Ynamide Coupling Reagents: Origin and Advances

Conspectus

Since the pioneering work of Curtius and Fischer, chemical peptide synthesis has witnessed a century’s development and evolved into a routine technology. However, it is far from perfect. In particular, it is challenged by sustainable development because the state-of-the-art of peptide synthesis heavily relies on legacy reagents and technologies developed before the establishment of green chemistry. Over the past three decades, a broad range of efforts have been made for greening peptide synthesis, among which peptide synthesis using unprotected amino acid represents an ideal and promising strategy because it does not require protection and deprotection steps. Unfortunately, C → N peptide synthesis employing unprotected amino acids has been plagued by undesired polymerization, while N → C inverse peptide synthesis with unprotected amino acids is retarded by severe racemization/epimerization owing to the iterative activation and aminolysis of high racemization/epimerization susceptible peptidyl acids. Consequently, there is an urgent need to develop innovative coupling reagents and strategies with novel mechanisms that can address the long-standing notorious racemization/epimerization issue of peptide synthesis.

This Account will describe our efforts in discovery of ynamide coupling reagents and their application in greening peptide synthesis. Over an eight-year journey, ynamide coupling reagents have evolved into a class of general coupling reagents for both amide and ester bond formation. In particular, the superiority of ynamide coupling reagents in suppressing racemization/epimerization enabled them to be effective for peptide fragment condensation, and head-to-tail cyclization, as well as precise incorporation of thioamide substitutions into peptide backbones. The first practical inverse peptide synthesis using unprotected amino acids was successfully accomplished by harnessing such features and taking advantage of a transient protection strategy. Ynamide coupling reagent-mediated ester bond formation enabled efficient intermolecular esterification and macrolactonization with preservation of α-chirality and the configuration of the conjugated α,β-C–C double bond. To make ynamide coupling reagents readily available with reasonable cost and convenience, we have developed a scalable one-step synthetic method from cheap starting materials. Furthermore, a water-removable ynamide coupling reagent was developed, offering a column-free purification of the target coupling product. In addition, the recycle of ynamide coupling reagent was accomplished, thereby paving the way for their sustainable industrial application.

As such, this Account presents the whole story of the origin, mechanistic insights, preparation, synthetic applications, and recycle of ynamide coupling reagents with a perspective that highlights their future impact on peptide synthesis.

Key References

• Hu L.; Xu S.; Zhao Z.; Yang Y.; Peng Z.; Yang M.; Wang C.; Zhao J.. Ynamides as Racemization-Free Coupling Reagents for Amide and Peptide Synthesis. J. Am. Chem. Soc. 2016, 138, 13135–13138 .¹The first demonstration of using ynamide to activate amino acids via stable α-acyloxyenamide active esters for racemization-free peptide bond formation.

• Xu S.; Jiang D.; Peng A.; Hu L.; Liu T.; Zhao L.; Zhao J.. Ynamide-Mediated Peptide Bond Formation: Mechanistic Study and Synthetic Applications. Angew. Chem., Int. Ed. 2022, 61, e202212247.²A systematic mechanistic study on ynamide-mediated peptide bond formation expanded its applications to fragment condensation and solid-phase peptide synthesis, as well as head-to-tail cyclization.

• Liu T.; Peng Z.; Lai M.; Hu L.; Zhao. J.. Inverse Peptide Synthesis Using Transient Protected Amino Acids. J. Am. Chem. Soc. 2024, 146, 4270–4280 .³The first practical N → C inverse peptide synthesis indirectly using unprotected amino acids as the building blocks.

• Yang J.; Wang C.; Xu S.; Zhao J.. Ynamide-Mediated Thiopeptide Synthesis. Angew. Chem., Int. Ed. 2019, 58, 1382–1386.⁴Use of ynamide as racemization-free coupling reagent for incorporating thioamide substitutions to the peptide backbone.

Introduction

Since the late 19th century, when the concept of proteins being composed of amino acids linked by peptide bonds was firmly established, peptide chemistry has entered its golden era.⁵ However, little attention was paid to peptide bond formation methods, as existing approaches like the aminolysis of acid azides of Curtius⁶ and the acid chlorides of Fischer⁷ were highly effective. It was not until the 1950s, with the elucidation of essential peptide structures such as those of insulin⁸ and oxytocin,⁹ and the study of reactive intermediates in biological acylation processes, interest in peptide bond formation was rekindled. This catalyzed the development of numerous peptide synthesis methods, some of which have become cornerstones in modern peptide synthesis.¹⁰ Nowadays, peptide synthesis, in particular, the solid phase peptide synthesis¹¹ (SPPS) has evolved into a routine technology. Despite these advancements, state-of-the-art peptide synthesis still heavily relies on legacy coupling reagents and technologies developed between the 1950–1980s. Notorious issues such as poor atom- and step-economy, racemization/epimerization, and unexpected side reactions persistently afflict conventional peptide synthesis.¹² In fact, it has been reported that producing just one kilogram of peptide would generate tons of chemical waste.¹³ Therefore, atom-economic peptide bond formation has been identified as one of the top 10 challenges of green chemistry over the past two decades.¹² In contrast to conventional C → N chemical peptide synthesis, inverse peptide synthesis using unprotected amino acids elongating in the N → C direction holds great promise for greening peptide synthesis. Various activated amino acid derivatives, including acyl azides,¹⁴ acyl fluorides,¹⁵N-hydroxysuccinimide esters,¹⁶ substituted phenol esters,¹⁷N-(Z-α-aminoacyl)benzotriazoles,¹⁸ and mixed anhydrides,¹⁹ have been employed for N → C elongation using unprotected amino acids. Unfortunately, such ideal strategies have been limited to the synthesis of dipeptides because N → C peptide elongation involves the iterative repetition of activation and aminolysis of peptidyl acids, the racemization/epimerization tendency of which is usually 35–110 times higher than that of the corresponding N-carbamate-protected amino acids.²⁰ While some optimizations have been performed, practical strategy remains unavailable.²¹ Only the novel racemization/epimerization-free peptide bond formation strategies and coupling reagents can implement this atom-economic N → C peptide synthesis strategy.

In this Account, we delve into an investigation conducted on the ynamide coupling reagent, a novel activation platform for carboxylic acids with α-acyloxyenamide active esters as stable intermediates with optimal reactivity to ensure a racemization-free condensation. Ynamide coupling reagents are not only amenable to amide bonds but also effective for facilitating ester and thioester bond formation. Expanding their utility, we have successfully applied them in a variety of applications, including linear peptide synthesis, peptide fragment condensation, peptide head-to-tail cyclization, and site-specific thioamide bond modifications. What sets ynamide coupling reagents apart is their remarkable superiority, which is illustrated in their ability to prevent racemization/epimerization during the activation and subsequent aminolysis of α-chiral carboxylic acids. This unique feature has finally enabled us to successfully accomplish N → C inverse peptide synthesis using unprotected amino acids as the building blocks via a transient protection strategy. Our exploration will begin with the initial discovery of the ynamide coupling reagent, progress through the development of a robust reaction design, and culminate in establishing the breadth of application scope.

Origin of the Ynamide Coupling Reagent

After more than one century’s development, hundreds of coupling reagents and strategies are available for forging peptide bonds.¹⁰ Developing innovative approaches for peptide bond formation that can truly stand out within this enormous crowd becomes an incredibly challenging task. To delve into the pursuit of racemization-free peptide bond formation strategies and coupling reagents with novel mechanisms, it is crucial to reflect on some significant milestones in the development of peptide bond formation (Scheme 1a). One such event occurred in 1955 when Arens and co-workers²² introduced ethoxyacetylene, a coupling reagent for peptide synthesis, at approximately the same time that the renowned dicyclohexylcarbodiimide²³ (DCC) coupling reagent was reported. Ethoxyacetylene seemed promising because harmless and easily removable ethyl acetate was the only byproduct. However, the moderate reactivity of ethoxyacetlyene necessitated transition metal catalysts for activating carboxylic acids. Additionally, the poor thermal stability of alkoxyacetylene further complicated its application. To remedy the disadvantages of ethoxyacetlyene, in 1964, Viehe and co-workers introduced an ynamine coupling reagent by replacing the alkoxy group on the acetylenic triple bond with a more electron-donating amino group which was proposed to enhance lone pair delocalizing ability to the alkynyl C–C triple bond (Scheme 1a).²⁴ As expected, ynamines were more reactive than alkoxyacetylene. However, due to the poor thermal stability, high sensitivity to moisture, and difficulties in handling, as well as a pronounced tendency to induce racemization during peptide bond formation, the practical utility of ynamine coupling reagents has been greatly constrained. Later, Gais²⁵ and Neuenschwander,²⁶ independently, anchored an electron-withdrawing group (EWG) to the C–C triple bond to stabilize ynamine by taking advantage of the electron push–pull effect. However, their complex preparation and difficult handling, along with racemization/epimerization issue, led the peptide community to ultimately abandon ynamine coupling reagents. While the development of alkyne-based coupling reagents did not yield practical success, it represented a novel activation model for peptide coupling reagents, especially considering the bottleneck encountered by conventional carbodiimide based coupling reagents.¹⁰

Scheme 1. (a) Timeline of the Development of Alkyne-Based Coupling Reagents; (b) Major Pathway for the Racemization Mechanism in Ynamine-Mediated Activation Step; and (c) Our Working Hypothesis on Ynamide Coupling Reagent.

Under basic reaction conditions, carboxylic acids with an α-chiral center containing an acidic proton are highly susceptible to racemization during the activation and aminolysis processes. Two mechanisms have been proposed for rationalizing racemization: (1) direct abstraction of the α-hydrogen (H^α) and (2) formation of oxazolone intermediates. Both racemization pathways are highly related to base-induced proton abstraction, in particular the intramolecular proton abstraction by the basic center of coupling reagents.²⁷ Mechanism analysis disclosed that the ynamine coupling reagent activated carboxyl acids via its 1,3-dipole resonance structure (Scheme 1b). The protonation of the 1,3-dipole species results in the formation of the keteniminium, which yields the corresponding α-acyloxyenamine active esters upon the addition of the carboxyl anion. The intramolecular H^α abstraction induced by the basic nitrogen atom of α-acyloxyenamine is the major contributor for the ynamine coupling reagents-caused racemization (Scheme 1b).²⁸ Thus, we proposed that introduction of an electron-withdrawing group (EWG) to the nitrogen atom of ynamine might be helpful not only to stabilize ynamine but also to decrease the basicity of the tertiary amine, thereby minimizing the risk of base-induced racemization (Scheme 1c). Indeed, ynamines bearing an EWG on the nitrogen atom known as ynamides have witnessed rapid development over the past three decades.²⁹ However, what might have seemed like a straightforward proposal was challenging because the key working hypothesis of the ynamide coupling reagent was the delocalization of the nitrogen lone pair to the alkynyl C–C triple bond. With an EWG attached to the nitrogen atom, it was uncertain whether activation would proceed. Furthermore, it was uncertain whether subsequent aminolysis of the α-acyloxyenamides intermediate would occur. A literature search disclosed that hydroacyloxylation of ynamide with carboxylic acids could proceed to generate α-acyloxyenamides albeit with transition metal catalysis or high temperatures.³⁰ We therefore first examined the feasibility of the aminolysis of α-acyloxyenamides. To our delight, the aminolysis of stable α-acyloxyenamides active ester proceeded smoothly at room temperature to give the target amide in a quantitative yield.¹ This encouraging result promoted us to explore the hydroacyloxylation of ynamide. Among the various ynamides that we screened, terminal ynamides N-methylynetoluenesulfonamide (MYTsA) and N-methylynemethylsulfonamide (MYMsA) were identified as the optimal choices (Scheme 2a). This ynamide-mediated two-step amide bond formation process could be performed in a one-pot manner because the excellent efficiency of the activation step enabled the α-acyloxyenamides active ester to be used directly for aminolysis without further purification (Scheme 2b). Importantly, no racemization/epimerization was observed when either MYTsA or MYMsA was used as the coupling reagent for peptide bond formation (Scheme 2c). The substrate shows excellent tolerance for common amine protecting groups (Boc, Cbz, and Fmoc) and remarkable selectivity for amino groups, even in the presence of side-chain functional groups such as −OH, −SH, −CONH₂, and NH of indole. Additionally, a sterically hindered coupling partner such as Aib, a typically challenging substrate for common peptide coupling, gives the target dipeptide in excellent yield. However, requisite attention should be paid when employing His as a carboxyl partner because incomplete activation that is likely due to the presence of the imidazole side-chain functional group may occur.

Scheme 2. (a) Optimization of Ynamide for Activation of a Carboxylic Acid; (b) Two Steps Approach vs the One-Pot, Two-Step Strategy; and (c) Ynamide-Mediated Peptide Bond Formation.

Mechanistic Aspects of Ynamide-Mediated Peptide Bond Formation

Ynamide coupling reagents showed significant promise in achieving racemization-free peptide synthesis. However, their further applications, in particular, in solid-phase peptide synthesis were retarded by their moderate activation capacity, which resulted in a prolonged reaction time. To enhance the efficiency and deepen understanding of the mechanism of ynamide-mediated peptide bond formation, kinetics studies were conducted with the assistance of in situ IR spectroscopy.² For the activation step, it was revealed that the activation of the carboxylic acid by MYTsA followed first-order and zero-order kinetics with respect to the carboxylic acid and ynamide, respectively. To gain further insights into this activation step, deuterated ynamide was reacted with benzoic acid under standard conditions. Three isomers including nondeuterated, monodeuterated, and dideuterated were obtained in a ratio of 8:84:8, indicating that the formation of α-acyloxyenamide active esters occurred stepwisely rather than concertedly.² For the aminolysis step, a linear relationship was observed when plotting the reaction rate against the concentration of α-acyloxyenamide, and the same trend was observed for the amino partner. These findings unequivocally indicated that the aminolysis process followed first-order kinetics concerning the concentrations of both α-acyloxyenamide and amine.

Furthermore, X-ray chromatography analysis (Scheme 3a) of α-acyloxyenamide of pivalic acid illustrated that the bond length between the acyl moiety and the leaving group of α-acyloxyenamides (the C(O)–OR bond) was lengthened compared to that of conventional alkyl esters. This elongation rendered the acyl group more electrophilic and prone to splitting. Additionally, the conjugation of the oxygen lone pair with the enamide motif led to a remarkable shortening of the C(O)O–R bond, which favored the departure of the leaving group. Importantly, a critical process was that, upon departure, the leaving enolate of α-acyloxyenamides underwent an energetically favored keto–enol tautomerization, accompanied by the release of 23.8 kcal/mol of free energy (Scheme 3b). This tautomerization caused a significant change in the energetic profile of the aminolysis reaction mechanism, distinguishing it from that of pentafluorophenyl esters with similar C(O)–OR and C(O)O–R bond lengths. Hence, the structural analysis provided compelling evidence supporting the notion that α-acyloxyenamides served as activated esters.

Scheme 3. (a) Geometry of the Ester Linkage: C(O)–OR and C(O)O–R Bond Lengths; (b) Gibbs Free Energy for Keto-Enol Tautomerization; and (c) Brönsted-Type Structure–Reactivity Study for the Aminolysis Reaction.

To further study the aminolysis step, the relationship between the reactivity of the amine and its basicity in the aminolysis step was studied. The rate-limiting step of the aminolysis process is closely related to the basicity of the attacking amine and the nature of the leaving group, as measured by the Brönsted parameter β (defined as the slope in plots of log k vs pKa).³¹ The aminolysis rates of various primary amines with different basicity were evaluated, and a β value of 0.23 (Scheme 3c) suggested that α-acyloxyenamides derived from carboxylic acids might fall into the concept of superactive esters, as proposed by Kamiński and Papini.³² For superactive esters, the formation of the tetrahedral intermediate is the rate-limiting step of the aminolysis reaction, which completely differs from the classic active esters, where the collapse of the tetrahedral intermediate is the rate-limiting step. This might be attributed to the energetically favored keto–enol tautomerization of the leaving enolate of α-acyloxyenamides, which lowers the kinetic barrier for the collapse of the tetrahedral intermediate, leading to the observed change in the rate-limiting step. These findings provide valuable insights into the unique behavior of α-acyloxyenamides and contribute to a better understanding of their reactivity in peptide bond formation.

Applications of the Ynamide Coupling Reagent

Peptide Bond Formation in Aqueous Media

Inspired by the mechanistic study, we investigated a series of factors that govern the kinetic barrier and reaction rate of the aminolysis step. Solvent screening experiments demonstrated that the reaction rate steadily increased with an increase in solvent polarity, which is consist with our previous conclusion regarding the rate-limiting role of the tetrahedral intermediate formation and the ability of polar solvents to stabilize the charge-separated intermediate (Scheme 4a). A noteworthy discovery was the significant acceleration of the reaction rate by employing water or a water/dimethyl sulfoxide mixture as the solvent. This optimization led to a dramatic shortening of the reaction time, from a laborious 24 to 1–2 h, unequivocally showcasing a substantial acceleration of the aminolysis reaction. Moreover, density functional theory (DFT) calculations reinforced these findings and shed light on the role of water in lowering the kinetic barrier of the aminolysis step through a hydrogen-bonding network by serving as a proton shuttle (Scheme 4b). In the updated protocol,² the use of water as the solvent or cosolvent not only accelerated the aminolysis process but also paved the way for a more environmentally friendly and versatile peptide synthesis, particularly in the context of green chemistry principles.

Scheme 4. (a) Solvent Effect in Aminolysis Reaction and (b) Computed Energy Profiles for the Aminolysis Mechanism.

Application in Solid-Phase Peptide Synthesis

The α-acyloxyenamide active esters could be easily produced and exhibited excellent stability in atmospheric environments, both in the presence of air and water. They hold great potential as promising building blocks for SPPS but are plagued by the sluggish aminolysis rate under heterogeneous reaction conditions. Fortunately, the use of water as a cosolvent significantly enhances the aminolysis reaction rate. As water is needed to accelerate the reaction, a water-swellable resin (Tentagel resin) was employed to explore the feasibility of α-acyloxyenamides as building blocks for SPPS. However, the initial results were still unattractive for SPPS. It is worth noting that the aminolysis time was dramatically shortened to 20–30 min upon adding 1-hydroxy-7-azabenzotriazole (HOAt) and diisopropylethylamine (DIEA) as additives, rendering this method competitive with conventional SPPS. Its application potential was exemplified in the synthesis of a well-known model difficult peptide, the Acyl Carrier Protein fragment 65–74 (ACP 65–74) which comprises sterically hindered amino acid residues and a β-sheet formed by internal association.³³ The coupling reaction on resin proceeded rapidly and could be completed within 20–30 min for each amino acid, including sterically hindered amino acids such as Val and Ile (Scheme 5a). Ultimately, the ACP (65–74) was obtained with an impressive crude purity of 90% after cleavage from the resin (Scheme 5b). The successful synthesis of ACP (65–74) unequivocally demonstrated that the α-acyloxyenamides of proteinogenic amino acids could be used as effective building blocks for SPPS.

Scheme 5. Solid-Phase Synthesis of ACP (65–74) with α-Acyloxyenamide Active Esters of Proteinogenic Amino Acids as the Building Blocks.

Ynamide-Mediated Peptide Head-to-Tail Cyclization and Fragment Condensation

Cyclic peptides have gained significant interest in peptide drug discovery owing to their unique properties, including their rigid skeleton, high target affinity, potential membrane permeability, and considerable protease stability.³⁴ Peptide head-to-tail cyclization, facilitated by coupling reagents, stands as a straightforward strategy for synthesizing cyclic peptides.³⁵ However, the conventional coupling reagent-mediated head-to-tail cyclization of a linear peptide has inherent limitations, such as C-terminal epimerization, cyclodimerization, and formation of linear oligomers.³⁶ Owing to the superiority of the ynamide coupling reagents in suppressing racemization/epimerization of the peptidyl acid, we applied them in peptide head-to-tail cyclization (Scheme 6a). The ynamide-mediated peptide head-to-tail cyclization proceeded smoothly with exceptional selectivity, resulting in a monocyclization product with no detectable epimerization. In contrast, significant epimerization and cyclodimerization side reactions were observed for conventional coupling reagents, which not only complicated the purification process but also resulted in low yields of the desired cyclopeptides (Scheme 6b). As shown in Scheme 6, a broad range of linear peptides, including naturally occurring cyclopeptides, cyclized effortlessly with excellent selectivity toward monocyclization, with no epimerization at the C-terminal α-chirality center for all substrates. Ynamide coupling reagents also exhibited remarkable tolerance to diverse cyclization junctions. The amidation selectivity toward amino groups in the presence of −OH, −CONH₂, and the NH of indole made the protection of these side-chain groups unnecessary. Even linear peptides devoid of turn-inducing elements could be cyclized smoothly with good yields, showcasing the versatility and robustness of the ynamide-mediated peptide head-to-tail cyclization platform.

Scheme 6. Ynamide-Mediated Peptide Head-to-Tail Cyclization and Fragment Condensation.

Convergent peptide synthesis through fragment condensation is an effective strategy for large peptide synthesis. Using a one-pot, two-step strategy, the peptide fragments condense smoothly with diverse ligation junctions to furnish the target peptides in excellent yields (Scheme 6d). Gratifyingly, no epimerization was observed for the entire fragment condensation process, thus providing a promising convergent peptide synthesis strategy. In addition, this method enables the efficient and convenient synthesis of peptide active pharmaceutical ingredients such as peptavlon, octreotide, the cholecystokinin peptide kit, and vasopressin by peptide fragment condensation. It features diverse ligation junctions, offering a useful platform for synthesizing medium-sized peptides through fragment condensation and overcoming the limitations of the conventional coupling reagents whose ligation sites are confined to Gly and Pro residues.

Ynamide-Mediated Inverse Peptide Synthesis Using Transient Protected Amino Acids

In current chemical peptide synthesis methods, peptide chain elongation is almost exclusively executed from the C-terminus to the N-terminus (C → N) by employing N^α-protected amino acids as the building blocks.^11,37 However, these methods are challenged by green chemistry nowadays. Sustainable and atom-economic methods for peptide bond formation are in great demand.¹² The bioinspired N → C inverse peptide synthesis using unprotected amino acids, which does not require protection and deprotection, has been proposed as an ideal greening strategy. Unfortunately, this strategy has been kept unsuccessful for 60 years because of the severe epimerization that occurs during N → C peptide elongation. Our previous studies unambiguously confirmed that ynamide coupling reagent is a promising tool for addressing the racemization/epimerization issue of N → C peptide elongation. By employing ynamide as the coupling reagent, the first practical inverse peptide synthesis using transient protected amino acids has been developed (Scheme 7).³ The robustness of this strategy is exemplified by successful syntheses of a series of peptide active pharmaceutical ingredients from cheap and readily available unprotected amino acids (Scheme 7a). In addition, this strategy is also applicable to inverse solid-phase peptide synthesis and is compatible with green solvents (Scheme 7b). The transient protection, activation, aminolysis, and in situ deprotection were performed in one pot, which enabled us to synthesize peptides using unprotected amino acids indirectly.

Scheme 7. Inverse Peptide Elongation with Unprotected Amino Acids as the Starting Materials.

Ynamide-Mediated Thioamide Substituted Peptide Synthesis

Thioamide substituted peptides, sulfur-based isologue of peptides in which the oxygen atom in the peptide backbone’s carbonyl group is replaced by a sulfur atom,³⁸ exhibit excellent antibiotic activity and enzymatic degradation resistance. Before our study, limited methods were available for site-specific introduction of a thioamide substitution into a peptide backbone.³⁹ Furthermore, due to the higher polarization of the thiocarbonyl group, the α-chiral center of thioamide bond is highly susceptible to epimerization/racemization, making the construction of thioamide-substituted peptides more challenging. Therefore, precisely incorporating thioamide bonds into the backbone of peptides and proteins remains a formidable task.

Monothiocarboxylic acid exists in an equilibrium between two tautomeric forms: thiocarboxylic acid and carboxylic thioacid (Scheme 8a).⁴⁰ Interestingly, a pioneering study by Oshima and Yorimitsu suggested that the thiocarboxylic tautomer of monothiocarboxylic acids could be trapped by ynamide.⁴¹ Inspired by this study, we treated monothiocarboxylic acids with MYTsA and found that excellent yields of the corresponding α-thioacyloxyenamides and vinyl thioesters could be obtained with a ratio of up to 2:1 (Scheme 8b). α-Thioacyloxyenamides of monothiocarboxylic acids derived from all 20 proteinogenic N^α-protected α-amino acids except histidine can be readily prepared using this method. The formation and aminolysis of these α-thioacyloxyenamides tolerates unprotected side-chain functional groups such as −OH and −CONH₂. Notably, the preparation of α-thioacyloxyenamides of proteinogenic amino acids, as well as the subsequent aminolysis for thioamide bond construction, proceeded smoothly without any racemization/epimerization (Scheme 8c). Additionally, the novel thioacylating reagents, α-thioacyloxyenamides, demonstrated remarkable stability and ease of purification, characterization, and long-term storage at low temperatures.

Scheme 8. Ynamide-Mediated Thioamide Substituted Peptide Synthesis and Other Applications.

The aminolysis of α-thioacyloxyenamides was typically completed within minutes, making them suitable for SPPS. By utilizing the proteinogenic amino acid based α-thioacyloxyenamides as building blocks, we successfully introduced site-specific monothioamide substitution and multiple thioamide bonds into a growing peptide backbone through SPPS, providing a practical and versatile method for thioamide-substituted peptide synthesis (Scheme 8d). This strategy is not limited to thioamide substituted peptides; it can also be applied to common thioamides.⁴² Similarly, thiocarbonyl esters could be obtained by employing O or S nucleophiles in the presence of a catalytic amount of Cs₂CO₃ (Scheme 8e).⁴³

Ynamide-Mediated Ester Bond Formation

Ester is a widely encountered and crucial functional group in pharmaceuticals, materials, and numerous bioactive natural products.⁴⁴ The most practical approaches involve pre- or in situ activation of carboxylic acids using stoichiometric “coupling” reagents, which convert the −OH group of the acid into a good leaving group, followed by alcoholysis. Although excellent amidation selectivity toward amino groups in the presence of the −OH group during ynamide-mediated peptide bond formation was obtained, we did observe esterification side product in some special cases. Later, we found that the presence of an acid or base catalyst was crucial for esterification due to the lower nucleophilicity of the hydroxyl group. After extensive studies, a one-pot, two-step esterification protocol was developed, which was similar to those of amide bond formation (Scheme 9a). The distinction lies in the need for a base catalyst during alcoholysis, and the solvent also plays an important role. Additionally, this method could also be extended to the thioesterification reaction, allowing us to prepare various thioesters using -SH nucleophiles, especially peptide thioesters (Scheme 9d).⁴⁵

Scheme 9. Ynamide-Mediated Esterification and Macrolactonization.

Encouraged by our success with intermolecular esterification, we shifted our focus to macrolactonization, a challenging task for constructing the macrolides widely found in antibiotics, pharmaceuticals, and bioactive natural products.⁴⁶ However, notorious challenges, including base-induced racemization of seco-acids containing an α-chirality center,⁴⁷Z/E isomerization of α, β-unsaturated seco-acids,⁴⁸ and a competitive reaction involving intermolecular esterification leading to the formation of diolide side products have rendered it a formidable task. Unlike in intermolecular esterification, an acid catalyst, p-toluenesulfonic acid monohydrate (PTSA·H₂O), has proven efficient in promoting the macrolactonization reactions of α-acyloxyenamides derived from seco-acids and MYTsA (Scheme 9b). The excellent efficiency of the first activation step enables the direct utilization of the aforementioned activated esters in the subsequent acid-catalyzed macrolactonization, offering a simple “two-step, one-pot” protocol that can be conducted under ambient conditions.⁴⁹ This protocol avoids the harsh conditions typically associated with traditional marcrolactonization strategies.⁵⁰ Importantly, this method can be conducted at relatively high concentrations (5–100 mM) without a significant diolide side product. Additionally, this approach facilitates the construction of medium-sized macrolides, which are challenging to synthesize using other strategies. This method offers distinct advantages for linear seco-acids containing α-chiral centers and linear α, β-unsaturated seco-acids with conjugated C–C double bonds by effectively preventing racemization of α-chiral centers and Z/E isomerization of C2–C3 double bonds, which are commonly encountered in conventional methods. Furthermore, this strategy can be applied to the synthesis of challenging cyclodepsipeptides containing both ester and amide bonds, providing an alternative route by employing macrolactonization as the final ring closure step (Scheme 9c).

Preparation and Recycling of Ynamide Coupling Reagents

Since the pioneering work of Viehe et al.,⁵¹ which marked the inception of ynamide chemistry, a diverse array of synthetic methods have been developed for ynamides.^29b However, with regard to the development of a reliable and industrially scalable synthesis method for terminal ynamides such as MYTsA and MYMsA from cheap and readily available chemical raw materials, the current landscape remains notably inadequate. Generally, at least two steps are involved in the synthesis of terminal ynamides.⁵² To make ynamides readily available coupling reagents, we have developed a robust one-step synthetic approach for producing MYTsA and MYMsA with 1,1- or 1,2-dichloroethylene, and N,4-dimethylbenzenesulfonamide (TsNHMe) or N-methylmethanesulfonamide (MsNHMe) as the starting materials (Scheme 10a).⁵³ (Caution!1,1-Dichloroethylene is a toxic and carcinogenic chemical agent with a boiling point of 32 °C. In particular, care should be taken when using this method for large scale preparation as a fire may occur!) More importantly, we have successfully accomplished the recycle of the corresponding hydrated byproduct of MYMsA or MYTsA by treating it in an alkaline aqueous solution, resulting in the quantitative production of TsNHMe or MsNHMe, respectively (Scheme 10b). Furthermore, both MYMsA and MYTsA exhibit excellent stability; no degradation was observed even after exposure to open air at room temperature for 10 days or storage in a refrigerator for 12 months. The easy preparation of ynamide and readily recycling of its hydrated byproduct pave the way for the industrial large-scale application of ynamide coupling reagents.

Scheme 10. Preparation and Recycling of Ynamide Coupling Reagent.

To simplify the purification process, we have developed a water-removable ynamide coupling reagent by introducing a temporarily masked adjacent dihydroxyl group (Scheme 11a).⁵⁴ Upon acidic aqueous workup, the protected vicinal dihydroxyl group of both excess ynamide and its hydrated byproduct is released, rendering them water-soluble at 260 g/L. By simply extracting the reaction mixture, the target amides, peptides, or esters could be obtained in excellent yields, while the water-soluble byproduct remains in the aqueous layer. The potential for its synthetic application was further demonstrated in the synthesis of peptoid carfilzomib via a rare stepwise N → C elongation because the epoxide warhead has to be introduced to the peptide chain in late step. Indeed, carfilzomib could be obtained with an exceptional 70% overall yield, surpassing previous literature results.⁵⁵ This approach eliminates the necessity for column chromatography purification and thus enhances its practicality and scalability, making it highly attractive for industrial larger-scale applications.

Scheme 11. Water-Removable Ynamide Coupling Reagent and Its Applications.

Conclusion and Outlook

In this Account, we present the origin and advances of ynamide coupling reagents. By elaborately introducing an EWG to the nitrogen atom of ynamine, we discovered a class of novel ynamide coupling reagents that activated carboxylic acids via the formation of stable α-acyloxyenamide active esters as isolable intermediates. Subsequent aminolysis of the aforementioned active esters offered the amides in excellent yields. The distinguishing feature of ynamide coupling reagents lies in their remarkable superiority in suppressing racemization during the activation and aminolysis of carboxylic acids containing an α-chiral center. This attribute enables them to be effective coupling reagents for peptide bond formation. Mechanistic study deepened our understanding of the working mechanism of ynamide coupling reagents and expanded their application to SPPS, fragment condensation and head-to-tail cyclization. By taking advantage of the capability of ynamide to trap monothiocarboxylic acids selectively to generate thiocarbonyl vinyl esters, an ynamide mediated thioamide bond formation strategy was developed and has been successfully applied to site-specific introduction of thioamide substitutions to the peptide backbone in both solution- and solid-phase peptide synthesis. Additionally, our study revealed that ynamide coupling reagents could also be used to facilitate the formation of ester and thioester bonds. Their application in the construction of intramolecular ester bonds enabled macrolactonization to proceed smoothly under mild acid-catalyzed conditions, circumventing the racemization of the α-chiral center and Z/E isomerization of conjugated α, β-C–C double bond encountered in conventional macrolactonization strategies. To ensure the cost-effective preparation and ready availability of ynamide coupling reagents, we developed a highly efficient one-step synthesis of ynamides from cheap starting materials and successfully implemented a recovery protocol for the coupling byproducts. Meanwhile, we designed a water-removable ynamide coupling reagent the byproduct of which could be easily removed by acidic aqueous workup, enhancing its potential for industrial applications. Most importantly, the advantage of ynamide coupling reagents in suppressing epimerization/racemization enabled peptide elongation to proceed in a rare N → C manner, which finally allowed us to accomplish the first practical inverse peptide synthesis indirectly using unprotected amino acids as the starting materials.^3,56 Inspired by the design concept of ynamide coupling reagents, we have also developed a new class of racemization-free allenone coupling reagents for mediating peptide bond formation.⁵⁷ The common feature of ynamide and allenone coupling reagents is that both of them take active vinyl esters as the stable intermediates, which offers an opportunity to independently optimize activation and aminolysis reaction conditions, effectively minimizing side reactions and impurities. The excellent formation efficiency and optimal reactivity of these active vinyl esters endowed ynamide and allenone coupling reagents the ability to merge the benefits of conventional coupling reagents and active esters, while eliminate their drawbacks. Such features suggested that coupling reagents employing the in situ formed active vinyl esters as stable intermediates could represent a new direction for coupling reagent design. In addition, this chemistry can also be applied to protein modification and drug development.^100,58 We believe that coupling reagents with stable active ester as isolable intermediate coupled with “the third wave of peptide synthesis”⁵⁹ might offer unexpected opportunities and solutions to the long-standing green chemistry challenges faced by peptide synthesis.

Biographies

Long Hu received his B.Sc. in 2013 from Jiangxi University of Traditional Medicine and M.Sc. in 2017 from Jiangxi Normal University. In 2021, he obtained his Ph.D. from The University of Heidelberg. Then, he conducted his postdoctoral research at The University of Heidelberg and Israel Institute of Technology. In November 2022, he joined Guangzhou Medical University as an associate professor. His research interests focus on the development of methodologies for biomolecular synthesis and modification.

Junfeng Zhao obtained his M.Sc. degree at Central China Normal University in 2005 and his PhD degree at Nanyang Technological University in 2010. Following his doctoral studies, he conducted postdoctoral research at Nanyang Technological University, University of Bonn, and University of Muenster from 2010 to 2013. He then joined The University of Hong Kong as a research assistant professor. He started his independent career at Jiangxi Normal University as a full professor in 2014. In 2021, his research group moved to Guangzhou Medical University. Currently, his research interest focuses on novel synthetic methodologies and chemical biology of peptide and protein.

Financial support from the National Natural Science Foundation of China (Grant No. 22277015, 22307023, 91853114, 21778025) and Guangzhou Municipal Science and Technology Bureau (GZ-STB, Grant No. 2024A04J02468) is greatly appreciated.

The authors declare no competing financial interest.