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  • Synthesis and evaluation of a series of 1,2,4,5-tetrazines for bioorthogonal conjugation.

Synthesis and evaluation of a series of 1,2,4,5-tetrazines for bioorthogonal conjugation.

Bioconjugate chemistry (2011-09-29)
Mark R Karver, Ralph Weissleder, Scott A Hilderbrand
ABSTRACT

1,2,4,5-Tetrazines have been established as effective dienes for inverse electron demand [4 + 2] Diels-Alder cycloaddition reactions with strained alkenes for over 50 years. Recently, this reaction pair combination has been applied to bioorthogonal labeling and cell detection applications; however, to date, there has been no detailed examination and optimization of tetrazines for use in biological experiments. Here, we report the synthesis and characterization of 12 conjugatable tetrazines. The tetrazines were all synthesized in a similar fashion and were screened in parallel to identify candidates most ideally suited for biological studies. In depth follow-up studies revealed compounds with varying degrees of stability and reactivity that could each be useful in different bioorthogonal applications. One promising, highly stable, and water-soluble derivative was used in pretargeted cancer cell labeling studies, confirming its utility as a bioorthogonal moiety.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
(E)-Cyclooct-4-enol
Sigma-Aldrich
TCO PEG4 succinimidyl ester
Sigma-Aldrich
TCO-amine HCl salt
Sigma-Aldrich
(E)-Cyclooct-4-enyl 2,5-dioxo-1-pyrrolidinyl carbonate
Sigma-Aldrich
(4-(1,2,4,5-Tetrazin-3-yl)phenyl)methanamine hydrochloride, 95%