- Optical Manipulation of Subcellular Protein Translocation Using a Photoactivatable Covalent Labeling System
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The photoactivatable chemically induced dimerization (photo-CID) technique for tag-fused proteins is one of the most promising methods for regulating subcellular protein translocations and protein–protein interactions. However, light-induced covalent protein dimerization in living cells has yet to be established, despite its various advantages. Herein, we developed a photoactivatable covalent protein-labeling technology by applying a caged ligand to the BL-tag system, a covalent protein labeling system that uses mutant β-lactamase. We further developed CBHD, a caged protein dimerizer, using caged BL-tag and HaloTag ligands, and achieved light-induced protein translocation from the cytoplasm to subcellular regions. In addition, this covalent photo-CID system enabled quick protein translocation to a laser-illuminated microregion. These results indicate that the covalent photo-CID system will expand the scope of CID applications in the optical manipulation of cellular functions.
- Kowada, Toshiyuki,Arai, Keisuke,Yoshimura, Akimasa,Matsui, Toshitaka,Kikuchi, Kazuya,Mizukami, Shin
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supporting information
p. 11378 - 11383
(2021/04/09)
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- Elucidating target specificity of the taccalonolide covalent microtubule stabilizers employing a combinatorial chemical approach
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The taccalonolide microtubule stabilizers covalently bind β-tubulin and overcome clinically relevant taxane resistance mechanisms. Evaluations of the target specificity and detailed drug–target interactions of taccalonolides, however, have been limited in part by their irreversible target engagement. In this study, we report the synthesis of fluorogenic taccalonolide probes that maintain the native biological properties of the potent taccalonolide, AJ. These carefully optimized, cell-permeable probes outperform commercial taxane-based probes and enable direct visualization of taccalonolides in both live and fixed cells with dramatic microtubule colocalization. The specificity of taccalonolide binding to β-tubulin is demonstrated by immunoblotting, which allows for determination of the relative contribution of key tubulin residues and taccalonolide moieties for drug–target interactions by activity-based protein profiling utilizing site-directed mutagenesis and computational modeling. This combinatorial approach provides a generally applicable strategy for investigating the binding specificity and molecular interactions of covalent binding drugs in a cellular environment.
- Du, Lin,Ramachandran, Karthik,Risinger, April L.,Yee, Samantha S.
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- Facile Large-Scale Synthesis of 5- and 6-Carboxyfluoresceins: Application for the Preparation of New Fluorescent Dyes
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A series of fluorescein dyes have been prepared from a common precursor through a very simple synthetic procedure, giving access to important precursors for fluorescent probes. The method has proven an efficient access to regioisomerically pure 5- and 6-carboxyfluoresceins on a large scale, in good yields, and with high regioisomeric purity. Furthermore, we have applied the method to the development of a new type of mixed fluorescein derivatives of 5-carboxyfluorescein. We have demonstrated the scope of the procedure by synthesizing a new type of double chromophore within the fluoro-Jade family.
- Hammershoj, Peter,Kumar, E. K. Pramod,Harris, Pernille,Andresen, Thomas L.,Clausen, Mads H.
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p. 7301 - 7309
(2015/11/25)
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- METHOD FOR THE PREPARATION OF INTERMEDIATES FOR CARBOXY-FLUORESCEINS AND NOVEL CARBOXY-FLUORESCEIN
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The invention provides a method for the preparation of regioisomerically pure intermediates which are useful for the preparation of carboxy-fluorescein-type compounds. Such compounds have broad applications within bio-conjugation and/or fluorescent imagin
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Page/Page column 8; 20; 21
(2015/11/27)
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- Rose Bengal analogs and vesicular glutamate transporters (VGLUTs)
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Vesicular glutamate transporters (VGLUTs) allow the loading of presynaptic glutamate vesicles and thus play a critical role in glutamatergic synaptic transmission. Rose Bengal (RB) is the most potent known VGLUT inhibitor (K i 25 nM); therefore we designed, synthesized and tested in brain preparations, a series of analogs based on this scaffold. We showed that among the two tautomers of RB, the carboxylic and not the lactonic form is active against VGLUTs and generated a pharmacophore model to determine the minimal structure requirements. We also tested RB specificity in other neurotransmitter uptake systems. RB proved to potently inhibit VMAT (Ki 64 nM) but weakly VACHT (Ki >9.7 μM) and may be a useful tool in glutamate/acetylcholine co-transmission studies.
- Pietrancosta, Nicolas,Kessler, Albane,Favre-Besse, Franck-Cyril,Triballeau, Nicolas,Quentin, Thomas,Giros, Bruno,Mestikawy, Salah El,Acher, Francine C.
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experimental part
p. 6922 - 6933
(2010/10/19)
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- Preparation of 5- and 6-carboxyfluorescein
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Condensation of resorcinol with 4-carboxyphthalic anhydride in methanesulfonic acid gave a mixture of 5- and 6-carboxyfluorescein stereoisomers. These were separated by recrystallization from methanol- or ethanol-hexane to give 5- and 6-carboxyfluorescein, each in over 98% purity.
- Ueno, Yuichiro,Jiao, Guan-Sheng,Burgess, Kevin
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p. 2591 - 2593
(2007/10/03)
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