A Minimal Tetrazine Linchpin for Cysteine Bridging and Bioorthogonal Ligation
4 affiliations
- 1Yusuf Hamied Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge, United Kingdom, CB2 1EW
- 2Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge, United Kingdom, CB2 1PD
- 3Engineering Biology Interdisciplinary Research Centre
- 4University of Cambridge
Abstract
Site-selective bridging of cysteines and native disulfide bonds represents a powerful strategy for peptide and protein modification. However, most bis-electrophilic reagents rely on azide-alkyne cycloadditions for downstream bioconjugation, while existing tetrazine-based cysteine linkers can generate undesired byproducts, such as peptide cyclodimers, or incorporate additional components that are not directly involved in conjugation or ligation. Here, we report divinyl- s -tetrazine ( DvTz ) as a minimal linchpin in which the electron-deficient tetrazine core activates the vinyl groups as Michael acceptors for efficient cysteine conjugation, while simultaneously serving as a bioorthogonal handle for subsequent inverse-electron-demand Diels-Alder (IEDDA) ligation. DvTz enables high-yielding, cysteine-selective stapling of unprotected peptides, tolerates all twenty canonical amino acids, and accommodates a broad range of sequences and stapling distances. A one-pot stapling-IEDDA sequence enables facile installation of functional tags without intermediate purification. The chemistry was extended to antibody Fab fragments, achieving site-specific disulfide rebridging and subsequent IEDDA ligation across three distinct Fab proteins, with exclusive formation of singly conjugated species. A Fab-drug conjugate was constructed with retained antigen-binding activity and selective potency against HER2-positive cell lines, showcasing the application of DvTz in therapeutically relevant settings. Collectively, this work establishes DvTz as a simple and versatile linchpin for peptide and protein bioconjugation.
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BibTeX
@misc{yang2026minimal,
title = {{A Minimal Tetrazine Linchpin for Cysteine Bridging and Bioorthogonal Ligation}},
author = {Tianyi Yang and Yujia Cui and Danye Chen and Daria K. Wanic and Linwei Zeng and Mark R. Howarth and David R. Spring},
howpublished = {ChemRxiv},
year = {2026},
month = oct,
doi = {10.26434/chemrxiv.15007631/v2},
url = {https://chemrxiv.org/doi/full/10.26434/chemrxiv.15007631/v2},
note = {CC BY-NC-ND},
}
RIS
TY - UNPB
TI - A Minimal Tetrazine Linchpin for Cysteine Bridging and Bioorthogonal Ligation
AU - Tianyi Yang
AU - Yujia Cui
AU - Danye Chen
AU - Daria K. Wanic
AU - Linwei Zeng
AU - Mark R. Howarth
AU - David R. Spring
PY - 2026
DA - 2026/10/27
PB - ChemRxiv
DO - 10.26434/chemrxiv.15007631/v2
UR - https://chemrxiv.org/doi/full/10.26434/chemrxiv.15007631/v2
AB - Site-selective bridging of cysteines and native disulfide bonds represents a powerful strategy for peptide and protein modification. However, most bis-electrophilic reagents rely on azide-alkyne cycloadditions for downstream bioconjugation, while existing tetrazine-based cysteine linkers can generate undesired byproducts, such as peptide cyclodimers, or incorporate additional components that are not directly involved in conjugation or ligation. Here, we report divinyl- s -tetrazine ( DvTz ) as a minimal linchpin in which the electron-deficient tetrazine core activates the vinyl groups as Michael acceptors for efficient cysteine conjugation, while simultaneously serving as a bioorthogonal handle for subsequent inverse-electron-demand Diels-Alder (IEDDA) ligation. DvTz enables high-yielding, cysteine-selective stapling of unprotected peptides, tolerates all twenty canonical amino acids, and accommodates a broad range of sequences and stapling distances. A one-pot stapling-IEDDA sequence enables facile installation of functional tags without intermediate purification. The chemistry was extended to antibody Fab fragments, achieving site-specific disulfide rebridging and subsequent IEDDA ligation across three distinct Fab proteins, with exclusive formation of singly conjugated species. A Fab-drug conjugate was constructed with retained antigen-binding activity and selective potency against HER2-positive cell lines, showcasing the application of DvTz in therapeutically relevant settings. Collectively, this work establishes DvTz as a simple and versatile linchpin for peptide and protein bioconjugation.
ER -