Abstract
Linkers that enable the site-selective synthesis of chemically modified proteins are of great interest to the field of chemical biology. Homogenous bioconjugates often show advantageous pharmacokinetic profiles and consequently increased efficacy in vivo. Cysteine residues have been exploited as a route to site-selectively modify proteins, and many successfully approved therapeutics make use of cysteine directed conjugation reagents. However, commonly used linkers, including maleimide–thiol conjugates, are not stable to the low concentrations of thiol present in blood. Furthermore, only a few cysteine-targeting reagents enable the site-selective attachment of multiple functionalities: a useful tool in the fields of theranostics and therapeutic blood half-life extension. Herein, we demonstrate the application of the pyridazinedione motif to enable site-selective attachment of three functionalities to a protein bearing a single cysteine residue. Extending upon previously documented dual modification work, here we demonstrate that by exploiting a bromide leaving group as an additional reactive point on the pyridazinedione scaffold, a thiol or aniline derivative can be added to a protein, post-conjugation. Thiol cleavability appraisal of the resultant C–S and C–N linked thio-bioconjugates demonstrated C–S functionalized linkers to be cleavable and C–N functionalized linkers to be noncleavable when incubated in an excess of glutathione. The plug-and-play trifunctional platform was exemplified by attaching clinically relevant motifs: biotin, fluorescein, a polyethylene glycol chain, and a model peptide. This platform provides a rare opportunity to combine up to three functionalities on a protein in a site-selective fashion. Furthermore, by selecting the use of a thiol or an amine for functionalization, we provide unique control over linker cleavability toward thiols, allowing this novel linker to be applied in a range of physiological environments.
Highlights
Linkers that enable the site-selective synthesis of chemically modified proteins are of great interest to the field of chemical biology
We have reported the extensive use of bromopyridazinediones (Br PDs) and dibromopyridazinediones (DiBr PDs) in the fields of single cysteine modification and functional disulfide rebridging, respectively.[16,21−24] The pyridazinedione (PD) moiety has been shown to address many of the drawbacks associated with commonly employed Michael acceptors in the context of cysteine modification, providing a linker that is stable in serum for several days (i.e., PD-derived bioconjugates do not react with high concentrations of human serum albumin (HSA) and low concentrations of glutathione (GSH)).[25,26]
Br PDs have typically been used for single cysteine modification,[16] but it is thought that by employing DiBr PDs for this purpose, a further reactive position on the PD scaffold could be exploited for the modular synthesis of trifunctional bioconjugates (Figure 1)
Summary
Through the use of cysteine conjugation, and subsequent postconjugation functionalization, we demonstrate pyridazinedione derivatives as first-in-class linkers that can provide a platform for the efficient synthesis of chemically diverse bioconjugates that can host up to three functionalities. We exploit an additional reactive center on the conjugated pyridazinedione scaffold to allow postconjugation reactions with thiols and aniline derivatives. Offering a further level of thiol cleavability control may allow this platform to be extended to intracellular applications (e.g., ex vivo cellular imaging through bioconjugation to cellpenetrating peptides (CPPs)). Authors Calise Bahou − Department of Chemistry, University College London, WC1H OAJ London, United Kingdom Peter A. Szijj − Department of Chemistry, University College London, WC1H OAJ London, United Kingdom Richard J.
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