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Title: Site-selective azide incorporation into endogenous RNase A via a "chemistry" approach
Authors: Chen, X.
Henschke, L.
Wu, Q.
Muthoosamy, K.
Neumann, B.
Weil, T. 
Issue Date: 14-Jan-2013
Citation: Chen, X., Henschke, L., Wu, Q., Muthoosamy, K., Neumann, B., Weil, T. (2013-01-14). Site-selective azide incorporation into endogenous RNase A via a "chemistry" approach. Organic and Biomolecular Chemistry 11 (2) : 353-361. ScholarBank@NUS Repository.
Abstract: Site-selective labeling of endogenous proteins represents a major challenge in chemical biology, mainly due to the absence of unique reactive groups that can be addressed selectively. Recently, we have shown that surface-exposed lysine residues of two endogenous proteins and a peptide exhibit subtle changes in their individual reactivities. This feature allows the modification of a single residue in a highly site-selective fashion if kinetically controlled labeling conditions are applied. In order to broaden the scope of the "kinetically-controlled protein labeling" (KPL) approach and highlight additional applications, the water-soluble bioorthogonal reagent, biotin-TEO-azido-NHS (11), is developed which enables the site-selective introduction of an azido group onto endogenous proteins/peptides. This bioconjugation reagent features a biotin tag for affinity purification, an azido group for bioorthogonal labeling, a TEO (tetraethylene oxide) linker acting as a spacer and to impart water solubility and an N-hydroxysuccinimidyl (NHS) ester group for reacting with the exposed lysine residue. As a proof of concept, the native protein ribonuclease A (RNase A) bearing ten available lysine residues at the surface is furnished with a single azido group at Lys 1 in a highly site-selective fashion yielding azido-(K1)RNase A. The K1 site-selectivity is demonstrated by the combined application and interpretation of high resolution MALDI-ToF mass spectroscopy, tandem mass spectroscopy and extracted ion chromatography (XIC). Finally, the water soluble azide-reactive phosphine probe, rho-TEO-phosphine (21) (rho: rhodamine), has been designed and applied to attach a chromophore to azido-(K1)RNase A via Staudinger ligation at physiological pH indicating that the introduced azido group is accessible and could be addressed by other established azide-reactive bioorthogonal reaction schemes. © 2013 The Royal Society of Chemistry.
Source Title: Organic and Biomolecular Chemistry
ISSN: 14770520
DOI: 10.1039/c2ob26561c
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