Rigid, highly reactive and stable DOTA-like tags containing a thiol-specific phenylsulfonyl pyridine moiety for protein modification and NMR analysis.

Site specific labeling of paramagnetic NMR tags generate valuable structural restraints such as pseudocontact shifts (PCSs) and residual dipolar couplings (RDCs) that can be used to characterize structures, interactions and dynamics of protein-ligand or nucleic acid complexes. Herein, two single-armed DOTA-like tags, BrPSPy-DO3M( S )A-Ln and BrPSPy-6M-DO3M( S )A-Ln, each containing a thiol-specific reacting group, phenylsulfonyl pyridine moiety, are demonstrated as rigid, reactive and stable paramagnetic tags for protein labeling by formation of a reductive resistant thiol-ether bond between the protein and the tag. The introduction of Br neighboring the 4-phenylsulfone group in pyridine enhances the reactivity towards the solvent exposed thiol group in a protein, whereas the methyl group in the sixth position in pyridine increases the rigidity of the tag in the protein conjugates. The high performance and tunable reactivity and rigidity of these two tags can be added in the toolbox of paramagnetic tags suitable for the high-resolution measurements of biomolecules.