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trans-Cyclooctene--a stable, voracious dienophile for bioorthogonal labeling - PubMed

Review

trans-Cyclooctene--a stable, voracious dienophile for bioorthogonal labeling

Ramajeyam Selvaraj et al. Curr Opin Chem Biol. 2013 Oct.

Abstract

Discussed herein is the development and advancement of trans-cyclooctene as a tool for facilitating bioorthogonal labeling through reactions with s-tetrazines. While a number of strained alkenes have been shown to combine with tetrazines for applications in bioorthogonal labeling, trans-cyclooctene enables fastest reactivity at low concentration with rate constants in excess of k2=10(6) M(-1) s(-1). In the present article, we describe advances in computation and synthesis that have enabled applications in chemical biology and nuclear medicine.

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Figures

Scheme 1
Scheme 1

trans-Cyclooctene (TCO) as a tool for chemical biology and in vivo imaging. (a) The advancement of TCO as a tool is a multidisciplinary effort involving high-level computation, new synthetic methodology, chemical biology and nuclear medicine. (b) The significance of high rate constants is illustrated by simple table of rate data and corresponding half-lives. (c) Mechanism of the tetrazine–TCO ligation.

Scheme 2
Scheme 2

Tetrazine–TCO ligation as a tool for nuclear medicine (a) Pretargeted SPECT imaging in live mice through initial administration of a TCO-antibody conjugate with later administration of a tetrazine–111In-DOTA conjugate. (b) 18F-labeled TCO 12 enables rapid construction of PET-probes. In the shown example, the importance of efficient labeling at nearly equimolar stoichiometry is emphasized by a blocking experiment, where coinjecting with a 5-fold excess of unlabeled exendin-4 resulted in a greatly reduced signal. (c) Tetrazines commonly utilized in bioconjugation studies.

Scheme 3
Scheme 3

(a) TCO conformation has a significant effect on strain energy in the ground state. (b) Computation correctly predicted that a conformationally strained TCO (‘s-TCO’) would display enhanced reactivity relative to parent TCO. (c) With more reactive tetrazine 21, s-TCO derivative reacts with a rate that is too quick to measure by stopped flow kinetics.

Scheme 4
Scheme 4

Tetrazine–TCO ligation with genetically encoded proteins (a) A tetrazine-derived unnatural amino acid can be genetically encoded site-specifically into proteins of interest. s-TCO derivatives can be used to tag the unnatural amino acids with fast rates in vivo. (b) A TCO-derivatized lysine has been site specifically incorporated into proteins in E. coli and mammalian cells, and used for rapid fluorogenic labeling in live cells. (c) Current limitations of s-TCO.

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