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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 <jats:italic>in vivo<\/jats:italic> applications.<\/jats:p>","DOI":"10.1038\/ncomms13128","type":"journal-article","created":{"date-parts":[[2016,10,26]],"date-time":"2016-10-26T11:18:43Z","timestamp":1477480723000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":174,"title":["Stoichiometric and irreversible cysteine-selective protein modification using carbonylacrylic reagents"],"prefix":"10.1038","volume":"7","author":[{"given":"Barbara","family":"Bernardim","sequence":"first","affiliation":[]},{"given":"Pedro M.S.D.","family":"Cal","sequence":"additional","affiliation":[]},{"given":"Maria J.","family":"Matos","sequence":"additional","affiliation":[]},{"given":"Bruno L.","family":"Oliveira","sequence":"additional","affiliation":[]},{"given":"Nuria","family":"Mart\u00ednez-S\u00e1ez","sequence":"additional","affiliation":[]},{"given":"In\u00eas S.","family":"Albuquerque","sequence":"additional","affiliation":[]},{"given":"Elizabeth","family":"Perkins","sequence":"additional","affiliation":[]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5597-8127","authenticated-orcid":false,"given":"Francisco","family":"Corzana","sequence":"additional","affiliation":[]},{"given":"Antonio C.B.","family":"Burtoloso","sequence":"additional","affiliation":[]},{"given":"Gonzalo","family":"Jim\u00e9nez-Os\u00e9s","sequence":"additional","affiliation":[]},{"given":"Gon\u00e7alo J. 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