Modular multifunctional 5HP2O linkers for the versatile assembly of protein-oligonucleotide conjugates

Abstract

Abstract As complex biotherapeutics continue to evolve, the demand for single-site, multi-functionalized protein conjugates intensifies. Current methods often suffer from maleimide-related hydrolytic instability or require multi-step invasive protein modifications. Here, we establish 5-hydroxy-1,5-dihydro-2H-pyrrol-2-ones (5HP2O) as modular, hydrolytically stable hubs for assembling multi-functional protein-oligonucleotide conjugates (POCs). Synthesized via one-pot photooxidation of 2-substituted furans, these linkers incorporate tunable spacers, 5HP2O Michael acceptor, a NHS active ester and secondary cargoes. We demonstrate that 5HP2O-functionalized amino-oligonucleotides (AON) maintain complete hydrolytic resistance across a broad pH range, streamlining intermediate storage. Subsequent bioconjugation yields constructs with superior retro-Michael stability compared to aliphatic maleimide analogues. Expanding this platform, we generated diverse heterobifunctional, trifunctional (click-reactive and fluorescent), and tetrafunctional (enzymatically cleavable) linkers. We validated these tools through in vitro conjugate tracking and the assembly of a dual-antisense oligonucleotide conjugate.