Leveraging the Aminothiol-Specific Phosphorogenic Response of Iridium(III) Thioester Complexes for the Development of Intracellular Sensors and Cancer Phototherapeutics

Abstract

Site-specific bioconjugation techniques are extensively utilized in biological and biomedical fields to precisely label biomolecules with luminescent tags for direct visualization of their intracellular dynamics or with cytotoxic agents for the development of novel anticancer therapeutics. In this work, a series of cyclometalated iridium-(III) polypyridine complexes featuring a thioester moiety was designed as novel phosphorogenic probes for labeling N-terminal cysteine (N-Cys)-containing biomolecules. These thioester complexes were weakly emissive in solutions due to the presence of a low-lying nonradiative distorted triplet intraligand (³IL) state localized on the thioester unit, as elucidated by computational analyses. However, their emission intensities and singlet oxygen (¹O₂)-photosensitization efficiencies substantially increased upon reaction with l-Cys due to the conversion of the quenching thioester moiety to a nonquenching amide unit. Additionally, the thioester complexes exhibited high selectivity toward N-Cys and displayed significantly enhanced reactivity due to the electron-withdrawing iridium-(III) polypyridine moiety. The remarkable aminothiol-induced emission and ¹O₂-photosensitization turn-on of the thioester complexes were exploited for the development of intracellular Cys sensors and Cys-activatable photosensitizers for cancer-targeted photodynamic therapy. Furthermore, one of the thioester complexes was selected to react with various N-Cys-modified tumor-targeting peptides, yielding photofunctional iridium-(III)-peptide conjugates with high ¹O₂ generation efficiencies. These conjugates retained the tumor-targeting capabilities of the original peptides and showed high specificity for MDA-MB-231 cells compared to MCF-7 and HEK-293 cells, resulting in selective photocytotoxicity toward this triple-negative breast cancer cell line. We believe that our design approach will inspire the development of novel luminogenic thioester-based reagents for bioconjugation, bioimaging, and therapeutic applications.

Keywords: N-terminal cysteine; bioconjugation; bioimaging; intracellular sensing; iridium; phosphorogenic; photosensitizers; thioester.

1. Structures of the Iridium­(III) Thioester Complexes 1a–4a and Ester Complexes 1b–4b

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Perspective view of the cation of complex 1a, [Ir­(pq)₂(bpy-COSBn)]⁺. Thermal ellipsoids are shown at the 30% probability level. Hydrogen atoms are omitted for clarity.

1. Conjugation of the Thioester Complexes to N-Cys-Containing Biomolecules via NCL

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(a) HPLC chromatograms of the reaction mixtures of complex 1a (25 μM) without (control) and with CSS (1 mM), SCS (1 mM), and SSC (1 mM) in potassium phosphate buffer (50 mM, pH 7.0)/DMSO (3:2, v/v) containing TCEP (10 mM) after incubation at 298 K for 1 h. The absorbance was monitored at 350 nm. (b) ESI mass spectrum of the eluent collected at t _R = 5.8 min of the reaction of complex 1a and CSS.

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Emission spectra of complexes 1a–4a (10 μM) before (black) and after (red) incubation with l-Cys (100 μM) in aerated potassium phosphate buffer (50 mM, pH 7.0)/CH₃CN (3:2, v/v) containing TCEP (1 mM) at 298 K for 1 h.

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Spin densities of the emissive ³MLCT state and nonemissive ³DS states at their optimized structures for complexes 1a-Me, 1a-Cys, and 1b. Computed energy levels (eV) with respect to the optimized S₀ state are provided.

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LSCM images of live HeLa cells incubated with complex 1a (10 μM, 1 h, λ_ex = 405 nm, λ_em = 560–660 nm) without or with pretreatment of NEM (100 μM, 20 min) or with pretreatment of NEM (100 μM, 20 min) and l-Cys (100 μM, 30 min). Scale bars = 25 μm.

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LSCM images of live MDA-MB-231, MCF-7, and HEK-293 cells incubated with conjugates 3a-Cys, 3a-CASP, 3a-CYNT, and 3a-CMYI (10 μM, 16 h, λ_ex = 488 nm, λ_em = 650–750 nm). Scale bars = 25 μm.

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LSCM images of live MDA-MB-231 cells incubated with conjugates 3a-Cys, 3a-CASP, 3a-CYNT, and 3a-CMYI (10 μM, 16 h, λ_ex = 488 nm, λ_em = 650–750 nm) and further incubated with LysoTracker Deep Red (100 nM, 1 h, λ_ex = 635 nm, λ_em = 660–680 nm). Pearson’s correlation coefficient (PCC) = 0.80 (3a-Cys), 0.72 (3a-CASP), 0.69 (3a-CYNT), and 0.81 (3a-CMYI). Scale bars = 25 μm.