preparation of high molecular weight peptide-based biopolymers,102 hydro-

involving the preparation of D -proteins (i.e., proteins synthesized entirely from D-amino acids) such as racemic protein crystallography78−82 or the identification of mirrorimage protein therapeutics.81,83,84 Because D-proteins cannot be produced by living systems, this is a purely synthetic application which best illustrates the usefulness of ligation-based approaches (for a recent review, see ref 85).63,86−89 Note also recent efforts to produce mirror-image DNA molecules, i.e., LDNA, by primer extension of a template L-DNA catalyzed by a fully synthetic D-polymerase.90 Beyond the total chemical synthesis of peptides and proteins, the NCL reaction has largely transcended its initial scope of application by enabling straightforward access to conjugates and biomolecules. The attractiveness of NCL as a synthetic method in fact not only lies in its ability to proceed in mild conditions compatible with a wide range of substrates but also stems from a high chemoselectivity allowing for specific (bio)conjugation. These properties were used early on to produce membrane-anchored proteins by reacting recombinant F


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gels,103,104 multivalent peptide-based dendrimers,105 or crosslinked micelles.106 With the development of nanotechnologies and surface chemistries, NCL reactions have found a new scope of application. More particularly, they have been extensively used for surface functionalization107 or polypeptide immobilization.108−111

examined in this section among other important aspects. Several experimental studies shed light on the individual steps involved in NCL and are nicely complemented by physical organic studies and recent computational works. The latter enable a thorough understanding of the parameters that govern reactivity and reaction pathways. The rise in computational resources, as well as the development of new computational strategies, provided chemists with powerful means to study specific molecular aspects and reaction pathways that would otherwise have remained inaccessible. The purpose of this review is to critically examine the results of these studies in relation to experimental results and to emphasize, illustrate, or explain specific aspects of the fundamental rules that govern the reactivity of thioesters and selenoesters, as well as the other chemical processes involved in NCL. Section 5 follows by presenting the mechanisms involved in the ligations relying on N,S-, N,Se-, and O,S-acyl shift systems. The field has recently witnessed the emergence of these novel thioester surrogates that have significantly enriched the toolbox of protein chemists. Figure 5 highlights the importance of these

1.3. Scope and Organization of the Review

The scope and organization of the review is represented schematically in Figure 4. The review aims to provide a comprehensive approach to NCL and extended methods by dissecting each fundamental step. We followed a dual approach, relying on both experimental and theoretical (computational) studies to provide a complete set of tools to rationalize mechanism and selectivity (sections 4 to 7). This is enriched by sections discussing in more detail specific aspects regarding peptide thio- and selenoesters (section 2), Cys peptides (section 3), and the common solvents/denaturants used for NCL (section 8). The last section, section 9, gives a prospective view of the field of protein chemical synthesis. Following this introduction, the review continues with section 2 presenting the structural properties of thioesters and selenoesters and their reactivities in general terms. In particular, the hydrolysis and aminolysis of thioesters and selenoesters is discussed in this section because they are paramount to NCL. Section 2 continues with the chemical methods developed for the synthesis of peptide thioesters and selenoesters. While Cys peptides are easily prepared by conventional solid-phase peptide synthesis (SPPS4), early developments of the NCL reaction were complicated by the paucity of chemical methods allowing the production of peptide thioesters. The 9fluorenylmethyloxycarbonyl (Fmoc) amine protecting group 112,113 and the Fmoc SPPS114−116 method were introduced in the 1970s, several years after the introduction of the tert-butyloxycarbonyl (Boc) SPPS method by Merrifield4 to overcome the strong acidic conditions used for removal of the Boc group and for the final peptide deprotection and cleavage steps.117−120 Nevertheless, initial SPPS strategies for peptide thioester synthesis relied primarily on Boc SPPS due to the sensitivity of the thioester functionality to the basic conditions used for removing the Fmoc group. The safer conditions required for performing peptide syntheses using Fmoc SPPS, however, led to the adoption of this method by a large majority of peptide chemists. Logically, this trend stimulated the development of a rich diversity of Fmoc SPPS strategies for peptide thioester synthesis, which merit special attention in this review. This section also includes the synthesis of peptide thioesters from O,S- and N,S-acyl shift systems and discusses the latent thioester surrogates that have been developed so far. The review goes on with section 3, which presents the properties of Cys and Sec that are important to consider in the context of the NCL reaction. This section also introduces the protecting groups which are used for the temporary masking of N-terminal Cys or Sec residues during protein synthesis using NCL. These Cys/Sec protection strategies facilitate the creation of ambitious assembly schemes targeting challenging protein targets.121 Section 4 discusses in depth the different steps involved in the NCL reaction. In particular, the thiol−thioester exchange reaction, the nucleophilic catalysis of the NCL reaction and the rearrangement of the thioester-linked intermediate are

Figure 5. Relative importance of the different acyl donors used for protein chemical synthesis (NCL, NCL-extended methods, and DSL in the period 2011−2018). The data were extracted from the Protein Chemical Synthesis (PCS) database (http://pcs-db.fr).18

systems for protein chemical synthesis relative to other types of acyl donors used in the field. These chemical systems can replace the peptide thioester in the NCL reaction. Importantly, some of these chemical systems behave as latent thioesters that can be activated on demand, therefore facilitating the synthesis of complex protein targets. The mechanisms involved in these reactions differ significantly from those intervening in the NCL with classical peptide thioesters. For this reason, they are discussed in a separate section by combining both experimental and mechanistical approaches. Section 6 details the scope and limitations of the NCL reaction and extended methods. The facilitated access to peptide thioesters and other acyl donors contributed to the clarification of the relationship between the C-terminal residue of the thioester component and the rates of these reactions or G


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identical.139 Logically, C(O)−S and the S−CH3 bond lengths in S-methyl thioacetate140 are similar while the C(O)−N and C(O)−O bonds in the corresponding amide141 and oxoester140 are shorter than the N−CH3 and O−CH3 bonds, respectively (Table 1). Significantly shorter C(O)−X bond lengths in

the occurrence of side reactions. This knowledge is of prime importance for designing protein synthetic schemes optimally. Section 7 shows how protein chemists overcome the low occurrence of Cys in proteins, their placement in nonstrategic positions, or even their absence in the target proteins. The frequency of Cys in proteins is low in comparison to other amino acid residues such as Gly or Ala. This fact stimulated the development of methods enabling the ligation to noncysteine amino acids which include auxiliary-mediated NCL,122 side chain assisted ligations,123 and NCL/dechalcogenation approaches.124,125 These chemical tools, which today are essential pillars of modern protein chemical synthesis, are thoroughly discussed in this section. The review continues with section 8, which discusses the role of phosphate buffer and of the most popular denaturants, detergents, and organic cosolvents used for running NCL. Finally, section 9 serves as an executive summary of the review and gives a prospective view of the field of protein chemical synthesis before concluding remarks (section 10).

Table 1. C(O)−X and X−CH3 Bond Lengths in N-Methyl Acetamide (X = NH), Methyl Acetate (X = O), and Methyl Thioacetate (X = S) (ΔX−C Represents the Difference between X−CH3 and C(O)−X Bond Lengths)

X a

NH (amide) O (ester)b S (thioester)b a

2. PEPTIDE THIOESTERS AND PEPTIDE SELENOESTERS: GENERAL PROPERTIES AND SYNTHESIS The thioester functional group is the cornerstone of important bioprocesses such as lipid biosynthesis,19 protein splicing,126 ubiquitin or ubiquitin-like protein conjugation,127 or proteolytic degradation by cysteine proteases.128 In these processes, the thioester carbonyl group reacts with a variety of nucleophiles, explaining why nucleophilic additions to thioesters has been the subject of so many experimental129−133 and computational studies134,135 in the past even before the introduction of the NCL reaction. For this reason, it is important to recapitulate the main structural characteristics of thioesters and the basics of their reactivity (section 2.1) before reviewing the various synthetic methods enabling the production of peptide thioesters (section 2.2). Not surprisingly, the section dedicated to the mechanism of the NCL reaction (section 4) will come back to some general concepts highlighted in section 2.1. Following the same approach, the properties of peptide selenoesters as well as their synthesis are presented in section 2.4.

C(O)−X

X−CH3

ΔX−C

1.386 1.360 1.781

1.469 1.442 1.805

0.083 0.082 0.024

Data taken from ref 141. bData taken from ref 140.

oxoesters and amides are evocative of orbital interactions of the X ns and np lone pairs with the antibonding σ* and π* molecular orbitals of the carbonyl group, leading to a significant stabilization through resonance. Typically, the resonance structure of type II (Figure 6) is responsible for the pronounced double bond character of the peptide bond and for its planar structure.142 In contrast, such a resonance effect is much weaker in thioesters for which mesomeric structure II only sparingly contributes to the ground state structure. The poor contribution of sulfur to electron delocalization relative to nitrogen or oxygen results in higher rotational freedom around the C(O)−S bond and an increase in the electrophilic character of the carbonyl carbon in thioesters.139 2.1.1.2. Conformation of Thioesters. The preference of acyclic thioesters to adopt a syn conformation (Figure 7A) has been clearly established experimentally, as shown by examination of the X-ray crystal structure of several low molecular weight thioesters. For example, S-benzyl ferrocenecarbothioate143 exhibits one conformation in the crystal which corresponds to a syn-periplanar orientation of the S−CH2Ph bond relative to the CO double bond (Figure 7B). The predominance of the syn-planar conformer has also been observed in the gas phase by gas electron diffraction (GED) of dimethyl monothiocarbonate144 and S-methyl thioacetate.140 The thioester bond is present in a few natural proteins and is usually found in the syn conformation too.145−148 For example, the Spy0125 protein features a solvent-exposed thioester between the side chain thiol of Cys426 and the side chain carbonyl group of a Glu575 (Figure 7C).148 It is believed to play a pivotal role in the adhesion of the bacteria to the host cells by forming covalent bonds with exposed nucleophiles. The above-mentioned experimental observations are in line with ab initio calculations which predict that acyclic alkyl thioesters should prefer the syn-planar conformer.149,150 For example, gas phase quantum chemical calculations (B3LYP/631G* and MP2/6-31G*) conducted on S-methyl thioacetate revealed the higher stability of the syn conformer (ΔE ∼ 5 kcal.mol−1) and estimated the internal rotation barrier linking the syn and the anti conformers as 12 kcal·mol−1.149 The factors involved in the conformational discrimination between syn and anti conformers could be identified by natural bond orbital (NBO) analysis of thioesters.135,140,144,151,152 These studies revealed specific stereoelectronic effects respon-

2.1. General Properties of Thioesters

2.1.1. General Structural Aspects of Thioesters. 2.1.1.1. Electronic Structure of Thioesters. Thioesters, oxoesters, and amides are carboxylic acid derivatives that differ by the type of heteroatom X linked to the carbonyl group (X = S, O, NH, respectively, in Figure 6). How electrons from the X heteroatom delocalize into the carbonyl C(O) moiety has a profound impact on the structure and reactivity of these compounds. Vibrational analyses have shown that the C(O)−X force constant is larger than the X−R 2 force constant in oxoesters136,137 and amides.138 In contrast, the force constants for the C(O)−S and the S−R2 bonds in thioesters are almost

Figure 6. Resonance structures for amides (X = NH), oxoesters (X = O), and thioesters (X = S). H


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Figure 7. (A) The syn (left) and anti (right) conformers of an acyclic thioester. The syn conformer is characterized by a syn-periplanar orientation of the CO and the SR2 fragments, while the anti conformer is characterized by an anti-periplanar orientation of the CO and the SR2 fragments. (B) X-ray crystal structure showing the preferential syn conformation of S-benzyl ferrocenecarbothioate in the crystal (CCDC entry 722639).143 (C) Xray crystal structure of Spy0125 protein (PDB 2XI9148). This protein contains an internal thioester bond which involves the side chain thiol of Cys426 and the side chain carbonyl group of Glu575.

addition to these stereoelectronic effects, steric hindrance further destabilizes the anti form. Regarding cyclic thioesters, i.e., thiolactones, small and medium-sized thiolactones (4 < n < 7), such as β-propiolactone (n = 4), 153 γ-butyrothiolactone (n = 5), 154 and δvalerothiolactone (n = 6)155 are constrained in the less favorable anti conformation (Figure 9). Computational analysis suggests that this is also the case for ε-caprothiolactone (n = 7). In contrast, larger cycles such as the dimers of εcaprothiolactone (n = 14) and ω-hexadecathiolactone (n = 34) have sufficient backbone flexibility to enable a syn conformation for the (CO)-S fragment. Thiodepsipeptides, a family of peptides characterized by a thiolactone structure essential for full biological activity156 (for a review see ref 157), were also shown to preferentially adopt a syn conformation. An example is given in Figure 9 with the X-ray crystal structure of a thiodepsipeptide AIP-4 analogue. Thus, once thiolactones are sufficiently large and unstrained to enable both types of conformations, these structures preferentially adopt the syn conformation as in low molecular weight acyclic thioesters.158−160 2.1.2. Reactivity of Thioesters toward Nucleophile Addition: General Trends. The thermodynamic stability of thioesters installs them at the center of a reactivity scale toward nucleophile addition, somewhere above unreactive amides and poorly reactive oxoesters but below overactivated carboxylic acid anhydrides and acyl chlorides. From a synthetic utility perspective, such an intermediate reactivity confers on them the capacity to transfer their acyl group under relatively mild conditions and on an acceptable time scale, two important features which are exploited to the full in NCL reactions.

sible for anomeric interactions. Interaction between the s-type lone pair, i.e., ns, located on the sulfur atom with an antiperiplanar antibonding molecular orbital, such as the σ*C−O orbital for the syn conformer or the σ*C−C orbital for the anti conformer, are highly comparable and discriminate both conformers only slightly (Figure 8A).140 In contrast, the interaction between the p-type lone pair np and the adjacent antibonding π*CO molecular orbital clearly favors the syn conformer over the anti by about 3 kcal·mol−1 (Figure 8B). In

Figure 8. Schematic view of (A) the orbital interaction between the ns lone pair of sulfur and the σ*C−O in the syn conformer (left) or the σ*C−C in the anti conformer (right) of S-methyl thioacetate. (B) The orbital interaction between the np lone pair of sulfur and the π*CO of S-methyl thioacetate. The conjugation energies are taken from ref 140. I


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reflects the greater susceptibility of peptide aryl thioesters to hydrolysis during synthesis and during NCL as compared to peptide alkyl thioesters. To conclude at this point, thioesters are significantly more reactive than oxoesters toward thiol or nitrogen nucleophiles while being relatively resistant to hydrolysis at neutral pH. These features contribute collectively to make NCL a powerful reaction in water, in addition to other aspects that will be discussed in section 4. The rate of the acid-catalyzed hydrolysis of thioesters is low, so that they can be manipulated in acidic aqueous media without major problems. For example, peptide thioesters show excellent stability during reversed-phase HPLC at pH ∼ 2, including the subsequent lyophilization step. In contrast, the pronounced reactivity of thioesters toward nitrogen-based nucleophiles has been seen as a serious constraint for designing synthetic approaches toward peptide thioesters. The special reactivity of thioesters and the challenge imposed by their synthesis using SPPS approaches have stimulated the creativity of peptide chemists for two decades. The next section intends to give a flavor of this work. 2.2. Chemical Synthesis of Peptide Thioesters

The production of peptide thioesters has been extensively reviewed in the past, and some seminal reviews by leading experts in the field can be recommended.162−167 Besides chemical methods, peptide or protein thioesters can be produced by biological means. In particular, four years after the introduction of the NCL reaction, the extension of this ligation method to the use of peptide or protein thioesters produced by intein technology was described independently by the groups of Muir162,168 and Xu.169 This method, named expressed protein ligation (EPL), considerably extended the size and type of proteins amenable to semisynthesis. The combination of peptide segments of biological and synthetic origin is a particularly powerful tool for accessing proteins featuring well-defined post-translational modifications (PTMs) at specific sites and therefore has been extensively used to study the role of PTMs in protein function.170 The work carried out for deciphering the chromatin code is particularly illustrative of the precision with which the complexity of such regulation mechanisms can be addressed with these techniques.58,171,172 The production of peptide thioesters by intein technology or other biological methods is not within the scope of this review, and we recommend recent reviews written by leaders in the field.162,166,173 The aim of this section is rather to present the main concepts underpinning the strategies developed for the chemical synthesis of peptide thio- and selenoesters. Although not exhaustive, this section gives a large overview of the field and emphasizes the most important methods available so far for accessing peptide thio- and selenoesters. 2.2.1. Classification of Peptide Thioester Production Methods. Thioesters are sensitive to a variety of oxygen, nitrogen, or sulfur nucleophiles. Typically, and besides some exceptions, thioesters do not survive the repetitive piperidine treatments used for Fmoc SPPS. In contrast, thioesters are particularly resistant to strong acids such as TFA or HF. These

Figure 9. Syn/anti conformation of thiolactone as a function of ring size (X-ray crystal structure of an AIP-4 peptide analogue extracted from PDB 3QG6158).

Another property of thioesters that has been shown in many studies is their relatively good stability toward hydrolysis. Often seen as “activated esters”, thioesters are effectively many orders of magnitude more reactive toward amines and thiolates than oxoesters.130,132,161 However, several experimental studies have reported that thioesters are as resistant as oxoesters to hydrolysis,129,130,132 a fact that is also predicted by computational analysis.135 Regarding the hydrolysis of thioesters, Bracher et al. decomposed the apparent rate constant kobs measured for the hydrolysis of S-methyl thioacetate (entry 1, Table 2) and Sphenyl 5-dimethylamino-5-oxo-thiopentanoate (entry 2, Table 2), two relevant model compounds for C-terminal peptide alkyl or aryl thioesters used for protein synthesis.133 The rate of hydrolysis can be expressed as v = kobs · [thioester] with kobs = ka[H+] + kw + kb[OH−], which represents the sum of terms that correspond to the acid-mediated (ka), the pH-independent (kw), and the base-mediated (kb) individual contributions to hydrolysis rate. The data collected in Table 2 reveal that, irrespective of the type of thioester, the alkaline rate constant kb significantly outweights the acid-catalyzed ka and the pH-independent kw constants. In these conditions, the hydrolysis rate becomes significant only at pH values greater than 8.0. At neutral pH, which is the classical pH for conducting NCL reactions, the rate of hydrolysis is the lowest. Table 2 also shows that the pH-independent and the basecatalyzed hydrolysis rates are significantly larger for the aryl thioester than for the alkyl thioester. In practice, this difference

Table 2. Rate Constants for the Hydrolysis of S-Methyl Thioacetate and S-Phenyl 5-Dimethylamino-5-oxo-thiopentanoatea entry 1 2 a

thioester CH3CO-SCH3 (CH3)2NCO(CH2)3CO-SPh

ka (M−1 s−1) −5

1.5 × 10 1.2 × 10−5

kw (s−1) −8

3.6 × 10 47 × 10−8

kb (M−1 s−1) 0.16 0.64

kobs = ka[H+] + kw + kb[OH−]. Data were taken from ref 133. J


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Figure 10. Synthesis of peptide thioesters, category I: the thioester functionality is generated following activation of a peptide acid.

inclination of chemists to synthesize modified proteins that biological systems cannot produce to any significant extent170 stimulated the design of linkers that can yield peptide thioesters using adapted Fmoc SPPS protocols. The most popular methods for the Fmoc SPPS of peptide thioesters belong to category I. 2.2.2. Synthesis of Peptide Thioesters: Category I. Two strategies were examined for introducing the thioester functionality after peptide elongation. The methods depicted in Figure 10 have in common the use of classical carboxylic acid activating procedures for introducing the thioester functionality on the peptide. The second set of methods relies on the use of solid-phase linkers that enable the attachement of the Cterminal residue through a stable bond of the amide type. Once the peptide is elongated by SPPS, the C-terminal amide bond is activated to enable the formation of the thioester function by thiolysis. Four main activation methods have been used: alkylation (Figure 11), acylation (Figure 12), oxidation (Figure 13), or condensation (Figure 14). 2.2.2.1. By Enthalpic Activation of Carboxylic Acids. A first strategy consists in generating the thioester group while the peptide remains attached to the solid support (Figure 10A,B). In this case, the activated ester is reacted with a presynthesized α-amino thioester. The reagents used to produce the thioester group can be easily removed by performing simple washing steps before subjecting the peptidyl thioester resin to the final cleavage and deprotection step in a strong acid. An example of this kind was described by Alsina et al.175 using the backbone

general rules of reactivity have strongly impacted the design of synthetic methods toward peptide thioesters which can be classified in two distinct categories. In category I, we included the methods which generate the thioester functionality after the peptide elongation or production step. The great advantage of these methods is that exposure of the thioester group to all the reagents required to assemble the polypeptide chain (e.g., piperidine for Fmoc SPPS) is avoided. In category II, the thioester functionality is introduced before the peptide elongation step, possibly in a protected form, and thus must survive during the SPPS and all subsequent chemical steps. A similar classification can be used for peptide selenoesters. Historically, the first methods that were developed for accessing peptide thioesters relied on peptide assembly using Boc SPPS and the design of TFA resistant thioester linkers. These methods, some of which are important to the field of protein chemical synthesis, are classified in category II and discussed in section 2.2.3. In comparison to Boc SPPS, the stringent criteria for linker design enabling the Fmoc SPPS of peptide thioesters undoubtedly hampered the emergence of practical solutions in this area for a long time. However, the safety issues posed by handling anhydrous HF required for the final deprotection and cleavage step in Boc SPPS, combined with the incompatibility of HF treatment with various modifications such as glycosylation,174 prompted the adoption of Fmoc SPPS by most peptide research groups. The increase in importance of the NCL reaction for protein chemical synthesis and the K


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Figure 11. Synthesis of peptide thioesters, category I: the thioester functionality is generated after peptide synthesis from N-alkyl N-acyl sulfonamides.

Figure 12. Synthesis of peptide thioesters, category I: the thioester functionality is generated after peptide synthesis from an imide precursor.

L


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Figure 13. Synthesis of peptide thioesters, category I: the thioester functionality is generated after peptide synthesis from an activated peptide obtained by oxidation.

amide linker (BAL176) strategy (Figure 10A). An alternative is to attach the peptide to the resin through a side chain functionality such as the carboxyl group of Asp/Glu, the hydroxyl group of Ser/Thr, the phenol group of Tyr, or the thiol group of Cys (Figure 10B).177 Both approaches use the Fmoc SPPS for the peptide elongation step and the selective unmasking of an allyl-protected carboxylic acid group to introduce the C-terminal amino acid equipped with a thioester functionality. Finally, the peptidyl resin is treated with TFA to release the unprotected peptide thioester in solution. An alternative strategy for introducing the thioester functionality on the peptide chain consists in activating a protected peptide acid in solution and in reacting the resulting activated ester with a thiol such as ethyl-3-mercaptopropionate178 or p-acetamidothiophenol179 (Figure 10C). The peptide thioester is obtained after deprotection and cleavage in TFA. One limitation of the method is the need to produce and handle protected peptide segments that are notoriously difficult to solubilize and purify. 2.2.2.2. By Thiolysis of Acyl Sulfonamides (Kenner SafetyCatch Sulfonamide Linker). In 1999, important reports published by Ingenito et al.180 and Shin et al.174 exploited the Kenner safety-catch sulfonamide linker181−184 for the Fmoc SPPS of peptide thioesters (Figure 11). N-Acyl sulfonamides are acidic species (pKa ∼ 2.5).185 Therefore, their deprotonated form is highly abundant in the basic conditions used for Fmoc removal, amino acid coupling, and acetylation. The negative charge of the N-acyl sulfonamide conjugate base is highly delocalized, thereby considerably decreasing the electrophilicity of the carbonyl attached to the sulfonamide. This property ensures good stability of the N-acyl sulfonamide linker in classical Fmoc SPPS protocols.174,180,186 The N-acyl sulfonamide group can be activated after the peptide elongation step by N-alkylation using TMS-CHN2180 or iodoacetonitrile.174,180 The protected peptidyl thioester is produced and detached from the solid support by thiolysis, a

step which can be facilitated by using 2 M LiBr/THF as a reaction mixture.187 Finally, the peptide thioester is fully deprotected in TFA.174,180 Interestingly, Burlina et al. showed that N-methyl N-peptidyl sulfonamides resist the TFA cleavagedeprotection step and can subsequently be used as thioester surrogates during NCL thanks to their in situ conversion into thioesters upon reaction with the thiol additive.188 In a complementary approach, Ollivier et al. showed that the N-acyl sulfonamide moiety can be alkylated with a βmercaptotriisopropylsilyl ethanol derivative using the Mitsunobu reaction (Figure 11).189 In this case, the protected thioester was produced by intramolecular thiolysis via a 5-membered intermediate after removal of the triisopropylsilyl group. In this approach, the protected peptide thioester remained attached to the solid support until the final cleavage and deprotection, thereby avoiding the manipulation of the protected peptides. The work of Mende et al.190,191 on the use of a Kenner linker for the solid-phase synthesis and self-purification of peptide thioesters is also worth mentioning (for a review, see ref 7). 2.2.2.3. By Thiolysis of Imides. The need to facilitate the production of thioesters using Fmoc SPPS has stimulated the development of another family of linkers which are converted to electrophilic imides by intramolecular acylation (Figure 12). Thiolysis of the intermediate peptidyl imide combined with TFA deprotection results in the formation of the unprotected peptide thioester.192−196 The diaminobenzoyl linker (Dbz) developed by BlancoCanosa et al. deserves a special mention due to its frequent use for peptide thioester synthesis.192,195,197,198 In this approach, the protected peptidyl o-aminoanilide resin is acylated with pnitrophenylchloroformate and then treated with TFA to produce an unprotected N-peptidyl benzimidazolinone (Nbz). Nbz peptides can be used for preparing peptide thioesters, typically aryl thioesters, by thiolysis. Because of the ease of peptide Nbz thiolysis at neutral pH, they can also be used directly in the NCL reaction. Initially, the Dbz linker was M


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some difficulties have been reported during the synthesis of peptide hydrazides featuring C-terminal Asp, Asn, and Gln residues or sometimes Lys residues,201,211,212 the ease of synthesizing peptide hydrazides by Fmoc SPPS combined with the high reactivity of thioesters derived from MPAA have undoubtedly contributed to the success of this chemistry. The possibility of accessing large peptide hydrazides by recombinant techniques is another asset of hydrazide ligation. This can be achieved by hydrazinolysis of intein fusion products,201,213 of an expressed protein with a genetically incorporated α-hydroxy acid,214 or by sortase-mediated hydrazinolysis of proteins with hydrazine.215 An alternative is the hydrazinolysis of a recombinantly expressed protein containing a C-terminal Cys residue.216 The production of N-peptidyl benzotriazoles from peptidyl Dbz resin is complementary to the above-mentioned hydrazide method (Figure 13).217 In this approach, a fully deprotected Cterminal Dbz peptide is treated with sodium nitrite in acidic 6 M Gn·HCl at low temperature to form a C-terminal peptidyl benzotriazole. As for the hydrazide method, the reaction is quenched by an excess of a thiol such as MPAA217 or 3mercaptopropionic acid,219 which displaces the benzotriazolyl group to produce a peptide thioester.218 2.2.2.5. By Thiolysis of N-Acyl Pyrazoles. The Knorr pyrazole synthesis from hydrazine derivatives has recently inspired an elegant activation method of category I which complements the approaches discussed so far (Figure 14).220

designed with no protecting group on the free aniline functionality.192 Acylation of aniline, however, has been observed during the repetitive coupling steps, explaining why new versions of the Dbz linker were designed. Either monomethylation (Figure 12B)195 or protection of aniline with allyloxycarbonyl group for Fmoc SPPS (Figure 12A)194 or 2-chlorobenzyloxycarbonyl group for Boc SPPS (R = 2-Cl-Z, Figure 12A)199 proved satisfactory to overcome this limitation. Similar approaches described in Figure 12 take advantage of side chain carboxylic acid,193 or alcohol196 functionalities for promoting imide formation and are reminiscent of the safetycatch linker designed by Sola et al.200 In these approaches, the protected peptide is first detached from the resin by thiolysis and subsequently deprotected in TFA. 2.2.2.4. By Thiolysis of Acyl Azides and N-Acyl Benzotriazoles. Another set of methods belonging to category I have in common the use of an oxidation step for the activation of the amide-type linker (Figure 13). While the previous methods based on imide chemistry rely on the exposure of an internal nucleophile to an acylating reagent and thus require working with a protected peptide chain, it is possible to activate some peptide amides or hydrazides by selective oxidation at the level of a deprotected peptide. This is a great advantage and in practice the most popular method for peptide thioester synthesis today, the peptide hydrazide method reported by Liu and co-workers,201 belongs to this subgroup. Pioneering studies in this area were reported by Millington et al.,202 who showed that unprotected peptides immobilized by an aryl hydrazide linker can be cleaved from the resin as peptide amides by exposing the peptidyl resin to catalytic Cu(II) in aerobic conditions in the presence of an amine nucleophile. The method was extended to the synthesis of peptide thioesters by Camarero et al. (Figure 13).203 Because thioesters tend to decompose in the presence of Cu(II),204 the oxidation was performed with N-bromosuccinimide which required maintaining the protecting groups on the peptide. The protected peptide thioester was then produced by aminolysis of the protected peptidyl diazene resin with preformed α-amino thioesters. Finally, the peptide thioester was deprotected in TFA as usual. A significant advance in the field of protein chemical synthesis was reported by Liu and co-workers in 2011 with the synthesis of peptide thioesters from peptide hydrazides (Figure 13).201,205,206 According to this method, the fully deprotected peptide hydrazide is converted into an acyl azide207 by the action of sodium nitrite in acidic 6 M guanidinium hydrochloride. One precaution is to perform this oxidation step at low temperature, typically below −5 °C, to avoid the Curtius rearrangement of the acyl azide to the corresponding isocyanate.208 The acyl azide intermediate is most of the time trapped by aryl thiol MPAA to produce the corresponding peptide thioester in situ. Note that the S-nitrosothiols or disulfides that can potentially form by exposing free Cys thiols to nitrous acid are reduced by the excess of added MPAA. Other thiols such as MESNa209 or TFET210 are also reported alternatives for the thiolysis of hydrazides. Importantly, the oxidation step and the formation of the aryl thioester can be done in the presence of the Cys peptide. In this case, the conversion of the peptide hydrazide into a thioester can be followed by the NCL reaction with the Cys peptide in one-pot. Since its introduction in 2011, the hydrazide-based ligation has become one of the most widely used methods for thioester formation and ligation (see Figure 5).18 Although

Figure 14. Synthesis of peptide thioesters, category I: the thioester functionality is generated after peptide synthesis from an activated peptide obtained by condensation.

Indeed, Flood et al. have shown that peptide hydrazides can be efficiently converted into peptide thioesters through the intermediate formation of N-acyl pyrazoles. The latter are produced in situ by a condensation reaction involving the hydrazide functionality and acetyl acetone at acidic pH and further displaced by the thiol additive to generate the thioester. The screening of various thiols showed the best results for MPAA. This mode of activation of peptide hydrazides mechanistically differs from the other approaches described so far and provides a chemoselective and mild process to generate thioesters in the absence of acylating or oxidizing reagents. 2.2.2.6. By Thiolysis of N,S- or O,S-Acyl Shift Systems. Synthesis of Thioesters f rom N,S-Acyl Shift Systems. Another subgroup of methods giving access to peptide thioesters and belonging to Category I exploit the capacity of some N-(2sulfanylethyl) amides or (2-sulfanylethyl) esters and derivatives thereof to rearrange spontaneously in water into transient thioesters (Figure 15). N


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A more in-depth discussion of the reactivity of these systems and how they rearrange into thioesters can be found in section 5. Here we wish to discuss some aspects that are specific to the use of these reagents as precursors of peptide thioesters. The interchange of N-(2-sulfanylethyl)amides or (2sulfanylethyl)esters by thiols proceeds through a thiol− thioester exchange involving the transient thioesters produced by X,S-acyl shift and the thiol. An important point to keep in mind is that this process is equilibrated. Therefore, the thiol must be used in excess to drive the equilibrium toward the desired product. In the case of N-(2-sulfanylethyl)amides, the high thermodynamic stability of the starting amides relative to the target thioesters must also be counterbalanced by inactivating the released 2-mercaptoethylamine handle by protonation.221 In practice, N-(2-sulfanylethyl)amides222−231 and related systems232 used for peptide thioester synthesis are indeed all exchanged in acidic conditions (Figure 16). The development of N,S-acyl shift systems for peptide thioester synthesis was pioneered by Hojo et al. with the introduction of N-alkylcysteine.223 This motif, as well as bis(2sulfanylethyl)amido (SEA)228,233,234 and SEAlide226 systems, are the most frequently used precursors for peptide thioester synthesis among the different N,S-acyl shift systems developed so far. In the case of the SEA-mediated formation of peptidyl

Figure 15. Interchange of O,S- and N,S-acyl shift systems by thiols gives access to peptide thioesters.

Figure 16. Synthesis of peptide thioesters, category I: the thioester functionality is produced by an N,S-acyl shift system−thiol interchange reaction. O


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thioester synthesis currently but are more favorably considered for their capacity to act as thioester surrogates in NCL. These points are discussed in more detail in section 5.4. 2.2.2.7. By Photolysis of Amides. Photochemical methods are always attractive when sensitive groups such as thioesters must be included in complex molecules. The use of these approaches in protein chemical synthesis has been examined by several groups, either for masking the reactivity of N-terminal Cys residues,240 of C-terminal thioesters surrogates,227,241 or in the design of removable thiol auxiliaries.242,243 The use of photochemistry for thioester synthesis is less common, and the work of Pardo et al. is a rare example in the field (Figure 18).244 The photolysis of N-acyl 7-nitroindolines at 350 nm triggers an intramolecular N,O-acyl migration. The resulting transient Opeptidyl intermediate is trapped by N-hydroxysuccinimide (HOSu) or N-hydroxybenzotriazole (HOBt) to produce an activated ester. The latter is converted into a thioester by exposure to thiophenol or alternately into a peptide hydrazide by reaction with hydrazine. The method has the advantage of generating the thioester group in neutral conditions. A limitation of the method is the need to keep the peptide protecting groups until synthesis terminates. 2.2.3. Synthesis of Peptide Thioesters: Category II. 2.2.3.1. Boc SPPS Using Thioester Linkers. An alternative approach to the late introduction of the thioester functionality is the design of thioester-based linkers compatible with the Boc or Fmoc protocols (Figure 19 and Figure 20, respectively). With some exceptions, the thioester group is usually sensitive to the repetitive piperidine treatments used for Fmoc SPPS. In contrast, it is stable to strong acidic solvents such as anhydrous HF or TFA. The first approaches to the SPPS of peptide thioesters were developed using Boc SPPS. The development of a 3-mercaptopropionamide linker (MPAL) by Hojo and Aimoto is an important contribution to the field (I in Figure 19).245 The need to access peptide alkyl thioesters for protein synthesis using NCL stimulated further adaptations of the 3mercaptopropionamide linker to various solid supports depending on the final application.246−248 For example, a considerable advantage of the MPAL method is the possibility of attaching the 3-mercaptopropionic acid residue to a solubilizing polypeptide tag, typically an oligoarginine peptide. Introduced by SPPS between the acid labile linker and the MPAL unit (II in Figure 19),249−252 the peptide tag becomes part of the thiol leaving group of the thioester peptide and thus is not incorporated in the final product after NCL. Peptide thioesters containing oligoarginine thiol handles of variable length (peptide−MPAL−Argn, n = 5,249 n = 6,250,252 n = 10251) were found to be useful for the chemical synthesis of hydrophobic polypeptides and in particular membrane proteins. In another mode of use, Brust et al.248 adapted Hojo’s linker to a safety-catch amide linker (SCAL,247,253 III in Figure 19). The SCAL linker is stable in anhydrous HF and enables the side chain protecting groups to be removed without cleaving the peptide from the resin. The peptide thioester can be detached from the solid support by TFA treatment in the presence of ammonium iodide.254 In these conditions, the electron-withdrawing sulfoxide groups of the SCAL linker are reduced to electron-donating thioether groups, thereby rendering the linker cleavable in TFA. Classical Boc SPPS protocols also enable the synthesis of peptide aryl thioesters using the 4-mercaptophenylamide linker designed by Bang et al. (V in Figure 19).255 An in situ

thioesters, the rate of the reaction was substantially enhanced in the presence of alkyl selenols that were shown to efficiently catalyze thiol-thioester exchanges at acidic pH.233 As previously mentioned, the interchange of N,S-acyl shift systems by thiols is an equilibrated reaction that requires special experimental conditions to drive the equilibrium toward the thioester product. In an elegant study, Zheng et al. found a way to produce the thioester irreversibly by enabling the formation of an enamine after N,S-acyl migration (Scheme 3).235 In acidic Scheme 3. Synthesis of Peptide Thioesters, Category I: The Thioester Functionality Is Generated after Peptide Synthesis by N,S-Acyl Shift Followed by Enamine Hydrolysis

conditions, the transient enamine intermediate hydrolyzes into a stable ketone product which can be isolated. The method was found to be tolerant to various C-terminal amino acid residues. In a different approach, Dheur et al. showed that the N,S-acyl shift equilibrium of the bis(2-sulfanylethyl)amide (SEA) group in aqueous acid can be displaced toward a stable and isolable thioester product by trapping the β-aminothiol functionality of the SEA thioester intermediate with glyoxylic acid (Scheme 4).236 The method relies on the high chemoselectivity of Scheme 4. Synthesis of Peptide Thioesters, Category I: The Thioester Functionality Is Generated after Peptide Synthesis by N,S-Acyl Shift Followed by Thiazolidine Formation

thiazolidine formation between β-aminothiols and aldehydes in aqueous acidic media.13 The reactivity of thiazolidine thioester (TT) peptides enables ligation at difficult junctions and is discussed in more detail in section 6.1. Synthesis of Thioesters f rom O,S-Acyl Shift Systems. Differences in thermodynamic stability between O- and S-esters are lower than those between amides and S-esters. In practice, (2sulfanylethyl)esters can be exchanged in a broad range of experimental conditions including in neutral aqueous media (Figure 17).237−239 However, in the latter conditions, the reaction can be complicated by the hydrolysis of the (2sulfanylethyl)ester. These systems are seldom used for peptide P


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Figure 17. Synthesis of peptide thioesters, category I: the thioester functionality is produced by an O,S-acyl shift system−thiol interchange reaction.

group enabled the duration of the deprotection step to be shortened and resulted in a lower proportion of epimerized product. The possibility of synthesizing similar S-tertiary peptide thioesters by Fmoc SPPS was confirmed recently by Raz and Rademann.260 During the same period, Clippingdale et al. reported the synthesis of S-primary peptide alkyl thioesters using the MPAL linker (II in Figure 20). In this case, a non-nucleophilic base, 7diazabicyclo[5.4.0]undec-7-ene (DBU) was combined with HOBt to remove the Fmoc groups.261 Bu et al. further optimized the DBU/HOBt deblocking mixture for optimal deprotection.262 2.2.3.3. Fmoc SPPS Using Protected Thioester Linkers. The use of thioester linkers met with a great deal of success for the Boc SPPS of peptide thioesters and continues to be regularly utilized. Comparatively, the combination of thioester linkers and Fmoc SPPS is less popular due to the risk of peptide thioester hydrolysis, aminolysis, and epimerization. A few approaches examined the feasibility of introducing the thioester in a protected form to facilitate the elongation of the peptide chain by Fmoc SPPS. Brask et al.263 used a trithioortho ester derived from glycine and a backbone anchoring strategy based on the BAL linker175,176 to synthesize peptidyl glycyl thioesters by Fmoc SPPS (Figure 21A). This method has not been extended to other C-terminal residues. The second approach described in Figure 21B relies on the stability of thioamides in the conditions used for Fmoc SPPS.264 The thioamide was converted after the peptide elongation step to a thioimine by S-alkylation with benzyl bromide. The treatment of the supported peptidyl Sbenzylthioimine with aqueous TFA enabled the removal of the protecting groups, the detachment of the peptide from the resin and the conversion of the S-benzylthioimine into the target benzyl thioester. This method was illustrated by the synthesis of peptide benzyl thioesters containing a C-terminal Phe or Gly residue. Note that, when present, methionine residues had to be protected in the form of their sulfoxide in order to resist the alkylation step. Consequently, Metcontaining peptides were cleaved and deprotected using TFA/NH4I-Me2S.265

Figure 18. Synthesis of peptide thioesters, category I: the thioester functionality is generated after peptide synthesis from an activated peptide obtained by photolysis.

neutralization protocol256 minimized diketopiperazine formation and the formation of a two-amino acid deletion side product. Recently, Raz et al. reinvestigated the Boc SPPS of peptide thioesters using the 3-mercaptopropionamide or 4mercaptophenylamide linkers and showed that TFA/TMSBr can advantageously replace HF, especially when acid-sensitive modifications have to be introduced in the peptide thioester.257 2.2.3.2. Fmoc SPPS Using Thioester Linkers. Several publications have described the use of adapted Fmoc SPPS protocols for peptide thioester synthesis. An early approach was reported by Li et al., who examined the synthesis of peptide alkyl thioesters using S-tertiary thioester linker I (Figure 20) and a mixture of 1-methylpyrrolidine, hexamethylenimine, and HOBt for the removal of the Fmoc groups.258 HOBt was used to reduce the basicity of the Fmoc deblocking mixture and thus thioester aminolysis and hydrolysis. However, a subsequent study also showed that this protocol induces the partial epimerization of the C-terminal residue.259 Replacing the Fmoc group by the more acidic 9-(2-fluoro)fluorenylmethoxycarbonyl (Fmoc-(2F)) amine protecting

2.3. Latent Thioester Surrogates

One of the factors that determine the number of peptide segments used for assembling a protein is the size of the Q


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Figure 19. Synthesis of peptide thioesters by Boc SPPS, category II: the thioester functionality is introduced before the peptide elongation step by SPPS.

Figure 20. Synthesis of peptide thioesters by Fmoc SPPS, category II: the thioester functionality is introduced before the peptide elongation step by SPPS.

repeated incorporation of bifunctional peptide blocks equipped with an N-terminal Cys residue and a C-terminal latent thioester. Once the NCL has been performed, the latent thioester must be activated into a thioester to continue the elongation process. Ideally, a latent thioester should not interfere during NCL before being activated. In practice, the latent thioester is a chemical group that can be totally inert (type I) or can have a very low reactivity (type II) in classical conditions for NCL with aryl or alkyl thioesters. These two situations are fundamentally different in that the result of the ligation in the presence of a latent thioester of type I is not influenced by

individual peptide segments produced by SPPS, which is usually less than 50 amino acids. Therefore, the chemical synthesis of proteins involving more than 100 AA is often conducted by assembling more than two peptide segments. Consequently, strategies that allow the sequential assembly of peptide segments are particularly important in the field. Sequential concatenation in the C-to-N direction is discussed in section 3.1 (see Scheme 19) because it relies on the temporary protection of N-terminal cysteines. In contrast, concatenation in the N-to-C direction is based on the use of Cterminal latent thioesters as described in Scheme 5. The elongation starts from a peptide thioester and is based on the R


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the rate of the reaction. In contrast, ligations performed in the presence of latent thioesters of type II are kinetically controlled and can potentially be complicated if the rate of the NCL is slow. This is typically the case when the junction formed by NCL involves a difficult amino acid residue such as Thr, Val, Ile, or Pro (see section 6.1). A point to mention is that concatenation in the N-to-C direction benefits from a self-purification process when the SPPS protocols used to produce the individual peptide segments include a capping step. The capping step, which is classically an acetylation reaction, avoids the formation of deleted peptides during SPPS and facilitates the purification of the peptide segment by HPLC. Nevertheless, the full-length peptide segments can be contaminated by shorter capped peptides even after performing a resolutive purification step. Interestingly, these capped peptide contaminants lack the critical N-terminal cysteine for participating in the NCL reaction. Therefore, they cannot be incorporated into the growing peptide chain during the N-to-C elongation as illustrated in Scheme 5.7,266,267 This section discusses the latent thioesters whose activation results in the formation of a classical peptide alkyl or aryl thioester. In particular, the studies that utilized N,S-acyl shift systems in this way are presented in this section. Note that several N,S-acyl shift systems were also used for N-to-C elongation by exploiting specific reactivities without being converted into classical thioesters. In this alternative mode of use, the latent thioester is activated and reacted directly with the Cys peptide. Because the mechanisms implicated in these latter reactions are markedly different from those discussed in this section, they are detailed more specifically in section 5. 2.3.1. Latent Thioesters of Type I. The use of the hydrazide method developed by Liu and co-workers201 for peptide thioester synthesis has been discussed in detail in section 2.2.2. The hydrazide group is a type I latent thioester because it is completely inert under classical NCL conditions. It has been used for the assembly of large peptide segments in the N-to-C direction according to the general method described in Scheme 6.268 The use of hydrazides for N-to-C peptide assembly requires the separation of the elongated peptide hydrazide from the thiol catalyst (e.g., MPAA, the gold standard catalyst for NCL, see section 4.3.1) used for the NCL step, as the latter is not compatible with the subsequent nitrous acid treatment enabling the transformation of the C-terminal hydrazide group into a thioester. In contrast, peptide hydrazide activation and ligation can be performed in one pot. Another latent thioester that has been used for N-to-C elongation is the SEA group (Figure 22). In the form of the cyclic disulfide called SEAoff, it is unreactive toward Cys peptides in the presence of MPAA and thus behaves as a latent

Figure 21. Synthesis of peptide thioesters by Fmoc SPPS, category II: the thioester functionality is introduced before the peptide elongation step by SPPS in a protected form.

Scheme 5. Latent Thioester Systems Are Particularly Useful for N-to-C Protein Synthesis

Scheme 6. N-to-C Elongation Using Peptide Hydrazides as Latent Thioesters (Type I)

S


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Figure 22. N-to-C elongation using SEA latent thioester: (A) in solution, (B) on solid phase.

thioester of type I during NCL.269 It is activated by reductive ring-opening with TCEP into the SEAon dithiol group, which is exchanged in situ by 3-mercaptopropionic acid to produce the corresponding thioester (see also section 2.2.2 and Figure 16).228 The whole process consisting in the NCL reaction and the subsequent exchange of the SEA group can be performed in one pot (Figure 22A).270 Further N-to-C elongation requires the reaction of the obtained peptide thioester with a novel bifunctional peptide. Therefore, the elongated peptide thioester must be separated from TCEP as the latter would activate the incoming SEAoff group during the subsequent ligation step. The process has been adapted to the solid phase with the advantage of simplifying the intermediate purification steps, which are reduced to simple washing steps (Figure 22B). Using this method up to five peptide segments could be ligated sequentially.7,271 A more detailed discussion of the SEA system and of other modes of use as a latent thioester can be found in section 5. 2.3.2. Latent Thioesters of Type II. The peptidyl N-acyl guanidines developed by the group of Kajihara are type II latent thioesters thanks to their low reactivity toward MPAA (Scheme 7).272,273 This property enables performing an NCL in the Nto-C direction with minimal interference from the N-acyl guanidino group as illustrated in Scheme 7A. The elongated peptidyl N-acyl guanidine can be converted to an alkyl thioester in one pot by adding a powerful thiol nucleophile such as MESNa to the ligation mixture. The obtained peptide thioester can potentially be engaged subsequently in another NCL reaction. Interestingly, a recent report showed that a peptide thioester can be converted to a peptidyl N-pivaloyl guanidine by treatment with an excess of N-pivaloyl guanidine at pH 8.0 (Scheme 7B).273 Thus, peptide thioesters and peptidyl N-

Scheme 7. (A) N-to-C Elongation Using Peptidyl N-Acyl Guanidines As Latent Thioesters (Type II); (B) Peptidyl NPivaloyl Guanidines Are Interconvertible with Peptide Thioesters

pivaloyl guanidine are interconvertible, a property that opens up various synthetic possibilities. T


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Having presented the properties of peptide thioesters, their synthesis and the design of latent thioesters, this section continues with peptide selenoesters following a similar organization. 2.4. Peptide Selenoesters: General Properties

The preference of thioesters for the syn conformer has been discussed in detail in section 2.1.1. The conformational behavior of model selenoester CH3−C(O)−XCH3 (X = Se) was also investigated using energy calculations, NBO, and atoms in molecules (AIM) analyses and was compared to those of oxygen (X = O) and sulfur (X = S) analogues.150 The syn conformer is favored over the anti for the three types of esters, but the difference in energy between the syn and anti conformers diminishes from oxygen to selenium (ΔG298 = 30.7, 25.0, and 15.1 kJ/mol for X = O, S, and Se, respectively). This study also revealed that the changes in resonance energies, C(O)−X and X−CH3 bond lengths, and syn/anti stabilities can be rationalized on the basis of the magnitudes of np(X) → π*CO, ns(X) → σ*CO electronic interactions which decrease from oxygen to selenium (illustrated in Figure 8 for thioesters). The rare experimental studies that support the preferential syn conformation of selenoesters comes from X-ray crystal structures of low molecular weight selenoesters.274,275 The structure shown in Figure 23 enables the significant difference in length between C−Se and simple C−C bonds to be appreciated.275

Figure 24. Compared hydrolysis and aminolysis of a series of cholinederived chalcogenoesters.

amines. Indeed, aminolysis of the same series of compounds in the presence of n-butylamine revealed a markedly different behavior for each chalcogenoester. The choline-derived selenoester reacted 75 times more rapidly than the corresponding thioester, whereas the oxoester remained inert (Figure 24).277 These findings were in agreement with previous work by the same authors in 1963 that compared the aminolysis rates of dibenzoylcysteamine and dibenzoylselenocysteamine by nbutylamine.276 Here again, selenoesters were identified as superior acyl donors compared to their sulfur counterparts. At the same period, Jakubke compared the aminolysis rates of Z-Gly-SPh and Z-Gly-SePh278 and exploited the enhanced reactivity of selenoesters toward amines as a mode of Cterminal activation in peptide coupling reactions for accessing di- to hexapeptides from peptidyl selenophenyl esters.278−280 The utility of selenophenyl esters as acyl donors continues to stimulate the development of novel methods for synthesizing these derivatives, as well as a variety of applications.281−284 Of course, and as for thioesters, the high susceptibility of peptidyl selenoesters to aminolysis has considerably influenced the synthetic approaches developed for their production, as will be evidenced in the next section of this review.

Figure 23. X-ray crystal structure of 4-acetyl-1-acetylseleno-2-phenylcyclobutene showing a syn conformation for the selenoester group (CCDC entry 236473275).

2.4.1. Reactivity toward Oxygen and Amine Nucleophiles. The increase in length of the C(O)−X (X = O, S, Se) bond in going from oxoesters (X = O) to selenoesters (X = Se) are echoed in their reactivity toward various nucleophiles, selenoesters being the most reactive toward nucleophilic attack by water or thiols. However, detailed studies of the rate constants for hydrolysis and thiol addition reactions in this series are rare.130,276,277 The alkaline hydrolysis of a series of choline-derived chalcogenoesters has been investigated by Chu and Mautner (Figure 24).277 Whereas the choline and thiocholine ester derivatives exhibited the same resistance to hydrolysis as previously observed by Connors and Benders, 130 the selenoester was shown to hydrolyze 3 to 10 times more rapidly depending on experimental conditions. Though significant, this difference in reactivity is small compared to that observed with

2.5. Chemical Synthesis of Peptide Selenoesters

Compared to peptide thioesters, the synthesis of peptide selenoesters has been developed to a much lesser extent, probably due to the even higher sensitivity of selenoesters to various reagents and above all to nitrogen nucleophiles. Nevertheless, we can predict important developments in this area, given the considerable potential of selenoesters for accelerating peptide bond formation using NCL285 or DSL.9 Not surprisingly, most of the synthetic methods for peptide selenoesters involve the late introduction of the selenoester functionality and therefore belong to category I. 2.5.1. Synthesis of Peptide Selenoesters. Category I. 2.5.1.1. By Enthalpic Activation of Carboxylic Acids. U


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Figure 25. Synthesis of peptide selenophenyl esters (category I) by enthalpic activation of carboxylic acids.

Selenophenyl esters are among the most popular selenoesters used for protein chemical synthesis owing to their exceptional reactivity. In his pioneering studies, Jakubke used selenophenyl esters for peptide coupling and developed efficient synthetic methods for the production of protected selenophenyl esters.278 The mixed anhydride method or carbodiimide activation proved particularly useful for producing protected peptide selenophenyl esters from protected peptide acids. More recent work involved the conversion of protected peptide acids into the corresponding peptide selenophenyl esters in DMF by treatment with an excess of diphenyldiselenide (DPDS)/nBu3P according to the method developed by Singh et al. (Figure 25A).9,286,287 The protected peptide selenophenyl ester was further deprotected in TFA. In a recent report, Hanna et al. adapted this method to generate selenophenyl esters on resin using a side chain anchoring strategy (Figure 25B).288 Allyl esters derived from αamino acids or dipeptides were anchored through the side chains of Asp, Glu, Ser, or Tyr. The peptides were elongated using Fmoc-SPPS. Then, the C-terminal allyl group was removed selectively and the selenophenyl ester functionality was introduced using diphenyldiselenide/nBu3P. The authors noticed that selenoesterification at room temperature induced substantial epimerization of the C-terminal residue, which could be reduced by working at a lower temperature. Finally, the peptide selenophenyl ester was cleaved and deprotected in TFA. 2.5.1.2. By Interchange from Thioesters or N,S-Acyl Shift Systems. With the recent development of a large variety of synthetic methods toward peptide alkyl thioesters, it is tempting to envisage the production of peptide selenoesters from their sulfur counterparts. However, the direct exchange of peptide alkyl thioesters by alkyl289 or aryl selenols233,285 is a poorly efficient process and has not been used so far for the production of peptide selenoesters. Nevertheless, peptide selenophenyl esters can be produced from peptide alkyl thioesters using the two-step method shown in Scheme 8.285 Peptide alkyl thioesters are first converted to peptide selenoacids by treatment with sodium hydrogen selenide. The

Scheme 8. Solution Synthesis of Peptide Alkyl Selenoesters from Peptide Alkyl Thioesters (Category I)

peptide selenoacid is not isolated but is alkylated in situ at pH 4.0 with iodoacetamide. The low pH of the alkylation step enables the occurrence of side reactions with peptide nucleophilic groups to be minimized. The main limitation of the method comes from the high nucleophilicity or reducing power of hydrogen selenide ions during the first step of the process, which was found to generate unidentified byproducts. Only one example of peptide selenoester synthesis by selenol/N,S-acyl shift interchange has been reported so far. Takei et al. have recently produced peptide selenophenyl esters by exchanging the N-ethyl cysteine N,S-acyl shift system by phenylselenol (Scheme 9).290 In practice, selenophenol was generated in situ by reduction of diphenyldiselenide with TCEP, and the exchange was performed in aqueous acetonitrile containing acetic acid and 6 M urea. 2.5.2. Synthesis of Peptide Selenoesters: Category II. The design of selenoester linkers for the Boc SPPS of peptide selenoesters is in its infancy, with only one example reported so far. The linker developed by Ghassemian et al.291 is reminiscent of the 3-mercaptopropionamide linker (MPAL, see Figure 19) designed by Hojo et al. (Scheme 10).245 The supported 3selenocyanatopropionamide was deprotected using sodium borohydride. Owing to the high propensity of selenols to oxidize into diselenides, the resin was first treated with DTT before being acylated with the C-terminal amino acid using classical activation procedures. The peptide chain was V


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and these fundamentally concern their acid−base and redox properties. In a complementary manner, this section presents the common additives used to maintain thiols and selenols in a reduced and active state throughout the ligation process and also introduces the various protecting groups designed to temporarily shutdown their reactivity.

Scheme 9. Synthesis of Peptide Selenoesters (Category I): Synthesis of Peptide Selenophenyl Esters by Selenophenol/ N-Ethyl Cysteine N,S-Acyl Shift Interchange

3.1. Cysteine

3.1.1. Nucleophilicity and Acid−Base Properties of the Cysteine Residue. Thiols (R-SH) equilibrate with thiolates (R-S−) by loss of a proton (Scheme 11). The Scheme 11. Ionization of Thiols

nucleophilicity of thiolates toward thioesters exceeds by several orders of magnitude that of thiols. Consequently, they are the effective nucleophilic species in NCL, a point which is supported by experimental and computational studies (see section 4). A direct consequence of the higher nucleophilicity of thiolates compared to thiols is that the participation of Cys thiols in NCL is dictated by their ease of ionization, i.e., their pKa and the pH of the ligation mixture. The pKa of internal Cys thiol in alanine pentapeptide is 8.5 ± 0.03,294 a value close to those repeatedly reported for unperturbed internal Cys thiols in proteins (Scheme 12A).295

Scheme 10. Boc-SPPS of Peptide Alkyl Selenoesters (Category II)

Scheme 12. Ionization Equilibria for Internal and NTerminal Cysteine

elongated using Boc SPPS and the peptide selenoester deprotected and cleaved in anhydrous HF. From the pioneering on-resin elongation of peptidyl thioesters to the synthesis of activatable thioester surrogates, the repertoire of acyl donors has been consequently enriched over the years. The tremendous efforts devoted to their development has certainly contributed to making NCL and extended methods the efficient and versatile chemical tools they now are. The second partner involved in the ligation process, i.e., the cysteinyl peptide, also occupies a prominent place in the performance of these reactions. As such, its properties and reactivity are discussed in the following section. However, the pKa of Cys thiol in folded proteins can be subject to significant variations depending on the conformational and electronic environment of cysteine (pKa = 6.8 ± 2.7, mean for 25 proteins).296 Because NCL is usually performed under strong denaturing conditions, the pKa value of 8.5 found for Cys thiol in short unstructured peptides undoubtedly represents a good estimate of the pKa of internal Cys thiols in unfolded polypeptides. N-Terminal cysteine is a case apart, and the extent to which this location modifies its acid−base properties, and hence its reactivity as a nucleophile is particularly relevant in the context of NCL (Scheme 12B). In 1955, the pioneering work of Benesch and Benesch shed light on the difficulties of providing a correct estimate for the acidity constants of free cysteine.297

3. PROPERTIES OF CYSTEINE OR SELENOCYSTEINE RESIDUES The chemoselectivity of NCL and extended methods primarily rests on the nucleophilic capture of thioesters by Cys thiols or Sec selenols. The influence of the physicochemical properties of thiols or selenols on the performance of the ligation process is a central issue for obtaining insights into the mechanism and improving the experimental design of these reactions. Of course, the basics of the chemical properties of Cys thiols and Sec selenols have been reviewed several times and discussing in detail these points is not within the scope of this review.292,293 In this section, we rather wish to point out the aspects of Cys and Sec chemistries that are important in the context of NCL W


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Indeed, the respective dissociation constants of the αammonium and β-thiol groups of cysteine are of the same order of magnitude, making their ionization interdependent (for a review, see ref 298). N-terminal cysteine residues are potentially subject to a similar interdependency of the ionization constants of α-ammonium and β-thiol groups, KNapp and KSapp, respectively, in Scheme 12B. The very limited data available in the literature tend to support this view with a contraction of pKa values between 6.0 and 7.0 for both the ammonium and thiol groups in the case of short cysteinyl peptides (Table 3, entries 2 and 3).

Scheme 13. Mechanism of Thiol Oxidation by Molecular Oxygen

Table 3. pKa for N-Terminal Cysteinyl Peptides peptide 1 Cys-Gly-OH 2 Cys-Gly-Gly-OH 3 Cys-Gly-Gly-Gly-Gly-OH a

reported pKas nd (−OH), 7.87 (−SH), 8.75 3.13 (−OH), 6.36, 6.95a 3.21 (−OH), 6.01, 6.87a

ref (−NH3+)

297 299 299

thiolates is particularly effective so that eqs 4 and 5 (Scheme 13) dominate at pH values that favor the ionization of the thiol. Therefore, this process occurs more rapidly when the pH of the aqueous solution is close to or above the pKa of the thiol. Conversely, thiols are protected from oxidation in highly acidic solutions. As discussed above, the pKa of N-terminal Cys thiol is estimated at around 7, while the pKa of classical thiol catalysts, i.e., aryl thiols, is about 6.6 (see section 4.3.1). Consequently, thiol oxidation is expected to be significant at the neutral pH used for NCL unless special precautions are taken. The formation of thiyl radicals and hence the oxidation of thiols by molecular oxygen can be catalyzed by metal ions such as Fe(II) or Fe(III).303,305 Most laboratory chemicals and buffers contain trace amounts of adventitious iron.306 In these conditions, and in the absence of any precaution to exclude oxygen, the half-life of common thiols used in biochemistry such as 2-mercaptoethanol, DTT, or 3-mercaptopropionic acid (MPA) at neutral pH is only a few hours.307 Logically, the addition of the metal chelator EDTA would significantly reduce the rate of metal-induced thiol oxidation. However, whereas this practice is very common for biological buffers, EDTA is rarely used as an additive during ligations.308 3.1.2.2. Disulfide Bond Reducing Agents Used for NCL. Reduction by Thiols. NCL is usually carried out under experimental conditions that permit thiol oxidation. Besides the working pH the role of which has been discussed above, other factors that facilitate the progressive conversion of thiols into disulfides during ligation are the small scales of synthesis (micromoles) and the difficulty in blocking the entry of oxygen into the reaction vessel over a period of hours. When ligations are carried out in a strictly inert atmosphere, the use of a strong reducing agent can be optional if NCL is performed in the presence of thiol additives used to catalyze the reaction (see section 4.3). Indeed, commonly used arylthiol catalysts are mildly reducing309 and capable of maintaining Cys residues in a reduced state when used in large excess. Alkylthiol additives such as benzylthiol,8 MESNa,310,311 or glutathione312 are more reducing than arylthiols and sometimes used alone or in combination with arylthiols in NCL. The mechanism of disulfide reduction by thiols proceeds through a classical thiol−disulfide exchange process that is depicted in Scheme 14. Reduction by DTT. Unless the entry of molecular oxygen into the reaction vessel can be prevented, it is recommended to combine NCL thiol catalysts with a stronger disulfide bond reducing agent. Dithiothreitol (DTT,313 Scheme 15) has been

The pKa values for −SH and −NH3+ could not be attributed.

Because the pKa of the α-amino group in short peptides is 8.0 on average,294 and that of an internal Cys thiol is 8.5 as mentioned above, this implies a shift of 1.5−2 pKa units for each group. Therefore, N-terminal Cys thiols can be considered 30−100 times more acidic than internal unperturbed Cys thiols. Their higher tendency to ionization at neutral pH, i.e., the usual working pH for NCL, compared to internal Cys thiols is expected to have significant implications for their reactivity as well as that of the thioester-linked intermediate formed in the capture step. These aspects of Cys thiol reactivity are further developed in section 4. 3.1.2. Controlling the Redox State of Cysteine during NCL. While thiolates are significantly more nucleophilic than thiols toward thioesters, they are also more susceptible to oxidation by molecular oxygen into disulfides, which as such are unproductive in NCL. This competitive reaction pathway leads to a depletion of thiol-based nucleophiles in the reaction mixture that can negatively affect both the rate and the yield of the ligation process. This is not only true for the Cys thiol involved in the formation of the peptide junction but also for all the exogenous thiol additives present in the mixture (e.g., thiols used as catalysts, see section 4.3). The oxidation state of the sulfhydryl group in general, and more particularly that of Cys, significantly influences the experimental design of NCL and related reactions. It is thus important to define the main features of these redox processes as well as the factors that can affect their kinetics and to review the solutions used to prevent or revert the formation of disulfides during ligation. 3.1.2.1. Mechanism of Cys Oxidation. The most predominant redox process at play during ligation reactions is the oxidation of thiols to disulfides by dissolved molecular oxygen. This reaction has been intensively studied in the past and takes place in the pH range 7−9 according to the general mechanism shown in Scheme 13.300−303 Oxidation to disulfides involves the intermediate formation of thiyl radicals (eq 1, Scheme 13).304 Thiyl radicals can combine to produce disulfides (eq 3, Scheme 13) or alternately react with thiolates to produce disulfide radical anions (eq 4, Scheme 13). The latter are powerful reductants and thus react readily with molecular oxygen to produce dioxygen radical anions (eq 5, Scheme 13), which ultimately can decompose with the production of thiyl radicals (eq 6, Scheme 13). The reaction of thiyl radicals with X


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Scheme 14. Mechanism of Reduction of Disulfides R′S−SR′ by Thiols RSH

Scheme 16. Formation, Rearrangement, and Degradation of DTT Thioesters

Scheme 15. Mechanism of Reduction of Dichalcogenides by DTT: The Reduction of Disulfides Is Taken as an Example

back to S-esters and potentially again participate in the ligation reaction, but this rearrangement can be slow.318 More problematic is the fact that peptide O-esters derived from DTT can decompose by an irreversible intramolecular mechanism leading to peptide acid byproducts (for a detailed mechanism, see sections 3.1.2.2 and 5.4.1) Reduction by TCEP. An alternative to DTT for maintaining thiols in reduced state during NCL is TCEP,319,320 which irreversibly reduces disulfides back to thiols once they are produced. Because of its excellent water solubility and high reducing power over a broad pH range, TCEP is a widely used reducing agent for both NCL and extended methodologies. TCEP is marketed as its odorless, stable in air, and highly water soluble (310 g·L−1) hydrochloride salt TCEP·HCl (Figure 26A).

frequently used as an additive in NCL for this purpose, although it has been largely supplanted by tris(2-carboxyethyl)phosphine (TCEP) in recent years (see the next section). DTT was introduced as a disulfide bond reducing agent by Cleland in 1964.314 The pKa values of the thiol groups are 9.2 and 10.1. With a redox potential of −0.33 V vs a standard hydrogen electrode at pH 7.0 and 25 °C,315 DTT is one of the most powerful reducing thiols characterized so far.309 It easily reduces Cys disulfides, mixed disulfides between Cys and alkyl or aryl thiols, and also a wide range of alkyl diselenides.315 The mechanism leading to the reduction of dichalcogenides by DTT is shown in Scheme 15 and relies on a series of reversible thiol−disulfide interchanges involving thiolate species where the cyclization of DTT to a 6-membered disulfide acts as a sink to displace the equilibrium. One important aspect to be aware of when using DTT as a reducing agent is its sensitivity to oxidation by air, either in the dry state or in solution. It is recommended to store DTT as a powder at −20 °C. The shelf life of solid DTT indicated by the manufacturers ranges from one to four years. The stability of DTT solutions is poor, and their preparation in advance or storage is not recommended. The half-life of DTT in 0.1 M potassium phosphate buffer at pH 7.5 and 20 °C is 10 h307 and drops to 1.4 h at pH 8.5. Analogous to the effect of metal ions on the rate of oxidation of simple thiols,303 the half-life of DTT is significantly reduced in the presence of metal ions such as Cu2+, Fe2+, Fe3+, or Ni2+.307,316,317 Stevens et al. reported that the half-life of DTT at pH 7.5 (20 °C) decreases from 10 h to only 0.6 h in the presence of 0.1 mM Cu2+. It is to be reminded that iron is a common contaminant of buffers and reagents.307 Because of the pKa of DTT thiols, DTT readily participates in thiol−thioester exchanges (Scheme 16). This reaction can be troublesome during NCL because S-esters derived from DTT spontaneously rearrange into O-esters. The latter can rearrange

Figure 26. (A) Structure of tris(2-carboxyethyl)phosphine (redox potential from ref 321, pKa values from ref 322). (B) Potential TCEP species involved in the reduction of disulfides around pH 3. (C) General mechanism of disulfides reduction by trialkylphosphines.

The capacity of TCEP to effectively reduce disulfide bonds in proteins was first reported by Levison et al., who compared 2mercaptoethanol, tris(hydroxymethyl)phosphine, and TCEP for their ability to reduce disulfide bonds in IgGs.323 The accepted mechanism by which TCEP reduces disulfides is similar to that of any other trialkylphosphine (Figure 26C).320 The attack of the phosphine on the disulfide results in the transient formation of a thiolate and a thiophosphonium Y


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piperazine-N′-(2-ethanesulfonic acid) (HEPES) buffers ( 300 nm. Despite the advantages of using photolabile protecting groups for the temporary masking of N-terminal Cys, such approaches are underexplored at the present time.

Figure 29. Temporary masking of N-terminal Cys is required for the elongation of peptides in the C-to-N direction using NCL. (A) Onepot assembly of two peptide segments for the synthesis of large Cys peptides, (B) One-pot assembly of three peptide segments.

structure databases shows that the highest frequency is found for domains composed of ∼50−150 amino acids.353 Thus, by providing simplified access to these protein domains, one-pot three-peptide segment assembly schemes cover many needs in academic and industrial laboratories studying protein function. The potential of one-pot approaches stimulated the adaptation of known amine or thiol protecting groups to reversible Cys protection. One important criterion for their selection is that the treatment used for removing the protecting group, as well as the derivatives resulting from removal, must not interfere with the subsequent NCL reaction. Those protecting groups that respond to this specification can be easily identified in Table 4 by looking at the “NCL/-PG/NCL” column. They can be gathered into three main classes according to the general conditions used for their removal, i.e., the addition of specific reagents, a change in pH, or by UV light irradiation, and are discussed in this order. Regarding the protecting groups that are removed upon addition of specific reagents, thiazolidine protection was the first to be used to design one-pot assembly schemes.349 This application relies on the relative compatibility of the thioester functionality with O-methylhydroxylamine, the reagent used in large excess for reversing thiazolidine (0.1−0.2 M, pH 3−4). Nevertheless, aminolysis of the thioester functionality by Omethylhydroxylamine has been reported in several studies which utilized peptidyl glycyl thioesters, 340 which are particularly sensitive to nucleophiles due to the low steric hindrance of the C-terminal glycyl carbonyl group but also for other C-terminal amino acid residues such as Tyr354 or Lys.251 As mentioned at the beginning of this section, the Acm group was among the first protecting groups used for the concatenation of peptide segments with the NCL reaction. One important limitation of its use concerned the harsh conditions AC


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Table 4. Protecting Groups for N-Terminal Cysa

AD


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Table 4. continued

a See Abbreviations. Green circles = yes; red x’s = no; − = not determined. bThese protecting groups are used to protect both N-terminal and internal Cys thiols.

3.1.3.2. Protection of Internal Cysteine Residues. Protecting groups for internal cysteine residues are usually included during SPPS of the peptide segments and must therefore survive the peptide deprotection and cleavage step in strong acid (entries 1−6, Table 5). In several cases, however, the Cys protecting group can be introduced after peptide production. Examples include the reversible trityl protecting group developed by Mochizuki et al. (entry 6, Table 5)375 or the 2carboxyethylthio (SCE) group (entry 14, Table 4) that has been utilized for facilitating the solubility of Cys-rich peptides as a result of the hydrophilicity of its pendant carboxylate group.354,373

Considering that the protection of internal Cys residues is overwhelmingly tied to ligation/desulfurization approaches, the use of the protecting groups presented in Table 5 is discussed in detail in section 7.2.3.1. 3.2. Selenocysteine

The search for more reactive groups toward acyl donors and the apparently similar reactivities of sulfur and selenium early stimulated the extension of NCL to N-terminal selenocysteinyl peptides (Sec peptides), a reaction that was reported by three different groups the same year (Scheme 22).382−384 AE


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Table 5. Protecting Groups (PG) for Internal Cys Thiolsa

See Abbreviations. Green circles = yes; red x’s = no; − = not determined. bReagent K TFA cleavage cocktail used at rt for 2 h resulted in the partial deprotection of Pocam protecting group. Deprotection at 4 °C for 4 h proved satisfactory. a

peptides are few and far between. In 2011, Mobli et al. exploited the sensitivity of the 77Se nucleus in NMR to determine the acidity constants of N-terminal and internal Sec selenols in a selenated analogue of vasopressin based on the different chemical shifts of selenol and selenolate (Figure 30).388 Similar to Cys thiols, Sec selenols were found to exhibit higher acidity when selenocysteine was located at the Nterminus of the peptide (pKa(Sec) = 3.3) than at an internal position (pKa(Sec) = 4.3), probably due to the proximity of the α-ammonium group. Note that the pKa of internal Sec selenol

Classical NCL and its Sec variant may be outwardly similar, but the markedly different physicochemical properties of Cys thiols and Sec selenols often require an adapted experimental design for Sec-based ligation. This section provides an overview of Sec properties that are crucial in this context. In addition, further information on selenocysteine can be found in important reviews written by leading experts in the field.385−387 3.2.1. Acid−Base Properties and Nucleophilicity of Selenocysteine. As for cysteinyl peptides, reports investigating the acidity of the N-terminal selenocysteine residue in AF


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in NCL. For example, Hondal et al. have shown that free selenocysteine reacted 3 orders of magnitude more rapidly than cysteine with activated aryl thioesters at pH 5.0.384 The fact that Sec selenols are significantly more reactive toward acyl donors in this case is not tied to the intrinsic nucleophilic properties of the selenium atom but simply reflects the higher acidity of selenols compared to that of thiols. At a neutral or mildly acidic working pH, the favorable, even quasi-exclusive, proportion of selenolates in solution makes Sec-based ligations more efficient than thiol-based ones in terms of acyl donor capture. 3.2.2. Controlling the Redox State of Selenocysteine during NCL. Compared to Cys thiols, the very low redox potential of Sec selenols complicates their isolation under aerobic conditions (E°′CysH/Cys−Cys = −0.23 V vs E°′SecH/Sec−Sec = −0.38 V). Consequently, Sec peptides are usually isolated as diselenides or as mixed sulfenyl−selenyl dichalcogenides.331 Even more than Cys, Sec residues can be inactivated during ligation by dichalcogenide formation if molecular oxygen is present in the reaction vessel. Therefore, NCLs with Sec peptides are best carried out in strictly inert conditions. Whereas weakly reducing aryl thiols used in large excess are sufficient to keep Cys thiols in a reduced state during NCL (see section 3.1.2), a strong reductant such as DTT or TCEP is preferably required to reduce diselenides in the starting material or those formed in situ due to the entry of molecular oxygen and maintain free selenol concentration at its maximum level throughout the ligation process. The major problem encountered with the joint use of Seccontaining peptides and TCEP is the deselenization reaction of the Sec residue to alanine with the concomitant formation of TCEP-derived selenophosphine (TCEP = Se) as described in Scheme 17 for X = Se.331 Although the application of Sec mediated ligation/deselenization approaches has resulted in significant achievements in the field of protein total synthesis, a point which is fully developed in section 7.2.2., TCEP-induced deselenization of Sec can be a serious problem when the Sec residue must be maintained in the final ligation product. In this case, the use of TCEP must be ideally complemented by the addition of a radical scavenger such as ascorbate which prevents the formation of selenyl radicals in the reaction mixture.390 Interestingly, TCEP = Se has also been shown to be an inhibitor of the TCEP-induced deselenization process when used in combination with MPAA at pH 5.5.391 These conditions enabled selenopeptides to be incubated for days in the presence of TCEP, while the use of MPAA alone resulted in substantial Sec deselenization. The inhibition was observed for concentrations of TCEP = Se above 150 mM. DTT can be a useful alternative to TCEP, especially for selenopeptide synthesis using NCL or related reactions because there is no risk of deselenization of Sec to Ala with this reducing agent.391,392 The risk of promoting thioester hydrolysis through the formation of O-esters as discussed for regular NCL (see Scheme 16) can be greatly minimized for Sec-mediated NCL, as in this case the ligation can be usually conducted below pH 7.384 Such conditions minimize thiol− thioester exchanges due to the low proportion of thiolates in the mixture and thus the formation of unwanted DTT O- or Sesters.391,392 3.2.3. Controlling the Reactivity of Selenocysteine by the Use of Protecting Groups. N-Terminal Sec protection strategies are significantly less diversified than those of their sulfur counterpart, probably owing to the moderate use of selenocysteine-mediated ligation as compared to classical NCL

Scheme 22. Selenocysteine-Mediated NCL Reaction

Figure 30. Selenocysteine analogue of vasopressin (reduced form) used for Sec selenol pKa determination using 77Se NMR.

in vasopressin is about one pKa unit lower than the selenol pKa in selenocysteine (pKa(Sec) = 5.24).389 Although the authors provide an accurate estimate of the acidity of Sec selenols in vasopressin, they recommend not generalizing these values because the acid−base properties of the Sec side chain seem highly dependent on the structural context of the Sec residue. As a result of the higher polarizability of the selenium atom compared to that of sulfur, selenols/selenolate pairs are roughly 2−5 times more nucleophilic than their respective thiols/ thiolates counterparts in substitution (SN2)389 or in carbonyl addition−elimination reactions,384 which is the type of reaction that yields the thio- or selenoester-linked intermediate in NCL (Table 6). Such a small difference in reactivity can however not account for the spectacular variations in reaction rates sometimes observed when a Sec peptide is used in place of a Cys peptide Table 6. Compared Nucleophilicity of Cys Thiol and Sec Selenol

a Maximum pseudo-first-order constants corresponding to working pH > pKa(XH) (X = S, Se). bRate constants established at working pH = pKa(Cys) = 8.30 for Cys and at pH = pKa(Sec) = 5.24 for Sec.

AG


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reactions.382−384 The protecting groups developed so far for selenocysteine were primarily developed to facilitate the synthesis of selenopeptides by SPPS (selenoethers, e.g., pmethoxybenzyl) or their handling post-SPPS (diselenides or mixed selenosulfides). These groups are, however, incompatible with multiple consecutive NCL steps as described in Scheme 19, thus precluding C-to-N concatenation of selenocysteinylpeptides. Recently, the thiazolidine protection of N-terminal cysteine residues has been extended to the development of the selenazolidine (Sez) protecting group for N-terminal selenocysteine (Figure 31).393 Similar to thiazolidines though over

the 125-residue human phosphohistidine phosphatase 1 (PHPT1) protein from three peptide segments. Sections 2 and 3 provide important information regarding the reactivity of thioesters and Cys peptides as well as those of their selenated analogues. This knowledge is essential for the comprehension of section 4, which concentrates on the mechanism of NCL.

4. DISSECTING THE INDIVIDUAL CHEMICAL STEPS IN NCL The canonical mechanism proposed for NCL in the presence of thiol additives in most publications is presented in Figure 32 and will form the basis of our discussion of this reaction. In brief, the starting thioester I or thioester III derived from it by thiol−thioester exchange through step 1 reacts with the Nterminal Cys peptide II to produce the thioester-linked intermediate IV (step 2). The latter rearranges to produce the ligation product V (step 3). This section starts with an analysis of the origin of the regioand chemoselectivity of this reaction (section 4.1) and continues with a presentation of the seminal experimental work of Hupe and Jencks on thiol−thioester exchanges (section 4.2).132 This work, as well as recent computational studies, shed light on the mechanisms involved in the catalysis of NCL by thiols (step 1, section 4.3) and the formation of the thioesterlinked intermediate (step 2, section 4.4), i.e., two processes which are basically thiol−thioester exchange reactions. Section 4.3 also discusses other types of catalysis, while the use of powerful acyl donors for accelerating the capture step is presented in section 4.4. The rearrangement of the thioesterlinked intermediate is analyzed in section 4.5. Finally, the adaptation of the NCL reaction to the synthesis of selenopeptides or selenoproteins is delineated in section 4.6.

Figure 31. Total synthesis of human phosphohistidine phosphatase 1 (PHPT1) protein using Sez protection for selenocysteine.

4.1. Origin of the Regio- and Chemoselectivity of NCL

One hallmark of the NCL reaction is its high regio- and chemoselectivity in water although the peptides are unprotected. Figure 32 presents the various functional groups in polypeptides that can potentially act as nucleophiles toward thioesters. Some of these groups have acid−base properties, i.e.,

longer reaction times, selenazolidines can be reversed by addition of O-methylhydroxylamine at pH 4−5. The synthetic utility of the Sez group was illustrated by the total synthesis of

Figure 32. Canonical mechanism of NCL. The pKas of ionizable groups are indicated in magenta (data taken from ref 294). AH


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limit the accumulation of branched thioesters VI or thiolactones VII in the mixture.268,404−407 Additionally, they also prevent the formation of cysteine-based disulfides by scavenging cysteine thiyl radicals (see section 3.1.2.1). To conclude, several factors contribute to the high regio- and chemoselectivity of NCL, among which the main ones are the reversibility of thiol−thioester exchanges, the high efficacy of the intramolecular S,N-acyl migration process at the N-terminal cysteine residue, and finally the experimental conditions (low peptide concentration, neutral pH) which disfavor the intervention of amine nucleophiles other than the α-amino group of N-terminal cysteine. Given the critical importance of thiol−thioester exchanges in NCL, a thorough examination of the mechanism of this reaction is essential for understanding its most intimate governing parameters, including the reactivity of the thioester and Cys peptide. These mechanistic aspects are presented in section 4.2.

the imidazole group of histidine, the phenol group of tyrosine, the ε-amino group of lysine, the α-amino group, the C-terminal carboxylic acid group, the side chain carboxylic acid groups of Asp and Glu, the thiol group of internal cysteines, and last but not least the thiol and amino groups of the N-terminal cysteine residue. Their pKas are indicated in magenta.294 Here we wish to discuss how the nucleophilicity of these groups toward thioesters, their ionization state at neutral pH, and the reversibility of the thiol−thioester exchange process together contribute to make this reaction a highly regio- and chemoselective chemical process. Among the nucleophiles present in the system, thiolates produced by ionization of the cysteine thiols are by far the most reactive toward thioesters, including amino groups. Therefore, mixing the peptide thioester and the Cys peptide results in a dynamic mixture of thiol and thioesters species (I, IV, VI, VII in Figure 32) that undergo reversible thiol−thioester exchange reactions. If thiol additives (e.g., aryl thiols) are included in the reaction, they also participate in the thiol−thioester exchange processes with the formation of thioester species of type III (Figure 32, step 1). It is important to bear in mind that thiol− thioester exchanges take place during NCL independently of the type of catalyst used. Each of the thioester species formed in these thiol−thioester exchanges can in principle react with the above-mentioned nucleophilic groups. However, side chain alcohol groups of serine or threonine (not shown), the phenol group of tyrosine and carboxylate groups of aspartic and glutamic residues are poor nucleophiles and do not interfere with the NCL reaction. The imidazole group of histidine probably reacts with the thioester functionality. However, the formation of N-acyl imidazole intermediates is thermodynamically unfavorable and reversible so that here again no interference is expected from histidine residues in the NCL reaction. Apart from thiolates, the other potent nucleophiles present on peptides are α- and εamino groups, which can irreversibly react with the thioester either inter- or intramolecularly to produce amide bonds. Indeed, and following the pioneering work of Wieland, the aminolysis of alkyl394 or aryl thioesters21−23,395−403 has been intensively utilized for amide coupling, including peptide cyclization.396 Such reactions are performed at high concentrations (e.g., >0.1 M) and in the presence of a base to ensure that the amine is mostly in an unprotonated form. In contrast, NCL is performed at low peptide concentrations (∼ mM) and at a pH where amino groups are mostly protonated and thus non-nucleophilic. In these conditions, intermolecular thioester aminolysis reactions are unlikely to occur to a significant extent. Although intramolecular aminolysis can potentially arise, the intramolecular S,N-acyl migration reaction that yields the native peptide V is so efficient, by proceeding through a kinetically favored 5-membered ring intermediate, that it acts as a sink and gradually directs all the thioester species toward the target product. Although some NCL reactions can be conducted in the absence of thiol additives, the presence of the latter can have several advantages. The thiol additives can catalyze the reaction when thioesters of type III, which are derived from I by thiol− thioester exchange, are more powerful acyl donors than the starting peptide thioester (Figure 32). They can also accelerate thiol−thioester exchanges and thereby facilitate the reversal of unproductive thioesters, i.e., the branched thioesters of type VI produced by acylation of an internal Cys thiol or thiolactones of type VII. One important role of these additives is therefore to

4.2. The Thiol−Thioester Exchange Reaction

As mentioned in the introductory remarks, thiol−thioester exchanges play a crucial role in NCL by permitting dynamic acyl transfers between Cys thiols and possibly thiol additives when present. Determining the intimate nature of the thiol− thioester exchange mechanism is challenging, yet many computational and experimental studies have been devoted to understanding S,S-acyl transfer reactions.408 The most widely accepted mechanism for reversible S,S-acyl transfer reactions between thiols and thioesters is a stepwise process involving thiolate nucleophiles with the formation of a tetrahedral intermediate (TI in Scheme 23). Scheme 23. Thiol−Thioester Exchange Reaction Is Reversible and Probably Proceeds through a Tetrahedral Intermediate

In a seminal paper, Hupe and Jencks analyzed the reaction of a series of alkyl and aryl thioesters derived from acetic acid with various alkyl and aryl thiols.132 The authors reported a Brønsted-type linear free energy relationships analysis which provided important insights into structure−reactivity relationships and the nature of the rate-determining steps (Figure 33). In this experiment, p-nitrothiophenyl acetate was reacted with a series of thiols with pKas ranging from 2.68 (C6F5SH) to 10.35 (EtSH), and the logarithm of the second-order rate constant k was expressed as a function of the pKa of the attacking thiol. The obtained Brønsted plot is obviously biphasic with a breakpoint at a pKa value of 4.50. The sensitivity factor βnuc is a measure of the relationship between the nucleophilicity of the thiol nucleophile and its basicity. It corresponds to the slope of the lines on both sides of the breakpoint. For thiols with a pKa < 4.50, the sensitivity factor is βnuc = 1, meaning that nucleophilicity completely reflects any change in basicity. A sharp change in slope is observed for thiols with a pKa > 4.50, for which the sensitivity AI


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Figure 33. Brønsted plot of the reaction of p-nitrothiophenyl acetate with various thiols. The logarithm of the second-order rate constant k is expressed as a function of the pKa of the attacking thiol. The continuous curve was produced by using the equation derived from the steady-state treatment of intermediate TI (see Scheme 23). The equation and the data were collected from ref 132.

Figure 34. Proposed reaction profile for a stepwise thiol−thioester exchange as a function of the pKa of attacking and departing thiols (black, symmetrical reaction profile; blue, step 1 is rate-determining; red, step 2 is rate-determining; R, reagents, P, products; TI, tetrahedral intermediate; TS′, transition state leading to the formation of TI; TS′′, transition state for the decomposition of TI to P.

factor βnuc decreased to 0.27. In this case, variation of thiol pKa has a less pronounced impact on nucleophilicity for acyl transfer. The biphasic Brønsted plot shows that a change in ratedetermining step occurs by decreasing the pKa of the attacking thiol below 4.50, which coincides with the pKa of the departing p-nitrophenylthiol. An equation derived from the steady-state treatment of intermediate TI, i.e., the continuous curve in Figure 33, permitted an excellent fit of the data, a fact that strongly supports the existence of the tetrahedral intermediate TI (Scheme 23). The authors further determined the effective charges developing on the sulfur atoms along the reaction coordinate. This analysis supports the existence of a tetrahedral intermediate in which “most of the electron-withdrawing character of the carbonyl group is lost”.132 Note, however, that a change in rate-determining step is not exclusive to reactions involving two consecutive steps with an intermediate but can also indicate an asymmetric reaction coordinate profile typical of concerted asynchronous mechanisms (also referred to as one-step multistage). To better visualize the impact of thiol pKas (thiol nucleophile, departing thiol) on the reaction coordinate, imagine a model thiol−thioester exchange in which both the nucleophile and the leaving group are identical thiols. In such a situation, the reaction profile is perfectly symmetrical, with equal reaction rates for the decomposition of the tetrahedral intermediate TI to the reagents R and to the products P (R1 = R2, k−1 = k2, black reaction coordinate in Figure 34). This is exactly the situation at the breakpoint in the Brønsted plot shown in Figure 33. Any structural change in the nucleophile or the leaving group would affect the entire reaction profile, and

either k−1 or k2 could become rate-determining, and hence induce an asymmetry in the kinetic profile (blue and red reaction coordinates, respectively, in Figure 34). An important conclusion is that the mechanism of the thiol−thioester exchange reaction is governed by the difference in pKa between the conjugated acids of the nucleophile and the leaving group and is independent of the nature (alkyl or aryl) of the thiol. These mechanistic considerations and reactivity rules are important to consider each time thiol−thioester exchanges are in play. They enable the rationalization of the reactivity of peptide thioesters toward thiol nucleophiles, the role of thiol catalysts in NCL and the difference in reactivity between Cys thiols, whose pKa can vary significantly depending of their environment. 4.3. Nucleophilic Catalysis

Historically and even now, peptide alkyl thioesters are popular acyl donors for protein synthesis due to their ease of synthesis and good stability. However, the intrinsic average reactivity of peptide alkyl thioesters and the mM concentrations of the peptide segments used in NCL generally do not enable the achievement of fast ligation rates unless special protocols are applied. Protein chemists have therefore explored different means of promoting the NCL with peptide alkyl thioesters to broaden the scope of this reaction and minimize byproduct formation. As will be discussed in more detail in section 4.5, the rearrangement of the thioester-linked intermediate, i.e., step 3 in Figure 32, is not rate-limiting in NCL. Therefore, accelerating NCL means accelerating the formation of the AJ


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Figure 35. Nucleophilic catalysis of the NCL reaction.

Thiophenol was the first aryl thiol used to catalyze the NCL reaction (entry 1, Table 8).409 It has the disadvantage of being sparingly soluble in water and has a pungent unpleasant smell. It was often used in combination with more reducing alkyl thiols such as benzylmercaptan (see entry 4, Table 9). Compared to thiophenol, MPAA is almost odorless and is highly soluble in water at neutral pH (entry 2, Table 8).310 It is often used in combination with TCEP as a reducing agent. MPAA is commercially available at an affordable price. Among the few aryl thiols catalysts that have been designed so far, MPAA is the most widely used and is now considered as the gold standard for performing NCL reactions. It nevertheless has some limitations, in particular, its presence can complicate the purification of the ligation product by RP-HPLC, especially when the target peptide and MPAA elute close by. It is therefore preferable to remove MPAA from the mixture prior to the purification step. This can be done by extracting the aqueous mixture with diethyl ether after acidification because MPAA is poorly soluble in water below pH 5.66,234,410 This protocol is, however, not applicable to all protein targets and must be evaluated case by case. 4-Hydroxythiophenol is another water-soluble catalyst of interest which has a pKa close to MPAA and is almost as efficient as MPAA in catalyzing the NCL reaction (entry 3, Table 8).310 Recently, a sulfonated analogue (3-MBSA, entry 4, Table 8) has been developed in an attempt to minimize the interference of the thiol catalyst during the HPLC purification step.311 3MBSA required a 5-step synthesis from p-nitrobenzyl chloride and was found to be less potent as a catalyst compared to MPAA. Nevertheless, 3-MBSA is less hydrophobic and elutes earlier by RP-HPLC than MPAA, a property which enabled the direct purification of a ligation mixture by HPLC. Regarding the mechanism of action of aryl thiol catalysts, the work of Hupe and Jencks discussed in section 4.2 suggests the stepwise process depicted in Scheme 24 for the in situ formation of the peptide thioaryl ester from a peptide thioalkyl ester in the presence of the aryl thiol. The pKa of the aryl thiol nucleophile is well below the pKa of the leaving alkyl thiol group. In these conditions and bearing in mind the general reaction coordinate profile described in Figure 34, the breakdown of the tetrahedral intermediate TI to produce the peptide thioaryl ester product is less favorable than its breakdown back into the reactants (k 2 < k −1 ). The concentration of the tetrahedral intermediate TI dictates the rate of peptide thioaryl ester product formation. The formation

thioester-linked intermediate. One successful strategy for achieving this goal consists in converting the starting peptide alkyl thioester into a more powerful acyl donor in situ during NCL. This can be achieved by performing the NCL in the presence of additives which act as nucleophilic catalysts (step 1 in Figure 32). Nucleophilic catalysis is defined by the International Union of Pure and Applied Chemistry (IUPAC) as the action of a Lewis base (thiol, imidazole...), which accelerates a reaction by forming a Lewis adduct with a Lewis acid (thioester). While Figure 32 presents the canonical mechanism for NCL in the presence of a thiol catalyst, it does not embrace all the types of nucleophilic catalysts developed so far for this reaction. A more general scheme is found in Figure 35. The aim of this section is to discuss the first step of this process, i.e., the in situ addition of nucleophiles to the thioester that results in the production of more powerful acylating species. 4.3.1. Catalysis by Aryl Thiols. Compared to peptide alkyl thioesters, peptide aryl thioesters are powerful acyl donors due to the enhanced leaving group capabilities of aryl thiols. As a consequence, the thiol−thioester exchange reaction of aryl thioesters with simple thiols, which can be considered approximately as mimics of Cys thiols, proceeds significantly faster than with alkyl thioesters (Table 7). Typically, the Table 7. Second-Order Rate Constants kex for the Thiol− Thioester Exchange Reaction of S-Methyl Thioacetate and SPhenyl 5-Dimethylamino-5-oxo-thiopentanoate with MESNaa,b entry

thioester

pKa of departing thiol

kex (M−1·s−1)

1 2

CH3CO-SCH3 (CH3)2NCO(CH2)3CO-SPh

10.34 6.43

1.7 120

See Abbreviations. bRateex = kex · [RS−] · [thioester]. The data were taken from ref 133. a

second-order rate constant for the exchange of S-phenyl 5dimethylamino-5-oxo-thiopentanoate with MESNa is 70-fold higher than that obtained with S-methyl thioacetate.133 The higher reactivity of thiophenyl esters toward alkyl thiols compared to alkyl thioesters has stimulated the development of several aryl thiol additives that enable the in situ formation of reactive aryl thioesters from alkyl thioesters by thiol−thioester exchange and thus catalyze the NCL reaction as shown in Figures 32 and 35. These reagents are listed in Table 8. AK


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Table 8. Aryl Thiol Additives Used for Catalyzing the NCL Reaction with Thioestersa

a

See Abbreviations.

Scheme 25. Thiol−Thioester Exchange of N-Formyl Glycine Thiomethyl Ester by Thiophenolate

Scheme 24. Proposed Mechanism of Action of Aryl Thiol Catalysts According to Hupe and Jencks Studies of S,S-Acyl Transfer Reactions

of the latter can be facilitated by the use of a large excess of the aryl thiol catalyst. This is exactly what is done in practice with concentrations of aryl thiol catalyst frequently in the range 100−200 mM, while peptides are ligated at a concentration of a few mM. Although kinetically favorable (k−2 > k2), the reverse attack of the alkyl thiolate on the aryl thioester III is probably disadvantaged by the low concentration of alkyl thiolate in the reaction mixture. The mechanism proposed in Scheme 24 can be compared to a computational study of the thiol−thioester exchange reaction using N-formyl glycine thiomethyl ester and thiophenol as model compounds (Scheme 25).414 The authors examined different pathways (neutral or anionic stepwise and neutral or anionic concerted mechanisms) at the B3LYP/6-31+G* level of theory using the polarized continuum model (PCM) for the implicit inclusion of water. Among these, only the concerted pathways permitted the isolation of TSs (Scheme 25). The TSs associated with the concerted neutral and anionic mechanisms

displayed distinct structural features, most significantly a much shorter ArylS---CO bond in the anionic TS than in the neutral TS. Given that the concerted neutral pathway has a significantly higher activation barrier (49.7 kcal·mol−1) than that of the anionic pathway (29.4 kcal·mol−1) and that the thiophenolate anion is the predominant species at pH 7 in water, the authors concluded that the anionic concerted pathway is more likely to occur. Interestingly, the structure of the anionic concerted TS shows significant differences in bond order to the carbonyl between the MeS− leaving group ((CO)−S bond length: 2.845 Å) and the nucleophile PhS− ((CO)−S bond length: 1.863 Å). This dissymetry mirrors the differences in nucleophilicity/leaving group ability between MeS− and PhS− AL


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Table 9. Alkyl Thiol Additives Used for Catalyzing the NCL Reaction

facilitated by using an excess of the alkyl thiol additive. Compared to aryl thiols, alkyl thiols are usually less acidic. Therefore, they are better nucleophiles, but the resulting peptide thioesters are less potent acyl donors than peptide aryl thioesters. MESNa is a widely used thiol additive in the field of protein chemical synthesis (entry 1, Table 9) and also widely used for preparing peptide thioesters.72,416 It is a modest leaving group due to its high pKa and thus a modest catalyst when compared to thiophenol or MPAA as mentioned by several studies.310,311 Recently, Huang et al. evaluated the interest of methyl thioglycolate (MTG) for catalyzing the NCL reaction (entry 2, Table 9).370 As expected from pKas, the rate of NCL in the presence of MTG was slower (∼1.8-fold) than in the presence of MPAA. In a different approach, Thompson et al. illustrated the positive value of trifluoroethanethiol (TFET) for catalyzing the NCL reaction (entry 3, Table 9).210 The pKa of TFET (7.30) is slightly higher than the pKa of thiophenol (6.43). Consequently, TFET is a slightly better nucleophile than thiophenol for the thiol−thioester exchange reaction with pnitrothiophenyl acetate (second-order rate constant at 25 °C, TFET 4.9 × 103 M−1·min−1, thiophenol 2.16 × 103 M−1· min−1).132 Conversely and as expected, the second-order rate constant for the reaction of mercaptoethanol with thiophenyl acetate (3.39 × 103 M−1·min−1) is slightly higher than with trifluoroethanethiol acetate (3.10 × 103 M−1·min−1).132 In practice, TFET proved to be a powerful catalyst, albeit not as effective as MPAA at the same concentration. Interestingly, the volatility of TFET was exploited advantageously for designing one-pot ligation metal-free desulfurization schemes. Finally, benzylmercaptan has often been used in combination with thiophenol in NCL reactions (entry 4, Table 9). It is unlikely that benzylmercaptan contributes to catalysis to a significant extent because of its high pKa. It was primarily used to increase the reducing power of the mixture, thereby minimizing the formation of disulfides with Cys thiols (benzylmercaptan is a better reducing agent than thiophe-

and hence the difference in pK a values between the corresponding conjugate acids. The isolation of a concerted transition state with asynchronous bond formation is compatible with the experimental analysis of Hupe and Jencks,132 despite the fundamental differences between stepwise and concerted mechanisms. Similar conclusions were drawn recently by Sun et al. using the Minnesota functional M06-2X with the 6-31G* basis set.415 However, it must be remembered that these computational studies were performed using implicit solvent models and thus must be regarded critically because water was not explicitly involved in the mechanism. Note that the NCL reaction is often performed using peptide thioaryl esters as acyl donors, such as peptide thioesters derived from MPAA, whose synthesis is greatly facilitated by the hydrazide method.201 In this case, the first step depicted in Figure 32 corresponds to an exchange between the peptide thioaryl ester and the exogenous aryl thiol. Unless the incoming and departing aryl thiols differ significantly in their structure and pKa, this is a perfectly symmetrical situation according to Hupe and Jencks theory and the exchanging peptide thioaryl esters have the same reactivity toward the Cys peptide in the capture step. 4.3.2. Catalysis by Alkyl Thiols. The theory of Hupe and Jencks is independent of the structure of the thiol components (see Figure 34) and therefore can be used to discuss the mechanism of action of alkyl thiol additives as well. An alkyl thiol can catalyze the NCL reaction if it is less basic than the alkyl thiol used to produce the peptide thioester. A way to achieve this is to use alkyl thiol additives that have an electronwithdrawing moiety close to the thiol group (pKa < 9.5), while the peptide thioester is derived from simple alkyl thiols with a pKa around 10.5, e.g., thioesters derived from 3-mercaptopropionic acid. This feature is indeed shared by all the alkyl thiol catalysts listed in Table 9. In this case, the mechanism of the thiol−thioester exchange reaction depicted in Scheme 24 for aryl thiol catalysts applies to these alkyl thiol catalysts as well: the tetrahedral intermediate preferably breaks down into the reactants and the formation of the thioester exchange product is AM


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Scheme 26. Potential Participation of “Unproductive” Thioesters to the NCL Reaction

nol309). Likewise, ethanethiol has been used as an alternative to benzylmercaptan in the synthesis of hydrophobic proteins.417 The renewed interest in the use of alkyl thiols such as MESNa, MTG, or TFET for catalyzing the NCL reaction stems in part from their modest thiyl radical scavenging properties in comparison to aryl thiols. Therefore, the catalysis of the NCL reaction by alkyl thiols has the potential advantage of simplifying the development of one-pot ligation/metal-free desulfurization protocols. This point is the focus of section 7.2. 4.3.3. Catalysis by Internal Cysteine Thiols. So far, we have discussed the performance of the NCL reaction in the presence of thiol additives acting as nucleophilic catalysts. Whether thiol additives are present or not, at least one other type of thiol is present during the reaction, i.e., the thiol from the N-terminal Cys residue in peptide segment II (Scheme 26). Note that this thiol undergoes a shift in pKa of about −1.5 units compared to that of internal Cys residues as discussed in section 3.1.1. The synthetic design of proteins frequently requires the assembly of peptide segments containing one or more internal Cys thiols in addition to the N-terminal one. In such a situation, all internal thiols can participate in thiol− thioester exchanges and thus to the formation of branched thioesters of type VI whose role and reactivity in NCL reactions has to be clarified (Scheme 26). The work of Heller et al. provides pertinent information regarding the reactivity of thioesters of type VI in comparison with thioesters derived from simple alkyl thiols.420 The authors compared the rate of hydrolysis of ethyl thioacetate CH3COSEt and Ac-Gly-Cys(Ac)-Gly and found that the latter hydrolyzed 20 times more rapidly than the former, suggesting that branched thioesters of type VI are potentially more reactive than classical peptide alkyl thioesters. The evidence for the capacity of internal thioesters to behave as powerful acyl donors in NCL was clearly established in a recent study by Tsuda et al. The authors showed that Cys-rich peptide segments can be ligated in the absence of catalyst at a rate comparable to those obtained in the presence of MPAA or thiophenol catalysts (100 mM) (Scheme 27A). 421 A thiolactone as well as a thioester derived from Ac-Cys-NH2 were used to investigate the acyl donor capabilities of the intermediates that are potentially formed during NCL in the absence of aryl thiol additives. These thioesters were significantly more reactive during NCL than a classical alkyl thioester, i.e., peptidyl-mercaptopropionyl-Leu-NH2 but nevertheless less reactive than a thioester derived from MPAA. The

Scheme 27. Acyl Donor Capabilities of Cys-Branched Thioesters in Aryl Thiols Catalyst Free NCL: (A) Comparison of Cys Derivatives with Thiolactone, Alkyl and Aryl Thioesters; (B) Compared Reactivity of PG-Cys-LeuNH2-Derived Thioesters

authors noted a good correspondence between the pKa of the departing thiol (mercaptopropionamide ∼10.5, Cys thiol ∼8.5, MPAA 6.6) and the rate of NCL, in agreement with Jencks’s studies.132 The good acyl donor properties of thioesters derived from Ac-Cys-NH2 stimulated the same team to further investigate the reactivity of peptide thioesters derived from PG-Cys-Leu-NH2 (PG = acetyl, trifluoroacetyl, mesyl, tosyl), AN


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the trifluoroacetyl analogue being among the most reactive (Scheme 27B).422 The enhanced reactivity of thioesters derived from Tfa-Cys-Leu-NH2 dipeptide compared to the other analogues is probably due to the superior electron-withdrawing power of the trifluoroacetyl group, which lowers the pKa of the nearby Cys thiol. Another study that is highly relevant for discussing the enhanced reactivity of branched thioesters of type VI (Scheme 26) in comparison with classical peptide thioesters has been published recently by Schmalisch et al. (Scheme 28).423 In this

Scheme 29. Imidazole Catalysis of Nucleophilic Addition to Thioesters

at neutral pH from peptide thioesters also motivated the use of imidazole or related compounds for promoting the NCL reaction. For example, Sakamoto et al. showed that imidazole can catalyze the NCL of peptide alkyl thioesters with Nterminal Cys peptides if used at high concentrations (2.5 M).428 The need for large concentrations of imidazole in order to obtain synthetically useful rates is due to the fact that the acyl imidazole intermediate is usually not thermodynamically favored over the thioester unless large concentrations of imizadole are added to the ligation mixture. In line with these observations, Sakamoto et al. showed that 1,2,4-triazole (2.5 M, pH 7.1, 37 °C) can also facilitate the reaction between Npeptidyl N′-methyl benzimidazolinones and cysteinyl peptides.429 The development of NCL catalysis by N-type nucleophiles is primarily stimulated by the need to simplify the implementation of one-pot sequential NCL-desulfurization protocols. Indeed, and contrary to gold standard aryl thiol catalysts such as MPAA, imizadole, and related compounds do not interfere with the metal-free desulfurization of Cys into Ala (see section 7.2). A nice application of this type has been recently reported by Chisholm et al., who could implement the sequential NCL/ desulfurization process in flow by using N-methylimidazole catalysis for the ligation step.430 This microfluidic technique is described in more detail in section 6.1.1 (Scheme 59) as it is primarily intended for the production of difficult junctions. The fact that imidazole is not a powerful nucleophile toward thioesters makes the intermolecular reaction of histidine residues with thioesters inefficient at the peptide millimolar concentrations used for ligation chemistries. This was confirmed by Zhang and Tam who showed that no reaction occurs when N-terminal histidyl peptides are mixed with peptide alkyl thioesters (Scheme 30).431 In other words, the Nterminal imidazole group of histidine cannot capture peptide thioesters in the same way as the side chain Cys thiol to produce X-His peptide bonds after rearrangement. In contrast, the use of more reactive peptide perthioesters produced by reacting peptide thioacids with Ellman’s reagent proved useful in this regard (Scheme 30). 4.3.4.2. Catalysis by Phosphines. Trialkyl phosphines are known to be powerful catalysts for acylation reactions. For example, Vedejs and Diver showed that tributyl phosphine catalyzes the acylation of alcohols by anhydrides432 and other acyl donors. This reaction most likely involves the formation of acyl phosphonium R3P+C(O)R species.433 The acylation of alcohols by aryl thioesters in the presence of trialkyl phosphines as catalysts is also a particularly efficient reaction, whereas about 10% only of the target ester is produced in the absence of catalyst (Scheme 31). The effect of TCEP concentration on the kinetics of NCL was recently reexamined by Tsuda et al.421 This study showed a significant increase of the rate of NCL up to 50 mM of TCEP, while the rate plateaued above this concentration. In addition,

Scheme 28. An Internal Cys Thiol Activates the Thioester Functionality by Intramolecular Thiol−Thioester Exchange

work, thioesters containing a 3-mercaptopropionyl cysteinyl thiol handle (I in Scheme 28) were found to be significantly more reactive in the NCL reaction than thioesters containing a 3-mercaptopropionyl alanyl thiol handle. The presence of a Cys thiol in the thiol limb of the starting peptide thioester I enables an intramolecular S,S-acyl migration to take place in situ, yielding peptide thioester VI′, which apparently is a more powerful acyl donor than its precursor I. The peptide thioester of type VI′ can be considered as a model of the branched peptides that can form in situ during NCL, i.e., VI in Scheme 26. Therefore, this work, as well as the other studies mentioned in this section, provides a strong indication that internal Cys thiols are active players in the NCL reaction by accelerating thiol−thioester exchanges. 4.3.4. Non-Thiol-Based Catalysts. The aim of this section is to discuss the nucleophilic catalysis of NCL by compounds other than thiols. Thus far, recapitulating the nucleophilic and leaving group properties of thiol catalysts by nonsulfur-based additives has seldom been explored. The main class of catalysts that have been utilized in this context is nitrogen-based heterocycles (e.g., imidazole), followed to a much lesser extent by phosphorus-based nucleophiles (e.g., phosphines). 4.3.4.1. Catalysis by Imidazole or Related Compounds. Imidazole catalysis of nucleophilic addition to thioesters has been the subject of numerous studies424,425 and is known to proceed through the formation of an N-acyl imidazole intermediate that subsequently reacts with the nucleophile (Scheme 29). The capacity of imidazole to promote the addition of N or O nucleophiles to thioesters has found useful applications in peptide chemistry, in particular for the synthesis of cyclic peptides.426,427 The knowledge of the powerful acylating properties of N-acyl imidazoles and of their ease of formation AO


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Scheme 30. Principle of Histidine Ligation Developed by Zhang and Tam431

Figure 36. Ligation of peptidyl thio- and selenoesters LYRAFS(Se)Alk to CFRANK.

thioesters. The main properties of selenophenol are depicted in Table 10. The higher reactivity of peptide alkyl selenoesters in comparison to peptide alkyl thioesters and the interest of selenophenol as an additive was also observed for peptides equipped with a C-terminal proline residue (see section 6.1). The greater reactivity of selenoesters versus thioesters in NCL is due to the fact that selenoesters are better acyl donors than thioesters toward thiol289 or amine nucleophiles (see section 2.4).276,278 In particular, the work of Makriyannis et al. on the reaction of benzoylselenocholine Ph-COSeCH2CH2N+(CH3)3 with model alkyl thiols (cysteine, homocysteine, choline thiol) clearly established that a very rapid Se,S-acyl transfer reaction takes place at neutral pH with the production of benzoyl thioesters.289 The reaction was so quick that the rate constants could not be measured. Apart from these important studies on selenoester reactivity toward S or N nucleophiles, little or no experimental or computational studies are available on Se,S-acyl transfer reactions. Therefore, a detailed discussion of the mechanism of these reactions cannot be made. Regarding the chemoselectivity of the acyl transfer step, observations tend to suggest that the reactivity of selenoesters in acyl transfer processes increases with the softness of the nucleophile. In other words, selenoesters are expected to preferably acylate thiol nucleophiles over oxygen or amine nucleophiles naturally present in the ligation mixture, including the solvent. Recent studies by Ghassemian et al.291 on the general behavior of peptidyl selenoesters have confirmed that, despite a slightly more pronounced susceptibility to hydrolysis, peptidyl selenoesters are highly attractive acyl donors in NCL reactions.285

Scheme 31. Acylation of Alcohols by Thioesters Is Catalyzed by Trialkyl Phosphines

the authors compared TCEP, thiophenol, and MPAA for their ability to promote the ligation of short Cys-rich peptide segments. The MPAA-free TCEP-catalyzed NCL proceeded at a rate comparable to MPAA-catalyzed NCL, suggesting a significant implication of phosphines in these conditions. In this case, however, the potential participation of internal Cys thiols cannot be excluded, a point that is discussed in more detail in section 4.3.3. 4.3.5. Nucleophilic Catalysis of Peptide SelenoesterBased NCL. The majority of NCLs are performed using peptide thioesters as acyl donors. The quest for more powerful acyl donors that could substitute thioesters in NCL while providing better rates is an important goal, especially for accelerating protein synthesis or facilitating the formation of difficult junctions (see section 6.1). Recent studies have shown that thioesters can be advantageously substituted by selenoesters in NCL reactions due to their higher reactivity toward Cys peptides. The first illustration of this effect was described by Alewood and Durek in 2011.285 In particular, the reactivity of alkyl thioand selenoesters was compared in model ligation experiments (Figure 36). The selenoester reacted only 1.9 times more rapidly than the thioester in the same experimental conditions using MPAA as an additive (condition A). In contrast, replacing MPAA by benzeneselenol resulted in a further ∼9-fold increase in the reaction rate (condition B). In this case, however, the reaction was complicated by the accumulation of a branched thioester involving the internal Cys residue. This side reaction is due to the poor nucleophilicity of selenophenol, which cannot react with alkyl thioesters and thus reverse unproductive

4.4. Formation of the Thioester-Linked Intermediate

4.4.1. The Capture of Peptide Aryl Thioesters. In the previous section 4.3, we analyzed in detail how a peptide alkyl thioester is transformed in situ into a more powerful acyl donor due to the presence of a nucleophilic catalyst in the reaction mixture. The vast majority of NCL reactions utilize aryl thiols in this regard, resulting in the formation of a peptide aryl thioester (see section 4.3.1, Scheme 24). Therefore, we will limit the discussion on the mechanism of the capture step, i.e., step 2 in Figure 32, to this particular case (Scheme 32). According to the Hupe and Jencks theory of thiol−thioester exchanges, the situation is balanced because the pKa of the nucleophile, i.e., the N-terminal Cys thiol, and that of the AP


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Table 10. Properties of Selenophenol for Catalyzing the NCL with Alkyl Selenoestersa

a See Abbreviations. bAmong the rare pKa values that have been reported for PhSeH, this value is probably the most accurate and was confirmed by a recent computational study.435 A pKa value of 5.9 for PhSeH was determined using an electrochemical method.436 However, this value has been questioned.437

thiol-thioester exchange of N-formyl glycine thiomethyl ester by thiophenol discussed in section 4.3.1 (Scheme 25), the transthioesterification step preferably proceeds through a concerted anionic mechanism. Importantly, the free energy of activation for this process (ΔG⧧ = 24.9 kcal·mol−1) is lower than for the reaction of N-formyl glycine thiomethyl ester with thiophenol (ΔG⧧ = 29.4 kcal·mol−1, Scheme 25), thereby confirming that the latter process is rate-limiting in NCL. 4.4.2. The Capture of Peptide Aryl Thioesters: The Special Case of Kinetically Controlled Ligations. As discussed in detail in section 4.4.1., the capture step leading to the formation of the thioester-linked intermediate (step 2 in Figure 32) proceeds significantly faster with a peptide aryl thioester than with a peptide alkyl thioester because an aryl thiol is a better leaving group than an alkyl thiol. In consequence, if both types of peptide thioesters are mixed with a Cys peptide at neutral pH, the peptide aryl thioester will react much faster with the Cys peptide than the peptide alkyl thioester. This difference in reactivity can be exploited for accessing peptide alkyl thioesters from two shorter peptide segments derivatized as aryl thioester and alkyl thioester functionalities (Scheme 33). This process is called a kinetically controlled ligation (KCL438).404,439 An important condition for reaching the best selectivities is to conduct ligation in the absence of an aryl thiol catalyst, which would otherwise activate the alkyl thioester. A second ligation step can optionally be triggered in one pot by adding the aryl thiol together with the third peptide segment.438 A problem that was observed during KCLs is the formation of branched products due to the reaction of the aryl thioester segment with internal Cys thiols (Scheme 34).405,440 Another complication arises from the formation of intermediate thiolactones by reaction of an internal Cys thiol with the Cterminal alkyl thioester moiety.405,407,438 Such thioester linkages are not usually observed during NCL because they are reversed by the exogeneous thiol additive such as thiophenol or MPAA. In the case of KCLs, a nucleophilic thiol additive (e.g., an alkyl thiol such as MESNa) can be added after the ligation to reverse branched peptides and thiolactones (Scheme 34). The Cterminal thioester group of the product is concomitantly exchanged during this step. Note that the efficiency of KCLs critically depends on the nature of the C-terminal amino acid residues bearing the thioester functionalities.441 The use of rapid-reacting amino acid residues for the alkyl thioester segment (glycine, histidine,

Scheme 32. Mechanism of the Capture Step with Peptide Aryl Thioesters

leaving thiophenolate group are similar. In this particular situation, we can predict that the breakdown of the tetrahedral intermediate into the starting species or into the products will occur at about the same rate, i.e., k−1 ∼ k2 in Scheme 32. More useful information is provided by the computational study of Wang et al., who modeled the transthioesterification step of N-formyl glycine thiophenyl ester by free cysteine at the B3LYP/6-31+G* level of theory using the polarized continuum model (PCM) (Figure 37).414 As in the case of the computed

Figure 37. Computational study of the reaction of N-formyl glycine thiophenyl ester with cysteine. The bond lengths indicated for the TS are in angstroms. AQ


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concentrations of the thioester and Cys functionalities. As long as the rate-limiting step of NCL is the in situ production of the thiophenyl ester, i.e., step 1 in Figure 32, increasing the rate of the capture step (step 2 in Figure 32) will have no effect on the global rate of the ligation process. However, in certain circumstances, such as low peptide concentration, the capture step can become rate-limiting. In this case, increasing the effective concentration of the reactive functionalities is a powerful means of accelerating the ligation process. This can be achieved by exploiting specific conformational or self-assembly properties of the ligated peptides which force the reactants to be in close proximity. Such reactions are called template- or folding-directed ligations. In template-assisted ligations, the two segments to be ligated do not interact with each other but with a template present in the ligation mixture (Figure 38). When the template is also the

Scheme 33. Principle of Kinetically Controlled Ligations (KCLs438) Relying on the Difference of Reactivity between Aryl and Alkyl Thioesters

Scheme 34. Side Reactions Occurring during KCLs with Aryl Thioester and Alkyl Thioester Functionalities

Figure 38. Principle of template-assisted ligations with autocatalysis by the ligation product.

product of the ligation as described in Figure 38, the reaction is autocatalyzed. The first example of a template-assisted and autocatalyzed ligation was described by Lee et al. in 1996,442 shortly after the introduction of the NCL reaction. In this study, a 32-residue peptide based on the leucine-zipper domain of GCN4 was shown to serve as a coiled coil-based template for its own assembly by NCL. Autocatalysis of the reaction by the ligation product was unambiguously demonstrated by performing several control experiments involving the addition of mutated templates or the use of a denaturant (Gn·HCl). At the same time, the self-replication of peptides through coiled-coil templating and autocatalysis was also investigated by Yao et al.443 In this work, the coiled-coil peptide system was pHdependent, with maximal self-association occurring at pH 4.0. Interestingly, autocatalysis was also found to be maximal at pH ∼ 4.0 and was inhibited by the addition of trifluoroethanol, a

or cysteine246) or of slow-reacting amino acids for the aryl thioester (valine, isoleucine, proline, or threonine) complicates the implementation of KCLs. In practice, KCLs are limited to the use of alkythioesters derived from sterically hindered amino acids that are combined with aryl thioesters derived from unhindered residues, i.e., Thr and Ala respectively in the work of Mandal et al.407 on the synthesis of a crambin analogue. 4.4.3. Folding or Template-Assisted Ligations. The rate of the capture step leading to the formation of the thioesterlinked intermediate, i.e., step 2 in Figure 32, depends on the AR


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GPCR with the thioester construct resulted in the rapid and selective labeling of the membrane receptor. The scope of the method was further extended to different dyes and GPCRs (for a recent review on the labeling and single-molecule methods to monitor G protein-coupled receptor dynamics, see ref 454), and the critical role of the aryl thioester for achieving high labeling yields was emphasized.455,456 4.4.4. Intramolecular Ligations. NCL is intensively used for the production of cyclic peptides as a way of improving the stability and/or the biological activity of linear polypeptides as well as creating novel protein architectures.46−52,407,457 The cyclization reaction has often to overcome the entropic cost for achieving disfavored precyclization conformations, and factors such as polypeptide chain length or local restrictions on flexibility (e.g., proline residues) can significantly affect the ease of macrocyclization. These aspects and the scope and limitations of NCL for accessing cyclic peptides and proteins has been discussed in leading reviews and will not be detailed here.53−55 Instead, we would like to highlight two specific cases by starting with the work of Tam and co-workers, who showed that internal Cys residues can make a positive contribution to the rate of peptide cyclization by NCL in the absence of an exogenous thiol additive, as exemplified by the synthesis of circulins A and B.46,458 In a control experiment, the protection of the internal Cys residues reduced the rate of cyclization of peptides corresponding to the sequences of circulins A and B by a factor of 100. Importantly, these experiments were carried out in denaturing conditions, thereby ruling out the potential intervention of conformational effects. This result was interpreted as the consequence of intramolecular thiol− thioester exchanges and the formation of transient thiolactones which ultimately led to the formation of a thiolactone with the N-terminal Cys residue through step-by-step ring expansions and thus to the backbone-cyclized peptide after S,N-acyl migration (Scheme 35). This mechanism, named thia-zip cyclization, is consistent with the fact that the end-to-end distance in a random polypeptide chain is larger than the distance between the C-terminus where the thioester group is located and any of the internal Cys thiols. Therefore, the formation of an internal thioester by reaction of the C-terminal thioester function with an internal Cys thiol is more probable than the direct formation of a thioester with the more distant N-terminal Cys residue. The thia-zip mechanism described above can be further discussed in the light of these recent investigations regarding the reactivity of branched thioesters derived from internal Cys residues (see section 4.3.3). The latter assist peptide cyclization by an entropically driven ring−chain expansion involving thiolactone formation of increasing ring size. In addition to decreasing the end-to-end distance, these intermediate thiolactones are probably more reactive toward thiol nucleophiles than the starting linear alkyl thioester. A combination of thiolactone ring size expansion and higher reactivity synergistically assists the formation of the cyclic peptide. Note that the thia-zip mechanism can assist peptide cyclization when the thiolactone intermediates are easily accessible to internal Cys thiols. This is not always the case and the steric crowding of thiolactone functionalities can sometimes complicate the backbone cyclization of Cys-rich peptides due to the accumulation of poorly reversible thiolactone species.459

solvent known to disrupt coiled-coil formation. More recently, strong templating and autocatalytic effects have also been noted in the replication of short β-sheet peptides.444 It is to be noted that the template-assisted NCL reaction has been successfully used in nucleic acid chemistry as well. In this case, the high specificity and efficiency of nucleic acid hybridization (PNA−RNA, PNA−DNA, or PNA−PNA) was used to bring the reactive ends in close proximity.445−450 In contrast, the use of a template is not necessary and no autocatalysis is involved when the peptide segments to be ligated have the capacity to naturally interact with each other. Different situations are possible depending on whether the interacting units are fully or only partly incorporated in the ligation product. Figure 39A is a general description of the

Figure 39. Folding-assisted ligations.

setup in which the two interacting peptides are integrated in the final product.451,452 An example of this kind is the assembly of chymotrypsin inhibitor CI2 from two peptide segments.451 The importance of the preassociation of the two peptide precursors was demonstrated by showing that the addition of a denaturant to the reaction mixture resulted in a dramatic decrease in the ligation rate. Recently, this strategy was used by Zhao et al. to trap weak protein−protein interactions.452 An alternative mode of use of folding-assisted ligation is described in Figure 39B. In this case, one of the peptide interacting units is placed in the thiol handle of the thioester component. The other one is placed in the Cys component between the Cys residue and the rest of the protein. The benefits of such a design combined with the favorable kinetics and selectivity provided by coiled-coil assisted NCL permitted the rapid and selective labeling of membrane proteins in live cells with a fluorescent dye.96 The membrane protein, the human GPCR receptor Y2R in green in Figure 39B, was modified accordingly at the N-terminus with a Cys-peptide motif derived from the heterodimeric coiled-coil system designed by Litowski and Hodges.453 On the other side, the dye label to be transferred to the membrane protein was modified by the complementary coil motif (peptide 1) through an aryl thioester bond. Treatment of the cells expressing the AS


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Scheme 35. Principle of Thia-Zip Cyclization

Scheme 37. Intramolecular Rearrangement of the ThioesterLinked Intermediate Results in the Formation of a Peptide Bond to Cys

enabling its formation. Because it is not rate-determining, this reaction has therefore not received as much attention as the other steps in efforts to improve the kinetics of the NCL reaction. Nevertheless, it is important to understand the detailed processes involved in the rearrangement of the thioester-linked intermediate when it comes to extend the NCL to other thiol capture moieties such as thiol amino acids and thiol-based auxiliaries (see section 7). Indeed, the intramolecular S,N-acyl migration step can become rate-limiting for some of these adaptations of the NCL reaction. Aside from the kinetic aspects of the rearrangement, its reversibility has not been much considered until recently with the design of novel reactions relying on this property,216,225 including the ligations based on the use of N,S-acyl shift thioester surrogates (see sections 5.2 and 5.3).41,121,165 Although the rearrangement of the thioester-linked intermediate has not been the subject of experimental investigations so far, a wealth of information can be found on the rearrangement of simple chemical systems derived from cysteamine or cysteine. These experimental studies published in the 1950s and the 1960s are discussed in relation to recent computational investigations conducted on simple models of the thioester-linked intermediate. Finally, the reversibility of the S,N-acyl transfer process is discussed more specifically at the end of this section. 4.5.1. Experimental Studies. The experimental study of the intramolecular S,N-acyl migration reaction occurring from the thioester-linked intermediate would require the isolation of thioesters containing a nucleophilic amino group in their structure. This is potentially difficult due to the high propensity of these compounds to rearrange into amides by S,N-acyl group transfer.24,462−465 The rearrangement can even proceed in relatively acidic conditions as is the case for S-acetylcysteine, which was found to rearrange at pH 3.466 Moreover, the monitoring of the rearrangement of S-acyl derivatives of βaminothiols can potentially be complicated by the fast rate of S,N-acyl migration reactions proceeding through 5-membered ring intermediates. For example, Wieland et al. showed that Sglycylcysteamine Gly-SCH2CH2NH2 completely rearranges at pH 5.2 in 2 min.464,467

The second case discussed in this section is the cyclization of proteins using NCL (Scheme 36). The backbone cyclization of Scheme 36. Folding-Assisted Protein Cyclization Illustrated with the Cyclization of the WW1 Domain of Yes KinaseAssociated Protein (Structure Adapted from PDB 2LTW461)

a protein can be greatly facilitated when the linear precursor adopts a defined fold in native conditions that places the reactive functionalities at a short distance. A nice example of this approach is the cyclization of a WW domain of the Yes kinase-associated protein,49 which was shown to occur 1 order of magnitude more rapidly in nondenaturing conditions compared to the ligation carried out in strongly denaturing conditions, i.e., in 6 M Gn·HCl. Such a configuration is not always achieved in precursor proteins and the cyclization strategy must be adapted case by case. In particular, the knowledge of the structure adopted by the protein can be valuable for optimizing the length of the linker connecting N and C extremities.460 4.5. Rearrangement of the Thioester-Linked Intermediate

Among the different steps involved in the mechanism of the NCL reaction, the formation of the native peptide bond to Cys by intramolecular rearrangement of the thioester-linked intermediate IV is certainly the least discussed in the literature (Scheme 37, see also step 3 in Figure 32). An explanation for this is likely to be found in the rate of this rearrangement which proceeds significantly faster than the thiol−thioester exchanges AT


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Scheme 38. (A) Mechanism Proposed by Barnett and Jencks131 for the Formation of S-Acetyl-mercaptoethylamine S or NAcetyl-mercaptoethylamine N from Intermediate I Produced by Acid Hydrolysis of 2-Methyl-Δ2-thiazoline T. (B) Concerted Proton Transfer Assisted by Hydrogen Carbonate.

If we consider that S-acetylcysteamine constitutes an appropriate model of the thioester-linked intermediate formed in the NCL reaction, useful mechanistic insights into the S,Nacyl migration reaction can be extracted from the work of Barnett and Jencks on the hydrolysis of 2-methyl-Δ2-thiazoline T (Scheme 38A).131 In aqueous acidic solutions, water addition to thiazoline T results in the formation of tetrahedral intermediate I. The latter evolves either by C−N or C−S bond breaking to respectively form S-acetyl-mercaptoethylamine S or N-acetyl-mercaptoethylamine N. Both the pHdependent distribution of S and N products and the pH rate profile of the reaction led Barnett and Jencks to postulate the existence of two other cyclic intermediates I+ and I+− in the reaction path linking S and N. The data suggest that above pH 2.3, the intramolecular aminolysis of S-acetyl-mercaptoethylamine S, yielding I+‑ presents the largest energy barrier. Another important conclusion is that the product distribution is kinetically controlled by the protonation of I+− to I+ by the acid HA. This is even the case for situations where the protonation is diffusion-controlled due to the large rate constant k−2 (6.6 × 108 s−1) for the expulsion of the amine from intermediate I+− (kHA[HA] < k−2). In other words, the thioester S cyclizes to produce intermediate I+−, but the latter is transformed back to thioester S unless the alkoxide moiety of I+− is protonated to produce I+. As shown in Figure 40, the Brønsted plot for the protonation step converting I+− into I+ (kHA in Scheme 38A) is biphasic and shows a curve break point at pKa ∼ 7.4, which corresponds to the pKa of I+. Obviously, the rate constant of the protontransfer step depends on the acidity of the proton donor relative to the acidity of I+. In the presence of acids stronger than I+, the protonation is diffusion-controlled and the rate of proton transfer is independent of the strength of the acid (dotted red line, Brønsted α value = 0). The solvated proton H3O+ is above this line, probably due to a contribution from water molecules that assist the protonation by a “proton jump” mechanism according to Barnett and Jencks.131 For acids weaker than I+, the rate of proton transfer is proportional to the strength of the acid (dotted blue line, Brønsted α value ∼1). Note that water and hydrogen carbonate deviate from the blue line in that the rate of the proton transfer for these weak acids is much higher than expected from the Brønsted plot. This positive deviation was ascribed to bifunctional catalysis as shown in Scheme 38B. In this case, the concerted mechanism that directly connects I+− and I provides a pathway of lower energy compared to the stepwise

Figure 40. Brønsted plot for the general acid catalysis of acetyl transfer of S-acetyl mercaptoethylamine S (50 °C, I = 1.0 M). The dotted red line corresponds to a Brønsted α value of zero, while the blue line corresponds to a Brønsted α value of ∼1.0. The data were taken from ref 131.

protonation mechanism linking I+− and I and proceeding through I+. Note also that the bifunctional catalysis mechanism does not operate for dihydrogen phosphate (Figure 40). In the same study, Barnett and Jencks showed that the rate of the S,N-acyl transfer in S-acetyl-mercaptoethylamine S was insensitive to high salt concentrations or to the addition of a cosolvent such as n-BuOH. In contrast, Cuccovia et al. reported that the rate of the S,N-acyl migration for the same compound was strongly inhibited (100-fold) by the negatively charged detergent sodium dodecyl sulfate (SDS),465 while positively charged or neutral detergents had no effect. The electrostatic interaction between the anionic surface of SDS micelles and the ammonium group was assumed to increase the pKa of S-acetylmercaptoethylamine from 9.1468 in water to about 11 in the presence of the SDS. This change in acido-basic properties of the amine results in a decrease of nucleophile concentration and slows the conversion of S to I+− according to the mechanism proposed by Barnett and Jencks. The use of detergents for improving the solubility of hydrophobic peptides during NCL is discussed in more detail in section 8.3. To conclude on this point, experimental data obtained from the hydrolysis of thiazoline T and the interconversion of thioester S and amide N tend to support a stepwise addition− elimination mechanism through three successive tetrahedral AU


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thioester group (i.e., S′ → (I+−)′) presents the highest energy barrier in the S,N-acyl transfer process. Both experimental and computational studies currently converge to propose a stepwise mechanism for the S,N-acyl transfer step, although differences exist between the published approaches. Regarding computational approaches, the chemical complexity of the NCL reaction and the difficulties in including explicit solvent molecules in the models (i.e., water) make the unequivocal prediction of the reaction parameters and mechanism of the sole S,N-acyl transfer step difficult. There is also a lack of an experimental reference result using model reactions that can recapitulate the reactivity of the species involved in NCL. In this regard, future experimental and theoretical studies on S,N-acyl shift reactions could be inspired by the extensive work that has been devoted to the aminolysis of oxoesters,469−472 and more particularly to the role of explicit water molecules in these reactions. Reversibility of the S,N-acyl shift. It is to be noted that the computed activation barriers involved in the S,N-acyl transfer step by Wang et al. are significantly lower than those determined for thiol-thioester exchanges (12.1 vs ∼30 kcal· mol−1) and thus explain why the steps converting the thioesterlinked intermediate to the amide are not rate-limiting in NCL.414 In contrast, the energy of activation for the reverse N,S-acyl shift process was estimated at 31.1 kcal.mol−1 (N′ → TS2′ in Figure 41), a high value which led the authors to conclude that the S,N-acyl shift is irreversible. In fact, this process is best presented as being reversible with a high activation energy for the reverse N,S-acyl shift process. This view is supported by previous studies that evidenced the capacity of simple N-(2-sulfanylethyl)amides to spontaneously rearrange into thioesters,131,473−475 and by recent studies showing that N-peptidyl cysteine or cysteine amide can undergo an intramolecular N,S-acyl migration reaction under forcing conditions (Scheme 39A).225,476 This property has been exploited for the synthesis of peptide thioesters225 or peptide hydrazides216 depending on the type of nucleophile used for trapping the transient thioester intermediate produced by rearrangement (Scheme 39A, see also Figure 16). Of note is the greater propensity of N-peptidyl cysteine derivatives over N-peptidyl cysteinamides to undergo an N,Sacyl shift process.477 This observation suggests that in the former case an intramolecular acid catalysis facilitates the N,Sacyl shift process, perhaps through the protonation of the scissile amide by the α-carboxyl group. Intramolecular acid catalysis is expected to facilitate the formation of (IN+)′′ from amide N′′ (Scheme 39B), that is, an important intermediate in the pathway toward the thioester S′′ according to the computational study of Wang et al. (see Figure 41). This type of intramolecular catalysis is believed to play an important role in the reactivity of several N,S-acyl shift systems and is therefore discussed again in section 6, which is dedicated to these thioester surrogates.

intermediates. The cyclization of thioester S into I+− is followed by a rate-limiting protonation step yielding intermediate I+. Provided such a mechanism also applies to NCL, the protonation of I+− is likely diffusion-controlled and involves acids present in high concentration at pH 7, such as dihydrogen phosphate, a common buffer used in NCL, or aryl thiol catalysts. Importantly, Barnett and Jencks have drawn attention to the fact that similar reactions though with different reagents would probably not proceed in exactly the same way. Typically, some of the intermediates proposed in their work may have an extremely short lifetime so that some successive steps may become concerted. 4.5.2. Computational Studies. The difficulties in designating a general mechanistic pattern for S,N-acyl migration reactions brought to light by experimental studies are also found in computational approaches applied to NCL. Wang et al. investigated the mechanism of the formation of Nformylglycyl cysteine N′ from thioester-linked intermediate S′ at the B3LYP/6-31+G* level of theory using the polarized continuum model (PCM) for the implicit inclusion of water (Figure 41).414 A transition state TS1′ linking the thioester S′ to tetrahedral intermediate (I+−)′ could be identified (Figure 41), the latter having the same core structure as intermediate

Figure 41. Mechanism proposed by Wang et al., see ref 414. The bond lengths indicated for TS2′ are in angstroms.

I+− proposed by Barnett and Jencks (Scheme 38). TS1′ has the structure of a late TS and resembles the 5-membered cyclic intermediate (I+−)′. According to this study, the breakdown of intermediate (I+−)′ by scission of the C−S bond proceeds through TS2′ to give the protonated amide intermediate (IN+)′, which was not envisaged by Barnett and Jencks. The large distance between the carbonyl carbon and the leaving sulfur atom shows that TS2′ is late with a significant rupture of the C−S bond. Regarding the activation barriers, the formation of (I+−)′ from S′ proceeds with an activation barrier of 12.7 kcal· mol−1, while the breakdown of (I+−)′ into (IN+)′ has a computed activation barrier of 7.1 kcal·mol−1. These results are in agreement with the former observation by Barnett and Jencks that intramolecular addition of the amine on the

4.6. NCL at Selenocysteine

Selenoproteins are a class of proteins that contain at least one Sec residue.478 The frequency of this residue is very low, i.e., 1.19 × 10−5% for proteins from the UniRef50 database, compared to the frequency of the other proteinogenic residues and in particular Cys (1.48%).18 Although the human genome encodes only 25 selenoproteins,478 they are essential to life by playing critical roles in redox biochemical processes. AV


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Scheme 39. (A) N,S-Acyl Shift of a C-Terminal Cys Residue and Its Use for the Synthesis of Peptide Thioesters or Peptide Hydrazides; (B) Proposed Mechanism for the Assistance of the α-Carboxyl Group to the N,S-Acyl Shift of N-Peptidyl Cysteine Derivatives

redox or biochemical processes implicating Cys residues,482,483 for accessing peptides endowed with novel properties484,485 or for controlling the folding of Cys rich peptides486−489 (for reviews, see refs 292, 386, 490, 491). Several intrinsic properties of Sec make the synthesis of Sec peptide fragments and selenoproteins more challenging than Cys analogues, among which are the ease of β-elimination of Se-protected selenocysteine derivatives,492,493 the ease of oxidation of Sec selenols (see section 3.2.2), the paucity of protecting groups for Sec applicable to the multistep assembly of proteins,393 and the propensity of Sec to desulfurize in the presence of TCEP.331 In contrast, the unique chemical reactivity of Sec provides opportunities for designing postligation treatments that broaden the scope of Sec-mediated NCL. A hallmark of Sec reactivity is its capacity to deselenize in the presence of phosphine reagents, a property which has frequently been exploited for the production of X-Ala junctions,331,393 as well as X-Ser junctions, albeit to a lesser extent.390,494 These recent extensions to NCL at selenocysteine are further discussed in section 7. So far only a dozen selenoproteins have been prepared by Sec-mediated NCL.18 The significant progress made during the past decade should facilitate the use of Sec-mediated NCL, the synthesis of selenoproteins, and the characterization of this class of proteins, which are difficult to prepare by biological methods. The commonly accepted mechanism for Sec-mediated NCL mirrors that of canonical NCL proposed in section 4.1 and can be decomposed into the same three main events: (i) the formation of a more reactive acyl donor through thiol-thioester exchange reactions (I → III), (ii) a capture step involving the N-terminal selenocysteine residue to form a transient selenoester-linked intermediate IV and (iii) a final Se,N-acyl transfer process leading to the formation of peptide V with a native peptide bond to selenocysteine (Scheme 40). The manner in which the rate of each of these steps qualitatively evolves from NCL to Sec-mediated NCL is critical for the discussion of the experimental results reported for the latter. The in situ activation of alkyl thioesters by aryl thiols, which is rate limiting in classical NCL, does not differ in Sec-mediated NCL and will not be discussed again (see section 4.3). In contrast, the capture step of the aryl thioester III is expected to proceed significantly faster in NCL at selenocysteine due to the higher acidity of selenols and their quasi-exclusive presence in the form of highly nucleophilic selenolates at neutral pH (see section 3.2.1).384 Regarding the rearrangement of the transient selenoester-linked intermediate IV, the study of Chu and

NCL at selenocysteine is an extension of the NCL reaction in which the capture element, i.e., N-terminal cysteine, is replaced by an N-terminal selenocysteine residue (Scheme 40). Developed simultaneously by three research groups in the early 2000s,382−384 Sec-mediated NCL is a powerful synthetic method for accessing natural selenoproteins,479,480 as well as also being a useful synthetic tool for producing selenocysteinecontaining analogues of natural peptides or proteins. In particular, the substitution of Cys by Sec is a way of facilitating the study of proteins by X-ray crystallography,481 investigating

Scheme 40. General Principle of NCL with Selenocysteinyl Peptides

AW


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require working in strong reducing conditions to keep the selenocysteine residue in a reduced state. In this work, TCEP was used in combination with a cocktail of inhibitors to counter TCEP-induced deselenization (ascorbate,390 TCEP = Se391). Compared to the other amide bond metathesis reactions reported so far, which are primarily based on N,S-acyl shift systems (see Scheme 47), selenocysteine has the unique advantage of allowing transamidation and metathesis reactions to be conducted at the level of the native peptide bond.496 In its current form, the reported selenopeptide metathesis process is unable to constitute a means for producing the type of highly dynamic peptide libraries that are required for screening campaigns. Nevertheless, this work provides important clues on the impact of Sec residues on the stability of the peptide backbone. This information can be useful for improving the production of selenopeptides or proteins in general and for optimizing the experimental conditions used for performing Sec-mediated NCL.

Mautner on the aminolysis of choline-derived thio- and selenoesters is in favor of a faster acyl transfer reaction in the case of Sec-mediated NCL (Figure 24, section 2.4).277 In consequence, the use of selenocysteine as a capture element is not expected to enhance the apparent rate of ligations under the operating conditions of classical NCL because the reaction remains kinetically controlled by the regime of thiol−thioester exchanges. In practice, the results obtained during the total synthesis of human selenoprotein M and PHPT1 by Metanis and co-workers support this view.393,479 The formation of various Gly-Sec junctions was achieved in about 4 h under standard conditions (50−200 mM MPAA, 50 mM TCEP, 100 mM ascorbate, pH 7.3), which is approximately the time required for completion of a classical NCL reaction involving a glycyl thioester.246 Some significantly slower ligation rates have, however, been reported for Secmediated NCL in the absence of a strong reducing agent such as TCEP.494,495 This highlights the importance of adjusting the reducing power of the reaction medium so that selenocysteine residues can be present in a reduced state and sustain an acceptable rate for the capture step. 4.6.1. Reversibility of the N,Se-Acyl Shift. Similar to the peptide bond to cysteine, that to selenocysteine can also undergo a reverse N,Se-acyl migration reaction that results in the formation of a selenoester-linked product in situ. The first mention of such a process was reported by Adams et al., who studied the rearrangement of N-peptidyl selenocysteinyl acids or amides into selenoesters (Scheme 41).477 The latter were

5. LIGATIONS UTILIZING N,S-, N,Se-, AND O,S-ACYL SHIFT SYSTEMS 5.1. General Presentation of N,S-, O,S-, and N,Se-Acyl Shift Systems

N,S-, N,Se-, or O,S- (grouped as X,Y) acyl shift systems can be used as precursors for peptide thioester or selenoester synthesis as discussed in section 2.2.2.6. However, several of these systems can act directly as acyl donors toward Cys peptides under mild conditions (Scheme 43). The mechanisms involved in these reactions differ significantly from those discussed for NCL both regarding the nature of the rate-limiting step and the effect of the reaction parameters on the ligation rate. Moreover, some of these systems can be maintained in a latent form by judiciously choosing the thiol additives, reductants, buffers, or the temperature used for ligation and subsequently activated on demand for performing another ligation reaction. This opens the possibility of designing simple and efficient assembly strategies without resorting to protecting groups. The aim of this section is to discuss specifically the mechanistic aspects involved in the control of the reactivity of X,Y-acyl shift systems. How the latent properties of these systems can be used for facilitating protein assembly has been discussed in recent reviews and will not be covered in detail here.121

Scheme 41. N,Se-Acyl Shift of N-Peptidyl Selenocysteines and Displacement of the Formed Selenoester by MESNa Produces Peptide Thioesters

5.2. N,S-Acyl Shift Systems

not isolated but trapped in situ by a thiol such as MESNa to produce peptide thioesters. The conversion to peptide thioesters proceeded faster with peptide acids than with peptide amides, suggesting that the rearrangement is facilitated by the C-terminal carboxylic acid group, as for N-peptidyl cysteine thioester surrogates (see Scheme 39B). Interestingly, kinetic studies also established that the conversion into peptide thioesters proceeded more rapidly and under milder acidic conditions with C-terminal Sec than with C-terminal Cys. The peptide bond to internal Cys is poorly reversible and constitutes the driving force in NCL, while the situation is different for Sec peptides. The capacity of internal selenocysteine to undergo N,Se-acyl transfers under mild conditions was demonstrated by Ollivier et al. (Scheme 42).391 In the presence of MPAA, selenopeptides can participate in transamidation or metathesis reactions, probably through the capture of the transient selenoester by the aryl thiol additive (for a more detailed presentation of transamidation and amide metathesis processes, see section 5.2.2, Scheme 46A). The reactions

The N,S-acyl systems designed so far share a common trait: they are all derived from the N-(2-sulfanylethyl)amide scaffold (Scheme 44; Table 11, entries 1−14), an exception to this rule being the N-(2-mercaptoethoxy) amide system recently introduced by Shelton et al.232 (Table 11, entry 15). The fact that some N-(2-sulfanylethyl)amides can rearrange spontaneously in water under mild conditions and act directly as acyl donors toward Cys peptides is a recent application of N,S-acyl shift systems. Pioneering publications include the study of N-peptidyl mercapto oxazolidinones,497 bis(2sulfanylethyl)amide (SEA),234,498 and SEAlide499 systems. More recent contributions to the field involve the direct use of the α-methyl cysteine,500 N-alkylcysteine,223,318 or N-Hnbcysteinamide501 N,S-acyl shift systems in NCL (Scheme 44 and Table 11). The conditions required for these systems to act as acyl donors are usually mild, with no risk of triggering a similar rearrangement with cysteine, therefore making them valuable tools as shown by their capacity to access challenging proteins. AX


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Scheme 42. Principle of the Selenopeptide Transamidation and Metathesis Reactions Designed by Ollivier et al.391

Scheme 43. Ligation with N,S-, N,Se-, or O,S-Acyl Shift Systems

acyl shift systems can be controlled by the choice of the reaction buffer, a property that was exploited in their use as latent thioesters. For all these reasons, these systems should not be regarded as just doing the same as classical peptide thioesters. 5.2.1. Mechanisms. The aim of this section is primarily to discuss the mechanisms involved in the formation of the transient thioesters from the (2-mercaptoethyl)amide thioester surrogates as depicted in Scheme 44. As discussed in section 4.5, the estimated activation barrier of the N,S-acyl transfer process for N-formyl-glycyl-cysteine is very high (see Figure 41).414 As a consequence, N-peptidyl cysteinamide derivatives can be rearranged only under forcing conditions.225 Because the N,S-acyl shift systems listed in Table 11 enable ligations to be performed under mild conditions while sharing some common features with N-peptidyl cysteinamides, one important question is what makes the acyl shift easier in these systems. In this regard, the greater propensity of N-peptidyl cysteine peptides over N-peptidyl cysteinamides to undergo an N,S-acyl shift process is informative and suggests the occurrence of an intramolecular acid catalysis (R = H, Scheme 45, see also Scheme 39B).477 A similar assistance to the N,S-acyl shift by the neighboring carboxylic acid group was proposed by Asahina et al. with an N-ethylcysteine thioester surrogate (R = Et, Scheme 45).507 Another interesting observation made on N-peptidyl cysteinamides is the favored role played by histidine as the penultimate residue. Indeed, peptides featuring a C-terminal His-Cys dyad were shown to undergo an N,S-acyl migration more readily than any other Xaa-Cys combination, raising questions about the role of the imidazole group of histidine in the reaction.225 Although not demonstrated in this particular case, the destabilization of histidyl peptide bonds by the side chain imidazole group was noticed in other studies. For example, the fast cis−trans isomerization of His-Pro junctions

Scheme 44. Most N,S-Acyl Shift Systems Are Based on the N-(2-Sulfanylethyl)amide Scaffold

In general, the use of N,S-acyl transfer systems for peptide ligation results in slower reaction rates than those observed in NCL with peptide thioesters. However, the combination of important positive aspects such as their ease of synthesis using Fmoc-SPPS and their high stability toward hydrolysis makes N,S-acyl shift systems an appealing alternative to preformed thioesters. Some N,S-acyl shift systems are also efficient acyl donors in mildly acidic conditions and therefore enable the synthesis of peptides that do not tolerate neutral conditions.233,502 Last but not least, the reactivity of some N,SAY


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Table 11. N,S-Acyl Shift Systems Used for the Ligation of Peptidesa

AZ


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Table 11. continued

a

See Abbreviations. bContrary to the soluble version, the solid supported SEAlide acted as a useful thioester surrogate in the absence of phosphate salts.

nitrogen.513 Such a type of hydrogen bond donation was proposed by Raibaut et al. on the basis of DFT calculations on the SEA N,S-acyl shift system (Figure 42A).270 The transition state of lowest energy consists of a monoanionic species with one (2-sulfanylethyl) appendage twisting the amide bond as a result of an intramolecular S−H−N interaction, while the amide carbonyl group is attacked by the thiolate of the second limb. Note that computational studies suggest that the rearrangement is concerted because a stable tetrahedral intermediate could not be identified in the reaction pathway. Another N,S-acyl shift system that features an acidic group is the N-Hnb-cysteinamide activating unit developed by Terrier et al. (Figure 42B).501,514,515 The para-nitrophenol group was found to be critical for achieving fast ligation rates. Recent DFT computational studies suggest that the phenol group activates the amide by hydrogen bond donation to the carbonyl oxygen and that the rate-limiting step of the process is not the formation of the neutral tetrahedral intermediate but the waterassisted “proton jump” yielding a zwitterionic tetrahedral intermediate. Unlike the SEA or N-Hnb-cysteinamide systems, the SEAlide system does not possess a nearby and internal acidic function able to assist thioester formation (Table 11, entry 6, and the related SECmide system Table 11, entry 11). Their rearrangement into thioesters can in fact be triggered by the addition of phosphate salts (Figure 43).231,499 The authors proposed that

Scheme 45. Intramolecular Acid Catalysis of the N,S-Acyl Shift of N-Peptidyl Cysteine or N-Peptidyl N-ethylcysteine by the C-Terminal Carboxylic Acid Group

was ascribed to an interaction between the acidic proton of the imidazolium ring and the amide nitrogen atom.510 More generally, the N-protonation of amides is known to weaken the nN → π*C=O conjugation and to induce the pyramidalization of the sp2 nitrogen atom.511 This protonation results in a partial loss of delocalization energy and in an increase of the carbonyl group electrophilicity. It must be noted that the N-protonation of amides (pK a N ∼ −7) is thermodynamically less favorable than O-protonation (pKa° ∼ 0).512 However, the former can occur within constrained systems that can favor a hydrogen bond donation to the amide BA


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Figure 42. (A) Proposed monoanionic transition state in the N,S-acyl transfer reaction of SEA system. (B) Proposed mechanism for the rearrangement of the N-Hnb-cysteinamide system.

amide oxygen (step 1 in Figure 43). In the second mode, phosphates might favor the proton transfer at the tetrahedral intermediate stage (step 2 in Figure 43). As discussed in section 4.5, a proton transfer at the level of the tetrahedral intermediate has been shown to be rate-limiting during the rearrangement of N-acetyl-mercaptoethylamine and can be accelerated by species that can facilitate the “proton jump” from oxygen to nitrogen. While the exact nature of the rate-limiting step and the role of phosphates in SEAlide-mediated ligations remain to be established, phosphate catalysis is a strong asset which enables the control of the reactivity of SEAlide systems on demand. Although the mechanistic studies on the above-mentioned systems are still in their infancy, it is clear that intra or intermolecular acid−base catalysis plays a major role in their reactivity. The reactivity of N-peptidyl α-methylcysteine thioester surrogates (Figure 44) reported by Burlina et al.

Figure 43. Catalysis of the rearrangement of SEAlide by phosphate salts.

Figure 44. Strain promoted acceleration of the N,S-acyl shift.

relies on a conceptually different approach. While N-peptidyl cysteamine is a poor acyl donor, the gem-dimethyl derivative (gdm) depicted in Figure 44 was found to ligate in neutral conditions with Cys peptides.500 N-Peptidyl α-methylcysteine analogues were found to be good acyl donors too and easier to prepare by conventional SPPS (Figure 44, Table 11, entry 9). The positive role of gem-disubstituents on the rate of

the catalysis by phosphates is due to their amphoteric properties. Bifunctional acid−base catalysis by phosphates might occur at two different stages. In the first mode of catalysis, phosphates might facilitate the formation of the tetrahedral intermediate by activating both the thiol and the carbonyl groups, the latter by hydrogen bond donation to the BB


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Scheme 46. (A) Principle of Transamidation; (B) The Transamidation Process Developed by Ruff et al. Relies on the Reversibility of the Peptide Bond to N-Methylcysteine

cyclization has been the subject of numerous mechanistic investigations and several theories were proposed to account for the observed effects.516 The rearrangement of N-peptidyl αmethylcysteine proceeds through the formation of a 5membered ring intermediate. In this case, the Thorpe−Ingold theory based on valency deviation has been questioned and other explanations were put forward (e.g., reactive rotamer theory).516 Although the exact role of gem-substituents in the present case remains to be established (Thorpe−Ingold effect vs intramolecular acid catalysis, see Figure 44 vs Scheme 45), the approach is of great interest and could perhaps serve to improve other N,S-acyl shift systems. 5.2.2. N,S-Acyl Shift Systems: Application to Peptide Transamidation and Metathesis. Beyond the demonstrated ability of N,S-acyl shift systems to efficiently generate thioesters, the feasibility of N,S-acyl transfer has triggered the emergence of new transamidation and amide bond metathesis reactions under mild conditions. In a transamidation reaction, an amide reacts with an amine to produce a novel couple of amide and amine products (Scheme 46A). Transamidation are usually performed under forcing conditions (high temperature, metal catalysis) due to the poor reactivity of classical amides. If the process is reversible, it allows the creation of dynamic mixtures of high chemodiversity whose composition can evolve in response to physical or (bio)chemical stimuli such as ionic strength, temperature, or the presence of a binder. Several chemistries have been applied to the creation of dynamic peptide mixtures,496 but the use of reversible amide bond forming reactions in this area is still underexplored and highly challenging. Formally, the ligation of (2-sulfanylethyl) amides, such as those discussed in the previous section with Cys peptides, is a transamidation process with the disadvantage of being poorly reversible. In contrast, replacing the Cys component by an Nmethyl cysteinyl peptide renders the process reversible because both starting amides and end amide products can easily undergo an N,S-acyl shift process. The proof of concept for such a transamidation process was reported by Ruff et al. using a N-peptidyl N-methyl cysteine thioester surrogate (Scheme 46B).318 The reactions were run at neutral pH in the presence of DTT which acted both as a disulfide bond reducing agent and a catalyst by promoting thiol−thioester exchanges. Recently, Brea et al. used similar concepts for designing lipid dynamic mixtures with the aim of mimicking the remodeling of phospholipid membranes (Figure 45).100 Transamidation was triggered by adding lipids featuring a free N-methyl cysteine residue and DTT. In this case, the formation of the amidophospholipid product 4 in substantial amount was

Figure 45. Design of dynamic lipid mixtures by using the N-methyl cysteine exchange unit in a transamidation reaction.

noticed after relatively short reaction times ( 8 (i = 7, Figure 54), while no rearrangement occurred for higher homologues. On the basis of the preceding, the approaches overcoming the low frequency of Cys in proteins can consequently be classified into two distinct categories according to whether the intramolecular acyl transfer is short-range and favorable (i = 5− 7) or long-range and less favorable (i > 7, Figure 54). In particular, auxiliary-mediated NCL (i = 5, Table 14; i = 6, Table 13 and Table 15) and the use of thiol or selenol amino acid surrogates are gathered in the first category (thiol amino acids: i = 5, Table 16; i = 6, Table 17; i = 7, Table 18; selenol amino acids: i = 5, Table 19; i = 6, Table 20). The second category includes side-chain assisted ligations (Table 22) and thioester aminolysis reactions assisted by internal cysteines. 7.1. Short Range Acyl Transfers: Auxiliary-Mediated NCL

Removable thiol-based auxiliaries are appended to the α-amino group of a peptide segment in order to mimic a Cys peptide in NCL (Figure 55). The S,N-acyl shift process which proceeds from the transient thioester-linked intermediate involves either a 5-membered ring intermediate (2-mercaptoethyl type, Table 14) or a 6-membered ring intermediate (2-mercaptobenzyl type, Table 13 and 2-mercaptoethyloxy type, Table 15). The auxiliaries are removed by application of a specific chemical treatment after ligation. In principle, such methods might potentially extend the scope of NCL reactions to the formation of virtually any type of peptide bond. In practice, however, converting a primary amine into a secondary one results in a significant decrease of nucleophilicity that limits the use of most thiol-based auxiliary approaches to the formation of unhindered junctions, typically X-Gly peptide bonds. The other limitations shared by several of the developed systems is the necessity to isolate the backbonemodified peptide after ligation and the harsh conditions required for removing the thiol auxiliary. However, recent studies made significant advances in the field and are specifically discussed hereafter. 7.1.1. Thiol Auxiliaries Based on the 2-Mercaptobenzyl Scaffold. Thiol auxiliaries built on the 2-mercaptobenzyl BP


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Figure 54. Methods extending the principle of NCL to non Cys sites can be classified into two distinct categories according to the size of the cyclic intermediate involved in the intramolecular thioester aminolysis step. i corresponds to the number of atoms composing the cyclic intermediate.

scaffolds (Table 13) can be removed in strong acidic conditions after ligation, provided that some electron-donating methoxy groups are introduced on the phenyl moiety (Table 13, entry 2). However, even in this case, several authors noticed that removal of the auxiliary was unexpectedly slow.573−575 Mechanistic studies conducted by Nakamura et al. established that the treatment of the ligation product (tertiary amide I in Scheme 66) with TFA resulted in its conversion into the aryl thioester II by N,S-acyl migration.574 In strong acidic conditions, the equilibrium favors the thioester to a large extent due to the protonation of the α-amino group, thereby slowing down the removal of the auxiliary which can occur only at the level of the tertiary amide. The difficulty in removing thiol-auxiliaries of the 2-mercaptobenzyl type might be the reason why they are less fully developed than those based on the 2-mercaptoethyl scaffold. The recent development of a photocleavable auxiliary by Nadler et al. is potentially a way to circumvent the above-mentioned problems because in this case deprotection is achieved in neutral conditions where N,S-acyl migration is insignificant (Table 13, entry 3).576 7.1.2. Thiol Auxiliaries Based on the 2-Mercaptoethyl Scaffold. The first 2-mercaptoethyl scaffold was introduced by Canne et al. but had the disadvantage of being noncleavable (Table 14, entry 1).579 This led the authors to develop the alternative 2-mercaptoethyloxy auxiliary (Table 15, entry 1), which can be removed by treatment with zinc in acidic conditions.579 After this pioneering work, Botti et al. developed 1-phenyl-2-mercaptoethyl auxiliaries equipped with methoxy substituents on the phenyl ring to enable their removal in strong acids (Table 14, entry 2).585 The dimethoxy derivative is labile in TFA and has been used recently by several groups for accessing ubiquitin conjugates by the formation of a Gly-Gly junction.63,580,581 Later on, Kawakami et al. showed that the introduction of a nitro group on the phenyl ring instead of methoxy groups enabled the removal of the auxiliary under photolytic conditions (Table 14, entry 4). A significant advance in this area is the recent introduction by Loibl et al. of the 2-mercapto-2-phenethyl auxiliary (Table 14, entry 5).582 It can be introduced on the peptidyl resin after SPPS by reductive alkylation. An important feature of this

Figure 55. Extension of the NCL to peptide segments equipped with thiol-based auxiliaries.

BQ


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Table 13. N-2-Mercaptobenzyl-Type Auxiliaries (6-Membered Ring Intermediate)a

a

See Abbreviations. bThe reaction was compatible with C-terminal glycyl, alanyl and phenylalanyl thioesters and N-terminal Gly and Ala surrogates. The conversion with N-terminal Val was less than 5%. The 2-mercapto-4-nitrobenzylamino auxiliary derivative failed to ligate. cGly/Gly, Lys/Gly, and Gly/Ala junctions could be formed but not the Ala/Ala junction. dThe formation of a Gly/Gly junction was reported.

mercaptoethyl scaffold.583 Therefore, it was suggested that the β substituent might favor the S,N-acyl shift through a Thorpe−Ingold effect. Another interesting feature of the 2-mercapto-2-phenethyl auxiliary is that it is removed after ligation using mild conditions, typically by treatment with TCEP and morpholine in slightly basic conditions (pH 8.5). Mechanistic investigations using NMR spectroscopy with a 13 C-labeled auxiliary enabled the identification of some products formed upon auxiliary cleavage, i.e., benzaldehyde, N-formyl morpholine, and formate. This led the authors to postulate the decomposition pathway depicted in Scheme 67, which involves the formation of a benzyl radical by reaction of the thiol with TCEP. The benzyl radical then combines with molecular oxygen to produce a peroxyradical which evolves into an alkoxy radical in the presence of TCEP. Fragmentation of the latter yields an amidoalkyl radical together with benzaldehyde. Repeating this sequence of reactions results in the formation of an amidomethyloxy radical, which is oxidized by molecular oxygen into an N-formyl peptide. The latter was indeed observed experimentally. Finally, displacement of the Nformyl peptide by morpholine or hydroxide ion yields N-formyl morpholine or formate, respectively. Another recent contribution to the field of auxiliary-mediated NCL is the method developed by Weller et al., who reinvestigated the potential of the 2-mercaptoethyloxy auxiliary for the synthesis of SUMO−histone conjugates (entry 2, Table 15).584 The importance of the method stems from the mild experimental conditions used for removing the auxiliary after ligation, compared to the initial zinc/acidic conditions reported by Canne et al. (Scheme 68).579 Indeed, the 2-mercaptoethyloxy auxiliary was found to be cleaved in the presence of high concentrations of MPAA at neutral pH in aerobic conditions.

Scheme 66. Peptides Modified by a 2-Mercapto-4,5dimethoxybenzyl on the Backbone Rearranged into Thioesters in Strong Acids

auxiliary is that ligation is not limited to the formation of peptide bonds to Gly and can be extended to more sterically demanding amino acid residues such as Arg, Met, or Glu (see note d in Table 14). Kinetic studies aimed at understanding the origin of the good reactivity of the 2-mercapto-2-phenethyl auxiliary in NCL revealed the favoring role of β substituents, i.e., phenyl group, on the ligation rate compared to the unsubstituted 2-mercaptoethyl analogue (Table 14, entry 1). The kinetic profile of the reaction argued for the S,N-acyl shift from the thioester-linked intermediate to be rate limiting, as already observed for other auxiliaries based on the 2BR


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Table 14. N-2-Mercaptoethyl-Type Auxiliaries (5-Membered Ring Intermediate)a

a See Abbreviations. bThe formation of Gly/Gly and Ala/Gly junctions was reported. The S,N acyl shift reaction did not occur when the N-terminal Gly residue was substituted by Ala. Nevertheless, transthioesterification proceeded efficiently because the thioester-linked intermediate was found to accumulate in this case. cGly/Gly and Ala/Gly junctions were readily formed, while the yields for Gly/Ala, Gly/Leu, and Ala/Ala were low (100 equiv) to accelerate the in situ formation of the more reactive aryl thioester by thiol−thioester exchange. In these conditions, the inhibition of the desulfurization process by the aryl thiol is particularly pronounced so that removing the catalyst prior to the desulfurization step is mandatory. MPAA can be removed by extraction at acidic pH66 or by dialysis.634 An alternative approach is to use solid-phase techniques for simplifying the removal of the thiol catalyst. The immobilization of the polypeptide chain on a solid support enables removal of the catalyst by simple washing steps and performing the desulfurization on the solid phase with no mass loss.352,363,635 Another strategy consists in using scavenger resins for removing the catalyst from the mixture, as

to the assembly of proteins that lack Cys residues in their sequence. The situation is different for proteins that have native Cys residues because in this case they will be desulfurized together with the mercapto amino acid surrogates introduced specifically for facilitating the assembly of the protein unless some special features enable a selective desulfurization as discussed in the previous sections. The selectivity problem can be solved by protecting the native Cys residues during the desulfurization step to avoid their conversion into Ala. One of the first thiol protecting groups that has been used for this purpose is the acetamidomethyl group due to its high stability in a wide range of experimental conditions (Table 5, entry 1).348,376 The Acm group is traditionaly removed by treatment with a silver or mercury salt in combination with a thiol. These conditions are harsh and require toxic reagents, a fact that stimulated the development of analogues that can be removed under milder conditions. These analogues include the Hqm and Hgm377 protecting groups (entries 2 and 3, respectively, Table 5), which are removed by treatment with hydrazine, and the Pocam380 group (entry 4, Table 5), which is removed by treatment with zinc in acetic acid. Interestingly, the Acm group and its analogues can be combined in a single polypeptide and deprotected sequentially with high selectivity. This property that has been advantageously used for the synthesis of cysteine-rich peptides such as the α-conotoxin SI380 or the human neutrophil defensin HNP-2377 with a full control of the disulfide bond pattern. Recently, resurgent interest in Acm has resulted from the discovery of its rapid removal in the presence of PdCl2 optionally in combination with magnesium salt additives (entry 1d, Table 5).355 Other traditional protecting groups for Cys thiols include benzyl or alkyl thioethers. Some of these are difficult to remove, thus severely limiting their application in protein synthesis. For example, the 4-methoxybenzyl630 or tert-butyl252 groups were employed during the synthesis of Iberiotoxin or NY-ESO-1 analogues by NCL, respectively, but their removal was not realized. The Hmboff system developed by Qi et al. is an elegant evolution of the 4-methoxybenzyl group (entry 5, Table 5, Scheme 75).381 It is as stable to TFA as the 4-methoxybenzyl group and therefore can be introduced in peptides by conventional Fmoc SPPS. However, it is converted to the Hmbon form during ligation at neutral pH and can be removed by treatment with TFA in a subsequent step. Another thiol protecting group of the thioether family is the trityl (Trt) group (entry 6, Table 5, X = H in Scheme 76).375 STritylation can be performed chemoselectively after SPPS by treating the peptide with trityl alcohol in 1,1,3,3,3-hexafluoro-2CA


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Table 19. Extension of the NCL Using Selenol Amino Acids (5-Membered Ring Intermediate)a

a

See Abbreviations. bPrereduction of the selenylsulfide with DTT allows reducing the quantity of TCEP and thus increasing the selectivity of the deselenization reaction. cMPAA reduces the rate of the deselenization process but minimize β-hydroxy phenylalanine byproduct formation.

hydrazone bond is preferably formed at mildly acidic pH (pH

demonstrated with the MPAA hydrazide derivative described in Scheme 77.636 This MPAA analogue can be captured from the ligation mixture by formation of a hydrazone bond with a benzaldehyde-functionalized polymer resin. Because the

4), the pH of the mixture has to be adjusted between the ligation and the desulfurization steps. CB


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Table 20. Extension of the NCL Using Selenol Amino Acids (6-Membered Ring Intermediate)

a

See Abbreviations.

Scheme 70. Principle of the TCEP-Induced Metal-Free Desulfurization Method

Another approach consists in performing the ligation step without added MPAA. To ensure fast ligation rates, the peptide alkyl thioester can be substituted by a more powerful acyl donor. If a peptide MPAA thioester is used, a maximum of 1 equiv of MPAA is released in the mixture during ligation, a quantity which is insufficient to significantly interfere with the desulfurization step (Scheme 78).636 The method was applied successfully to the synthesis of ubiquitin and ubiquitin-like proteins.355,636 Peptidyl β-thiolactones are also promising in this area as they are powerful acyl donors without the limitation of inducing the release of a thiol component in the mixture

Scheme 71. Principle of the TCEP-Borohydride Desulfurization Applied to Peptide Deuteration

CC


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Scheme 73. Mechanism of the Light-Induced Deselenization of Diethyldiselenide by Diphenylmethylphosphine

Scheme 74. Principle of the TCEP-Induced Metal-Free Deselenization Method

Figure 57. Selective desulfurization based (A) on differences in the ease of carbon radical formation or (B) on differences in accessibility.

during ligation that might interfere with the desulfurization step.539,552 Another option for performing one-pot NCL/desulfurization reactions is to substitute the aryl thiol by a catalyst which is a poor scavenger of thiyl radicals. A first possibility is to use alkyl

thiols such as MESNa, MTG, or TFET (entries 1, 2, and 3, respectively, Table 9). These thiols are less powerful in

Scheme 72. SUMO-2 TAMRA Conjugates Selective Desulfurization Driven by Conformational Effects (SUMO-2 Structure PDB 1WM3)

CD


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Scheme 76. S-Tritylation for Protecting Cys Thiols during Desulfurization

Table 21. Bond Dissociation Energies for Some Bonds to Sulfur and Selenium

a

Experimental values. bValues obtained by computational studies.

Scheme 77. Principle of the One-Pot Ligation/ Desulfurization Process Employing an Aldehyde Resin for the Capture of the Aryl Thiol Catalyst

Figure 58. Calculated activation barriers of the deselenization and hydrogen abstraction steps for selenocysteine and β-seleno-aspartic acid. The data were taken from ref 614.

Scheme 75. Principle of Hmbon/off System for Cys Protection during Desulfurization

catalyzing the NCL reaction than aryl thiols such as MPAA (see section 4.3.2). However, they share the property of being less efficient in scavenging thiyl radicals than aryl thiols335 due to the higher S−H bond dissociation energy of alkyl thiols (87 kcal·mol−1)304 relative to those of thiophenol (77−83 kcal· mol−1).632,633 In addition, these alkyl thiols have the capacity to efficiently transfer a hydrogen to carbon radicals like tert-butyl mercaptan125 or GSH,588 i.e., two hydrogen donors classicaly used as additives during metal-free desulfurization of Cys CE


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down by MPAA so that in this case the removal of the aryl thiol catalysts is recommended as well.331 7.2.4. Chemical Treatments Other than Dechalcogenation. We have thus far discussed the wide use of dechalcogenation reactions for extending NCL to the formation of nearly all types of peptide junctions. Other chemoselective transformations involving thiols or selenols can also be useful to produce proteinogenic amino acid residues or amino acid mimetics. For example, the development of mild and selective alkylation methods of Cys thiol has been pursued for a long time independently of NCL and is still the subject of intense research. Logically, Cys alkylation has been combined with NCL to produce peptide bonds to pseudohomoglutamate,639 pseudohomoglutamine,405,640−643 pseudolysine,644,645 or pseudoglycosylated residues (Figure 59, left).646,647 Analogously, hCys thiol methylation yielded Xaa-Met junctions (Figure 59, right; entry 11, Table 17),379,609,648,649 while hSec methylation enabled the production of selenomethionine peptides.650 In the latter case, hSec methylation could be performed selectively in the presence of a Cys residue due to the large difference in pKa between thiols and selenols.389 The conversion of a Cys residue to Ser was also achieved by Cys methylation followed by CNBr oxidation.648 Some authors have also described the conversion of Cys to dehydroalanine after NCL using bis alkylating reagents developed by Davis and co-workers (Figure 59, reaction e),651,652 a method which has been recently extended to the incorporation of a non-natural α,β-unsaturated amide linkage into large protein scaffolds.653 Many more Cys modifications are at the disposal of protein chemists as discussed in detail in leading reviews.654−656

Scheme 78. Principle of One-Pot Ligation/Desulfurization Based on the Use of a Peptide Thioester Derived from MPAA

peptides. These properties make alkyl thiols such as MESNa or MTG compatible with one-pot NCL/desulfurization schemes.370,378 Compared to MESNa or MTG, TFET has the additional advantage of having a low boiling point so that it can be removed from the ligation mixture simply by flushing with an inert gas, thereby reducing the risk of interfering with the desulfurization process. This property was advantageously exploited for the formation of different junctions including XaaAla,210 Xaa-Asp,210 Xaa-Glu,210 Xaa-Phe,592 and Xaa-Asn589 (for recent reviews, see 637,638). Note that an alternative to the above-mentioned alkyl thiols is to use a nonthiol catalyst such as imidazole.428 Regarding the development of one-pot NCL/deselenization methods, the deselenization of Sec by TCEP is also slowed

7.3. Long-Range Acyl Transfers

7.3.1. Long Range S,N-Acyl Transfer Assisted by Internal Cysteines. Several peptide bond forming reactions relying on a thioester capture step followed by a long-range S,N-acyl transfer step were developed to extend the NCL reaction to non Cys junctions. We discuss herein the aminolysis

Figure 59. Common derivatizations of cysteine, homocysteine, and homoselenocysteine after chemical ligation. CF


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Ala)-OH (i = 9) showed that the S,N-acyl migration proceeding through an 8- or 9-membered ring is highly disfavored. S,NAcyl migrations proceeding through larger macrocyclic TSs (e.g., i = 11 and 14) could be performed under forcing conditions.657 The analysis of the results obtained for the other S-acyl isopeptides examined in these studies is complex given the multiplicity of the byproducts, the variability of the conditions, and the lack of quantitative data.661 Complementary data were reported by Haase et al., who determined the rate acceleration of the aminolysis reaction for i = 8, 11, 14, 17, 20, and 23 vs a control peptide lacking the internal Cys residue (Figure 60).662 The maximal rate

of peptide thioesters assisted by an internal Cys residue (Scheme 79). In these reactions, the intramolecular thioester Scheme 79. Thioester Aminolysis Assisted by an Internal Cys Residue

aminolysis step from the thioester-linked intermediate proceeds through the formation of a macrocyclic transition state involving i atoms with i ≥ 8. Several experimental and computational studies examined long-range S,N-acyl transfers for clarifying the relationships between the size of the cyclic transition state, i.e., the number of residues separating the Cys from the α-amino group, and the efficacy of acyl transfer.657−667 One important motivation for these studies was to define the synthetic scope for such acyl transfers. Another question of interest was to know if these macrolactamization reactions follow the classical tendency for macrolactonization that was reported in a series of seminal papers.668−673 In this classical picture of reaction rate versus ring size formation, macrolactonization proceeds at the fastest reaction rate via 5- and 6-membered ring transition states.672 A progressive increase in ring size is first associated with a significant decrease in rate, with a minimum for i = 8, and then undergoes a progressive increase leading to a leveling-off from i > 13. Katritzky and co-workers studied the reactivity of various Sacyl isopeptides containing internal S-acylated cysteine units with increasing chain lengths.657−661,664 These isopeptides covered macrocyclic TSs upon S,N-acyl migrations with i ranging from 8 to 20 and constitute good mimics of the thioester-linked intermediate formed in the capture step during internal Cys-assisted ligations. The studies with S-acyl isopeptide Gly-Cys(Cbz-Ala)-OH (i = 8) or β-Ala-Cys(Cbz-

Figure 60. Thioester aminolysis assisted by an internal cysteine. Effect of the size i of the macrocyclic transition state on the rate acceleration vs control peptide AGGARAEYS-NH2 and HPLC yield.

acceleration was observed for i = 17, while almost no assistance could be measured for i = 8. This work shows also that S,N-acyl transfers involving macrocyclic TSs with i = 8, 11, 14, 20, and 23 are difficult to perform, with i = 8 probably being the worst case as in the Katritzky studies. These experimental studies can be discussed in the light of a computational and statistical analysis of the intramolecular S,Nacyl transfer reaction performed by Katritzky and co-workers, who calculated the activation parameters and the geometrical strain related to intramolecular long-range S,N-acyl migrations (Figure 61A).663,665 The calculations showed that long-range intramolecular S,N-acyl migrations are primarily under enthalpic control (ΔH⧧).665 A statistical treatment was applied to the obtained macrocyclic transitions states for intramolecular long-range S,N-acyl migrations with increasing ring size. It emphasized the importance of the preorganization of structures,674−678 supported by the emergence of stabilizing hydrogen bonding interactions and leading to the formation of intramolecular complexes featuring a near attack conformation (NAC).674−676,679 In such a NAC, the two reacting atoms are CG


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Figure 61. (A) Long range S,N-acyl shifts from S-acyl isopeptides. (B) Evolution of the relative activation enthalpy (ΔΔH⧧, in kcal mol−1) vs ring size for long-range S,N-acyl shifts for macrocyclic TSs ranging from i = 5−20. Local maxima can be seen for i = 8, 11, and 14. Computational results were obtained at the HF/6-31G* level of theory. Adapted from ref 665.

at a distance of van der Waals contact and at an angle similar to the bond to be formed in the TS. The data presented in Figure 61B show the variation of the relative activation enthalpy (ΔΔH⧧, in kcal·mol−1) vs ring size for long-range S,N-acyl migrations.665 S,N-Acyl migrations involving medium ring sizes (i = 5−10) are characterized by a classical evolution of ΔH⧧ as a function of i,131,526,668−673,680,681 that is a progressive increase of ΔH⧧ for i ranging from 5 to 8, with a maximum in ΔH⧧ associated with the 8-membered macrocyclic transition state. Then this extremum is followed by a significant decrease in ΔH⧧ for i = 9, 10. For larger macrocycles (i > 10), the trend is rather unexpected with a succession of extrema (maxima at i = 11, 14 and 20) without the expected leveling-off of ΔH⧧ at i > 13 that is observed classically for macrolactonizations. Interestingly, the lowest ΔH⧧ was obtained for i = 17, that is the value of i which gave the best rate accelerations in the Seitz studies (Figure 60).662 Overall, this computational study is consistent with the abovediscussed experimental studies from the Seitz and Katritzky laboratories.657−662,665−667 7.3.2. Side-Chain Assisted Ligations. An alternative to the use of an internal Cys residue for favoring intermolecular thioester aminolysis reactions consists in introducing a thiol handle on an amino acid side chain (Figure 62). These methods called side chain assisted ligations were developed with amino acids such as Asp/Glu and Ser/Thr, which facilitate the introduction of the thiol handle through the side chain carboxylic acid or alcohol functionalities, respectively. For the majority of the side chain assisted ligations developed so far, the modified amino acid was internal to the peptide sequence. An exception is the Ser/Thr analogue designed by Hojo et al., which was placed at the N-terminus of the peptide segment (entry 13, Table 22). Apart from the latter case for which thioester aminolysis proceeds through a short-range 7-membered transition state, all the other entries in Table 22 involve long-range S,N-acyl

Figure 62. Side-chain assisted ligations.

transfers with i ranging from 14 to 29. Of note is the fact that most of the systems designed for side chain assisted ligations feature a conformationally constrained scaffold such as a CH


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Table 22. Side Chain-Assisted Ligationa

CI


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Table 22. continued

a

See Abbreviations. bTrans- and cis-cyclohexane auxiliaries behave similarly for Asp. Ligation proceeds more rapidly with the Asp modified peptide than with the Glu one. cLigation proceeds more rapidly for X-His/Asp junctions due to intramolecular base-catalysis of the acyl transfer. Ligation at Ala-Val or Val-Gly failed, with accumulation of the thioester-linked intermediate in both cases. In general, ligation proceeds with some hydrolysis of the thioester. dGly-Gly, His-Gly, Ala-Gly, Phe-Gly, Gly-His, His-His, Ala-His, Tyr-His, Gly-Asp, His-Asp, Ala-Asp, and Tyr-Asp. eGly-Gly, Gly-Asp, CJ


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Table 22. continued Gly-His, Tyr-Asp, His-Asp, and Ala-Asp. fGly-Gly, Gly-Asp, and Gly-His. Low yields were obtained for large amino acids, e.g., His, due to unfavorable steric interactions with the extended glycans in the ligation. gDiethyl ether is used to extract PhSH during the ligation and maximize the formation of the thioester-linked intermediate, the product of the ligation reaction. hThe treatment is realized at the level of the thioester-linked intermediate and triggers the S,N-acyl shift and the removal of the auxiliary.

cyclohexyl (entry 1, 2, Table 22), cyclopentyl (entry 3, Table 22) or sugar framework (entries 4−12, Table 22) that restricts the average distance between the reactive ends and probably favors the intramolecular aminolysis step. The studies with the sugar system described in entry 10 of Table 22 showed that increasing the number of amino acid residues and thus the distance between the thiol and the α-amino group of the peptide nucleophile resulted in progressively slower rates.682 The thiol auxiliary can be removed after ligation to yield a proteinogenic amino acid (Figure 62, entries 1−3, 6, 13 of Table 22). Alternatively, the different examples listed in Table 22 show that the thiol handle can be modified chemically or enzymatically to produce a peptide featuring a glycosylated Ser, Thr, or Asn residue (entries 4, 5, and 7−12 of Table 22). In this case, the sugar moiety itself contributes to the thiol handle and thus to the ligation step (hence the name “sugar-assisted ligation” also given to these methods) but is conserved in the final product. To conclude on this part, a large diversity of chemical methods derived from NCL are available for assembling proteins through different junctions. Meanwhile, the chemist is constantly faced with the need to solubilize the ligated peptide segments and all the intermediates until the target protein is isolated. While Gn·HCl is a powerful solubilizing additive in many cases, many other options have been examined and are used sometimes in replacement or in combination with Gn·HCl for enhancing the solubility of the peptide fragments. The various solvents and additives that are used for protein synthesis are detailed in the next section.

Scheme 80. Phosphates Can Promote the Hydrolysis of Activated Esters through the Formation of Acyl Phosphates

8. THE ROLE OF PHOSPHATE BUFFER, DENATURANTS, DETERGENTS, AND ORGANIC SOLVENTS IN THE NCL REACTION

thioacetate, this value rising to 80−93% for the hydrolysis of 4nitrophenyl thioacetate. These data suggest a strong relationship between the thiol leaving group capability and the contribution of nucleophilic catalysis by phosphate dianion to peptide thioester hydrolysis. Interestingly, the incubation of an authentic sample of acetyl phosphate in phosphate buffer (0.1 M, pH 8.5) in the presence of a large excess of thiophenol did not result in the formation of detectable quantities of phenyl thioacetate, showing the poor reversibility of acetyl phosphate formation as observed by others.693 Of course, the abovementioned reactions can potentially occur with C-terminal peptide thiophenyl esters. In addition, recent studies show that peptide phosphate mixed anhydrides of type II can equilibrate with 5(4H)oxazolone intermediates IVa (Scheme 82).694 Owing to their low pKa (∼9, H2O, 25 °C695), the latter are prone to basecatalyzed epimerization into 5(4H)-oxazolones IVb. Type IV compounds are known for their good acylating properties in aqueous mixtures and can evolve to hydrolysis byproducts IIIa,b by attack of a water molecule. They can also react with thiol nucleophiles to produce partially epimerized peptide thioesters of type I.111 This process is highly relevant with regard to epimerization issues that can occur during presolubilization of peptide thioesters in phosphate buffer or during NCL. For example, peptide aryl thioesters derived from MPAA spontaneously undergo hydrolysis and epimerization in

Scheme 81. Hydrolysis of Phenyl or 4-Nitrophenyl Thioacetate Is Catalyzed by Phosphate Dianion

8.1. Role of Phosphate Buffer

Phosphate buffers are frequently used for conducting NCL and extended ligation techniques. It is thus important to be aware of the role of phosphate ions in mechanisms of side product formation such as hydrolysis and epimerization. The capacity of phosphate ions to promote the hydrolysis of activated esters has been known for a long time. Several studies established the formation of acyl phosphate II as an intermediate in this type of reaction, which is produced by nucleophilic addition of phosphate dianion on the activated ester (Scheme 80).689−691 Once formed, acyl phosphates II undergo a monomolecular elimination reaction, leading to the formation of a carboxylate intermediate III along with metaphosphoric acid IV.692,693 Finally, metaphosphoric acid IV combines with water to produce phosphoric acid V. The fact that this mechanism also applies to the phosphate promoted hydrolysis of thioesters was established by Gill et al. using phenyl thioacetates as model coumpounds.691 1H NMR analysis of the hydrolysis mixtures showed the involvement of phosphate dianion both as a nucleophile (knuc) and as a general base (kgb), as shown in Scheme 81. knuc was found to contribute up to 40−50% to the hydrolysis rate in the case of phenyl CK


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Scheme 82. Reaction of Peptide Thioesters with Phosphate Can Potentially Lead to Peptide Phosphate Mixed Anhydrides, Peptidyl 5(4H)-Oxazolones and Peptide Thioester Hydrolysis and Epimerization

phosphate buffer at pH 7 (∼15% of epimerization for a peptidyl alanyl thiophenyl ester within 1 h).611 Likewise, peptidyl seryl MPA thioesters were recently shown to epimerize in phosphate buffer. In line with the above discussion, the rate of epimerization was reduced by decreasing the concentration of phosphate (16% after 20 h in 100 mM phosphate buffer versus 4% at 10 mM buffer concentration).696 Epimerization of the serine residue was also observed during NCL at 37 °C and could be reduced to background levels by conducting the ligation at 4 °C.

guanidinium cations to anions such as phosphate, sulfate, or carboxylate (for a review of noncovalent binding between guanidinium and anions, see ref 705).706 The guanidinium ion can also participate in direct hydrogen bonding with backbone carbonyl groups or promote hydrogen bonding of water to backbone carbonyls by changing the hydrogen bonding network of water.706,707 Although the precise mechanism of Gn·HCl-induced denaturation of polypeptides is still a subject of discussion, it is believed that the phenomena discussed above collectively contribute to the denaturation of polypeptides by Gn·HCl.707 Thioesters are stable in Gn·HCl at neutral or mildly acidic pH. In contrast, the use of a basic pH leads to thioester aminolysis by guanidine and thus to the formation of Npeptidyl guanidine side products (Scheme 83) in addition to the classical hydrolysis product.532

8.2. Role of Denaturants

The reactivity of NCL partners (i.e., the thioester and the cysteinyl peptide) is mainly contained in the chemical properties of their respective reacting extremities but can be significantly modulated by the conformation and the solubility of peptide segments in the reaction medium. Among the different additives employed for improving the solubility of peptides, denaturants (Gn·HCl, urea), detergents, organic cosolvents, and combinations thereof are the most commonly used and are the focus of the next sections. Note that although highly useful, numerous solubility problems have been reported even in the presence of denaturants and detergents. An additional tool to improve the solubility of peptides in aqueous solvents is to modify them with hydrophilic tags such as PEGs,697 poly-Lys, or poly-Arg on amino acid side chains,631,698,699 on the backbone,700,701 or on the thioester appendage.249,251,252,520,702 These aspects were recently reviewed and are not detailed here.703 8.2.1. Guanidine Hydrochloride. As mentioned above, 6 M guanidine hydrochloride (Gn·HCl704) is intensively used as a medium for NCL and extended methods. Gn·HCl often significantly improves the solubility of the peptide segments, additives, and of the ligation product. Most polypeptides are also fully denatured in 6 M Gn·HCl, thereby avoiding the partial folding of peptide segments that could bury the reactive sites and thus alter reaction kinetics. Molecular dynamics simulations suggest that guanidinium does not significantly modify hydrophobic interactions but alters ionic interactions due to the strong binding of

Scheme 83. Thioester Aminolysis by Guanidine at Basic pH

Another point worth discussing is the effect of Gn·HCl on the pH. The pH of the ligation mixture has a significant impact on the reaction rate of NCL and extended ligation methods. A detailed study of the effect of Gn·HCl on the pH and pKa of carboxylic acids was reported by Garcia-Mira and SanchezRuiz.708 In water−cosolvent mixtures, the glass-electrode pH meter which is initially calibrated with standard aqueous buffers, gives an apparent value for the pH, i.e., pHapp, which differs from the true pH of the mixture, pH*. pH* is defined by eq 1: pH* = − log10(γ *H ·[H+])

(1)

where γ*H is the proton activity coefficient in the solvent mixture. CL


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Garcia-Mira and Sanchez-Ruiz have shown that the difference between the true and measured pH, i.e., δpH* = pH* − pHapp, varies with Gn·HCl concentration and can be adequately described by the equation given in Figure 63. This study

Scheme 84. Equilibrium between Urea and Ammonium Cyanate and the Formation of Carbamylated or Thiazolidin2-one Byproducts by Reaction with Amine and/or Thiol Nucleophiles

8.3. Detergents for Improving the Solubility of Hydrophobic Peptide Segments and Proteins

A particularly efficient approach for improving the solubility of peptide segments rich in apolar residues relies on the use of detergents249,344,417,710,714 or lipid bilayer systems.715 Thanks to their amphipathic structure, detergents associate with hydrophobic peptides and additives to form water-soluble complexes. Beyond a certain concentration threshold (i.e., CMC, critical micellar concentration), detergent molecules associate into spheroidal aggregates with a polar surface and a hydrophobic heart known as micelles. These structures are able to differentially sequestrate reactants based on their lipophilicity compared to the complexes formed below the CMC. Consequently, the detergent concentration is an important factor to optimize case by case. β-n-Octylglucoside (OG, Table 23, entry 1) is a nonionic and dialyzable detergent that has attracted much attention as an additive in NCL owing to its high CMC value.716,717 For example, Bianchi et al. synthesized the HCV protease cofactor protein NS4A by ligating two peptide segments in HEPES buffer in the presence of 2% (v/v) OG because the use of 2−6 M Gn·HCl in the presence or absence of acetonitrile, dioxane, or DMF had proved unsuccessful.714 In a seminal work, Lahiri et al. reported the total synthesis of diacylglycerol kinase, an integral membrane enzyme of Escherichia coli, by performing the ligations in 8 M urea in the presence of 20 mM OG.417 Likely owing to its low CMC value, the use of dodecyl-β-Dmaltoside has seldom been reported (Table 23, entry 2). A rare example of its use for protein synthesis was described by Becker et al. for accessing the protooncogene H-Ras.344 The usefulness of n-dodecylphosphocholine (DPC, Table 23, entry 3) in NCL was reported by Chu and Becker.710 This zwitterionic detergent was combined with 8 M urea for facilitating the synthesis of prion protein analogues and the ligation product was directly purified by RP-HPLC despite the presence of the detergent. In a recent study, Kwon et al. reported that the ligation of influenza M2-derived peptide segments also proceeded efficiently in 8 M urea in the presence of DPC. 709 The final ligation product was, however, contaminated by DPC which could not be completely removed from the product. It is noteworthy that ligation was unsuccessful when OG was used as a detergent. Sodium dodecyl sulfate (SDS, Table 23, entry 4) is a useful anionic detergent for challenging NCL reactions with hydro-

Figure 63. Effect of Gn·HCl on δpH*. The figure was prepared using the equation reported in ref 708: δ pH* = −0.182 C + 0.161C + 5.5 × 10−3C 2 , where C corresponds to Gn·HCl concentration.

highlights that a pH correction factor of δpH* ∼ +0.72 must be applied to the measured pH to obtain the true pH in 6 M Gn· HCl. Given the large effect of 6 M Gn·HCl on pH and pKa values, great care must be taken in comparing kinetic data obtained from experiments carried out in the presence or absence of Gn·HCl. 8.2.2. Urea. Urea has been successfully used as a denaturant alone or in combination with organic cosolvents or detergents for improving the solubility of hydrophobic peptide segments and ligation products during NCL.417,709,710 It is, however, seldom used in comparison with Gn·HCl, perhaps due to the fact that urea is significantly less effective than Gn·HCl as a denaturant.711 For example, the midpoint of the denaturation curve of ribonuclease is 6.96 M in urea and only 3.01 M in Gn· HCl. Another point that can potentially complicate its utilization in NCL is the equilibrium between urea and ammonium cyanate in water at neutral pH (Scheme 84).712 Cyanate produced from urea can react with amine and thiol nucleophiles such as Cys thiols to yield carbamylated side products.713 The reaction of cyanate with an N-terminal cysteine residue can eventually result in the formation of a thiazolidin-2-one byproduct, which is unable to participate in the ligation.520 The compatibility of urea with NCL must be evaluated case by case as cyanate formation depends of many factors such as urea concentration, the temperature, or the presence of cosolvents. CM


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tags to improve the solubility of hydrophobic peptides. For example, the synthesis of the full-length influenza virus M2 protein or a truncated variant was realized in TFE/Gn·HCl723 or TFE/urea709 solvent mixtures, respectively. The assembly of the truncated variant in TFE/Gn·HCl proved unsuccessful, showing the importance of adapting the organic solvent/ denaturant combination case by case. The NCL reaction can also be carried out in DMF in anhydrous conditions in the presence of an organic base such as triethylamine.724−726 For example, Sohma et al. showed that the NCL reaction with a MPAA thioester proceeded well in DMF in the presence of TCEP, MPAA and triethylamine.724 In the absence of water, thiol−thioester exchanges and thioester aminolysis reactions are slowed down. Therefore, the combination of a more reactive MPAA thioester and a tertiary amine catalyst are required to reach an acceptable ligation rate. Dittman et al. also performed NCL reactions in DMF in the presence of triethylamine.725 Compared to NMP or TFE, DMF gave the best kinetics while no reaction was observed in DMSO. The combination of thiophenol/benzylmercaptan was significantly more effective than MPAA for catalyzing the NCL reaction. Moreover, addition of LiCl prevented the precipitation of the hydrophobic peptides while allowing the ligation to proceed chemoselectively at a practical rate without significant epimerization of the thioester component.726 The capacity of LiCl or other lithium salts to significantly improve the solubility of unprotected peptides in aprotic organic solvents such as THF was indeed demonstrated by the group of Seebach.727 Dittman et al. verified that the NCL reaction in DMF in the presence of triethylamine (20 mM), thiophenol (20 mM), and LiCl (0.18 M) proceeded with minimal epimerization (3%). A higher concentration of thiophenol (0.3 M) yielded an almost fully epimerized ligation product, probably due to the accumulation of the thiophenyl ester in situ. The reaction was also chemoselective with no interference by Lys residues and free α-amino groups other than those of Cys.726

Table 23. Detergents Used for Improving the Solubility of Hydrophobic Peptides during NCL

phobic peptides.249,718 Valiyaveetil et al. used SDS for the semisynthesis of the membrane-spanning region of the potassium channel KcsA.718 The protein was produced from two peptide segments that were lyophilized in the presence of SDS following the purification step and prior to the ligation step. This resulted in higher solubility of the peptide reactants in the ligation buffer. The target protein was purified by HPLC. In another study, Sato et al. examined the influence of the thiol catalyst (thiophenol or MESNa) and the SDS concentration (35 or 7 mM) on the efficiency of the ligation process to produce transmembrane regions derived from opioid receptor-like 1.249 The authors used a peptide alkyl thioester derived from 3-mercaptopropionic acid (peptide-COSCH2CH2CO-Gly-Arg5-Leu). Thiophenol was a poor catalyst in the presence of 35 mM SDS. The thiophenyl ester produced by thiol−thioester exchange accumulated in the mixture without being consumed by the Cys peptide. Using SDS at 35 mM and substituting thiophenol by the more hydrophilic thiol MESNa allowed ligation to proceed partially. Moreover, ligation was further improved by decreasing the SDS concentration from 35 to 7 mM, i.e., below the CMC. This study highlights the importance of adapting the detergent/thiol catalyst couple for optimal efficiency in the ligation of hydrophobic peptides.

9. NCL MECHANISTIC OVERVIEW AND PERSPECTIVES The NCL reaction8,15 and extended methods represent a set of powerful, synthetically recognized tools (Figure 64), yet these reactions show some limitations restricting their scope. The main strentghs and limitations are gathered into eight different groups defined in Figure 64 and are detailed in Table 24, which includes references for redirecting the reader toward the corresponding sections in the manuscript. Concerning the NCL reaction itself, the slow kinetic rates experienced with some classical peptide thioesters, especially those equipped with C-terminal Val, Ile, or Pro residues (group 1, Figure 64, Table 24),246 urge the development of novel acyl donors that are more appropriate for the formation of difficult junctions. A solution to this problem is the use of increasingly powerful acyl donors (e.g., peptide aryl thioesters,132,310,409 peptide selenoesters9,285). Although of interest, this strategy comes with inherent limitations due to a higher susceptibility of these peptide derivatives to epimerization, hydrolysis, and side product formation during synthesis, storage, or ligation. It is expected that the emergence of new process technologies such as microfluidics and related continuous flow strategies will contribute to broaden the synthetic scope of these powerful acyl donors by minimizing the above-mentioned side reactions and offering a greater control of the reaction parameters.430,459

8.4. Organic Solvents

In addition to denaturant or detergent additives, the NCL reaction has been conducted in various solvent conditions. In particular, a number of organic cosolvents are well tolerated, such as trifluoroethanol (TFE)709 or DMSO,328 and can be used in combination with classical denaturants or hydrophilic CN


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Figure 64. Schematic view of the distinct steps involved in NCL and extended methods. Dotted boxes define nine groups that detail the various strengths and limitations of such reactions (see Table 24).

derived from simple thiols in the NCL reaction can be predicted with confidence from Jencks’ seminal work,132 we must admit the limited understanding of the reactivity of more elaborated thioesters. A remaining yet very important question to address is how heteroatoms or functional groups close to the thioester group can promote its reactivity (group 3, Figure 64/ Table 24). For example, the abnormally high reactivity of alkyl thioesters featuring basic or acidic groups in their thiol limb has been noticed.550,728 Recent work has also shed light on the abnormally high reactivity of some strained thiolactone intermediates (group 3.1, Table 24).538,552 Understanding which internal factors can promote the reactivity of thioesters and how they act should facilitate future progress in this area. Computational studies provide useful information for rationalizing the various reaction pathways involved in NCL and are thus very supportive for predicting structure-(re)activity relationships for each NCL reaction partner. An important conclusion which can be drawn from the reading of previous literature is that the computational study of the thiol−thioester exchange reaction involving thiol catalysts, i.e., the rate-limiting step of NCL, is still in its infancy (group 4, Figure 64, Table 24). The role of explicit water molecules in this process is largely unexplored, as well as the contribution of specific acido-basic equilibria, which potentially activate or deactivate the reactivity of some pivotal functions. The difficulties in exploring these reactions in water by computational methods is in part due to the high number of atoms and degrees of freedom to consider, which implies having access to powerful computational resources to perform the calculations.

In addition to potential reactivity and/or selectivity issues, the size and the ever increasing complexity of synthetic targets requires the setup of complex multistep reaction schemes. The design of stable precursors to peptide thioesters or selenoesters (e.g., peptide hydrazides,201,220 N,S(Se)-acyl shift systems,42,165) that can be easily activated prior to or during ligation into powerful acyl donors is currently a preferred approach in the field (groups 1.2 and 1.3, Table 24). Such precursors bear the advantage of being easily prepared by conventional, widely accessible Fmoc SPPS. This, in addition to their excellent stability, makes them more adapted to the industrial production of pharmaceutical peptides. Peptide chemists can now rely on a palette of such systems and having at their disposal such tools potentially simplifies the synthesis of complex proteins. For instance, recent reports have shown that several of these systems can be activated sequentially in one pot for accessing large polypeptides without intermediate purifications, thereby saving time and yield.75,270,362 Moreover, the selective activation of these systems opens new possibilities for producing complex peptide scaffolds such as cyclic or branched peptides.517 The development of novel acyl donors for NCL inevitably raises the question of the relationship between thioester structure and reactivity. Up to now, the few mechanistic studies of the NCL reaction were carried out with peptide thioesters derived from simple alkyl or aryl thiols. This is partly due to the complexity of producing peptide thioesters, which has limited the possible variations in the thioester structure up to very recently. While the reactivity of peptide thioesters CO


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Table 24. Strengths and Limitations of NCL and Extended Methods: The Different Groups Are Defined in Figure 64

Another constraint on the computation of reaction pathways is the involvement of various electrostatic interactions in the

transition states that are often underestimated or neglected with conventional computational methods. Nevertheless, more CP


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interest of these systems for protein synthesis.582,587 Undoubtedly, the development of novel removable traceless auxiliaries that provide faster ligation rates while being removed using mild conditions is still an important goal to pursue. Hundreds of proteins have been prepared by chemical synthesis over the past 20 years. The large amount of data accumulated constitutes a considerable reservoir of information that helps chemists to anticipate, fix, or at least minimize potential hurdles in synthesizing a given protein target. This knowledge would be even more useful if the data were organized to enable rapid and relevant information recovery, with the aim of providing decision support to protein chemists. In this regard, we interpret the recent development of a database (Protein Synthesis Database, PCS, http://pcs-db.fr)18 and of a predictive informatic tool (Aligator software)731 as a new trend in the field. The last point that we would like to discuss here is the adaptation of NCL and extended methods to solid-phase protein synthesis (group 9, Figure 64, Table 24). Although synthesizing proteins on a water compatible support can provide several important advantages that have led to the success of SPPS,4,732 most of the proteins were produced in solution. We estimate that only 2.5% of the junctions have been formed on a solid phase since the introduction of NCL.18 Such approaches are potentially complex because monitoring solidphase reactions is still a challenge. Thus, measuring the effect of the solid support on the rate of NCL and on other chemical steps such as thioester surrogate activation or Cys deprotection is a difficult task; no information is available on how unprotected peptide segments diffuse inside the matrix of common solid supports and how diffusion is affected by the peptide composition and size as well as the nature of the aqueous solvent. In this context, transposing and optimizing assembly strategies from solution to solid phase can be cumbersome. Other limitations are certainly in play to explain the reluctance of chemists to adapt their protocols to solidphase protein synthesis. In particular, these methods are highly dependent on the design of efficient linker technologies and elongation protocols, the design of which is challenging.247,266,267,271,350,363,733 Although there is still a long journey for making these approaches more popular, solid-phase protein synthesis alone or in combination with solution chemistry has a strong potential, especially for accessing large peptide thioesters and Cys peptides (see groups 1.1 and 2.1, Figure 64, Table 24).267 Solid-phase protein synthesis on hydrophilic solid supports should also facilitate the synthesis of proteins that are poorly soluble in conventional aqueous media. As such it can be regarded as complementary to the hydrophilic tags that are intensively developed to facilitate the synthesis of hydrophobic proteins in solution (see section 8).703 Last but not least, solidphase protein synthesis enables to reduce the number of highresolutive purifications steps (typically HPLC) usually required during protein assembly in solution and therefore has a great potential for increasing the yields and reduce the times of synthesis.271,734 The need to overcome the above-mentioned limitations will certainly continue to stimulate major research efforts in the future. This is especially important in a context where peptide and protein drugs constitute a fast growing class of pharmaceuticals,735−738 whose development is highly dependent on the availability of efficient production methods among which the Fmoc SPPS and NCL are central tools.

accurate understanding of the intimate elemental steps at stake during catalysis as well as their intrinsic mechanism is an important step toward the development of novel thiol or selenol additives (group 4 in Figure 64, Table 24). Besides kinetic considerations, these catalysts could potentially address some limitations of the NCL and extended methods by providing operational advantages. For example, they should minimize the formation of byproducts, be easily removed prior to purification, and/or enable one-pot ligation desulfurization approaches to facilitate the setup of multistep assembly strategies. Computational studies should also complement efforts toward the design of optimized acyl donors (group 1 in Figure 64, Table 24). Besides the need for more powerful acyl donors, a strong demand resides in the limitation of the side reactions encountered with some C-terminal amino acids such as Asp, Asn, Glu, and Pro residues (group 1 in Figure 64, Table 24). This is a serious issue as these amino acids represent globally about 20% of the amino acids naturally linked to Cys or Ala, thereby significantly restricting the scope of NCL. Byproduct formation with these amino acids proceeds through an intramolecular attack of an internal nucleophile on the thioester functionality (side chain carboxamide for Asn, side chain carboxylic acid function for Asp and Glu, and backbone amide for Pro). The design of novel acyl donors and/or the development of smart protection strategies for C-terminal Asn, Asp, and Glu residues might provide some help in this area.562 Another point that merits specific developments is the problem of aspartimide formation during protein synthesis.729 Several tools are now available for minimizing this side reaction during SPPS such as optimized Fmoc deprotection cocktails, side chain protecting groups for aspartic acid, or backbone protecting groups for the residue following Asp or Asn.730 However, these protecting groups are removed during the peptide cleavage and deprotection step in TFA so that aspartimide formation can occur afterward during ligation. This is a general problem in ligation chemistry as it can happen regardless of the type of ligation used. We can estimate that the occurrence of Asp/Asn-Gly dipeptides in proteins, which are particularly prone to aspartimide formation, is on average 2.7 units per protein (1.17 for Asp-Gly, 1.54 for Asn-Gly, data extracted from UniRef50570). Therefore, such aspartimideprone sites are frequent and can considerably complicate the synthesis of most proteins. The development of chemical tools that can avoid this problem will certainly have a profound impact in the field of protein synthesis. NCL requires a Cys residue at the ligation site. The low frequency of Cys in proteins has stimulated the development of various extended methods that are extensively covered within this review. A great step forward has been made with the development of mercapto or seleno amino acid surrogates (group 2 in Figure 64, Table 24) combined with efficient dechalcogenation protocols (group 8 in Figure 64, Table 24), so that now almost all types of junctions can potentially be made using NCL chemistry. However, the preparation of the protected mercapto or seleno amino acid surrogates for use in SPPS usually requires multistep procedures that significantly restrict their use. It is therefore not surprising that one of the most popular approaches involves the dechalcogenation of Cys for accessing X-Ala junctions.124,125,331 The development of mercapto or seleno removable auxiliaries is a valuable alternative and recent studies have significantly extended the CQ


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10. CONCLUSION NCL and extended methods use a wide variety of concepts and chemical methods that are summarized in this review. Although the reactivity and applications of thioester chemistry have stimulated the creativity of peptide chemists for a long time, two important conclusions can be drawn after reading this review. The first is that thioester chemistry, in particular aspects related to NCL, has never attracted as much attention from peptide and protein chemists as it has over recent years. The search for novel peptide thioester or selenoester surrogates, catalysts, mercapto, or seleno amino acid surrogates, solubilizing auxiliaries or optimized experimental conditions is clearly recognized as an important goal, driven by the need to continually access more synthetically challenging proteins. The second conclusion concerns our understanding of the chemical events occurring in NCL and related reactions. Our comprehension of apparently simple reactions such as thioester aminolysis or thiolysis is fragmentary, including how water participates in these processes. Computational methods have provided insights into some of the chemical events occurring in these reactions, but the complexity of even the simplest model reactions brings us to the limits of what can be studied in silico, especially if water as solvent is explicitly taken into account. Although our understanding of how the chemical systems work is incomplete, the diversity of proteins accessible through chemical synthesis is stunning. Proteins composed of several hundred amino acids are now prepared using fully synthetic methods, and the array of modifications that can be introduced by chemical synthesis is limitless. These new possibilities open new avenues for studying protein function and for developing novel therapeutics. The applications of NCL and related methods extend far beyond just chemistry or the life sciences because they also enable the design of novel micro and nanotechnologies, polymers, or materials. The applications are rapidly expanding and will certainly benefit from future fundamental advances in the field.

team (Lille, France) for two years as postdoctoral research fellow where she developed novel biofunctionalization chemistries for hybrid nanoparticle synthesis. In 2003, she was appointed assistant professor at the University of Sidi Mohamed Ben Abdellah (Fez City, Morocco) and was promoted to full professor in 2017 at the same University. She is interested in chemical ligation methods for the synthesis of peptides and proteins. Vincent Diemer obtained his engineer’s degree in 2003 from the Ecole Nationale Supérieure de Chimie de Mulhouse (ENSCMu, France). Four years later, he received his Ph.D in chemistry from the University of Haute-Alsace. During this period, he synthesized sterically hindered zwitterionic biaryls in the context of a structure−activity relationship in nonlinear optics. After several postdoctoral fellowships, he was recruited by the Centre National de la Recherche Scientifique (CNRS) in 2018 and joined the group of Dr. Oleg Melnyk (Lille, France). His current research activities are focused on the design of new synthetic strategies leading to valuable peptides or proteins in chemical biology. Marine Cargoët obtained her engineer’s degree in 2014 from the Ecole Nationale Supérieure de Chimie de Rennes (ENSCR, France). She received her Ph.D. degree in 2017 from the University of Lille (Lille, France), where she developed novel selenium-based chemical methods for protein synthesis in solution and solid phase. Jean-Christophe M. Monbaliu studied chemistry at the Université Catholique de Louvain (Belgium) and obtained his Ph.D. in organic chemistry in 2008. After a first postdoctoral fellowship in process chemistry at the Ghent University (Belgium, 2008−2010), he next moved to the USA, where he was appointed at the University of Florida (2010−2012) and then at the Massachusetts Institute of Technology (2012−2013). He returned to Belgium and settled at the University of Liège, where he created the Center for Integrated Technology and Organic Synthesis (CiTOS). His research activity is at the interface of synthetic organic chemistry, physical organic chemistry, and new process technologies and focussed on the preparation of high value-added molecules, including peptides. Oleg Melnyk graduated as an engineer in chemistry in 1989 from the Ecole Nationale de Chimie de Paris (ENSCP, France). He received his Ph.D. degree in 1994 from the University of Paris VI (France). During his thesis work, he developed a semisynthetic route to cortisone. After postdoctoral training from 1994 to 1996 in the group of Prof. A. Tartar (Lille, France), he was recruited by the Centre National de la Recherche Scientifique (CNRS) in Lille. He is now director of research at CNRS, and his main research interests are the development of chemical methods for protein synthesis and the study of protein function.

AUTHOR INFORMATION Corresponding Authors

*O.M.: phone, 33(0)320871214; E-mail, [email protected]. fr. *J.-C.M.M.: E-mail, [email protected]. ORCID

Jean-Christophe M. Monbaliu: 0000-0001-6916-8846 Oleg Melnyk: 0000-0002-3863-5613 Notes

ACKNOWLEDGMENTS V.A. and O.M. gratefully acknowledge the financial support from European Union and Région Hauts-de-France (grant OPTEINS). J.-C.M.M. gratefully acknowledges the University of Liège (Wellcome grant WG-13/03 and FSR 2016 μPEPS) and the F.R.S.-FNRS (CDR 29148827). We are grateful to Dr. R. J. Pierce for proofreading the manuscript.

The authors declare no competing financial interest. Biographies Vangelis Agouridas obtained his Ph.D. in organic chemistry from the University of Versailles Saint-Quentin (France) in 2006. After a first postdoctoral fellowship in natural product synthesis at Boston University (USA), he returned to France for two additional fellowships in medicinal chemistry at Paris V and Paris XI faculties of pharmacy. In 2010, he was appointed assistant professor at the Ecole Nationale Supérieure de Chimie de Lille (ENSCL, France) and joined the group of Dr. Oleg Melnyk in 2013. His main research activity is focused on developing amide-bond metathesis reactions applied to peptides and proteins.

ABBREVIATIONS 2-Cl-Z = 2-chlorobenzyloxycarbonyl 3-MBSA = 3-mercaptobenzyl sulfonate α-MeC = α-methylcysteine Ac = acetyl AcA = acetoacetyl Acm = acetamidomethyl

Ouafâa El Mahdi obtained her Ph.D. degree in organic chemistry from the University of Montpellier in 1997. She then joined Oleg Melnyk’s CR


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HEPPS = 3-[4-(2-hydroxyethyl)piperazin-1-yl]propane-1sulfonic acid HFIP = hexafluoro-2-isopropanol Hgm = N,N-bis(2-acetoxyethyl)glycinamidomethyl His, H = histidine Hmb = N-2-hydroxy-4-methoxybenzyl Hmp = 2-hydroxy-3-mercaptopropionic acid Hnb = 2-hydroxy-5-nitrobenzyl HOBt = hydroxybenzotriazole HOOBt = 3-hydroxy-1,2,3-benzotriazin-4(3H)-one HOSu = N-hydroxysuccinimide (RP-)HPLC = (reverse phase-) high performance liquid chromatography Hqm = hydroxyquinolinemethyl hSec = homoselenocysteine IgG = immunoglobulin G Ile, I = isoleucine KAHA = ketoacid-hydroxylamine ligation KCL = kinetically controlled ligation LC-MS = liquid chromatography−mass spectrometry Leu, L = leucine LG = leaving group Lys, K = lysine Mapoc = 4-(dimethylamino)phenacyloxycarbonyl MBPA = 4-mercaptobenzyl phosphonic acid Me = methyl MEGA = N-mercaptoethoxyglycinamide MESNa = sodium 2-mercaptoethylsulfonate Met, M = methionine MFD = metal-free desulfurization MICA = 5-methylisoxazole-3-carbonyl Mop = 1-methyl-2-oxo-2-phenylethyl MOPS = 3-morpholinopropane-1-sulfonic acid MPA = 3-mercaptopropionic acid MPAA = 4-mercaptophenylacetic acid MPAL = 3-mercaptopropionamide linker Ms = mesyl Msc = 2-methylsulfonylethyloxycarbonyl MTG = methyl thioglycolate NAC = near attack conformation NBO = natural bond orbital Nbz = N-acyl benzimidazolinone NCL = native chemical ligation NCL/-PG = NCL/Cys deprotection sequence NCL/-PG/NCL = NCL/Cys deprotection/NCL sequence nd = not determined Nin = ninhydrin thiazolidine NMP = N-methyl-2-pyrrolidone NMR = nuclear magnetic resonance nr = not reported Nu = nucleophile Nvoc = 6-nitroveratryloxycarbonyl OG = β-n-octylglucoside ORTEP = Oak Ridge thermal-ellipsoid plot program PBS = phosphate buffer saline PCM = polarized continuum model PCS = protein chemical synthesis database PDB = Protein Data Bank Pen = penicillamin PEG = polyethylene glycol PG = protecting group Ph = phenyl Phe, F = phenylalanine

AcOH = acetic acid ADO = 8-amino-3,6-dioxa-octanoic acid AIM = atoms in molecules (analysis) Ala, A = alanine All = allyl Alloc = allyloxycarbonyl Arg, R = arginine AscH2 = ascorbic acid Asn, N = asparagine Asp, D = aspartic acid ATP = adenosine triphosphate BAL = backbone amide linker BDE = bond dissociation energy BMEA = N,N-bis(2-mercaptoethyl)-amide Bn = benzyl Boc = tert-butyloxycarbonyl Bu = butyl CASET-Weinreb amide = carboxamide 2-sulfanylethyl Weinreb amide derivative Cbz, Z = benzyloxycarbonyl CCDC = Cambridge Crystallographic Data Center CMC = critical micelle concentration CPE = cysteinyl prolyl ester Cys, C = cysteine DBU = 7-diazabicyclo[5.4.0]undec-7-ene Dbz = 3,4-diaminobenzoyl linker DFT = density functional theory DHA = dehydroascorbic acid DIEA = N,N-diisopropylethylamine DKP = diketopiperazine DMF = N,N-dimethylformamide DMNB = dimethoxynitrobenzyl DMSO = dimethyl sulfoxide DNA = deoxyribonucleic acid Dobz = p-boronobenzyloxycarbonyl DPC = n-dodecylphosphocholine DPDS = diphenyldiselenide DSL = diselenide selenoester ligation DTNP = 2,2′-dithio-bis-5-nitropyridine DTT = dithiothreitol EDC = N-(3-(dimethylamino)propyl)-N′-ethylcarbodiimid EDTA = ethylenediaminetetraacetic acid EPL = expressed protein ligation Et = ethyl Fmoc = 9-fluorenylmethyloxycarbonyl Fmoc-(2F) = 9-(2-fluoro)fluorenylmethoxycarbonyl gdm = gem-dimethyl GED = gas electron diffraction Glc = D-glucose Gln, Q = glutamine Glu, E = glutamic acid Gly, G = glycine Gn·HCl = guanidine hydrochloride GPCR = G protein-coupled receptor GSH = glutathione GSSG = glutathione disulfide HBTU = 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate HCV = hepatitis C virus hCys = homocysteine HEPES = 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid CS


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REFERENCES

PhFl = 9-(9-phenylfluorenyl) PHPT1 = phosphohistidine phosphatase 1 PMB = p-methoxybenzyl PNA = peptide nucleic acid PNGase A = peptide N-glycosidase A Pocam = N-methyl-phenacyloxycarbamidomethyl Pro, P = proline Proc = propargyloxycarbonyl Pse = phenylsulfonylethyl PTM = post-translational modification PyBOP = benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (t)RNA = (transfer) ribonucleic acid SCAL = safety-catch amide linker SCE = 2-carboxyethylthio SDS = sodium dodecyl sulfate SEA = bis(2-sulfanylethyl)amide SEAE = S-(2-((2-sulfanylethyl)amino)ethyl) SEAlide = N-(2-sulfanylethyl)anilide SEAoxy = N-sulfanylethylaminooxybutyramide Sec = selenocysteine SECmide = N-sulfanylethylcoumarinyl amide SeEA = bis(2-selenylethyl)amide Ser, S = serine SET-Weinreb amide = 2-sulfanylethyl Weinreb amide derivative Sez = selenazolidine SN2 = bimolecular nucleophilic substitution SPPS = solid-phase peptide synthesis StBu = tert-butylsulfenyl STL = serine/threonine ligation SUMO = small ubiquitin-like modifier TAMRA = 5-carboxytetramethylrhodamine Tbeoc = 2-(tert-butyldisulfanyl)ethyloxycarbonyl tBu = tert-butyl TCEP = tris(2-carboxyethyl)phosphine TFA = trifluoroacetic acid Tfa = trifluoroacetyl Tfacm = trifluoroacetamidomethyl TFET = 2,2,2-trifluoroethanethiol TfOH = trifluoromethanesulfonic acid THF = tetrahydrofuran Thr, T = threonine Thz = 1,3-thiazolidine-4-carboxo TI = tetrahedral intermediate TIPS = triisopropylsilane Tmob = 2,4,6-trimethoxybenzyl TMS = trimethylsilyl Tris = 2-amino-2-hydroxymethyl-1,3-propanediol Trp, W = tryptophan Trt = trityl Ts = tosyl TS = transition state TT = thiazolidine thioester Tyr, Y = tyrosine Ub = ubiquitin UV = ultraviolet VA-50 = 2,2′-azo-bis(2-methylpropanimidamide) dihydrochloride VA-044 = 2,2′-azo-bis-[2-(2-imidazolin-2-yl)propane] dihydrochloride Val, V = valine

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Native Chemical Ligation and Extended Methods: Mechanisms

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