Development of new ligation methodologies to access challenging protein targets

Abstract

Protein ligation methodologies have advanced protein engineering by enabling the synthesis of larger, more complex proteins with precise site-specific modifications. These developments have improved control, efficiency, and selectivity—crucial for drug development, the study of protein interactions, and the design of therapeutic proteins. Despite substantial progress, further innovations in ligation chemistry remain essential to drive breakthroughs in biotechnology and synthetic biochemistry. A novel DEAMC photolabile protecting group for selenocysteine (Sec) was developed, allowing efficient LED-induced deprotection at 450 nm under mild, reagent-free conditions. This enabled an iterative DSL approach for one-pot protein assembly. Incorporation of DEAMC-Sec into peptides yielded clean deprotection and facilitated the four-step synthesis of ApoCIII in 60% yield. A selenium-based ligation strategy was also employed for synthesizing lipoprotein and glycolipoprotein vaccine candidates for tuberculosis (TB), with lipidated LprA and Mpt83 variants exhibiting potent TLR2 agonism. To enhance expressed protein ligation (EPL), a continuous flow chemistry platform was established, improving yields and reaction rates relative to batch methods. This was demonstrated by the semisynthesis of the sulfoprotein ACA-01. A one-pot EPL–photodesulfurization strategy was further applied to the synthesis of unmodified and phosphorylated β-synuclein using a demulsifying buffer, enabling investigation of serine phosphorylation in β-Syn inhibition of α-Syn aggregation. These developments offer robust and versatile tools for the generation of engineered and native proteins, with broad utility in both research and therapeutic contexts.