ABSTRACT
We studied the specific labeling of streptavidin using the modular method for affinity labeling (MoAL) that we developed based on a catalytic amide-forming reaction using 2-chloro-4,6-dimethoxy-1,3,5-triazine (CDMT) and a tertiary amine catalyst. The primary structures of avidin and streptavidin are significantly different from each other, and streptavidin does not possess an acidic amino acid equivalent to Asp108 of avidin, which is the target acidic amino acid that was labeled using MoAL. However, using biotinylated modular ligand catalysts (MLC) originally designed for labeling avidin, the labeling of streptavidin was found to successfully proceed at Glu51, which is located in a different region. The present study indicates that MoAL is readily applicable to protein labeling without a precise design for MLC. The most important factor for the design of MLC is to ensure that the linker is of sufficient length to connect the ligands to a catalytic site.
Subject(s)
Amides/chemistry , Streptavidin/chemistry , Affinity Labels/chemistry , Amino Acid Sequence , Avidin/chemistry , Biotinylation , Ligands , Models, Molecular , Molecular Sequence Data , Spectrometry, Mass, Matrix-Assisted Laser Desorption-IonizationABSTRACT
We studied 1,3,5-triazine-based amide-forming reactions that are mediated or catalyzed by various tert-amines. The representative tert-amine was trimethylamine, which has amido, 1,2,3-triazolyl, aryl, and alkyl linkers. It was found that electron-deficient aryl and heteroaryl linkers, particularly 1,2,3-triazolyl linkers, are superior. On the basis of our findings, we synthesized ligand catalysts, including a 1,2,3-triazolyl linker that connects a protein ligand to a trimethylamine moiety, and found that fluorescent-labeling of a targeting protein using the ligand catalysts proceeded in good yields.
Subject(s)
Amides/chemistry , Amines/chemistry , Methylamines/chemistry , Triazines/chemistry , Catalysis , Ligands , Molecular StructureABSTRACT
We studied the specific labeling of avidin with biotinylated modular ligand catalysts via MoAL, which we recently established. The labeling yield was found to depend on the linker length connecting the catalytic site to biotin in the modular ligand catalyst 1, and the maximum yield was obtained with 1d possessing octamethylene linker. The labeling reaction reached a maximum rate with only 4 equiv of the ligand catalyst. Presumably, all the subunits of avidin with homotetrameric structure formed a stable complex with 4 equiv of the catalyst because of the extremely high affinity. The ligand catalyst bound to avidin first catalyzed N-triazinylation of the ε-amino group of Lys111, and the resulting regenerated catalyst then catalyzed the reaction of Asp108 and CDMT.
Subject(s)
Affinity Labels/chemistry , Avidin/chemistry , Biotin/chemistry , Biotinylation , Catalysis , Catalytic Domain , Ligands , Methods , Structure-Activity RelationshipABSTRACT
A modular methodology for affinity labeling, in which three essential elements generally constituting affinity probes are prepared separately as individual molecules, has been developed based on a catalytic amidation.
