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dc.contributor.authorFreedy, Allyson Men
dc.contributor.authorMatos, Maria Jen
dc.contributor.authorBoutureira, Omaren
dc.contributor.authorCorzana, Franciscoen
dc.contributor.authorGuerreiro, Anaen
dc.contributor.authorAkkapeddi, Padmaen
dc.contributor.authorSomovilla, Víctor Jen
dc.contributor.authorRodrigues, Tiagoen
dc.contributor.authorNicholls, Karlen
dc.contributor.authorXie, Bangwenen
dc.contributor.authorJiménez-Osés, Gonzaloen
dc.contributor.authorBrindle, Kevinen
dc.contributor.authorNeves, Andreen
dc.contributor.authorBernardes, Goncaloen
dc.date.accessioned2018-02-07T10:25:50Z
dc.date.available2018-02-07T10:25:50Z
dc.date.issued2017-12-05en
dc.identifier.issn0002-7863
dc.identifier.urihttps://www.repository.cam.ac.uk/handle/1810/271767
dc.description.abstractChemical 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 homogenous 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.
dc.format.mediumPrint-Electronicen
dc.languageengen
dc.publisherAmerican Chemical Society
dc.rightsAttribution 4.0 International*
dc.rights.urihttp://creativecommons.org/licenses/by/4.0/*
dc.subjectCell Line, Tumoren
dc.subjectAnimalsen
dc.subjectHumansen
dc.subjectMiceen
dc.subjectDisulfidesen
dc.subjectAminesen
dc.subjectPyrazolesen
dc.subjectPyridinesen
dc.subjectAlanineen
dc.subjectAlbuminsen
dc.subjectAnnexin A5en
dc.subjectAntibodiesen
dc.subjectCell Deathen
dc.subjectCell Survivalen
dc.subjectMolecular Structureen
dc.subjectDose-Response Relationship, Drugen
dc.subjectKineticsen
dc.subjectQuantum Theoryen
dc.subjectModels, Molecularen
dc.subjectSynaptotagmin Ien
dc.subjectHEK293 Cellsen
dc.titleChemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation.en
dc.typeArticle
prism.endingPage18375
prism.issueIdentifier50en
prism.publicationDate2017en
prism.publicationNameJournal of the American Chemical Societyen
prism.startingPage18365
prism.volume139en
dc.identifier.doi10.17863/CAM.18761
dcterms.dateAccepted2017-11-21en
rioxxterms.versionofrecord10.1021/jacs.7b10702en
rioxxterms.versionVoR*
rioxxterms.licenseref.urihttp://www.rioxx.net/licenses/all-rights-reserveden
rioxxterms.licenseref.startdate2017-12-05en
dc.contributor.orcidFreedy, Allyson M [0000-0002-2953-8133]
dc.contributor.orcidBoutureira, Omar [0000-0002-0768-8309]
dc.contributor.orcidCorzana, Francisco [0000-0001-5597-8127]
dc.contributor.orcidRodrigues, Tiago [0000-0002-1581-5654]
dc.contributor.orcidJiménez-Osés, Gonzalo [0000-0003-0105-4337]
dc.contributor.orcidBrindle, Kevin [0000-0003-3883-6287]
dc.contributor.orcidNeves, Andre [0000-0003-2740-5166]
dc.contributor.orcidBernardes, Goncalo [0000-0001-6594-8917]
dc.identifier.eissn1520-5126
rioxxterms.typeJournal Article/Reviewen
pubs.funder-project-idEuropean Commission (626890)
pubs.funder-project-idEngineering and Physical Sciences Research Council (EP/M003647/1)
pubs.funder-project-idThe Royal Society (uf110046)
pubs.funder-project-idEuropean Research Council (676832)
pubs.funder-project-idCancer Research UK (CB4100)
pubs.funder-project-idCancer Research UK (C14303/A17197)
rioxxterms.freetoread.startdate2100-01-01


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Attribution 4.0 International
Except where otherwise noted, this item's licence is described as Attribution 4.0 International