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

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.

Selective Modification of Ribosomally Synthesized and Post‐Translationally Modified Peptides (RiPPs) through Diels–Alder Cycloadditions on Dehydroalanine Residues

“…[10] The electrophilic nature of dehydroamino acids has made them attractive functionalities for biorthogonal reactions. [11][12][13][14][15][16][17][18][19][20] In recent years, these dehydroamino acids have emerged as interesting targets for the late-stage modification of RiPPs, throughM ichael additions, [21][22][23][24] hydrogenations, [25] cross-coupling reactions, [26,27] photoredox catalysis, [28] cyclopropanations, [29] and 1,3-dipolar cycloadditions. [30] These studies have highlighted the potential of dehydroamino acid modification in RiPPs, but also illustrate the challenge of achieving selectivity due to the high structural complexity of RiPPs and the difficulties of discriminating between the various dehydroamino acids present.…”

A Chemical Probe for Dehydrobutyrine

“…When using biocompatible conditions, all of the thiol‐containing compounds that were tested yielded extensive modification of peptide A after 24 hours but resulted in only minimal modification of peptide B (0–11.5 % modified). The aza‐Michael reaction was recently reported as a robust method for the modification of Dha‐bearing proteins ––. A variety of primary and secondary amines were tested, but none could label peptide B under more stringent, buffered conditions.…”

Chemical Methods for Selective Labeling of Proteins

“…In this perspective, Davis and coworkers demonstrated a few remarkable examples of thio‐Michael addition for generating the mimics of post‐translational modification (Scheme a) . More recently, Bernardes and co‐workers reported a chemoselective aza‐Michael addition reaction with Dha (Scheme b) . It operates efficiently at high concentration of amine without perturbing the disulfide bond.…”