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

## TL;DR

This study introduces a new method to track a toxic iridium-based drug in live cells, showing it accumulates in the nucleus.

## Contribution

A bioorthogonal postlabeling strategy for live-cell imaging of a half-sandwich Ir(III) complex without altering its properties.

## Key findings

- Five half-sandwich Ir(III) complexes were synthesized and characterized, including X-ray structures of three.
- The most cytotoxic complex shows a preference for methionine over cysteine in binding.
- Live-cell imaging reveals nuclear localization of the complex, likely due to covalent interactions with nuclear proteins.

## 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.

This study reports the synthesis of five tetrazine‐coordinated, half‐sandwich iridium complexes and extensively describe their reactivity. Live cell fluorescent bioorthogonal labeling reveals the nuclear tropism of the most cytotoxic complex.© 2025 WILEY‐VCH GmbH