Modulating Excited State Properties and Ligand Ejection Kinetics in Ruthenium Polypyridyl Complexes Designed to Mimic Photochemotherapeutics.

{"full_text": " pubs.acs.org/IC Article\n\n\n\n Modulating Excited State Properties and Ligand Ejection Kinetics in\n Ruthenium Polypyridyl Complexes Designed to Mimic\n Photochemotherapeutics\n Faith N. Robinette, Nathaniel P. Valentine, Konrad M. Sehler, Andrew M. Medeck, Keylon E. Reynolds,\n Skylar N. Lane, Averie N. Price, Ireland G. Cavanaugh, Steven M. Shell, and Dennis L. Ashford*\n Cite This: Inorg. Chem. 2024, 63, 8426\u22128439 Read Online\nSee https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.\n\n\n\n\n ACCESS Metrics & More Article Recommendations *\n s\u0131 Supporting Information\n Downloaded via MOSCOW STATE UNIV on May 12, 2026 at 11:26:51 (UTC).\n\n\n\n\n ABSTRACT: Ruthenium(II) polypyridyl complexes have gained\n significant interest as photochemotherapeutics (PCTs) due to their\n synthetic viability, strong light absorption, well understood excited\n state properties, and high phototoxicity indexes. Herein, we report\n the synthesis, characterization, electrochemical, spectrochemical,\n and preliminary cytotoxicity analyses of three series of ruthenium-\n (II) polypyridyl complexes designed to mimic PCTs. The three\n series have the general structure of [Ru(bpy)2(N\u2212N)]2+ (Series\n 1), [Ru(bpy)(dmb)(N\u2212N)]2+ (Series 2), and [Ru(dmb)2(N\u2212\n N)]2+ (Series 3, where N\u2212N is a bidentate polypyridyl ligand, bpy\n = 2,2\u2032-bipyridine, and dmb = 6,6\u2032-dimethyl-2,2\u2032-bipyridine). In the\n three series, the N\u2212N ligand was systematically modified to\n incorporate increased conjugation and/or electronegative heteroatoms to increase d\u03c0-\u03c0* backbonding, red-shifting the lowest energy\n metal-to-ligand charge transfer (MLCT) absorptions from \u03bbmax = 454 to \u03bbmax = 580 nm, nearing the therapeutic window for PCTs\n (600\u22121100 nm). In addition, steric bulk was systematically introduced through the series, distorting the Ru(II) octahedra, making\n the dissociative 3dd* state thermally accessible at room and body temperatures. This resulted in a 4 orders of magnitude increase in\n photoinduced ligand ejection kinetics, and demonstrates the ability to modulate both the MLCT* and dd* manifolds in the\n complexes, which is critical in PCT drug design. Preliminary cell viability assays suggest that the increased steric bulk to lower the\n 3\n dd* states may interfere with the cytotoxicity mechanism, limiting photoinitiated toxicity of the complexes. This work demonstrates\n the importance of understanding both the MLCT* and dd* manifolds and how they impact the ability of a complex to act as a PCT\n agent.\n\n\n \u25a0 INTRODUCTION\n Nearly half of all chemotherapeutics administered today are\n tumor cells and lack of strong light absorption in the near-IR,\n therapeutic window (600\u22121100 nm).14\u221221 PCTs overcome\n derived from platinum-based drugs, commonly referred to as the limitation of hypoxia in tumorous cells by relying on the\n platins.1\u22123 While these drugs remain widely used, they still photoinduced release of a therapeutic reagent, making them\n suffer from major drawbacks, most notably harsh side-effects oxygen-independent.20,22\u221230 Ru(II) polypyridyl complexes\n due to limited selectivity of malignant cells over healthy have received significant attention as PCTs due their synthetic\n ones.4\u22129 Photochemotherapy (PCT) and photodynamic viability, relatively long-lived triplet metal-to-ligand charge\n therapy (PDT) attempt to overcome these drawbacks by transfer excited states (3MLCT*), and well understood\n utilizing a compound that is minimally or nontoxic in the dark photophysical behavior.10,31\u221240\n and in its native state, but becomes cytotoxic under Ru(II) PCTs act by either releasing a known cytotoxic\n illumination due to a photoinduced reaction. This provides reagent from the coordination sphere23,41\u221245 or by generating\n spatiotemporal control of toxicity within the body, ultimately a disolvated activated metal species that can have cisplatin-like\n limiting the harsh side-effects associated with traditional\n platins.10\u221213\n PDTs rely on the photoinduced excited state electron Received: March 5, 2024\n transfer (type I) or excited state triplet energy transfer (type Revised: April 10, 2024\n II) to generate reactive oxygen species inside the cellular Accepted: April 15, 2024\n microenvironment, triggering oxidative cell death.10 While this Published: April 25, 2024\n technique has found use in clinical oncology, the widespread\n use of PDTs has been limited due to the hypoxic nature of\n\n \u00a9 2024 American Chemical Society https://doi.org/10.1021/acs.inorgchem.4c00922\n 8426 Inorg. Chem. 2024, 63, 8426\u22128439\n\fInorganic Chemistry pubs.acs.org/IC Article\n\ninteractions with DNA at the newly opened coordination sites\n(Figure 1).22,28,46\u221248 For successful clinical implementation,\n\n\n\n\nFigure 1. Photoinduced ligand ejection from Ru(II) center to Figure 2. General excited state diagram of Ru(II) polypyridyl\ngenerate disolvated species. complexes showing electronic excitation (red), rapid intersystem\n crossing (ISC, orange), thermal or electronic relaxation (green), and\n an activation barrier for photoinduced ligand ejection (\u0394Epld).\nPCTs must absorb light and undergo their photoinduced\nreactions in the near-IR region, as this light penetrates skin\nmuch deeper than blue light due to scattering from biological which distorts the Ru(II) octahedral, making the dissociative\n 3\nmolecules.21 While near-IR ligand ejection has been reported dd* states more thermally accessible. This demonstrates the\nfor several Ru(II) PCTs, these complexes still have relatively importance of understanding ground and excited state energies\nlow oscillator strengths at wavelengths >500 nm and typically and how they can impact the ability of a complex to act as a\nfocus on the ejection of a monodentate ligand as opposed to PCT agent. Finally, preliminary cell viability studies suggest\nbidentate ligand ejection required for metalation of additional steric bulk around the Ru(II) metal center and on\nDNA.20,24,29,30,41,46,49\u221252 Finally, the compounds must be the photoreleased ligand can inhibit cytotoxic behavior.\nstable in the dark in solution for extended periods and be\nsoluble in biological (aqueous) medium for both clinical\nviability and photoreactivity with water.\n \u25a0 RESULTS AND DISCUSSION\n Ligand Synthesis. 2,2\u2032-Bipyridine (bpy), 6,6\u2032-dimethyl-\n With this in mind, we recently reported a detailed analysis of 2,2\u2032-bipyridine (dmb), and 2,2\u2032-biquinoline (bqn) were\na series of Ru(II) polypyridyl complexes designed to absorb purchased form Fischer Scientific and used without further\nnear the therapeutic window while also introducing steric bulk purification. 2,2\u2032-Bipyrizine59 (bpz) and 2,2\u2032-biquinoxaline37\naround the metal center to trigger photoinduced ligand loss.37 (bqx) were synthesized as previously reported. 2,2\u2032-Bi(1,8-\nHowever, we demonstrated that traditional ligand design naphthyridine) (bnn) was synthesized in 12% yield using a\nstrategies to red-shift MLCT absorptions by lowering the Friedla\u0308nder condensation of 2-amino-3-formylpyridine and\nenergy of the \u03c0* orbitals also impacted ligand dissociation 2,3-butandione in the presence of a base catalyst (Scheme 1).\nkinetics by limiting the accessibility of the dissociative 3dd* Detailed experimental information is provided in the\nstates. Photoinduced ligand ejection in Ru(II) octahedral Supporting Information.\npolypyridyl complexes requires thermal population of the Complex Synthesis. All complex syntheses and manipu-\nformally antibonding 3dd* states from the 3MLCT* excited lations were carried out in the dark to eliminate photo-\nstates.31,53\u221255 This is typically achieved by increasing steric reactions. In addition, all of the complexes were isolated as\nbulk around the Ru(II) center, which distorts the pseudo- their chloride salts, which were used for all of the analysis\noctahedral, lowering the energy of the 3dd* states. However, if reported herein except for the electrochemical experiments\nthe 3MLCT* state is lowered enough, it can make the thermal where the complexes were converted to their PF6 salts via\nbarrier (\u0394Epld, see Figure 2) to the 3dd* states inaccessible, metathesis to avoid chloride interference in the electro-\neffectively turning off photoinduced ligand ejection and chemistry. Purity of the complexes was confirmed by NMR\nultimately the ability of the complex to act as a PCT agent.37 and EA with the data reported in the Supporting Information.\n Expanding upon this understanding and interplay between It is also important to note that all of the complexes are readily\nthe MLCT* manifold energies and dissociative 3dd* states, we soluble in aqueous media (>200 \u03bcM), which is an important\nset out to design three new series of Ru(II) complexes that aspect for biological applications.\nallow control over light absorption, thermal stability, photo- Series 1 complexes were prepared following a previously\nactivation kinetics, and ultimately photoinduced cytotoxicity. reported procedure as their chloride salts by the reaction of\nThe complexes have the general structure of [Ru(bpy)2(N\u2212 Ru(bpy)2Cl260 with 1 equiv of the corresponding N\u2212N ligand\nN)]2+ (Series 1), [Ru(bpy)(dmb)(N\u2212N)]2+ (Series 2), and in 1:1 EtOH/H2O in a microwave oven reactor at 140 \u00b0C for 1\n[Ru(dmb)2(N\u2212N)]2+ (Series 3, where N\u2212N is a bidentate h.37 The reaction progress of all of the series of complexes was\npolypyridyl ligand, bpy = 2,2\u2032-bipyridine, and dmb = 6,6\u2032- monitored by UV\u2212vis spectroscopy by the disappearance of\ndimethyl-2,2\u2032-bipyridine, see Figure 3). The N\u2212N ligand was the Ru(X)(Y)Cl2 (where X and Y are bpy or dmb) starting\ndesigned to incorporate increased conjugation and/or electro- material and the appearance of the absorption peaks for the\nnegative heteroatoms within the ligand framework which tris-ligated complexes. All complexes were then purified by size\nlowers the energy of ligand \u03c0* orbitals, increasing their \u03c0*- exclusion chromatography (Sephadex LH-20 or Sorbadex S-25\nacceptor ability, and resulting in lower MLCT energy fine) using 1:1 H2O:CH3OH as eluent, where like fractions,\nabsorptions.31,56\u221258 Through the series, steric bulk is also identified by UV\u2212vis, were combined to yield the pure\nsystematically introduced by the addition of dmb ligands, complexes as their chloride salts.\n 8427 https://doi.org/10.1021/acs.inorgchem.4c00922\n Inorg. Chem. 2024, 63, 8426\u22128439\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 3. Structures of [Ru(bpy)2(N\u2212N])2+ (S1), [Ru(bpy)(dmb)(N\u2212N)]2+ (S2), and [Ru(dmb)2(N\u2212N)] (S3) complexes investigated in this\nstudy.\n\nScheme 1. Synthesis of bnn Ligand tion for full details). However, despite multiple attempts using\n a variety of reaction conditions and solvents, S3-bqx was not\n successfully synthesized in quantifiable yields. This suggests\n that the strong electron-withdrawing nature of the bqx ligand,\n along with the added steric bulk of the two dmb ligands,\n resulted in a relatively unstable complex.\n Figure 4 shows representative 1H NMR data of S1-bnn, S2-\n bnn, and S3-bnn in D2O with all NMR data reported in the\n Supporting Information. Both S1-bnn and S3-bnn shifts are\n consistent with C2 symmetry with corresponding protons in\n the aromatic region appearing at the same chemical shifts for\n The tris-heteroleptic nature of Series-2 complexes made the bpy and dmb ligands, respectively. In addition, this\ntheir synthesis more challenging and required a step-by-step symmetry is also observable with the methyl resonances in S3-\napproach of putting each polypyridyl ligand on the Ru(II) bnn with two equal resonances, one where the attached\ncenter (Scheme 2). This was achieved by first reacting [Ru(\u03b76- pyridine ring is opposite the other dmb ligand and the other\nbenzene)(Cl)2]261 with 2 equiv of bpy in MeOH according to opposite the bnn ligand. However, as expected, this symmetry\na reported procedure to generate the [Ru(\u03b76-benzene)(bpy)- is broken in heteroleptic S2-bnn with all aromatic and methyl\n(Cl)]Cl intermediate.60 The dmb ligand was then added by a\n resonances appearing at different chemical shifts. The aromatic\nmodified reported procedure24 in the presence of excess LiCl\n resonances of S1-bnn are slightly more deshielded than those\nto prevent the formation of tris-ligated species, giving the\n of S2-bnn and S3-bnn due to the electron donating nature of\nRu(bpy)(dmb)Cl2 intermediate in 66% yield. Finally, the\ncorresponding N\u2212N ligand was then added to the Ru(II) the methyl groups.\ncenter following the same procedure as that for Series 1 Electrochemistry. The electrochemical properties of each\ncomplexes. To the best of our knowledge, this is one of the complex were analyzed in dry CH3CN (0.1 M TBAPF6\nmost straightforward and high-yielding synthetic preparations supporting electrolyte, where TBAPF6 = tetrabutyl ammonium\nfor tris-heteroleptic ruthenium compounds reported. Full hexafluorophosphate) by cyclic and square-wave voltammetry.\ndetails are provided in the Supporting Information. It should be noted that each complex was first converted to its\n S3-bpy, S3-bpz, and S3-bqn complexes were synthesized by PF6 salt prior to analysis to remove interference from the Cl\u2212\na slightly modified procedure as that for the Series 1 ions (see Supporting Information for details). All of the\ncomplexes. For these complexes, the dichlororuthenium(II) electrochemical data are reported in Table 1.\ncyclooctadiene polymer62 was reacted with two equivalents of All complexes exhibit reversible [Ru]3+/2+ redox couples (eq\ndmb in o-dichlorobenzene at 190 \u00b0C to generate the 1) with E1/2 values ranging from 1.18 V for S1-bnn to 1.64 V\nRu(dmb)2Cl2 intermediate in 71% yield. Ru(dmb)2Cl2 was for S2-bqx (vs SCE). For the same N\u2212N ligand across the\nthen reacted with one equivalent of bpy, bpz, or bqn to series, there is little impact on the [Ru]3+/2+ redox potential,\ngenerate their respective Series 3 complexes (Scheme 3). demonstrating that the addition of the dmb ligands across the\nHowever, this final step did not work with the final two ligands series does not significantly impact the Ru(II) d\u03c06 energy\nbqx and bnn. S3-bnn was successfully synthesized in 18% yield levels. As an example, the redox potential of the [Ru]3+/2+\nby reacting Ru(dmb)2Cl2 with one equivalent of bnn in the couple in the bqn complexes across the series only varies by\npresence of two equivalents AgNO3 to abstract the chloride 0.05 V, as shown in Figure 5A. The same trend across the\nions from the coordination sphere (see Supporting Informa- series is also observed in the three ligand-based reductions,\n\nScheme 2. General Synthesis of Heteroleptic S2 Complexes\n\n\n\n\n 8428 https://doi.org/10.1021/acs.inorgchem.4c00922\n Inorg. Chem. 2024, 63, 8426\u22128439\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nScheme 3. General Synthesis of S3 Complexes\n\n\n\n\nFigure 4. 1H NMR spectra in D2O at 22 \u00b0C of S1-bnn (red, bottom), S2-bnn (green, middle), and S3-bnn (blue, top) at 400 MHz.\n\nwhere the addition of the dmb ligands does not significantly the Ru(II) center as opposed to away from in the case of bqx.\nimpact reduction potentials. This is demonstrated by all three This allows the additional nitrogens in bnn to donate electron\nof the reversible ligand-based reduction potentials for the bqn density to the Ru(II) center, stabilizing the Ru(III) oxidation\ncomplexes appearing at similar potentials (see Figure 5B and state, and lowering the [Ru]3+/2+ redox potential (see Figure 6\nTable 1). and Table 1).67 This is also supported by the fact that for the\n bnn complexes, the [Ru]3+/2+ potential increases in the order\n [Ru III(bpy)2 (N N)]3 + + e [Ru II(bpy)2 (N N)]2 + of S1-bnn < S2-bnn < S3-bnn where the additional methyl\n (1) groups in Series 2 and 3 limit the interactions of the extra\n In contrast, redox potentials within a single series vary nitrogens in the bnn ligand with the Ru(II) center (see Table\nsignificantly as the N\u2212N ligand changes. As an example, the 1).\n[Ru]3+/2+ redox couple in Series 2 varies from 1.25 V for S2- Ligand-based reduction potentials also vary significantly\nbpy to 1.64 V for S2-bqx (vs SCE, see Figure 6A). This shift is within a series as the N\u2212N bond is changed. Specifically, the\ndue to the increased conjugation and/or addition of electro- variation in the first reduction potential ([Ru]2+/+, 0.98 V) is\nnegative heteroatoms across the N\u2212N ligands. This lowers the considerably larger than the differences in the second\nenergy of the \u03c0* orbitals of the N\u2212N ligand, resulting in ([Ru]+/0+, 0.49 V) and third reduction ([Ru]0/1\u2212, 0.16 V)\nincreased d\u03c0-\u03c0* backbonding from the Ru(II) center to the for Series 2 (see Figure 6 and Table 1). This demonstrates, as\nN\u2212N ligand, stabilizing the d\u03c06 electronic configuration, and expected, that the first reduction is largely N\u2212N ligand\nultimately increasing the redox potential for the [Ru]3+/2+ centered (eq 2) due to the lower lying \u03c0* orbitals of the N\u2212N\ncouple.31,64\u221266 However, the same trend is not observed for ligand, where the second and third reductions occur mainly on\nthe bnn ligand across all three series. While the bnn ligand has the bpy and dmb ligands of the complexes (eqs 3 and 4).37 All\na significant amount of conjugation and electronegative of the complexes have more positive [Ru]2+/+ redox potentials\nheteroatoms, the additional nitrogens are oriented toward compared to the [Ru(bpy)3]2+ analogs in the series, again due\n 8429 https://doi.org/10.1021/acs.inorgchem.4c00922\n Inorg. Chem. 2024, 63, 8426\u22128439\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 1. Electrochemical Properties of all Complexes in N2 Unexpectedly, the first reduction potentials of S1-bnn and\nDeaerated CH3CN (0.1 M TBAPF6 Electrolyte)a S2-bnn are \u223c0.3 V more negative than those of the\n corresponding S1-bqx and S2-bqx complexes, despite both\n E1/2 E1/2 E1/2 E1/2\ncompound ([Ru]3+/2+) ([Ru]2+/+) ([Ru]+/0) ([Ru]0/1\u2212) ligands having the same amount of conjugation and electro-\n S1-bpy 1.29 \u22121.35 \u22121.55 \u22121.78\n negative heteroatoms. The free bnn ligand has a slightly more\n S1-bpz 1.54 \u22120.88 \u22121.44 \u22121.68 negative reduction potential (\u22121.44 V vs SCE) compared to\n S1-bqn 1.37 \u22120.92 \u22121.38 \u22121.69 the free bqx ligand (\u22121.29 V vs SCE, see Figure S1). In\n S1-bqx 1.60 \u22120.46 \u22121.08 \u22121.61 addition, there is likely increased d\u03c0-\u03c0* backbonding from the\n S1-bnn 1.18 \u22120.76 \u22121.24 \u22121.84 Ru(II) metal center into the bnn ligand compared to the bqx\n S2-bpy 1.26 \u22121.39 \u22121.59 \u22121.85 ligand given the orientation of the extra nitrogens in bnn,\n S2-bpz 1.54 \u22120.88 \u22121.47 \u22121.70 resulting in a more negative [Ru]2+/+ redox couple. This is\n S2-bqn 1.37 \u22120.94 \u22121.46 \u22121.78 supported by the observed lowering of the [Ru]3+/2+ redox\n S2-bqx 1.64 \u22120.46 \u22121.10 \u22121.70 potential in the bnn complexes compared to bqx complexes,\n S2-bnn 1.23 \u22120.73 \u22121.23 \u22121.86 suggesting significant interactions between the Ru(II) metal\n S3-bpy 1.32 \u22121.39 \u22121.64 \u22121.92 center and the four bnn nitrogens.\n S3-bpz 1.57 \u22120.85 \u22121.47 \u22121.71 UV\u2212vis Absorption. All of the complexes in water yield\n S3-bqn 1.42 \u22120.94 \u22121.43 \u22121.84 intense \u03c0 \u2192 \u03c0* absorption features below 350 nm (\u03b5 \u2248 4.0 \u00d7\n S3-bnn 1.24 \u22120.79 \u22121.32 \u22122.00 104\u22127.0 \u00d7 104 M\u22121 cm\u22121). The complexes that incorporate\na\n In CH3CN deaerated with N2 for 10 min, 1 mM in complex and 0.1 bqn, bqx, and bnn ligands also feature lower energy and\nM TBAPF6 supporting electrolyte. The complexes were converted to structured absorption transitions between 350\u2212450 nm that\nthe PF6 salt prior to analysis. GC working electrode, graphite rod are attributed to the [d\u03c06 ] \u2192 [d\u03c05 \u03c02*]1 transitions of the N\u2212\ncounter electrode, and Ag/AgNO3 (0.01 M AgNO3 with 0.1 M N ligands.31 All complexes also exhibit broad, lower-energy\nTBAPF6 in CH3CN) reference (values were adjusted to agree with\n MLCT bands that range from 400 to 600 nm (\u03b5 \u2248 4.1 \u00d7 103\u2212\nliterature values for [Ru(bpy)3]3+/2+ at 1.29 V vs SCE).34,57,63,68 E1/2\nvalues from differential pulse voltammetry. 4.1 \u00d7 103 M\u22121 cm\u22121), which as mentioned, is an important\n feature of moving absorption into the therapeutic window\n (600\u22121100 nm) for PCTs. These transitions are assigned as\nto the lower lying \u03c0*-orbitals of the N\u2212N ligand compared to [d\u03c06] \u2192 [d\u03c05 \u03c01*]1 excitations from the Ru(II) metal center to\nbpy or dmb. In general, the more conjugation and/or the polypyridyl ligands.31,55,58,69 Complexes that incorporate\nelectronegative heteroatoms on the N\u2212N ligand, the more the bpz, bqn, bqx, and bnn ligands all demonstrate splitting of\npositive the [Ru]2+/+ redox potential due to the lowering of the their MLCT manifolds due to transitions from the Ru(II)\n\u03c0*-orbitals. center to the bpy (or dmb, eq 5) ligands and N\u2212N (eq 6)\n [Ru II(bpy)(dmb)(N N)]2 + + e ligand \u03c0*-orbitals for the higher and lower energy transitions,\n respectively.31,37\n [Ru II(bpy)(dmb)(N N\u2022 )]+ (2)\n [Ru II(bpy)2 (N N)]2 + + h\n [Ru II(bpy)(dmb)(N N\u2022 )]+ + e\n [Ru III(bpy \u2022 )(bpy)(N N)]2 + * (5)\n [Ru II(bpy \u2022 )(dmb)(N N\u2022 )]0 (3)\n\n [Ru II(bpy \u2022 )(dmb)(N N\u2022 )]0 + e [Ru II(bpy)2 (N N)]2 + + h\n [Ru II(bpy \u2022 )(dmb\u2022 )(N N\u2022 )]1 (4) [Ru III(bpy)2 (N N\u2022 )]2 + * (6)\n\n\n\n\nFigure 5. (A) Cyclic voltammograms acquired at 100 mV/s for 1 mM solutions of S1-bqn, S2-bqn, and S3-bqn in N2 deaerated CH3CN with 0.1\nM TBAPF6 supporting electrolyte. GC working electrode, graphite rod counter electrode, and Ag/AgNO3 reference electrode (0.01 M AgNO3 with\n0.1 M TBAPF6 in CH3CN). Values were adjusted to agree with literature values for [Ru(bpy)3]3+/2+ at 1.29 V vs SCE.63\n\n 8430 https://doi.org/10.1021/acs.inorgchem.4c00922\n Inorg. Chem. 2024, 63, 8426\u22128439\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 6. Cyclic voltammograms acquired at 100 mV/s for 1 mM solutions of S2-bpy, S2-bpz, S2-bqn, S2-bqx, and S2-bnn in N2 deaerated\nCH3CN with 0.1 M TBAPF6 supporting electrolyte. GC working electrode, graphite rod counter electrode, and Ag/AgNO3 reference electrode\n(0.01 M AgNO3 with 0.1 M TBAPF6 in CH3CN). Values were adjusted to agree with literature values for [Ru(bpy)3]3+/2+ at 1.29 V vs SCE.63\n\n Similar to the electrochemical behavior, through the series all series being bpy > bpz > bqn > bqx > bnn. Figure 8 shows\nfor the same N\u2212N ligand, the energies of the MLCT the UV\u2212vis spectra for all of the Series 2 complexes, with the\ntransitions do not shift significantly. This is shown in Figure spectra of all of the complexes shown in Supporting\n7 where the bnn complexes only vary from \u03bbmax = 578 nm for Information, and major features listed in Table 2.\n The ground state absorption differences between the\n complexes that incorporate bqx and bnn ligands deserve\n more discussion. For example, S2-bqx shows a \u03bbmax,MLCT = 560\n nm with S2-bnn being \u03bbmax,MLCT = 575 nm. Despite both the\n bqx and bnn ligands having the same amount of conjugation\n and electronegative nitrogen atoms, they demonstrate differ-\n ences in both their electrochemical and their absorption\n properties. This is because the nitrogens on the bnn ligand are\n oriented toward the Ru(II) center, allowing them to donate\n electron density, ultimately increasing the energy level of the\n Ru d\u03c06 electron configuration. This results in a less positive\n oxidation potential for the Ru3+/2+ redox couple (see Table 1)\n for the bnn complexes compared to the bqx complexes, and\n also decreases the energy gap between the Ru-d\u03c06 and bnn-\u03c0*\n orbitals. As a result, the [d\u03c06] \u2192 [d\u03c05 \u03c01*]1 MLCT absorption\n feature for the bnn ligand complexes are lower in energy\n throughout the series (Table 2).\n Photoinduced Ligand Ejection. A key aspect of the PCT\nFigure 7. UV\u2212vis spectra of S1-bnn, S2-bnn, and S3-bnn in H2O at phototoxicity mechanism is the loss of a bidentate ligand under\n22 \u00b0C. illumination to trigger DNA-metalation and ultimately\n apotosis.22,48,71,72 We previously reported and discussed the\nS1-bnn to \u03bbmax = 578 nm for S3-bnn (also see Table 2). This critical interplay between therapeutic window absorption and\nsuggests that the incorporation of the dmb ligands through the photoinduced ligand ejection.37 The series reported herein\nseries, in addition to the increased steric bulk around the extends this work by moving the MLCT absorptions near the\nRu(II) metal center, does not significantly impact the Ru(II) therapeutic window, while also systematically introducing\nd\u03c06 nor the N\u2212N \u03c0*energy states. However, the intensity of steric bulk around the Ru(II) metal center to modulate\nthe bands does decrease with the addition of dmb ligands. This photoinduced ligand ejection kinetics. The rate constant for\nis attributed to slightly weaker coordinative bonds to the photoinduced ligand dissociation (kpld) was measured at both\nRu(II) center due to the increased steric bulk, resulting in less 22 and 37 \u00b0C (average body temperature) using the apparatus\nefficient charge transfer.70 shown in Figure S5. The UV\u2212vis spectra of each complex were\n In contrast, the energies of the MLCT transitions are taken at known time intervals during illumination with white\nsignificantly altered within a series by varying the N\u2212N ligand light to determine kpld (see Supporting Information for full\nthrough the increase in conjugation and/or introduction of details).\nelectronegative heteroatoms. These variations lower the energy Representative UV\u2212vis spectra of S1-bnn, S2-bnn, and S3-\nof the N\u2212N \u03c0*-orbitals, which increases the \u03c0*-acceptor bnn under illumination at 37 \u00b0C are shown in Figure 9 with all\nability of the ligand, resulting in increased d\u03c0-\u03c0* back- of the spectra shown in the Supporting Information (Figures\nbonding.31,54,69 This results in lower energy MLCT absorption S6\u2212S8). As shown in Figures 9 and S6\u2212S8, there is a strong\nfeatures throughout the series, with the general trend through dependence on both the steric bulk around the Ru(II) metal\n 8431 https://doi.org/10.1021/acs.inorgchem.4c00922\n Inorg. Chem. 2024, 63, 8426\u22128439\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nTable 2. Spectroscopic and Photoinduced Ligand Ejection\nProperties for all Complexes\n absorbance \u03bb (nm) (\u03b5, kpld (s\u22121)b@ kpld (s\u22121)b@ kpld \u03bb fit\ncompound \u00d7103 M\u22121 cm\u22121)a 22\u00b0C 37\u00b0C (nm)c\n S1-bpy 454 (11.6)\n 417 (8.7)\n 286 (69.9)\n S1-bpz 486 (7.3)\n 406 (8.5)\n 281 (47.2)\n S1-bqn 527 (5.6) 4.5 \u00d7 10\u22125 5.2 \u00d7 10\u22125 527d\n 440 (5.8)\n 287 (40.6)\n S1-bqx 563 (5.9)\n 405 (16.7)\n 383 (17.4)\n 285 (41.2)\n Figure 8. UV\u2212vis absorption spectra of S2-bpy, S2-bpz, S2-bqn, S2-\n S1-bnn 575 (7.4) bqx, and S2-bnn in H2O at 22 \u00b0C.\n 440 (8.0)\n 349 (26.9)\n 285 (54.6) extent and rate of photoinduced ligand ejection. A brief\n S2-bpy 455 (11.9) 7.0 \u00d7 10\u22123 7.3 \u00d7 10\u22123 457 summary of all of the spectra reveal; (1) for Series 1, only the\n 425 (8.7) S1-bqn complex shows measurable photodissociation, (2) in\n 285 (67.9) Series 2 the S2-bpy and S2-bqn complex show complete\n S2-bpz 485 (9.5) dissociation in 60 s with S2-bnn showing minor dissociation at\n 410 (11.1) 37 \u00b0C, and (3) all Series 3 complexes demonstrate measurable\n 285 (56.7) dissociation at 22 and 37 \u00b0C (see Table 2 and Figures S6\u2212S8).\n S2-bqn 526 (6.7) 5.3 \u00d7 10\u22123 1.1 \u00d7 10\u22122 379 In addition, all complexes (except S3-bqn) have a faster kpld at\n 440 (4.9) higher temperatures. S3-bqn repeatedly showed slightly slower\n 291 (34.5) kinetics at elevated temperatures suggesting the reaction was\n S2-bqx 560 (7.6) near barrierless at the temperatures studied herein or the\n 405 (21.2) differences in rates were within the experimental error. It\n 385 (22.0) should also be noted that all of the complexes showed no\n 275 (39.2) dissociation in water and no or minor dissociation in the cell\n S2-bnn 575 (8.7) <10\u22125 582 culture medium for at least 30 days (Figure S12).\n 440 (9.7) In general and as expected, the rate of photoinduced ligand\n 346 (29.8) dissociation increases through the series for the same N\u2212N\n 285 (57.4) ligand as the steric bulk around the Ru(II) center increases.\n S3-bpy 460 (10.1) 2.2 \u00d7 10\u22123 2.8 \u00d7 10\u22123 460 For example, S1-bnn shows no observable ligand ejection over\n 428 (7.6) the hour-long photolysis, S2-bnn undergoes minimal ligand\n 295 (48.1) ejection over the hour, where S3-bnn shows complete ligand\n S3-bpz 500 (9.3) 9.5 \u00d7 10\u22124 2.2 \u00d7 10\u22123 415 ejection in less than 60 s (Figure 9). The increased steric bulk\n 415 (11.6) in S3-bnn lowers the energy of the formally antibonding 3dd*\n 290 (43.0) state, making it thermally accessible at the temperatures\n S3-bqn 520 (6.7) 1.9 \u00d7 10\u22123 1.7 \u00d7 10\u22123 356e analyzed (see Figure 2). A more detailed description of the\n 440 (6.7) kinetics and excited state processes will be presented in the\n 300 (45.4) next section. Analysis of the spectra of S3-bnn under\n S3-bnn 580 (4.1) 5.0 \u00d7 10\u22123 6.4 \u00d7 10\u22123 580 irradiation shows a decrease in intensity of the [d\u03c06] \u2192 [d\u03c05\n 440 (5.5) \u03c01*(bnn)]1 MLCT transition at 580 nm as well a decrease in\n 325 (19.2) the [d\u03c06] \u2192 [d\u03c05 \u03c02*(bnn)]1 transitions around 380 nm. The\n 300 (29.1) resulting spectrum for S3-bnn, as well as all of the other\na\n Measured in H2O. bMeasured in H2O monitored over time during compounds that undergo photoinduced ligand ejection, are\nirradiation with light from a GLORIOUS-LITE 30 W LED (240 W similar to those reported for diaquated ruthenium(II)\nhalogen equivalent, 3000 LM, broad spectrum) placed 5 cm above the complexes.22,37,73\u221275 This suggest ejection of one of the\nsample while stirring (see Figure S5). cWavelength chosen for kinetic bulkier N\u2212N or dmb ligands from the coordination sphere\nfits to reduce the interferences from the photoproduct absorption and under irradiation (eq 7).37\nmaximize sensitivity of the measurement (highest \u03b5). dFit over the\nfull-hour photolysis due to the reaction being much slower. eNote this [Ru II(bpy)2 (N N)]2 + + hv\nwas the best wavelength to monitor despite significant overlap\nbetween the starting material and photoproduct. [Ru II(bpy)2 (OH 2)2 ]2 + + N N (7)\n Photolyzed samples of S1-bqn, S2-bqn and S3-bqn were\ncenter (Series 1 \u2192 Series 3) and the amount of conjugation subjected to high-resolution electrospray mass spectrometry in\nand/or electronegative heteroatoms on the N\u2212N ligand on the an attempt to fully characterize the photoproducts. Analysis of\n 8432 https://doi.org/10.1021/acs.inorgchem.4c00922\n Inorg. Chem. 2024, 63, 8426\u22128439\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 9. Absorption spectra of complexes S1-bnn (A), S2-bnn (B), and S3-bnn (C) in 3 mL H2O at 37 \u00b0C monitored over time during\nirradiation with light from a GLORIOUS-LITE 30 W LED (240 W halogen equivalent, 3000 LM, broad spectrum) placed 5 cm above the sample\n(see Figure S5).\n\n\n\n\nFigure 10. First-order fits (red lines) for photoinduced ligand dissociation of ln(concentration) versus time for S2-bqn at 22 and 37 \u00b0C (A), S1-\nbnn, S2-bnn, and S3-bnn at 37 \u00b0C (B), and S2-bpy and S2-bpz at 22 \u00b0C (C).\n\nthe S1-bqn spectra shows a signal at m/z = 257.1068, example, S1-bnn shows no observable photoinduced ligand\ncorresponding to the protonated photoejected bqn ligand and ejection, with S2-bnn having kpld < 10\u22125 s\u22121 (the small changes\nno evidence of free bpy (see Figure S11). Spectra of S2-bqn in the spectra made quantifying kpld not possible) and S3-bnn\nand S3-bqn photolyzed samples display both a signal at m/z = kpld = 6.4 \u00d7 10\u22123 s\u22121 at 37 \u00b0C. This is due to increased steric\n257.1069 as well as a larger one at m/z = 185.1071, bulk around the Ru(II) center distorting the pseudo-\ncorresponding to the protonated photoejected dmb ligand. octahedral, lowering the energy of the formally antibonding\nThis demonstrates that for the Series 2 and 3 complexes, both 3\n dd* states and making them thermally accessible from the\nthe dmb and N\u2212N ligand are able to be photoejected from the 3\n MLCT* excited states.31,53\u221255\ncoordination sphere. Attempts to characterize the ruthenium However, steric bulk and the energies of the 3dd* states are\nspecies following illumination proved to be more challenging not the only considerations for ligand ejection kinetics, as\nusing this method. This is possibly due to continued 3\n MLCT* excited-state energies also play a critical role.37 As an\nphotoreaction of the diaquated species, such as the formation\n example, S2-bpy undergoes complete ligand ejection within 60\nof neutral, high-valent, ruthenium oxo species from the Ru(II)-\n s under these conditions with kpld = 4.5 \u00d7 10\u22123 s\u22121 at 22 \u00b0C.\naqua photoproducts, under extended photolysis condi-\n However, S2-bpz, despite having little to no difference\ntions. 75,76 This is supported by the appearance of\n[RuIV(bpy)2(OH)2]2+ (m/z = 448.0460) in the photolysis sterically, and thus similar 3dd* energies, shows no measurable\nsample of S1-bqn and [RuVI(bpy)(dmb)(O)2]2+ (m/z = photoreaction under the same conditions (see Figure 10C and\n474.0619) in the photolysis sample of S2-bqn (see Figure Table 2). The bpz ligand has lower lying \u03c0*-orbitals due to the\nS11). Future studies will focus on fully characterizing the extra electronegative nitrogen atoms compared to those of bpy.\nruthenium species throughout the photolysis period to better This lowers the energy of 3MLCT* excited state, increasing the\nunderstand speciation during illumination. activation energy barrier (\u0394Epld, energy gap for photoinduced\n The kinetics for photoinduced ligand ejection were fit to a ligand dissociation, see Figure 2) between the 3MLCT* and\nfirst order reaction equation for all of the complexes that\n 3\n dd* states, making them inaccessible at these temperatures.37\nunderwent quantifiable ligand dissociation with rate constants A more detailed description of this phenomenon is presented\n(kpld) reported in Table 2 at 22 and 37 \u00b0C and representative in the next section.\nfits shown in Figure 10 (all fits are shown in the Supporting Unexpectedly, both S3-bpy and S3-bqn demonstrated\nInformation). In general and as expected, kpld increases with slightly lower kpld values compared to those of S2-bpy and\nincreasing temperature. For example, kpld for S2-bqn is 5.3 \u00d7 S2-bqn. This could be attributed to the 3MLCT* \u2192 3dd*\n10\u22123 and 1.1 \u00d7 10\u22122 s\u22121 at 22 and 37 \u00b0C, respectively (see electron transfer moving into the Marcus inverted region,\nFigure 10A). Also, as expected, kpld generally increases across lowering kpld despite having a larger free energy loss in the\nthe series from Series 1 to Series 3 (see Figure 10B). As an photoreaction.34 However, more experimentation is needed to\n 8433 https://doi.org/10.1021/acs.inorgchem.4c00922\n Inorg. Chem. 2024, 63, 8426\u22128439\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 11. Representative excited state diagrams of the ruthenium polypyridyl complexes through the series with the same N\u2212N ligand. Note the\nrelative energy of the 3MLCT* state does not change, but there is a lowering of the 3dd* state through increased steric bulk.\n\nconfirm this hypothesis and is outside the scope of the present\nstudy.\n Controlling Photoinduced Ligand Dissociation. The\ndemands on clinically used PCTs are high, including high\nthermal stability in the dark in both the solid form and in\nsolution, facile ligand ejection kinetics under illumination,\naqueous solubility, and high cytotoxicity activity upon\nillumination. Due to these demands, the aim of this study\nwas to not only move the MLCT absorptions near and into the\ntherapeutic window but also control photoinduced ligand Figure 12. Representative excited state diagrams of the ruthenium\nejection kinetics while balancing thermal stability. This has polypyridyl complexes through the series with varying N\u2212N ligand.\nbeen achieved through the series described herein by carefully Note that the relative energy of the 3dd* state does not change, but\n lowering of the 3MLCT* manifold through introduction of electro-\ncontrolling both the MLCT manifolds, the 3dd* state energies,\n negative heteroatoms and/or conjugation.\nand ultimately the activation energy (\u0394Epld) between these two\nstates.\n Upon initial photoexcitation into the 1MLCT* excited state, Cytotoxicity Assays. Preliminary MTT (3-[4,5-dime-\nthe complexes rapidly undergo intersystem crossing into a thylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide) dye\n3\n MLCT* state, which can then relax back to the ground state reduction assays were used to analyze the potential biological\nor thermally populate the formally antiboding metal-centered activity of all of the complexes against human embryonic\n3\n dd* state (sometimes referred to as the 3MC state), triggering kidney (HEK293T) cell cultures with and without illumina-\nligand dissociation (see Figure 2). Note that the extent of tion. Briefly, HEK293T were seeded into 96-well plates and\nthermal relaxation into the 3MLCT* state is expected to be dosed with varying concentrations of the complexes. The\nsimilar across the series.37 The final electron transfer into the cultures were then either exposed to white light for 10 min or\n3\n dd* state can be described as a d\u03c05d\u03c0*1 \u2192 d\u03c05d\u03c3*1 covered from irradiation in situ (see Supporting Information\ntransition.54,55,77\u221279 Ligand ejection from the 3dd* state is for full experimental details). All of the cell viability assays for\nrapid compared to relaxation back to the MLCT manifold each complex in the dark and under illumination are shown in\nand/or ground state.54,58,80,81 Through the series with the Figure S12 and summarized in Table S1. Figure 13 shows\nsame N\u2212N ligand, kpld increases significantly with increasing representative data of each complex at 100 \u03bcM with and\nsteric bulk. For example, kpld increases at least 4 orders of without illumination.\nmagnitude through the series for the bnn ligand in the order Analysis of the cell viability data shows that all of the\nS1-bnn (\u226a10\u22126 s\u22121) < S2-bnn (<10\u22125 s\u22121) < S3-bnn (6.4 \u00d7 complexes reported herein are minimally toxic in the dark and\n10\u22123 s\u22121) at 37 \u00b0C. As discussed above, this is due to distorting in their native state. In addition, as expected, complexes that\nthe pseudo-octahedral, lowering the 3dd* states and making do not undergo facile photoinduced ligand ejection do not\nthem thermally accessible (decreasing \u0394Epld) from the show increased toxicity under illumination. However, un-\n expectedly, not all of the complexes that undergo facile\n3\n MLCT* excited states (Figure 11).31,53\u221255 Note that the\n3 photoinduced ligand ejection demonstrated toxicity under\n MLCT* energies do not change significantly through the illumination. Control experiments with cisplatin and etoposide\nseries for the same N\u2212N ligand, as supported by both the did not show light dependence on toxicity, suggesting that\nelectrochemical and absorption data. illumination alone would not trigger cell death.\n A similar phenomenon is also observed within a series for Only S2-bpy (PTI = 3.9), S3-bpy (PTI = 1.6), S2-bqn (PTI\nvarying N\u2212N ligands and the accessibility of the 3dd* states. = 2.4), and S3-bqn (PTI = 3.1) showed a statistically\nHowever, in this case, the steric considerations and therefore significant photoinduced toxicity index (PTI, ratio of IC50,dark\nthe 3dd* state energy levels stay relatively the same, but the over IC50,light, see Table S1). S2-bpy showed the highest PTI of\nMLCT* manifolds shift with varying ligands. For instance, S2- all of the complexes, although our measured PTI\u2019s are\nbqn has kpld = 1.1 \u00d7 10\u22122 s\u22121 at 37 \u00b0C where S2-bqx shows no significantly lower than those reported for the same complex\nobservable photodissociation. While both complexes have the on HL60 leukemia (PTI = 188) and A549 lung (PTI = 136)\nsame steric bulk around the Ru(II) metal center, the additional cancer cells by Glazer and co-workers.22 The PTIs observed for\nnitrogens on the bqx ligand lowers the energy of the MLCT the complexes reported herein are also lower than other\nmanifolds by \u223c0.14 eV, increasing the activation energy recently reported Ru(II) PCTs such as the Ru(II) cytochrome\n(\u0394Epld) into the dissociative 3dd* state, and shutting off P450 3A4 inhibitor reported by Toupin et al. (PTI = 9),82 a\nphotodissociation at these temperatures (Figure 12). trisheteroleptic Ru(II) complex that inhibited conjunctival\n 8434 https://doi.org/10.1021/acs.inorgchem.4c00922\n Inorg. Chem. 2024, 63, 8426\u22128439\n\fInorganic Chemistry pubs.acs.org/IC Article\n\n\n\n\nFigure 13. Percent change in HEK293T cell viability of 100 \u03bcM of each complex either illuminated with a GLORIOUS-LITE 30 W LED (240 W\nhalogen equivalent, 3000 LM, broad spectrum) placed 10 cm above the sample (light, red bars) or covered in situ from illumination (dark, black\nbars). Etoposide and cisplatin (20 \u03bcM) were included as positive controls.\n\nmelanoma growth in zebrafish reported by Chen et al. (PTI = illumination. In addition, the smallest metal photoproduct\n31),83 and [Ru(bpy)2(pz)2]2+ derivatives (where pz = likely released in all of the complexes is [Ru(bpy)2(OH2)2]2+\npyrazole) against A549 cells reported by Hirahara et al. (PTI from the S2-bpy complex, which resulted in the highest PTI in\n= 7.3).84 However, a direct comparison between PTIs is this study.22 This suggests that the diaquated-Ru(II) metal\ndifficult given the wide range of experimental variables center needs to be relatively unobstructed to interact with the\nincluding cell line, growth medium, irradiation source, DNA base pairs, which leads to cellular apopotosis.22,28 Finally,\ndistance, flux, and time. This is demonstrated by the lowered both S2-bqn and S3-bqn show increased toxicity under\nPTIs for S2-bpy in our experiment compared to the conditions illumination where the other complexes with similar kpld do\nused by Glazer and co-workers. not, suggesting the bqn ligand may act as a DNA intercalator\n In an effort to elucidate the cytotoxicity mechanism of the causing cell death. Further studies are needed to better\ncomplexes with the highest PTI\u2019s, Western blot and understand the cytotoxic behavior of these complexes and to\nimmunofluorescence microscopy experiments were completed. determine why the bulkier complexes may be inhibited within\nHistone H2AX phosphorylation mediated by the ATR/ATM the cellular microenvironment. While these data demonstrate\nkinase cascade is a common marker of damaged DNA in that addition of steric bulk around the Ru(II) metal center\ncells.85\u221287 H2AX phosphorylation was analyzed by Western facilitates photoinduced ligand ejection, it may also lead to\nblot as a function of treatment with cisplatin, S1\u2212S3 bpy, bqn, inhibited PCT behavior.\nand bnn complexes under irradiation. As shown in Figure S14,\nH2AX phosphorylation is relatively low in mock-treated cells\nbut increases upon the addition of cisplatin. Incubation of cells\nwith the Ru(II) complexes following irradiation resulted in\n \u25a0 CONCLUSIONS\n We have described here the synthesis, characterization,\nincreases in H2AX phosphorylation relative to mock treatment, electrochemical, spectrochemical, and preliminary cytotoxicity\nwith the S3-bqn sample resulting in the highest level of analyses of three series of ruthenium(II) polypyridyl complexes\nphosphorylation. In addition, immunofluorescence microscopy designed to mimic PCTs. The complexes were designed to\nwas used to investigate the histone phosphorylation phenotype incorporate increased conjugation and/or electronegative\nfor cells treated with the compounds that showed the highest heteroatoms within the ligand frameworks to lower the ligand\nPTIs. Treatment of the cells with cisplatin, S2-bpy, S2-bqn, \u03c0* orbitals and move absorptions near the therapeutic window.\nand S3-bqn resulted in formation of distinct nuclear foci of The lowest energy MLCT absorption maximum was red-\nH2AX phosphorylation, indicative of DNA damage centers shifted from \u03bbmax = 454 nm for S1-bpy to \u03bbmax = 580 nm for\n(Figure S15). Taken together, these results suggest that the S3-bnn. In addition, steric bulk was also systematically\ncompounds with the highest PTIs cause DNA damage under introduced throughout the series by the incorporation of\nirradiation, which likely contributes to their cytotoxic effects. dmb ligands, distorting the Ru(II) octahedral structure and\n Interestingly, S2-bpy demonstrated a 2.4\u00d7 increase in PTI making the dissociative 3dd* state thermally accessible at room\ncompared to S3-bpy, despite both complexes showing and body temperature. Photoinduced ligand ejection kinetics\ncomplete ligand dissociation in less than 1 min under increased by at least 4 orders of magnitude throughout the\nillumination. This suggests that the additional steric bulk in series. Furthermore, the incorporation of the sterically bulky\nthe photoproducts, both the Ru(II)-diaquated species and the dmb ligands did not significantly alter the ground state or\nejected ligand, can inhibit toxicity and ultimately PCT excited state manifolds involved in redox chemistry and light\nbehavior. This is supported by the fact that majority of the absorption. These findings are important in understanding the\ncomplexes that exhibit facile photoinduced ligand ejection due ground and excited state energies with respect to PCT agents.\nto increased steric bulk around the Ru(II) center (Series 2 and However, it appears that the addition of steric bulk around the\nSeries 3) do not show significant increased toxicity under Ru(II) metal center inhibits the cytotoxic behavior of the\n 8435 https://doi.org/10.1021/acs.inorgchem.4c00922\n Inorg. Chem. 2024, 63, 8426\u22128439\n\fInorganic Chemistry pubs.acs.org/IC Article\n\nphotoproducts, ultimately limiting the PCT activity of these was carried out by S.M.S. at the University of Virginia College\ncomplexes. at Wise. Mass spectrometric analyses were performed at the\n\n\u25a0 ASSOCIATED CONTENT\n* Supporting Information\n s\u0131\n University of Tennessee, Knoxville Biological and Small\n Molecule Mass Spectrometry Core with the assistance of\n Nick Trybala, Dr. Hector F. Castro, and Vernon Stafford.\nThe Supporting Information is available free of charge at\nhttps://pubs.acs.org/doi/10.1021/acs.inorgchem.4c00922.\n Experimental and synthetic procedures, characterization\n \u25a0 REFERENCES\n (1) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer statistics, 2020. CA\n data of complexes, UV\u2212visible spectra of all complexes, A Cancer J. 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P.; Lilge, L.; Cameron, C. G.; McFarland, S. A.\n Keylon E. Reynolds \u2212 Department of Natural Sciences, Transition metal complexes and photodynamic therapy from a tumor-\n Tusculum University, Greeneville, Greeneville, Tennessee centered approach: challenges, opportunities, and highlights from the\n 37745, United States development of TLD1433. Chem. Rev. 2019, 119 (2), 797\u2212828.\n Skylar N. Lane \u2212 Department of Natural Sciences, Tusculum (11) Imberti, C.; Zhang, P.; Huang, H.; Sadler, P. J. New designs for\n University, Greeneville, Greeneville, Tennessee 37745, United phototherapeutic transition metal complexes. Angew. Chem., Int. Ed.\n States Engl. 2020, 59 (1), 61\u221273.\n (12) Heinemann, F.; Karges, J.; Gasser, G. Critical overview of the\n Averie N. Price \u2212 Department of Natural Sciences, Tusculum use of Ru(II) polypyridyl complexes as photosensitizers in one-\n University, Greeneville, Greeneville, Tennessee 37745, United photon and two-photon photodynamic therapy. Acc. Chem. Res. 2017,\n States 50 (11), 2727\u22122736.\n Ireland G. Cavanaugh \u2212 Department of Natural Sciences, (13) Zhang, Y.; Zhou, Q.; Tian, N.; Li, C.; Wang, X. Ru(II)-\n Tusculum University, Greeneville, Greeneville, Tennessee complex-based DNA photocleaver having intense absorption in the\n 37745, United States phototherapeutic window. Inorg. Chem. 2017, 56 (4), 1865\u22121873.\n Steven M. Shell \u2212 Department of Natural Sciences, University (14) Jarvi, M. T.; Patterson, M. S.; Wilson, B. C. Insights into\n of Virginia College at Wise, Wise, Virginia 24293, United photodynamic therapy dosimetry: simultaneous singlet oxygen\n States luminescence and photosensitizer photobleaching measurements.\n Biophys. J. 2012, 102 (3), 661\u2212671.\nComplete contact information is available at: (15) Clement, S.; Deng, W.; Camilleri, E.; Wilson, B. C.; Goldys, E.\nhttps://pubs.acs.org/10.1021/acs.inorgchem.4c00922 M. X-ray induced singlet oxygen generation by nanoparticle-\n photosensitizer conjugates for photodynamic therapy: determination\nNotes of singlet oxygen quantum yield. Sci. Rep. 2016, 6 (1), 19954.\nThe authors declare no competing financial interest. (16) Weersink, R. A.; Bogaards, A.; Gertner, M.; Davidson, S. R. H.;\n Zhang, K.; Netchev, G.; Trachtenberg, J.; Wilson, B. C. Techniques\n\u25a0 ACKNOWLEDGMENTS\nThis work was supported by the Arthur Vining Davis\n for delivery and monitoring of TOOKAD (WST09)-mediated\n photodynamic therapy of the prostate: clinical experience and\n practicalities. J. Photochem. Photobiol. B Biol. 2005, 79 (3), 211\u2212222.\nFoundations under grant no. G-2206-23043 and Eastman (17) Kohler, L.; Nease, L.; Vo, P.; Garofolo, J.; Heidary, D. 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