Fluorine-18 and Radiometal Labeling of Biomolecules via Disulfide Rebridging

Abstract

Biomolecules labeled with positron-emitting radionuclides like fluorine-18 or radiometals like copper-64 and zirconium-89 are increasingly employed in nuclear medicine for diagnosis purposes. Given the fragility and complexity of these compounds, their labeling requires mild conditions. Besides, it is essential to develop methods inducing minimal modification of the tertiary structure, as it is fundamental for the biological activity of such complex entities. Given these requirements, disulfide rebridging represents a promising possibility since it allows protein modification as well as conservation of the tertiary structure. In this context, we have developed an original radiofluorinated dibromopyridazine dione prosthetic group for labeling of disulfide-containing biomolecules via rebridging. We employed it to radiolabel octreotide, a somatostatin analogue, and to radiolabel fragment antigen binding (Fab) targeting programmed death-ligand 1 (PD-L1), whose properties were then evaluated in vitro and in vivo by positron emission tomography (PET) imaging. We next extended our strategy to the radiolabeling of cetuximab, a monoclonal antibody, with various radiometals commonly used in PET imaging (zirconium-89, copper-64) by developing various rebridging molecules bearing the appropriate chelators. The stabilities of the radiolabeled antibody conjugates were assessed in biological conditions.

Phenylpropynones as Selective Disulfide Rebridging Bioconjugation Reagents

“…These trends were also confirmed by density functional theory calculations performed by us, as well as by Costa, Villarasa, and co-workers . We thus decided to explore this “linchpin-type” reactivity of alkynones toward thiols in more detail, as we expected that the non-oxidative and atom economical cross-linking of thiols could be a valuable technology for the purpose of bioconjugation . By investigating various alkynone monomers, we found that the double addition of a thiol to simple alkynone reagents is indeed also feasible in aqueous buffered media.…”

“…The outcome of the model bioconjugation studies (Tables –) next prompted us to investigate the utility of the phenylpropyone reagents in disulfide rebridging in more complex biomolecules (Scheme ), , as an alternative to the well-known dibromomaleimide reagents . We were pleased to find that lipoic acid can be converted in high yield to 1,3-dithiane 14 by a one-pot reductive cleavage with the phosphine reagent TCEP and subsequent treatment with phenylpropynone 2 in an aqueous buffer.…”