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Design, synthesis, conjugation and reactivity of novel trans,trans-1,5-cyclooctadiene-derived bioorthogonal linkers

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journal contribution
posted on 31.07.2020, 13:04 by Beatrice Longo, Chiara Zanato, Monica Piras, Sergio Dall'Angelo, Albert D Windhorst, Danielle Vugts, Massimiliano Baldassarre, Matteo Zanda
The tetrazine/trans-cyclooctene (TCO) inverse electron-demand Diels–Alder (IEDDA) reaction is the fastest bioorthogonal “click” ligation process reported to date. In this context, TCO reagents have found widespread applications; however, their availability and structural diversity is still somewhat limited due to challenges connected with their synthesis and structural modification. To address this issue, we developed a novel strategy for the conjugation of TCO derivatives to a biomolecule, which allows for the creation of greater structural diversity from a single precursor molecule, i.e., trans,trans-1,5-cyclooctadiene [(E,E)-COD] 1, whose preparation requires standard laboratory equipment and readily available reagents. This two-step strategy relies on the use of new bifunctional TCO linkers (5a–11a) for IEDDA reactions, which can be synthesized via 1,3-dipolar cycloaddition of (E,E)-COD 1 with different azido spacers (5–11) carrying an electrophilic function (NHS-ester, N-succinimidyl carbonate, p-nitrophenyl-carbonate, maleimide) in the ω-position. Following bioconjugation of these electrophilic linkers to the nucleophilic residue (cysteine or lysine) of a protein (step 1), the resulting TCO-decorated constructs can be subjected to a IEDDA reaction with tetrazines functionalized with fluorescent or near-infrared (NIR) tags (step 2). We successfully used this strategy to label bovine serum albumin with the TCO linker 8a and subsequently reacted it in a cell lysate with the fluorescein-isothiocyanate (FITC)-derived tetrazine 12. The same strategy was then used to label the bacterial wall of Gram-positive Staphylococcus aureus, showing the potential of these linkers for live-cell imaging. Finally, we determined the impact of structural differences of the linkers upon the stability of the bioorthogonal constructs. The compounds for stability studies were prepared by conjugation of TCO linkers 6a, 8a, and 10a to mAbs, such as Rituximab and Obinutuzumab, and subsequent labeling with a reactive Cy3-functionalized tetrazine.


University of Aberdeen

European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 675417



  • Science


  • Chemistry

Published in

Bioconjugate Chemistry






2201 - 2210


American Chemical Society (ACS)


AM (Accepted Manuscript)

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© American Chemical Society

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This document is the Accepted Manuscript version of a Published Work that appeared in final form in Bioconjugate Chemistry, copyright © American Chemical Society after peer review and technical editing by the publisher. To access the final edited and published work see

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Prof Matteo Zanda. Deposit date: 31 July 2020