Active ester-based peptide bond formation and its application in peptide synthesis

Abstract

Peptide bonds, the amide bonds formed between α-amino acids, constitute the backbone of peptides and proteins. Although peptide bonds can be constructed efficiently under mild reaction conditions in nature, their chemical approach imposes great challenges on chemists. The notorious racemization/epimerization issue caused by the “overactivation” of the carboxylic groups by using conventional coupling reagents always complicated peptide synthesis and purification. Alternatively, active esters with well-defined structures and moderate reactivity, formed in situ or prior prepared, play a crucial role in suppressing racemization/epimerization. Herein, we systematically and critically summarized the pros and cons of using active esters in peptide syntheses with a hope of stimulating novel innovations for disruptive peptide synthesis strategies.

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1. Introduction

The fundamental chemistry of protein and peptide synthesis is amide bond formation, which itself is an essential basic reaction of organic chemistry. As a ubiquitous functional group, the amide bond is widely found in polymers, pharmaceuticals, cosmetics, agrochemicals, materials, and other fine chemicals. The Comprehensive Medicinal Chemistry database shows that amide bonds are found in up to 25% of currently marketed drugs and two-thirds of drug candidates.¹ Moreover, the acylation of an amine is engaged in approximately 16% of reactions in the synthesis of pharmaceuticals.² As a consequence, a broad range of amide bond formation strategies, e.g., acyl halides,³ acyl azides,⁴ and anhydrides,⁵ have been developed.^6–9 However, most of these methods are not amenable to peptide bond formation because of the limited availability of the corresponding chiral starting materials. The most reliable and widely used method for peptide synthesis is still the coupling reagent-mediated dehydration condensation between the carboxylic group of the N^α-protected α-amino acid and the amino group of the other α-amino acid. More than hundreds of coupling reagents have been developed and commercialized over the past decades.^10–13 Many thanks to Merrifield for his great contribution of inventing solid-phase peptide synthesis (SPPS), which has revolutionized peptide synthesis and plays a mainstay role for peptide synthesis by combining with coupling reagents and peptide synthesizers. However, SPPS is far from perfect. Particularly, SPPS has been criticized because of its low atom-economy in the context of environmentally sustainable development.¹⁴ In addition, extensive reverse-phase high-performance liquid chromatography (HPLC) purification is endlessly necessary because the overactivation of conventional coupling reagents results in epimers and other side products which have similar properties to that of the target peptides. During the past two decades, peptide drug discovery has witnessed a rapid renaissance with innovations in peptide drug formulation and delivery technologies. With the growing request for peptide therapeutics, highly efficient and environmentally friendly peptide manufacturing processes are in great demand. To highlight the importance and urgency of this topic, general methods for sustainable peptide bond formation have twice been identified as one of the top ten challenges in the green chemistry research area of synthetic organic chemistry over the past two decades.¹⁵ It is noted that these problems could not be addressed by optimization of current peptide synthesis strategies and technologies, which were developed in the 1950–1980s when the green chemistry concept was not established yet. The loss of chiral integrity of α-amino acids not only requires extensive HPLC purification but also results in the tremendous loss of target peptides, which is an enduring and notorious issue encountered by practitioners. The use of an extra racemization suppressor, which can form a transient active ester intermediate in situ during the coupling process, is an efficient strategy to suppress racemization/epimerization. Although the aminolysis reaction of common esters cannot take place spontaneously,¹⁶ active esters with a good leaving group by taking advantage of a thermodynamically favorable process are susceptible to the attack of an amine. In fact, the active ester strategy with structure well-defined stable esters as the key intermediates was an effective method widely used for peptide synthesis before the advent of coupling reagents (Scheme 1).^17–20 For example, the first chemical total synthesis of oxytocin was accomplished by employing the p-nitrophenyl (PNP) esters of α-amino acids.²¹ However, the prior preparation of active esters requires not only extra steps but also a stoichiometric coupling reagent, a hydroxyl nucleophile, and even a racemization suppressor. In terms of step- and atom-economy as well as operational convenience, there is no advantage for the conventional two-step active ester method compared with coupling reagent-mediated one-pot peptide bond formation. Thus, the active ester method gradually faded with the rapid development of coupling reagents, particularly the bifunctional peptide coupling reagents which play dual roles in activating the carboxylic acid and suppressing racemization. However, after decades of optimization, current peptide synthesis strategies with conventional coupling reagents are reaching a very high standard and their inherent limits. In this context, the active ester strategies employing the structure well-defined active esters would provide a promising solution for the predicament of conventional coupling reagent-mediated peptide synthesis if their disadvantages could be addressed. We herein provide a historical overview and critical analysis of the pros and cons of using active esters for peptide syntheses as well as the state-of-the-art of active ester methods, with a hope of inspiring innovations in developing novel active esters for peptide synthesis.

Scheme 1 Timeline for peptide synthesis using active ester methods discussed in this review.

The stable active esters with well-defined structures will be the focus of this review. However, to maintain the integrity of the overview of the role played by active esters in peptide synthesis, some transient active esters will also be discussed when necessary. It should be noted that transient active esters involved in conventional coupling reagents, such as carbodiimides,²² phosphonium salts,²³ aminium/uronium salts,²⁴ and bifunctional coupling reagents [O-(benzotriazol-1-yl)-1,1,3,3-tetramethyluro-nium hexafluoro-phosphate (HBTU),²⁴O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyl-uronium hexafluorophosphate (HATU),²⁵ benzo-triazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP),²⁶ benzotriazol-1-yloxytri(pyrrolidino) phosphonium hexafluorophosphate (PyBop),²⁷ 3-(diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one (DEPBT),²⁸ diphenylphosphoryl azide (DPPA),^29,30etc.], mediated peptide bond formation, native chemical ligation (NCL),^31–35 imine/hemiaminal-type aldehyde capture ligation,³⁶ Ser/Thr ligation,^37,38 peptide thioester,^39–44 and peptide hydrazides,^45,46 which have been extensively reviewed elsewhere, will not be covered. In addition, enzyme-catalyzed peptide syntheses by employing amino/peptide esters are also beyond the scope of this review.^47–50 Interested readers are referred to several excellent reviews on these exciting topics.^{3,6,10,11,33,51,52} We will focus on the stable active esters with an emphasis on recently developed novel active esters for peptide bond formation. The active esters will be divided into three typical categories in terms of their preparation strategies, i.e., formed in situ with the assistance of a catalyst, synthesized from carboxylic acids and nucleophilic hydroxyl components in the presence of activating reagents, and formed spontaneously from addition reactions of coupling reagents and carboxylic acids directly.

2. Transient active esters

Protein biosyntheses in ribosomes involve enzyme-catalyzed aminolysis of the aminoacyl-tRNA (the tRNA ester of proteinogenic amino acids) for peptide elongation (Scheme 2). Similar to nature, organic chemists have also taken the aminolysis of esters as an effective strategy for synthesizing amides and peptides,⁵³ and a variety of active esters have been exploited for peptide bond formation because of their moderate reactivity, which is beneficial to avoid racemization/epimerization and other undesired side reactions.

Scheme 2 Protein synthesis in ribosomes.

2.1 Common esters activated in situ by a Lewis acid or base catalyst

Alkyl esters are commonly inert and can only be attacked by strong nucleophiles like hydroxylamine and hydrazine.¹⁶ Until 1902, in his seminal contribution to peptide synthesis, Fischer reported that the aminolysis of ethoxycarbonyl diglycine ethyl ester (Eoc-Gly-Gly-OEt) with leucine ethyl ester (H-Leu-OEt) could be adopted for Eoc-Gly-Gly-Leu-OEt synthesis (Scheme 3) under heating conditions.⁵⁴ Nevertheless, low reactivity toward aminolysis restricted their practical application. Catalyst or heating is crucial to promote the aminolysis of N^α-protected amino acid esters.

Scheme 3 Aminolysis of ethyl ester for peptide bond formation.

A series of catalysts have been developed to facilitate the aminolysis of alkyl esters. Transition metal complexes were initially chosen as well-designed catalysts to promote the aminolysis of α-amino acid esters.^55–57 In 1967, Buckingham⁵⁸ and Collman⁵⁹ independently reported cobalt(III) complex-catalyzed peptide bond formation via the aminolysis of amino acid esters. Subsequently, Nakahara et al. discovered a copper(II) complex-catalyzed peptide bond formation reaction.⁶⁰ In 1970, Yamada et al. reported another copper(II)-catalyzed peptide bond-forming reaction.⁶¹ Unfortunately, such copper(II) catalysis resulted in the polymerization of amino esters and gave peptide mixtures (Scheme 4).

Scheme 4 Cobalt(III) and copper(II)-catalyzed peptide formation.

In 2012, Ohshima and Mashima reported that NaOMe was an efficient catalyst for peptide bond formation. However, an extra addition of acidic alcohol or phenol was required to alleviate the basic reaction conditions, which caused severe racemization. Furthermore, a high reaction temperature and prolonged reaction time restricted its application in peptide synthesis (Scheme 5).⁶²

Scheme 5 Peptide coupling reaction catalyzed by NaOMe (adapted with permission from ref. 62, Chem. Commun., 2012, 48, 5434. Copyright (2012) Royal Society of Chemistry).

In 2016, Tsuji and Yamamoto developed a novel strategy for the chemoselective synthesis of amides using a tantalum-catalyzed hydroxy-directed amidation of β-hydroxycarboxylic acid esters (Scheme 6).⁶³ The β-hydroxy group initiated the site-selective activation of the carbonyl group via coordinating with the tantalum catalyst. The reaction proceeded with high chemoselectivity, obtaining the corresponding β-hydroxyamides in moderate to high yields. This strategy could also be extended to the synthesis of dipeptide derivatives. However, a hydroxy directing group at the β-position of the carbonyl group is necessary. In this regard, this method is limited to specific amino acids such as threonine (Thr) and serine (Ser). To circumvent such limitations, an oxime functionality was introduced to the α-position of the ester group. By doing so, the hydroxy-directed strategy has evolved into a solvent-free amidation of N-hydroxyimino esters by using a niobium catalyst (Scheme 7).⁶⁴ Theoretically, the following reduction of the C–N double bond and consequent cleavage of the N–OH bond would offer peptide with a free N-terminal amino group that could be used for the next peptide bond formation. Unfortunately, the unstable N-hydroxyimino esters were immediately transformed into stable isoxazolones.⁶⁴ Afterwards, the third-generation Lewis-acid catalyzed substrate-directed peptide bond formation was reported to resolve the limited substrate scope. The carboxyl groups of methyl ester were “remotely” activated via coordination between the protecting group and the Ta catalyst (Scheme 8, eqn (1) and (2)).^65–67 This reaction system has a broad functional group tolerance and is amenable not only for α-amino acid esters but also β-homoamino acid esters.

Scheme 6 Hydroxy-directed synthesis of dipeptides (adapted with permission from ref. 63, J. Am. Chem. Soc., 2016, 138, 14218. Copyright (2016) American Chemical Society).

Scheme 7 N-Hydroxyimino ester-directed synthesis of peptide derivatives (adapted with permission from ref. 64, ACS Catal., 2018, 8, 2181. Copyright (2018) American Chemical Society).

Scheme 8 Substrate-directed Lewis-acid-catalyzed peptide synthesis.

2.2 Alkyl ester intermediate bearing electron-withdrawing group

In 2014, Nguyen and Lyons established a novel tropylium-based reagent to activate carboxylic acids for amidation via the dearomatization/rearomatization of 1,1-dichlorocycloheptatriene (TropCl₂) systems.⁶⁸ The TropCl₂ could conveniently and reversibly convert to the positively charged aromatic system, which could react with the carboxylic acid to offer an active ester (Scheme 9).^68,69 Aminolysis of such tropylium esters furnished amides or peptides upon releasing a better leaving group tropone. The catalytic version of this strategy has also been achieved by employing stoichiometric oxalyl chloride which regenerated the TropCl₂in situ. Although a low level of racemization was observed when it was used for peptide bond formation, its further application in long peptide synthesis deserved further exploration.

Scheme 9 TropCl₂-mediated peptide formation.

A 4-dimethylamino-pyridine (DMAP)-alkyl halide charge-transfer complex was designed by Eichen and Szpilman group as a novel coupling reagent.⁷⁰ This in situ formed reagent reacted with carboxylic acids to afford the hemiaminal active esters (Scheme 10), which could couple with the amines or amino acid esters to form amides or peptides, respectively.⁷⁰ The reaction proceeds smoothly with common N^α-protected amino acids without any detectable racemization/epimerization. A series of dipeptides and tripeptides have been constructed using this strategy. However, the loading of DMAP (10 equiv.) and CBrCl₃ (10 equiv.) for the generation of the charge-transfer complex is relatively high.

Scheme 10 Peptide synthesis using hemiaminal active esters (adapted with permission from ref. 70, Angew. Chem., Int. Ed., 2021, 60, 12406. Copyright (2021) Wiley-VCH).

Very recently, Wang and Sun have innovatively introduced a recyclable coupling reagent 5-nitro-4,6-dithiocyanatopyrimidine (NDTP) for amide and peptide bond formation under mild conditions.⁷¹ The mechanism indicated that NDTP activated the carboxylic acid via acyl thiocyanide, which could be viewed as an active thioester bearing an electron-withdrawing group (EWG). Various racemization-free dipeptides could be prepared from protected amino acids and methyl esters of amino acid hydrochloride by using NDTP as the coupling reagent (Scheme 11). The subsequent experiment indicated that NDTP was practical as well for SPPS. This novel coupling reagent might open a new avenue for peptide synthesis.

Scheme 11 NDTP-mediated peptide synthesis (adapted with permission from ref. 71, Org. Lett., 2022, 24, 1169. Copyright (2022) American Chemical Society).

2.3 Silicon reagent-promoted peptide bond formation

In 2011, Liebeskind and coworkers reported a novel approach for in situ activation of thioacids via the silatropic switch process. The proposed mechanism indicated that the sulfur atom of the thioacid was kinetically trapped by the trimethylsilyl group to form the S-trimethylsilylthiol esters, which could spontaneously transform into O-silylthionoesters via a thermodynamically driven tautomerization. The silylation of thioacid was accomplished smoothly by treating thioacid with bistrimethylsilylacetamide (BSA). Interestingly, the aminolysis of the O-silylthionoesters afforded an amide bond exclusively rather than a thioamide bond (Scheme 12). It could be used for peptide bond formation with little racemization, which was positively influenced by the nature of the silylating agent.⁷²

Scheme 12 BSA-mediated peptide synthesis.

Based on Lewis acid-catalyzed peptide bond formation, a silicon-mediated approach to peptides via an in situ-generated silyl ester was reported by Yamamoto and Muramatsu to circumvent the use of rare and expensive metals.⁷³ The HSi[OCH(CF₃)₂]₃ reacted with N-protected amino acids to afford the active silyl esters in situ. Such esters then reacted with the amino acid esters or N-silylated amino acid esters to furnish the peptides (Scheme 13).⁷³ This strategy has also been extended to the unprotected or partially protected amino acids using two silylating reagents with different reactivities (Scheme 14).⁷⁴ In such a one-pot peptide bond formation strategy, dipeptides and tripeptides could be obtained smoothly using unprotected amino acids as the acyl donor. Recently, a novel type of peptide bond formation utilizing imidazolylsilane derivatives and unprotected amino acids has been reported by Hattori and Yamamoto (Scheme 15).⁷⁵ The diimidazolylsilane derivatives are effective reagents to construct five-membered ring active esters, which react with amino acid tert-butyl (^tBu) esters smoothly to offer the desired dipeptides.

Scheme 13 Hydrosilane-mediated peptide synthesis (adapted with permission from ref. 73, ACS Catal., 2020, 10, 9594. Copyright (2020) American Chemical Society).

Scheme 14 Peptide synthesis by transient masking with silylating reagents (adapted with permission from ref. 74, J. Am. Chem. Soc., 2021, 143, 6792. Copyright (2021) American Chemical Society).

Scheme 15 Diimidazolylsilane derivative-mediated peptide synthesis (adapted with permission from ref. 75, J. Am. Chem. Soc., 2022, 144, 1758. Copyright (2022) American Chemical Society).

Furthermore, deprotection of the ^tBu ester at the C-terminus affords silicon-containing fused cyclic dipeptides. Remarkably, these fused cyclic dipeptides are sufficiently stable to be isolated with silica gel chromatography and handled under atmospheric conditions. In addition, the fused cyclic dipeptides turn out to be perfect active esters to react with both nucleophiles (elongation at the C-terminus) and electrophiles (elongation at the N-terminus). Besides unnecessary purification of the in situ formed silyl active ester intermediates, the silyl active ester strategies possess other features, including high functional group tolerance, convenient operation, and offering peptides with high optical purity. In addition, by employing unprotected amino acids as the starting material, these silyl active ester strategies merged the protection and activation of amino acids in one operation, and thus shortened the tedious procedure and remarkably reduced chemical waste as well as the time, energy, chemicals, and solvents, making this protocol a potential paradigm for bioactive peptide synthesis. However, the application of this method for larger peptide syntheses and SPPS needs to be further explored.

2.4 Boron ester promoted peptide bond formation

The first boron reagent for promoting the coupling of N^α-protected amino acid and α-amino ester for peptide bond formation was reported in 1970.⁷⁶ However, the efficient boronic acid catalyzed amidation was disclosed in 1996 by Yamamoto.⁷⁷ The original mechanistic proposal suggested that the carboxylic acid was activated by boronic acid via an in situ-formed “monomeric” mixed anhydride (heteroatom active ester) (Scheme 16). Later, Whiting⁷⁸ and Ishihara⁷⁹ revised the mechanism and suggested a feasible “diboroxane” mixed anhydride intermediate based on spectroscopic observations. After the first report of arylboronic acid-catalyzed amide bond formation, various modified aromatic aryl boronic acids, including (2-(thiophen-2-ylmethyl)phenyl)boronic acid,⁸⁰ 5-methoxy-2-iodophenylboronic acid,⁸¹ and 3,5-bis(trifluoromethyl)phenyl-boronic acid,⁷⁹ have been developed for the catalytic peptide syntheses (Scheme 16).^6,9,82 Although important progress has been achieved in the boronic acid-catalyzed peptide synthesis, the reactions still suffer from inefficient catalysis. Recently, Takemoto and co-workers reported that the formation of the “diboroxane” mixed anhydride intermediate might be hampered, preventing the further development of aryl boronic acids as catalysts for peptide bond formation.⁸³ To circumvent this dilemma, gem-diboronic acid (gem-DBA, Scheme 17)⁸³ and diboronic acid anhydride (DBAA)⁸⁴ with a bidentate B–O–B skeleton activation of α-amino acids were designed as peptide condensation catalysts. These reagents are compatible with various functionalized α-amino acids, albeit with a slight racemization.

Scheme 16 Boron acid-catalyzed peptide bond formation.

Scheme 17 gem-DBA-mediated peptide synthesis (adapted with permission from ref. 83, ACS Catal., 2019, 10, 683. Copyright (2019) American Chemical Society).

2.5 Organophosphorus ester intermediate-promoted peptide bond formation

Phosphorus species have been widely adopted as coupling reagents since the systematic study by Castro, who first introduced BOP as a peptide coupling reagent.²⁶ In seeking new coupling reagents, Ni and co-workers recently reported an efficient racemization-free amidation and esterification reaction by employing a Ph₃PO(cat.)/(COCl)₂ system.⁸⁵ The in situ-generated acyl phosphonium salt served as an active ester to afford amides and esters in the presence of the corresponding amines and alcohols (Scheme 18). The reaction was easy to handle and could be used to synthesize dipeptides without racemization. The success of the 100 mmol reaction showcased a great potential for scale application. Thanks to the recent advancements in photoredox catalysis, a photocatalytic redox condensation strategy for amide/peptide synthesis was designed by Zhao and co-workers using a trivalent phosphine (PPh₃) as the reductant.⁸⁶ During the reaction, PPh₃ was transformed into an acyloxyphosphonium species, which was readily attacked by amines (Scheme 19). The compatibility with functionalized amino acids and assembly of peptides through SPPS, especially the utility of a continuous-flow photoreactor, illustrated its potential application.

Scheme 18 Ph₃PO(cat.)/(COCl)₂-mediated peptide synthesis (adapted with permission from ref. 85, Org. Lett., 2021, 23, 7497. Copyright (2021) American Chemical Society).

Scheme 19 Photocatalytic redox condensation for peptide synthesis (adapted with permission from ref. 86, Angew. Chem., Int. Ed., 2022, 61, e202112668. Copyright (2022) Wiley-VCH).

3 The prior prepared active esters

Although unactivated alkyl esters of N^α-protected amino acids could be employed for peptide bond formation via in situ activation by catalysts or additives, these methods continue to face serious problems, including thermodynamic and kinetic barriers. In addition, the scope of these methods has been mostly limited to simple dipeptide or tripeptide substrates. Thus, it is necessary to explore their applications in the synthesis of larger peptides, especially in SPPS. Current mainstream peptide syntheses are accomplished by converting the –OH groups of the amino acids into better leaving groups, namely activated carboxylic acid derivatives. Although hundreds of activating reagents have been developed to realize this purpose, these activated carboxylic acid derivatives tend to undergo undesired side reactions such as racemization/epimerization due to their overactivation.^{3,6,7,10,11,51} Therefore, additives are always necessary to convert the higher active intermediate into the mild acyl donors, whereby racemization/epimerization could be suppressed or avoided (Scheme 20). Among them, several strong hydroxyl nucleophiles were identified as effective additives to convert the highly reactive acyl donor species into active esters in situ or prior prepared. The classical prior prepared active esters, including EWG-substituted alkyl esters, lactones, phenyl ester derivatives, and hydroxamic active esters, have been widely used for peptide synthesis because they offered an opportunity for addressing the notorious racemization/epimerization issue.

Scheme 20 Prior prepared active ester.

3.1 Alkyl esters activated by electron-withdrawing substituents

Once an EWG is incorporated into an alkyl moiety, the alkyl esters will be activated and can be attacked by amine to execute the amide bond formation. The acylation efficiency of methyl esters substituted with one or more EWGs was thoroughly examined by Schwyzer et al.,⁸⁷ among which cyanomethyl ester was identified as an effective acylating reagent for peptide synthesis (Scheme 21).^87–90 Although the racemization could be avoided using the cyanomethyl ester strategy,⁹⁰ their applications in peptide manufacture are limited because of their low reactivity.⁹¹

Scheme 21 Peptide synthesis using cyanomethyl esters.

3.2 Lactones

β-Lactones formed from the intramolecular esterification of the β-hydroxy carboxylic acid are more reactive than common alkyl esters because of the strain of the four-membered ring. Thus, Ser-β-lactone has been used to prepare dipeptides (Scheme 22, eqn (1)).⁹² It is known that the sulfonium derivatives of methionine easily undergo elimination of the MeS-functional group with concomitant formation of homoserine (Hse)-γ-lactone.⁹² Using this method, γ-lactones have been prepared by cyanogen bromide cleavage with methionine-containing peptides.⁹³ However, owing to their low reactivity toward nucleophiles, ring-opening of the γ-lactones was only observed in the intramolecular acylation case (Scheme 22, eqn (2)).⁹⁴ Bifunctional reagents have been employed to construct five-membered heterocyclic lactones not only for protecting the amino group but also for activating the carboxyl group.⁹⁵ Such lactones can be attacked by a sort of nucleophile, in particular the amino group, to construct an amide bond. Among these bifunctional reagents, hexafluoroacetone (HFA) derivatives represented the most successful one for constructing tailor-made building blocks for peptide, depsipeptide, and diketopiperazine syntheses (Scheme 23).^96–99 An impressive HFA strategy was reported to give a two-step synthesis of the sweetener aspartame, which typically took at least four steps by conventional methods (Scheme 23).¹⁰⁰ Similar to the silyl active ester strategies, this method avoided the protection and deprotection steps, which were necessary for conventional peptide synthesis strategies and thus were promising for developing atom-economical peptide synthesis strategies. However, the elongation of peptide chains using HFA-amino acids was accompanied by the formation of undesired diketopiperazines (DKPs).⁹⁸

Scheme 22 Peptide synthesis using the lactone method.

Scheme 23 Examples of the application of HFA derivatives.

3.3 Active aryl esters

A systematic study of the aminolysis of esters RCOOR′ indicated that the reactivity and electronic effects of the R and R′ groups are densely interwoven.¹⁰¹ In light of this, attention has been paid to ester derivatives with π-electron systems, which displayed greater reactivity than alkyl esters by several magnitudes. The first attempt to boost ester reactivity was realized by using thiophenyl esters (Scheme 24).¹⁰² The amino acid thioesters and sodium salt of unprotected amino acids were boiled in methanol for several hours to furnish the sodium salt of the dipeptides with concurrently release of the thiophenol. Inspired by this, various electron-deficient thiophenyl esters and other chalcogen-substituted phenol esters were studied.^103–107 Such modifications resulted in higher aminolysis reactivity than that of the corresponding phenyl esters. However, the released chalcogen-substituted phenol derivatives such as thiophenols are unpleasant to handle because of their malodorous smell.

Scheme 24 Use of amino acid thioester for peptide synthesis.

It was soon recognized that the aromatic ring rather than the chalcogen atom is responsible for activation. Bodanszky embarked on a systematic investigation of substituted phenyl esters, in which electron-withdrawing substituents were installed in the phenyl group to further increase its leaving ability.¹⁰⁸ Preliminary studies involved four nitrophenol esters with different substitution styles, whose reactivities were far greater than that of the phenyl ester and even the thiophenyl ester (Scheme 25, 25-1–25-4).^103,108 The 2,4-dinitrophenyl esters (Scheme 25, 25–4) provide excessive reactivity, rendering themselves susceptible to hydrolysis. Compared with acyl chlorides, mixed anhydrides, acid azides, and coupling reagent-mediated amide bond formation, nitrophenyl esters could efficiently avoid racemization/epimerization and other undesired side reactions. Meanwhile, the stability of the nitrophenyl esters allowed for preparation and storage on a large scale. These active esters proved to be preferable agents for a stepwise approach to peptides in the 1950s. p-Nitrophenyl (PNP) esters of proteinogenic amino acids were identified as the optimal building blocks for synthesizing long peptide chains such as oxytocin (Scheme 26),^21,109,110 gramicidin-S,¹¹¹ secretin,¹¹² and the B-chain of insulin in practice.¹¹³

Scheme 25 Active aryl esters for peptide bond formation.

Scheme 26 Total synthesis of oxytocin using PNP esters.

The positive feedback of using electron-deficient phenyl esters to enhance the reactivity of aryl esters stimulated further investigations on aryl esters carrying various electron-withdrawing substituents. Boissonnas and co-workers¹¹⁴ employed the more electron-deficient phenyl esters such as 2,4,6-trichloro- and pentachlorophenyl (PCP) esters (Scheme 25, 25-5, 25-6), which were highly compatible with N-benzyloxycarbonyl (Cbz) and ^tBu protecting groups for peptide synthesis. Tripeptides could also be constructed via chain elongation from either the N- or C-terminus using PCP esters, further highlighting the flexibility of the active ester strategy (Scheme 27).^114–116

Scheme 27 Synthesis of a tripeptide using PCP esters.

Meanwhile, the impact of halogen substituents on the substituted phenol esters was explored. Pless and Boissonnas conducted an extensive investigation regarding the reaction rates and dissociation constants of diversely substituted phenols.¹¹⁴ Their study suggested that the pK_a of phenol derivatives did impact the reactivity of the aryl esters. Moreover, the steric hindrance caused by substituents offset the electron-withdrawing effects. The steric hindrance effects of the phenyl esters are ranked in the order of Cl < Br < I. The reactivity of monochloro aryl esters with different substitution positions on the phenyl ring is ranked in the order of p < m < o. Regardless of the steric hindrance effects, the electronic effects of multiple halogen substituents are superimposed to enhance the electrophilicity of the ester carbonyl group. This investigation led to the identification of 2,4,5-trichlorophenyl esters (Scheme 25, 25-7), which resolved the steric issues of the 2,4,6-trichloro- and PCP esters as the optimal active ester for peptide synthesis. However, it remains limited in practice due to its slow ammonolysis rate, especially in SPPS.^114,117 A variety of aryl esters have been examined to gain additional insight,¹¹⁸ among which the pentafluorophenyl (PFP) esters (Scheme 25, 25-8) appeared to be of significant advance.^119–121 The van der Waals radius of fluorine is smaller than that of chlorine and slightly larger than that of hydrogen, yet its electronegativity far surpasses chlorine and hydrogen atoms. Given a better electron-withdrawing effect delivered by the fluorine atom without causing bulkiness, PFP esters are efficient even in a crowded environment.^120,121 The advances of these active esters in solution-phase peptide synthesis prompted chemists to explore their applicability in SPPS.¹²² A decapeptide ACP(65–74) and a dodecapeptide were prepared via SPPS with Fmoc-amino-acid PFP esters as the building blocks (Scheme 28).^123,124

Scheme 28 Solid phase synthesis of ACP(65–74) using PFP esters.

Similar enhancements in reactivity are achievable using EWGs, other than nitro groups and halogens, such as cyano (Scheme 25, 25-9)¹²⁵ and sulfonyl groups (Scheme 25, 25-10).^126,127 Aromatic heterocycles had also proved to be viable substitutes for the phenyl ring of phenol ester. The electronegative nitrogen and oxygen atoms in these heteroaromatic rings may diminish the electron density at the ester carbonyl group, thus prompting its reactivity toward nucleophiles. The esters of 5-hydroxypyrazole (Scheme 25, 25-11)¹²⁸ and 2-hydroxypyridine (Scheme 25, 25-12)¹²⁹ had been employed in peptide syntheses. Although the application of active aryl esters in peptide syntheses prospered for a long time and most aryl esters could be purified and stored, and PFP esters were even commercially available, the preparation of these esters using standard ester-formation strategies such as dicyclohexylcarbodiimide (DCC)-mediated coupling was tedious. Thus, the aryl active esters were only used in some special cases. The in situ-formed benzyne trapping strategy may mitigate this dilemma in the future.¹³⁰

3.4 Hydroxamic active esters

In 1961, a well-known type of active ester was proposed by Nefkens and Tesser, in which N-hydroxyphthalimide rather than the phenol derivatives was used as the hydroxyl component of the active ester (Scheme 29, 29-1).¹³¹ The aminolysis of N-hydroxyphthalimide esters took place rapidly. Being insoluble in water, the released N-hydroxyphthalimide can be removed by a basic solution treatment. However, serious racemization makes this method inapplicable for peptide segment condensation.¹³² More robust N-hydroxy-succinimide (HOSu) esters (Scheme 29, 29-2) were soon designed to mitigate such defects.¹³³ The HOSu esters of proteinogenic amino acids displayed satisfactory properties in peptide synthesis owing to their higher reactivity, easy preparation, high stability against hydrolysis, and compatibility with aqueous reaction conditions. Furthermore, the released HOSu is water-soluble and can be straightforwardly removed at the purification stage (Scheme 30).¹³⁴ The HOSu esters have also been widely adopted to modify lysine residues in proteins¹³⁵ and living cells.¹³⁶ Despite their extraordinary potential, Gross and Bilk's study demonstrated that these esters suffered from a side reaction through Lossen rearrangement (Scheme 31).^11,137–140 To address this issue, Fujino et al. introduced the N-hydroxy-5-norbornene-endo-2,3-dicarboxyimide (HONB) esters (Scheme 29, 29-3) containing a rigid backbone, which is beneficial for suppressing the formation of the β-alanine-type by-product. The successful application of HONB esters in various peptide syntheses prompted the synthesis of porcine or bovine luteinizing hormone-releasing hormone (LH-RH, Scheme 32).¹⁴¹ This strategy showcased a high degree of flexibility. Both C to N and N to C elongation strategies are compatible.¹⁴¹

Scheme 29 Hydroxamic active esters used in peptide syntheses.

Scheme 30 Peptide synthesis using the HOSu ester method.

Scheme 31 The mechanism for the side reaction during the Fmoc protection of amino acids with Fmoc-OSu (adapted with permission from ref. 11, Chem. Rev., 2011, 111, 6557. Copyright (2011) American Chemical Society).

Scheme 32 Synthesis of LH-RH using HONB esters.

The former works focused on the N,N-diacylhydroxylamine derivatives, but these active esters typically suffered from poor solubility, side reactions, and tedious preparation. The O-acyl hydroxylamine esters were recommended as potential substitutes to circumvent these issues. The investigation of O-acyl hydroxylamine derivatives indicated that the neighbor nitrogen atom enables the hydroxyl group to be readily acylated; more importantly, the electronegativity of the nitrogen atom could effectively activate the acylated derivatives as well.^142,143 Therefore, N-monoacylhydroxyl-amine was employed to construct the activated ester of N-protected amino acids. 2-Pyridone derivatives prepared by Paquette were the first batch of N-monoacylhydroxylamine active esters, which coupled with amino esters to generate dipeptides (Scheme 29, 29-4).¹⁴⁴ In 1965, Bittner and co-workers reported a kind of novel activated amino acid ester via the esterification of N,N-diethylhydroxylamine and N-carboxy-α-amino acid anhydrides (Scheme 29, 29-5).¹⁴⁵

However, the reactivity of this kind of ester was relatively low, “some days” were required for completing the aminolysis reaction. Coincidentally, Young et al. developed similar 1-hydroxypiperidine (PIP) esters for peptide synthesis (Scheme 29, 29-6),¹⁴⁶ which were stable intermediates and could be activated by adding a certain amount of acetic acid. In this case, the N,N-dialkylhydroxylamine esters afforded acidic conditions for peptide synthesis, avoiding the risk of base-induced racemization/epimerization (Scheme 33).¹⁴⁷ In addition, the aminolysis of PIP esters is revealed to be accelerated by the neighboring nitrogen atom, which can form a hydrogen bond with the incoming amine and accept a proton to boost the subsequent aminolysis step.¹⁴⁸

Scheme 33 Synthesis of peptides using PIP esters.

In 1970, more than 30 other N-hydroxy amino acid esters were prepared by König and Geiger, among which the amino acid esters of 1-hydroxybenzotriazole (HOBt) were favorable acylating and racemization-resistant active esters (Scheme 29, 29-7).¹⁴⁹ The preformed HOBt esters displayed a striking advance in suppressing racemization during peptide fragment condensation (FC) in solution.^149,150 Later, the preactivation of peptide fragments in the presence of HOBt with DCC as the coupling reagent was successfully expanded to solid-phase fragment condensation (SPFC);¹⁵¹ however, a flaw of the DCC/HOBt method is that the excess activated peptide fragments cannot be recovered unchanged. Racemization/epimerization contaminates unreacted and potentially valuable excess fragments.¹⁵¹ Shortly thereafter, different HOBt derivatives were introduced.^152–155 Those 1-hydroxy-7-azabenzotriazole-based (HOAt, Scheme 29, 29-8) active esters were recognized to be superior to others, and accelerated coupling reactions with reduced loss of chiral integrity in both solution- and solid-phase peptide synthesis.¹⁵⁶ The mechanistic study suggests that the nitrogen atom located at the 7-position of the 7-aza benzotriazole improves its leaving ability by taking advantage of the neighboring group effect, which is generally acknowledged to enhance reactivity and reduce the risk of losing chiral integrity.¹⁵³ Though HOBt and its derivatives have been used widely as racemization suppressors or peptide coupling reagents, potentially explosive properties limit their applications.¹⁵⁷

3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benzotriazine (HODhbt), a six-membered analogous of HOBt, is not only a superior racemization suppressor but also a better additive to improve the coupling efficacy (Scheme 29, 29-9).¹⁴⁹ It had also been used in SPPS (Scheme 34).^158–160 Inspired by the earlier studies of HOAt, Carpino and co-workers synthesized aza-derivatives (HODhat and HODhad) of HODhbt (Scheme 29, 29-10, 29-11).¹⁶¹ Their experimental results showed that an unexpected peptide chain termination occurred during the peptide synthesis, which might be attributed to o-azidobenzoic acid ester, a by-product produced in HODhbt ester preparation, although HODhat esters are slightly more reactive than the HOAt esters.¹⁶¹ To reduce the impact of this by-product, the DCC used in the preparation of these esters has to be controlled rigorously, and the crude products should be recrystallized carefully.^158,159,162

Scheme 34 Synthesis of endoplasmin(1–18) using HODhbt esters.

The additive ethyl 2-cyano-2-(hydroxyimino)acetate (Oxyma), which was first reported as a racemization suppressor for peptide synthesis by Itoh in 1973,¹⁶³ was reinvestigated by El-Faham and Albericio to overcome the limitations of benzotriazole-based and HODhbt active esters (Scheme 29, 29-12).¹⁶⁴ Oxyma displays greater superiority in suppressing racemization/epimerization and improving coupling efficiency than HOBt in both automated and manual peptide synthesis. Moreover, the excellent thermal stability of Oxyma makes it a promising candidate with lower thermal risk.

4. Active esters formed upon reaction with activating reagents

As described in the third part, the preparations of conventional active esters required not only stoichiometric coupling reagents but also stoichiometric hydroxyl nucleophiles, such as HOPFP, HOSu, and HOAt. Most active esters necessitate extra preparation steps rendered such methods neither atom- nor step-economical. In addition, the risk of explosion,¹⁵⁷ life-threatening anaphylaxis,¹⁶⁵ or generating toxic HCN gas¹⁶⁶ was always intertwined with these active esters. Fortunately, the in situ-formed active esters demonstrated an excellent ability to alleviate the above problems and have emerged as potentially green and ideal substitutes. The advantages and disadvantages of the in situ-formed active esters are discussed in detail in this section (Scheme 35).

Scheme 35 Active esters formed upon reaction with activating reagents.

4.1 Alkoxyacetylenes

Methoxyacetylene, a coupling agent for peptide synthesis, was developed by Arens and co-workers in 1955, almost at the same time as that of DCC (Scheme 36).¹⁶⁷ Mechanistically, methoxyacetylene first reacts with a carboxylic acid, producing acyl enol esters, which undergo aminolysis or alcoholysis to produce the corresponding amides or esters, respectively.^168–175 These active acyl enol esters are quite attractive for peptide synthesis because the only by-product of the aminolysis reaction is ethyl acetate, which is harmless, volatile, and can be removed easily. However, long reaction times were required for carboxylic acids with moderate acidity. Although transition metal catalysts such as Hg(II),¹⁷⁰ Ru(II),¹⁷³ Ag(I),¹⁷⁶ and Au(I)¹⁷⁷ complexes are helpful for facilitating the activation of carboxylic acids, the low thermal stability of alkoxyacetylene and the serious racemization that occurred during the coupling circumscribed its further application in peptide synthesis.¹⁷⁸ Recently, Breinbauer and co-workers have demonstrated that enol esters of peptide fragments prepared via the Ru(II)-catalyzed addition of peptide acid and alkyne could be used as an acyl donor for enzyme-catalyzed racemization-free peptide fragment condensation.¹⁷⁹ Inspired by such work, Gooßen and co-workers disclosed that acetylene, in the presence of Ru(II) catalyst, could also be used as a coupling reagent for amide bond formation (Scheme 37).¹⁷⁸ The only by-product was the versatile acetaldehyde. However, remarkable racemization occurred when it was used for peptide synthesis. As the smallest coupling reagent, acetylene would become an ideal coupling reagent if its flaws could be resolved.

Scheme 36 Methoxyacetylene-mediated peptide bond formation.

Scheme 37 Ruthenium-catalyzed acetylene or alkoxyacetylene-mediated peptide synthesis (adapted with permission from ref. 178, Nat. Commun., 2016, 7, 11732. Copyright (2016) Springer Nature).

4.2 α-Chlorobenzaldoxime and imidoyl halide

In 1964, Widdowson and Sutherland reported the synthesis of O,N-dibenzoylhydroxylamine esters from α-chlorobenzaldoxime and silver benzoate without the use of other reagents.¹⁸⁰ The nitrile oxide liberated by the α-chlorobenzaldoxime reacts with the carboxylic acid spontaneously to yield the desired active esters (Scheme 38, eqn (1)). Subsequently, various nitrile oxide derivatives were evaluated for peptide synthesis.¹⁸¹ Among these, N-trimethylacetyl-hydroxylamine proved to be the optimal choice for preparing active esters of proteinogenic amino acids (Scheme 38).¹⁸² This method was applied for the synthesis of di- and tripeptides but was not amenable for larger peptides due to the competing Lossen rearrangement reaction, which gave isocyanate by-product.^183,184 Similarly, the imidoyl halide reagents underwent rapid unimolecular ionization to give the nitrilium ions, which react rapidly with carboxylate ions to afford stable O-acylisoamide active esters (Scheme 38, eqn (2)).¹⁸⁵ The aminolysis of these active esters completed rapidly. The potential racemization of the α-amino acid could be suppressed by adjusting the pH ≤ 7 or modifying the substituent of imidoyl halide reagents.

Scheme 38 Peptide syntheses using N-monoacylhydroxylamine esters.

4.3 Woodward reagents

In 1961, Woodward and Olofson reinvestigated the reactions between isoxazolium salts and carboxylic acids,^186–188 with a hope of developing a coupling reagent that could compete with DCC. After extensive studies, N-ethyl-S-phenylisoxazolium-3′-sulfonate (Woodward's Reagent K) was identified as an optimal choice.¹⁸⁹ Mechanistically, the hydrogen atom at C-3 of isoxazolium 39-1 is first abstracted by a base, opening the ring in a concerted manner to offer α-ketoketenimine 39-2 (Scheme 39). Then an N-protected amino acid is added to such a reactive intermediate, forming an unstable adduct (39-4) that rapidly rearranges to enol ester 39-5. As an appealing acylating agent with high reactivity, the enol ester 39-5 enables spontaneous aminolysis to furnish a peptide bond (Scheme 39). Despite the high efficacy of the first-generation Woodward coupling reagent and the water-solubility of the by-product, a competitive rearrangement of the enol ester results in the formation of the imide, which is a less reactive acylating agent and challenging to remove.¹⁸⁶ To address this issue, a bulky substituent was installed on the nitrogen atom to suppress such uninvited rearrangement.^190,191 The enol ester derived from 39-1b was sufficiently stable for storage and resistant to rearrangement. However, the formation of enol esters from 39-1b was relatively slow and suffered from side reactions in polar solvents.^190,191 Later, N-alkylbenzisoxazolium cation was used to circumvent this issue, in which the enol esters derived from these reagents were stable and did not rearrange.^192–194 Unfortunately, this approach suffered from racemization and low reactivity during the aminolysis step, thus eventually resulting in Woodward reagents being abandoned by the peptide community.

Scheme 39 Peptide synthesis using Woodward coupling reagents.

4.4 Triazine-based coupling reagents (TBCRs)

In 1994, to rationalize the drastically high reactivity of their triazine-based coupling reagents (TBCRs),¹⁹⁵ Kamiński coined a new term “superactive ester” to differentiate it from classic “active ester” with different rate-limiting steps for aminolysis.¹⁹⁶ In general, the aminolysis of active ester proceeds via a stepwise mechanism involving a nucleophilic attack of an amine on the ester carbonyl group to furnish a tetrahedral intermediate (TI) and a subsequent expulsion of a leaving group (Scheme 40).^197,198 The rate-limiting step of aminolysis reaction is closely related to the basicity of the amines and the reactivity of active esters. This relationship can be measured by the Brønsted parameter β_nuc (defined as the slope in a plot of log k vs. pK_a).¹⁹⁶ For a large number of active esters, β_nuc = 0.9 ± 0.2 has been assigned to the aminolysis reactions with a rate-limiting breakdown of TI, while β_nuc = 0.2 ± 0.1 for the others with a rate-limiting attack of the amine on the ester carbonyl group (Scheme 40).^196,199 To differentiate these two types of active ester, Kamiński classified esters bearing a good leaving group with a β_nuc = 0.2 ± 0.1 as “superactive esters”.¹⁹⁹ As shown in Fig. 1, lowering the energy barrier in TS2 formation below that of the TS1 required the leaving group to participate in an additional energetically favored process, such as rearrangement, to offer further stabilization synchronized with its departure. Accordingly, the “superactive ester” has the merits of coupling smoothly with not only strong nucleophilic amines but also moderate basic amino components. 2-Alkylacetyloxy-4,6-dimethoxy-1,3,5-triazine ester formation is likely to proceed in a way that the tertiary amine is substituted for the chlorine atom and forms a quaternary ammonium species (Scheme 41). This step proceeded with extreme sensitivity toward the steric hindrance of the amine. The “superactive ester” is then afforded by the S_NAr reaction of the quaternary ammonium species and a carboxylic acid, in which the steric bulkiness of the acid is compatible (Scheme 41).^196,200 Unfortunately, the triazine reagents are irritating to the eyes and nose; thus, proper protection is necessary during the operation.^201,202 In addition, the tertiary amine for the activation of carboxylic acids by CDMT is essential, and only certain tertiary amines, such as N-methylpiperidine (NMM), are able to activate the reaction.²⁰³ The formation rate of triazinylammonium salts is dependent on the steric bulkiness of the N-substituents. The success of this two-step amide bond formation procedure heavily relied on the completion of the carboxylic acid activation step.^201,203 To resolve this issue, Kamiński et al. integrated the CDMT–NMM system to obtain 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMTMM).²⁰³ However, the initial DMTMM suffered from a demethylation side reaction in aprotic solvents during storage.^201,203 The chloride anion was thus switched to the non-nucleophilic tetrafluoroborate anion to block the demethylation.¹⁹⁹ This tetrafluoroborate salt coupling reagent (DMTMMBF₄) was widely adopted in the synthesis of dipeptides derived from natural and unnatural hindered amino acids. Although TBCRs have been demonstrated to be efficient in assembling peptides, head-to-tail peptide cyclization (Scheme 42), and SPPS (Scheme 43), the indispensable presence of base increases the epimerization risk.¹⁹⁹

Scheme 40 Mechanism of the aminolysis of an active ester and Brønsted relation for amide bond formation (shadowed area) with a classic active ester (left panel) or “superactive ester” (right panel) (adapted with permission from ref. 199, J. Am. Chem. Soc., 2005, 127, 16912. Copyright (2005) American Chemical Society).

Fig. 1 Energetic profile for the aminolysis reaction of a classic active ester (left, rate-limiting TI breakdown) and “superactive ester” (right, fast TI breakdown, rate-limiting attack of amine on an acylating reagent) (adapted with permission from ref. 199, J. Am. Chem. Soc., 2005, 127, 16912. Copyright (2005) American Chemical Society).

Scheme 41 Mechanism of carboxylic acid activation with 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) (adapted with permission from ref. 199, J. Am. Chem. Soc., 2005, 127, 16912. Copyright (2005) American Chemical Society).

Scheme 42 Synthesis of peptides using the “superactive ester” method.

Scheme 43 Solid phase synthesis of ACP(65–74) with DMTMMBF₄ as the coupling reagent.

4.5 Isonitriles

While probing preferable methods for the synthesis of larger size peptides and glycopeptides, Danishefsky and his co-workers reinvestigated the reactivities of isonitriles and developed a two-component amide bond formation strategy by taking advantage of the condensation of a carboxylic acid and an isonitrile.^204–207 The amide bond formation was accomplished by the reaction of carboxylic acid and isonitrile to generate a high-energy formimidate carboxylate mixed anhydride (FCMA), which undergoes a 1,3-O → N acyl transfer to deliver an N-formyl moiety to the nitrogen atom.²⁰⁸ Being reversible, the formation of oxy-FCMA is disfavored under usual reaction conditions. Only high temperatures with microwave irradiation can trigger the 1,3-O → N acyl transfer of oxy-FCMA. Conversely, the generation of the thio-FCMA and the corresponding S → N rearrangement is more rapid and facile than that of the oxy-FCMA counterpart (Scheme 44, type I). The glycopeptide N-glycosyl asparagine was synthesized based on the type I method.²⁰⁹ Given the sluggishness and inefficiency of the type II intermolecular nucleophilic reaction (Scheme 44, type II), the FCMA, as an active ester, is more likely to undergo an intramolecular acyl transfer in oxyacid cases. The reaction of the thio-FCMA proceeds competitively between the intramolecular 1,3-S → N acyl transfer and the intermolecular acylation. Later, sterically demanding groups were introduced to the isonitriles, which were beneficial to effectively suppress the type I intramolecular S → N acyl migration pathway while favoring the type II bimolecular acylation (Scheme 44).²¹⁰ Founded on hindered isonitriles (e.g., tert-butylisonitrile), thio-FCMA intermediates, as powerful acyl donors, accommodated a broad range of substrates, including bulky thioacids, secondary amines, and alcohols.²¹⁰ However, expanding this coupling method to even a dipeptidyl thioacid failed to provide satisfactory results. The dipeptide thio-FCMA underwent rapid conversion to the non-productive oxazolone intermediate.²¹¹ To remedy this situation, HOBt was added to the reaction mixture to convert the active thio-FCMA intermediate into an HOBt ester.²¹¹ Although the pathway was not definitively proved in the solution phase, the mechanism was well elucidated under SPPS conditions.²¹² Both the type I and II methodologies have been exploited for simple amide synthesis and peptide fragment condensations,^211,212 as well as the synthesis of natural products (Scheme 44)²¹³ such as cyclosporine A²⁰⁹ and human granulocyte colony-stimulating factor (G-CSF).²¹⁴

Scheme 44 Coupling mechanism for isonitrile reagents and applications to the synthesis of G-CSF and cyclosporine A (adapted with permission from ref. 209, J. Am. Chem. Soc., 2010, 132, 4098. Copyright (2010) American Chemical Society).

4.6 Ynamides

Given the low reactivity of the C–C triple bond of alkoxyacetylenes, Viehe and co-workers designed an ynamine coupling reagent by replacing the oxygen atom of alkoxyacetylenes with a stronger electron-donating nitrogen atom with the hope of addressing the low reactivity of alkoxyacetylenes (Scheme 45, a).^215–217 By doing so, the reactivity of the alkynyl-based coupling reagents increased dramatically, thus making the transition metal catalyst used for alkoxyacetylenes not necessary for ynamine coupling reagents. However, ynamines were too reactive to handle. Despite continued efforts, ynamines have proved to be of little utility as peptide coupling reagents due to their poor thermal stability, moisture sensitivity, and high susceptibility to racemization during peptide bond formation.^217–219 The high sensitivity and reactivity of ynamines are attributed to the strong delocalization ability of the nitrogen atom lone pair toward the alkynyl C–C triple bond. To retain the advantages while mitigating the instability of ynamine coupling reagents, Gais and Neuenschwander, independently, anchored an EWG to the opposite side of the C–C triple bond of ynamine by taking advantage of a “push–pull” effect. Indeed, the “push–pull” effect worked well to increase the stability of ynamine (Scheme 45, b).^220–222 The mechanism study implies that the carboxyl group is initially added to the C–C triple bond of the ynamine (Scheme 45, 45-3). This primary adduct is unstable and rapidly rearranges to the active ester 45-4.^223,224 Although the “push–pull” effect stabilizes ynamine coupling reagents remarkably and demonstrates broad compatibility with amino acid side chain functional groups,²²³ ynamine coupling reagents were finally abandoned by the peptide community, which might be attributed to their difficult preparation and handling, and racemization/epimerization occurred during peptide bond formation.

Scheme 45 The ynamine- or ynamide-mediated amide bond formation.

Ynamides, a special class of ynamines bearing an EWG on the nitrogen atom, have attracted much attention over the past three decades.^225,226 The EWG attaching to the nitrogen atom diminished the electron-donating ability of the nitrogen atom toward the alkynyl moiety. Thus, these electron-deficient ynamines (ynamides) display remarkable stability compared with ynamines. With this knowledge in mind, Zhao and his co-workers successfully developed a class of novel ynamide coupling reagents.²²⁷ The EWG on the nitrogen atom plays a crucial role in attenuating the basicity of the ynamide, thus enabling the ynamide as a racemization-free coupling reagent for peptide bond formation.²²⁷ Among the ynamides screened, N-methylynemethylsulfonamide (MYMsA) and N-methylynetoluene-sulfonamide (MYTsA) displayed superior reactivity in facilitating amide and peptide bond formation in a racemization/epimerization-free manner.^227,228 Ynamide coupling reagent-mediated amide bond formation proceeded via a two-step process with α-acyloxyenamides as stable active ester intermediates.²²⁹ The addition of carboxylic acids to the ynamide proceeded smoothly to deliver the α-acyloxyenamides in quantitative yields. It should be noted that the α-acyloxyenamides are stable and can be purified and stored for months without any deterioration. Upon treatment with an amino component, the aminolysis of α-acyloxyenamides afforded the corresponding amides in excellent yields. Both the formation and the aminolysis of the α-acyloxyenamides proceeded spontaneously under very mild reaction conditions, thus enabling a convenient two-step, one-pot protocol. The α-acyloxyenamide active esters are compatible with amino acid side chain functional groups, including -OH, -SH, -CONH₂, and indole -NH (Scheme 46). With this strategy, the full scope of proteinogenic α-amino acids could act as both acyl and amine components for racemization/epimerization-free peptide synthesis, and peptide segments could also condensate smoothly. Although the ynamide coupling reagent proved to be a promising racemization-free coupling reagent, the moderate reactivity of α-acyloxyenamide active esters plagued their application potential. To overcome the inherent limitation, recently, a systematic mechanistic study including kinetics studies, Brønsted-type structure–reactivity study, and density functional theory (DFT) calculation had been performed. The X-ray crystallography analysis of the α-acyloxyenamides of pivalic acid esters indicated that the C(O)-OR bond length between the acyl moiety and the leaving group of the α-acyloxyenamide was longer than that of the common alkyl esters (1.355 Å vs. 1.340 Å, Table 1). This hinted that the acyl group of α-acyloxyenamide was more electrophilic and favorable for splitting. The Brønsted parameter β_nuc = 0.23 suggested that the α-acyloxyenamides belong to a “superactive ester”, for which the formation of the tetrahedral intermediate is the rate-limiting step of the aminolysis step. In addition, the energetically favored keto–enol tautomerization of the leaving enolate decreased the kinetic barrier for the breakdown of the tetrahedral intermediate.²³⁰ The DFT calculations elucidated that the H₂O-aided hydrogen transfer proceeded via a favorable six-membered-ring geometry. The energy barrier of the intermolecular hydrogen-bonding model was 7.0 kcal mol⁻¹ lower than that of the model without H₂O. The kinetics studies, Brønsted-type structure–reactivity relationship study, and DFT calculation results were consistent and implied that polar solvents were favored for the aminolysis reaction. Based on the mechanistic studies, H₂O or a H₂O/DMSO mixture was identified as a better solvent, and could drastically shorten the aminolysis reaction time from 24 h to 1–2 h. This significant improvement successfully expanded the applicability scope of ynamide coupling reagent to peptide fragment condensation (Scheme 47), head-to-tail cyclization (Scheme 48), and SPPS as well (Scheme 49).²³⁰

Scheme 46 Peptide synthesis using the MYTsA reagent (adapted with permission from ref. 227, J. Am. Chem. Soc., 2016, 138, 13135. Copyright (2016) American Chemical Society).

Scheme 47 MYMsA-mediated peptide fragment condensation (adapted with permission from ref. 230, Angew. Chem., Int. Ed., 2022, 61, e202212247. Copyright (2022) Wiley-VCH).

Scheme 48 MYMsA-mediated peptides head-to-tail cyclization (adapted with permission from ref. 230, Angew. Chem., Int. Ed., 2022, 61, e202212247. Copyright (2022) Wiley-VCH).

Scheme 49 Solid-phase synthesis of ACP(65–74) with α-acyloxyenamide esters (adapted with permission from ref. 230, Angew. Chem., Int. Ed., 2022, 61, e202212247. Copyright (2022) Wiley-VCH).

Table 1 Geometry of the ester linkage: C(O)-OR and C(O)O-R bond lengths of selected esters (adapted with permission from ref. 230, Angew. Chem., Int. Ed., 2022, 61, e202212247. Copyright (2022) Wiley-VCH)

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Zhao's subsequent investigation suggested that ynamide could also be used as a coupling reagent for thioamide bond formation, which was highly prone to racemization/epimerization, by employing monothiocarboxylic acid as the thioacyl donor.²³¹ The novel thioacylating reagent α-thioacyloxyenamides could be obtained selectively from the addition reaction of monothiocarboxylic amino acids and ynamides. Notably, α-thioacyloxyenamides acted as active thiocarbonyl esters to furnish thioamide bonds when treated with various primary and secondary amines under mild reaction conditions. Most importantly, the α-thioacyloxyenamides of the proteinogenic amino acids could be used to construct thiopeptide bonds without any detectable racemization/epimerization, a notorious issue that plagues the application of thioamide-substituted peptides in protein chemical biology (Scheme 50). Trithioamide-substituted Leu-enkephalin and closthioamide were constructed smoothly using this strategy.²³¹ α-Thioacyloxyenamides could also be used as efficient thioacylating reagents for site-specific incorporation of a thioamide bond into a growing peptide backbone via SPPS. Except for His, the monothiocarboxylic acids derived from 19 of 20 proteinogenic α-amino acids worked well to afford the thioamide-substituted peptide with excellent efficiency. This robust protocol could introduce continuous multi-thioamide bonds to the peptides on SPPS, thus offering a practical approach for thioamide-substituted peptides (Scheme 51).²³²

Scheme 50 Thioamide-substituted peptide synthesis using the MYTsA reagent (adapted with permission from ref. 231, Angew. Chem., Int. Ed., 2019, 58, 1382. Copyright (2019) Wiley-VCH).

Scheme 51 Synthesis of thioamide-substituted peptides in SPPS (adapted with permission from ref. 232, J. Org. Chem., 2020, 85, 1484. Copyright (2020) American Chemical Society).

4.7 Allenones

Although efficient for solution-phase peptide synthesis, ynamide coupling reagents are not attractive for standard SPPS because of their moderate reactivity. In their efforts to search for more reactive vinyl esters, Zhao and co-workers discovered that α-carbonyl vinyl esters derived from allenone are promising candidates.²³³ A common feature of DCC,²² diphenylketenimine,²³⁴ Woodward's Reagent K,¹⁸⁶ 1,3-dipole resonance structures of ethoxyacetylene²³⁵ and ynamide coupling reagent²²⁷ was that all of them contained an sp-hybridized electron-deficient carbon center. Inspired by such features and based on their success with the ynamide coupling reagent, they envisioned that it would be feasible to design a more reactive coupling reagent from allene derivatives whose linear structure also contains an sp carbon center. In addition, there is no basic center in the vinyl ester intermediates, which might be beneficial for avoiding racemization/epimerization. After extensive studies, Zhao and co-workers disclosed that allenones are the optimal choice.²³³ Allenone activated carboxylic acids via a spontaneous addition/rearrangement cascade reaction to furnish active vinyl ester intermediates, which underwent rapid aminolysis reaction with primary or secondary amines (Scheme 52). Compared with the aminolysis reaction of the α-acyloxyenamides, which required 2–20 h, the vinyl esters derived from allenone proceeded faster and reached completion in 20 min – 2 h with excellent yields. As shown in Scheme 52, broad substrate scopes of both the electrophilic carboxy and nucleophilic amine partners were observed. The functional groups in the side chains of Ser and Thr (–OH), Asn and Gln (–CONH₂), and Trp (NH) were all tolerated in the aminolysis reaction. Not only was this strategy effective for the synthesis of simple amides and dipeptides, but it was also amenable for peptide fragment condensation (Scheme 52). Similar to that of ynamide coupling reagents, no racemization/epimerization was detected for allenone-mediated peptide bond formation. Easy preparation and rapid aminolysis make the α-carbonyl vinyl esters appealing for SPPS. Using proteinogenic amino acid-based α-carbonyl vinyl esters as the building blocks, the difficult peptide ACP(65–74) could be obtained via SPPS with an excellent crude purity of 98% after cleavage from the resin (Scheme 53). However, with the use of the same loading of amino acids, a crude purity of 86%, 77%, or 60% was obtained when PyBOP, HBTU, or PFP ester was employed as the coupling reagent or active ester, respectively. The superiority of the allenone coupling reagent in suppressing the racemization/epimerization of peptide acids was further demonstrated in carfilzomib synthesis (Scheme 54). The total synthesis of carfilzomib was accomplished via a N to C peptide elongation strategy, which was seldom used for peptide synthesis due to the severe epimerization. The superiority of allenone in suppressing the racemization/epimerization of peptide acids afforded an opportunity for peptide syntheses, which were incompatible with the classical C to N peptide elongation mode. Stability, without racemization/epimerization, and few undesired side reactions unambiguously illustrated that the α-carbonyl vinyl esters originating from allenone and proteinogenic amino acids would come into the spotlight as an attractive alternative for conventional peptide syntheses.

Scheme 52 Allenone-mediated peptide bond formation (adapted with permission from ref. 233, J. Am. Chem. Soc., 2021, 143, 10374. Copyright (2021) American Chemical Society).

Scheme 53 Solid-phase synthesis of ACP(65–74) using α-carbonyl vinyl esters as the building blocks (adapted with permission from ref. 233, J. Am. Chem. Soc., 2021, 143, 10374. Copyright (2021) American Chemical Society).

Scheme 54 Synthesis of carfilzomib with allenone (52-2) as the coupling reagent (adapted with permission from ref. 233, J. Am. Chem. Soc., 2021, 143, 10374. Copyright (2021) American Chemical Society).

Inspired by the allenone coupling reagent, Li et al. reported that propargyl sulfoniums, which have similar behavior to that of allenones under basic conditions, could also act as a coupling reagent.²³⁶ They had been successfully applied to peptide synthesis and protein amidation. The mechanistic study indicated that the active thioesters prepared by the addition of thioacids and propargyl sulfoniums are the key intermediates, which could be applied in SPPS and protein labeling studies (Scheme 55).

Scheme 55 Sulfonium-promoted peptide and protein amidation (adapted with permission from ref. 236, Org. Lett., 2022, 24, 581. Copyright (2022) American Chemical Society).

5. Conclusion

In conclusion, we have systematically summarized the origin, development, pros and cons of using active ester strategies for peptide synthesis. The significant advantage of an active ester strategy is their ability to suppress racemization/epimerization during peptide bond formation. In addition, active esters are commonly inert to the side chains of amino acids such as –OH, –SH, –COOH, –CONH₂, and indole NH, making the least protection strategy feasible. However, the passion for using active esters faded with the development of coupling reagents because of their extra preparation step, additional operation and chemicals, moderate reactivity, and long aminolysis reaction time. In the past two decades, the growing request for peptides, the stringent requirement of sustainable development, and the inherent limits and drawbacks of conventional coupling reagent-mediated peptide synthesis, in turn, promoted a reinvestigation of active ester strategies. The in situ-formed transient active esters with the assistance of catalysts illustrated the benefit of active esters for peptide synthesis methodology innovations, albeit its application in relatively larger peptide synthesis as well as SPPS is waiting to be addressed. The prior prepared stable active esters, represented by PFP esters and HOSu esters, with well-defined structures, had always played a major part in peptide synthesis. However, nowadays they are only used occasionally for some special cases because of their tedious preparation and long aminolysis reaction time. The in situ-formed active esters from the addition reaction between coupling reagents and carboxylic acids would be an attractive and practical alternative because their concise preparation avoided the use of extra hydroxyl nucleophiles. For examples, coupling reagents such as alkoxyacetylenes, Woodward reagents, TBCR, and isonitriles facilitated peptide bond formation via the in situ-formed active esters with crucial intermediates. Unfortunately, active esters derived from these coupling reagents are not stable enough, resulting in complex reaction systems, racemization/epimerization, and other undesired side reactions. Gratifyingly, recently developed ynamide and allenone coupling reagents overcome the disadvantages mentioned above because they could afford bench-stable active vinyl esters upon activation of carboxylic acids. These stable vinyl esters could be used to construct amide bonds in a two-step, one-pot manner or a step-by-step manner with great flexibility. The energy released in the tautomerization of the enolate leaving group of the vinyl esters into acylate drove the aminolysis reaction to complete in a reasonably short time. More importantly, no racemization/epimerization was detected when they were used for peptide bond formation, enabling ynamides and allenones to be successfully used for dipeptide synthesis, peptide fragment condensation, peptide head-to-tail cyclization, and SPPS. Importantly, both ynamide and allenone coupling reagents are not only effective for conventional C to N peptide synthesis but also amenable for N to C peptide synthesis, which generally cannot be achieved by conventional coupling reagents, thus offering infinite possibilities for peptide synthesis. The design concept of ynamide and allenone coupling reagents with stable vinyl esters as active intermediates integrated the advantages of traditional coupling reagents and active esters for racemization-free peptide bond formation while avoiding their disadvantages. Owing to their ready preparation, stability, superiority in addressing racemization, flexibility and convenience for operation, the broad application of active esters in both SPPS and liquid-phase peptide synthesis (the third wave of peptide synthesis)²³⁷ can be expected in the future. It is foreseeable that this review will inspire new ideas about the design of novel active esters and spur the future application of active ester strategies for peptide synthesis and other coupling reactions.

Author contributions

Junfeng Zhao conceived the idea and directed the literature survey. Jinhua Yang and Huanan Huang composed the manuscript.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (22277015, 22067008), and the Natural Science Foundation of Jiangxi Province (20202ACBL203004).

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Footnote

† These authors contributed equally.

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