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

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 ( 3 IL) state localized on the thioester unit, as elucidated by computational analyses. However, their emission intensities and singlet oxygen ( 1 O 2 )-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 1 O 2 -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 1 O 2 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. ## Introduction Introduction Bioconjugation holds immense importance in the fields of biochemistry and biomedicine. This process entails the covalent attachment of biomolecules to generate compounds with augmented functionalities. Its versatility is demonstrated across a wide range of applications, such as site-specific labeling of biomolecules for bioimaging and biosensing, targeted drug delivery, and immunoassays. However, traditional bioconjugation techniques, such as those involving the modification of lysine (Lys) and cysteine (Cys) residues, are constrained by drawbacks including the heterogeneous labeling of biomolecules. This complexity complicates purification and has adverse effects on the biophysical and pharmacokinetic properties of the conjugates. Other biocompatible reactions, such as the Staudinger ligation, ketone–hydrazide reaction, and numerous bioorthogonal reactions like the inverse electron-demand Diels–Alder cycloaddition, necessitate the integration of unnatural functional groups into biomolecules or intricate genetic manipulations. N-Terminal cysteine (N-Cys) residues have been employed for site-specific labeling of peptides and proteins due to several key factors. These include the unique reactivity of the 1,2-aminothiol group and the low natural abundance of N-Cys residues, enabling the selective labeling of N-Cys over internal residues. Additionally, the modification of N-Cys residues causes minimal structural perturbation, thereby preserving the native conformation of the biomolecule. Thioesters have conventionally been utilized in ligation reactions with N-Cys residues of unprotected peptides via native chemical ligation (NCL) under physiological conditions for protein synthesis. , The process involves transthioesterification followed by a spontaneous S → N acyl shift to yield a stable amide bond. Additionally, thioester-functionalized fluorophores have been developed as NCL-based probes for detecting biothiols − and labeling peptides and proteins. − Luminescent transition metal complexes have been pivotal in the realm of biological research due to their remarkable photophysical and biological properties. − Our group has a long-standing interest in the development of transition metal complexes as bioconjugation reagents, particularly in the preparation of photofunctional bioconjugates for bioimaging and targeted photodynamic therapy (PDT) applications. − Recently, we have developed iridium­(III) 2-formylphenylboronic acid and 2-cyanobenzothiazole complexes that enable selective modification of N-Cys-containing biomolecules. However, these complexes lack the capability to display phosphorogenic response (i.e., phosphorescence turn-on) upon the conjugation reaction, hindering their potential applications in imaging N-Cys-containing biomolecules in the cellular environment with high sensitivity. Given that thioesters exhibit distinctive emission quenching capabilities in thioester-modified stilbenes, − we postulate that the incorporation of a thioester moiety into transition metal complexes will generate a unique class of phosphorogenic bioconjugation reagents for labeling N-Cys-containing biomolecules, providing a new platform for bioimaging and phototherapeutic applications. In this work, we designed and synthesized a series of cyclometalated iridium­(III) polypyridine complexes featuring a thioester group [Ir­(N^C) 2 (bpy-COSBn)]­(PF 6 ) (bpy-COSBn = S -benzyl 4’-methyl-2,2’-bipyridine-4-carbothioate; HN^C = 2-phenylquinoline (Hpq) ( 1a ), 2-(1-naphthyl)­benzothiazole (Hbsn) ( 2a ), 1-(benzo­[ b ]-thiophen-2-yl)­isoquinoline (Hiqbt) ( 3a ), and 6-(benzo­[ b ]­thiophen-2-yl)­phenanthridine (Hbtph) ( 4a )) (Chart ). Their ester counterparts [Ir­(N^C) 2 (bpy-COOMe)]­(PF 6 ) (bpy-COOMe = methyl 4’-methyl-2,2’-bipyridine-4-carboxylate; HN^C = Hpq ( 1b ), Hbsn ( 2b ), Hiqbt ( 3b ), and Hbtph ( 4b )) were also prepared for comparison studies. The photophysical, photochemical, and electrochemical properties of the complexes were studied. Additionally, the reactivity, selectivity, and possible phosphorogenic response of the thioester complexes toward l -Cys were examined. The utilization of the thioester complexes for intracellular Cys sensing and cancer-targeted PDT was also investigated. Furthermore, various tumor-targeting peptide conjugates were constructed from the reaction of the thioester complex and N-Cys-modified peptides, and their photophysical and photochemical properties, cellular uptake, intracellular localization, and (photo)­cytotoxicity were studied. 1 Structures of the Iridium­(III) Thioester Complexes 1a – 4a and Ester Complexes 1b – 4b ## Results and Discussion Results and Discussion Synthesis and Characterization of the Iridium­(III) Complexes The synthesis of the thioester ligand bpy-COSBn involved the reaction of 4-succinimidylcarboxy-4’-methyl-2,2’-bipyridine (bpy-NHS) with benzyl mercaptan in an anhydrous THF solution containing N , N -diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP). The synthesis of the ester ligand bpy-COOMe was performed following procedures in the literature. The iridium­(III) complexes were prepared by the reaction of iridium­(III) dimers [Ir 2 (N^C) 4 Cl 2 ] (HN^C = Hpq, Hbsn, Hiqbt, and Hbtph) with bpy-COSBn or bpy-COOMe in CH 2 Cl 2 /MeOH, followed by anion exchange with KPF 6 , and purification by column chromatography and recrystallization from CH 2 Cl 2 /Et 2 O to afford orange to deep red crystals. The complexes were characterized by HR-ESI-MS, 1 H and 13 C NMR, and IR spectroscopy, and gave satisfactory elemental analyses. Single crystals of complex 1a were obtained by vapor diffusion of Et 2 O into a concentrated solution of the complex in CH 3 CN. Crystallographic data, selected bond lengths, and bond angles are listed in Tables S1 and S2 . The perspective view of the complex cation is shown in Figure . 1 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. The iridium­(III) center of the complex adopts a distorted octahedral geometry, and the trans angles at the metal center range from 170.7 to 172.1°. The Ir–C bonds of the cyclometalating ligands are coordinated to the metal center in a cis orientation. The trans influence of the carbon donors renders slightly longer Ir–N bond lengths for the bpy-COSBn ligand (2.169 Å and 2.179 Å) than those for the pq ligands (2.094 and 2.119 Å). The bite angles of the pq ligands (79.2 and 79.5°) are larger than that of the bpy-COSBn ligand (75.06°), which is similar to those of related cyclometalated iridium­(III) polypyridine systems, [Ir­(N^C) 2 (N^N)] + . − Photophysical, Photochemical, and Electrochemical Properties The electronic absorption spectra and data of the thioester complexes 1a – 4a and ester complexes 1b – 4b are presented in Figures S1 and S2 and Table S3 , respectively. All the complexes displayed intense spin-allowed intraligand ( 1 IL) (π → π*) (N^N and N^C) absorption bands at ca. 250–350 nm and weaker spin-allowed metal-to-ligand charge-transfer ( 1 MLCT) (dπ­(Ir) → π*­(N^N and N^C)) absorption bands/shoulders at ca. 360–550 nm. − The weak absorption tail beyond ca. 560 nm is assigned to spin-forbidden 3 MLCT (dπ­(Ir) → π*­(N^N and N^C)) transitions. Upon photoexcitation, all the complexes exhibited orange-red to near-infrared (NIR) emission in solutions under ambient conditions and in low-temperature alcohol glass. The emission spectra and photophysical data of the thioester and ester complexes are presented in Figures S3 and S4 and Table S4 , respectively. Importantly, the thioester complexes 1a – 4a (Φ em = 0.002–0.025 in CH 3 CN) exhibited significantly lower emission quantum yields than their ester counterparts 1b – 4b (Φ em = 0.010–0.13 in CH 3 CN), indicative of emission quenching associated with the thioester moiety. The pq complexes ( 1a , b ) displayed positive solvatochromism and short emission lifetimes in fluid solutions at 298 K and a significant blue shift upon cooling the samples to 77 K, suggestive of a predominant 3 MLCT (dπ­(Ir) → π*­(N^N)) emissive state. However, there should be mixing of some 3 IL (π → π*) (pq) character due to their structured emission bands and long emission lifetimes (5.07 and 4.62 μs) in 77-K glass. In contrast, the bsn ( 2a , b ), iqbt ( 3a , b ), and btph ( 4a , b ) complexes showed a structured NIR emission band with low solvent dependency in fluid solutions at 298 K and long emission lifetimes (2.88–5.49 μs) in 77-K glass, suggestive of a predominant 3 IL (π → π*) (N^C) excited state. − The 1 O 2 generation efficiencies of all complexes were evaluated by monitoring the emission band of 1 O 2 centered at ca. 1270 nm , in aerated CH 3 CN ( Table S5 ). The thioester complexes 1a – 4a showed lower 1 O 2 generation quantum yields (0.13–0.80) than the ester complexes 1b – 4b (0.51–0.98), indicating quenching of the complexes by the thioester group. Among the ester complexes 1b – 4b , the bsn ( 2b ), iqbt ( 3b ), and btph ( 4b ) complexes displayed substantially higher 1 O 2 generation efficiencies (Φ Δ = 0.82–0.98) than the pq complex ( 1b ) (Φ Δ = 0.51), primarily due to the presence of a low-lying, long-lived 3 IL excited state (τ o = 1.28–3.79 μs; Table S4 ) for 1 O 2 photosensitization. The electrochemical properties of the thioester complexes 1a – 4a were studied by cyclic voltammetry, and the electrochemical data are listed in Table S6 . These complexes exhibited a quasi-reversible oxidation couple at +1.12 to +1.38 V versus SCE, which is assigned to a metal-centered iridium­(IV)/(III) oxidation process. , Based on the first reduction potentials (−0.92 to −0.99 V versus SCE, Table S6 ) and the low-temperature emission energy ( E 00 = 1.79–2.28 eV, Table S4 ) of the thioester complexes, the excited-state redox potentials ( E °[Ir 2+/+ *]) of complexes 1a – 4a were determined to range from −0.63 to −0.96 V versus SCE ( Figure S5 ). These potentials are less negative than the reduction potential of bpy-COSBn (−1.00 V versus SCE; Table S6 ), suggesting that the redox reaction between the excited iridium­(III) complexes and the appended thioester moiety is not thermodynamically favorable (Δ G ° = +0.05 to +0.37 eV). This eliminates the possibility of photoinduced electron transfer (PeT) as the quenching mechanism. Reactivity, Selectivity, and Phosphorogenic Response toward N-Cys The reactivity of the thioester complexes 1a – 4a toward N-Cys-containing biomolecules in potassium phosphate buffer (50 mM, pH 7.0)/DMSO (3:2, v / v ) containing tris­(2-carboxyethyl)­phosphine (TCEP) (250 μM) at 298 K ( Scheme ) were investigated using l -Cys as a model. As revealed by high-performance liquid chromatography (HPLC) analyses, the reaction of the thioester complexes (20 μM) with l -Cys (25 μM) completed within 1 h, with conversion yields exceeding 95% ( Figure S6 ). Using complex 1a as an example, the initial peak at t R = 9.7 min disappeared, and a new peak at t R = 6.8 min emerged in the chromatogram after incubation with l -Cys for 1 h ( Figure S6 ). The formation of the conjugation products 1a-Cys – 4a-Cys was validated by ESI-MS analyses ( Figure S7 ). The isolated conjugate 1a-Cys was further characterized by 1D and 2D COSY 1 H NMR. The signals at δ = 8.18 and 1.89 ppm in the 1 H NMR spectra ( Figures S8 and S9 ) correspond to the CONH and SH protons, respectively, confirming the successful labeling of l -Cys with the thioester complex via the NCL reaction involving transthioesterification and an S → N acyl shift. The reaction kinetics of complexes 1a – 4a with l -Cys was studied in buffer solutions at 298 K by monitoring the reaction at different time intervals using HPLC. The second-order rate constants ( k 2 ) for the reactions range from 189.2 to 2,385.5 M –1 s –1 , following the order: 4a < 2a < 3a < 1a ( Figure S10 ). These values are one to two orders of magnitude higher than that of the ligand bpy-COSBn (11.3 M –1 s –1 ), illustrating that the direct conjugation of the thioester moiety to the cationic iridium­(III) polypyridine unit significantly enhances the reactivity. , , − In particular, complexes 1a and 3a demonstrated remarkable reactivity at a rate of 10 3 M –1 s –1 , which facilitates the rapid labeling of N-Cys-containing biomolecules. To evaluate the selectivity and stability of the thioester complexes, complex 1a was selected as a model and the reactions were monitored by HPLC and ESI-MS. The complex remained intact and showed negligible reaction upon incubation with a large excess of amino acids, including l -Lys, l -histidine ( l -His), l -serine ( l -Ser), and l -threonine ( l -Thr) (2 mM) ( Figure S11 ). Importantly, upon incubation with peptide models containing a Cys at the N-terminus (CSS), center (SCS), and C-terminus (SSC), complex 1a selectively reacted with CSS ( Figure a), forming the conjugate 1a-CSS ( t R = 5.8 min) as evidenced by ESI-MS analysis ( Figure b). These results highlight the high reactivity and excellent chemo- and regioselectivity of the thioester complexes toward N-Cys-containing biomolecules. 1 Conjugation of the Thioester Complexes to N-Cys-Containing Biomolecules via NCL 2 (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. Notably, upon incubation of the thioester complexes (10 μM) with l -Cys (100 μM) in TCEP-containing buffer solutions at 298 K for 1 h, substantial emission enhancement was observed in the solutions ( I / I o = 10.7–31.8) ( Table S7 and Figure ) due to conversion of the quenching thioester moiety into a nonquenching amide group during the NCL reaction. Interestingly, complex 1a displayed a bathochromic shift in its emission maximum from ca. 556 to 606 nm upon reaction with l -Cys ( Figure , Table S7 , and Figure S12 ). The distinct photophysical changes of complex 1a upon the NCL reaction compared to complexes 2a – 4a can be attributed to the larger involvement of 3 MLCT (dπ­(Ir) → π*­(N^N)) character in its emissive state, which renders it more sensitive to the structural changes on the bpy ligand (i.e., from thioester to amide) associated with the NCL reaction. The observed photophysical changes brought about by the NCL reaction highlight the potential applications of the thioester complexes not only for precise labeling of biomolecules to yield photofunctional conjugates, but also for imaging l -Cys and N-Cys-containing biomolecules in live cells to examine their functions and dynamics. 3 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)/CH 3 CN (3:2, v / v ) containing TCEP (1 mM) at 298 K for 1 h. To determine the effect of bioconjugation on the photophysical and photochemical properties of the complexes, their Cys conjugates 1a-Cys – 4a-Cys were isolated and purified by semipreparative HPLC. All Cys conjugates exhibited high emission quantum yields (Φ em = 0.016–0.12) ( Table S8 and Figure S13 ) and 1 O 2 generation quantum yields (Φ Δ = 0.53–0.98) ( Table S9 ) in CH 3 CN, comparable to the ester complexes (Φ em = 0.010–0.13, Φ Δ = 0.51–0.98; Tables S4 and S5 ). Importantly, complex 3a displayed the most pronounced change in the 1 O 2 generation quantum yield after reacting with l -Cys (from 0.13 to 0.92; Tables S5 and S9 ), showcasing the controllable 1 O 2 -photosensitization behavior of the thioester complexes via the NCL reaction. Computational Studies To gain more insights into the emission enhancement of thioester complexes upon reaction with l -Cys, density functional theory (DFT) and unrestricted density functional theory (UDFT) calculations were performed on the thioester complex 1a-Me (an S -methyl thioester analogue of complex 1a ), its NCL reaction product 1a-Cys , and ester counterpart 1b . The benzyl group in the thioester moiety of complex 1a is simplified to a methyl group in 1a-Me for the comparison with the methyl ester complex 1b . For all three complexes, the emissive triplet (T 1 ) state is dominated by the 3 MLCT (dπ­(Ir) → π*­(bpy-COSMe), dπ­(Ir) → π*­(bpy-CONH-Cys), and dπ­(Ir) → π*­(bpy-COOMe) for complexes 1a-Me , 1a-Cys , and 1b , respectively) character ( Figure ). The emission energy of complex 1a-Me computed at the relaxed 3 MLCT structure is 1.82 eV ( Table S10 ), which is in good agreement with the experimental emission measured in CH 2 Cl 2 (λ em = 648 nm, 1.91 eV; Table S4 ). The emission bands are computed to be 1.96 and 1.92 eV for complexes 1a-Cys and 1b , respectively, which are also in line with the experimentally observed blue shift in λ em . It should be noted that the distortion of the bpy-based ligands can lead to triplet distorted states ( 3 DS) that deactivate the radiative 3 MLCT → S 0 process. , A metal-centered ( 3 MC) state, featuring a five-coordinate iridium center through the dissociation of an Ir–N bond, is energetically higher-lying than the ground (S 0 ) state by ca. 2.8 eV ( Figure ). This state has been identified for all three complexes, and might not be directly related to the emission enhancement upon conversion of complex 1a-Me into 1a-Cys . Interestingly, in contrast to the amide and ester groups in complexes 1a-Cys and 1b , respectively, the planar thioester moiety (θ­(OC–S–C) = 0.0°) in complex 1a-Me can undergo a structural distortion to form a 3 IL state with an OC–S–C torsion angle of −86.6°. As compared to the large vertical T 1 –S 0 gap (1.82 eV) at the relaxed 3 MLCT structure, the T 1 –S 0 gap at the relaxed 3 IL structure is decreased to 1.24 eV, which might facilitate nonradiative decay through the 3 IL → S 0 intersystem crossing (ISC) process. Based on Marcus theory ( Table S11 ), the rate constant ( k ISC ) of the 3 IL → S 0 ISC is computed to be 1.65 × 10 8 s –1 , which is several orders of magnitude greater than the rate constant for the radiative decay process ( k r P = 3.41 × 10 2 s –1 ). Thus, the distorted 3 IL state localized on the bpy-COSMe ligand is suggested to be nonemissive. From our computational interpretations, we believe that energy transfer from the emissive 3 MLCT state to the nonemissive 3 IL (thioester) state leads to the emission quenching observed in the iridium­(III) thioester complexes. 4 Spin densities of the emissive 3 MLCT state and nonemissive 3 DS states at their optimized structures for complexes 1a-Me , 1a-Cys , and 1b . Computed energy levels (eV) with respect to the optimized S 0 state are provided. Intracellular Cysteine Sensing The intriguing aminothiol-specific phosphorogenic response of the thioester complexes motivated us to explore their applications in sensing intracellular Cys and N-Cys-containing proteins. Complex 1a was used as a model compound due to (1) its high reactivity ( k 2 = 2,385.5 M –1 s –1 ; Figure S10 ); (2) distinctive emission profiles before and after the NCL reaction with Cys ( Figure ); and (3) high selectivity toward Cys over other thiols such as ethanethiol and glutathione (GSH) (the most abundant biothiol in cells) ( Table S12 and Figure S14 ). Laser-scanning confocal microscopy (LSCM) images of live HeLa cells incubated with complex 1a (10 μM, 1 h) revealed intense cytoplasmic emission ( Figure ), which is due to the reaction of the complex with intracellular Cys to give the emissive amide product 1a-Cys . Additionally, the intracellular emission spectrum of cells treated with complex 1a closely resembles that of cells treated with conjugate 1a-Cys ( Figure S16 ), confirming the reaction of complex 1a with intracellular Cys. However, when the cells were pretreated with the thiol scavenger N -ethylmaleimide (NEM) (100 μM, 20 min), the emission intensity significantly decreased, which is attributed to the reduced intracellular Cys level. The emission was restored upon further incubation of the NEM-pretreated cells with l -Cys (100 μM, 30 min), supporting that the observed intracellular emission was due to the specific reaction of the thioester complex with Cys. These results demonstrate that complex 1a can function as a sensor for intracellular Cys. This also suggests the potential for detecting N-Cys-containing proteins in live cells. 5 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. Cancer-Targeted PDT Despite its role as an essential biothiol in maintaining intracellular redox homeostasis, Cys is known as an important cancer biomarker due to its overexpression in cancer tissues compared to normal tissues. Given the significant increase in the 1 O 2 generation efficiency of complex 3a upon reaction with l -Cys (from 0.13 to 0.92 in CH 3 CN; Tables S5 and S9 ), we anticipate that complex 3a can serve as a Cys-activatable photosensitizer for cancer-targeted PDT. Thus, we examined the (photo)­cytotoxic activity of complex 3a in cancerous MDA-MB-231 (high intracellular Cys level) and normal HEK-293 (low intracellular Cys level) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The complex was essentially noncytotoxic in the dark (IC 50,dark > 50 μM) ( Table S13 ) in both cell lines. However, upon irradiation at 450 nm (light dosage = 14.6 mW cm –2 ) for 10 min, the complex exhibited remarkable photocytotoxic effects, with greater potency in MDA-MB-231 cells (IC 50,light = 0.57 μM) compared to HEK-293 cells (IC 50,light = 1.7 μM) ( Table S13 ). This discrepancy in potency can be ascribed to (1) the higher cellular uptake of the complex in MDA-MB-231 cells (0.18 fmol/cell) than HEK-293 cells (0.087 fmol/cell) ( Table S13 ); and (2) the higher intracellular Cys level in MDA-MB-231 cells leading to the greater in situ formation of conjugate 3a-Cys . These results highlight the potential of complex 3a in cancer-selective PDT. Preparation and Characterization of Tumor-Targeting Iridium­(III)–Peptide Conjugates Considering that the thioester complex 3a showed rapid reaction kinetics toward l -Cys ( k 2 = 1,658.3 M –1 s –1 ; Figure S10 ) and its Cys conjugate 3a-Cys showed a high 1 O 2 generation quantum yield (Φ Δ = 0.92 in CH 3 CN; Table S9 ), complex 3a was used to modify various tumor-targeting peptides to yield photofunctional metal–peptide conjugates for theranostic applications. Three peptides were selected: (1) CASPSGALRSC (CASP), which demonstrates high targeting specificity toward the triple-negative breast cancer MDA-MB-231 cell line; (2) YNTNHVPLSPKY (YNT), which targets the enzyme carbonic anhydrase IX (CAIX) that is often overexpressed in various cancer cell types under hypoxic conditions; and (3) CMYIEALDKYAC (CMYI), which targets the epidermal growth factor receptor (EGFR), a critical transmembrane receptor that is overexpressed in numerous cancer cell lines. All three peptides were modified with an N-Cys residue for conjugation. The iridium­(III)–peptide conjugates 3a-CASP , 3a-CYNT , and 3a-CMYI were prepared by incubation of complex 3a with the peptides CASP, CYNT, and CMYI, respectively, in TCEP-containing buffer solutions at 298 K for 12 h. Notably, despite the presence of an intrinsic Cys residue at the C-terminus of both CASP and CMYI peptides, the reactions only yielded one major product ( Figure S17 ), highlighting the excellent chemo- and regioselectivity of the thioester complexes. The conjugates were purified by semipreparative HPLC, and the purified products were characterized by HPLC and ESI-MS analyses ( Figures S18 and S19 ). Upon photoexcitation, the conjugates displayed moderate emission intensities and lifetimes in aqueous solutions ( Table S8 and Figure S20 ). Additionally, the peptide conjugates exhibited comparable 1 O 2 photosensitization capabilities (Φ Δ = 0.20–0.28) to conjugate 3a-Cys (Φ Δ = 0.26) in aqueous solutions ( Table S14 ). The enhanced emission quantum yields, extended lifetimes, and increased 1 O 2 photosensitization efficiencies of the conjugates, as compared to the thioester complex 3a , indicate the elimination of the thioester-mediated quenching pathway upon the NCL reaction. Cellular Uptake, Localization, and (Photo)­cytotoxicity of the Peptide Conjugates To evaluate the cellular uptake and localization of the iridium­(III)–peptide conjugates, two cancer cell lines MDA-MB-231 (high expression levels of CAIX and EGFR) and MCF-7 (low expression levels of CAIX and EGFR), and one normal cell line HEK-293 were utilized as the models. , As revealed by ICP-MS analyses, the cellular uptake of all three peptide conjugates was substantially higher in MDA-MB-231 cells (0.21–1.8 fmol/cell) compared to MCF-7 (0.074–0.10 fmol/cell) and HEK-293 cells (0.033–0.10 fmol/cell) ( Table S15 ). This observation aligns with the higher abundance of the target CAIX and EGFR proteins in MDA-MB-231 cells and the specific targeting capabilities of the peptide conjugates. In contrast, the peptide-free conjugate 3a-Cys showed similar levels of cellular uptake in all three cell lines (0.040–0.30 fmol/cell; Table S15 ), indicating its lack of selectivity. The cellular uptake mechanism and pathways of the conjugates were further studied. For conjugate 3a-CASP , treatment of MDA-MB-231 cells with the conjugate (10 μM) at 4 °C led to a notable decrease in intracellular iridium content by ca. 70% ( Figure S21 ), suggesting that the conjugate was internalized through an energy-dependent mechanism. The cellular uptake efficiency of the conjugate was reduced by ca. 30% when the cells were pretreated with a clathrin-mediated endocytosis inhibitor chlorpromazine (30 μM, 1 h) ( Figure S22 ). In contrast, it remained largely unchanged when the cells were pretreated with other endocytosis inhibitors, such as a macropinocytosis inhibitor 5-( N -ethyl- N -isopropyl)­amiloride (EIPA) (50 μM, 1.5 h) and a caveolin-mediated endocytosis inhibitor methyl-β-cyclodextrin (Me-β-CD) (5 mM, 1 h). These findings suggest that conjugate 3a-CASP was internalized into the cells through an energy-dependent pathway, likely involving clathrin-mediated endocytosis. For conjugates 3a-CYNT and 3a-CMYI , pretreatment of the cells with a CAIX inhibitor acetazolamide (1 mM, 6 h) or an EGFR inhibitor gefitinib (50 μM, 1 h) only reduced the uptake of the conjugates by ca. 20 and 10% ( Figures S23 and S24 ), respectively. These findings implied that the modification of the tumor-targeting peptides with an iridium­(III) complex modulated their uptake pathways, which is probably associated with their cationic charge and high lipophilicity. The intracellular distribution of the conjugates was studied by LSCM. Live MDA-MB-231, MCF-7, and HEK-293 cells were incubated with conjugates 3a-Cys , 3a-CASP , 3a-CYNT , and 3a-CMYI (10 μM) for 16 h. As depicted in the LSCM images, after incubation with the peptide conjugates, MDA-MB-231 cells displayed more pronounced intracellular emission compared to MCF-7 and HEK-293 cells ( Figure ). However, treatment with the Cys conjugate 3a-Cys resulted in similar emission intensities across the three cell lines ( Figure ). These observations correlate well with the respective cellular uptake efficiencies of the conjugates ( Table S15 ). Co-staining experiments demonstrate that the conjugates were primarily localized in the lysosomes of MDA-MB-231 cells after uptake ( Figure and Figure S25 ). 6 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. 7 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. The (photo)­cytotoxicity of the conjugates was studied using the MTT assay. Conjugates 3a-CASP , 3a-CYNT , and 3a-CMYI all exhibited minimal dark cytotoxicity (IC 50,dark > 25 μM; Table ) across the three cell lines. Upon irradiation (450 nm, 14.6 mW cm –2 , 10 min), all conjugates showed the highest photocytotoxicity toward MDA-MB-231 cells, with IC 50,light values ranging from 0.18 to 0.41 μM ( Table ). The photocytotoxic effects of the conjugates were consistent among MCF-7 and HEK-293 cells (IC 50,light = 0.65–1.4 μM and 0.56–1.4 μM, respectively; Table ), correlating with their comparatively lower cellular uptake efficiencies in these two cell lines ( Table S15 ). These results collectively demonstrate that conjugates 3a-CASP , 3a-CYNT , and 3a-CMYI retained the tumor-targeting properties of the original peptides, and the combined action of photocytotoxic iridium­(III) complexes and tumor-targeting peptides in these conjugates facilitates precise and effective cancer-targeted PDT. 1 (Photo)­cytotoxicity of the Peptide Conjugates of Complex 3a toward MDA-MB-231, MCF-7, and HEK-293 Cells conjugate MDA-MB-231 MCF-7 HEK-293   IC 50,dark /μM IC 50,light /μM PI IC 50,dark /μM IC 50,light /μM PI IC 50,dark /μM IC 50,light /μM PI 3a-CASP >25 0.41 ± 0.01 >61 >25 1.4 ± 0.1 >18 >25 1.4 ± 0.1 >18 3a-CYNT >25 0.18 ± 0.01 >139 >25 0.65 ± 0.03 >38 >25 0.56 ± 0.02 >45 3a-CMYI >25 0.23 ± 0.01 >109 >25 1.0 ± 0.1 >25 >25 0.90 ± 0.08 >28 a PI is the ratio IC 50,dark /IC 50,light under different conditions. The cells were first incubated with the conjugates in the dark for 24 h, replaced with fresh medium, and then incubated in the dark or irradiated at 450 nm (light dosage = 14.6 mW cm –2 ) for 10 min, and subsequently incubated in the dark for 24 h. ## Synthesis and Characterization of the Iridium­(III) Complexes Synthesis and Characterization of the Iridium­(III) Complexes The synthesis of the thioester ligand bpy-COSBn involved the reaction of 4-succinimidylcarboxy-4’-methyl-2,2’-bipyridine (bpy-NHS) with benzyl mercaptan in an anhydrous THF solution containing N , N -diisopropylethylamine (DIPEA) and 4-dimethylaminopyridine (DMAP). The synthesis of the ester ligand bpy-COOMe was performed following procedures in the literature. The iridium­(III) complexes were prepared by the reaction of iridium­(III) dimers [Ir 2 (N^C) 4 Cl 2 ] (HN^C = Hpq, Hbsn, Hiqbt, and Hbtph) with bpy-COSBn or bpy-COOMe in CH 2 Cl 2 /MeOH, followed by anion exchange with KPF 6 , and purification by column chromatography and recrystallization from CH 2 Cl 2 /Et 2 O to afford orange to deep red crystals. The complexes were characterized by HR-ESI-MS, 1 H and 13 C NMR, and IR spectroscopy, and gave satisfactory elemental analyses. Single crystals of complex 1a were obtained by vapor diffusion of Et 2 O into a concentrated solution of the complex in CH 3 CN. Crystallographic data, selected bond lengths, and bond angles are listed in Tables S1 and S2 . The perspective view of the complex cation is shown in Figure . 1 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. The iridium­(III) center of the complex adopts a distorted octahedral geometry, and the trans angles at the metal center range from 170.7 to 172.1°. The Ir–C bonds of the cyclometalating ligands are coordinated to the metal center in a cis orientation. The trans influence of the carbon donors renders slightly longer Ir–N bond lengths for the bpy-COSBn ligand (2.169 Å and 2.179 Å) than those for the pq ligands (2.094 and 2.119 Å). The bite angles of the pq ligands (79.2 and 79.5°) are larger than that of the bpy-COSBn ligand (75.06°), which is similar to those of related cyclometalated iridium­(III) polypyridine systems, [Ir­(N^C) 2 (N^N)] + . − ## Photophysical, Photochemical, and Electrochemical Properties Photophysical, Photochemical, and Electrochemical Properties The electronic absorption spectra and data of the thioester complexes 1a – 4a and ester complexes 1b – 4b are presented in Figures S1 and S2 and Table S3 , respectively. All the complexes displayed intense spin-allowed intraligand ( 1 IL) (π → π*) (N^N and N^C) absorption bands at ca. 250–350 nm and weaker spin-allowed metal-to-ligand charge-transfer ( 1 MLCT) (dπ­(Ir) → π*­(N^N and N^C)) absorption bands/shoulders at ca. 360–550 nm. − The weak absorption tail beyond ca. 560 nm is assigned to spin-forbidden 3 MLCT (dπ­(Ir) → π*­(N^N and N^C)) transitions. Upon photoexcitation, all the complexes exhibited orange-red to near-infrared (NIR) emission in solutions under ambient conditions and in low-temperature alcohol glass. The emission spectra and photophysical data of the thioester and ester complexes are presented in Figures S3 and S4 and Table S4 , respectively. Importantly, the thioester complexes 1a – 4a (Φ em = 0.002–0.025 in CH 3 CN) exhibited significantly lower emission quantum yields than their ester counterparts 1b – 4b (Φ em = 0.010–0.13 in CH 3 CN), indicative of emission quenching associated with the thioester moiety. The pq complexes ( 1a , b ) displayed positive solvatochromism and short emission lifetimes in fluid solutions at 298 K and a significant blue shift upon cooling the samples to 77 K, suggestive of a predominant 3 MLCT (dπ­(Ir) → π*­(N^N)) emissive state. However, there should be mixing of some 3 IL (π → π*) (pq) character due to their structured emission bands and long emission lifetimes (5.07 and 4.62 μs) in 77-K glass. In contrast, the bsn ( 2a , b ), iqbt ( 3a , b ), and btph ( 4a , b ) complexes showed a structured NIR emission band with low solvent dependency in fluid solutions at 298 K and long emission lifetimes (2.88–5.49 μs) in 77-K glass, suggestive of a predominant 3 IL (π → π*) (N^C) excited state. − The 1 O 2 generation efficiencies of all complexes were evaluated by monitoring the emission band of 1 O 2 centered at ca. 1270 nm , in aerated CH 3 CN ( Table S5 ). The thioester complexes 1a – 4a showed lower 1 O 2 generation quantum yields (0.13–0.80) than the ester complexes 1b – 4b (0.51–0.98), indicating quenching of the complexes by the thioester group. Among the ester complexes 1b – 4b , the bsn ( 2b ), iqbt ( 3b ), and btph ( 4b ) complexes displayed substantially higher 1 O 2 generation efficiencies (Φ Δ = 0.82–0.98) than the pq complex ( 1b ) (Φ Δ = 0.51), primarily due to the presence of a low-lying, long-lived 3 IL excited state (τ o = 1.28–3.79 μs; Table S4 ) for 1 O 2 photosensitization. The electrochemical properties of the thioester complexes 1a – 4a were studied by cyclic voltammetry, and the electrochemical data are listed in Table S6 . These complexes exhibited a quasi-reversible oxidation couple at +1.12 to +1.38 V versus SCE, which is assigned to a metal-centered iridium­(IV)/(III) oxidation process. , Based on the first reduction potentials (−0.92 to −0.99 V versus SCE, Table S6 ) and the low-temperature emission energy ( E 00 = 1.79–2.28 eV, Table S4 ) of the thioester complexes, the excited-state redox potentials ( E °[Ir 2+/+ *]) of complexes 1a – 4a were determined to range from −0.63 to −0.96 V versus SCE ( Figure S5 ). These potentials are less negative than the reduction potential of bpy-COSBn (−1.00 V versus SCE; Table S6 ), suggesting that the redox reaction between the excited iridium­(III) complexes and the appended thioester moiety is not thermodynamically favorable (Δ G ° = +0.05 to +0.37 eV). This eliminates the possibility of photoinduced electron transfer (PeT) as the quenching mechanism. ## Reactivity, Selectivity, and Phosphorogenic Response toward N-Cys Reactivity, Selectivity, and Phosphorogenic Response toward N-Cys The reactivity of the thioester complexes 1a – 4a toward N-Cys-containing biomolecules in potassium phosphate buffer (50 mM, pH 7.0)/DMSO (3:2, v / v ) containing tris­(2-carboxyethyl)­phosphine (TCEP) (250 μM) at 298 K ( Scheme ) were investigated using l -Cys as a model. As revealed by high-performance liquid chromatography (HPLC) analyses, the reaction of the thioester complexes (20 μM) with l -Cys (25 μM) completed within 1 h, with conversion yields exceeding 95% ( Figure S6 ). Using complex 1a as an example, the initial peak at t R = 9.7 min disappeared, and a new peak at t R = 6.8 min emerged in the chromatogram after incubation with l -Cys for 1 h ( Figure S6 ). The formation of the conjugation products 1a-Cys – 4a-Cys was validated by ESI-MS analyses ( Figure S7 ). The isolated conjugate 1a-Cys was further characterized by 1D and 2D COSY 1 H NMR. The signals at δ = 8.18 and 1.89 ppm in the 1 H NMR spectra ( Figures S8 and S9 ) correspond to the CONH and SH protons, respectively, confirming the successful labeling of l -Cys with the thioester complex via the NCL reaction involving transthioesterification and an S → N acyl shift. The reaction kinetics of complexes 1a – 4a with l -Cys was studied in buffer solutions at 298 K by monitoring the reaction at different time intervals using HPLC. The second-order rate constants ( k 2 ) for the reactions range from 189.2 to 2,385.5 M –1 s –1 , following the order: 4a < 2a < 3a < 1a ( Figure S10 ). These values are one to two orders of magnitude higher than that of the ligand bpy-COSBn (11.3 M –1 s –1 ), illustrating that the direct conjugation of the thioester moiety to the cationic iridium­(III) polypyridine unit significantly enhances the reactivity. , , − In particular, complexes 1a and 3a demonstrated remarkable reactivity at a rate of 10 3 M –1 s –1 , which facilitates the rapid labeling of N-Cys-containing biomolecules. To evaluate the selectivity and stability of the thioester complexes, complex 1a was selected as a model and the reactions were monitored by HPLC and ESI-MS. The complex remained intact and showed negligible reaction upon incubation with a large excess of amino acids, including l -Lys, l -histidine ( l -His), l -serine ( l -Ser), and l -threonine ( l -Thr) (2 mM) ( Figure S11 ). Importantly, upon incubation with peptide models containing a Cys at the N-terminus (CSS), center (SCS), and C-terminus (SSC), complex 1a selectively reacted with CSS ( Figure a), forming the conjugate 1a-CSS ( t R = 5.8 min) as evidenced by ESI-MS analysis ( Figure b). These results highlight the high reactivity and excellent chemo- and regioselectivity of the thioester complexes toward N-Cys-containing biomolecules. 1 Conjugation of the Thioester Complexes to N-Cys-Containing Biomolecules via NCL 2 (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. Notably, upon incubation of the thioester complexes (10 μM) with l -Cys (100 μM) in TCEP-containing buffer solutions at 298 K for 1 h, substantial emission enhancement was observed in the solutions ( I / I o = 10.7–31.8) ( Table S7 and Figure ) due to conversion of the quenching thioester moiety into a nonquenching amide group during the NCL reaction. Interestingly, complex 1a displayed a bathochromic shift in its emission maximum from ca. 556 to 606 nm upon reaction with l -Cys ( Figure , Table S7 , and Figure S12 ). The distinct photophysical changes of complex 1a upon the NCL reaction compared to complexes 2a – 4a can be attributed to the larger involvement of 3 MLCT (dπ­(Ir) → π*­(N^N)) character in its emissive state, which renders it more sensitive to the structural changes on the bpy ligand (i.e., from thioester to amide) associated with the NCL reaction. The observed photophysical changes brought about by the NCL reaction highlight the potential applications of the thioester complexes not only for precise labeling of biomolecules to yield photofunctional conjugates, but also for imaging l -Cys and N-Cys-containing biomolecules in live cells to examine their functions and dynamics. 3 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)/CH 3 CN (3:2, v / v ) containing TCEP (1 mM) at 298 K for 1 h. To determine the effect of bioconjugation on the photophysical and photochemical properties of the complexes, their Cys conjugates 1a-Cys – 4a-Cys were isolated and purified by semipreparative HPLC. All Cys conjugates exhibited high emission quantum yields (Φ em = 0.016–0.12) ( Table S8 and Figure S13 ) and 1 O 2 generation quantum yields (Φ Δ = 0.53–0.98) ( Table S9 ) in CH 3 CN, comparable to the ester complexes (Φ em = 0.010–0.13, Φ Δ = 0.51–0.98; Tables S4 and S5 ). Importantly, complex 3a displayed the most pronounced change in the 1 O 2 generation quantum yield after reacting with l -Cys (from 0.13 to 0.92; Tables S5 and S9 ), showcasing the controllable 1 O 2 -photosensitization behavior of the thioester complexes via the NCL reaction. ## Computational Studies Computational Studies To gain more insights into the emission enhancement of thioester complexes upon reaction with l -Cys, density functional theory (DFT) and unrestricted density functional theory (UDFT) calculations were performed on the thioester complex 1a-Me (an S -methyl thioester analogue of complex 1a ), its NCL reaction product 1a-Cys , and ester counterpart 1b . The benzyl group in the thioester moiety of complex 1a is simplified to a methyl group in 1a-Me for the comparison with the methyl ester complex 1b . For all three complexes, the emissive triplet (T 1 ) state is dominated by the 3 MLCT (dπ­(Ir) → π*­(bpy-COSMe), dπ­(Ir) → π*­(bpy-CONH-Cys), and dπ­(Ir) → π*­(bpy-COOMe) for complexes 1a-Me , 1a-Cys , and 1b , respectively) character ( Figure ). The emission energy of complex 1a-Me computed at the relaxed 3 MLCT structure is 1.82 eV ( Table S10 ), which is in good agreement with the experimental emission measured in CH 2 Cl 2 (λ em = 648 nm, 1.91 eV; Table S4 ). The emission bands are computed to be 1.96 and 1.92 eV for complexes 1a-Cys and 1b , respectively, which are also in line with the experimentally observed blue shift in λ em . It should be noted that the distortion of the bpy-based ligands can lead to triplet distorted states ( 3 DS) that deactivate the radiative 3 MLCT → S 0 process. , A metal-centered ( 3 MC) state, featuring a five-coordinate iridium center through the dissociation of an Ir–N bond, is energetically higher-lying than the ground (S 0 ) state by ca. 2.8 eV ( Figure ). This state has been identified for all three complexes, and might not be directly related to the emission enhancement upon conversion of complex 1a-Me into 1a-Cys . Interestingly, in contrast to the amide and ester groups in complexes 1a-Cys and 1b , respectively, the planar thioester moiety (θ­(OC–S–C) = 0.0°) in complex 1a-Me can undergo a structural distortion to form a 3 IL state with an OC–S–C torsion angle of −86.6°. As compared to the large vertical T 1 –S 0 gap (1.82 eV) at the relaxed 3 MLCT structure, the T 1 –S 0 gap at the relaxed 3 IL structure is decreased to 1.24 eV, which might facilitate nonradiative decay through the 3 IL → S 0 intersystem crossing (ISC) process. Based on Marcus theory ( Table S11 ), the rate constant ( k ISC ) of the 3 IL → S 0 ISC is computed to be 1.65 × 10 8 s –1 , which is several orders of magnitude greater than the rate constant for the radiative decay process ( k r P = 3.41 × 10 2 s –1 ). Thus, the distorted 3 IL state localized on the bpy-COSMe ligand is suggested to be nonemissive. From our computational interpretations, we believe that energy transfer from the emissive 3 MLCT state to the nonemissive 3 IL (thioester) state leads to the emission quenching observed in the iridium­(III) thioester complexes. 4 Spin densities of the emissive 3 MLCT state and nonemissive 3 DS states at their optimized structures for complexes 1a-Me , 1a-Cys , and 1b . Computed energy levels (eV) with respect to the optimized S 0 state are provided. ## Intracellular Cysteine Sensing Intracellular Cysteine Sensing The intriguing aminothiol-specific phosphorogenic response of the thioester complexes motivated us to explore their applications in sensing intracellular Cys and N-Cys-containing proteins. Complex 1a was used as a model compound due to (1) its high reactivity ( k 2 = 2,385.5 M –1 s –1 ; Figure S10 ); (2) distinctive emission profiles before and after the NCL reaction with Cys ( Figure ); and (3) high selectivity toward Cys over other thiols such as ethanethiol and glutathione (GSH) (the most abundant biothiol in cells) ( Table S12 and Figure S14 ). Laser-scanning confocal microscopy (LSCM) images of live HeLa cells incubated with complex 1a (10 μM, 1 h) revealed intense cytoplasmic emission ( Figure ), which is due to the reaction of the complex with intracellular Cys to give the emissive amide product 1a-Cys . Additionally, the intracellular emission spectrum of cells treated with complex 1a closely resembles that of cells treated with conjugate 1a-Cys ( Figure S16 ), confirming the reaction of complex 1a with intracellular Cys. However, when the cells were pretreated with the thiol scavenger N -ethylmaleimide (NEM) (100 μM, 20 min), the emission intensity significantly decreased, which is attributed to the reduced intracellular Cys level. The emission was restored upon further incubation of the NEM-pretreated cells with l -Cys (100 μM, 30 min), supporting that the observed intracellular emission was due to the specific reaction of the thioester complex with Cys. These results demonstrate that complex 1a can function as a sensor for intracellular Cys. This also suggests the potential for detecting N-Cys-containing proteins in live cells. 5 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. ## Cancer-Targeted PDT Cancer-Targeted PDT Despite its role as an essential biothiol in maintaining intracellular redox homeostasis, Cys is known as an important cancer biomarker due to its overexpression in cancer tissues compared to normal tissues. Given the significant increase in the 1 O 2 generation efficiency of complex 3a upon reaction with l -Cys (from 0.13 to 0.92 in CH 3 CN; Tables S5 and S9 ), we anticipate that complex 3a can serve as a Cys-activatable photosensitizer for cancer-targeted PDT. Thus, we examined the (photo)­cytotoxic activity of complex 3a in cancerous MDA-MB-231 (high intracellular Cys level) and normal HEK-293 (low intracellular Cys level) cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The complex was essentially noncytotoxic in the dark (IC 50,dark > 50 μM) ( Table S13 ) in both cell lines. However, upon irradiation at 450 nm (light dosage = 14.6 mW cm –2 ) for 10 min, the complex exhibited remarkable photocytotoxic effects, with greater potency in MDA-MB-231 cells (IC 50,light = 0.57 μM) compared to HEK-293 cells (IC 50,light = 1.7 μM) ( Table S13 ). This discrepancy in potency can be ascribed to (1) the higher cellular uptake of the complex in MDA-MB-231 cells (0.18 fmol/cell) than HEK-293 cells (0.087 fmol/cell) ( Table S13 ); and (2) the higher intracellular Cys level in MDA-MB-231 cells leading to the greater in situ formation of conjugate 3a-Cys . These results highlight the potential of complex 3a in cancer-selective PDT. ## Preparation and Characterization of Tumor-Targeting Iridium­(III)–Peptide Conjugates Preparation and Characterization of Tumor-Targeting Iridium­(III)–Peptide Conjugates Considering that the thioester complex 3a showed rapid reaction kinetics toward l -Cys ( k 2 = 1,658.3 M –1 s –1 ; Figure S10 ) and its Cys conjugate 3a-Cys showed a high 1 O 2 generation quantum yield (Φ Δ = 0.92 in CH 3 CN; Table S9 ), complex 3a was used to modify various tumor-targeting peptides to yield photofunctional metal–peptide conjugates for theranostic applications. Three peptides were selected: (1) CASPSGALRSC (CASP), which demonstrates high targeting specificity toward the triple-negative breast cancer MDA-MB-231 cell line; (2) YNTNHVPLSPKY (YNT), which targets the enzyme carbonic anhydrase IX (CAIX) that is often overexpressed in various cancer cell types under hypoxic conditions; and (3) CMYIEALDKYAC (CMYI), which targets the epidermal growth factor receptor (EGFR), a critical transmembrane receptor that is overexpressed in numerous cancer cell lines. All three peptides were modified with an N-Cys residue for conjugation. The iridium­(III)–peptide conjugates 3a-CASP , 3a-CYNT , and 3a-CMYI were prepared by incubation of complex 3a with the peptides CASP, CYNT, and CMYI, respectively, in TCEP-containing buffer solutions at 298 K for 12 h. Notably, despite the presence of an intrinsic Cys residue at the C-terminus of both CASP and CMYI peptides, the reactions only yielded one major product ( Figure S17 ), highlighting the excellent chemo- and regioselectivity of the thioester complexes. The conjugates were purified by semipreparative HPLC, and the purified products were characterized by HPLC and ESI-MS analyses ( Figures S18 and S19 ). Upon photoexcitation, the conjugates displayed moderate emission intensities and lifetimes in aqueous solutions ( Table S8 and Figure S20 ). Additionally, the peptide conjugates exhibited comparable 1 O 2 photosensitization capabilities (Φ Δ = 0.20–0.28) to conjugate 3a-Cys (Φ Δ = 0.26) in aqueous solutions ( Table S14 ). The enhanced emission quantum yields, extended lifetimes, and increased 1 O 2 photosensitization efficiencies of the conjugates, as compared to the thioester complex 3a , indicate the elimination of the thioester-mediated quenching pathway upon the NCL reaction. ## Cellular Uptake, Localization, and (Photo)­cytotoxicity of the Peptide Conjugates Cellular Uptake, Localization, and (Photo)­cytotoxicity of the Peptide Conjugates To evaluate the cellular uptake and localization of the iridium­(III)–peptide conjugates, two cancer cell lines MDA-MB-231 (high expression levels of CAIX and EGFR) and MCF-7 (low expression levels of CAIX and EGFR), and one normal cell line HEK-293 were utilized as the models. , As revealed by ICP-MS analyses, the cellular uptake of all three peptide conjugates was substantially higher in MDA-MB-231 cells (0.21–1.8 fmol/cell) compared to MCF-7 (0.074–0.10 fmol/cell) and HEK-293 cells (0.033–0.10 fmol/cell) ( Table S15 ). This observation aligns with the higher abundance of the target CAIX and EGFR proteins in MDA-MB-231 cells and the specific targeting capabilities of the peptide conjugates. In contrast, the peptide-free conjugate 3a-Cys showed similar levels of cellular uptake in all three cell lines (0.040–0.30 fmol/cell; Table S15 ), indicating its lack of selectivity. The cellular uptake mechanism and pathways of the conjugates were further studied. For conjugate 3a-CASP , treatment of MDA-MB-231 cells with the conjugate (10 μM) at 4 °C led to a notable decrease in intracellular iridium content by ca. 70% ( Figure S21 ), suggesting that the conjugate was internalized through an energy-dependent mechanism. The cellular uptake efficiency of the conjugate was reduced by ca. 30% when the cells were pretreated with a clathrin-mediated endocytosis inhibitor chlorpromazine (30 μM, 1 h) ( Figure S22 ). In contrast, it remained largely unchanged when the cells were pretreated with other endocytosis inhibitors, such as a macropinocytosis inhibitor 5-( N -ethyl- N -isopropyl)­amiloride (EIPA) (50 μM, 1.5 h) and a caveolin-mediated endocytosis inhibitor methyl-β-cyclodextrin (Me-β-CD) (5 mM, 1 h). These findings suggest that conjugate 3a-CASP was internalized into the cells through an energy-dependent pathway, likely involving clathrin-mediated endocytosis. For conjugates 3a-CYNT and 3a-CMYI , pretreatment of the cells with a CAIX inhibitor acetazolamide (1 mM, 6 h) or an EGFR inhibitor gefitinib (50 μM, 1 h) only reduced the uptake of the conjugates by ca. 20 and 10% ( Figures S23 and S24 ), respectively. These findings implied that the modification of the tumor-targeting peptides with an iridium­(III) complex modulated their uptake pathways, which is probably associated with their cationic charge and high lipophilicity. The intracellular distribution of the conjugates was studied by LSCM. Live MDA-MB-231, MCF-7, and HEK-293 cells were incubated with conjugates 3a-Cys , 3a-CASP , 3a-CYNT , and 3a-CMYI (10 μM) for 16 h. As depicted in the LSCM images, after incubation with the peptide conjugates, MDA-MB-231 cells displayed more pronounced intracellular emission compared to MCF-7 and HEK-293 cells ( Figure ). However, treatment with the Cys conjugate 3a-Cys resulted in similar emission intensities across the three cell lines ( Figure ). These observations correlate well with the respective cellular uptake efficiencies of the conjugates ( Table S15 ). Co-staining experiments demonstrate that the conjugates were primarily localized in the lysosomes of MDA-MB-231 cells after uptake ( Figure and Figure S25 ). 6 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. 7 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. The (photo)­cytotoxicity of the conjugates was studied using the MTT assay. Conjugates 3a-CASP , 3a-CYNT , and 3a-CMYI all exhibited minimal dark cytotoxicity (IC 50,dark > 25 μM; Table ) across the three cell lines. Upon irradiation (450 nm, 14.6 mW cm –2 , 10 min), all conjugates showed the highest photocytotoxicity toward MDA-MB-231 cells, with IC 50,light values ranging from 0.18 to 0.41 μM ( Table ). The photocytotoxic effects of the conjugates were consistent among MCF-7 and HEK-293 cells (IC 50,light = 0.65–1.4 μM and 0.56–1.4 μM, respectively; Table ), correlating with their comparatively lower cellular uptake efficiencies in these two cell lines ( Table S15 ). These results collectively demonstrate that conjugates 3a-CASP , 3a-CYNT , and 3a-CMYI retained the tumor-targeting properties of the original peptides, and the combined action of photocytotoxic iridium­(III) complexes and tumor-targeting peptides in these conjugates facilitates precise and effective cancer-targeted PDT. 1 (Photo)­cytotoxicity of the Peptide Conjugates of Complex 3a toward MDA-MB-231, MCF-7, and HEK-293 Cells conjugate MDA-MB-231 MCF-7 HEK-293   IC 50,dark /μM IC 50,light /μM PI IC 50,dark /μM IC 50,light /μM PI IC 50,dark /μM IC 50,light /μM PI 3a-CASP >25 0.41 ± 0.01 >61 >25 1.4 ± 0.1 >18 >25 1.4 ± 0.1 >18 3a-CYNT >25 0.18 ± 0.01 >139 >25 0.65 ± 0.03 >38 >25 0.56 ± 0.02 >45 3a-CMYI >25 0.23 ± 0.01 >109 >25 1.0 ± 0.1 >25 >25 0.90 ± 0.08 >28 a PI is the ratio IC 50,dark /IC 50,light under different conditions. The cells were first incubated with the conjugates in the dark for 24 h, replaced with fresh medium, and then incubated in the dark or irradiated at 450 nm (light dosage = 14.6 mW cm –2 ) for 10 min, and subsequently incubated in the dark for 24 h. ## Conclusions Conclusions Selective and site-specific bioconjugation plays a pivotal role in biochemistry and biomedical research. In this work, we developed a series of iridium­(III) thioester complexes as phosphorogenic labeling reagents specific for N-Cys-containing biomolecules. Although NCL involving thioesters and N-Cys has been investigated for chemical synthesis of peptides and proteins, the role of thioesters as an emission quenching moiety remains underexplored, and most thioester-based fluorogenic probes for N-Cys typically employ thioester as a responsive linker, connecting a fluorophore to a quencher to enable quenching through PeT or Förster resonance energy transfer. In contrast, our work demonstrates that integrating a thioester moiety into transition metal complexes can effectively modulate their emission and reactive oxygen species photosensitization capabilities. These complexes displayed weak emission in solutions due to the presence of a low-lying nonradiative distorted 3 IL state localized on the thioester moiety that quenches the emission, as discerned through computational analyses. However, upon NCL reaction with N-Cys, the complexes exhibited substantially increased emission intensities and 1 O 2 -photosensitization capabilities due to the conversion of the quenching thioester unit to a nonquenching amide linkage. Notably, the emission quenching induced by the 3 IL (thioester) state is effective across a relatively broad energy range (550–700 nm). Although our computational mechanistic study focuses on iridium­(III) thioester complexes, the proposed strategy should be applicable to other systems beyond these complexes. Specifically, our strategy should be more effective in systems featuring an emissive state of sufficient triplet state energy and efficiency for energy transfer to the designed quenching state involving a large structural distortion. Potential extension of our strategy to other systems may form the basis for future independent work. Additionally, the thioester complexes showed remarkable selectivity toward N-Cys and significantly enhanced reactivity due to the electron-withdrawing iridium­(III) polypyridine moiety. These promising attributes led to the successful applications of complexes 1a and 3a as an intracellular Cys sensor and Cys-activatable photosensitizer for cancer-targeted PDT, respectively. Furthermore, complex 3a was reacted with various N-Cys-modified tumor-targeting peptides to afford photofunctional peptide conjugates that showed high specificity and selective photocytotoxicity toward MDA-MB-231 cells compared to MCF-7 and HEK-293 cells. We believe that our design approach can extend to various organic and inorganic systems, inspiring the development of novel luminogenic thioester-based reagents for bioconjugation, bioimaging, and therapeutic applications. ## Methods Methods Physical Measurements and Instrumentation 1 H and 13 C NMR spectra were recorded on a Bruker 300, 400, or 600 MHz AVANCE III spectrometer at 298 K using deuterated solvents. Chemical shifts (δ, ppm) were reported relative to tetramethylsilane (TMS) or the residual peak of the deuterated solvent ((CD 3 ) 2 CO, 2.05 ppm; CD 3 CN, 1.94 ppm). Positive-ion ESI mass spectra were recorded on a SCIEX API-3200 Triple-Q MS/MS mass spectrometer at 298 K. HR-ESI mass spectra were recorded on a SCIEX X500R Q-TOF at 298 K. IR spectra of the samples in KBr pellets were recorded in the range of 4000–400 cm –1 using a PerkinElmer Spectrum 100 FTIR spectrometer. Elemental analyses were carried out on an Elementar Analysensysteme GmbH Vario MICRO elemental analyzer. HPLC was performed on an Agilent 1260 Infinity II system coupled with a diode array detector WR using H 2 O containing 0.1% ( v / v ) trifluoroacetic acid (TFA) (solvent A) and CH 3 CN containing 0.1% ( v / v ) TFA (solvent B) as the solvents, and the detector was set to 220, 250, and 350 nm. Preparation of Iridium­(III)–Cys Conjugates 1a-Cys – 4a-Cys A mixture of the iridium­(III) thioester complex (4 μmol) and l -Cys (200 μmol) in CH 3 CN/potassium phosphate buffer (50 mM, pH 7.0) (10:1, v / v , 11 mL) was stirred at 298 K in the dark for 18 h. The mixture was diluted with CH 2 Cl 2 (20 mL), and the organic layer was washed with H 2 O (20 mL × 3), dried over anhydrous MgSO 4 , and filtered. The solvent was removed under reduced pressure and the residual solid was purified by semipreparative reversed-phase HPLC (RP-HPLC). The HPLC purification was carried out on an Agilent semipreparative column (ZORBAX Eclipse XDB-C18 column: 9.4 × 250 mm, 5 μm) using solvent A and solvent B as the solvents, with a linear gradient of 50–100% solvent B over 30 min and a flow rate of 2 mL min –1 . Fractions containing the product were combined and lyophilized. The orange purified conjugates were characterized by analytical RP-HPLC and ESI-MS. The HPLC analysis was carried out using an Agilent analytical column (ZORBAX Eclipse Plus C18 column: 4.6 × 150 mm, 5 μm) with a linear gradient of 40–100% solvent B over 12 min and a flow rate of 1 mL min –1 . 1a-Cys . Yield: 3.4 mg (82%). t R = 6.8 min. 1 H NMR (300 MHz, CD 3 CN, 298 K): δ 8.51 (d, J = 1.2 Hz, 1H, H3 of bpy), 8.40–8.27 (m, 5H, H3 and H4 of quinolinyl ring of pq, and H6 of bpy), 8.24–8.10 (m, 4H, CONH, H3′ of bpy, and H3 of phenyl ring of pq), 8.03 (d, J = 5.7 Hz, 1H, H6’ of bpy), 7.85–7.77 (m, 3H, H8 of quinolinyl ring of pq and H5 of bpy), 7.39 (td, J = 7.6, 2.9 Hz, 2H, H7 of quinolinyl ring of pq), 7.34–7.25 (m, 3H, H5′ of bpy and H5 of quinolinyl ring of pq), 7.17 (t, J = 7.6 Hz, 2H, H4 of phenyl ring of pq), 7.13–7.00 (m, 2H, H6 of quinolinyl ring of pq), 6.81 (td, J = 7.6, 3.4 Hz, 2H, H5 of phenyl ring of pq), 6.53–6.47 (m, 2H, H6 of phenyl ring of pq), 4.67 (q, J = 6.2 Hz, 1H, CH of Cys), 3.07–2.95 (m, 2H, CH 2 of Cys), 2.40 (s, 3H, CH 3 of bpy), 1.89 (br, 1H, SH of Cys). MS (ESI, positive mode, m / z ): 918.5 [M – CF 3 COO – ] + . 2a-Cys . Yield: 3.7 mg (80%). t R = 9.7 min. MS (ESI, positive mode, m / z ): 1030.5 [M – CF 3 COO – ] + . 3a-Cys . Yield: 3.7 mg (80%). t R = 9.3 min. MS (ESI, positive mode, m / z ): 1030.5 [M – CF 3 COO – ] + . 4a-Cys . Yield: 3.9 mg (79%). t R = 9.9 min. MS (ESI, positive mode, m / z ): 1130.5 [M – CF 3 COO – ] + . Preparation of Peptide Conjugates of Complex 3a A mixture of complex 3a (2 μmol) and N-Cys-containing peptide (CASP, CYNT, and CMYI) (3 μmol) in potassium phosphate buffer (50 mM, pH 7.0)/DMSO (3:2, v / v , 2 mL) containing TCEP (10 mM) was stirred at 298 K in the dark for 12 h. The solvent was removed under reduced pressure and the residual dark orange solid was purified by semipreparative RP-HPLC. The HPLC purification was carried out on an Agilent semipreparative column (ZORBAX Eclipse XDB-C18 column: 9.4 × 250 mm, 5 μm) using solvent A and solvent B as the solvents, with a linear gradient of 20–100% solvent B over 20 min and a flow rate of 2 mL min –1 . Fractions containing the product were combined and lyophilized. The dark orange purified conjugates were characterized by analytical RP-HPLC and ESI-MS. The HPLC analysis was carried out using an Agilent analytical column (ZORBAX Eclipse Plus C18 column: 4.6 × 150 mm, 5 μm) with a linear gradient of 40–100% solvent B over 12 min and a flow rate of 1 mL min –1 . 3a-CASP . Yield: 3.0 mg (72%). t R = 7.3 min. MS (ESI, positive mode, m / z ): 979.7 [M – CF 3 COO – + H + ] 2+ . 3a-CYNT . Yield: 3.6 mg (70%). t R = 6.9 min. MS (ESI, positive mode, m / z ): 1223.4 [M – CF 3 COO – + H + ] 2+ , 816.0 [M – CF 3 COO – + 2H + ] 3+ . 3a-CMYI . Yield: 3.3 mg (68%). t R = 7.7 min. MS (ESI, positive mode, m / z ): 1166.9 [M – CF 3 COO – + H + ] 2+ . X-ray Structural Analysis for Complex 1a Single crystals of the complex were obtained by slow diffusion of Et 2 O into a concentrated CH 3 CN solution of the complex at 298 K. Single-X-ray data were collected on a Rigaku Oxford Diffraction, Synergy Custom system, HyPix diffractometer using Cu Kα radiation (1.54184 Å). Cell refinement, data collection, and data reduction were done using CrysAlisPro 1.171.43.127a (Rigaku OD, 2024) program. The structure was resolved by direct methods and refined using full-matrix least-squares on F 2 with the SHELXL program through the OLEX2 interface. All nonhydrogen atoms of the complexes were refined with anisotropic thermal parameters. Hydrogen atoms were placed in idealized positions and refined with fixed geometry with respect to their carrier atoms. The structure features a positional disorder on the bpy-COSBn ligand with a 4:1 ratio under C 2 symmetry. The predominant conformation (80%) is depicted in Figure . Stability Studies The thioester complex 1a (10 μM) was incubated in potassium phosphate buffer (50 mM, pH 7.0)/CH 3 CN (3:2, v / v ) containing TCEP (1 mM) at 37 °C in the dark for 1 h. The solution was extracted with CH 2 Cl 2 (1 mL × 3). The organic extract was dried over anhydrous MgSO 4 , filtered, and the solvent was removed under reduced pressure. The residue was dissolved in CH 3 CN and analyzed by ESI-MS. Selectivity Studies For the chemoselectivity studies, a mixture of complex 1a (20 μM) and l -Lys (2 mM), l -His (2 mM), l -Ser (2 mM), or l -Thr (2 mM) was incubated in potassium phosphate buffer (50 mM, pH 7.0)/DMSO (3:2, v / v ) containing TCEP (10 mM) at 298 K in the dark for 1 h. For the regioselectivity studies, a mixture of complex 1a (25 μM) and the tripeptide CSS, SCS, or SSC (1 mM) was incubated in potassium phosphate buffer (50 mM, pH 7.0)/DMSO (3:2, v / v ) containing TCEP (10 mM) at 298 K in the dark for 1 h. An aliquot of the reaction mixture (20 μL) was analyzed by RP-HPLC. The HPLC analysis was carried out using an Agilent analytical column (ZORBAX Eclipse Plus C18 column: 4.6 × 150 mm, 5 μm) with a linear gradient of 40–100% solvent B over 12 min and a flow rate of 1 mL min –1 . Phosphorogenic Response of Thioester Complexes toward Thiols The thioester complexes 1a – 4a (10 μM) were incubated with l -Cys (100 μM) in aerated potassium phosphate buffer (50 mM, pH 7.0)/CH 3 CN (3:2, v / v ) containing TCEP (1 mM) at 298 K in the dark for 1 h. The emission spectra were recorded on a HORIBA FluoroMax-4 spectrofluorometer and the emission lifetimes were measured on an Edinburgh Instruments FLS980 spectrometer. The emission intensities were determined by the areas under the emission spectra before and after incubation with l -Cys. An emission titration experiment was conducted by the gradual addition of l -Cys to a solution of complex 1a (10 μM) in aerated potassium phosphate buffer (50 mM, pH 7.0)/CH 3 CN (3:2, v / v ) containing TCEP (1 mM) at 298 K. The emission spectra were recorded on a HORIBA FluoroMax-4 spectrofluorometer. Each emission spectrum was acquired 4 min after mixing. For the selectivity studies, a mixture of complex 1a (10 μM) and l -Cys (100 μM), ethanethiol (100 μM), or GSH (100 μM) was incubated in aerated potassium phosphate buffer (50 mM, pH 7.0)/CH 3 CN (3:2, v / v ) containing TCEP (1 mM) at 298 K in the dark for 1 h. The emission spectra were recorded on a HORIBA FluoroMax-4 spectrofluorometer. The emission intensities were determined by the areas under the emission spectra before and after incubation with thiols. Cell Cultures HeLa, MDA-MB-231, MCF-7, and HEK-293 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C under a 5% CO 2 atmosphere. They were subcultured every 2 to 3 days. Live-Cell Confocal Imaging Complex 1a : HeLa cells in growth medium were seeded in a 35-mm confocal dish and grown at 37 °C under a 5% CO 2 atmosphere for 48 h. The growth medium was removed, and the cells were washed with PBS (1 mL × 3) and incubated with complex 1a (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C for 1 h. After the treatment, the medium was removed, and the cells were washed with PBS (1 mL × 3) and imaged using a Leica TCS SPE confocal microscope (inverted configuration) with a 63× oil-immersion objective lens. The excitation wavelength of complex 1a was 405 nm. For the control experiments, the cells were treated with NEM (100 μM) at 37 °C for 20 min with or without posttreatment with l -Cys (100 μM) at 37 °C for 30 min prior to incubation with complex 1a . The intracellular emission spectra were recorded using a λ-scanning mode. HeLa cells were treated with complex 1a (10 μM, 1 h) or conjugate 1a-Cys (20 μM, 6 h). The incubation period was longer for the conjugate due to its poorer cellular uptake efficiency compared with complex 1a . Conjugates 3a-Cys , 3a-CASP , 3a-CYNT , and 3a-CMYI : MDA-MB-231, MCF-7, or HEK-293 cells in growth medium were seeded in a 35-mm confocal dish and grown at 37 °C under a 5% CO 2 atmosphere for 48 h. The growth medium was removed, and the cells were washed with PBS (1 mL × 3) and incubated with the conjugates (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C for 16 h. After the treatment, the medium was removed, and the cells were washed with PBS (1 mL × 3) and imaged using a Leica TCS SPE confocal microscope (inverted configuration) with a 63× oil-immersion objective lens. The excitation wavelength of the conjugates was 488 nm. For the co-staining experiments in MDA-MB-231 cells, after treatment with the conjugates, the medium was removed, and the cells were washed with PBS (1 mL × 3) and incubated with LysoTracker Deep Red (100 nM) for 1 h or MitoTracker Green (100 nM) for 20 min in growth medium at 37 °C. The medium was then removed, and the cells were washed with PBS (1 mL × 3) and imaged using a Leica TCS SPE confocal microscope (inverted configuration) with a 63× oil-immersion objective lens. The excitation wavelength of LysoTracker Deep Red and MitoTracker Green were 635 and 488 nm, respectively. The PCC values were determined using the program ImageJ (Version 1.4.3.67). MTT Assays HeLa or HEK-293 cells were seeded in two 96-well flat-bottomed microplates (ca. 10,000 cells per well) in growth medium (100 μL) and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. The growth medium was removed and replaced with medium/DMSO (100 μL, 99:1, v / v ) containing complex 3a with concentrations ranging from 10 –5 to 10 –8 M. Wells containing untreated cells were used as blank control. The microplates were incubated at 37 °C in the dark under a 5% CO 2 atmosphere for 2 h. After the treatment, the medium was removed and replenished with phenol red-free growth medium (100 μL). One of the microplates was irradiated at 450 nm (14.6 mW cm –2 ) for 10 min with an LED cellular photocytotoxicity irradiator (PURI Materials, Shenzhen, China) and the other microplate was kept in the dark. Then the growth medium was replaced with fresh medium and the cells were further incubated at 37 °C under a 5% CO 2 atmosphere for 24 h. The medium in each well was then replaced with fresh medium (90 μL) and 10 μL of MTT (5 mg mL – 1 ) in PBS. The microplates were incubated at 37 °C under a 5% CO 2 atmosphere for 4 h. The growth medium was then removed, and DMSO (100 μL) was added to each well. After 20 min, the absorbance of the solutions at 570 nm was measured with an Multiskan SkyHigh Microplate Spectrophotometer (Thermo Scientific). The IC 50 values of the complex were determined from dose dependence of surviving cells after the treatment using the OriginPro 8.0 software package. For conjugates 3a-CASP , 3a-CYNT , and 3a-CMYI , the procedure was similar to that of complex 3a , except that MDA-MB-231, MCF-7, and HEK-293 cells were used and the conjugates were incubated for 24 h instead of 2 h before irradiation. Cellular Uptake Measurements HeLa or HEK-293 cells were seeded in a 35-mm tissue culture dish and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. The culture medium was removed and replaced with complexes 1a and 3a (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 2 h. After the treatment, the medium was removed, and the cells were washed with PBS (1 mL × 3). The cells were then trypsinized and harvested with PBS (1 mL). The cell numbers were obtained by a Logos Biosystems LUNA-II automated cell counter. The resultant solution was digested with 65% HNO 3 (1 mL) at 70 °C for 2 h, allowed to cool to room temperature, and analyzed by a NexION 2000 ICP-MS (PerkinElmer SCIEX Instruments). For conjugates 3a-Cys , 3a-CASP , 3a-CYNT , and 3a-CMYI , the procedure was similar to that of complexes 1a and 3a , except that MDA-MB-231, MCF-7, and HEK-293 cells were used and the conjugates were incubated for 16 h instead of 2 h before the measurements. Cellular Uptake Mechanism Studies Conjugate 3a-CASP : MDA-MB-231 cells were seeded in a 35-mm tissue culture dish and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. The culture medium was removed and replaced with conjugate 3a-CASP (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 4 or 16 h. In the low-temperature experiments, the cells were preincubated at 4 °C for 1 h prior to incubation with conjugate 3a-CASP (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 4 h. In the chemical inhibition experiments, the cells were pretreated with EIPA (50 μM) for 1.5 h, Me-β-CD (5 mM) for 1 h, or chlorpromazine (30 μM) for 1 h at 37 °C under a 5% CO 2 atmosphere. The cells were then washed with PBS (1 mL × 3) and incubated with conjugate 3a-CASP (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 16 h. Conjugate 3a-CYNT : MDA-MB-231 cells were seeded in a 35-mm tissue culture dish and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. The culture medium was removed and replaced with conjugate 3a-CYNT (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 16 h. In the CAIX inhibition experiments, the cells were pretreated with acetazolamide (1 mM) at 37 °C under a 5% CO 2 atmosphere for 6 h. The cells were then washed with PBS (1 mL × 3) and incubated with conjugate 3a-CYNT (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 16 h. Conjugate 3a-CMYI : MDA-MB-231 cells were seeded in a 35-mm tissue culture dish and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. The culture medium was removed and replaced with conjugate 3a-CMYI (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 16 h. In the EGFR inhibition experiments, the cells were pretreated with gefitinib (50 μM) at 37 °C under a 5% CO 2 atmosphere for 1 h. The cells were then washed with PBS (1 mL × 3) and incubated with conjugate 3a-CMYI (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 16 h. For all the experiments, after incubation with the conjugates, the medium was removed, and the cells were washed with PBS (1 mL × 3). The cells were then trypsinized and harvested with PBS (1 mL). The cell numbers were measured by a Logos Biosystems LUNA-II automated cell counter. The resultant solution was digested with 65% HNO 3 (1 mL) at 70 °C for 2 h, allowed to cool to room temperature, and analyzed by a NexION 2000 ICP-MS (PerkinElmer SCIEX Instruments). Additional experimental procedures and data processing methods are detailed in the Supporting Information . ## Physical Measurements and Instrumentation Physical Measurements and Instrumentation 1 H and 13 C NMR spectra were recorded on a Bruker 300, 400, or 600 MHz AVANCE III spectrometer at 298 K using deuterated solvents. Chemical shifts (δ, ppm) were reported relative to tetramethylsilane (TMS) or the residual peak of the deuterated solvent ((CD 3 ) 2 CO, 2.05 ppm; CD 3 CN, 1.94 ppm). Positive-ion ESI mass spectra were recorded on a SCIEX API-3200 Triple-Q MS/MS mass spectrometer at 298 K. HR-ESI mass spectra were recorded on a SCIEX X500R Q-TOF at 298 K. IR spectra of the samples in KBr pellets were recorded in the range of 4000–400 cm –1 using a PerkinElmer Spectrum 100 FTIR spectrometer. Elemental analyses were carried out on an Elementar Analysensysteme GmbH Vario MICRO elemental analyzer. HPLC was performed on an Agilent 1260 Infinity II system coupled with a diode array detector WR using H 2 O containing 0.1% ( v / v ) trifluoroacetic acid (TFA) (solvent A) and CH 3 CN containing 0.1% ( v / v ) TFA (solvent B) as the solvents, and the detector was set to 220, 250, and 350 nm. ## Preparation of Iridium­(III)–Cys Conjugates Preparation of Iridium­(III)–Cys Conjugates 1a-Cys – 4a-Cys A mixture of the iridium­(III) thioester complex (4 μmol) and l -Cys (200 μmol) in CH 3 CN/potassium phosphate buffer (50 mM, pH 7.0) (10:1, v / v , 11 mL) was stirred at 298 K in the dark for 18 h. The mixture was diluted with CH 2 Cl 2 (20 mL), and the organic layer was washed with H 2 O (20 mL × 3), dried over anhydrous MgSO 4 , and filtered. The solvent was removed under reduced pressure and the residual solid was purified by semipreparative reversed-phase HPLC (RP-HPLC). The HPLC purification was carried out on an Agilent semipreparative column (ZORBAX Eclipse XDB-C18 column: 9.4 × 250 mm, 5 μm) using solvent A and solvent B as the solvents, with a linear gradient of 50–100% solvent B over 30 min and a flow rate of 2 mL min –1 . Fractions containing the product were combined and lyophilized. The orange purified conjugates were characterized by analytical RP-HPLC and ESI-MS. The HPLC analysis was carried out using an Agilent analytical column (ZORBAX Eclipse Plus C18 column: 4.6 × 150 mm, 5 μm) with a linear gradient of 40–100% solvent B over 12 min and a flow rate of 1 mL min –1 . 1a-Cys . Yield: 3.4 mg (82%). t R = 6.8 min. 1 H NMR (300 MHz, CD 3 CN, 298 K): δ 8.51 (d, J = 1.2 Hz, 1H, H3 of bpy), 8.40–8.27 (m, 5H, H3 and H4 of quinolinyl ring of pq, and H6 of bpy), 8.24–8.10 (m, 4H, CONH, H3′ of bpy, and H3 of phenyl ring of pq), 8.03 (d, J = 5.7 Hz, 1H, H6’ of bpy), 7.85–7.77 (m, 3H, H8 of quinolinyl ring of pq and H5 of bpy), 7.39 (td, J = 7.6, 2.9 Hz, 2H, H7 of quinolinyl ring of pq), 7.34–7.25 (m, 3H, H5′ of bpy and H5 of quinolinyl ring of pq), 7.17 (t, J = 7.6 Hz, 2H, H4 of phenyl ring of pq), 7.13–7.00 (m, 2H, H6 of quinolinyl ring of pq), 6.81 (td, J = 7.6, 3.4 Hz, 2H, H5 of phenyl ring of pq), 6.53–6.47 (m, 2H, H6 of phenyl ring of pq), 4.67 (q, J = 6.2 Hz, 1H, CH of Cys), 3.07–2.95 (m, 2H, CH 2 of Cys), 2.40 (s, 3H, CH 3 of bpy), 1.89 (br, 1H, SH of Cys). MS (ESI, positive mode, m / z ): 918.5 [M – CF 3 COO – ] + . 2a-Cys . Yield: 3.7 mg (80%). t R = 9.7 min. MS (ESI, positive mode, m / z ): 1030.5 [M – CF 3 COO – ] + . 3a-Cys . Yield: 3.7 mg (80%). t R = 9.3 min. MS (ESI, positive mode, m / z ): 1030.5 [M – CF 3 COO – ] + . 4a-Cys . Yield: 3.9 mg (79%). t R = 9.9 min. MS (ESI, positive mode, m / z ): 1130.5 [M – CF 3 COO – ] + . ## Preparation of Peptide Conjugates of Complex Preparation of Peptide Conjugates of Complex 3a A mixture of complex 3a (2 μmol) and N-Cys-containing peptide (CASP, CYNT, and CMYI) (3 μmol) in potassium phosphate buffer (50 mM, pH 7.0)/DMSO (3:2, v / v , 2 mL) containing TCEP (10 mM) was stirred at 298 K in the dark for 12 h. The solvent was removed under reduced pressure and the residual dark orange solid was purified by semipreparative RP-HPLC. The HPLC purification was carried out on an Agilent semipreparative column (ZORBAX Eclipse XDB-C18 column: 9.4 × 250 mm, 5 μm) using solvent A and solvent B as the solvents, with a linear gradient of 20–100% solvent B over 20 min and a flow rate of 2 mL min –1 . Fractions containing the product were combined and lyophilized. The dark orange purified conjugates were characterized by analytical RP-HPLC and ESI-MS. The HPLC analysis was carried out using an Agilent analytical column (ZORBAX Eclipse Plus C18 column: 4.6 × 150 mm, 5 μm) with a linear gradient of 40–100% solvent B over 12 min and a flow rate of 1 mL min –1 . 3a-CASP . Yield: 3.0 mg (72%). t R = 7.3 min. MS (ESI, positive mode, m / z ): 979.7 [M – CF 3 COO – + H + ] 2+ . 3a-CYNT . Yield: 3.6 mg (70%). t R = 6.9 min. MS (ESI, positive mode, m / z ): 1223.4 [M – CF 3 COO – + H + ] 2+ , 816.0 [M – CF 3 COO – + 2H + ] 3+ . 3a-CMYI . Yield: 3.3 mg (68%). t R = 7.7 min. MS (ESI, positive mode, m / z ): 1166.9 [M – CF 3 COO – + H + ] 2+ . ## X-ray Structural Analysis for Complex X-ray Structural Analysis for Complex 1a Single crystals of the complex were obtained by slow diffusion of Et 2 O into a concentrated CH 3 CN solution of the complex at 298 K. Single-X-ray data were collected on a Rigaku Oxford Diffraction, Synergy Custom system, HyPix diffractometer using Cu Kα radiation (1.54184 Å). Cell refinement, data collection, and data reduction were done using CrysAlisPro 1.171.43.127a (Rigaku OD, 2024) program. The structure was resolved by direct methods and refined using full-matrix least-squares on F 2 with the SHELXL program through the OLEX2 interface. All nonhydrogen atoms of the complexes were refined with anisotropic thermal parameters. Hydrogen atoms were placed in idealized positions and refined with fixed geometry with respect to their carrier atoms. The structure features a positional disorder on the bpy-COSBn ligand with a 4:1 ratio under C 2 symmetry. The predominant conformation (80%) is depicted in Figure . ## Stability Studies Stability Studies The thioester complex 1a (10 μM) was incubated in potassium phosphate buffer (50 mM, pH 7.0)/CH 3 CN (3:2, v / v ) containing TCEP (1 mM) at 37 °C in the dark for 1 h. The solution was extracted with CH 2 Cl 2 (1 mL × 3). The organic extract was dried over anhydrous MgSO 4 , filtered, and the solvent was removed under reduced pressure. The residue was dissolved in CH 3 CN and analyzed by ESI-MS. ## Selectivity Studies Selectivity Studies For the chemoselectivity studies, a mixture of complex 1a (20 μM) and l -Lys (2 mM), l -His (2 mM), l -Ser (2 mM), or l -Thr (2 mM) was incubated in potassium phosphate buffer (50 mM, pH 7.0)/DMSO (3:2, v / v ) containing TCEP (10 mM) at 298 K in the dark for 1 h. For the regioselectivity studies, a mixture of complex 1a (25 μM) and the tripeptide CSS, SCS, or SSC (1 mM) was incubated in potassium phosphate buffer (50 mM, pH 7.0)/DMSO (3:2, v / v ) containing TCEP (10 mM) at 298 K in the dark for 1 h. An aliquot of the reaction mixture (20 μL) was analyzed by RP-HPLC. The HPLC analysis was carried out using an Agilent analytical column (ZORBAX Eclipse Plus C18 column: 4.6 × 150 mm, 5 μm) with a linear gradient of 40–100% solvent B over 12 min and a flow rate of 1 mL min –1 . ## Phosphorogenic Response of Thioester Complexes toward Thiols Phosphorogenic Response of Thioester Complexes toward Thiols The thioester complexes 1a – 4a (10 μM) were incubated with l -Cys (100 μM) in aerated potassium phosphate buffer (50 mM, pH 7.0)/CH 3 CN (3:2, v / v ) containing TCEP (1 mM) at 298 K in the dark for 1 h. The emission spectra were recorded on a HORIBA FluoroMax-4 spectrofluorometer and the emission lifetimes were measured on an Edinburgh Instruments FLS980 spectrometer. The emission intensities were determined by the areas under the emission spectra before and after incubation with l -Cys. An emission titration experiment was conducted by the gradual addition of l -Cys to a solution of complex 1a (10 μM) in aerated potassium phosphate buffer (50 mM, pH 7.0)/CH 3 CN (3:2, v / v ) containing TCEP (1 mM) at 298 K. The emission spectra were recorded on a HORIBA FluoroMax-4 spectrofluorometer. Each emission spectrum was acquired 4 min after mixing. For the selectivity studies, a mixture of complex 1a (10 μM) and l -Cys (100 μM), ethanethiol (100 μM), or GSH (100 μM) was incubated in aerated potassium phosphate buffer (50 mM, pH 7.0)/CH 3 CN (3:2, v / v ) containing TCEP (1 mM) at 298 K in the dark for 1 h. The emission spectra were recorded on a HORIBA FluoroMax-4 spectrofluorometer. The emission intensities were determined by the areas under the emission spectra before and after incubation with thiols. ## Cell Cultures Cell Cultures HeLa, MDA-MB-231, MCF-7, and HEK-293 cells were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin at 37 °C under a 5% CO 2 atmosphere. They were subcultured every 2 to 3 days. ## Live-Cell Confocal Imaging Live-Cell Confocal Imaging Complex 1a : HeLa cells in growth medium were seeded in a 35-mm confocal dish and grown at 37 °C under a 5% CO 2 atmosphere for 48 h. The growth medium was removed, and the cells were washed with PBS (1 mL × 3) and incubated with complex 1a (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C for 1 h. After the treatment, the medium was removed, and the cells were washed with PBS (1 mL × 3) and imaged using a Leica TCS SPE confocal microscope (inverted configuration) with a 63× oil-immersion objective lens. The excitation wavelength of complex 1a was 405 nm. For the control experiments, the cells were treated with NEM (100 μM) at 37 °C for 20 min with or without posttreatment with l -Cys (100 μM) at 37 °C for 30 min prior to incubation with complex 1a . The intracellular emission spectra were recorded using a λ-scanning mode. HeLa cells were treated with complex 1a (10 μM, 1 h) or conjugate 1a-Cys (20 μM, 6 h). The incubation period was longer for the conjugate due to its poorer cellular uptake efficiency compared with complex 1a . Conjugates 3a-Cys , 3a-CASP , 3a-CYNT , and 3a-CMYI : MDA-MB-231, MCF-7, or HEK-293 cells in growth medium were seeded in a 35-mm confocal dish and grown at 37 °C under a 5% CO 2 atmosphere for 48 h. The growth medium was removed, and the cells were washed with PBS (1 mL × 3) and incubated with the conjugates (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C for 16 h. After the treatment, the medium was removed, and the cells were washed with PBS (1 mL × 3) and imaged using a Leica TCS SPE confocal microscope (inverted configuration) with a 63× oil-immersion objective lens. The excitation wavelength of the conjugates was 488 nm. For the co-staining experiments in MDA-MB-231 cells, after treatment with the conjugates, the medium was removed, and the cells were washed with PBS (1 mL × 3) and incubated with LysoTracker Deep Red (100 nM) for 1 h or MitoTracker Green (100 nM) for 20 min in growth medium at 37 °C. The medium was then removed, and the cells were washed with PBS (1 mL × 3) and imaged using a Leica TCS SPE confocal microscope (inverted configuration) with a 63× oil-immersion objective lens. The excitation wavelength of LysoTracker Deep Red and MitoTracker Green were 635 and 488 nm, respectively. The PCC values were determined using the program ImageJ (Version 1.4.3.67). ## MTT Assays MTT Assays HeLa or HEK-293 cells were seeded in two 96-well flat-bottomed microplates (ca. 10,000 cells per well) in growth medium (100 μL) and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. The growth medium was removed and replaced with medium/DMSO (100 μL, 99:1, v / v ) containing complex 3a with concentrations ranging from 10 –5 to 10 –8 M. Wells containing untreated cells were used as blank control. The microplates were incubated at 37 °C in the dark under a 5% CO 2 atmosphere for 2 h. After the treatment, the medium was removed and replenished with phenol red-free growth medium (100 μL). One of the microplates was irradiated at 450 nm (14.6 mW cm –2 ) for 10 min with an LED cellular photocytotoxicity irradiator (PURI Materials, Shenzhen, China) and the other microplate was kept in the dark. Then the growth medium was replaced with fresh medium and the cells were further incubated at 37 °C under a 5% CO 2 atmosphere for 24 h. The medium in each well was then replaced with fresh medium (90 μL) and 10 μL of MTT (5 mg mL – 1 ) in PBS. The microplates were incubated at 37 °C under a 5% CO 2 atmosphere for 4 h. The growth medium was then removed, and DMSO (100 μL) was added to each well. After 20 min, the absorbance of the solutions at 570 nm was measured with an Multiskan SkyHigh Microplate Spectrophotometer (Thermo Scientific). The IC 50 values of the complex were determined from dose dependence of surviving cells after the treatment using the OriginPro 8.0 software package. For conjugates 3a-CASP , 3a-CYNT , and 3a-CMYI , the procedure was similar to that of complex 3a , except that MDA-MB-231, MCF-7, and HEK-293 cells were used and the conjugates were incubated for 24 h instead of 2 h before irradiation. ## Cellular Uptake Measurements Cellular Uptake Measurements HeLa or HEK-293 cells were seeded in a 35-mm tissue culture dish and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. The culture medium was removed and replaced with complexes 1a and 3a (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 2 h. After the treatment, the medium was removed, and the cells were washed with PBS (1 mL × 3). The cells were then trypsinized and harvested with PBS (1 mL). The cell numbers were obtained by a Logos Biosystems LUNA-II automated cell counter. The resultant solution was digested with 65% HNO 3 (1 mL) at 70 °C for 2 h, allowed to cool to room temperature, and analyzed by a NexION 2000 ICP-MS (PerkinElmer SCIEX Instruments). For conjugates 3a-Cys , 3a-CASP , 3a-CYNT , and 3a-CMYI , the procedure was similar to that of complexes 1a and 3a , except that MDA-MB-231, MCF-7, and HEK-293 cells were used and the conjugates were incubated for 16 h instead of 2 h before the measurements. ## Cellular Uptake Mechanism Studies Cellular Uptake Mechanism Studies Conjugate 3a-CASP : MDA-MB-231 cells were seeded in a 35-mm tissue culture dish and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. The culture medium was removed and replaced with conjugate 3a-CASP (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 4 or 16 h. In the low-temperature experiments, the cells were preincubated at 4 °C for 1 h prior to incubation with conjugate 3a-CASP (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 4 h. In the chemical inhibition experiments, the cells were pretreated with EIPA (50 μM) for 1.5 h, Me-β-CD (5 mM) for 1 h, or chlorpromazine (30 μM) for 1 h at 37 °C under a 5% CO 2 atmosphere. The cells were then washed with PBS (1 mL × 3) and incubated with conjugate 3a-CASP (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 16 h. Conjugate 3a-CYNT : MDA-MB-231 cells were seeded in a 35-mm tissue culture dish and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. The culture medium was removed and replaced with conjugate 3a-CYNT (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 16 h. In the CAIX inhibition experiments, the cells were pretreated with acetazolamide (1 mM) at 37 °C under a 5% CO 2 atmosphere for 6 h. The cells were then washed with PBS (1 mL × 3) and incubated with conjugate 3a-CYNT (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 16 h. Conjugate 3a-CMYI : MDA-MB-231 cells were seeded in a 35-mm tissue culture dish and incubated at 37 °C under a 5% CO 2 atmosphere for 48 h. The culture medium was removed and replaced with conjugate 3a-CMYI (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 16 h. In the EGFR inhibition experiments, the cells were pretreated with gefitinib (50 μM) at 37 °C under a 5% CO 2 atmosphere for 1 h. The cells were then washed with PBS (1 mL × 3) and incubated with conjugate 3a-CMYI (10 μM) in medium/DMSO (99:1, v / v ) at 37 °C under a 5% CO 2 atmosphere for 16 h. For all the experiments, after incubation with the conjugates, the medium was removed, and the cells were washed with PBS (1 mL × 3). The cells were then trypsinized and harvested with PBS (1 mL). The cell numbers were measured by a Logos Biosystems LUNA-II automated cell counter. The resultant solution was digested with 65% HNO 3 (1 mL) at 70 °C for 2 h, allowed to cool to room temperature, and analyzed by a NexION 2000 ICP-MS (PerkinElmer SCIEX Instruments). Additional experimental procedures and data processing methods are detailed in the Supporting Information . ## Supplementary Material Supplementary Material