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  3. Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents

  • Nat Commun. 2016 Oct 26;7:13128. doi: 10.1038/ncomms13128.
Barbara Bernardim 1 2,  Pedro M S D Cal 1 3,  Maria J Matos 1,  Bruno L Oliveira 1,  Nuria Martínez-Sáez 1,  Inês S Albuquerque 3,  Elizabeth Perkins 4,  Francisco Corzana 1 5,  Antonio C B Burtoloso 2,  Gonzalo Jiménez-Osés 5 6,  Gonçalo J L Bernardes 1 3
Affiliations

Affiliations

  • 1 Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge UK.
  • 2 Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, São Paulo CEP 13560-970, Brazil.
  • 3 Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.
  • 4 Albumedix Ltd, Castle Court, 59 Castle Boulevard, Nottingham NG7 1FD, UK.
  • 5 Departamento de Química, Universidad de La Rioja, Centro de Investigacioón en Síntesis Química, 26006 Logroño, Spain.
  • 6 Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, BIFI-IQFR (CSIC), 50018 Zaragoza, Spain.
PMID: 27782215 DOI: 10.1038/ncomms13128
Abstract

Maleimides remain the reagents of choice for the preparation of therapeutic and imaging protein conjugates despite the known instability of the resulting products that undergo thiol-exchange reactions in vivo. Here we present the rational design of carbonylacrylic reagents for chemoselective cysteine bioconjugation. These reagents undergo rapid thiol Michael-addition under biocompatible conditions in stoichiometric amounts. When using carbonylacrylic reagents equipped with PEG or fluorophore moieties, this method enables access to protein and antibody conjugates precisely modified at pre-determined sites. Importantly, the conjugates formed are resistant to degradation in plasma and are biologically functional, as demonstrated by the selective imaging and detection of apoptotic and HER2+ cells, respectively. The straightforward preparation, stoichiometric use and exquisite cysteine selectivity of the carbonylacrylic reagents combined with the stability of the products and the availability of biologically relevant cysteine-tagged proteins make this method suitable for the routine preparation of chemically defined conjugates for in vivo applications.

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