Abstract
Site-selective bioconjugation of cysteine-containing peptides and proteins is currently achieved via a maleimide–thiol reaction (Michael addition). When maleimide-functionalized chelators are used and the resulting bioconjugates are subsequently radiolabeled, instability has been observed both during radiosynthesis and post-injection in vivo, reducing radiochemical yield and negatively impacting performance. Recently, a phenyloxadiazolyl methylsulfone derivative (PODS) was proposed as an alternative to maleimide for the site-selective conjugation and radiolabeling of proteins, demonstrating improved in vitro stability and in vivo performance. Therefore, we have synthesized two novel PODS-bearing bifunctional chelators (NOTA-PODS and NODAGA-PODS) and attached them to the EGFR-targeting affibody molecule ZEGFR:03115. After radiolabeling with the aluminum fluoride complex ([18F]AlF), both conjugates showed good stability in murine serum. When injected in high EGFR-expressing tumor-bearing mice, [18F]AlF-NOTA-PODS-ZEGFR:03115 and [18F]AlF-NODAGA-PODS-ZEGFR:03115 showed similar pharmacokinetics and a specific tumor uptake of 14.1 ± 5.3% and 16.7 ± 4.5% ID/g at 1 h post-injection, respectively. The current results are encouraging for using PODS as an alternative to maleimide-based thiol-selective bioconjugation reactions.
Highlights
Reactive sulfhydryl groups of cysteine residues are attractive sites for the chemical attachment of dyes, chelators, or drugs to biomolecules
The natural low abundance of accessible and reduced cysteine residues prevents the formation of heterogeneous mixtures of the bioconjugates
Instead of the conventional maleimide, thiol-reactive bifunctional chelators for the conjugation to the cysteine-containing small protein were developed by functionalizing the macrocycles with Phenyloxadiazolyl methylsulfone (PODS)
Summary
Reactive sulfhydryl groups of cysteine residues are attractive sites for the chemical attachment of dyes, chelators, or drugs to biomolecules. Targeting cysteine residues on biomolecules has several key benefits. The presence of the thiol group, a highly reactive nucleophile, allows for a fast and selective reaction at physiological pH. The natural low abundance of accessible and reduced cysteine residues prevents the formation of heterogeneous mixtures of the bioconjugates. A customized site-specific incorporation of cysteine residues into a biomolecule can be achieved [1]. Different classes of thiol-targeting electrophilic compounds have been used, with maleimides being the most common choice [2,3,4]. Maleimide conjugates have shown instability mostly as a consequence
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