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Thio-NHS esters are non-innocent protein acylating reagents

Author

Listed:
  • Weibing Liu

    (The Rosalind Franklin Institute
    University of Oxford)

  • Aziz Khan

    (The Rosalind Franklin Institute
    University of Oxford)

  • Yana Demyanenko

    (The Rosalind Franklin Institute)

  • Shabaz Mohammed

    (The Rosalind Franklin Institute
    University of Oxford
    University of Oxford)

  • Benjamin G. Davis

    (The Rosalind Franklin Institute
    University of Oxford
    University of Oxford)

Abstract

N-Hydroxysuccinimide (NHS)-ester derivatives are widely used reagents in biological chemistry and chemical biology. Their efficacy relies critically on the exclusive chemoselectivity of activated acyl over that of the imidic acyl moieties in the succinimide. Here, through systematic structural variation that modulates acyl reactivity, coupled with a statistically controlled ultra-rapid screen for unknown modifications in tandem mass spectra as well as lysine profiling across complex lysine environments, including those within proteomes containing many thousands of proteins, we reveal that ring-opening to afford N-succinamide derivatives is a present, sometimes dominant, side-reaction. The extent of side-reaction is shown to be site-dependent, with side-reaction and desired reaction occurring within the same protein substrate. The resulting formation of bioconjugates with unintended, unstable linkages and modifications suggests the re-evaluation of: (i) known commercial reagents; and (ii) functional conclusions previously drawn using NHS esters in areas as diverse as antibody-drug biotherapy, vaccination and cross-link-enabled structural analyses.

Suggested Citation

  • Weibing Liu & Aziz Khan & Yana Demyanenko & Shabaz Mohammed & Benjamin G. Davis, 2025. "Thio-NHS esters are non-innocent protein acylating reagents," Nature Communications, Nature, vol. 16(1), pages 1-12, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-60527-5
    DOI: 10.1038/s41467-025-60527-5
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    1. Gail D. Lewis & Guangmin Li & Jun Guo & Shang-Fan Yu & Carter T. Fields & Genee Lee & Donglu Zhang & Peter S. Dragovich & Thomas Pillow & BinQing Wei & Jack Sadowsky & Douglas Leipold & Tim Wilson & A, 2024. "The HER2-directed antibody-drug conjugate DHES0815A in advanced and/or metastatic breast cancer: preclinical characterization and phase 1 trial results," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
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