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We describe furan as a triggerable ‘warhead’ for site-specific cross-linking using the actin and thymosin β4 (Tβ4)-complex as model of a weak and dynamic protein-protein interaction with known 3D structure...

Chemical cross-linking is well-established for investigating protein-protein interactions. Traditionally, photo cross-linking is used but is associated with problems of selectivity and UV toxicity in a biological context. We here describe, with live cells and under normal growth conditions, selective cross-linking of a furan-modified peptide ligand to its membrane-presented receptor with zero toxicity, high efficiency, and spatio-specificity. Furan-modified kisspeptin-10 is covalently coupled to its glycosylated membrane receptor, GPR54(KISS1R). This newly expands the applicability of furan-mediated cross-linking not only to protein-protein cross-linking but also to cross-linking in situ. Moreover, in our earlier reports on nucleic acid interstrand cross-linking, furan activation required external triggers of oxidation (via addition of N-bromo succinimide or singlet oxygen). In contrast, we here show, for multiple cell lines, the spontaneous endogenous oxidation of the furan moiety with concurrent selective cross-link formation. We propose that reactive oxygen species produced by NADPH oxidase (NOX) enzymes form the cellular source establishing furan oxidation.

Flexible in vitro translation (FIT) was used as a screening method to uncover a new methodology for peptide constraining based on the attack of a nucleophilic side-chain functionality onto an oxidized furylalanine side chain. A set of template peptides, each containing furylalanine as furan-modified amino acid and a nucleophilic residue (Cys, His, Lys, Arg, Ser, or Tyr), was produced through FIT. The translation mixtures were treated with N-bromosuccinimide (NBS) to achieve selective furan oxidation and subsequent MALDI analysis demonstrated Lys and Ser as promising residues for cyclisation. Solid-phase peptide synthesis (SPPS) was used to synthesize suitable amounts of material for further in-depth analysis and characterisation. It was found that in the case of the peptide containing lysine next to a furylalanine residue, a one-pot oxidation and reduction reaction leads to the generation of a cyclic peptide featuring a pyrrole moiety as cyclisation motif, resulting from the attack of the lysine side chain onto the oxidized furylalanine side chain. Structural evidence was provided via NMR and the generality of the methodology was explored. We hereby expand the scope of our previously developed furan-based peptide labeling and crosslinking strategy.

Unprotected peptides equipped with property enhancing moieties and a Cys(Scm) residue can be site-selectively conjugated to proteins via crossed-disulfide formation.

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