62062-43-5Relevant articles and documents
Native FKBP12 engineering by ligand-directed tosyl chemistry: Labeling properties and application to photo-cross-linking of protein complexes in vitro and in living cells
Tamura, Tomonori,Tsukiji, Shinya,Hamachi, Itaru
, p. 2216 - 2226 (2012/03/12)
The ability to modify target "native" (endogenous) proteins selectively in living cells with synthetic molecules should provide powerful tools for chemical biology. To this end, we recently developed a novel protein labeling technique termed ligand-directed tosyl (LDT) chemistry. This method uses labeling reagents in which a protein ligand and a synthetic probe are connected by a tosylate ester group. We previously demonstrated its applicability to the selective chemical labeling of several native proteins in living cells and mice. However, many fundamental features of this chemistry remain to be studied. In this work, we investigated the relationship between the LDT reagent structure and labeling properties by using native FK506-binding protein 12 (FKBP12) as a target protein. In vitro experiments revealed that the length and rigidity of the spacer structure linking the protein ligand and the tosylate group have significant effects on the overall labeling yield and labeling site. In addition to histidine, which we reported previously, tyrosine and glutamate residues were identified as amino acids that are modified by LDT-mediated labeling. Through the screening of various spacer structures, piperazine was found to be optimal for FKBP12 labeling in terms of labeling efficiency and site specificity. Using a piperazine-based LDT reagent containing a photoreactive probe, we successfully demonstrated the labeling and UV-induced covalent cross-linking of FKBP12 and its interacting proteins in vitro and in living cells. This study not only furthers our understanding of the basic reaction properties of LDT chemistry but also extends the applicability of this method to the investigation of biological processes in mammalian cells.
Trifunctional Reagents for Derivatizing Sulfhydryl Groups
Finn, Frances M.,Yamanouchi, Keitaro,Titus, Gail,Hofmann, Klaus
, p. 152 - 168 (2007/10/02)
The syntheses of four trifunctional reagents for alkylating sulfhydryl groups in proteins are described: N-γ-maleimidobutyrylbiocytinyl-tyramine (compound I) and its sulfone, N-γ-maleimidobutyrylbiocytinyltyrosine (compound II), and Nα-4-maleimidobutyrylbiocytinamido-2'-ethane (compound III).Each reagent contains a maleimide function capable of reacting with SH groups, a p-hydroxyphenyl group that can be iodinated, and a "biotin handle" to facilitate purification of the derivatized proteins or peptides derived from them by biotin-avidin affinity chromatography.Detailed conditions for obtaining the pure di-iododervatives of the compounds have been developed.The biotin is attached to all the reagents via the ε-amino group of lysine (biocytin) to provide sufficient space for optimum binding to avidin.The half-times (t1/2) for dissociation of compound I from succinoyl avidin (36.7 days), its monoiodo (26.1 days) and di=iodo derivatives (21.4 days), and compound I sulfone (29.8 days), demonstrate that iodination does not significantly interfere with binding of the biotin residue to succinoyl avidin and that these reagents can be used effectively as affinity ligands> Remarkably, all the reagents can be iodinated without loss of their sulfhydryl alkylating capacity.Alkylation of highly purified human placental insulin receptor with the di-iodo derivatives of the reagents resulted in significant incorporation of 125I into the β-subunit of the receptor and the alkylation was prevented by prior exposure of the receptor to NEM.The advantages of these reagents over those previously available are that the parent molecules (1) are inexpensive to prepare, (2) are solids that can be stored indefinitely without degradation, (3) and can be radiolabeled to specific activity levels over seventy times higher with 125I than the specific activity available for 3H derivatives.
