oxSTEF Reagents Are Tunable and Versatile Electrophiles for Selective Disulfide-Rebridging of Native Proteins

Site-selectivedisulfide rebridging has emerged as a powerful strategyto modulate the structural and functional properties of proteins.Here, we introduce a novel class of electrophilic reagents, designatedoxSTEF, that demonstrate excellent efficiency in disulfide rebridgingvia double thiol exchange. The oxSTEF reagents are prepared usingan efficient synthetic sequence which may be diverted to obtain arange of derivatives allowing for tuning of reactivity or steric bulk.We demonstrate highly selective rebridging of cyclic peptides andnative proteins, such as human growth hormone, and the absence ofcross-reactivity with other nucleophilic amino acid residues. TheoxSTEF conjugates undergo glutathione-mediated disintegration undertumor-relevant glutathione concentrations, which highlights theirpotential for use in targeted drug delivery. Finally, the alpha-dicarbonylmotif of the oxSTEF reagents enables second phaseoxime ligation, which furthermore increases the thiol stability ofthe conjugates significantly.

Automated summary

Site-selective disulfide rebridging is a powerful strategy for modulating protein structure and function. We introduce oxSTEF, a novel class of electrophilic reagents, which efficiently rebridge disulfide bonds via double thiol exchange. The oxSTEF reagents show highly selective rebridging of cyclic peptides and native proteins, as well as no cross-reactivity with other nucleophilic amino acid residues. Additionally, the oxSTEF conjugates disintegrate under tumor-relevant glutathione concentrations and its α-dicarbonyl motif enables second phase oxime ligation, significantly increasing the thiol stability of the conjugates.