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

Abstract

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...

Visualization of multiple localizations of GLUT4 by fluorescent probes of PYP-tag with designed unnatural warhead

“…These results demonstrated that the N -glycan of PYP-GLUT4 is involved in quality control of PYP-GLUT4 and dictates the pathway PYP-GLUT4 takes within the cell. While these observations are in agreement with previous reports that indicated that the N -glycan plays an important role in quality control of PYP-GLUT4 WT , 34 this is the first report that actually visualized the distribution of different types of PYP-GLUT4 molecules within a live cell using fluorescent probes of different wavelengths.…”

“…Based on these results, we concluded that the most suitable ligand structure for the new probe would be 5, which employed a chloroacetyl group at the reactive site and a trifluoromethyl group within the ligand portion. The reason 5 exhibited high reactivity against PYP-tag was thought to be due to the electron-withdrawing trifluoromethyl functionality elevating the reactivity of the chloroacetyl group 34 and noncovalent interaction between PYP-tag and 5 that is absent in glutathione. Thus, incorporation of those structural features into the novel probe design was expected to accelerate the labeling reaction rate of the probe.…”

Affinity Labeling with 4-Azidophthalimide (AzPI): Relation between Labeling Rate and Fluorescence Intensity

“…[1][2][3][4][5][6][7][8] However, design and synthesis of highly functionalized chemical probes are often time-consuming. [9][10][11] To address this issue, we recently presented a simple design strategy for chemical probes bearing only a small alkoxy nitrobenzoxadiazole (O-NBD, 180 Da), 12) 2,3-dichloromaleimide (diCMI, 164 Da) 13) or 4-azidophthalimide (AzPI, 189 Da) 14) tag, each of which can serve as a combined reactive group and tracer unit. The AzPI tag is particularly useful for identifying target proteins of bioactive small molecules because it generates highly reactive nitrene species under photo-irradiation conditions and is capable of forming a covalent bond with various amino acids.…”