Chemo and regioselective lysine modification on native proteins

Maria Matos, Bruno Oliveira, Nuria Martínez-Sáez, Ana Guerreiro, Pedro Cal, Jean Bertoldo, Maria Maneiro, Elizabeth Perkins, Julie Howard, Michael Deery, Justin Chalker, Francisco Corzana, Gonzalo Jiménez-Osés, Gonçalo Bernardes

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    63 Citations (Scopus)

    Abstract

    Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest pKa was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein-antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.

    Original languageEnglish
    Pages (from-to)4004-4017
    Number of pages14
    JournalJournal of The American Chemical Society
    Volume140
    Issue number11
    DOIs
    Publication statusPublished - 2018

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  • Cite this

    Matos, M., Oliveira, B., Martínez-Sáez, N., Guerreiro, A., Cal, P., Bertoldo, J., Maneiro, M., Perkins, E., Howard, J., Deery, M., Chalker, J., Corzana, F., Jiménez-Osés, G., & Bernardes, G. (2018). Chemo and regioselective lysine modification on native proteins. Journal of The American Chemical Society, 140(11), 4004-4017. https://doi.org/10.1021/jacs.7b12874