Repository logo
 

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

Published version
Peer-reviewed

Change log

Authors

Freedy, Allyson M 
Matos, Maria J 
Guerreiro, Ana 

Abstract

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.

Description

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

Journal Title

J Am Chem Soc

Conference Name

Journal ISSN

0002-7863
1520-5126

Volume Title

139

Publisher

American Chemical Society (ACS)
Sponsorship
European Commission (626890)
Engineering and Physical Sciences Research Council (EP/M003647/1)
The Royal Society (uf110046)
European Research Council (676832)
Cancer Research UK (CB4100)
Cancer Research UK (C14303/A17197)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (675007)
European Commission (EC) (852985)
Cancer Research UK (17242)