(413f) Development of a New, Orthogonal Chemistry for the Megamolecule Assembly Strategy

The synthesis of precisely defined molecules with molecular weights greater than 100,000 Da is a current challenge in the field of bioconjugation chemistry. One approach to overcome this is through the rational design of targeted covalent inhibitors that selectively react with a nucleophilic residue in the active site of a monomeric protein domain. By engineering a toolbox of both (i) fusion proteins of several different enzymes as genetic building blocks and (ii) the synthesis of orthogonal polymeric linkers terminated with orthogonal covalent inhibitors, our lab has demonstrated the development of a multi-protein assembly method – known as the megamolecule strategy – to prepare atomically precise molecules with molecular weights of up to one megadalton. In this work, we present the newest covalent chemistry in our toolbox based on the inhibition of the human-derived cellular retinoic acid binding protein II (CRABP2) domain. We synthesized a synthetic retinoid bearing an arylfluorosulfate warhead as our targeted covalent inhibitor for use with Sulfur Fluoride Exchange (SuFEx) click chemistry. Arylfluorosulfates have high kinetic stability for CRABP2 and have also been found to be inactive by alternative chemical modifications, making these groups highly specific for orthogonal protein bioconjugation reactions. After anchoring the ligand to the active site, two arginine residues act as hydrogen binding donors and allow for the necessary geometric and electrostatic stabilization for the nucleophilic tyrosine phenolic group to undergo SuFEx with the selectively reactive sulfur-fluoride bond. We conjugated the ligand to a polyethylene glycol backbone and measured the second-order rate constant for the reaction between the protein and ligand to be 3626 ± 503 M^-1s^-1. We evaluated the stability of the protein-inhibitor complex at room temperature and found no degradation of the reaction over two weeks. Further, we studied the orthogonality of this inhibitor-protein system with the other chemistries in our megamolecule toolbox. We found that our other two chemistries – using cutinase and SnapTag – along with their covalent inhibitors do not interact with the CRABP2 protein domain, nor its synthetic retinoid inhibitor. Introducing a third, orthogonally reactive protein-inhibitor pair to our assembly toolbox, enables exact control over the specificity, orientation, and stoichiometry of protein domains within atomically precise nanostructures.