175885-18-4Relevant articles and documents
Dichloromaleimide (diCMI): A small and fluorogenic reactive group for use in affinity labeling
Chiba, Kosuke,Hashimoto, Yuichi,Yamaguchi, Takao
, p. 1647 - 1653 (2016)
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.
Covalently functionalized amide cross-linked hydrogels from primary amines and polyethylene glycol acyltrifluoroborates (PEG-KATs)
Schauenburg, Dominik,Osuna Gálvez, Alberto,Bode, Jeffrey W.
, p. 4775 - 4782 (2018)
A new method for the rapid preparation of chemically cross-linked hydrogels based on a multi-arm polyethylene glycol (PEG) bearing potassium acyl trifluoroborate (KAT) functional groups with multi-dentate amines is described. These scaffolds-prepared in aqueous buffer-give strong, transparent hydrogels. At pH 3, the gel formation is complete within seconds, and the reaction rate can be tuned by modulating the pH. Rheology measurements show that the hydrogel properties can be tuned as a function of both the weight percent of solids in the gel and the denticity of amine cross-linker, allowing for predictable formation of gels with desired traits. This process relies on a rapid amide-forming reaction of KATs and in situ generated N-chloroamines. Numerous commercially available amines, including di-, tri- and tetra-functional amines as well as peptides and carbohydrates serve as effective cross-linkers. Monodentate amines included in the gelation mixture are covalently linked into the gel matrix by amide-bonds, allowing gels containing immobilized molecules including dyes, sensors, or biotin amine, to be prepared in a single step from simple starting materials. The ability to induce gelation only upon addition of equimolar amounts of an inexpensive inducer (N-chlorosuccinimide) allows premixed components to be stored as an aqueous solution for weeks and converted to gels on demand.
One-Step Synthesis of Photoaffinity Probes for Live-Cell MS-Based Proteomics
Fallon, David J.,Lehmann, Stephanie,Chung, Chun-wa,Phillipou, Alex,Eberl, Christian,Fantom, Ken G. M.,Zappacosta, Francesca,Patel, Vipulkumar K.,Bantscheff, Marcus,Schofield, Christopher J.,Tomkinson, Nicholas C. O.,Bush, Jacob T.
supporting information, p. 17880 - 17888 (2021/09/16)
We present a one-step Ugi reaction protocol for the expedient synthesis of photoaffinity probes for live-cell MS-based proteomics. The reaction couples an amine affinity function with commonly used photoreactive groups, and a variety of handle functionalities. Using this technology, a series of pan-BET (BET: bromodomain and extra-terminal domain) selective bromodomain photoaffinity probes were obtained by parallel synthesis. Studies on the effects of photoreactive group, linker length and irradiation wavelength on photocrosslinking efficiency provide valuable insights into photoaffinity probe design. Optimal probes were progressed to MS-based proteomics to capture the BET family of proteins from live cells and reveal their potential on- and off-target profiles.
Rapid and Selective Labeling of Endogenous Transmembrane Proteins in Living Cells with a Difluorophenyl Ester Affinity-Based Probe
Chan, Hsin-Ju,Lin, Xin-Hui,Fan, Syuan-Yun,Ru Hwu, Jih,Tan, Kui-Thong
, p. 3416 - 3420 (2020/10/02)
The long-term stability of affinity-based protein labeling probes is crucial to obtain reproducible protein labeling results. However, highly stable probes generally suffer from low protein labeling efficiency and pose significant challenges when labeling low abundance native proteins in living cells. In this paper, we report that protein labeling probes based on an ortho-difluorophenyl ester reactive module exhibit long-term stability in DMSO stock solution and aqueous buffer, yet they can undergo rapid and selective labeling of native proteins. This novel electrophile can be customized with a wide range of different protein ligands and is particularly well-suited for the labeling and imaging of transmembrane proteins. With this probe design, the identity and relative levels of basal and hypoxia-induced transmembrane carbonic anhydrases were revealed by live cell imaging and in-gel fluorescence analysis. We believe that the extension of this difluorophenyl ester reactive module would allow for the specific labeling of various endogenous membrane proteins, facilitating in-depth studies of their distribution and functions in biological processes.
Introducing aldehyde functionality to proteins using ligand-directed affinity labeling
Fung, Yi Man Eva,Huang, Yiran,Li, Xiaoyu,Peng, Jianzhao,Song, Yinan,Xiong, Feng
supporting information, p. 6134 - 6137 (2020/06/10)
Aldehyde is a versatile chemical handle for protein modification. Although many methods have been developed to label proteins with aldehyde, target-specific methods amenable to endogenous proteins are limited. Here, we report a simple affinity probe strategy to introduce aldehydes to native proteins. Notably, the probe contains a latent aldehyde functionality that is only exposed upon target binding, thereby enabling a one-pot labeling procedure.
Cell surface clicking of antibody-recruiting polymers to metabolically azide-labeled cancer cells
Uvyn, Annemiek,De Coen, Ruben,De Wever, Olivier,Deswarte, Kim,Lambrecht, Bart N.,De Geest, Bruno G.
, p. 10952 - 10955 (2019/09/18)
Triggering antibody-mediated innate immune mechanisms to kill cancer cells is an attractive therapeutic avenue. In this context, recruitment of endogenous antibodies to the cancer cell surface could be a viable alternative to the use of monoclonal antibodies. We report on antibody-recruiting polymers containing multiple antibody-binding hapten motifs and cyclooctynes that can covalently conjugate to azides introduced onto the glycocalyx of cancer cells by metabolic labeling with azido sugars.
A Chemical Probe for Protein Crotonylation
Bos, Jeffrey,Muir, Tom W.
supporting information, p. 4757 - 4760 (2018/04/16)
Protein lysine crotonylation has emerged as an important post-translational modification (PTM) in the regulation of gene transcription through epigenetic mechanisms. Here we introduce a chemical probe, based on a water-soluble phosphine warhead, which reacts with the crotonyl modification. We show that this reagent is complementary to antibody-based tools allowing detection of endogenous cellular proteins such as histones carrying the crotonylation PTM. The tool is also used to show that the histone acylation activity of the transcriptional coactivator, p300, can be activated by pre-existing lysine crotonylation through a positive feedback mechanism. This reagent provides a versatile and sensitive probe for the analysis of this PTM.
Specific fluorescence labeling of target proteins by using a ligand-4-azidophthalimide conjugate
Chiba, Kosuke,Asanuma, Miwako,Ishikawa, Minoru,Hashimoto, Yuichi,Dodo, Kosuke,Sodeoka, Mikiko,Yamaguchi, Takao
, p. 8751 - 8754 (2017/08/10)
We herein propose a simple affinity-labeling method using a ligand-4-azidophthalimide (AzPI) conjugate. As a proof of concept, we show that two different ligand-AzPI conjugates enabled highly specific fluorescence labeling of their individual target proteins even in crude cell lysates. This method was also applied to label endogenous target proteins inside living cells.
A covalent G-site inhibitor for glutathione S-transferase Pi (GSTP1-1)
Shishido, Yuko,Tomoike, Fumiaki,Kimura, Yasuaki,Kuwata, Keiko,Yano, Takato,Fukui, Kenji,Fujikawa, Haruka,Sekido, Yoshitaka,Murakami-Tonami, Yuko,Kameda, Tomoshi,Shuto, Satoshi,Abe, Hiroshi
supporting information, p. 11138 - 11141 (2017/10/17)
We herein report the first covalent G-site-binding inhibitor for GST, GS-ESF (1), which irreversibly inhibited the GSTP1-1 function. LC-MS/MS and X-ray structure analyses of the covalently linked GST-inhibitor complex suggested that 1 reacted with Tyr108 of GSTP1-1. The mechanism of covalent bond formation was discussed based on MD simulation results.
Affinity labeling with 4-azidophthalimide (AzPI): Relation between labeling rate and fluorescence intensity
Chiba, Kosuke,Hashimoto, Yuichi,Yamaguchi, Takao
, p. 994 - 996 (2018/10/31)
We recently developed 4-azidophthalimide (AzPI) as a compact fluorogenic photoreactive tag that can be attached to ligands to achieve selective fluorescence labeling of target proteins even in the presence of a large excess of non-target proteins. To further establish the utility of the AzPI tag, we focused here on streptavidin labeling with biotin–AzPI conjugates, and evaluated the relation between the amount of covalently labeled streptavidin (labeling rate) and fluorescence intensity. The labeling rate was proportional to the fluorescence intensity under standardized photo-irradiation conditions. Prolongation of the photo-irradiation time led to a marked increase in the labeling rate, but this was accompanied by a gradual decrease in the fluorescence intensity, which appeared to be due at least in part to photo-induced degradation of the target streptavidin. These findings should be helpful for achieving sensitive fluorescence detection of target proteins by using the AzPI tag.
