posted on 2020-06-10, 12:11authored byArthur
H. A. M. van Onzen, Ron M. Versteegen, Freek J. M. Hoeben, Ivo A. W. Filot, Raffaella Rossin, Tong Zhu, Jeremy Wu, Peter J. Hudson, Henk M. Janssen, Wolter ten Hoeve, Marc S. Robillard
The high rate of the ‘click-to-release’
reaction
between an allylic substituted trans-cyclooctene
linker and a tetrazine activator has enabled exceptional control over
chemical and biological processes. Here we report the development
of a new bioorthogonal cleavage reaction based on trans-cyclooctene and tetrazine, which allows the use of highly reactive trans-cyclooctenes, leading to 3 orders of magnitude higher
click rates compared to the parent reaction, and 4 to 6 orders higher
than other cleavage reactions. In this new pyridazine elimination
mechanism, wherein the roles are reversed, a trans-cyclooctene activator reacts with a tetrazine linker that is substituted
with a methylene-linked carbamate, leading to a 1,4-elimination of
the carbamate and liberation of a secondary amine. Through a series
of mechanistic studies, we identified the 2,5-dihydropyridazine tautomer
as the releasing species and found factors that govern its formation
and subsequent fragmentation. The bioorthogonal utility was demonstrated
by the selective cleavage of a tetrazine-linked antibody–drug
conjugate by trans-cyclooctenes, affording efficient
drug liberation in plasma and cell culture. Finally, the parent and
the new reaction were compared at low concentration, showing that
the use of a highly reactive trans-cyclooctene as
the activator leads to a complete cycloaddition reaction with the
antibody–drug conjugate in seconds vs hours for the parent
system. Although the subsequent release from the IEDDA adduct is slower,
we believe that this new reaction may allow markedly reduced click-to-release
reagent doses in vitro and in vivo and could expand the application scope to conditions wherein the trans-cyclooctene has limited stability.