benzoylacrylic-acid and acetonitrile

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.

Topics: Acetonitriles; Acrylates; Amino Acid Sequence; Annexin A5; Benzoates; Cross-Linking Reagents; Cysteine; HEK293 Cells; Humans; Immunoconjugates; Maleimides; Protein Conformation, alpha-Helical; Protein Conformation, beta-Strand; Protein Stability; Receptor, ErbB-2; Staining and Labeling; Stereoisomerism; Sulfides; Trastuzumab