The Photocatalyzed Thiol‐ene reaction: A New Tag to Yield Fast, Selective and reversible Paramagnetic Tagging of Proteins

Abstract

Paramagnetic restraints have been used in biomolecular NMR for the last three decades to elucidate and refine biomolecular structures, but also to characterize protein-ligand interactions. A common technique to generate such restraints in proteins, which do not naturally contain a (paramagnetic) metal, consists in the attachment to the protein of a lanthanide-binding-tag (LBT). In order to design such LBTs, it is important to consider the efficiency and stability of the conjugation, the geometry of the complex (conformational exchanges and coordination) and the chemical inertness of the ligand. Here we describe a photocatalyzed thiol-ene reaction for the cysteine-selective paramagnetic tagging of proteins. As a model, we designed an LBT with a vinyl-pyridine moiety which was used to attach our tag to the protein GB1 in fast and irreversible fashion. Our tag T1 yields magnetic susceptibility tensors of significant size with different lanthanides and has been characterized using NMR and relaxometry measurements.

Rigid, Highly Reactive and Stable DOTA‐like Tags Containing a Thiol‐Specific Phenylsulfonyl Pyridine Moiety for Protein Modification and NMR Analysis**

“…The reactivity of these DOTA-like tags with a solvent exposed thiol group in a protein was analyzed in the 15 N labeled ubiquitin G47C mutant by the 2D 15 N-HSQC spectrum at Scheme 1. Site-specific labeling of proteins via thioether bond formation between the 4-(phenylsulfonyl) pyridine tag and the solvent exposed thiol group in a protein.…”

“…PCSs of backbone amide protons were calculated by the chemical shift differences between the paramagnetic and diamagnetic samples as determined in the 15 N-HSQC spectra. The assignment of the paramagnetic species was assisted by the fact that 1 H and 15 N dimensions for the backbone amide groups exhibit similar chemical shift changes, because the proton and nitrogen atoms of each amide group have similar distance and orientation with respect to the paramagnetic center especially for the nucleic spins further way from the paramagnetic center. The T2-Ln tagged ubiquitin and GB1 presented overall narrower NMR signals than the T1-Ln tagged proteins, and only one single set of paramagnetic resonances were observed for all residues in T2-Ln tagged ubiquitin.…”

“…The widely used maleimide moiety in protein modifications is not suitable for NMR study, because the protein-tag conjugate generated by the maleimide derivatives produces a new stereogenic center and it generates more than one paramagnetic species in the NMR spectra. [12] Alternative thiol-ene addition reaction without generation of a stereogenic carbon center was proposed, but the high flexibility of two-methylene tether between the protein and the tag average magnetic anisotropy as determined in the protein conjugates, [12][13][14][15] resulting small PCSs and RDCs. An interesting reaction between 4-phenyl(or methyl) sulfonyl pyridine with a thiol group in generation of a thioether bond was reported for site-specific modification of proteins, [16][17] and this reaction is thiol-specific and only proceeds in the aqueous solution, [16] which is highly suitable for protein modifications under mild conditions.…”

Development of a Borane–(Meth)acrylate Photo‐Click Reaction

“…[4] In particular,thiol-ene [5][6][7] and thiol-yne [8] reactions have been employed in photopolymerizations.T hey proceed by as tep-growth mechanism involving the radical propagation by addition of photogenerated thiyl radicals-using atype Ior type II photoinitiator-onto av inyl monomer,f ollowed by achain-transfer reaction (Figure 1). Thiol-ene reactions have for example recently been used for the synthesis of hydrogels, [9][10][11][12] bioorganic-functionalized materials, [13,14] polymerdispersed liquid-crystalline phases, [15,16] 3D-printing, [17][18][19] telechelic polyethylenes, [20] etc. However,i ft hiol-ene and -yne reactions have been adopted by many academics,t hiols often smell bad, and are generally too unstable for industrial applications,inparticular in (meth)acrylate formulations because of the competitive Michael addition.…”

Development of a Borane–(Meth)acrylate Photo‐Click Reaction

“…They proceed by a step‐growth mechanism involving the radical propagation by addition of photogenerated thiyl radicals—using a type I or type II photoinitiator—onto a vinyl monomer, followed by a chain‐transfer reaction (Figure 1). Thiol‐ene reactions have for example recently been used for the synthesis of hydrogels, [9–12] bioorganic‐functionalized materials, [13, 14] polymer‐dispersed liquid‐crystalline phases, [15, 16] 3D‐printing, [17–19] telechelic polyethylenes, [20] etc.…”