Chemo- and Regioselective Lysine Modification on Native Proteins

  1. Matos, M.J. 2
  2. Oliveira, B.L. 2
  3. Martínez-Sáez, N. 2
  4. Guerreiro, A. 6
  5. Cal, P.M.S.D. 6
  6. Bertoldo, J. 2
  7. Maneiro, M. 4
  8. Perkins, E. 1
  9. Howard, J. 2
  10. Deery, M.J. 2
  11. Chalker, J.M. 3
  12. Corzana, F. 5
  13. Jiménez-Osés, G. 5
  14. Bernardes, G.J.L. 26
  1. 1 Albumedix Ltd, Castle Court, 59 Castle Boulevard, Nottingham, United Kingdom
  2. 2 University of Cambridge
    info

    University of Cambridge

    Cambridge, Reino Unido

    ROR https://ror.org/013meh722

  3. 3 Flinders University
    info

    Flinders University

    Adelaida, Australia

    ROR https://ror.org/01kpzv902

  4. 4 Universidade de Santiago de Compostela
    info

    Universidade de Santiago de Compostela

    Santiago de Compostela, España

    ROR https://ror.org/030eybx10

  5. 5 Universidad de La Rioja
    info

    Universidad de La Rioja

    Logroño, España

    ROR https://ror.org/0553yr311

  6. 6 Universidade de Lisboa
    info

    Universidade de Lisboa

    Lisboa, Portugal

    ROR https://ror.org/01c27hj86

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Zeitschrift:
Journal of the American Chemical Society

ISSN: 0002-7863

Datum der Publikation: 2018

Ausgabe: 140

Nummer: 11

Seiten: 4004-4017

Art: Artikel

DOI: 10.1021/JACS.7B12874 SCOPUS: 2-s2.0-85044197995 WoS: WOS:000428356000030 GOOGLE SCHOLAR

Andere Publikationen in: Journal of the American Chemical Society

Zusammenfassung

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. © 2018 American Chemical Society.