Chemo- and Regioselective Lysine Modification on Native Proteins.
Authors
Matos, Maria J
Oliveira, Bruno L
Martínez-Sáez, Nuria
Guerreiro, Ana
Cal, Pedro MSD
Bertoldo, Jean
Maneiro, María
Perkins, Elizabeth
Howard, Julie
Publication Date
2018-03-21Journal Title
J Am Chem Soc
ISSN
0002-7863
Publisher
American Chemical Society (ACS)
Volume
140
Issue
11
Pages
4004-4017
Language
eng
Type
Article
This Version
VoR
Physical Medium
Print-Electronic
Metadata
Show full item recordCitation
Matos, M. J., Oliveira, B. L., Martínez-Sáez, N., Guerreiro, A., Cal, P. M., Bertoldo, J., Maneiro, M., et al. (2018). Chemo- and Regioselective Lysine Modification on Native Proteins.. J Am Chem Soc, 140 (11), 4004-4017. https://doi.org/10.1021/jacs.7b12874
Abstract
Site-selective chemical conjugation of synthetic molecules to proteins expands their functional and therapeutic capacity. Current protein modification methods, based on synthetic and biochemical technologies, can achieve site selectivity, but these techniques often require extensive sequence engineering or are restricted to the N- or C-terminus. Here we show the computer-assisted design of sulfonyl acrylate reagents for the modification of a single lysine residue on native protein sequences. This feature of the designed sulfonyl acrylates, together with the innate and subtle reactivity differences conferred by the unique local microenvironment surrounding each lysine, contribute to the observed regioselectivity of the reaction. Moreover, this site selectivity was predicted computationally, where the lysine with the lowest p Ka was the kinetically favored residue at slightly basic pH. Chemoselectivity was also observed as the reagent reacted preferentially at lysine, even in those cases when other nucleophilic residues such as cysteine were present. The reaction is fast and proceeds using a single molar equivalent of the sulfonyl acrylate reagent under biocompatible conditions (37 °C, pH 8.0). This technology was demonstrated by the quantitative and irreversible modification of five different proteins including the clinically used therapeutic antibody Trastuzumab without prior sequence engineering. Importantly, their native secondary structure and functionality is retained after the modification. This regioselective lysine modification method allows for further bioconjugation through aza-Michael addition to the acrylate electrophile that is generated by spontaneous elimination of methanesulfinic acid upon lysine labeling. We showed that a protein-antibody conjugate bearing a site-specifically installed fluorophore at lysine could be used for selective imaging of apoptotic cells and detection of Her2+ cells, respectively. This simple, robust method does not require genetic engineering and may be generally used for accessing diverse, well-defined protein conjugates for basic biology and therapeutic studies.
Keywords
Humans, Acrylates, Lysine, Proteins, Molecular Structure, Stereoisomerism, Computer-Aided Design, Hep G2 Cells
Sponsorship
Engineering and Physical Sciences Research Council (EP/M003647/1)
The Royal Society (uf110046)
European Commission Horizon 2020 (H2020) Marie Sk?odowska-Curie actions (702574)
European Research Council (676832)
European Commission (EC) (852985)
Embargo Lift Date
2100-01-01
Identifiers
External DOI: https://doi.org/10.1021/jacs.7b12874
This record's URL: https://www.repository.cam.ac.uk/handle/1810/275722
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