Mechanism for the enhanced reactivity of 4-mercaptoprolyl thioesters in native chemical ligation

Shah, Muhammad; Xu, Zheyuan; Liu, Lei; Jiang, Yuan‐Ye; Shi, Jing

doi:10.1039/c6ra13793h

Cited by 15 publications

(4 citation statements)

References 88 publications

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“… The free thiol moiety, which is located cis relative to the thioester, undergoes an intramolecular addition/elimination reaction to generate a strained thiolactone which precludes any n → π* interaction. This mechanism is supported by recent theoretical calculations . Subsequent ring opening of the bicyclic system with a cysteinyl peptide or other N-terminal β- or γ-thioamino acid derivatives results in the formation of a native peptide bond with reaction rates of the same order of magnitude as those reported for alanyl thioesters.…”

Section: Role Of the C-terminal Residue On The Reactivity Of C-termin...mentioning

confidence: 99%

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

et al. 2019

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The native chemical ligation reaction (NCL) involves reacting a C-terminal peptide thioester with an N-terminal cysteinyl peptide to produce a native peptide bond between the two fragments. This reaction has considerably extended the size of polypeptides and proteins that can be produced by total synthesis and has also numerous applications in bioconjugation, polymer synthesis, material science, and micro-and nanotechnology research. The aim of the present review is to provide a thorough mechanistic overview of NCL and extended methods. The most relevant properties of peptide thioesters, Cys peptides, and common solvents, reagents, additives, and catalysts used for these ligations are presented. Mechanisms, selectivity and reactivity are, whenever possible, discussed through the insights of computational and physical chemistry studies. The inherent limitations of NCL are discussed with insights from the mechanistic standpoint. This review also presents a palette of O,S-, N,S-, or N,Se-acyl shift systems as thioester or selenoester surrogates and discusses the special molecular features that govern reactivity in each case. Finally, the various thiol-based auxiliaries and thiol or selenol amino acid surrogates that have been developed so far are discussed with a special focus on the mechanism of long-range N,S-acyl migrations and selective dechalcogenation reactions.

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Section: Role Of the C-terminal Residue On The Reactivity Of C-termin...mentioning

confidence: 99%

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

et al. 2019

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“…Originally developed by Kent et al in 1994 [76] for protein synthesis [77], native chemical ligation (NCL) was more recently applied for peptide-polymer conjugation.…”

Section: Native Chemical Ligationmentioning

confidence: 99%

Bottom-up strategies for the synthesis of peptide-based polymers

Martin

Desfoux

Martínez

et al. 2021

Progress in Polymer Science

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“…NCL using 4 R -mercaptoproline and 4 R -selenoproline (Figure b) as a proline surrogate has been described in work by Danishefsky, Otaka, Payne, and others. − Alternatively, a C-terminal proline thioester could be applied for NCL at junctions with proline. However, proline C-terminal thioesters typically exhibit low reactivity due to the thioester being the electron acceptor of an n → π* interaction with the pre-prolyl carbonyl, which slows the rate of thioester exchange. , This limitation can be overcome using the proline surrogate 4 S -mercaptoproline at the C-terminal thioester, in which the 4 S -thiolate internally activates the proline carbonyl thioester to generate a bicyclic intermediate that accelerates thioester exchange with the coupling thiol partner. ,− However, the cost of 4 S -mercaptoproline ($400/g as Fmoc-4 S -mcp(Trt)-OH, $245/mmol) has, to date, limited its applications.…”

Section: Introductionmentioning

confidence: 99%

N-Terminal Proline Editing for the Synthesis of Peptides with Mercaptoproline and Selenoproline: Mechanistic Insights Lead to Greater Efficiency in Proline Native Chemical Ligation

Ludwig,

Forbes,

Zondlo

2024

ACS Chem. Biol.

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Native chemical ligation (NCL) at proline has been limited by cost and synthetic access. In addition, prior examples of NCL using mercaptoproline have exhibited stalling of the reaction after thioester exchange, due to inefficient S → N acyl transfer. Herein, we develop methods, using inexpensive Boc-4R-hydroxyproline, for the solid-phase synthesis of peptides containing Nterminal 4R-mercaptoproline and 4R-selenoproline. The synthesis proceeds via proline editing on the N-terminus of fully synthesized peptides on the solid phase, converting an N-terminal Boc-4R-hydroxyproline to the 4S-bromoproline, followed by an S N 2 reaction with potassium thioacetate or selenobenzoic acid. After cleavage from the resin and deprotection, peptides with functionalized N-terminal proline amino acids were obtained. NCL reactions with mercaptoproline proceeded slowly under standard NCL conditions, with the S-acyl transthioesterification intermediate observed as a major species. Computational investigations indicated that the bicyclic intermediates and transition states for S → N acyl transfer are sufficiently low in energy (10−15 kcal mol −1 above starting material) that ring strain cannot explain the slow S → N acyl transfer. Instead, the bicyclic zwitterionic tetrahedral intermediate has a low barrier for reversion to the S-acyl intermediate, causing reversion to the thioester (reverse reaction) to occur preferentially over elimination to generate the amide (forward reaction). We hypothesized that a buffer capable of general acid and/ or general base catalysis could promote S → N acyl transfer and thus achieve greater efficiency in proline NCL. In the presence of 2 M imidazole at pH 6.8, NCL with mercaptoproline proceeded efficiently to generate the peptide with a native amide bond. NCL with selenoproline also proceeded efficiently to generate the desired products when a thiophenol thioester was employed as a ligation partner. After desulfurization or deselenization, the products obtained were identical to those synthesized directly, confirming that the solid-phase proline editing reactions proceeded stereospecifically and without epimerization.

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Mechanism for the enhanced reactivity of 4-mercaptoprolyl thioesters in native chemical ligation

Abstract: Ring-strain-precluded strategy benefiting from entropy effects and n → π* orbital interaction, enhances the reactivity of C-terminal prolyl thioesters in NCL.

Cited by 15 publications

References 88 publications

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

Native Chemical Ligation and Extended Methods: Mechanisms, Catalysis, Scope, and Limitations

Bottom-up strategies for the synthesis of peptide-based polymers

N-Terminal Proline Editing for the Synthesis of Peptides with Mercaptoproline and Selenoproline: Mechanistic Insights Lead to Greater Efficiency in Proline Native Chemical Ligation

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