In Situ Synthesis of Alkenyl Tetrazines for Highly Fluorogenic Bioorthogonal Live‐Cell Imaging Probes

Abstract

In spite of the wide application potential of 1,2,4,5-tetrazines, particularly in live-cell and in-vivo imaging, a major limitation has been the lack of practical synthetic methods. Here we report the in situ synthesis of (E)-3-substituted-6-alkenyl-1,2,4,5-tetrazine derivatives via an elimination-Heck cascade reaction. Using this strategy, we provide 24 examples of π-conjugated tetrazine derivatives that can be conveniently prepared from tetrazine building blocks and related halides. These include tetrazine analogs of biological small molecules, highly conjugated buta-1,3-diene substituted tetrazines, and a diverse array of fluorescent probes suitable for live-cell imaging. These highly conjugated probes show dramatic fluorescent turn-on (up to 400-fold) when reacted with dienophiles such as cyclopropenes and trans-cyclooctenes, and we demonstrate their application for live-cell imaging. This work provides an efficient and practical synthetic methodology for tetrazine derivatives and will facilitate the application of conjugated tetrazines, particularly as fluorogenic probes for live-cell imaging.

Click Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation

“…Increasing the strain in the trans -cyclooctene moiety through cyclopropyl fusion resulted in a 50-fold rate enhancement (Lang et al, 2012b; Seitchik et al, 2012). Additionally, π-conjugated tetrazines exhibit strong fluorescence upon cycloadditions with dienophiles such as cyclopropenes and trans-cycloctene (Wu et al, 2014). Applications of tetrazine-TCO ligations have included labeling of newly synthesized proteins (Lang et al, 2012a) and cancer cells (Devaraj et al, 2010; Devaraj et al, 2009), in vivo cancer imaging with 111 In (Rossin et al, 2010) and 18 F radiolabeling (Keliher et al, 2011; Li et al, 2010; Reiner et al, 2011), cancer cell detection (Haun et al, 2010), fluorescent imaging of cytoskeletal proteins within living mammalian cells (Liu et al, 2012), and recently the methodology has been used with amino acids modified by tetrazine (Seitchik et al, 2012) and trans -cyclooctene (Lang et al, 2012b) for genetic incorporation into proteins.…”

Systemic Fluorescence Imaging of Zebrafish Glycans with Bioorthogonal Chemistry

“…Several groups have reported fluorogenic tetrazine probes that fluoresce upon reacting with strained alkenes and alkynes. [8] We were drawn to a recent study in which Devaraj and co-workers reported para-vinylenemethyltetrazines that exhibit up to 400-fold fluorescence turn-on; [9] we expected these tetrazines to be stable for at least several hours in serum, [10] making them good candidates for in vivo imaging experiments. Tetrazines are known to react with a variety of strained unsaturated hydrocarbons, including norbornene, [11] cyclopropene, [8c, 12] cyclooctyne, [8b,13] and trans-cyclooctene.…”

“…[16] Furthermore, several studies across different classes of fluorophores have shown that the pyridazine products resulting from alkyne-tetrazine cycloadditions are brighter than the dihydropyridazine products resulting from alkene-tetrazine cycloadditions. [9,17] Aside from its beneficial attributes, cyclooctyne has a liability as a chemical reporter: It is large relative to a monosaccharide substrate, unlike azides and terminal alkynes, the more common chemical reporters for glycan imaging. Indeed, unnatural monosaccharides containing modifications larger than a few atoms are generally poor substrates for the biosynthetic enzymes that enable their incorporation into glycans.…”

“…Probe 4 is virtually nonfluorescent, but successful reaction with a cyclooctyne yields a brightly fluorescent Oregon green moiety. [9] Embryos were injected with BCNSia at the 1-8 cell stage and were allowed to develop to various stages prior to imaging experiments. The caudal vein plexus is not fully formed until 30 hpf (hours post fertilization); [24] therefore, an equimolar mixture of 4 and Alexa Fluor (AF) 647 cadaverine, which we added as a nonspecific tracer of the vasculature, was injected into embryos at 30, 48, or 72 hpf.…”

Fluorous Analogue of Chloramine-T: Preparation, X-ray Structure Determination, and Use as an Oxidant for Radioiodination and s-Tetrazine Synthesis

“…Not only can the fluorous oxidant reported here be used to label arylstannanes and proteins, it was also shown to be very effective for producing functionalized tetrazines, which are emerging as highly effective synthons for bioorthogonal labeling reactions. [25][26][27][28] …”