Chemical Protein Modification through Cysteine

Gunnoo, Smita B.; Madder, Annemieke

doi:10.1002/cbic.201500667

Cited by 326 publications

(342 citation statements)

References 296 publications

(296 reference statements)

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“…[7] Um die Stabilitätu nserer Phosphonamidat-Thiol-Addukte zu untersuchen haben wir Fluoreszenzlçschungsuntersuchungen durchgeführt, bei dem ein fluoreszierendes Signal durch die Spaltung der Konjugate generiert wurde (Abbildung 3a). [7] Um die Stabilitätu nserer Phosphonamidat-Thiol-Addukte zu untersuchen haben wir Fluoreszenzlçschungsuntersuchungen durchgeführt, bei dem ein fluoreszierendes Signal durch die Spaltung der Konjugate generiert wurde (Abbildung 3a).…”

Section: Angewandte Chemieunclassified

“…[2] Ersteres verlangt oftmals aufwendige biochemische Manipulationen, wie zum Beispiel Amber-Codon-Unterdrückung oder zusätzliche enzymatische Tr ansformationen, während letzteres nur selektiv füre ine Aminosäurefunktion ist, wodurch Produktgemische mit unterschiedlichen Modifikationsgraden und mehreren Regioisomeren entstehen kçnnen. [7] Darüber hinaus wurden die einzigartigen nukleophilen Eigenschaften der Sulfhydrylgruppe fürdie Entwicklung einer Vielzahl von cysteinselektiven Reaktionen, wie ausgenutzt. [7] Darüber hinaus wurden die einzigartigen nukleophilen Eigenschaften der Sulfhydrylgruppe fürdie Entwicklung einer Vielzahl von cysteinselektiven Reaktionen, wie ausgenutzt.…”

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Cysteinselektive phosphonamidatbasierte Elektrophile für modulare Biokonjugationen

et al. 2019

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Wirb eschreiben eine neue Methode zur Proteinsynthese,d ie das bestehende Repertoire an Mçglichkeiten zur Proteinmodifikation erweitert:E ine chemoselektive Reaktion induziert Reaktivitätf üre ine nachfolgende Biokonjugation. Hierbei reagiert ein azidmodifizierter Baustein zunächst mit einem Ethinylphosphonit in einer Staudinger-Phosphonit-Reaktion (SPhR) zu einem Ethinylphosphonamidat. Die so entstehende elektronenarme Dreifachbindung modifiziert anschließend in einer cysteinselektiven Reaktion Proteine und Antikçrper.W ir zeigen, dass Ethinylphosphonamidate eine herausragende SelektivitätfürCysteine aufweisen, gepaart mit einer überragenden Stabilitätd er Thiol-Addukte im Vergleich zu klassischen Maleimid-Konjugaten. Diese Erkenntnis macht unsere Technik zu einer vielseitigen und leistungsstarken Methode fürdie mühelose Herstellung von stabilen, funktionalen Proteinkonjugaten.

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Section: Angewandte Chemieunclassified

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Cysteinselektive phosphonamidatbasierte Elektrophile für modulare Biokonjugationen

et al. 2019

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“…[5][6][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.…”

Section: Molecular Design Model Reaction and Spectrometric Analysismentioning

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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“…4,47–51 Control of charge provided a strong handle to control interactions. Previously, 4 we have reported modification of GOx with TEPA where the net charge was flipped from net negative to net positive to trigger enzyme binding to anionic nanosheets.…”

Section: Discussionmentioning

confidence: 99%

Tuning Enzyme/α-Zr(IV) Phosphate Nanoplate Interactions via Chemical Modification of Glucose Oxidase

et al. 2017

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Using glucose oxidase (GOx) and α-Zr(IV) phosphate nanoplates (α-ZrP) as a model system, a generally applicable approach to control enzyme–solid interactions via chemical modification of amino acid side chains of the enzyme is demonstrated. Net charge on GOx was systematically tuned by appending different amounts of polyamine to the protein surface to produce chemically modified GOx(n), where n is the net charge on the enzyme after the modification and ranged from −62 to +95 electrostatic units in the system. The binding of GOx(n) with α-ZrP nanosheets was studied by isothermal titration calorimetry (ITC) as well as by surface plasmon resonance (SPR) spectroscopy. Pristine GOx showed no affinity for the α-ZrP nanosheets, but GOx(n) where n ≥ −20 showed binding affinities exceeding (2.1 ± 0.6) × 106 M−1, resulting from the charge modification of the enzyme. A plot of GOx(n) charge vs Gibbs free energy of binding (ΔG) for n = +20 to n = +65 indicated an overall increase in favorable interaction between GOx(n) and α-ZrP nanosheets. However, ΔG is less dependent on the net charge for n > +45, as evidenced by the decrease in the slope as charge increased further. All modified enzyme samples and enzyme/α-ZrP complexes retained a significant amount of folding structure (examined by circular dichroism) as well as enzymatic activities. Thus, strong control over enzyme–nanosheet interactions via modulating the net charge of enzymes may find potential applications in biosensing and biocatalysis.

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Chemical Protein Modification through Cysteine

Cited by 326 publications

References 296 publications

Cysteinselektive phosphonamidatbasierte Elektrophile für modulare Biokonjugationen

Cysteinselektive phosphonamidatbasierte Elektrophile für modulare Biokonjugationen

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

Tuning Enzyme/α-Zr(IV) Phosphate Nanoplate Interactions via Chemical Modification of Glucose Oxidase

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