Isonitrile-responsive and bioorthogonally removable tetrazine protecting groups

Abstract

In vivo compatible reactions have a broad range of possible applications in chemical biology and the pharmaceutical sciences. Here we report tetrazines that can be removed by exposure to isonitriles under very mild conditions. Tetrazylmethyl derivatives are easily accessible protecting groups for amines and phenols. The isonitrile-induced removal is rapid and near-quantitative. Intriguingly, the deprotection is especially effective with (trimethylsilyl)methyl isocyanide, and serum albumin can catalyze the elimination under physiological conditions. NMR and computational studies revealed that an imine-tautomerization step is often rate limiting, and the unexpected cleavage of the Si-C bond accelerates this step in the case with (trimethylsilyl)methyl isocyanide. Tetrazylmethyl-removal is compatible with use on biomacromolecules, in cellular environments, and in living organisms as demonstrated by cytotoxicity experiments and fluorophore-release studies on proteins and in zebrafish embryos. By combining tetrazylmethyl derivatives with previously reported tetrazine-responsive 3-isocyanopropyl groups, it was possible to liberate two fluorophores in vertebrates from a single bioorthogonal reaction. This chemistry will open new opportunities towards applications involving multiplexed release schemes and is a valuable asset to the growing toolbox of bioorthogonal dissociative reactions.

Fig. 1. Proposed reaction to achieve the dual release of biological effectors from previously reported 3-isocyanopropyl (ICPr) and tetrazylmethyl (TzMe) derivatives developed herein.

Fig. 2. Isonitrile-mediated uncaging of amines and phenols from Tzmoc and TzMe derivatives. (a) Structures of tetrazylmethyloxycarbonyl (Tzmoc) and tetrazylmethyl (TzMe) groups used to cage amines and phenols, respectively. (b) Synthesis of Tzmoc or TzMe-caged probes (conditions and yields described in the ESI†). (c) Structures of reporter probes and isonitrile triggers used in this study. (d) Kinetics of pNA release from 4a triggered by different isonitriles (c(4a) = 0.2 mM, c(R–NC) = 2 mM, DMSO : PBS pH 7.4 (4 : 1, v/v), T = 37 °C, λ = 435 nm, n = 3). (e) Kinetics of pNA release from 4a triggered by n-BuNC catalysed by serum albumin (c(4a) = 8 μM, c(n-BuNC) = 6 mM, c(HSA) = 2 mg mL^–1, DMSO : PBS pH 7.4 (1 : 4, v/v), T = 37 °C, λ = 385 nm, n = 3). (f) Kinetics of O-carboxymethyl fluorescein release from 4b triggered by TMS-MeNC or n-BuNC (c(4b) = 8 μM, c(R–NC) = 6 mM, DMSO : PBS pH 7.4 (1 : 4, v/v), T = 37 °C, λ_ex = 488 nm, λ_em = 520 nm, n = 3).

Fig. 3. Effect of structural modifications to tetrazines on isonitrile-induced removal. (a) Structures of modified Tzmoc probes with a pNA reporter molecule. (b) Half-lives of the TMS-MeNC mediated Tzmoc deprotection (t_1/2 of release of pNA) from probes 4a, 4c, 4d, and 4e (c(4a–e) = 0.2 mM, c(TMS-MeNC) = 2 mM, DMSO : PBS pH 7.4 (4 : 1, v/v), T = 37 °C). (c) Release yields of pNA or O-carboxymethyl fluorescein from 4a or 4b, respectively, triggered by several dienophiles (structures shown in Fig. S10 in the ESI†); pNA release: see Fig. 2d; t = 8 h; O-carboxymethyl fluorescein release: c(4b) = 8 μM, c(dienophile) = 2 mM, DMSO : PBS pH 7.4 (1 : 4, v/v), T = 37 °C, λ_ex = 488 nm, λ_em = 520 nm, t = 8 h.

Fig. 4. ¹H-NMR analysis of reactions between isonitriles n-BuNC or TMS-MeNC and 4a to liberate pNA. (a and d) Proposed intermediates in the reaction between n-BuNC (a) or TMS-MeNC (d) and 4a leading to the release of pNA. (b and e) Time-dependent ¹H-NMR of the reaction progress between n-BuNC (b) or TMS-MeNC (e) and 4a (c(4a) = 6 mM, c(R–NC) = 15 mM, DMSO-d₆ : D₂O (9 : 1, v/v), T = 25 °C, expanded spectra in ESI). (c and f) Normalized amount of starting material (4a), subsequent intermediates, and pNA formed as a function of time.

Fig. 5. Investigated mechanistic pathways. (a) Cleavage of TMS from A1 by water with subsequent protonation. (b) Tautomerization of B1 induced by abstraction of a proton by water. (c) Cleavage of TMS from A1 by protonation followed by abstraction by water. (d) Tautomerization of B1 induced by protonation. (e) Predicted S_N2 transition state TS_A1>A2. (f) Cleavage of TMS from TMS-MeNC by water.

Fig. 6. TMS-MeNC mediated removal of TzMe-modified molecules on proteins, in the presence of cells, and in zebrafish embryos. (a) In-gel analysis of the fluorescent turn-on signal on SNAP protein labelled with 4b-BG (10 μM) and subsequent deprotection of the TzMe group with TMS-MeNC (100 μM); lanes 1 and 6 contain the protein ladder (for an expanded view of the fluorescence and Coomassie-stained gel and mass spectroscopy verification, see Fig. S34–S37 in the ESI†). (b) Structure of Tzmoc-caged doxorubicin prodrug (5) and dose–response curves for cytotoxicity studies with A549 cells after 72 h. (c) Cartoon representation of experiment to demonstrate the release of O-carboxymethyl fluorescein and fluorescence turn-on upon incubation with TMS-MeNC. (d) Visualization of the fluorescence signal in live zebrafish (scale bar = 200 μm) after a 2 h incubation with either 20 μM TMS-MeNC or DMSO.

Fig. 7. Dual release of two orthogonal fluorophores from ICPr- and TzMe-caged dyes in live zebrafish. (a) Cartoon representation of experiment demonstrating the dual release and fluorescence turn-on of O-carboxymethyl fluorescein and resorufin upon reaction of 4b and ICPr-rsf, and the corresponding control experiment with non-injected fish. (b) Visualization of fluorescein and resorufin fluorescence signal in live zebrafish (scale bar = 200 μm) injected with 4b after a 2 h incubation with 10 μM ICPr-rsf. (c) Visualization of fluorescein and resorufin fluorescence signal in non-4b injected control live zebrafish (scale bar = 200 μm) after a 2 h incubation with 10 μM ICPr-rsf.