Modular multifunctional 5HP2O linkers for the versatile assembly of protein-oligonucleotide conjugates
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.
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BibTeX
@article{meffert2026modular,
title = {{Modular multifunctional 5HP2O linkers for the versatile assembly of protein-oligonucleotide conjugates}},
author = {Jan H. Meffert and Alexandros Kostakis and Joshua McHale and Sara Mangsbo and Carlo Vascotto and Martin Bollmark and Annemieke Madder},
journal = {Communications Chemistry},
year = {2026},
month = oct,
doi = {10.1038/s42004-026-02238-6},
url = {https://www.nature.com/articles/s42004-026-02238-6},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Modular multifunctional 5HP2O linkers for the versatile assembly of protein-oligonucleotide conjugates
AU - Jan H. Meffert
AU - Alexandros Kostakis
AU - Joshua McHale
AU - Sara Mangsbo
AU - Carlo Vascotto
AU - Martin Bollmark
AU - Annemieke Madder
PY - 2026
DA - 2026/10/10
JO - Communications Chemistry
DO - 10.1038/s42004-026-02238-6
UR - https://www.nature.com/articles/s42004-026-02238-6
AB - 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.
SN - 2399-3669
ER -