oxSTEF Reagents Are Tunable and Versatile Electrophiles for Selective Disulfide-Rebridging of Native Proteins

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

doi:10.1021/acs.bioconjchem.3c00005

Cited by 7 publications

(10 citation statements)

References 46 publications

(92 reference statements)

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“…As seen in the in vivo biodistribution results (see SI, Figure 15A,B), accumulation of [ 18 F]-Fab in the tumors reached 0.58 ± 0.18%ID/cc and 0.23 ± 0.03%ID/cc 50 min and 4 h post injection, respectively. These results are in accordance with previous experiments carried out by Bouleau et al and Tran et al For instance, for the same Fab-labeled with fluorine-18 via a site-specific enzymatic reaction ([ 18 F]FPyOctA-Fab), the uptake was 0.47 ± 0.12%ID/cc 50 min post injection and 0.29 ± 0.12%ID/cc 4 h postinjection (for 23 μg of Fab). As seen in Figure , PET/CT images enabled the visualization of the PD-L1–positive H1975 xenografts 50 min and 4 h post injection (Figure A,B) and the specificity of [ 18 F]-Fab for H1975 tumors was demonstrated by the tumor/muscle and tumor/blood ratios, reaching respectively 5.34 ± 1.27 and 1.42 ± 0.32 after 4 h (Figure C,D).…”

Section: Resultssupporting

confidence: 93%

“…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. , 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: Resultsmentioning

confidence: 99%

“…Alkynes have also been employed for rebridging via thiol–yne coupling or via their incorporation in diethynyl phosphinates . Other examples of rebridging reagents include organic arsenicals, divinylpyrimidines, − arylene dipropiolonitriles (ADPN), di(bromomethyl)pyridines, diPODS, or oxSTEF reagents …”

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: Resultssupporting

confidence: 93%

Section: Resultsmentioning

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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“…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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“…Ketene dithioacetals are an attractive motif in organic synthesis with a wide range of reactivity (Scheme A) . In particular, ketene dithioacetals bearing an α-carbonyl group display biological activities, such as Leishmania donovani growth inhibition . In addition, the push–pull polarization of the double bond produced by the sulfur atoms and the carbonyl group makes these compounds versatile building blocks for the preparation of bioactive molecules or materials .…”

mentioning

confidence: 99%