Conjugation-Induced Fluorescent Labeling of Proteins and Polymers Using Dithiomaleimides

Robin, Mathew P.; Wilson, Paul; Mabire, Anne B.; Kiviaho, Jenny K.; Raymond, Jeffery E.; Haddleton, David M.; O’Reilly, Rachel K.

doi:10.1021/ja3105494

Cited by 109 publications

(135 citation statements)

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“…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][11][12][13][14][15][16][17][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 were recently reported to exhibit strong fluorescence at >400 nm with large Stokes shifts (>100 nm).…”

Section: Molecular Design Model Reaction and Spectrometric Analysismentioning

confidence: 99%

“…Bands were visualized using UV light or appropriate reagents followed by heating. Flash chromatography was carried out with silica gel (Silica gel 60N, 40-50 µm particle size) purchased from Kanto Chemical Co., Inc. NMR spectra were recorded on a JEOL JNM-GX500 or JNMECA-500 spectrometer, operating at 500 MHz for 1 H-NMR and at 125 MHz for 13 C-NMR. High-resolution mass spectra (HR-MS) were obtained using a BRUKER micrOTOF II mass spectrometer.…”

Section: Synthetic Proceduresmentioning

confidence: 99%

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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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Section: Molecular Design Model Reaction and Spectrometric Analysismentioning

confidence: 99%

Section: Synthetic Proceduresmentioning

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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“…10 Thus, the next step towards creating 'smart' polyHIPE materials that can respond to an external stimulus in the surrounding environment is to identify potential strategies for surface modification. 24 and polymeric nanoparticles, 28 all of which retain the fluorescent properties exhibited by DBMs/DTMs. Herein, for the first time we describe the conjugation of DTMs to thiol-acrylate polyHIPE materials as a facile route to polyHIPE surface modification with responsive polymers, poly(ethylene glycol) (PEG) and poly(N-isopropylacrylamide) (pNIPAM).…”

Section: A Note On Versionsmentioning

confidence: 99%

“…22,23 Synthesis of α-functional DTM polymers has been previously reported using reversible-addition fragmentation transfer (RAFT) polymerisation, 24 atom transfer radical polymerisation (ATRP) 25 and single electron transfer living radical polymerisation (SET-LRP). 26 Such polymers have been employed as functional handles for the preparation of complex polymer architectures, 27 protein-polymer conjugates, 24 and polymeric nanoparticles, 28 all of which retain the fluorescent properties exhibited by DBMs/DTMs. Herein, for the first time we describe the conjugation of DTMs to thiol-acrylate polyHIPE materials as a facile route to polyHIPE surface modification with responsive polymers, poly(ethylene glycol) (PEG) and poly(N-isopropylacrylamide) (pNIPAM).…”

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confidence: 99%

Reversible surface functionalisation of emulsion-templated porous polymers using dithiophenol maleimide functional macromolecules

et al. 2017

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(2017) Reversible surface functionalisation of emulsiontemplated porous polymers using dithiophenol maleimide functional macromolecules. Chemical Communications, 53 (70). pp. 9789-9792. Permanent WRAP URL:http://wrap.warwick.ac.uk/92002 Copyright and reuse:The Warwick Research Archive Portal (WRAP) makes this work of researchers of the University of Warwick available open access under the following conditions. Copyright © and all moral rights to the version of the paper presented here belong to the individual author(s) and/or other copyright owners. To the extent reasonable and practicable the material made available in WRAP has been checked for eligibility before being made available.Copies of full items can be used for personal research or study, educational, or not-for-profit purposes without prior permission or charge. Provided that the authors, title and full bibliographic details are credited, a hyperlink and/or URL is given for the original metadata page and the content is not changed in any way. Publisher statement:First published by Royal Society of Chemistry 2017 http://dx.doi.org/10.1039/C7CC03811A A note on versions:The version presented here may differ from the published version or, version of record, if you wish to cite this item you are advised to consult the publisher's version. Please see the 'permanent WRAP url' above for details on accessing the published version and note that access may require a subscription. Macroporous polymers are increasingly used in a wide range of applications including reaction supports, 1-3 separation processes 4 and tissue engineering scaffolds. [5][6][7] Highly porous polymers with a well-defined morphology and a fully interconnected network of pores can be prepared by emulsiontemplating whereby a high internal phase emulsion (HIPE) is created in which the major, "internal" phase, usually defined as constituting more than 74% of the volume, is dispersed within the continuous, minor, "external" phase. Most polyHIPE materials are prepared by radical polymerisation, initiated either thermally or photochemically; however, other methods have been reported. 8,9 Nevertheless, a lack of functionality on the surface of polyHIPE materials can be a limitation in expanding their applications. For example, it has been shown that a galactose-functionalised styrene-based polyHIPE material surpassed the unfunctionalised material in its performance as a scaffold for 3D culture of mammalian hepatocytes. 10 Thus, the next step towards creating 'smart' polyHIPE materials that can respond to an external stimulus in the surrounding environment is to identify potential strategies for surface modification. 24 and polymeric nanoparticles, 28 all of which retain the fluorescent properties exhibited by DBMs/DTMs. Herein, for the first time we describe the conjugation of DTMs to thiol-acrylate polyHIPE materials as a facile route to polyHIPE surface modification with responsive polymers, poly(ethylene glycol) (PEG) and poly(N-isopropylacrylamide) (pNIPAM). Conjugation of DTM end-capped po...

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“…In addition to the control over molecular weight and molecular weight distribution, CRPs gave access to the control of the chain-end functionality allowing, for example, for the coupling between polymer-polymer [28][29][30] and polymerprotein 31,32 . Several bromoisobutyrate derivatives were selected to demonstrate that a wide range of functional group can be inserted at the polymer chain-end ( Figure S8): ethyl-2-bromoisobutyrate (EBiB), cholesteryl-2-bromoisobutyrate (Chol-BiB), pyrenyl-2-bromisiobutyrate (Py-BiB), Nsuccinimidyl-2-bromoisobutyrate (Succ-BiB), 2-bromo-2-methyl propionic acid 2-(3,5-dioxo-10-oxa-4-azatricyclo[5.2.1.02,6]dec-8-en-4-yl) ethyl ester (Mal-BiB), adamantyl-2-bromoisobutyrate (Adam-BiB) and dihydroxypropyl-2-bromoisobutyrate (diOH-BiB).…”

Section: Effect Of the Initiatormentioning

confidence: 99%

Cu(0)-mediated living radical polymerisation in dimethyl lactamide (DML); an unusual green solvent with limited environmental impact

et al. 2015

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Conjugation-Induced Fluorescent Labeling of Proteins and Polymers Using Dithiomaleimides

Cited by 109 publications

References 56 publications

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

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

Reversible surface functionalisation of emulsion-templated porous polymers using dithiophenol maleimide functional macromolecules

Cu(0)-mediated living radical polymerisation in dimethyl lactamide (DML); an unusual green solvent with limited environmental impact

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