title: Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents.
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.
authors: Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge UK.|Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, São Paulo CEP 13560-970, Brazil.
authors: Barbara
authors: B
authors: Bernardim
authors: Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge UK.|Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.
authors: Pedro M S D
authors: PM
authors: Cal
authors: Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge UK.
authors: Maria J
authors: MJ
authors: Matos
authors: Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge UK.
authors: Bruno L
authors: BL
authors: Oliveira
authors: Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge UK.
authors: Nuria
authors: N
authors: Martínez-Sáez
authors: Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.
authors: Inês S
authors: IS
authors: Albuquerque
authors: Albumedix Ltd, Castle Court, 59 Castle Boulevard, Nottingham NG7 1FD, UK.
authors: Elizabeth
authors: E
authors: Perkins
authors: Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge UK.|Departamento de Química, Universidad de La Rioja, Centro de Investigacioón en Síntesis Química, 26006 Logroño, Spain.
authors: Francisco
authors: 0000-0001-5597-8127
authors: F
authors: Corzana
authors: Instituto de Química de São Carlos, Universidade de São Paulo, São Carlos, São Paulo CEP 13560-970, Brazil.
authors: Antonio C B
authors: AC
authors: Burtoloso
authors: Departamento de Química, Universidad de La Rioja, Centro de Investigacioón en Síntesis Química, 26006 Logroño, Spain.|Institute of Biocomputation and Physics of Complex Systems (BIFI), University of Zaragoza, BIFI-IQFR (CSIC), 50018 Zaragoza, Spain.
authors: Gonzalo
authors: G
authors: Jiménez-Osés
authors: Department of Chemistry, University of Cambridge, Lensfield Road, CB2 1EW Cambridge UK.|Instituto de Medicina Molecular, Faculdade de Medicina, Universidade de Lisboa, Avenida Professor Egas Moniz, 1649-028 Lisboa, Portugal.
authors: Gonçalo J L
authors: GJ
authors: Bernardes
mesh_terms: D000097:Acetonitriles / Q000737:chemistry; D000179:Acrylates / Q000737:chemistry*; D000595:Amino Acid Sequence; D017304:Annexin A5 / Q000737:chemistry; D001565:Benzoates / Q000737:chemistry*; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D057809:HEK293 Cells; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D008301:Maleimides / Q000737:chemistry; D000072756:Protein Conformation, alpha-Helical; D000072757:Protein Conformation, beta-Strand; D055550:Protein Stability; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D013237:Stereoisomerism; D013440:Sulfides / Q000138:chemical synthesis*; D000068878:Trastuzumab / Q000737:chemistry
chemical_list: D000097:Acetonitriles; D000179:Acrylates; D017304:Annexin A5; D001565:Benzoates; D003432:Cross-Linking Reagents; D003545:Cysteine; D018796:Immunoconjugates; D008301:Maleimides; D018719:Erb-b2 Receptor Tyrosine Kinases; D013440:Sulfides; D000068878:Trastuzumab; C400967:benzoylacrylic acid; C508053:ERBB2 protein, human; C032159:acetonitrile