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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 SN acyl transfer. Herein, we develop methods, using inexpensive Boc-4R-hydroxyproline, for the solid-phase synthesis of peptides containing N-terminal 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 SN2 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 SN acyl transfer are sufficiently low in energy (10-15 kcal mol–1 above starting material) that ring strain cannot explain slow SN 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 SN 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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Acyltransferase families that act on thioesters: Sequences, structures, and mechanisms
Abstract Acyltransferases (AT) are enzymes that catalyze the transfer of acyl group to a receptor molecule. This review focuses on ATs that act on thioester‐containing substrates. Although many ATs can recognize a wide variety of substrates, sequence similarity analysis allowed us to classify the ATs into fifteen distinct families. Each AT family is originated from enzymes experimentally characterized to have AT activity, classified according to sequence similarity, and confirmed with tertiary structure similarity for families that have crystallized structures available. All the sequences and structures of the AT families described here are present in the thioester‐active enzyme (ThYme) database. The AT sequences and structures classified into families and available in the ThYme database could contribute to enlightening the understanding acyl transfer to thioester‐containing substrates, most commonly coenzyme A, which occur in multiple metabolic pathways, mostly with fatty acids.
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- Award ID(s):
- 2001385
- PAR ID:
- 10485710
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- Proteins: Structure, Function, and Bioinformatics
- Volume:
- 92
- Issue:
- 2
- ISSN:
- 0887-3585
- Format(s):
- Medium: X Size: p. 157-169
- Size(s):
- p. 157-169
- Sponsoring Org:
- National Science Foundation
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