Intramolecular tetrazine-acryloyl cycloaddition: chemistry and applications

Abstract

An unprecedented intramolecular [4 + 2] tetrazine-olefin cycloaddition with α,β-unsaturated substrates was discovered. The reaction produces unique coumarin-dihydropyridazine heterocycles that exhibited strong fluorescence with large Stokes shifts and excellent photo- and pH-stability. This property can be used for reaction analysis. The rate of cycloaddition was found to be solvent dependent and was determined using experimental data with a kinetic modeling software (COPASI) as well as DFT calculations (k ₁ = 0.64 ± 0.019 s^-1 and 4.1 s^-1, respectively). The effects of steric and electronic properties of both the tetrazine and α,β-unsaturated carbonyl on the reaction were studied and followed the known trends characteristic of the intermolecular reaction. Based on these results, we developed a "release-then-click" strategy for the ROS triggered release of methylselenenic acid (MeSeOH) and a fluorescent tracer. This strategy was demonstrated in HeLa cells via fluorescence imaging.

Scheme 1. The design of intramolecular tetrazine-acryloyl cycloaddition.

Scheme 2. Initial studies of the intramolecular tetrazine-acryloyl cycloaddition.

Fig. 1. Substrate scope containing various O-hydroxyphenyl tetrazines. Tetrazine (2a–h, 0.1 mmol), triethylamine (0.2 mmol), acryloyl chloride (0.2 mmol), CH₃CN (1.0 mL). ^a Reaction was done under argon for 1 h. ^b Reaction was allowed to run for 24 h.

Scheme 3. Methyl-decorated acryloyl substrates in cycloaddition.

Fig. 2. Formation of 4h from 15 monitored by fluorescence. Compound 15 (100 μM) was incubated in 10% DMSO and [NaOH] at 25 °C. λ_em: 425 nm, λ_ex: 320 nm. (a) 1.0 M NaOH, (b) 0.1 M NaOH, (c) 0.01 M NaOH, (d) 0.001 M NaOH.

Fig. 3. DFT calculations for the intramolecular cycloaddition of 16. Calculations were done on SMD(H₂O)/M06-2X/6-311G(d).

Fig. 4. Increasing [H₂O₂]: 19 (100 μM), (a) 100.0 mM H₂O₂, (b) 50.0 mM H₂O₂ (c) 10.0 mM H₂O₂, (d) 1.0 mM H₂O₂ in 10% DMSO and 10 mM PBS buffer (pH = 7.4) at 25 °C. Oxidant Screen: 19 (100 μM) and oxidant (200 μM) in 10% DMSO and 10 mM PBS buffer (pH = 7.4) incubated at 37 °C for 1 h (+) Control: the positive control is reported as the average maximum intensities with pure compound 4h in 10% DMSO and 10.0 mM PBS buffer (pH = 7.4) incubated with each oxidant (200 μM) individually at 37 °C for 1 h.

Fig. 5. Fluorescent images of 4h release from 19 in HeLa cells: (a and b) cells were pre-treated with NAC (100 μM) and then treated with 19 (200 μM) for 1 h at 37 °C, (c and d) cells were treated with 19 for 1 h at 37 °C, (e and f) cells were treated with 19 for 1 h then washed and treated with H₂O₂ (200 μM) for 30 min at 37 °C, (g and h) cells were treated with 19 for 1 h then washed and treated with HOCl (200 μM) for 30 min at 37 °C.