Site‐Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines

Canovas, Coline; Moreau, Mathieu; Bernhard, Claire; Oudot, Alexandra; Guillemin, M.; Denat, Franck; Gonçalves, Victor

doi:10.1002/ange.201806053

Cited by 10 publications

(1 citation statement)

References 37 publications

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“…This translates into a perfect homogeneity of the obtained conjugate and avoids any impairment of the immunoreactivity. Although this combination of site-specific grafting and MOMIP has been used on a few simple proteins, 27,28 it has never been applied to full-length immunoglobulins. In the study at hand, we produced a PET/NIRF MOMIP compatible with site-specific conjugation, bearing the DFO chelatorfor 89 Zr chelationthe NIR fluorophore IRDye800CW and a bicyclononyne (BCN) for SPAAC-based bioconjugation.…”

Section: Introductionmentioning

confidence: 99%

Site-Specific, Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bimodal PET/NIRF Imaging of HER2-Positive Tumors

et al. 2022

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Because positron emission tomography (PET) and optical imaging are very complementary, the combination of these two imaging modalities is very enticing in the oncology field. Such bimodal imaging generally relies on imaging agents bearing two different imaging reporters. In the bioconjugation field, this is mainly performed by successive random conjugations of the two reporters on the protein vector, but these random conjugations can alter the vector properties. In this study, we aimed at abrogating the heterogeneity of the bimodal imaging immunoconjugate and mitigating the impact of multiple random. A trivalent platform bearing a DFO chelator for 89 Zr labeling, a NIR fluorophore -IRDye800CWand a bioconjugation handle was synthesized. This bimodal probe was site-specifically grafted to trastuzumab via glycan engineering. This new bimodal immunoconjugate was then investigated in terms of radiochemistry, in vitro and in vivo, and compared to the clinically relevant random equivalent. In vitro and in vivo, our strategy provides several improvements over the current clinical standard. The combination of site-specific conjugation with the monomolecular platform reduced the heterogeneity of the final immunoconjugate, improved the resistance of the fluorophore toward radiobleaching and reduced the non-specific uptake in the spleen and liver compared to the standard random immunoconjugate. To conclude, the strategy developed is very promising for the synthesis of better defined dual labeled immunoconjugates, although there is still room for improvement. Importantly, this conjugation strategy is highly modular and could be used for the synthesis of a wide range of dual labeled immunoconjugates.

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Section: Introductionmentioning

confidence: 99%

Site-Specific, Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bimodal PET/NIRF Imaging of HER2-Positive Tumors

et al. 2022

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Bridge‐Clamp Bis(tetrazine)s with [N]₈ π‐Stacking Interactions and Azido‐s‐Aryl Tetrazines: Two Classes of Doubly Clickable Tetrazines

et al. 2019

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Click chemistry at a tetrazine core is useful for bioorthogonal labeling and crosslinking. Introduced here are two new classes of doubly clickable s‐aryl tetrazines synthesized by Cu‐catalyzed cross‐coupling. Homocoupling of o‐brominated s‐aryl tetrazines leads to bis(tetrazine)s structurally characterized by tetrazine cores arranged face‐to‐face. [N]8 π‐stacking interactions are essential to the conformation. Upon inverse electron demand Diels–Alder (iEDDA) cycloaddition, the bis(tetrazine)s produce a unique staple structure. The o‐azidation of s‐aryl tetrazines introduces a second proximal intermolecular clickable function that leads to double click chemistry opportunities. The stepwise introduction of fluorophores and then iEDDA cycloaddition, including bioconjugation to antibodies, was achieved on this class of tetrazines. This method extends to (thio)etherification, phosphination, trifluoromethylation and the introduction of various bioactive nitrogen‐based heterocycles.

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Contemporary Approaches for Site‐Selective Dual Functionalization of Proteins

Kuan

Weil

2021

Angewandte Chemie

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Site‐selective protein functionalization serves as an invaluable tool for investigating protein structures and functions in complicated cellular environments and accomplishing semi‐synthetic protein conjugates such as traceable therapeutics with improved features. Dual functionalization of proteins allows the incorporation of two different types of functionalities at distinct location(s), which greatly expands the features of native proteins. The attachment and crosstalk of a fluorescence donor and an acceptor dye provides fundamental insights into the folding and structural changes of proteins upon ligand binding in their native cellular environments. Moreover, the combination of drug molecules with different modes of action, imaging agents or stabilizing polymers provides new avenues to design precision protein therapeutics in a reproducible and well‐characterizable fashion. This review aims to give a timely overview of the recent advancements and a future perspective of this relatively new research area. First, the chemical toolbox for dual functionalization of proteins is discussed and compared. The strengths and limitations of each strategy are summarized in order to enable readers to select the most appropriate method for their envisaged applications. Thereafter, representative applications of these dual‐modified protein bioconjugates benefiting from the synergistic/additive properties of the two synthetic moieties are highlighted.

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Site‐Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines

Cited by 10 publications

References 37 publications

Site-Specific, Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bimodal PET/NIRF Imaging of HER2-Positive Tumors

Site-Specific, Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bimodal PET/NIRF Imaging of HER2-Positive Tumors

Bridge‐Clamp Bis(tetrazine)s with [N]₈ π‐Stacking Interactions and Azido‐s‐Aryl Tetrazines: Two Classes of Doubly Clickable Tetrazines

Contemporary Approaches for Site‐Selective Dual Functionalization of Proteins

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Site‐Specific Dual Labeling of Proteins on Cysteine Residues with Chlorotetrazines

Cited by 10 publications

References 37 publications

Site-Specific, Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bimodal PET/NIRF Imaging of HER2-Positive Tumors

Site-Specific, Platform-Based Conjugation Strategy for the Synthesis of Dual-Labeled Immunoconjugates for Bimodal PET/NIRF Imaging of HER2-Positive Tumors

Bridge‐Clamp Bis(tetrazine)s with [N]8 π‐Stacking Interactions and Azido‐s‐Aryl Tetrazines: Two Classes of Doubly Clickable Tetrazines

Contemporary Approaches for Site‐Selective Dual Functionalization of Proteins

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Product

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Bridge‐Clamp Bis(tetrazine)s with [N]₈ π‐Stacking Interactions and Azido‐s‐Aryl Tetrazines: Two Classes of Doubly Clickable Tetrazines