Fast and Selective Reaction of 2-Benzylacrylaldehyde with 1,2-Aminothiol for Stable N-Terminal Cysteine Modification and Peptide Cyclization

Wu, Yanfu; Liu, Cong; Fan, Shihui; Zhao, Yibing; Wu, Chuanliu

doi:10.1021/acs.bioconjchem.1c00378

Cited by 11 publications

(7 citation statements)

References 48 publications

(97 reference statements)

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“…After purification, the N-terminal proline can be removed in vitro by treating the protein with a prolyl aminopeptidase (ProAP). The resulting N-terminal cysteine can then be specifically reacted with a thioester, a CBT derivative, or other reagents. − ,− …”

Section: Resultsmentioning

confidence: 99%

“…A particularly attractive approach takes advantage of the unique properties of an N-terminal cysteine, which selectively reacts with aldehydes to form thiazolidines, − thioesters to form amides (i.e., native chemical ligation), and 2-cyanobenzothiazole (CBT) or 2-((alkylthio)(aryl)methylene)malononitrile (TAMM) to form 2-thiazolines. The 1,2-aminothiol moiety of an N-terminal cysteine can also be bio-orthogonally modified with 2-benzylacrylaldehyde (BAA) or monosubstituted cyclopropenone (CPO)-containing reagents . This approach, of course, necessitates the effective production of proteins with a free N-terminal cysteine.…”

Section: Introductionmentioning

confidence: 99%

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Generation of Proteins with Free N-Terminal Cysteine by Aminopeptidases

et al. 2022

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Efficient, site-specific, and bio-orthogonal conjugation of chemical functionalities to proteins is of great utility in fundamental research as well as industrial processes (e.g., the production of antibody–drug conjugates and immobilization of enzymes for biocatalysis). A popular approach involves reacting a free N-terminal cysteine with a variety of electrophilic reagents. However, current methods for generating proteins with N-terminal cysteines have significant limitations. Herein we report a novel, efficient, and convenient method for producing recombinant proteins with free N-terminal cysteines by genetically fusing a Met-Pro-Cys sequence to the N-terminus of a protein of interest and subjecting the recombinant protein to the sequential action of methionine and proline aminopeptidases. The resulting protein was site-specifically labeled at the N-terminus with fluorescein and a cyclic cell-penetrating peptide through native chemical ligation and a 2-cyanobenzothiazole moiety, respectively. In addition, the optimal recognition sequence of Aeromonas sobria proline aminopeptidase was determined by screening a combinatorial peptide library and incorporated into the N-terminus of a protein of interest for most efficient N-terminal processing.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Generation of Proteins with Free N-Terminal Cysteine by Aminopeptidases

et al. 2022

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“…Examples of these newly developed reagents include the N-hydroxysuccinimide (NHS)-activated acrylamides by Gois [55] and 2-benzylacrylaldehyde (BAA) by Wu. [56] Both the NHS-activated acrylamides and 2-benzylacrylaldehyde showed exceptional fast kinetics (k 2 = 1540 M À 1 s À 1 and 2700 M À 1 s À 1 , respectively) and form a 7member ring conjugation product (Figure 13). The NHS-activated acrylamide 50 initiates the reaction with a nucleophilic attack of the thiol group to the Michael acceptor, followed by macrocyclization of the amino group with an activated ester to form the cyclized conjugate 53 (Figure 13, equation 1).…”

Section: Activated Michael Acceptorsmentioning

confidence: 99%

“…Activated bifunctional Michael acceptors react with 1,2‐aminothiol were developed for fast NCys conjugation. Examples of these newly developed reagents include the N‐hydroxysuccinimide (NHS)‐activated acrylamides by Gois [55] and 2‐benzylacrylaldehyde (BAA) by Wu [56] . Both the NHS‐activated acrylamides and 2‐benzylacrylaldehyde showed exceptional fast kinetics ( k 2 =1540 M −1 s −1 and 2700 M −1 s −1 , respectively) and form a 7‐member ring conjugation product (Figure 13).…”

Section: Introductionmentioning

confidence: 99%

Fast Cysteine Bioconjugation Chemistry

Chen

Gao

2022

Chemistry A European J

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Cysteine bioconjugation serves as a powerful tool in biological research and has been widely used for chemical modification of proteins, constructing antibody-drug conjugates, and enabling cell imaging studies. Cysteine conjugation reactions with fast kinetics and exquisite selectivity have been under heavy pursuit as they would allow clean protein modification with just stoichiometric amounts of reagents, which minimizes side reactions, simplifies purification and broadens functional group tolerance. In this concept, we summarize the recent advances in fast cysteine bioconjugation, and discuss the mechanism and chemical principles that underlie the high efficiencies of the newly developed cysteine reactive reagents.

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“…Several electrophilic groups were developed to selectively target proteins with N-terminal Cys, − e.g., thioesters, O -salicylaldehyde esters, activated aldehydes, activated nitriles, 2-((alkylthio)(aryl)methylene)malononitriles, N -hydroxysuccinimide-activated acrylamides, 2-benzylacrylaldehydes, 2-formylphenylboronic acids, − and monosubstituted cyclopropenones . Here, we focused on activated heteroaromatic nitriles, which were found to have a wide range of applicability in recent years. ,− Inspired by the final step in the biosynthesis of d -luciferin, Rao and co-workers utilized a click reaction between 2-cyanobenzothiazole and N-terminal Cys for protein labeling .…”

Section: Introductionmentioning

confidence: 99%

Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine

et al. 2023

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The binucleophilic properties of 1,2-aminothiol and its rare occurrence in nature make it a useful reporter for tracking molecules in living systems. The 1,2-aminothiol moiety is present in cysteine, which is a substrate for a biocompatible click reaction with heteroaromatic nitriles. Despite the wide range of applications for this reaction, the scope of nitrile substrates has been explored only to a limited extent. In this study, we expand the chemical space of heteroaromatic nitriles for bioconjugation under physiologically relevant conditions. We systematically assembled a library of 116 2-cyanobenzimidazoles, 1-methyl-2cyanobenzimidazoles, 2-cyanobenzothiazoles, and 2-cyanobenzoxazoles containing electron-donating and electron-withdrawing substituents at all positions of the benzene ring. The compounds were evaluated for their stability, reactivity, and selectivity toward the N-terminal cysteine of model oligopeptides. In comparison to the benchmark 6-hydroxy-2-cyanobenzothiazole or 6-amino-2cyanobenzothiazole, we provide highly selective and moderately reactive nitriles as well as highly reactive yet less selective analogs with a variety of enabling attachment chemistries to aid future applications in bioconjugation, chemical biology, and nanomaterial science.

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Fast and Selective Reaction of 2-Benzylacrylaldehyde with 1,2-Aminothiol for Stable N-Terminal Cysteine Modification and Peptide Cyclization

Cited by 11 publications

References 48 publications

Generation of Proteins with Free N-Terminal Cysteine by Aminopeptidases

Generation of Proteins with Free N-Terminal Cysteine by Aminopeptidases

Fast Cysteine Bioconjugation Chemistry

Tunable Heteroaromatic Nitriles for Selective Bioorthogonal Click Reaction with Cysteine

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