Zwitterionic organoboron complexes for overcoming the concentration barrier in chemical protein synthesis

Summary

Chemical protein synthesis builds proteins by chemoselectively joining unprotected peptide segments, but the leading ligations (native chemical ligation and the alpha-ketoacid-hydroxylamine, or KAHA, ligation) need millimolar concentrations. That requirement fails for large, hydrophobic, or aggregation-prone segments that cannot be dissolved that concentrated. KAT ligation between potassium acyltrifluoroborates (KATs) and hydroxylamines is fast at low concentration, but had never been usable for protein synthesis because no protecting group could carry a KAT through Fmoc solid-phase peptide synthesis (SPPS). This work solves that problem with ONNO, a tetradentate ligand that masks a KAT as a bench-stable, SPPS- and acid-stable zwitterionic organoboron complex, then releases it under mild conditions.

Key findings

• ONNO, a tetradentate ligand made in one reductive amination from commercial 8-hydroxyquinoline-2-carbaldehyde and 3-amino-4-hydroxybenzoic acid, masks both the carbonyl and the trifluoroborate of a KAT as one zwitterionic organoboron complex.
• ONNO-masked Fmoc-amino-acid KATs survive standard Fmoc solid-phase peptide synthesis and trifluoroacetic acid cleavage, which no previous KAT protecting group could do.
• The KAT is unmasked under mild conditions with the commercial reagent DMPU-HF, with complete retention of stereochemistry.
• KAT ligation joined peptide segments at 100 to 200 micromolar, roughly 50% conversion was still reached at 20 micromolar - about three orders of magnitude below the concentrations native chemical ligation and KAHA ligation require.
• The method delivered the 57-residue human apolipoprotein C-I and the aggregation-prone immunoglobulin V domain of human PD-L2 (hPD-L2) by convergent segment ligation.
• ONNO can also serve as a temporary hydrophilic handle that aids purification of otherwise intractable hydrophobic peptide segments.

Questions this paper answers

How can chemical protein synthesis be done at low (micromolar) concentration?

Schilling, Steiner, and Bode (Science 2026) mask potassium acyltrifluoroborates with the tetradentate ONNO ligand so they survive solid-phase peptide synthesis, then unmask them and use KAT ligation, which couples segments at 100 to 200 micromolar and still reaches about 50% conversion at 20 micromolar - far below the millimolar concentrations native chemical ligation and KAHA ligation need.

What is the ONNO ligand?

ONNO is a tetradentate ligand, 4-hydroxy-3-(((8-hydroxyquinolin-2-yl)methyl)amino)benzoic acid, that chelates boron to mask a potassium acyltrifluoroborate as a stable zwitterionic organoboron complex during Fmoc solid-phase peptide synthesis, and is removed with DMPU-HF with retention of configuration.

Read the paper

• Publisher version (doi:10.1126/science.aea7511)
• PubMed 41643010
• Preprint (ChemRxiv, 2025)

How to cite

Schilling, P. E.; Steiner, S.; Bode, J. W. Zwitterionic organoboron complexes for overcoming the concentration barrier in chemical protein synthesis. Science 2026, 391, 598–603. https://doi.org/10.1126/science.aea7511

@article{Schilling2026Zwitterionic,
  author = {Schilling, Philipp E. and Steiner, Samuel and Bode, Jeffrey W.},
  title = {{Zwitterionic organoboron complexes for overcoming the concentration barrier in chemical protein synthesis}},
  journal = {Science},
  volume = {391},
  number = {6785},
  pages = {598--603},
  year = {2026},
  doi = {10.1126/science.aea7511},
  url = {https://doi.org/10.1126/science.aea7511}
}