oxSTEF reagents are tunable and versatile electrophiles for selective disulfide-rebridging of native proteins

03 October 2022, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

Site-selective disulfide rebridging has emerged as a powerful strategy to modulate structural and functional properties of proteins. Here, we introduce a novel class of electrophilic reagents, designated oxSTEF, that demonstrate excellent efficiency in disulfide rebridging via double thiol-exchange. The oxSTEF reagents are prepared using an efficient synthetic sequence which may be diverted to obtain a range of structural derivatives. We demonstrate highly selective rebridging of cyclic peptides and native proteins, such as human growth hormone, and absence of cross-reactivity with other nucleophilic amino acid residues. The oxSTEF-conjugates undergo glutathione-mediated disintegration under tumor relevant glutathione concentrations, which highlights the potential for use in targeted drug delivery. Finally, the α-dicarbonyl motif of the oxSTEF reagents enables “second phase” oxime ligation which furthermore increases the thiol-stability of the conjugates significantly.

Keywords

Bioconjugation
disulfide rebridging
electrophiles
native proteins
antibodies

Supplementary materials

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Supporting Information
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Supplementary Figures Methods Compound characterization data Copies of NMR spectra
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