Site-Specific Conjugation of Peptides and Proteins via Rebridging of Disulfide Bonds Using the Thiol–Yne Coupling Reaction

Abstract

Herein, we describe an extension of our previously reported photomediated disulfide rebridging methodology to the conjugation of peptides and proteins. The methodology proved to be reproducible with various alkynes and different peptides. This study includes the first rebridging of the disulfide bond of a peptide through a thiol-yne reaction with a cyclooctyne. In all cases, the rebridging was proven by MS analyses and confirmed by the absence of olefinic protons on (1)H NMR spectra of the resulting products. Finally, this one-pot reduction thiol-yne conjugation was successfully applied to an antibody Fab fragment with a promising conversion, which set a good ground for the future syntheses of new protein and antibody conjugates.

Synthesis and Applications of Protein/Peptide-Polymer Conjugates

“…The pioneering work of Brocchini et al, who employed bis ‐sulfone functional PEG to modify immunotherapeutic protein interferon, was followed by contributions from the groups and Caddick and Baker, who introduced mono‐substituted (MSM) and di‐substituted maleimide (DSM) derivatives as irreversible and reversible linkers respectively. More recently, Caddick et al have also employed dibromopyridazinediones which have two additional sites for functionalization, conferring homo‐ or hetero‐bifunctional modification, and alkyne reagents have been employed for radical thiol‐yne chemistry at reduced disulfides. Haddleton, Davis and Wilson, have combined the efficiency of the DSM chemistry with the precision of RDRP and also introduced organic arsenicals as benign alternatives to DSM's, exhibiting comparable conjugation efficiency, reversibility and enhanced specificity.…”

Site‐Selective Disulfide Modification of Proteins: Expanding Diversity beyond the Proteome

“…A photochemical thiol‐yne coupling approach was recently reported . Commercially available bifunctional alkyne (Figure A, VI) can be used in the presence of a water‐soluble radical initiator, lithium phenyl‐2,4‐6‐trimethylphosphinate and irradiated with UV‐light (365 nm) to achieve disulfide rebridging to form a two‐carbon bridged bioconjugate.…”

“…Variation of terminal alkynes and peptides only improve the yield marginally (10–22 %). The approach can also be applied using cyclooctyne as a model for internal alkyne but similarly suffers low yield and a mixture of isomers are obtained which cannot be separated by chromatography . Further improvement in conjugation yields and purification needs to be achieved in order to stimulate broader applications.…”

Characterization of Disulfide Bond Rebridged Fab–Drug Conjugates Prepared Using a Dual Maleimide Pyrrolobenzodiazepine Cytotoxic Payload

“…Rebridging of the native interchain disulfide bond has been used as a strategy to prepare Fab–drug conjugates (FDCs) and ADCs . For example, Godwin and colleagues developed a rebridging conjugation approach using a bis‐sulfone linker–drug, in which interchain disulfide bonds in either full‐length antibodies or Fab fragments were first partially reduced, followed by bis‐alkylation to conjugate both thiols of the two native cysteines.…”

“…Caddick and colleagues contributed significantly over the past few years to the development of several rebridging approaches to prepare well‐defined FDCs and ADCs . Finally, rebridging of disulfide bonds through a thiol–yne reaction with a cyclooctyne has also been successfully applied to an antibody Fab …”