Capture-Tag-Release: A Strategy for Small Molecule Labeling of Native Enzymes.
A strategy for labeling native enzymes in a manner that preserves their activity is reported: capture-tag-release (CTR). Key to this approach is the small molecule CTR probe that contains an enzyme inhibitor, benzophenone crosslinker, and aryl phosphine ester. After UV-derived capture of the enzyme, addition of an azide-containing tag triggers a Staudinger ligation that labels the enzyme. A further consequence of the Staudinger ligation is fragmentation of the CTR probe, thus releasing the inhibitor and restoring enzymatic activity. As a proof-of-principle, the CTR strategy was applied to the hydrolase β-galactosidase. The enzyme was efficiently labeled with biotin, and the kinetic data for the biotinylated enzyme were comparable to those for unlabeled β-galactosidase. The CTR probe exhibits excellent targeting specificity, as it selectively labeled β-galactosidase in a complex protein mixture.
Department of Chemistry and Biochemistry, Fairfield University, 1073 N. Benson Road, Fairfield, CT, 06824, USA. avandyke@fairfield.edu.
Aaron R
AR
Van Dyke
Department of Chemistry and Biochemistry, Fairfield University, 1073 N. Benson Road, Fairfield, CT, 06824, USA.
Lily S
LS
Etemad
Department of Chemistry and Biochemistry, Fairfield University, 1073 N. Benson Road, Fairfield, CT, 06824, USA.
Michael J
MJ
Vessicchio
Department of Chemistry and Biochemistry, Fairfield University, 1073 N. Benson Road, Fairfield, CT, 06824, USA.
George A
GA
Naclerio
Department of Chemistry and Biochemistry, Fairfield University, 1073 N. Benson Road, Fairfield, CT, 06824, USA.
Victoria
V
Jedson
D001710:Biotin / Q000032:analysis / Q000737:chemistry; D007700:Kinetics; D015394:Molecular Structure; D054852:Small Molecule Libraries / Q000032:analysis* / Q000138:chemical synthesis / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*
D054852:Small Molecule Libraries; D001710:Biotin; D001616:beta-Galactosidase
Staudinger ligation; click chemistry; hydrolases; photoaffinity labeling; protein modifications