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.
Highlights
For a variety of important diagnostic and therapeutic applications, there is considerable interest in the covalent modification of proteins.[1−6] This field has grown to include a diverse range of reactions that modify a variety of different proteins with unique functions
We first investigated the potential of Dha as an aza-Michael acceptor within a small molecule context. This was performed by examining the reaction between a Boc-Dha methyl ester 1 and a variety of small molecule nitrogen nucleophiles 2a−9a with different N-hybridizations, which are representative of common motifs found in drug fragments, spectroscopic probes, linkers and PTMs (Figure 1b)
We have described a thorough evaluation of N-nucleophile additions to internal Dha residues as a chemoselective and biocompatible protein site-selective modification methodology for the construction of homogeneous protein conjugates
Summary
For a variety of important diagnostic and therapeutic applications, there is considerable interest in the covalent modification of proteins.[1−6] This field has grown to include a diverse range of reactions that modify a variety of different proteins with unique functions. Despite the great interest in the field of covalent chemical modification of proteins over the past decade, many of the documented methods lack site-selectivity within the chemically complex protein environment. For this reason, there is a pressing need for the development of reactions that modify defined amino acids within a protein’s structure. For this application as well as many others, the development of a site-selective conjugation methodology is essential
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