Protein Organic Chemistry and Applications for Labeling and Engineering in Live‐Cell Systems

Takaoka, Yousuke; Ojida, Akio; Hamachi, Itaru

doi:10.1002/anie.201207089

Cited by 292 publications

(204 citation statements)

References 157 publications

(143 reference statements)

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“…Further, a reaction carried out at neutral pH would allow our DOSY study to be directly applicable to the reaction conditions. Lipid precursor 4 contains a maleimide, widely used in bioconjugation reactions 41 (Fig. 5).…”

Section: Resultsmentioning

confidence: 99%

Physical autocatalysis driven by a bond-forming thiol–ene reaction

2014

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Autocatalysis has been extensively studied because it is central to the propagation of living systems. Chemical systems which self-reproduce like living cells would offer insight into principles underlying biology and its emergence from inanimate matter. Protocellular models feature a surfactant boundary, providing compartmentalization in the form of a micelle or vesicle and any model of the emergence of cellular life must account for the appearance, and evolution of, such boundaries. Here, we describe an autocatalytic system where two relatively simple components combine to form a more complex product. The reaction products aggregate into micelles that catalyse molecular self-reproduction. Study of the reaction kinetics and aggregation behaviour suggests a mechanism involving micelle-mediated physical autocatalysis and led to the rational design of a second-generation system. These reactions are driven by irreversible bond formation and provide a working model for the autocatalytic formation of protocells from the coupling of two simple molecular components.

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Section: Resultsmentioning

confidence: 99%

Physical autocatalysis driven by a bond-forming thiol–ene reaction

2014

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“…This appendage, which includes small functional groups, such as hydroxylamines, azides, alkynes, alkenes, or halides, is subsequently reacted with an exogenous bioorthogonal functional probe through a highly selective chemical reaction (Figure 1) [6,7].…”

Section: Introductionmentioning

confidence: 99%

Palladium-mediated chemistry in living cells

Chankeshwara

Indrigo

Bradley

2014

Current Opinion in Chemical Biology

104

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“…1,2) This method generally utilizes a chemical probe composed of a ligand moiety, a reactive group and a tag unit (Fig. 1a).…”

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confidence: 99%

Dichloromaleimide (diCMI): A Small and Fluorogenic Reactive Group for Use in Affinity Labeling

Chiba

Hashimoto

Yamaguchi

2016

CHEMICAL & PHARMACEUTICAL BULLETIN

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Chemical probes comprising a ligand moiety, a reactive group (e.g. epoxide, haloacetyl or photoreactive group) and a tag unit (e.g. fluorophore or radioisotope) are widely used in affinity labeling to identify the target proteins of bioactive molecules. However, design and synthesis of highly functionalized chemical probes are often time-consuming. In this paper, we propose a simple design strategy for chemical probes bearing a small 2,3-dichloromaleimide (diCMI) unit, which serves as a combined reactive group and tag unit by reacting with a nucleophilic lysine residue near the ligand-binding site of the target protein to generate the 2-amino-3-chloromaleimide fluorophore. Model ligand-protein experiments confirmed that the diCMI unit has suitable reactivity and fluorogenic capability for efficient affinity labeling.Key words affinity labeling; dichloromaleimide; protein modification Affinity labeling is a powerful method to label and visualize target proteins of bioactive molecules.1,2) This method generally utilizes a chemical probe composed of a ligand moiety, a reactive group and a tag unit (Fig. 1a). The reactive group serves to form a covalent bond with the target protein. For efficient and specific target labeling, the reactive group should be stable in aqueous media, have low reactivity towards non-target molecules, and react appropriately with the target protein after ligand-target binding. The tag unit serves to visualize the target protein. Fluorophores are often used as a tag unit because of the ease of detection. However, a major obstacle to affinity labeling is often synthesis of the highly functionalized chemical probe. More importantly, the ligand must retain its binding affinity after introduction of these two functional groups (reactive group and tag unit). For these reasons, a small alkyne tag is often used in affinity labeling, since it can be subsequently conjugated with a detection unit (e.g. azide-fluorophore, azide-biotin), despite the inconvenience of the additional conjugation step(s).3) We recently reported a small alkoxy nitrobenzoxadiazole (O-NBD, 180 Da) unit as a fluorogenic reactive group for affinity labeling.4) Here, we show that 2,3-dichloromaleimide (diCMI, 164 Da) can serve as an even smaller fluorogenic reactive group (Fig. 1b). Results and DiscussionMolecular Design, Model Reaction and Spectrometric Analysis The maleimide motif is a highly reactive Michael acceptor and has been widely used for chemical modification of thiols of biomolecules.5-7) For instance, fluorophore-conjugated maleimides have been employed for the modification of cysteine residues. 8,9) Recently, 2-bromomaleimide and 2,3-dibromomaleimide were developed as novel cysteine-labeling reagents, which react with thiol in an addition-elimination sequence (nucleophilic substitution) to afford thiomaleimide. 10-18)In an organic solvent such as tetrahydrofuran (THF), 2,3-dibromomaleimide also reacts with amine, affording 2-amino-3-bromomaleimide as an amine-conjugation product. Interestingly, 2-aminomaleimides we...

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Protein Organic Chemistry and Applications for Labeling and Engineering in Live‐Cell Systems

Cited by 292 publications

References 157 publications

Physical autocatalysis driven by a bond-forming thiol–ene reaction

Physical autocatalysis driven by a bond-forming thiol–ene reaction

Palladium-mediated chemistry in living cells

Dichloromaleimide (diCMI): A Small and Fluorogenic Reactive Group for Use in Affinity Labeling

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