An Amphibious Bifunctional Probe for Protein Chemical Cross-Linking

Chemical cross-linking mass spectrometry (CXMS) has emerged as a powerful and well-established method for probing protein structure, conformational dynamics, and protein-protein interactions, particularly in cases where classical high-resolution techniques face intrinsic limitations. The development of new cross-linking reagents with defined reactivity, activation, and compatibility with native conditions remains essential for expanding the scope and reliability of CXMS analyses. Here, we report the design and synthesis of two novel bis-hypervalent iodine reagents, called Togni cross-linking reagents TR1 and TR2, which function as efficient covalent protein cross-linkers. These reagents feature hypervalent iodine-fluoroalkyl motifs on both termini, enabling intra- and intermolecular cross-link formation. They selectively target aromatic amino acids and cysteine residues and can be activated either by ascorbate or through redox activation by thiol groups of cysteines. Moreover, these bifunctional probes can undergo transformation to form acyl fluoride, acting as a reactive acylator toward nucleophilic amino acid residues, in particular lysines. The performance of both reagents and activation modes is demonstrated on a set of structurally and functionally diverse proteins, including apomyoglobin/holomyoglobin and the small GTPase RHOA. Together, these results establish Togni cross-linking agents as versatile additions to the CXMS toolbox and highlight the potential of amphibious bis-hypervalent iodine-fluoroalkyl chemistry for studying protein structure and dynamics under mild aqueous conditions. Data is accessible via the ProteomeXchange server with the data set identifier PXD074784.