Arylethynyltrifluoroborate Dienophiles for on Demand Activation of IEDDA Reactions

Abstract

Strained alkenes and alkynes are the predominant dienophiles used in inverse electron demand Diels-Alder (IEDDA) reactions. However, their instability, cross-reactivity, and accessibility are problematic. Unstrained dienophiles, although physiologically stable and synthetically accessible, react with tetrazines significantly slower relative to strained variants. Here we report the development of potassium arylethynyltrifluoroborates as unstrained dienophiles for fast, chemically triggered IEDDA reactions. By varying the substituents on the tetrazine (e.g., pyridyl- to benzyl-substituents), cycloaddition kinetics can vary from fast (k₂ = 21 M^-1 s^-1) to no reaction with an alkyne-BF₃ dienophile. The reported system was applied to protein labeling both in the test tube and fixed cells and even enabled mutually orthogonal labeling of two distinct proteins.

Figure 1

IEDDA reaction between a dienophile and a tetrazine. (a) Strained and (b) unstrained dienophiles as tetrazine coupling partners in IEDDA reactions. (c) Reaction between vinylboronic acid and a dipyridyl tetrazine. (d) Chemically triggered, fast and selective IEDDA reaction between potassium arylethynyltrifluoroborates and pyridyl tetrazines. Py = pyridyl.

Figure 2

Aqueous reaction between HBF₃ and dPy-Tz. (a–d) Screening of additives. Reaction conditions: dPy-Tz (4 mM), HBF₃ (4 mM), and additives (100 mM) in MeOH/PBS (40%). Monitored at 530 nm at 30 °C. See the Supporting Information (Figure S4) for results with MgCl₂ and ZnCl₂ as additives.

Figure 3

Kinetic and computational studies. (a) Half-life of the reaction between arylethynyltrifluoroborate salts (4 mM), dPy-Tz (4 mM), and AlCl₃·6H₂O (20 mM) in a mixture of 40% MeOH/PBS followed at 530 nm at 30 °C. (b and c) Absorbance decay upon reaction of OBF₃ with different tetrazines. (d) Complete energy profile for the reaction of arylethynyltrifluoroborate HBF₃ and tetrazine dPy-Tz calculated at PCM(H₂O)/M06-2X/6-31+G(d,p) level. The activation (i.e., defluorination, in red), IEDDA reaction (in blue) and retro-DA leading to the final pyridazine (in yellow) are all fast and thermodynamically very favored. Activation (ΔG^‡) and reaction energies (ΔG) are in kcal mol^–1. (e) Comparison of reported second-order rate constants. (f) Reaction between optimal ethynyltrifluoroborate OBF₃ (24 mM) and tetrazine dPy-Tz (20 mM) triggered by AlCl₃·6H₂O.

Figure 4

(a) Dependence of relative rate of model reaction between dPy-Tz and HBF₃ on pH. (b) Theoretical relative rate constants (k_obs) calculated at different pH values for the parallel, competitive reactions of HBF₃ with dPy-Tz in different protonation states (dPy-Tz: neutral; dPyH⁺-Tz: protonated; dPyH₂²⁺-Tz: doubly protonated). The curve was generated using the intrinsic reaction rate constants (k₁, k₂, and k₃) derived from the corresponding calculated activation barriers (ΔG^‡), and the equilibrium constants (pK_a1 = 2.0; pK_a2 = 1.4) estimated with Marvin 19.19.0, 2019, ChemAxon (http://chemaxon.com).

Figure 5

Click reaction between norbornene derivatives (1 and 2) and dPy-Tz measured under identical conditions as reaction with HBF₃.

Figure 6

Protein site-selective modification by Al³⁺-triggered IEDDA reaction. (a) Carbonylacrylic linkers with OBF₃ or dPy-Tz. (b) Protein scaffolds used. (c–e) Installation of caa-dPy-Tz on Ub followed by IEDDA reaction with OBF₃, and deconvoluted mass spectra of respective products. (f–h) Installation of caa-OBF₃ on Ub followed by IEDDA reaction with dPy-Tz, and deconvoluted mass spectra of respective products. See Chapter 6 in the SI for labeling of C2Am and 2RB17C. (i) Structural ensemble obtained from 0.5 μs MD simulations of the conjugate prepared by reacting Ub-caa-OBF₃ with dPy-Tz. The numbers indicate the rmsd for heavy-atom superimposition of the backbone of the protein relative to the starting structure. DMF = N,N-dimethylformamide, DMSO = dimethyl sulfoxide.

Figure 7

(a) Structures of tetrazine fluorophores. (b) Selective labeling of Ub-caa-OBF₃ with two tetrazine fluorophores as analyzed by SDS-polyacrylamide gel electrophoresis.

Figure 8

Mutually orthogonal labeling of Lyz-Nor and Ub-caa-BF₃ with tetrazine fluorophores PhMe-Tz-Cy3 and dPy-Tz-BODIPY. (a) Representative scheme. (b) SDS–polyacrylamide gel electrophoresis analysis. From left to right: BODIPY channel, Cy3 channel, fluorescence overlap.

Figure 9

Epifluorescent microscopy of SK-BR-3 cells treated with dPy-Tz-BODIPY. (a) Prior to the treatment with the fluorophore, the cells were treated with nanobody 2RB17C-caa-OBF₃. (b) Control experiment–nanobody treatment was omitted. Scale bar represents 40 μm. For better comparison and contrast, the pictures were thresholded from the original range 0–255 to 2–30.