Metabolic Incorporation of latent fast reacting thioeSTERs

Single protein building blocks, so-called protein monomers, can be genetically engineered so that they polycondensate into rubber-like elastic (elastomeric) protein biopolymers. These elastomeric protein biopolymers are innovative raw materials for promising biomedical applications and for materials science. In recent years, the production of elastomeric protein biopolymers has been the subject of intense research. Despite significant advances, we still need versatile and efficient approaches to further improve and diversify the properties of the targeted materials. The goal of this project is to combine concepts from chemistry and synthetic biology to develop a fully integrated approach for the polycondensation of protein monomers. To achieve this goal, protein monomers will be equipped with activatable and extremely fast- reacting thioester groups at defined positions. We expect that the tremendous reactivity and chemoselectivity of the thioester groups will improve the single-chain polycondensation of the monomeric protein building blocks. In particular, the robustness of the approach will be challenged through the recombinant production of a titin protein monomer containing the thioester groups, which polymerizes to form a biopolymer endowed with muscle-like mechanical properties.

Metabolic incorporation of fastreacting oxalyl thioesters: an integrated solution toward the production of labeled proteins and biopolymers Project at a glance Proteins can be turned into ecofriendly, biocompatible materials-well suited for medicine and industry-but their diversity has been limited. The MISTER project connects chemistry with synthetic biology to create new "docking sites" in proteins, enabling precise labeling, linking, and assembly into robust proteinbased biopolymers. The idea Thioesters are valuable connectors for joining (poly)peptides by native chemical ligation. However, their use in conjugation remains limited by an unfavorable trade-off between stability and reactivity in aqueous media. MISTER addresses this by introducing a novel, activatable thioester switch called oxoSEA. This group is highly reactive toward -aminothiols but stays stable until activation. Then, it delivers highspeed, highselectivity reactions-streamlining protein labeling and linkage for research, medicine, and biomaterials. What was achieved? New building blocks at scale: A stable Lys(oxoSEA) building block was produced at gram scale, as both a protected precursor and in free form. Fast and selective reactions: Couplings proceed quickly at very low concentrations, including in complex samples, with ondemand activation under gentle, reducing conditions. Precise installation strategies: Chemical/enzymatic: Proteins (e.g., ubiquitin) were selectively equipped and efficiently linked; using Sortase A, conversions above 90% were achieved. Biological: The oxoSEA building block was genetically incorporated into bacterial proteins-an important step toward producing labeled proteins directly in cells. First materials: Chainlike protein biopolymers inspired by the muscle protein titin were built from bifunctional protein domains. A known hurdle-undesired ring formation-was significantly reduced using alternative, especially enzymatic, approaches. Why it matters For medicine: Proteins can be labeled or joined with pinpoint precision, supporting better diagnostics, targeted drug delivery, and tailored biomaterials. For sustainable materials: New protein polymers are biocompatible and degradable, with potential to partially replace petroleumbased plastics. For research: The integrated platform unites fast, controllable chemistry with synthetic biology, creating a versatile toolbox for many applications. What's next The chemistry-biology interface will be expanded, installation into more complex proteins streamlined, and polymer architectures optimized to reach higher molecular weights and new properties. In parallel, core reaction principles will be further clarified, paving the way for robust, scalable applications in biotechnology, protein chemistry, and materials science.