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

Nišavić, Marija; Wørmer, Gustav J.; Nielsen, Cecilie Marie; Jeppesen, Sofie; Palmfeldt, Johan; Poulsen, Thomas B.

doi:10.26434/chemrxiv-2022-7pkxq

Cited by 4 publications

(6 citation statements)

References 38 publications

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“…41 Other examples of rebridging reagents include organic arsenicals, 42 divinylpyrimidines, 43−45 arylene dipropiolonitriles (ADPN), 46 di(bromomethyl)pyridines, 47 diPODS, 48 or oxSTEF reagents. 49 Considering the promising characteristics of the rebridging approach and the scarcity of one-step site-specific methods for labeling of biomolecules, we aimed to (i) develop an original radiofluorinated prosthetic group for labeling via rebridging, (ii) employ this original radiofluorination method for the labeling of two model biomolecules, and (iii) expand the method to the labeling with radiometals. Dibromomaleimide derivatives have recently been used for radiolabeling of biomolecules with copper-64 and zirconium-89, 28 but, to our knowledge, radiofluorination via disulfide rebridging has never been reported before.…”

Section: ■ Introductionmentioning

confidence: 99%

“…Fragment antigen binding (Fab) derived from anti-PD-L1 IgG1 complement 4 (C4) was chosen as a model biomolecule since its biological properties have been previously analyzed by PET/CT in our group. 11,49 To that end, we selected the human non-small cell lung carcinoma (NSCLC) cell line H1975, considering its overexpression of PD-L1. Detailed procedures are provided in the Supporting Information (see Chapter V-Cell binding assay and PET imaging).…”

Section: ■ Introductionmentioning

confidence: 99%

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Fluorine-18 and Radiometal Labeling of Biomolecules via Disulfide Rebridging

Richard,

Martin Aubert,

Denis

et al. 2023

Bioconjugate Chem.

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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.

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Section: ■ Introductionmentioning

confidence: 99%

Section: ■ Introductionmentioning

confidence: 99%

Fluorine-18 and Radiometal Labeling of Biomolecules via Disulfide Rebridging

Richard,

Martin Aubert,

Denis

et al. 2023

Bioconjugate Chem.

View full text Add to dashboard Cite

show abstract

“…32 Recently an example of a one-carbon bridging reagent was also described, referred to as an oxSTEF motif, via two conjugate addition−elimination mechanisms. 33 Such linkers are desirable as they minimize the imposed distance extension between the two sulfur atoms. While this is unlikely to be of significance in large, structurally stable motifs such as antibodies, this is still a favorable design feature and may have greater significance when applied in the context of more structurally sensitive peptides or proteins.…”

Section: ■ Introductionmentioning

confidence: 99%

The Nitrile Bis-Thiol Bioconjugation Reaction

Patel,

Forte,

Bishop

et al. 2023

J. Am. Chem. Soc.

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Electron-poor aryl nitriles are promising reagents for bioconjugation due to their high electrophilicity and selectivity for reaction with thiols, albeit generally in a reversible manner. A transient species has previously been observed in such reactions, involving the addition of two thiols to the nitrile functional group, forming a tetrahedral amino dithioacetal (ADTA). In this work, the reaction of heteroaryl nitriles with bis-thiols is explored in an attempt to generate stable ADTAs, which could facilitate new bioconjugation protocols. By use of a 1,2-dithiol, or the incorporation of an electrophilic trap into the aryl nitrile design, the formation of stable products is achieved. The resultant "nitrile bis-thiol" (NBT) reaction is then explored in the context of protein modification, specifically to carry out antibody conjugation. By addition of these nitriles to the reduced disulfide bond of an antibody fragment, it is shown that, depending on the reagent design, cysteine-to-lysine transfer or disulfide bridged NBT products can be generated. Both represent site-selective conjugates and are shown to be stable when challenged with glutathione under physiological conditions and upon incubation in serum. Furthermore, the NBT reaction is tested in the more challenging context of a full antibody, and all four disulfide bonds are effectively modified by these new one-carbon bridging reagents. Overall, this reaction of heteroaryl-nitriles with bis-thiols is shown to be highly efficient and versatile, of tunable reversibility, and offers enticing prospects as a new addition to the toolbox of biocompatible "click"-type reactions.

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“…32 Recently an example of a one-carbon bridging reagent was also described, referred to as an oxSTEF motif, via two conjugate addition elimination mechanisms. 33 Such linkers are desirable as they minimise the imposed distance extension between the two sulfur atoms. Whilst this is unlikely to be of significance in large, structurally stable motifs such as antibodies, this is still a favourable design feature and may have greater significance when applied in the context of more structurally sensitive peptides or proteins.…”

Section: Introductionmentioning

confidence: 99%

The Nitrile Bis-Thiol Bioconjugation Reaction

Patel

Forte

Bishop

et al. 2023

Preprint

View full text Add to dashboard Cite

Electron-poor aryl nitriles are promising reagents for bioconjugation, due to their high electrophilicity and selectivity for reaction with thiols, albeit generally in a reversible manner. A transient species has previously been observed in such reactions, involving the addition of two thiols to the nitrile functional group forming a tetrahedral amino dithioacetal (ADTA). In this work, the reaction of heteroaryl nitriles with bis-thiols is explored in an attempt to generate stable ADTAs, which could facilitate new bioconjugation protocols. By use of a 1,2-dithiol, or the incorporation of an electrophilic trap into the aryl nitrile design, the formation of stable products is achieved. The resultant ‘nitrile bis-thiol’ (NBT) reaction is then explored in the context of protein modification, specifically to carry out antibody conjugation. By addition of these nitriles to the reduced disulfide bond of an antibody fragment it is shown that, depending on the reagent design, cysteine-to-lysine transfer or disulfide bridged NBT products can be generated. Both represent site-selective conjugates and are shown to be stable when challenged with glutathione under physiological conditions, and upon incubation in serum. Furthermore, the NBT reaction is tested on the more challenging context of a full antibody, and all four disulfide bonds are effectively modified by these new one-carbon bridging reagents. Overall, this reaction of heteroaryl-nitriles with bis-thiols is shown to be highly efficient and versatile, of tunable reversibility, and offers enticing prospects as a new addition to the toolbox of biocompatible ‘click’-type reactions.

show abstract

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

Cited by 4 publications

References 38 publications

Fluorine-18 and Radiometal Labeling of Biomolecules via Disulfide Rebridging

Fluorine-18 and Radiometal Labeling of Biomolecules via Disulfide Rebridging

The Nitrile Bis-Thiol Bioconjugation Reaction

The Nitrile Bis-Thiol Bioconjugation Reaction

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