CUED Publications database

Chemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation

Freedy, AM and Matos, MJ and Boutureira, O and Corzana, F and Guerreiro, A and Akkapeddi, P and Somovilla, VJ and Rodrigues, T and Nicholls, K and Xie, B and Jiménez-Osés, G and Brindle, KM and Neves, AA and Bernardes, GJL (2017) Chemoselective Installation of Amine Bonds on Proteins through Aza-Michael Ligation. Journal of the American Chemical Society, 139. pp. 18365-18375. ISSN 0002-7863

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Chemical 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 homogeneous 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.

Item Type: Article
Uncontrolled Keywords: Alanine Albumins Amines Animals Annexin A5 Antibodies Cell Death Cell Line, Tumor Cell Survival Crizotinib Disulfides Dose-Response Relationship, Drug HEK293 Cells Humans Kinetics Mice Models, Molecular Molecular Structure Pyrazoles Pyridines Quantum Theory Synaptotagmin I
Depositing User: Cron Job
Date Deposited: 21 Jun 2019 20:15
Last Modified: 10 Apr 2021 22:26
DOI: 10.1021/jacs.7b10702