Development of symmetrical cyclononynes for intracellular generation of protein-protein interaction inhibitors by isomer-free, strain-promoted cycloadditions

• Applicant: Professor Dr. Thorsten Berg
• Subject Area: Biological and Biomimetic Chemistry
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 540991815

Protein-protein interactions are of fundamental importance for the majority of cellular processes. Inhibition of these interactions offers the possibility of significantly expanding the currently rather limited target spectrum of small organic molecules. Because of the large size of protein-protein interfaces, however, high-affinity inhibitors of protein-protein interactions often display a high molecular weight. This has negative consequences for their cell permeability and/or bioavailability. We recently presented isomer-free, strain-promoted azide-alkyne cycloaddition (iSPAAC) as a method by which to generate large, chemically uniform bioactive molecules inside living cells, from two smaller components with higher cell permeability. Key to this approach was our design and synthesis of the symmetrical pyrrolocyclooctynes PYRROC, SYPCO, and TRIPCO, as well as the azacyclononyne Fmoc-ACN. SYPCO and Fmoc-ACN were used to generate large triazole inhibitors of protein-protein interactions mediated by the anti-apoptotic protein Bcl-xL. The advantage of azacyclononynes over pyrrolocyclooctynes is the lower spatial requirement of the bicyclic ring system formed in isomer-free strain-promoted cycloadditions, as compared to the larger tricyclic ring system formed by pyrrolocyclooctynes. This makes azacyclononynes more versatile agents for creating large high-affinity inhibitors of biological targets. However, while the SYPCO-based triazole inhibitor of Bcl-xL could be generated by iSPAAC in living cells, formation of the significantly more potent Bcl-xL inhibitor based on the less strained cyclononyne Fmoc-ACN proceeded too slowly for cell-based applications. The central aim of the work proposed here is to develop methodology for isomer-free strain- promoted cycloadditions with azacyclononynes that proceed sufficiently fast for use in living cells. To this end, we will explore the use of Fmoc-ACN and two newly designed azacyclononynes in strain-promoted cycloadditions, both in SPAAC and in inverse electron demand Diels-Alder reactions (IEDDA) with 1,2,4,5-tetrazines. We will identify the most promising reactions for isomer-free intracellular generation of large inhibitors of protein-protein interactions mediated by Bcl-xL. The successful execution of this research project will significantly expand and improve the molecular toolbox for generating large chemically uniform, high-affinity ligands of intracellular biological targets in living cells, from smaller, more cell-permeable building blocks.

• DFG Programme: Research Grants
• Co-Investigators: Professor Dr. Ralf Hoffmann; Professor Dr. Harald Krautscheid