Bioorthogonal Postlabeling Reveals Nuclear Localization of a Highly Cytotoxic Half-Sandwich Ir(III) Tetrazine Complex in Live Cells

Abstract

Intracellular imaging of anticancer metallodrugs often relies on prelabeling with organic fluorophores, which significantly affects their physicochemical properties and intracellular distribution. On the other hand, the reported postlabeling strategies based on click-chemistry reactions require cell fixation and permeabilization. Here, this study presents a postlabeling approach based on the catalyst-free, inverse electron-demand Diels-Alder reaction (iEDDA) between a strained fluorescein-tagged bicyclononyne derivative (BCN-FAM) and half-sandwich Ir(III) complexes containing bidentate ligands comprising a tetrazine (Tz-R,R') entity. Five half-sandwich Ir(III) complexes with formula [Cp*Ir(Tz-R,R')Cl]^0/+ have been synthesized and fully characterized, including the X-ray crystal structures of three of the five derivatives. Investigations of their stability and their reactivity in aqueous solution and in a model iEDDA reaction reveal the strong influence of the tetrazine ligand structure on the chemical properties of the corresponding complexes. A highly cytotoxic metallodrug candidate (Ir-C,N_Ph,Me) is identified from biological studies, and chemical reactivity studies disclose an unusual preference for binding of methionine over cysteine. Postlabeling of Ir-C,N_Ph,Me in live HeLa cells highlights its preferential accumulation within the nucleus, suggesting its retention through covalent modifications of nuclear proteins in good agreement with other half-sandwich iridium(III) complexes.

Keywords: antitumor agents; cellular imaging; fluorescent probes; inverse electron‐demand Diels–Alder; iridium; solvolysis; strained alkynes.

Figure 1

Representative examples of half‐sandwich Ir(III) complexes conjugated to organic fluorophores (BODIPY for I, II, and V; coumarin for III; and rhodamine for IV).

Scheme 1

Postlabeling strategy developed in this work.

Scheme 2

Synthetic routes to the Ir(III) tetrazine complexes. Procedure A: (i) AcONa (3 eq.), MeOH, RT, 15 min; (ii) Tz‐Ph,R, RT, 24–72 h. Procedure B: (i) Tz‐Py,R, MeOH, RT, 24 h; (ii) NH₄PF₆ (3 eq.).

Figure 2

Molecular structures of Ir‐N,N _Py,Me (top left), Ir‐C,N _Ph,Me (top right), and Ir‐N,N _Py,Py (bottom). Hydrogen atoms and counterions were omitted for clarity.

Figure 3

HPLC traces of Ir‐C,N _Ph,Me (10 μm in DMEM + 0.1% DMSO) immediately after dilution and after 30 min at room temperature. The intense peak observed around t _R = 2 min is due to DMEM components.

Figure 4

In vivo imaging of Ir‐C,N _Ph,Me in HeLa cells using BCN‐FAM as a sensor. HeLa cells were sequentially exposed to 2 μm Ir‐C,N _Ph,Me or Ir‐N,N _Py,Me for 30 min, and then to 8 μm BCN‐FAM for 4 h. After several washes to remove unbound freely diffusible BCN‐FAM, live confocal microscopy was then immediately performed to acquire the fluorescence signal (shown here as the sum of five confocal planes). Cells were also visualized by transmitted light to identify nuclei and nuclear bodies including nucleoli, which appeared darker. A robust fluorescence signal was detected in cells exposed to Ir‐C,N _Ph,Me but not in cells exposed to Ir‐N,N _Py,Me or the DMSO vehicle, indicating the presence of Ir‐C,N _Ph,Me in cells and its tracking with BCN‐FAM.