Precise Probing of Residue Roles by Post-Translational β,γ-C,N Aza-Michael Mutagenesis in Enzyme Active Sites

Abstract

Biomimicry valuably allows the understanding of the essential chemical components required to recapitulate biological function, yet direct strategies for evaluating the roles of amino acids in proteins can be limited by access to suitable, subtly-altered unnatural variants. Here we describe a strategy for dissecting the role of histidine residues in enzyme active sites using unprecedented, chemical, post-translational side-chain-β,γ C–N bond formation. Installation of dehydroalanine (as a “tag”) allowed the testing of nitrogen conjugate nucleophiles in “aza-Michael”-1,4-additions (to “modify”). This allowed the creation of a regioisomer of His (iso-His, Hisiso) linked instead through its pros-Nπ atom rather than naturally linked via C4, as well as an aza-altered variant aza-Hisiso. The site-selective generation of these unnatural amino acids was successfully applied to probe the contributing roles (e.g., size, H-bonding) of His residues toward activity in the model enzymes subtilisin protease from Bacillus lentus and Mycobacterium tuberculosis pantothenate synthetase.

Palladium prompted on-demand cysteine chemistry for the synthesis of challenging and uniquely modified proteins

“…HPLC and mass analysis showed that under standard conditions, TCEP reacts with the DHA residue such that > 50% of the probe is deactivated within 24 h, probably via the formation of a stable C-P bond (Supplementary Fig. 27 - 30 ) 50 , 51 . We reasoned that excluding TCEP from the buffer would provide greater stability of the probe for several weeks.…”

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

“…There are very few examples of the reaction of Dha with N -nucleophiles on peptides, 26 − 28 and to an even lesser extent on proteins—in a single example, Dha has been used as a precursor for the installation of an isomer of histidine through the reaction with imidazole. 29 − 32…”

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

“…Many thiopeptides and lanthipeptides contain one or more uniquely reactive dehydroamino acids such as dehydroalanine (Dha) and dehydrobutyrine (Dhb), which are the result of post‐translational enzymatic dehydration of Ser and Thr residues, respectively . The electrophilic nature of dehydroamino acids has made them attractive functionalities for biorthogonal reactions . In recent years, these dehydroamino acids have emerged as interesting targets for the late‐stage modification of RiPPs, through Michael additions, hydrogenations, cross‐coupling reactions, photoredox catalysis, cyclopropanations, and 1,3‐dipolar cycloadditions .…”

“…[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.…”