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

journal contribution
posted on 31.07.2020 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 -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 S. 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 labelling with a reactive Cy3-functionalized tetrazine.

Funding

University of Aberdeen

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

History

School

  • Science

Department

  • Chemistry

Published in

Bioconjugate Chemistry

Publisher

American Chemical Society (ACS)

Version

AM (Accepted Manuscript)

Rights holder

© American Chemical Society

Publisher statement

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 https://pubs.acs.org/doi/10.1021/acs.bioconjchem.0c00375.

Publication date

2020-07-30

Copyright date

2020

ISSN

1043-1802

eISSN

1520-4812

Language

en

Depositor

Prof Matteo Zanda. Deposit date: 31 July 2020

Article number

acs.bioconjchem.0c00375

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