Mechanistic study of CBT-Cys click reaction and its application for identifying bioactive N-terminal cysteine peptides in amniotic fluid

Zheng, Zhen; Chen, Peiyao; Li, Gongyu; Zhu, Yunxia; Shi, Zhonghua; Luo, Yufeng; Zhao, Chun; Fu, Ziyi; Cui, Xianwei; Wang, Fuqiang; Huang, Guangming; Liang, Gaolin

doi:10.1039/c6sc01461e

Cited by 41 publications

(36 citation statements)

References 55 publications

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“…From the mechanistic point of view, the thiol group is initially involved in the attack of the CBT cyano group for the formation of the luciferin ring. Our observation implied that the increasing nucleophilicity of the thiol group could accelerate the first step of the cyclization …”

Section: Figurementioning

confidence: 77%

Early‐Stage Incorporation Strategy for Regioselective Labeling of Peptides using the 2‐Cyanobenzothiazole/1,2‐Aminothiol Bioorthogonal Click Reaction

2018

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Herein, we describe a synthetic strategy for the regioselective labeling of peptides by using a bioorthogonal click reaction between 2‐cyanobenzothiazole (CBT) and a 1,2‐aminothiol moiety. This methodology allows for the facile and site‐specific modification of peptides with various imaging agents, including fluorophores and radioisotope‐containing prosthetic groups. We investigated the feasibility of an early‐stage incorporation of dipeptide 1 into targeting vectors, such as c[RGDyK(C)] and HER2 pep, during solid‐phase peptide synthesis. Then, the utility of the click reaction to label bioactive peptides with a CBT‐modified imaging agent (FITC–CBT, 9) was assessed. The ligation reaction was found to be highly selective and efficient under various conditions. The fluorescently labeled peptides 2 and 3 were obtained in respective yields of 88 and 82 % under optimized conditions.

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Section: Figurementioning

confidence: 77%

Early‐Stage Incorporation Strategy for Regioselective Labeling of Peptides using the 2‐Cyanobenzothiazole/1,2‐Aminothiol Bioorthogonal Click Reaction

2018

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“…Luciferin, a 2‐thiazoline, can be produced from a condensation reaction between CHBT and cysteine. According to the category of N‐terminal cysteine involved in click reactions, the condensation of CHBT and cysteine is a click condensation reaction and has a high second‐order rate constant . This indicates that condensation can occur readily in aqueous solutions, including the complex aqueous environment of cells .…”

Section: Resultsmentioning

confidence: 99%

“…As shown in Figure A, the energy barrier (2.4 kcal mol −1 ) for forming Piii1 / Riii2 is much lower than that (19.6 kcal mol −1 ) for consuming Piii1 / Riii2 , which indicates that Piii1 / Riii2 is easier to be captured. Recently, this Int was indeed captured and structurally characterized by mass spectrometry . By employing induced nanoelectrospray ionization‐mass spectrometry (InESI‐MS) and a home‐built micro‐reactor, the aminoluciferin product ion ( m/z 280) and Int ion ( m/z 297) can be detected upon the injection of amino‐CHBT ( m/z 176) into cysteine ( m/z 122).…”

Section: Resultsmentioning

confidence: 99%

“…Therefore, it is reasonable for us to theoretically investigate the reaction in DMSO or an aqueous solution to mimic the reaction in living fireflies. Additionally, as a click condensation reaction, this reaction has various applications, such as the self‐assembly modulation of supramolecular nanofibres, the labelling of N‐terminal cysteine residues, and in vivo bioluminescence detection . Step 3 is the stereoisomeric bio‐inversion of L‐luciferin into D‐luciferin in the presence of Coenzyme A (CoA) and thioesterase .…”

Section: Introductionmentioning

confidence: 99%

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Luciferin Regeneration in Firefly Bioluminescence via Proton‐Transfer‐Facilitated Hydrolysis, Condensation and Chiral Inversion

Cheng

Liu

2019

ChemPhysChem

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Firefly bioluminescence is produced via luciferin enzymatic reactions in luciferase. Luciferin has to be unceasingly replenished to maintain bioluminescence. How is the luciferin reproduced after it has been exhausted? In the early 1970s, Okada proposed the hypothesis that the oxyluciferin produced by the previous bioluminescent reaction could be converted into new luciferin for the next bioluminescent reaction. To some extent, this hypothesis was evidenced by several detected intermediates. However, the detailed process and mechanism of luciferin regeneration remained largely unknown. For the first time, we investigated the entire process of luciferin regeneration in firefly bioluminescence by density functional theory calculations. This theoretical study suggests that luciferin regeneration consists of three sequential steps: the oxyluciferin produced from the last bioluminescent reaction generates 2‐cyano‐6‐hydroxybenzothiazole (CHBT) in the luciferin regenerating enzyme (LRE) via a hydrolysis reaction; CHBT combines with L‐cysteine in vivo to form L‐luciferin via a condensation reaction; and L‐luciferin inverts into D‐luciferin in luciferase and thioesterase. The presently proposed mechanism not only supports the sporadic evidence from previous experiments but also clearly describes the complete process of luciferin regeneration. This work is of great significance for understanding the long‐term flashing of fireflies without an in vitro energy supply.

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“…[69] Recently,L iang and co-workers have employed an ew emerging induced nanoelectrospray ionization-masss pectrometric (InESI-MS) method to detect the key intermediates existed in this reaction, revealing at wo-step condensation process. [70] They found that atom Ni nt he new formingt hiazolin came from the amine in Cys, but not from the CN group in CBT.T his uniqueb iocompatible reaction was latter extended for the in situ synthesis of oligomers/polymers using am onomer (2)c ontaining both CBT and Cys moieties ( Figure 1b). [71] It was demonstrated that the reaction could be controlled by the constituents of cells( e.g.…”

Section: Introductionmentioning

confidence: 99%

Firefly Luciferin‐Inspired Biocompatible Chemistry for Protein Labeling and In Vivo Imaging

Wang

Luo

et al. 2017

Chemistry A European J

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Biocompatible reactions have emerged as versatile tools to build various molecular imaging probes that hold great promise for the detection of biological processes in vitro and/or in vivo. In this Minireview, we describe the recent advances in the development of a firefly luciferin-inspired biocompatible reaction between cyanobenzothiazole (CBT) and cysteine (Cys), and highlight its versatility to label proteins and build multimodality molecular imaging probes. The review starts from the general introduction of biocompatible reactions, which is followed by briefly describing the development of the firefly luciferin-inspired biocompatible chemistry. We then discuss its applications for the specific protein labeling and for the development of multimodality imaging probes (fluorescence, bioluminescence, MRI, PET, photoacoustic, etc.) that enable high sensitivity and spatial resolution imaging of redox environment, furin and caspase-3/7 activity in living cells and mice. Finally, we offer the conclusions and our perspective on the various and potential applications of this reaction. We hope that this review will contribute to the research of biocompatible reactions for their versatile applications in protein labeling and molecular imaging.

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Mechanistic study of CBT-Cys click reaction and its application for identifying bioactive N-terminal cysteine peptides in amniotic fluid

Abstract: CBT-Cys click condensation reaction has a high second-order reaction rate constant and has found wide applicability in recent years.

Cited by 41 publications

References 55 publications

Early‐Stage Incorporation Strategy for Regioselective Labeling of Peptides using the 2‐Cyanobenzothiazole/1,2‐Aminothiol Bioorthogonal Click Reaction

Early‐Stage Incorporation Strategy for Regioselective Labeling of Peptides using the 2‐Cyanobenzothiazole/1,2‐Aminothiol Bioorthogonal Click Reaction

Luciferin Regeneration in Firefly Bioluminescence via Proton‐Transfer‐Facilitated Hydrolysis, Condensation and Chiral Inversion

Firefly Luciferin‐Inspired Biocompatible Chemistry for Protein Labeling and In Vivo Imaging

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