Chemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation

  1. Freedy, A.M. 3
  2. Matos, M.J. 3
  3. Boutureira, O. 3
  4. Corzana, F. 23
  5. Guerreiro, A. 4
  6. Akkapeddi, P. 4
  7. Somovilla, V.J. 23
  8. Rodrigues, T. 4
  9. Nicholls, K. 1
  10. Xie, B. 5
  11. Jiménez-Osés, G. 2
  12. Brindle, K.M. 25
  13. Neves, A.A. 5
  14. Bernardes, G.J.L. 34
  1. 1 Albumedix Ltd., Castle Court, 59 Castle Boulevard, Nottingham, United Kingdom
  2. 2 Universidad de La Rioja
    info

    Universidad de La Rioja

    Logroño, España

    ROR https://ror.org/0553yr311

  3. 3 University of Cambridge
    info

    University of Cambridge

    Cambridge, Reino Unido

    ROR https://ror.org/013meh722

  4. 4 Universidade de Lisboa
    info

    Universidade de Lisboa

    Lisboa, Portugal

    ROR https://ror.org/01c27hj86

  5. 5 Cancer Research UK Cambridge Institute
    info

    Cancer Research UK Cambridge Institute

    Cambridge, Reino Unido

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Zeitschrift:
Journal of the American Chemical Society

ISSN: 0002-7863

Datum der Publikation: 2017

Ausgabe: 139

Nummer: 50

Seiten: 18365-18375

Art: Artikel

DOI: 10.1021/JACS.7B10702 SCOPUS: 2-s2.0-85038611908 WoS: WOS:000418783600038 GOOGLE SCHOLAR

Andere Publikationen in: Journal of the American Chemical Society

Zusammenfassung

Chemical modification of proteins is essential for a variety of important diagnostic and therapeutic applications. Many strategies developed to date lack chemo- and regioselectivity as well as result in non-native linkages that may suffer from instability in vivo and adversely affect the protein's structure and function. We describe here the reaction of N-nucleophiles with the amino acid dehydroalanine (Dha) in a protein context. When Dha is chemically installed in proteins, the addition of a wide-range N-nucleophiles enables the rapid formation of amine linkages (secondary and tertiary) in a chemoselective manner under mild, biocompatible conditions. These new linkages are stable at a wide range of pH values (pH 2.8 to 12.8), under reducing conditions (biological thiols such as glutathione) and in human plasma. This method is demonstrated for three proteins and is shown to be fully compatible with disulfide bridges, as evidenced by the selective modification of recombinant albumin that displays 17 structurally relevant disulfides. The practicability and utility of our approach is further demonstrated by the construction of a chemically modified C2A domain of Synaptotagmin-I protein that retains its ability to preferentially bind to apoptotic cells at a level comparable to the native protein. Importantly, the method was useful for building a homogeneous antibody-drug conjugate with a precise drug-to-antibody ratio of 2. The kinase inhibitor crizotinib was directly conjugated to Dha through its piperidine motif, and its antibody-mediated intracellular delivery results in 10-fold improvement of its cancer cell-killing efficacy. The simplicity and exquisite site-selectivity of the aza-Michael ligation described herein allows the construction of stable secondary and tertiary amine-linked protein conjugates without affecting the structure and function of biologically relevant proteins. © 2017 American Chemical Society.