Diethynyl Phosphinates for Cysteine‐Selective Protein Labeling and Disulfide Rebridging

Stieger, Christian E.; Franz, Luise; Körlin, Frieder; Hackenberger, Christian P. R.

doi:10.1002/anie.202100683

Cited by 44 publications

(48 citation statements)

References 38 publications

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“…PBS, cell lysates, serum and highly reductive environment). Elaborating on this P(V) platform, the same group recently reported phosphinates 42 , incorporating two terminal alkynes and thus offering protein cross-linking possibilities [ 69 ]. Applied to the disulfide rebridging of trastuzumab, half-antibody was observed as the main conjugation product; however, a side-reaction invariably competing with the expected inter-chain conjugation in these strategies (see §3.2.2.…”

Section: Chemoselective Strategiesmentioning

confidence: 99%

An overview of chemo- and site-selectivity aspects in the chemical conjugation of proteins

et al. 2022

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The bioconjugation of proteins—that is, the creation of a covalent link between a protein and any other molecule—has been studied for decades, partly because of the numerous applications of protein conjugates, but also due to the technical challenge it represents. Indeed, proteins possess inner physico-chemical properties—they are sensitive and polynucleophilic macromolecules—that make them complex substrates in conjugation reactions. This complexity arises from the mild conditions imposed by their sensitivity but also from selectivity issues, viz the precise control of the conjugation site on the protein. After decades of research, strategies and reagents have been developed to address two aspects of this selectivity: chemoselectivity—harnessing the reacting chemical functionality—and site-selectivity—controlling the reacting amino acid residue—most notably thanks to the participation of synthetic chemistry in this effort. This review offers an overview of these chemical bioconjugation strategies, insisting on those employing native proteins as substrates, and shows that the field is active and exciting, especially for synthetic chemists seeking new challenges.

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Section: Chemoselective Strategiesmentioning

confidence: 99%

An overview of chemo- and site-selectivity aspects in the chemical conjugation of proteins

et al. 2022

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“…N‐Hydroxysuccinimide (NHS)‐ modified phosphonamidates have been further developed for the stable conjugation of thiols and amines and applied to the construction of Protein‐Protein conjugates and ADCs [27] . Moreover, diethynyl phosphinates 2 have been used for a subsequent addition of two cysteine residues in a row and rebridging of native protein disulfides after reduction [28] …”

Section: Introductionmentioning

confidence: 99%

“…[27] Moreover, diethynyl phosphinates 2 have been used for a subsequent addition of two cysteine residues in a row and rebridging of native protein disulfides after reduction. [28] In the present work, we extend the repertoire of available unsaturated phosphonamidate reagents to bis-ethynylphosphonamidates 3, which we apply to construct protein-protein conjugates and ADCs from sulfhydryl containing drugs by the subsequent addition of two thiols in a row. We report four bisamidates that carry different functional O-substituents, such as a short ethylene glycol unit as well as a terminal alkyne.…”

Section: Introductionmentioning

confidence: 99%

Bis‐ethynylphosphonamidates as an Modular Conjugation Platform to Generate Multi‐Functional Protein‐ and Antibody‐Drug‐Conjugates

Kasper

Lassak

Vogl³

et al. 2022

Eur J Org Chem

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Bis‐ethynylphosphonamidates allow for a simple chemoselective addition of two thiol‐containing modules in a row. We describe four such bis‐electrophiles that carry different functional O‐substituents with tunable hydrophilicity and enable further subsequent conjugations, thus facilitating a simple protocol for constructing protein‐protein conjugates. An increased spacer length between the two ethynylphosphonamidates simplifies the formation of a conjugate from two bulky proteins. We apply these reagents to obtain homogeneous Antibody‐Drug‐Conjugates (ADCs) from DM1 and trastuzumab with excellent cytotoxicity and selectivity for the targeted cell line. Moreover, a bis‐ethynylphosphonamidate, carrying an additional alkyne for a chemoselective triple conjugation, has been subjected to fluorescent labeling of an ADC specifically at the drug site give an Antibody‐Drug‐Fluorophore‐Conjugate (ADFC), allowing for the observation of intracellular trafficking after ADC uptake into the targeted cell.

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“…Partial reduction of disulfide bridges, followed by Michael-type modification of any arising thiol groups, may cause disulfide bond scrambling and break the native quaternary antibody structure [ 13 , 14 , 15 ]. In addition, there are many complex chemical and chemo-enzymatic techniques for site-specific antibody modification using intricate modification reagents [ 12 , 16 , 17 , 18 , 19 , 20 ].…”

Section: Introductionmentioning

confidence: 99%

Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach

Sapozhnikova

Misyurin²,

Ryazantsev

et al. 2021

IJMS

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Bioconjugation of antibodies with various payloads has diverse applications across various fields, including drug delivery and targeted imaging techniques. Fluorescent immunoconjugates provide a promising tool for cancer diagnostics due to their high brightness, specificity, stability and target affinity. Fluorescent antibodies are widely used in flow cytometry for fast and sensitive identification and collection of cells expressing the target surface antigen. Nonetheless, current approaches to fluorescent labeling of antibodies most often use random modification, along with a few rather sophisticated site-specific techniques. The aim of our work was to develop a procedure for fluorescent labeling of immunoglobulin G via periodate oxidation of antibody glycans, followed by oxime ligation with fluorescent oxyamines. Here, we report a novel technique based on an in situ oxime ligation of ethoxyethylidene-protected aminooxy compounds with oxidized antibody glycans. The approach is suitable for easy modification of any immunoglobulin G, while ensuring that antigen-binding domains remain intact, thus revealing various possibilities for fluorescent probe design. The technique was used to label an antibody to PRAME, a cancer-testis protein overexpressed in a number of cancers. A 6H8 monoclonal antibody to the PRAME protein was directly modified with protected-oxyamine derivatives of fluorescein-type dyes (FAM, Alexa488, BDP-FL); the stoichiometry of the resulting conjugates was characterized spectroscopically. The immunofluorescent conjugates obtained were applied to the analysis of bone marrow samples from patients with oncohematological diseases and demonstrated high efficiency in flow cytometry quantification. The approach can be applied for the development of various immunofluorescent probes for detection of diagnostic and prognostic markers, which can be useful in anticancer therapy.

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Diethynyl Phosphinates for Cysteine‐Selective Protein Labeling and Disulfide Rebridging

Cited by 44 publications

References 38 publications

An overview of chemo- and site-selectivity aspects in the chemical conjugation of proteins

An overview of chemo- and site-selectivity aspects in the chemical conjugation of proteins

Bis‐ethynylphosphonamidates as an Modular Conjugation Platform to Generate Multi‐Functional Protein‐ and Antibody‐Drug‐Conjugates

Sensitive Immunofluorescent Detection of the PRAME Antigen Using a Practical Antibody Conjugation Approach

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