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Photoactivation of Boronic Acid Prodrugs via a Phenyl Radical Mechanism: Iridium(III) Anticancer Complex as an Example.
University of Texas at Arlington
MavMatrix
Chemistry & Biochemistry Dissertations
Department of Chemistry and Biochemistry
2023
Ru(II) Polypyridyl Photosensitizers for Phototherapy
Houston Cole
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Recommended Citation
Cole, Houston, "Ru(II) Polypyridyl Photosensitizers for Phototherapy" (2023). Chemistry & Biochemistry
Dissertations. 276.
https://mavmatrix.uta.edu/chemistry_dissertations/276
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RU(II) POLYPYRIDYL PHOTOSENSITIZERS FOR PHOTOTHERAPY
by
Houston Cole
DISSERTATION
Submitted to the Faculty of The Graduate School at The University of Texas at Arlington in
Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy
December 2023
Arlington, TX
Supervising Committee:
Dr. Sherri A. McFarland, Supervising Professor
Dr. Frederick MacDonnell
Dr. Frank Foss
Dr. He Dong
TABLE OF CONTENTS
CHAPTER 1. INTRODUCTION ..................................................................................................1
1.1 REFERENCES ..................................................................................................................2
CHAPTER 2. RU(II) PHENANTHROLINE-BASED OLIGOTHIENYL COMPLEXES AS
PHOTOTHERAPY AGENTS ......................................................................................................9
2.1 ABSTRACT .......................................................................................................................9
2.2 INTRODUCTION ............................................................................................................. 11
2.3 MATERIALS AND METHODS ........................................................................................ 12
2.3.1 Instrumentation. ....................................................................................................... 12
2.3.2 Synthesis and Characterization ............................................................................... 13
2.3.3 Computational Details .............................................................................................. 16
2.3.4 Electrochemistry ...................................................................................................... 16
2.4 RESULTS AND DISCUSSION ........................................................................................ 17
2.4.1 Synthesis and Characterization ............................................................................... 17
2.4.2 Computation ............................................................................................................ 19
2.4.3 Spectroscopy ........................................................................................................... 25
2.4.4 Electrochemistry ...................................................................................................... 22
2.4.5 Photobiological activity ............................................................................................ 36
2.5 CONCLUSIONS .............................................................................................................. 42
2.6 ASSOCIATED CONTENT ............................................................................................... 43
2.6.1 Author Information ................................................................................................... 43
2.6.2 Notes ....................................................................................................................... 44
2.6.3 Acknowledgements ................................................................................................. 45
2.7 REFERENCES ............................................................................................................... 45
2.8 SUPPORTING INFORMATION ...................................................................................... 62
2.8.1 Description of Methodology ..................................................................................... 62
2.8.2 Synthetic Characterization ....................................................................................... 68
2.8.3 Computational Studies ............................................................................................ 93
2.8.4 Spectroscopic Characterization ............................................................................. 106
2.8.5 Electrochemical Characterization .......................................................................... 109
2.8.6 Biological and Photobiological Characterization .................................................... 111
2.8.7 References ............................................................................................................ 118
CHAPTER 3. RU(II) TRIFLUOROMETHYL BIPYRIDINE-BASED OLIGOTHIENYL
COMPLEXES FOR PHOTODYNAMIC THERAPY ................................................................. 119
3.1 ABSTRACT ................................................................................................................... 119
3.2 INTRODUCTION .......................................................................................................... 120
3.3 MATERIALS AND METHODS ...................................................................................... 122
3.3.1 Instrumentation ...................................................................................................... 122
3.3.2 Synthesis and Characterization ............................................................................. 123
3.3.3 Electrochemistry .................................................................................................... 127
3.4 RESULTS AND DISCUSSION ...................................................................................... 127
3.4.1 Synthesis and Characterization ............................................................................. 127
3.4.2 Spectroscopy ......................................................................................................... 130
3.4.3 Electrochemistry .................................................................................................... 136
3.4.4 In Vitro Photobiological Activity .............................................................................. 139
3.5 CONCLUSIONS ............................................................................................................ 144
3.6 ASSOCIATED CONTENT............................................................................................. 145
3.7 ACKNOWLEDGEMENTS ............................................................................................. 145
3.8 REFERENCES ............................................................................................................. 145
3.9 SUPPORTING INFORMATION .................................................................................... 155
3.9.1 Synthesis and Characterization ............................................................................. 155
3.9.2 Spectroscopy ......................................................................................................... 158
3.9.3 Photobiology .......................................................................................................... 160
3.9.4 Synthetic Characterization ..................................................................................... 167
3.9.5 Spectroscopic Characterization ............................................................................. 190
3.9.6 Photobiological Evaluation ..................................................................................... 194
CHAPTER 4. CHIRALITY MATTERS: ENANTIOMERICALLY RESOLVED RU(II)
OLIGOTHIENYL COMPLEXES FOR PHOTODYNAMIC THERAPY ..................................... 205
4.1 ABSTRACT ................................................................................................................... 205
4.2 INTRODUCTION .......................................................................................................... 205
4.3 RESULTS AND DISCUSSION...................................................................................... 207
4.3.1 Resolution of enantiomers via HPLC ..................................................................... 207
4.3.2 In Vitro Photobiological Activity.............................................................................. 207
4.4 SUMMARY AND FUTURE DIRECTIONS..................................................................... 211
4.5 ASSOCIATED CONTENT ............................................................................................. 211
4.5.1 Acknowledgements ............................................................................................... 211
4.6 REFERENCES ............................................................................................................. 212
4.7 SUPPORTING INFORMATION .................................................................................... 215
4.7.1 Materials and Methods........................................................................................... 215
4.7.2 Synthetic Characterization ..................................................................................... 222
4.7.3 Photobiological Evaluation ..................................................................................... 231
CHAPTER 5. CONCLUSIONS AND PERSPECTIVES .......................................................... 241
5.1 REFERENCES .............................................................................................................. 242
ABSTRACT
Certain metal complexes, notably those containing the IP-4T ligand, demonstrate remarkable
efficiency in sensitizing singlet oxygen (>99%) and exhibit unprecedented photocytotoxicity in
normoxic conditions, with notable activity under hypoxic conditions as well. This pronounced
efficacy is potentially attributed to the involvement of 3ILCT states in photoredox reactions, a
characteristic influenced by the inherent ability of certain oligothiophenes to generate polarons
and bipolarons. This dissertation details the conceptualization, creation, and photobiological
evaluation of principal molecules, focusing on modifying three critical aspects: 1) the length of
the IP-nT 'PDT' ligand, 2) the electronic attributes of ancillary ligands like bipyridine or
phenanthroline, and 3) the chirality of the coordination complex. The primary goal of this study
is to investigate the hypothesis that molecules possessing low-lying and enduring 3ILCT states
contribute to this remarkable photocytotoxic potency. Moreover, this research aims to deepen
the understanding of which structural elements, beyond the IP-nT ligand, contribute to enhanced
photobiological properties.
CHAPTER 1. INTRODUCTION
As of 2022, cancer is the second leading cause of death in the United States.1 Alternate and adjuvant
strategies are urgently needed. One such strategy is photodynamic therapy (PDT). PDT is a light-based
form of cancer therapy that uses a photosensitizer (PS), light, and
oxygen to destroy tumors, and has been shown to induce an
Scheme 1. Jablonski diagram
for Photofrin.
ππ*
1
immune response that can protect against recurrence.2–13
ππ*
3
Activation of the PS by certain wavelengths of light produces triplet
excited states that interact with oxygen to generate reactive oxygen
1
O2
species (ROS) such as singlet oxygen, which is is thought to be the
most important ROS for mediating the PDT effect. The only FDAapproved PS for cancer therapy is Photofrin, a mixture of oligomeric
3
PS
O2
porphyrins that is activated with 630-nm light to produce triplet
excited states that interact with oxygen to generate reactive oxygen species (ROS) such as singlet
oxygen (1O2) (Scheme 1). There is ongoing interest in developing non tetrapyrrole-based PSs that could
exert their photocytotoxic effects through alternate mechanisms to treat hypoxic tumors, which are some
of the most aggressive and drug-resistant neoplasms.14–19
Photocytotoxicity in hypoxia has proven extremely difficult to achieve,20,21 necessitating entirely new
approaches for manipulating the reactivity of excited states.22,23 Traditional PSs generate ROS from triplet
ππ* excited states that exploit oxygen-dependent mechanistic pathways. In contrast, metal complexes
possess a variety of excited state configurations that can be accessed through different ligand and metal
combinations. These excited states may be localized to the metal (MC) or the ligands (LC), may involve
charge transfer between the metal and a ligand(s) (MLCT/LMCT) or even two different ligands (LLCT).24–
26
As such, each of these excited states has its own characteristic reactivity, which can include singlet
oxygen sensitization or oxygen-independent photochemical reactions as well as redox chemistry.
Our lab has shown that it is possible to achieve potent photocytotoxic effects with Ru(II) and Os(II) metalorganic dyads, where the metal center is chelated to an imidazo[4,5-f][1,10]phenanthroline ligand that is
appended to an oligothiophene chain (nT).27–34 In these scaffolds, the role of the metal is to facilitate the
formation of triplet states through intersystem system crossing, and the role of the ligand is to establish
an “organic” triplet state that is lower in energy than the metal-to-ligand charge transfer (MLCT) states
1
that usually dominate Ru(II) and Os(II) polypyridyl photophysics.24,25 The organic chromophore is also
responsible for prolonging the triplet excited state lifetime to allow enough time for bimolecular quenching
reactions (such as singlet oxygen sensitization) to take place, which results in a greater photocytotoxic
effect. While we have used a variety of organic chromophores in the past, the nT moiety is especially
important because it introduces charge transfer character to otherwise nonpolar intraligand (IL) excited
states. The resulting intraligand charge transfer (ILCT) excited states are capable of sensitizing singlet
oxygen even in hypoxia20,21 but may also facilitate oxygen-independent photoredox reactions that may
contribute to the overall photocytotoxicity. This scaffold is exemplified by TLD1433, the first Ru(II)-based
PS to advance to human clinical trials.27,35 Since TLD1433, we have rationally designed and developed
PSs that are even more potent in normoxia and hypoxia by installing an additional thiophene ring and by
combining the organic chromophore with various other coligands on Ru(II) or Os(II).20,21,27,36
Certain metal complexes incorporating the IP-4T ligand sensitize 1O2 in very high yield (>99%) and exert
unprecedented photocytotoxic effects in normoxia, and are also active in hypoxia.27,28 The unusually
potent activity may stem, in part, from 3ILCT states that participate in photoredox reactions due to the
known capacities of certain oligothiophenes to form polarons and bipolarons.27 This dissertation
describes the design, synthesis, and photobiological characterization of key target molecules that modify
the 1) length of the IP-nT “PDT” ligand, 2) electronic nature of the bipyridine or phenanthroline type
ancillary ligands, and 3) chirality of the coordination complex. The targets and subsequent studies will
test the hypothesis that molecules with low-lying and long-lived 3ILCT states are responsible for this
unusually high photocytotoxic potency and seeks to further understand what structural features (aside
from the IP-nT ligand) lead to more favorable photobiological properties.
1.1
(1)
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(45) Hu, X.; Yang, D.; Yao, T.; Gao, R.; Wumaier, M.; Shi, S. Regulation of Multi-Factors
(Tail/Loop/Link/Ions) for G-Quadruplex Enantioselectivity of Δ- and Λ- [Ru(Bpy) 2 (DppzIdzo)] 2+. Dalton Trans. 2018, 47 (15), 5422–5430. https://doi.org/10.1039/C8DT00501J.
8
CHAPTER 2. RU(II) PHENANTHROLINE-BASED OLIGOTHIENYL COMPLEXES AS
PHOTOTHERAPY AGENTS
Houston D. Cole,a Abbas Vali,a John A. Roque III,a,b Ge Shi,a Gurleen Kaur,a Rachel O. Hodges,b
Antonio Francés-Monerris,c Marta E. Alberto,d* Colin G. Cameron,a* Sherri A. McFarlanda*
a Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, Texas, 76019-0065 USA
b Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, North Carolina
27402 USA
c Institut de Ciència Molecular, Universitat de València, 46071 València, Spain
d Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria, Arcavacata di Rende, 87036 Italy
*Corresponding authors: C.G.C. <colin.cameron@uta.edu> ORCID 0000-0003-0978-0894, S.A.M.
<sherri.mcfarland@uta.edu> ORCID 0000-0002-8028-5055
2.1 ABSTRACT
Ru(II) polypyridyl complexes have gained widespread attraction as photosensitizers for photodynamic
therapy (PDT). Herein, we systematically investigate a series of the type [Ru(phen)2(IP-nT)]2+, featuring
1,10-phenanthroline (phen) coligands and imidazo[4,5-f][1,10]phenanthroline ligands tethered to n= 0–4
thiophene rings (IP-nT). The complexes were characterized and investigated for their electrochemical,
spectroscopic, and (photo)biological properties. The electrochemical oxidation of the nT unit shifted by 350 mV as n=1→4 (+920 mV for Ru-1T, +570 mV for Ru-4T); nT reductions were observed in complexes
Ru-3T (−2530 mV) and Ru-4T (−2300 mV). Singlet oxygen quantum yields ranged from 0.53–0.88, with
Ru-3T and Ru-4T being equally efficient (~0.88). The time-resolved absorption spectra of Ru-0T–1T
were dominated by metal-to-ligand charge transfer (3MLCT) states (τTA=0.40–0.85 µs), but long-lived
intraligand charge transfer (3ILCT) states were observed in Ru-2T–4T (τTA=25–148 μs). The 3ILCT
energies of Ru-3T and Ru-4T were computed to be 1.6 eV and 1.4 eV, respectively. Phototherapeutic
efficacy against melanoma cells (SK-MEL-28) under broad-band visible light (400–600 nm) increases as
n=0→4: Ru-0T was inactive up to 300 µM, Ru-1T–2T were moderately active (EC50 ~600 nM, PI=200),
and Ru-3T (EC50=57 nM, PI=>1100) and Ru-4T (EC50=740 pM, PI=114,000) were the most phototoxic.
Activity diminishes with longer wavelengths of light and is completely suppressed for all complexes
except Ru-3T and Ru-4T in hypoxia. Ru-4T is the more potent and robust PS in 1% O2 over seven
biological replicates (avg EC50=1.3 µm, avg PI=985). Ru-3T exhibited hypoxic activity in five out of seven
replicates, underscoring the need for biological replicates in compound evaluation. Singlet oxygen
9
sensitization is likely responsible for phototoxic effects of the compounds in normoxia, but the presence
of redox-active excited states may facilitate additional photoactive pathways for complexes with 3 or more
thienyl groups. The 3ILCT state with its extended lifetime (30–40x longer than the 3MLCT state for Ru-3T
and Ru-4T) implicates its predominant role in photocytotoxicity.
Keywords: Ruthenium polypyridyl complexes, photosensitizers, photobiology, photodynamic therapy,
metal-to-ligand charge transfer (MLCT), intraligand charge-transfer (ILCT), ligand-to-ligand charge
transfer (LLCT), melanoma, phenanthroline (phen), hypoxia
10
2.2 INTRODUCTION
Cancer remains the second most common cause of death globally, surpassed only by cardiovascular
disease.1 Despite significant advancements in treatment over the past few decades, particularly in the
realms of immunotherapy2–4 and targeted therapy,5,6 there remains a pressing need for novel treatments
and adjuvant therapies to complement surgery, radiation, and chemotherapy. In this regard, light-driven
treatment modalities present a compelling alternative.
Photodynamic therapy (PDT) represents a unique and promising approach to targeted cancer treatment,
which leverages a nontoxic photosensitizer (PS), benign light, and molecular oxygen to generate
cytotoxic reactive oxygen species (ROS) for destroying tumors. PDT offers the advantage of localized
intervention and minimal invasiveness, yielding fewer adverse effects and enhanced patient quality of
life.7,8
PDT leverages two layers of precision: (1) the selective uptake and retention of PSs in malignant tissues
and (2) the use of light to trigger toxicity. The result is that phototoxicity is confined to regions where the
PS, light, and oxygen overlap spatiotemporally. The PDT effect can be maximized by optimization of the
light regimen, including wavelength, fluence, irradiance, and dosimetry as well as the drug-to-light interval
(DLI).
The intrinsic reliance of PDT on oxygen to generate ROS is problematic for treating hypoxic tumours. In
addition, PDT can induce hypoxia as oxygen is consumed during irradiation.9,10 Decreased generation of
ROS limits the damage to cancerous cells. To address this, there is motivation to develop light-triggered
compounds that exploit oxygen-independent mechanisms for phototoxicity.11–59 In this context, metal
complexes such as Ru(II) polypyridyl systems have attracted considerable attention. 7,11,13,60–73 Judicious
choice of ligand-metal combinations provides access to access a variety of excited state configurations
with characteristic photophysical properties and reactivities. Strategies have included photorelease of
bulky
ligands
to
reveal
phototoxic
metals
and/or
ligands,11,15,21,22,63,74–77
photocaging
of
chemotherapeutics and enzyme inhibitors,13,14,16,71,74,78–95 photoredox reactions,96,97 and increasing ROS
yields (to maintain 1O2 generation at low oxygen tension).23,24,63
Our group has a longstanding interest in metal complexes as PSs, not just for alternate modes of action.
Their modular architectures and straightforward assembly allow rapid tuning of physicochemical,
photophysical, and biological properties, which facilitates our tumor-centered approach to PS design. Our
guiding premise is that an ideal PS does not exist, and PS design and optimization should consider the
11
specific application. Our TLD1433, a terthienyl-containing Ru(II) polypyridyl complex, is exemplary and
is currently in Phase II clinical trials for treating non-muscle invasive bladder cancer (NMIBC) with PDT
(Clinicaltrials.gov identifier NCT03945162).7,98 It has a high quantum yield for 1O2 generation and is
phototoxic toward cancer cells with minimal dark toxicity. It is preferentially activated in the clinic with
green light to avoid any damage to underlying muscle tissue.
To better understand the properties of oligothiophene-based metal complexes such as TLD1433, and to
also develop additional PSs, we are exploring different metal ions, coligands, thienyl groups, counter
ions, and coordination geometries.7,15,23,24,63,76,99 The longer-term goal is to establish structure-activity
relationships (SARs) for photoactive oligothiophene-containing metal complexes that consider their
physicochemical, photophysical, electrochemical, and biological characteristics. We are motivated by the
remarkable activities of some of these complexes containing longer thienyl chains. In this study, we
describe a new family of Ru(II) PSs bearing two ancillary 1,10-phenanthroline (phen) ligands and an
imidazo[4,5-f][1,10]phenanthroline (IP) ligand tethered to thienyl groups (nT) with n=0–4. The five
members of the [Ru(phen)2(IP-nT)]2+ family and the reference compound [Ru(phen)3]2+ were investigated
for their photocytotoxic effects toward melanoma cells using different wavelengths of light in normoxia
and in hypoxia. Their lipophilicities, ground state absorption and emission properties, excited state
configurations and lifetimes, and redox characteristics are systematically compared. The study provides
a framework for understanding photophysical properties and biological activities, offering a robust
platform to probe the fundamental dynamics that underpin PDT efficacy across a variety of
oligothiophene-containing metal complexes with future biological studies. It also introduces two new
hypoxia-active PSs that could be further developed.
2.3 MATERIALS AND METHODS
All complexes in this series were thoroughly characterized synthetically, spectroscopically,
electrochemically, and (photo)biologically. Additional procedural details and characterization data may
be found in the Supplementary Information.
Instrumentation.
Microwave reactions were performed in a CEM Discover microwave reactor. NMR spectra were collected
using a JEOL ECA 500 NMR spectrometer (1H) at UNCG’s NMR facility or Agilent 700 MHz NMR
spectrometer (1H, 1H‒1H COSY, 13C–1H HSQC, 13C–1H HMBC) at the Joint School of Nanoscience and
Nanoengineering (JSNN). ESI mass spectra were obtained using a Thermo Fisher LTQ Orbitrap XL
coupled to a Water’s Acquity Ultra-high Performance Liquid Chromatography (UPLC) stack using a BEH
12
C18 column at UNCG’s Triad Mass Spectrometry facility. HPLC analyses were carried out on an
Agilent/Hewlett Packard 1100 series instrument (ChemStation Rev. A. 10.02 software) using a Hypersil
GOLD C18 column (Thermo 25005-254630, guard 25003-014001) with an A–B gradient (40 min run; 2%
→ 95% B; A=0.1% formic acid in H2O, B=0.1% formic acid in MeCN). Reported retention times are
accurate to within ±0.1 min. Flash chromatography relied on the Teledyne Isco CombiFlash EZ Prep
system with Silicycle SiliaSep silica flash cartridges (FLH-R10030B-ISO25).
Synthesis and Characterization
To the best of our knowledge, Ru-0T‒Ru-4T have not been previously published. [Ru(phen)3](Cl)2 was
synthesized using a modified literature procedure100 that is described in detail below. Unless otherwise
specified, all reagents and solvents were purchased from commercial sources and used without further
purification. Water used for all biological experiments was deionized to a resistivity ≥ 18.2 MΩ using either
a Barnstead or Milli-Q® filtration system. Ru(phen)2Cl2•2H2O101 and IP-based ligands102 were prepared
according to adapted literature procedures. The synthesis of IP-based ligands follows that described
below for IP-4T. [2,2′:5′,2″:5″,2‴-quaterthiophene]-5-carbaldehyde (4T-CHO) was prepared as previously
described.103,104 Final products are synthetically characterized in Figure S1‒Figure S22 via 1H NMR, 1H–
H COSY NMR, HPLC, and ESI+–MS. Ru-4T required additional 13C, 13C–1H HSQC, and 13C–1H HMBC
1
NMR experiments for full assignment of the quaterthiophene-containing complex (Figure S9‒Figure S10).
The Cl− salts of final complex products were obtained via anion metathesis on HCl-treated Amberlite IRA410 resin with methanol as eluent and isolated in vacuo. Final complexes are a mixture of Δ/Λ isomers.
[Ru(phen)3](Cl)2. Ru(Cl)3∙~3H2O (58 mg, 0.20 mmol) and 1,10-phenanthroline (115 mg, 0.64 mmol) was
added to a microwave vessel containing argon-purged ethylene glycol (3 mL), then the mixture was
subjected to microwave irradiation at 180°C for 15 min with stirring. The resulting dark red solution was
then transferred to a separatory funnel with deionized water (25 mL) and CH2Cl2 (25 mL). After gentle
agitation, the CH2Cl2 was drained, and the remaining aqueous layer was washed with CH2Cl2 until the
CH2Cl2 layer was colorless (3x 25 mL portions). Then, CH2Cl2 (25 mL) and saturated aqueous KPF6 (5
mL) was added, and the mixture was shaken gently. The CH2Cl2 layer was drained, and the product was
further extracted from the aqueous layer using CH2Cl2 until the aqueous layer was colorless (4x25 mL
portions). The CH2Cl2 extracts were then combined and concentrated under reduced pressure. The
product was then eluted from a silica gel flash column chromatography cartridge with a gradient of MeCN
to 10% water in MeCN, followed by 7.5% water in MeCN with 0.5% KNO 3. The dark red, productcontaining fractions, which eluted only in the presence of KNO3, were then combined and concentrated
13
under vacuum, then transferred to a separatory funnel with CH2Cl2 (25 mL), deionized water (25 mL),
and saturated aqueous KPF6 (1 mL). The resulting mixture was gently agitated and the CH2Cl2 layer was
drained. Additional CH2Cl2 (2x25 mL portions) was used to extract the remaining product until the
aqueous layer was colorless. The CH2Cl2 layers were then combined and dried under vacuum to yield
[Ru(phen)3](PF6)2, which was then converted to the corresponding Cl- salt in quantitative yield using
Amberlite IRA-410 with MeOH as the eluent, then purified further using Sephadex LH-20 with MeOH as
the eluent, affording product [Ru(phen)3](Cl)2 as a dark red solid (107 mg, 58%).1H NMR (700 MHz,
MeOD-d3, ppm): δ 8.67 (d, J = 8.1 Hz, 6H, 4,7), 8.30 (s, 6H, 5,6), 8.10 (d, J = 5.2 Hz, 6H, 2,9), 7.70 (dd,
J = 8.3, 5.2 Hz, 6H, 3,8). HRMS (ESI+) m/z for [M-2Cl-]2+ calcd: 321.0547; Found: 321.0547. HPLC
retention time 9.27 min (99.5% purity by peak area).
[Ru(phen)2(IP)](Cl)2 (Ru-0T). Ru(phen)2Cl2∙2H2O (57 mg, 0.1 mmol) and IP (22 mg, 0.1 mmol) were
added to a microwave vessel containing argon-purged ethylene glycol (4 mL) and subjected to microwave
irradiation at 180°C for 15 min. The resulting dark red mixture was then isolated and purified in the same
manner as [Ru(phen)3](Cl)2, yielding the desired product Ru-0T as a dark red solid (48 mg, 64%). 1H
NMR (700 MHz, MeOD-d3, ppm): δ 9.00 (broad s, 2H, c), 8.70 (d, J = 8.3 Hz, 4H. 4,7), 8.67 (s, 1H, d),
8.33 (s, 4H, 5,6), 8.16 (dd, J = 5.3, 1.3 Hz, 2H, 2), 8.11 (dd, J = 5.2, 1.3 Hz, 2H, 9), 8.07 (dd, J = 5.3, 1.3
Hz, 2H, a), 7.77 (dd, J = 8.3, 5.3 Hz, 2H, b), 7.72 (m, 4H, 8,3). HRMS (ESI+) m/z for [M-2Cl-]2+ calcd:
341.0578; Found: 341.0582. [M-2Cl--H]+ calcd: 681.1084; Found: 681.1110. HPLC retention time: 9.07
min (98% purity by peak area).
[Ru(phen)2(IP-1T)](Cl)2 (Ru-1T). Ru(phen)2Cl2∙2H2O (57 mg, 0.1 mmol) and IP-1T (30 mg, 0.1 mmol)
were added to a microwave vessel containing argon-purged ethylene glycol (4 mL) and subjected to
microwave irradiation at 180°C for 15 min. The resulting dark red mixture was then isolated and purified
in the same manner as compound [Ru(phen)3](Cl)2, yielding the desired product Ru-1T as a dark red
solid (49 mg, 59%). 1H NMR (700 MHz, MeOD-d3, ppm): δ 9.09 (broad s, 2H, c), 8.70 (dd, J = 8.4, 1.4
Hz, 4H, 4,7), 8.33 (s, 4H, 5,6), 8.18 (dd, J = 5.3, 1.3 Hz, 2H, 2), 8.12 (dd, J = 5.2, 1.3 Hz, 2H, 9), 8.05
(dd, J = 5.2, 1.3 Hz, 2H, a), 8.01 (dd, J = 3.7, 1.2 Hz, 1H, d), 7.76 (dd, J = 8.3, 5.3 Hz, 2H, b), 7.75 – 7.72
(m, 5H, f,3,8), 7.30 (dd, J = 5.1, 3.7 Hz, 1H, e). HRMS (ESI+) m/z for [M-2Cl-]2+ calcd: 382.0517; Found:
382.0523. [M-2Cl--H]+ calcd: 763.0961; Found: 763.0974. HPLC retention time 10.62 min (99.5% purity
by peak area).
[Ru(phen)2(IP-2T)](Cl)2 (Ru-2T). Ru(phen)2Cl2∙2H2O (57 mg, 0.1 mmol) and IP-2T (38 mg, 0.1 mmol)
were added to a microwave vessel containing argon-purged ethylene glycol (4 mL) and subjected to
14
microwave irradiation at 180°C for 15 min. The resulting dark red mixture was then isolated and purified
in the same manner as compound [Ru(phen)3](Cl)2, yielding the desired product Ru-2T as a dark red
solid (58 mg, 39%). 1H NMR (700 MHz, MeOD-d3, ppm): δ 9.03 (broad s, 2H, c), 8.71 (d, J = 8.30, 4H,
4,7), 8.34 (s, 4H, 5,6), 8.21 (dd, J = 5.4, 1.3 Hz, 2H, 2), 8.12 (dd, J = 5.2, 1.3 Hz, 2H, 9), 8.05 (dd, J =
5.3, 1.3 Hz, 2H, a), 7.92 (d, J = 3.9 Hz, 1H, d), 7.78 – 7.71 (m, 6H, b,3,8), 7.44 (dd, J = 5.1, 1.2 Hz, 1H,
f), 7.36 (d, J = 3.9 Hz, 1H, e), 7.33 (d, J = 3.5 Hz, 1H, h), 7.09 (dd, J = 5.1, 3.6 Hz, 1H, g). HRMS (ESI+)
m/z for [M-2Cl-]2+ calcd: 423.0455; Found: 423.0458. [M-2Cl--H]+ calcd: 845.0838; Found: 845.0852.
HPLC retention time 21.19 min (99.5% purity by peak area).
[Ru(phen)2(IP-3T)](Cl)2 (Ru-3T). Ru(phen)2Cl2∙2H2O (114 mg, 0.2 mmol) and IP-3T (76 mg, 0.164 mmol)
were added to a microwave vessel containing argon-purged ethylene glycol (4 mL) and subjected to
microwave irradiation at 180°C for 15 min. The resulting dark red mixture was then isolated and purified
in the same manner as [Ru(phen)3](Cl)2, yielding the desired product Ru-3T as a dark red solid (59 mg,
59%). 1H NMR (700 MHz, MeOD-d3, ppm): δ 9.03 (d, J = 8.2 Hz, 2H, c), 8.70 (d, J = 8.9 Hz, 2H, 4), 8.68
(d, J = 8.4 Hz, 2H, 7), 8.34 (s, 4H, 5,6), 8.22 (d, J = 5.6 Hz, 2H, 2), 8.12 (dd, J = 5.2, 1.3 Hz, 2H, 9), 8.04
(d, J = 5.6 Hz, 2H, a), 7.90 (d, J = 3.9 Hz, 1H, d), 7.77 (dd, J = 8.2, 5.4 Hz, 2H, 3, 7.72 (dd, J = 8.53 Hz,
5.33 Hz, 2H, 8), 7.70 (dd, J = 8.20 Hz, 5.38 Hz, 2H, b), 7.35 (d, J = 5.4 Hz, 1H, h), 7.27 (d, J = 3.8 Hz,
1H, e), 7.22 (d, J = 3.5 Hz, 1H, j), 7.19 (d, J = 3.8 Hz, 1H, g), 7.11 (d, J = 3.7 Hz, 1H, f), 7.05 (dd, J = 5.1,
3.6 Hz, 1H, i). HRMS (ESI+) m/z for [M-2Cl-]2+ calcd: 464.0394; Found: 464.0405. [M-2Cl--H]+ calcd:
927.0715; Found: 927.0769. HPLC retention time 22.75 min (96% purity by peak area).
[Ru(phen)2(IP-4T)](Cl)2 (Ru-4T). Ru(phen)2Cl2∙2H2O (114 mg, 0.2 mmol) and IP-4T (90 mg, 0.164 mmol)
were added to a microwave vessel containing argon-purged ethylene glycol (4 mL) and subjected to
microwave irradiation at 180°C for 15 min. The resulting dark red mixture was then isolated and purified
in the same manner as compound [Ru(phen)3](Cl)2, yielding the desired product Ru-4T as a dark red
solid (49 mg, 28%). 1H NMR (700 MHz, MeOD-d3, ppm): δ 9.04 (s, 2H, c), 8.72 (d, J = 8.77 Hz, 2H, 4),
8.70 (d, J = 8.89 Hz, 2H, 7), 8.34 (s, 4H, 5,6), 8.22 (dd, J = 5.4, 1.3 Hz, 2H, 2), 8.12 (dd, J = 5.2, 1.3 Hz,
2H, 9), 8.05 (dd, J = 5.2, 1.3 Hz, 2H, a), 7.89 (d, J = 3.9 Hz, 1zH, d), 7.77 (dd, J = 8.2, 5.4 Hz, 2H, 3),
7.75 – 7.70 (m, 4H, 8,b), 7.33 (d, J = 3.9 Hz, 1H, e), 7.29 (dd, J = 5.1, 1.2 Hz, 1H, l), 7.24 (d, J = 3.7 Hz,
1H, f), 7.17 (dd, J = 3.6, 1.3 Hz, 1H, j), 7.12 (d, J = 3.8 Hz, 1H, h), 7.09 (d, J = 3.7 Hz, 1H, g), 7.04 (d, J
= 3.7 Hz, 1H, i), 7.00 (dd, J = 5.1, 3.5 Hz, 1H, k). 13C NMR (700 MHz, MeOD-d3, ppm): δ 153.96 (2),
153.77 (9), 151.57 (a), 150.08 (10), 149.29 (21), 149.19 (20), 147.36 (22), 141.46 (12), 138.31 (4,7,14),
138.14 (16), 137.78 (17), 136.36 (15), 136.24 (13), 132.57 (18), 132.54 (19), 131.89 (c, 11), 129.65 (d),
129.49 (6), 129.48 (5), 129.11 (k), 127.40 (8), 127.37 (3), 127.13 (b), 126.82 (f), 126.05 (l), 126.01 (h),
15
125.85 (e), 125.71 (g), 125.51 (i), 125.08 (j). HRMS (ESI+) m/z for [M-2Cl-]2+ calcd: 505.0333; Found:
505.0312. [M-2Cl--H]+ calcd: 1009.0593; Found: 1009.0663. HPLC retention time 23.92 min (99.5% purity
by peak area).
Computational Details
The computational protocol used to investigate the Ru(II)-complexes herein presented is based on a
combination of DFT and TDDFT105 as methods as implemented in the Gaussian16106 code and widely
tested in previous studies involving metallic photosensitizers for PDT107–114 and successfully adopted for
our related Os(II)- and Ru-compounds.23,24,63
The PBE0 exchange-correlation functional (XC)115 in conjunction with the 6-31+G(d,p) basis set was
chosen for the singlet ground and lowest triplet excited states optimizations in water adopting the quasirelativistic Stuttgart-Dresden pseudopotential to treat the Ru(II) center.116 The integral equation formalism
polarizable continuum model117,118 (IEFPCM) was used to simulate the water solvent environment by
using a dielectric constant equal to ɛ=80 by means of the polarizable conductor model (PCM).119
The M06 exchange-correlation (XC)-functional and the Tamm-Dancoff approximation (TDA)120 were used
to compute the UV-Vis absorption spectra in water on top of the corresponding S0 equilibrium geometries.
We recently adopted this method for optimizing the lowest triplet metal-to-ligand charge transfer (3MLCT)
and ligand-based mixed triplet intraligand charge transfer (3ILCT) / ligand-to-ligand charge transfer
(3LLCT) excited states and computing the emission energies.63 The TDA circumvents the general
underestimation of the triplet state energies from the conventional TDDFT approach,121 as was also
observed in our earlier investigations on other oligothiophene-based Ru(II) and Os(II) complexes for
which the vertical lowest triplet excited states were underestimated.23,24,110 The nature of the excited
states was determined in all cases by computing the corresponding natural transition orbitals (NTOs) with
the Chemissian 4.67 software,122 and through Gaussian output post-processing conducted with the
TheoDORE 3.1.1 program.123
Electrochemistry
Voltammetry was performed in dimethylformamide (DMF, Fisher HPLC grade) that had been dried and
deoxygenated with an Inert PureSolv MD7 solvent purification system, with 100 mM tetrabutylammonium
hexafluorophosphate (TBAPF6) (Fisher) as the supporting electrolyte, in a two-compartment low volume
cell with the three-electrode configuration under argon. A 3 mm glassy carbon disc was used as the
working electrode with a platinum wire counter electrode and a Ag/AgCl/4M KCl reference electrode.
16
Ferrocene (Fc) was used as an internal standard. The complex solutions were approximately 4 mM for
oxidation sweeps and 0.25 mM for reduction sweeps.
Measurements were conducted at room temperature using a WaveNow potentiostat (Pine Research
Company) with Aftermath software. Cyclic differential-pulse voltammetry (CDPV) measurements used a
sweep rate of 2 mV∙s-1 with a modulation amplitude varying from 12.5 to 100 mV. For reversible
processes, the formal redox potential E°′ was taken as the average of Epa (anodic peak potential) and Epc
(cathodic peak potential). For quasi-reversible processes, only Epa or Epc is reported.
2.4 RESULTS AND DISCUSSION
Synthesis and Characterization
[Ru(phen)3](Cl)2 and Ru-nT were synthesized using our previously published method for related Ru(II)
phenanthroline-based complexes.63 The complexes were isolated as PF6− salts and purified using flash
chromatography on silica. The PF6− salts were then converted to their corresponding Cl− salts in
quantitative yields via anion metathesis with Amberlite IRA-410 and further purified using size-exclusion
chromatography on Sephadex LH-20. The final yields were ~60% for [Ru(phen)3](Cl)2, Ru-0T, Ru-1T,
and Ru-3T, ~40% for Ru-2T, and ~30% for Ru-4T. The complexes were characterized by 1D and 2D 1H
NMR spectroscopy (Figure 1, Figure S1–Figure S10), with assignment of signals for [Ru(phen)3](Cl)2 and
Ru-0T–Ru-3T made using 1H–1H COSY NMR. Ru-4T was additionally analysed by 1H–13C HSQC and
17
Figure 1. Aromatic region of the 1H NMR spectra for [Ru(phen)3](Cl)2 and Ru-nT (n=0−4) in MeOD-d3 (Cl− salts;
298 K). All spectra were collected at 500 MHz, except for Ru-4T, which was collected at 700 MHz.
1
H–13C HMBC NMR to assign the hydrogens of the quaterthiophene unit. The assignments were
consistent with our related, previously reported compounds.23,63,76 The complexes were also
18
characterized by high-resolution ESI+ mass spectrometry (Figure S11–Figure S16). HPLC analyses
indicated that the complexes were ≥95% pure by integration (Figure S17–Figure S22).
The lipophilicities of [Ru(phen)3](Cl)2 and Ru-nT as their chloride salts were evaluated experimentally by
calculating their log Do/w values from partitioning between 10 mM phosphate buffer solution (pH 7.4) and
1-octanol (99.9%) (Figure 2 and Table S1). A negative log Do/w value indicates hydrophilicity whereas a
positive log Do/w value indicates higher lipophilicity.124 [Ru(phen)3](Cl)2 and Ru-nT up to n=2 were
relatively hydrophilic overall, with log Do/w values becoming increasingly more positive with additional
thiophene rings. An abrupt change in aqueous solubility occurred at n=3, with Ru-3T showing a clear
preference for 1-octanol. Ru-4T also preferred 1-octanol, but its log Do/w could not be determined due to
precipitation between the two layers that left no measurable amount of compound in the aqueous phase.
Precipitation at the octanol-buffer interface was also observed for the analogous Os(II) complex that selfassociates in PBS to form particles of up to 1–2 µm in diameter.23 This is not uncommon for Ru(II) and
Os(II) complexes containing the IP-4T ligand.
Complex
Ru-3T
Ru-2T
Ru-1T
Ru-0T
Ru(phen)3
-2
-1
0
1
log Do/w
Figure 2. Lipophilicities of [Ru(phen)3](Cl)2 and Ru-nT (n=0–3) in 1-octanol and phosphate buffer using the shakeflask method. The log Do/w value for Ru-4T was undefined due to precipitation at the octanol:phosphate buffer
interface that left no measurable amount of Ru-4T in the phosphate buffer phase.
Computation
Singlet states. Figure 3 shows the optimized singlet ground state structures of [Ru(phen) 3]2+ and Ru-nT
(n=0–4) in water at the DFT/PBE0 level of theory, and the main geometric parameters are reported in
Table S2 The central Ru(II) ion adopts an octahedral geometry with similar Ru-N bond distances across
the series, with the first thiophene ring being coplanar with the coordinated IP ligand. Each subsequent
ring introduces more conformational flexibility, with the fourth thienyl ring of Ru-4T being twisted out of
19
plane by approximately 18°. The nT chain length has a major impact on the frontier orbitals. Similar to
structurally related families we have reported,23,24,63 complexes with n≥2 have progressively higherenergy HOMOs that give rise to a systematic reduction of the H-L gaps as the % nT contribution increases
(Figure 4 and Figure S23, Table S3. The HOMO for Ru-2T extends over both the IP and nT unit, whereas
the HOMOs are localized primarily to the nT chain for Ru-3T and Ru-4T, where the nT contribution to the
HOMO is about 46% and 61%, respectively. In contrast, the LUMOs across the series are primarily phenbased (>95% for all complexes) and not affected significantly by nT.
[Ru(phen)3]2+
Ru-0T
Ru-1T
Ru-2T
Ru-3T
Ru-4T
Figure 3. Optimized geometries of [Ru(phen)3]2+ and Ru-nT (n=0-4) in a water environment at the PBE0/631+G(d,p)/SDD/ level of theory. The two phen ligands are shown in grey for the Ru IP-nT complexes for clarity.
The computed lowest-energy, spin-allowed singlet-singlet absorption transitions shift to longer
wavelengths with increasing n (Figure 5). The NTOs are predominantly 1MLCT (Ru˧phen/IP) for
20
[Ru(phen)3]2+, Ru-0T and Ru-1T. Their computed transitions are similar near 432–438 nm and slightly
higher in energy than the experimental bands (vide infra). The lowest energy transitions for Ru-2T, Ru3T, and Ru-4T are red-shifted with n, in agreement with experimental spectra. The lowest energy
absorption was computed at 455 nm and was mixed 1MLCT/1LLCT character for Ru-2T. In the case of
Ru-3T and Ru-4T, the lowest energy transitions were 1ILCT/1IL/1LLCT character (mostly localized to the
IP-nT ligand) and computed at 466 nm and 488 nm, respectively. Ru-4T has twice as much 1ILCT/1IL
character compared to Ru-3T. Here, LLCT mainly refers to CT between nT and IP (but does include very
minor involvement of the phen coligands); ILCT involves CT within nT; IL is ππ* localized to nT or IP.
Further details are summarized in Table S4 and Figure S24.
-2.0
-2.5
-3.0
-6.0
-6.5
Ru-0T
Ru-1T
Ru-2T
Ru-3T
Ru-4T
Figure 4. Computed HOMO and LUMO orbital energies (solid black lines) and percent contribution of the nT chain
to the HOMO (dashed line, red filled circles) and of the phen coligands to the LUMO (dashed line, green filled
triangles), for Ru-nT (n=0-4) in the singlet ground state, at the M06/6-31+G(d,p)/SDD level of theory, in water.
Images of Ru(II)-based HOMOs for Ru-0T and Ru-1T, the nT-based HOMOs for n=2-4, and the phen-based
LUMOs for all compounds, obtained at the same level of theory. Additional details can be found in Figure S23.
Triplet states. The optimized structures of the lowest excited triplet states (T1) for the Ru-nT family involve
a fully planar arrangement of the nT chain that maximizes the π-conjugation, with successive nT groups
21
antiplanar to one another. The geometrical parameters of the T1 states for [Ru(phen)3]2+ and Ru-nT (n=0–
4) are listed in Table S2 alongside the data for S0, and the optimized T1 state structures are shown in
Figure S25. The lowest-energy triplet excited state configurations of this series are either 3MLCT for
complexes without thiophenes (or only one thiophene as in the case of Ru-1T) or mixed 3ILCT/3LLCT
states for complexes with two or more thiophenes. The lowest-energy 3MLCT states lie near 2.2 eV for
all complexes in the series regardless of the thiophene chain length and whether the 3MLCT state is T1.
The energies of the mixed 3ILCT/3LLCT states depend on n and decrease systematically in energy from
1.82 eV for Ru-2T to 1.44 eV for Ru-4T. The triplet metal-centered (3MC) and intraligand (3IL) excited
states localized to the phen/IP coligands are much higher in energy and contribute very little to the
computed NTOs for T1. The energies and configurations of the computed triplet states are presented in
Figure 6 and complied in Table 1 and Table S5. The occupied and virtual NTOs are plotted in Figure
S26.
22
Figure 5. Occupied and Virtual NTOs of the computed lowest-energy singlet-singlet transitions in water (λ) with
the predominant character indicated. The experimental longest wavelength absorption maxima (λ exp) are reported
in parentheses. Additional NTOs are reported in Figure S24.
23
Although the T1 triplet state undergoes a significant drop in energy with increasing n, all are still sufficiently
energetic to sensitize 1O2.125,126 Mulliken spin densities (MSD) close to 1 on the Ru(II) center further
support that T1 is predominantly 3MLCT for [Ru(phen)3]2+, Ru-0T, and Ru-1T. The MSD values of 0 on
the Ru(II) center for complexes with n≥2 indicate that the metal is not involved in T1. The predominant
character of T1 for Ru-2T is mixed 3ILCT/3LLCT, where 3ILCT involves CT within the nT unit (nT˧ nT)
and 3LLCT involves CT between nT and IP (nT˧IP), and each contributes equally (§40%) to the transition
according to the topology analysis (Figure 6b). For Ru-3T and Ru-4T, T1 is >50% 3ILCT. The drop in T1
energy on going from n=2 to 4 is accompanied by diminishing 3LLCT character (from ~40% down to
~20%). Such behavior is in agreement with the related Ru(II) and Os(II) families we reported
previously,23,24,63 where T1 involves the IP-nT ligand for n=2–4 and is increasingly more localized to the
nT portion with increasing n. The higher-lying 3MLCT state (T2) for these complexes is similar in energy
(~2.2 eV) to those with n<2 having 3MLCT states as T1.
Table 1. Computed T1 adiabatic energies, configurations, and Mulliken spin densities (MSD) on the Ru(II) metal
center for [Ru(phen)3]2+ and Ru-nT (n = 0–4). A single configuration is listed if that character was >50%.
T1 energy (eV)
Configuration
MSD
[Ru(phen)3]2+
2.21
3MLCT
0.86
Ru-0T
2.18
3MLCT
0.86
Ru-1T
2.18
3MLCT
0.91
Ru-2T
1.82
3ILCT/3LLCT
0
Ru-3T
1.57
3ILCT
0
Ru-4T
1.44
3ILCT
0
24
Figure 6. (a) Computed T1 adiabatic energies for [Ru(phen)3]2+ and Ru-nT. (b) Molecular fragments (left) defined
to quantify the molecular topology of the T1 excited states and their configurations (right). The NTOs are reported
in Figure S26 and triplet excited state energies in Table S5.
Spectroscopy
UV-Vis absorption and emission spectroscopy
The electronic absorption spectra of the series collected on the hexafluorophosphate salts in MeCN are
shown in Figure 7, and the corresponding molar extinction coefficients are listed in Table 2. [Ru(phen)3]2+
has been previously reported, and our data are in agreement with published values.127 The spectra can
be generalized by two distinct regions. The sharper peaks below 300 nm, with maxima around 223 and
263 nm, are similar across the series and can be ascribed to π→π* transitions involving the phen
coligands and possibly the phen portion of the IP/IP-nT ligands that are proximal to the metal center.
These peaks occur at the same energy in related complexes23 and are not significantly affected by the
length of the pendant nT chain.
25
Figure 7: UV-vis spectra of [Ru(phen)3]2+ and the Ru-nT series as PF6‒ salts in MeCN.
At wavelengths between 300 to 500 nm, the absorption spectra for [Ru(phen)3]2+ and Ru-0T are similar
and typical of Ru(II) polypyridyl type complexes with Ru2+(dπ)→LL(π*) MLCT transitions involving phen
or phen/IP, respectively, as the π* acceptor orbitals. The complexes with IP-nT ligands have additional
contributions from 1LLCT (nTĺIP) transitions as well as 1ILCT (nTĺnT) for Ru-2T to Ru-4T. These
isolated transitions can be seen in the absorption spectra of the analogous uncomplexed IP-nT ligands
and free oligothiophenes128 but do experience some shifting when incorporated into the metal complexes.
Our computational studies considering the occupied and virtual NTOs of the Ru-nT complexes highlight
the predominant configurations of the computed absorption transitions occurring >400 nm and support
these ligand-based contributions. The lowest energy singlet-singlet transitions were computed to be
mixed 1MLCT/1LLCT for Ru-2T and 1ILCT/1LLCT for Ru-3T and Ru-4T (Figure 5 and Figure S24, Table
S4). These computed and experimental energies were lowest for Ru-4T, as expected for the more
extended π system, and the 1ILCT character was almost two-fold higher (Figure 5).
Table 2: Molar Extinction Coefficients at Various Absorption Peak Maxima for the Ru-nT series.
Compound
λabs (nm) (log (ε / M−1 cm−1))
[Ru(phen)3]2+
444 (4.20), 263 (4.91), 221 (4.84)
Ru-0T
450 (4.46), 263 (5.17), 223 (5.09)
Ru-1T
457 (4.49), 332 (4.56), 289 (4.95), 264 (5.15), 223 (5.08)
Ru-2T
457 (4.44), 384 (4.69), 263 (5.08), 223 (4.97)
Ru-3T
460 (4.57), 413 (4.75), 263 (5.05), 222 (4.94)
Ru-4T
465 (4.75), 436 (4.84), 264 (5.03), 223 (4.93)
26
All of the complexes in the series exhibited red emission near 610–620 nm as a single, broad and
featureless band in argon-sparged MeCN at room temperature (Figure 8, Table 3 and S5). The number
of appended thiophene rings did not affect the emission energies, suggesting that the luminescence
originates from a common 3MLCT state with similar ligand acceptor orbitals across the series. The
computed adiabatic 3MLCT energies matched the experimental room temperature 3MLCT emission
Figure 8: Normalized emission spectra of [Ru(phen)3]2+ and the Ru-nT series as PF6‒ salts at room temperature
(left) and at 77 K (right) in MeCN. The room temperature spectra used argon-sparged MeCN and the 77 K
spectra used a 4:1 EtOH:MeOH glass. The excitation wavelengths are noted in parentheses. Emission from Ru4T was weak and superimposed on scatter in the room temperature measurement (blue curves).
energies at around 2.2 eV. For complexes lacking thienyl groups and Ru-1T, this 3MLCT state was
computed as the lowest-energy triplet state (T1). For Ru-2T to Ru-4T, the emissive 3MLCT state was T2.
The room temperature emission for [Ru(phen)3]2+ was in agreement with that previously reported,129,130
with a quantum yield near 3% and lifetime of approximately 0.5 µs at room temperature. The rest of the
complexes in the series also had emission lifetimes between 0.5 and ~1 µs (Table 3 and Figure S27), but
quantum yields dropped progressively on going from Ru-0T to Ru-4T. For complexes with up to two
thiophene rings, quantum yields were still between 3 and 8.5%. However, emission from complexes with
longer thiophene chains was considerably weaker, falling to about 0.4% for Ru-3T and only 0.02% for
Ru-4T. The spectra in Figure 8 are normalized to emphasize similar 3MLCT emission energies, but the
much lower quantum yields for Ru-3T and Ru-4T are reflected in the poorer signal-to-noise ratios evident
in the spectra. The emission from Ru-4T is extremely weak and should be regarded as almost nonemissive with an extremely high error on the quantum yield as a result.
27
Assignment of the emission to 3MLCT states was corroborated by measurements at 77 K, where the
emission shifted to shorter wavelengths with increased quantum yields and exhibited vibronic character
typical of 3MLCT states (Figure 8). The vibronic intervals of around 1350 cm-1 are consistent with diimine
involvement in the emissive state,131 and did not vary significantly throughout the series. The thermally
induced Stokes shifts (ΔES) of around 1100 cm-1 compare well to the related model complex [Ru(bpy)3]2+
(ΔES= 1127 cm−1).132 These 3MLCT emission energies were computed at around 2.0 eV, in agreement
with the experimental 77 K energies (Table 3 and Table S5).
Singlet oxygen sensitization
All of the complexes have triplet excited states of sufficient energy to sensitize 1O2 with an energy of
approximately 0.97 eV.133 The quantum yields for 1O2 formation (ΦΔ) were calculated for the PF6− salts in
air-saturated MeCN calculated using the integrated 1O2 emission centered near 1276 nm with
[Ru(bpy)3](PF6)2 as the standard (ΦΔ,s=0.56) according to Equation 1.133 The results are compiled in Table
3. The 1O2 quantum yields for Ru(phen)32+ and Ru-0T, the compounds lacking any thiophene rings, were
very similar to the reference at 53 and 56%, respectively. Generation of 1O2 increased with thienyl chain
length, plateauing around 88% at n=3. For comparison, the related complexes [Ru(bpy)2(IP-nT)]2+ and
[Ru(4,4′-dmb)2(IP-nT)]2+ exhibit near unity quantum yields for n=3,4 and around 75% for n=2.98
Table 3: Photophysical properties of the series, measured as (PF6)‒ salts in MeCN. Excitation wavelengths are
noted in parentheses. *Too weak to accurately quantify.
RT emission
77 K emission
Compound
λem. (λex) / nm
Φem
τem / µs
[Ru(phen)3]2+
602 (448)
3.1×10−2
0.47
Ru-0T
617 (455)
8.4×10−2
0.70
Ru-1T
606 (458)
5.7×10−2
0.73
Ru-2T
612 (467)
3.0×10−2
1.1
Ru-3T
614 (468)
3.5×10−3
0.97
Ru-4T
614 (463)
*1.6×10−4
0.85
λem. (λex) / nm
568, 616, 672
(452)
571, 620, 679
(452)
573, 621, 678
(460)
576, 625, 692
(458)
573, 620, 685
(462)
574, 619
(457)
28
ΦΔ
τTA / μs
Φem,77 K
(λex / nm)
5.5×10−1
0.53 (450)
0.40
5.6×10−1
0.56 (456)
0.85
4.4×10−1
0.62 (461)
3.2×10−2
0.73 (462)
148
1.7×10−2
0.88 (457)
34–36
8.6×10−4
0.87 (462)
25
0.18, 0.79 (410, 460)
0.16 (610)
Transient Absorption
Nanosecond transient absorption (TA) spectroscopy was used to examine the triplet excited states.
Differential excited state absorption (ESA) spectra were measured in degassed MeCN following
excitation with a 355 nm laser with a 5-ns pulse width, with correction for luminescence. Selected time
slices are shown in Figure 9 and the full set of TA spectra are compiled in Figure S28. Transient lifetimes
were measured at ESA maxima or bleach minima and are compiled in Table 3. The corresponding timeresolved spectra and fits are shown in Figure S29.
The TA profiles of [Ru(phen)3]2+ and Ru-0T are typical of what might be expected of the 3MLCT state for
a Ru(II) polypyridyl complex, with a bleach in the 400 to 500 nm region arising from loss of the strong
MLCT ← 1A1 ground state absorption. Part of the ESA due to the ligand phen− transitions can be seen
1
at shorter wavelengths, and the extremely weak and broad absorption at longer wavelengths due to
phen− or LMCT transitions involving Ru(II) is also observed. Their TA lifetimes matched their emissive
lifetimes and lacked any involvement of the higher-lying ligand-based triplet excited states.
Figure 9: Transient absorption (TA) spectra of [Ru(phen)3]2+ and the Ru-nT series in deoxygenated MeCN
integrated over the indicated time slice following the excitation pulse. ΔO.D.=0 is indicated by a dotted line. Data
for the complexes with nanosecond lifetimes are shown on the left, and those with microsecond lifetimes are
shown on the right.
The transient profile of Ru-1T is more complex. A strong ground state bleach appears near 350 nm
alongside a strong ESA near 410 nm that overlaps the weaker 1MLCT ← 1A1 ground state bleach in the
400 to 500 nm region and another ESA beyond 475 nm (Figure 9 and Figure S28). The ESA at longer
wavelengths is more intense than that for [Ru(phen)3]2+ or Ru-0T but not nearly as strong as typical 3ILCT
states involving two or more thiophenes. The kinetics measured at 410 and 460 nm both exhibited a fast
29
decay (0.2 µs) of the ESA component and a slower decay (0.8 µs) of a bleach component. The slower
decay was in good agreement with the 3MLCT decay from the emission experiment, and the TA spectrum
collected at 0.5–1.0 µs after the laser pulse exhibits the typical 3MLCT signature (Figure S30). The ESA
at longer wavelengths (e.g., 610 nm) decayed with a single time constant of 0.2 µs. The strong overlap
between the IP-1T ligand-localized absorption and the excitation laser pulse (355 nm) may give rise to
the short decay associated with the broad ESA.
Ru-2T has the longest triplet lifetime of the family, and its TA spectrum is dominated by IP-2T ligandlocalized transitions. An intense ESA, with an onset near 450 nm and characteristic signature of the
oligothiophene-based 3ILCT state, obscured the 1MLCT ground state bleach in the 400-500 nm region.
The intense bleach in the region under 400 nm, with a minimum near 380 nm, involves the loss of the
1
IL/1ILCT ground state absorption. The decay kinetics in both the bleach and ESA regions are
monoexponential with a lifetime of 148 µs.
The TA spectra of Ru-3T and Ru-4T are also dominated by the oligothiophene-based 3ILCT triplets. Ru3T exhibited a bleach around 410 nm and a strong ESA near 625 nm, while Ru-4T produced these
corresponding transients at slightly longer wavelengths. The bleach for Ru-4T has its minimum around
440 nm, and the ESA is centered around 675 nm. Both the bleach and the ESA for both compounds
decayed monoexponentially with a lifetime of 36 µs for Ru-3T and 25 µs for Ru-4T. The 3ILCT state that
was observed by TA and the emitting 3MLCT state were decoupled as observed in the case of Ru-2T,
suggesting that the 3ILCT state is the lowest-energy triplet for n=2–4. Indeed, the computational studies
estimate T1 as predominantly 3ILCT/3LLCT for Ru-2T and 3ILCT for Ru-3T and Ru-4T (Table 1). The
systematic decrease in the 3ILCT state lifetime on going from Ru-2T to Ru-4T, with τTA dropping from 148
to 36 to 25 µs, is consistent with the shortening of triplet lifetimes in free oligothiophenes owing to the
decrease in the T1–S0 energy gap with increasing number of thiophenes.128 The absence of 3MLCT
contributions to the decays is consistent with the extremely weak 3MLCT emission quantum yields that
suggest the 3ILCT state dominates the relaxation dynamics on the nanosecond to microsecond
timescales.
Triplet Energies and Excited State Pathways
The energies of the oligothiophene-based 3ILCT states cannot be obtained directly because these states
are non-emissive, nor can they be estimated from the corresponding free IP-nT ligands and nT units
because they also do not emit. However, the 3ILCT energies can be estimated based on the shortening
of their TA lifetimes in the presence of suitable excited state quenchers, in accordance with the modified
30
Stern-Volmer relationship presented in Equation S2. The complexes were excited at 532 nm to avoid
directly exciting the quencher. The appearance of a new long-lived signal in the ESA spectra of the
mixtures confirmed that the triplet state of the quencher had indeed formed via energy transfer from the
excited complex.
The rate constants (kq) for triplet-triplet energy transfer between selected organic sensitizers of known
ππ* energies67,134 (ET) and the excited complexes are compiled in Table 4. Values for kq were determined
3
by examining the TA lifetimes of the complexes (measured at 620 nm and at 660 nm for Ru-3T and Ru4T, respectively) as a function of quencher concentration. The values for kq were largest when the 3ππ*
acceptor energy was near 1.53 eV. Therefore, the 3ILCT energies were estimated to lie near 1.5 eV
above the ground state. These values are in good agreement with the computed 3ILCT energies of 1.57
for Ru-3T and 1.44 eV for Ru-4T.
Table 4: Stern-Volmer triplet-triplet energy transfer rate constants for Ru-3T and Ru-4T in the presence of
quenchers with known 3ππ* energies (ET). n.d.=not determined.
Quencher
ET (eV)
Ru-3T kq (M-1 s-1)
Ru-4T kq (M-1 s-1)
tetracene
1.3
9.4 × 108
n.d.
perylene
1.5
5.2 × 109
3.7 × 109
diBr-anthracene
1.7
2.9 × 109
6.2 × 108
phenazine
1.9
1.2 × 108
3.1 × 108
pyrene
2.1
2.3 × 107
n.d.
fluorene
2.9
0
n.d.
From the spectroscopic data combined with computational studies and Stern-Volmer quenching
experiments, Jablonski diagrams modelling the excited state pathways for the two complexes with
predominant 3ILCT states are shown in Scheme 1. Excitation of Ru-3T and Ru-4T with visible light
produces singlet excited states of mixed 1MLCT, 1LLCT, or 1ILCT configurations, where the computed
singlet-singlet transitions have higher 1ILCT character for the longest wavelength absorption bands
(Table S3). For example, Ru-3T has 58% 3ILCT character for its 466 nm transition and Ru-4T has 77%
for its 488 nm transition. These initially formed excited states ultimately relax to their lower-lying triplets
of 3MLCT (T2) or 3ILCT (T1) configuration where T1 has a small amount of 3LLCT mixing (27% for Ru-3T
and 19% for Ru-4T). The 3MLCT emission is weak, and the excited state dynamics of Ru-3T and Ru-4T
on the nanosecond to microsecond timescales in the TA experiments are dominated by lowest-lying
31
3
ILCT states with longer lifetimes (25–40 µs). These dynamics are similar to those we have encountered
previously in other oligothienyl-containing complexes.23,24,99,135–139 While both 3MLCT and 3ILCT states
can generate 1O2, it is expected that the 3ILCT states with their longer lifetimes may play the larger role
in ROS production. Because oligothiophenes are known to be redox active, these states may also be
deactivated via electron transfer pathways in the presence of suitable electron donors or acceptors. The
electrochemical properties of Ru-3T and Ru-4T along with the rest of the series were investigated to gain
a better understanding of these characteristics.
Scheme 1: Jablonksi diagram depicting the excited state pathways of Ru-3T and Ru-4T. Energies are not to
scale and 1LLCT contribution to initially formed excited states not shown.
Electrochemistry
Oxidation of [Ru(phen)2(IP-nT)](PF6)2 complexes
Representative cyclic differential pulse voltammetry (CDPV) traces for oxidation of the complexes
measured relative to Ag/AgCl (4M KCl) are presented in Figure S31, and the formal redox potentials are
listed in Table 5 relative to ferrocene as the internal standard. The trends are compared in Figure 10. As
is typical of Ru(II) polypyridyl complexes, a single reversible wave appears due to the Ru2+/ Ru3+ process,
occurring near +820 to +880 mV vs. ferrocene in all the complexes. The potential for the Ru2+/ Ru3+
couple is largely unaffected by the length of the thienyl chain. For compounds Ru-1T through Ru-4T, a
second, quasi-reversible wave appears, due to the oxidation of the thiophene chain. For two thienyl
groups and longer, nT is more easily oxidized than the Ru(II) center. In contrast, the thiophene oxidation
is less favorable than the metal oxidation by about 100 mV for Ru-1T. The change with each successive
thiophene group is most pronounced on going from one to two thiophenes, with a difference of about 180
32
mV. Thereafter, the changes are around 80 to 90 mV. The difference in the nT oxidation potentials of Ru4T versus Ru-1T is about 350 mV. This trend is consistent with the behaviour of free oligothiophenes,
with oxidation occurring more readily for longer nT.140
Reduction of [Ru(phen)2(IP-nT)](PF6)2 complexes
The electrochemistry of Ru(II) polypyridyl complexes of this type is generally typified by three reversible
reduction waves as one electron is added to each ligand in succession.129 The reduction of [Ru(phen)3]2+
is known to be complicated by adsorption on the electrode,141 but we found this problem could be
mitigated using DMF as the solvent and a lower concentration (0.25 mM) on the reduction sweep.
Figure 10: Formal redox potentials (vs the ferrocene internal reference) and proposed assignments of the
(a) oxidation and (b) reduction processes, as measured by CDPV in DMF containing TBAPF6.
The cyclic differential pulse voltammograms are shown in Figure S32 and the formal potentials are
tabulated in Table 5 and compared graphically in Figure 10. The first two reduction waves for the Ru-nT
series shift to slightly more negative potentials compared to the model compound [Ru(phen) 3]2+. In
contrast, the potential of third reduction changes more dramatically, shifting negative by around 250 mV,
when phen is replaced by IP but is also largely unaffected by n. This indicates that the first two reductions
involve the phen ligands, and the third reduction involves the IP-nT ligand.
The potential of the third reduction of the Ru-nT complexes does not change much, becoming more
positive by only around 40 mV on going from Ru-0T to Ru-3T. This suggests that the reduction is
localized to the IP portion of the IP-nT ligand and is influenced only slightly by the number of thiophenes.
A fourth reduction appears only in the case of Ru-3T and Ru-4T. This fourth reduction potential shifts
positive by 240 mV on going from Ru-3T to Ru-4T, in agreement with smaller HOMO-LUMO gaps
33
associated with increasing π-conjugation in oligothiophenes.142 Although Ru-3T is the first complex in the
series where the oligothiophene unit can be reduced in the measurable potential window, 3T remains
harder to reduce than IP. Ru-4T shows a marked departure and represents the first point at which the
oligothiophene unit is reduced more readily than IP (Figure 10b).
Table 5. Formal redox potentials measured by CDPV in DMF containing 0.1 M TBAPF 6, referenced in volts against
ferrocene as the internal standard. The concentration of the complexes was 4 mM for the oxidation and 0.25 mM
for the reduction scans. The working and reference electrodes were glassy carbon and Ag/AgCl/4M KCl,
respectively. The Pt wire was used as a counter electrode. Overlapping waves were deconvoluted mathematically.
The error on these measurements is ±0.02 V.
′
′
′
′
′
𝑬𝟎𝒓𝒆𝒅 (𝟐)
𝟎
𝑬𝒓𝒆𝒅
(𝟏)
𝟎
𝑬𝒐𝒙
(𝟏)
[Ru(phen)3]2+
−2.22
−1.92
−1.78
+0.83
Ru-0T
−2.46
−2.00
−1.81
+0.83
Ru-1T
−2.45
−1.99
−1.81
+0.82
+0.92b,c
Ru-2T
−2.45
−1.99
−1.81
+0.74b,c
+0.88
−2.53a
−2.44
−1.99
−1.81
+0.65b,c
+0.87
−2.47
−2.30a
−1.99
−1.81
+0.57b,c
+0.87
Ru-3T
Ru-4T
𝑬𝟎𝒓𝒆𝒅 (𝟒)
′
𝑬𝟎𝒓𝒆𝒅 (𝟑)
Compound
𝟎
𝑬𝒐𝒙
(𝟐)
areduction of oligothiophene unit. bquasi-reversible. coxidation of oligothiophene unit.
Excited state redox potentials
The excited state redox potentials of Ru(II) polypyridyl complexes have been approximated from the
ground state oxidation and reduction potentials and E00, the energy difference between the thermally
equilibrated excited state and the ground state zeroth vibrational level.143 These earlier studies use the
77 K emission to estimate E00. In the present series, the capacities of the oligothiophene units of Ru-3T
and Ru-4T to also be oxidized and reduced in the ground state prompted us to estimate the redox power
of the 3ILCT state in addition to the 3MLCT state (Table 6). Since the long-lived 3ILCT state is nonemissive in this series, its computed energy from Table S5 was used. The E00 of the 3MLCT is taken from
the most intense emission peak energy as measured in a 4:1 ethanol:methanol glass at 77 K. The
potentials for oxidation (Equation 1) and reduction (Equation 2) of the excited states were estimated from
simple thermodynamic considerations, where 3PS* denotes the 3MLCT or longer-lived 3ILCT state.129,143
34
𝐸(3 PS ∗ ← PS + + 𝑒 − ) = 𝐸(PS ← PS + + 𝑒 − ) − 𝐸00
Equation 1
𝐸(3 PS ∗ + 𝑒 − → PS − ) = 𝐸(PS + 𝑒 − → PS − ) + 𝐸00
Equation 2
The ground state redox potentials related to the 3MLCT state correspond to the Ru3+/2+ oxidation and the
first phen0/- reduction voltametric waves and are similar throughout the series. This is consistent with an
3
MLCT excited state that involves ligands proximal to the Ru(II) center and thus largely unaffected by the
presence and number of thiophenes. For all compounds, the Ru3+/2+ oxidation potentials in the excited
state were near ‒1.29 V and the first phen0/- reduction potentials in the excited state were around 0.35 V.
Since the 3ILCT state involves the nT unit,23,63 the waves corresponding to oligothiophene oxidation and
reduction were used to estimate E*ox and E*red, respectively. E*red was also estimated using the IP
reduction since T1 has a small amount of 3LLCT character. These values were estimated for Ru-3T and
Ru-4T, the only two compounds in the series that showed both oxidations and reductions involving the
IP-nT ligand and for which T1 was predominantly 3ILCT.
Although the nT unit is more easily oxidized compared to the Ru(II) center in the ground state (Figure
10), the Ru(II) center is the better reducing species in the 3MLCT excited state (‒1.29 V versus ‒0.92 for
Ru-3T and ‒0.87 for Ru-4T). The reason for this difference is due to the higher energy stored in the
3
MLCT state compared to the 3ILCT states (2.16 eV versus 1.57 for Ru-3T and 1.44 for Ru-4T) that
offsets the differences in reducing power of the ground states. The phen ligand is the most easily reduced
ligand in both the ground and the excited states, resulting in the 3MLCT state being much more oxidizing
than the 3ILCT state (0.35 V versus ‒0.96 for Ru-3T or ‒0.86 for Ru-4T). Nevertheless, any excited state
redox processes contributing to photocytotoxicity could involve the 3ILCT state given that it may be
formed in much higher yield and with a lifetime that is 30 to 40× longer.
Table 6: Excited state redox potentials for the 3MLCT and 3ILCT states of Ru-3T and Ru-4T, vs. ferrocene in DMF.
E*red for the 3ILCT state was estimated two ways: using E0′red involving nT or IP. The latter number is in parentheses.
3MLCT
3ILCT
E00
E*ox
E*red
E00
E*ox
E*red
(eV)
(V vs. Cp2Fe)
(V vs. Cp2Fe)
(eV)
(V vs. Cp2Fe)
(V vs. Cp2Fe)
Ru-3T
2.16
‒1.29
0.35
1.57
‒0.92
‒0.96 (‒0.87)
Ru-4T
2.16
‒1.29
0.35
1.44
‒0.87
‒0.86 (‒1.03)
Complex
35
Photobiological activity
Figure 11: Summary of in vitro cytotoxicity and photocytotoxicity reported as log (EC50 ± SEM) values (a) and PI
values (b) obtained from dose−response curves in the SK-MEL-28 melanoma cell line with [Ru(phen)3](Cl)2 and
Ru-0T−Ru-4T. Treatments included dark (0 J cm−2; black circles) and 100 J cm−2 doses of 633 nm (red triangles),
523 nm (green inverted triangles), and visible light (400−700 nm, blue squares). The irradiance was
approximately 20 mW cm-1. Hypoxic (1% O2) results are shown with open symbols, and normoxic (∼18.5% O2)
data are shown with closed symbols.
The complexes in this series were evaluated for their dark and light-triggered cytotoxicities against human
skin melanoma cells (SK-MEL-28) cultured as 2D monolayers under normoxic (~18.5% O2) and hypoxic
(~1% O2) conditions (Figure 11). Details can be found in our previously published procedures63 and also
in the SI. Stock solutions of [Ru(phen)3](Cl)2 and Ru-0T−Ru-3T were prepared at 5 mM in water
containing 10% DMSO with solubilization first in DMSO followed by addition of water. Ru-4T was
prepared in 100% DMSO.
Normoxia.
Briefly, melanoma cells growing in log phase were seeded into two sets of 384-well plates: one set for
cytotoxicity (dark plates) and one set for photocytotoxicity (light plates) evaluation. Cells were allowed to
adhere to the wells at 37 °C over a period of 3−5 hours and then treated with varying concentrations of
PS (1 nM to 300 µM for all compounds, 1 aM to 300 µM for Ru-4T) serially diluted in DPBS. Following a
13−20 h drug-to-light-interval (DLI), the light plates were irradiated while the dark plates were kept in the
incubator. The light treatment used LEDs emitting broadband visible (400−700 nm, 21 mW cm-2) or
narrower green (523 nm, 18 mW cm-2) or red (633 nm, 18 mW cm-2) light with a fluence of 100 J cm-2.
The spectral outputs of the light sources are given in Figure S33. Both dark and light-treated plates were
then incubated at 37 °C for an additional 24 h before assessing cell viability with a resazurin-based assay.
The effective concentrations to reduce cell viability by 50% (EC50 values) were calculated from sigmoidal
36
fits of the dose-response curves for the dark and light-treated conditions based on three technical
replicates. The phototherapeutic indices (PIs), representing light-triggered amplification of cytotoxic
effects, were tabulated as ratios of the dark to light EC50 values.
The complexes of this series were relatively nontoxic to SK-MEL-28 cells in the absence of a light trigger
(Figure 11a, Table S6). Only Ru-3T and Ru-4T had dark EC50 values <100 µM, which were still
considered nontoxic (66.4 and 84.0 µM, respectively). [Ru(phen)3](Cl)2 and Ru-0T had dark EC50 values
that were beyond the highest concentration tested in the assay and were tabulated as >300 µM. As a
consequence, their PI values are undefined but reported as a lower limit using 300 µM as the dark
cytotoxicity.
Broad-band visible light produced photocytotoxicity from all compounds in the series (Figure 11, Table
S6). Systematic π-expansion from phen to IP-nT (n=1−4 thienyl groups) resulted in progressively higher
potency using visible light, spanning four orders of magnitude. The visible light EC50 values in normoxic
conditions ranged from 22 µM (PI>10) for the least active reference compound [Ru(phen)3](Cl)2 to as low
as 740 pM (PI>105) for the most active compound Ru-4T. Replacing a phen ligand with IP (Ru-0T)
increased the photocytotoxicity 3-fold (EC50=6.8 µM, PI>40). Appending one (Ru-1T) or two (Ru-2T)
thienyl groups to IP improved the potency another 10-fold, shifting the EC50 values into sub-micromolar
Figure 12. Dose−response (±SD) of Ru-4T in (a) normoxic Ң18.5% O2 or (b) hypoxic 1% O2-treated SK-MEL-28
melanoma cells. Treatments included dark (0 J cm−2; black circles) and 100 J cm−2 doses of 633 nm (red
triangles), 523 nm (green inverted triangles), and visible (400−700 nm, blue squares) light.
regime near 0.6 µM with PIs on order of 200. Another 10-fold enhancement in photocytotoxicity was
37
accomplished on going to three thiophene rings (Ru-3T; EC50=0.057 µM, PI~ 103), but the greatest
change occurred with four thiophenes (Ru-4T; EC50=740 pM, PI>105).
All compounds were inactive with red light, with the exception that Ru-3T (EC50=16.3 µM; PI=5) and Ru4T (EC50=16.3 µM; PI=5) exhibited marginal responses. This is in line with what would be expected for
compounds having little absorption of red light.69 [Ru(phen)3](Cl)2 and Ru-0T were also inactive with
green light. Therefore, the observed effects with broadband visible light were likely generated
preferentially by the shorter wavelengths, which is further supported by the attenuated EC50 values and
corresponding PIs for the thienyl-containing complexes with green light. The only exception was Ru-2T
which maintained its EC50 value near 0.5 µM and PI of ~200 with green light. The PIs for both Ru-1T and
Ru-3T were reduced ten-fold (PI=23 and 270, respectively), while that for Ru-4T was reduced by several
orders of magnitude (PI=~103). The photocytotoxic responses elicited by Ru-4T toward SK-MEL-28 cells
under both oxygen conditions with the different light parameters are compared in Figure 12.
Hypoxia.
The hypoxia assays were carried out as described for normoxia except that the dark and light plates with
adhered cells were moved to a hypoxia chamber (1% O2) for 2–3 h before compound addition. At the end
of the DLI in hypoxia, dissolved oxygen was measured using an immersive optical probe to confirm
hypoxic condition before sealing the light plates with highly transparent, low gas permeable qPCR film.
The light plates were illuminated outside of the hypoxia chamber alongside the normoxic plates. The films
were then removed, and all plates were incubated under normoxia (37°C, 5% CO2, ≥90% RH) for 20–23
h before cell viability determination.
As observed for the normoxic condition, the compounds were nontoxic to cells in the dark and with red
light under hypoxia (Figure 11, Table S6). [Ru(phen)3](Cl)2 and Ru-0T−Ru-3T lost all of their
photocytotoxicity with visible and green light in hypoxia, while Ru-4T gave modest activity with a visible
EC50 values of approximately 1–2 µM and PIs on the order of 40–60. This marked reduction in activity for
Ru-4T and inactivity for the rest of the series suggests that the largest contributor to the normoxic
photocytotoxicity for this family likely involves oxygen-dependent photophysical pathways.
Biological replicates.
The data shown in Figure 11 and Table S6 represent our initial results and are the average of technical
replicates performed in triplicate on cells of the same batch and identical passage number and have low
standard deviation as a result. Biological replicates will have more variation, and thus we validated our
38
results for Ru-3T and Ru-4T over seven biological replicates run in triplicate (Figure 13 and Table S7–
Table S10). Repeat 0 is the data from Figure 11 and Table S6 that was discussed above. Repeats 1–6
represent biological replicates with variations as described previously.63
Both Ru-3T and Ru-4T were completely nontoxic over all biological replicates, with mean EC50 values
just under 100 µM in both normoxia and hypoxia and standard error of the mean (SEM) being within ±25
µM for Ru-3T and ±10 µM Ru-4T. The visible EC50 values for Ru-3T in normoxia ranged from about 60
to 80 nM with a mean of 64 nM; the corresponding visible PIs ranged from 1200 to 2500 with a mean of
1600. The EC50 value for Ru-4T under the same conditions exhibited a much larger variance, ranging
from 40 fM to about 8.6 nM with a mean of 2.2 nM. Nevertheless, five of the seven replicates were within
an order of magnitude of each other. Three were between 0.64 and 0.80 nM and two were around 4.8 to
8.6 nM, giving rise to PIs on the order of 104 to 105. Only two of the seven biological replicates for Ru-4T
were well outside of this range at 40 and 320 fM with unusually large PIs of 108–109. Of note, only the
most potent IP-4T complexes under the most potent light condition (visible) in the normoxic condition
produce more than several orders of magnitude variation in photocytotoxicity between biological
replicates, with EC50 values extending into the ubertoxin range (fM and lower) with visible light.63 In the
case of the related analog ML19C01, [Ru(2,9-dmp)2(IP-4T)](Cl)2, four of six biological replicates fell in
this range and produced PIs as large as 1012. To date, this behavior has only been observed for certain
IP-4T complexes of Ru(II) and (to a lesser degree) of Os(II). Herein, Ru-4T adds one more example that
may help us better understand this phenomenon in the future.
39
Figure 13. Interassay performance (± log(SEM)) of Ru-3T (top, a + b) and Ru-4T (bottom, c + d) in normoxic
(filled symbols, solid lines, ~18.5% O2) and hypoxic (open symbols, dashed lines, 1% O2) SK-MEL-28 melanoma
cells. Treatments included dark (no light; black circles) and 100 J cm-2 treatments at ~20 mW cm-2 of visible (400700 nm) light (blue square), 523 nm (green inverted triangle) and 633 nm (red triangle). SEM = standard error of
the mean.
With green light in normoxia, there was little variance in the activity of Ru-3T. The green EC50 values
varied from 0.16 to 0.64 µM and PIs from 140 to 570, with the mean being 0.30 µM and 370. For Ru-4T,
five of seven replicates gave green EC50 values between 30 and 80 nM (PIs ranged from 1100 to 3300).
The remaining two were between 0.15 and 0.26 µM (PIs ranged from 360 to 720). On average Ru-4T
was more active than Ru-3T but by only about five-fold.
With red light normoxia, Ru-3T was inactive in four of seven replicates and only moderately active in the
remaining three with red EC50 values around 3 to 4 µM and PIs around 30 to 40. Ru-4T was moderately
better, with red EC50 values of 1 to 2 µM (PIs 64 to 77) in three of the seven replicates but 10 to 50 µM
(PIs 2 to 11) in the remainder.
Despite losing all activity in hypoxia in the initial evaluation, Ru-3T was phototoxic with visible and green
light in five of the seven biological replicates under hypoxia. Three of the replicates gave EC50 values
near 1 µM and two were between 0.22 and 0.45 µM. The resulting PIs ranged from about 70 to 500.
40
Overall, the activity of Ru-3T was reduced by five to fifteen-fold in hypoxia with visible light but only about
two-fold for green light (because there was a larger difference in the visible and green EC50 values in
normoxia). There was no significant activity for Ru-3T with red light over seven biological replicates.
Ru-4T was also generally much more active with visible light in hypoxia than the initial evaluation showing
single-digit µM photocytotoxicity. Three of the replicates produced EC50 values between 32 and 70 nM,
while two were near 0.25–0.30 µM. The corresponding PIs were between about 290 and 3000. EC50
values in the two remaining replicates were 2.1 to 6.4 µM (PIs 15 and 42). The large variance in
photocytotoxicity for Ru-4T in normoxia was reduced to only a few orders of magnitude in hypoxia,
making the attenuation in activity on going from normoxia to hypoxia much more pronounced for Ru-4T
compared to Ru-3T. Nevertheless, Ru-4T was slightly more potent on average than Ru-3T in hypoxia.
With green light, the difference between EC50 values in normoxia and hypoxia for Ru-4T was much less.
In five of the seven hypoxic replicates, the green EC50 values were between 0.20 and 0.81 µM (PIs 110
to 480) compared to the mean EC50 value of about 0.10 µM in normoxia (mean PI 1600). With red light,
Ru-4T was inactive in four of seven replicates and only marginally active in the rest with EC50 values
between 4 and 20 µM (PIs between 4 and 20). Again, Ru-4T was only slightly more potent on average
than Ru-3T.
To summarize, Ru-4T is superior to Ru-3T over the seven biological replicates when activated with visible
light in normoxia. The light EC50 values and corresponding PIs for both compounds are attenuated on
going from visible to green to red light, suggesting that the observed effects with broadband visible light
are generated primarily by the shorter, bluer wavelengths. The light EC50 values and PIs are also
attenuated on going from normoxia to 1% hypoxia, with the greatest differences observed with visible
light as the most potent condition. For both compounds, the visible light-triggered activity in hypoxia was
similar to that with green light in normoxia, and the differences between green light activity in normoxia
and hypoxia were relatively small. The compounds were relatively inactive with red light, although Ru-4T
did show modest activity in normoxia presumably due to some low probability of directly populating the
lowest-lying but spin-forbidden triplet state. Of note, Ru-4T marks another example that follows our
recently published ML19C01, with evidence of phototoxic effects at concentrations on the order of fM in
several of the biological replicates.
41
2.5 CONCLUSIONS
The complexes of this family were designed to vary the number of thienyl groups nT attached to the IP
ligand in a family of Ru(II) polypyridyl complexes based on 1,10-phenanthroline as the coligand. The
motivation is part of a larger initiative to correlate structural variations with photobiological activities
across different coordination complex families where we are considering: metal ion, coligands, thienylappended ligands, thienyl groups and number of thiophenes, counter ions, ionizable groups and
protonation states, and coordination number and geometry. Within the phen family of IP-nT complexes,
the extension of the thiophene chain systematically increased the lipophilicity and shifted the
(oligo)thienyl-localized ππ* transitions to lower energy. The electrochemical properties of the complexes
were similar and reminiscent of Ru(II) polypyridyl complexes in general with regard to metal oxidation
and ligand reduction. However, complexes with at least one thiophene or more exhibited an additional
oxidation, involving the thienyl group(s), that occurred more readily than metal oxidation and with
increasing n. Ru-3T and Ru-4T could also be reduced on the thienyl chain, which was the 4th reduction
for Ru-3T but 3rd for Ru-4T.
The MLCT states for the complexes were similar in energy, with 3MLCT emission in agreement with
typical Ru(II) polypyridyl complexes but quantum yields dropping by one to two orders of magnitude for
Ru-3T and Ru-4T, respectively. The reduced phosphorescence was accompanied by an increase in the
1
O2 quantum yields and access to 3ILCT states with prolonged lifetimes. The 3ILCT state was the lowest-
lying triplet for Ru-2T to Ru-4T and decoupled from the 3MLCT states. T1 was computed to be of mixed
3
ILCT/3LLCT character for Ru-2T, whereas T1 was predominantly 3ILCT (>50%) for Ru-3T and Ru-4T.
The % contribution of 3LLCT to T1 decreased with increasing n, with 65% 3ILCT and <20% 3LLCT
character for Ru-4T. The triplet lifetime of Ru-2T was the longest at 148 μs and decreased with additional
thiophenes as would be expected for a radiationless process governed by the energy gap law. The 1O2
quantum yields were highest for Ru-3T and Ru-4T at about 88%.
The high ROS production for the complexes with extended thiophene chains resulted in potent
phototoxicity in vitro. With visible light activation, Ru-3T consistently yielded EC50 values between 10 and
100 nM and PIs greater than 103. Despite having a slightly lower 1O2 quantum yield and shorter 3ILCT
lifetime, Ru-4T was considerably more potent under the same conditions. On average its light EC50 values
were sub-nanomolar with PIs in the 104 to 105 range, but the higher variability in activity led to some
measurements in the femtomolar regime and PIs as large as 109. This activity was attenuated with longer
wavelengths of light and in 1% hypoxia, but notably Ru-4T gave reliable sub-micromolar activity in
42
hypoxia with PI values as high as 3,000. The trends for the most potent compounds Ru-3T and Ru-4T
were verified over seven biological replicates performed in triplicate. The fact that Ru-3T could be
generally classified as a hypoxia-active photosensitizer underscores the importance of biological
replicates as this activity was missed in the initial assessment.
From these studies, a lowest-lying 3ILCT state appears to be key to potent phototoxicity and activity in
hypoxia. While the prolonged excited state lifetime of the nT-localized triplet is important, its precise
magnitude and 1O2 quantum yield are not sole determinants of potency since (i) Ru-2T has the longest
lifetime but is not the most phototoxic, and (ii) Ru-3T and Ru-4T have similar 1O2 yields but Ru-4T is
superior (Figure S34). Alternate pathways could involve other ROS and oxygen-independent electron
transfer processes. Although the 3MLCT states were estimated to be more highly oxidizing and reducing
compared to the lowest-lying 3ILCT states, any excited state redox processes contributing to phototoxicity
could involve the 3ILCT state given that it is the lowest energy triplet with a lifetime that is 30 to 40× longer
and the nT group is redox active. In addition, higher-lying and conformationally distinct 3ILCT states
cannot be excluded and have been implicated in the picosecond dynamics of similar families.144
Our study focused on the photophysical drivers of activity and did not consider biological factors such as
cellular uptake and localization, subcellular targets, and cell death pathways that may affect cytotoxicity
and potentiate phototoxicity. Future studies are aimed at reconciling both photophysical and biological
characteristics to explain the unusual potency of certain oligothienyl-based PSs and building structureactivity relationship (SAR) databases for light-responsive transition metal complexes.
2.6 ASSOCIATED CONTENT
Additional method information and characterization data may be found in the Supplementary Information.
This material is available free of charge via the Internet at https://pubs.acs.org.
Author Information
Corresponding authors
Colin G. Cameron – Department of Chemistry and Biochemistry, The University of Texas at Arlington,
Arlington, Texas 76019-0065, United States; orcid.org/0000-0003-0978-0894; Email:
colin.cameron@uta.edu
43
Sherri A. McFarland – Department of Chemistry and Biochemistry, The University of Texas at Arlington,
Arlington, Texas 76019-0065, United States; orcid.org/0000-0002-8028-5055; Email:
sherri.mcfarland@uta.edu
Authors
Houston D. Cole − Department of Chemistry and Biochemistry, The University of Texas at Arlington,
Arlington, Texas 76019-0065, United States
Abbas Vali − Department of Chemistry and Biochemistry, The University of Texas at Arlington,
Arlington, Texas 76019-0065, United States
John A. Roque III − Department of Chemistry and Biochemistry, The University of Texas at Arlington,
Arlington, Texas 76019-0065, United States; Department of Chemistry and Biochemistry, The
University of North Carolina at Greensboro, Greensboro, North Carolina 27402, United States; Present
Address: University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599
Ge Shi − Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington,
Texas 76019-0065, United States
Gurleen Kaur − Department of Chemistry and Biochemistry, The University of Texas at Arlington,
Arlington, Texas 76019-0065, United States
Rachel O. Hodges − Department of Chemistry and Biochemistry, The University of North Carolina at
Greensboro, Greensboro, North Carolina 27402, United States; Present Address: Wake Forest
University School of Medicine, 307 Winston-Salem, NC 27109
Antonio Francés-Monerris – Institut de Ciència Molecular, Universitat de València, 46071 València,
Spain
Marta E. Alberto – Dipartimento di Chimica e Tecnologie Chimiche, Università della Calabria,
Arcavacata di Rende, 87036 Italy
Notes
S.A.M. has a potential research conflict of interest due to a financial interest with Theralase Technologies,
Inc. and PhotoDynamic, Inc. A management plan has been created to preserve objectivity in research in
accordance with UTA policy.
44
Acknowledgements
The authors would like to thank the National Cancer Institute (NCI) of the National Institutes of Health
(NIH) (Award R01CA222227) as well as the National Science Foundation (NSF) (Award 2102459) for
support. A.F.M. thanks the grant PID2021-127554NA-I00 funded by the Spanish Ministry of Science
and Innovation (MCIN/AEI/10.13039/501100011033) and by “ERDF A way of making Europe”.
M.E.A. acknowledges the CINECA award under the ISCRA initiative (HyPS4DAT project) for the
availability of high-performance computing resources. The content in this work is solely the responsibility
of the authors and does not necessarily represent the official views of the National Institutes of Health or
National Science Foundation. The authors also thank Dr. Daniel Todd as UNCG’s Triad Mass
Spectrometry Facility manager and his assistants Jennifer Simpson and Diane Wallace. S.A.M. likewise
thanks Dr. Franklin Moy (UNCG) and Dr. Brian Edwards (UTA) for their experimental support and
instrument maintenance as NMR facility managers. The authors also thank Susan Monro, Colin Spencer,
Ryan DeCoste, Huimin Yin, and Lance Chamberlain for additional synthesis and characterization support
for these complexes that was not reported here.
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2.8 SUPPORTING INFORMATION
Description of Methodology
Lipophilicity
The lipophilicities of the complexes were assessed as log distribution coefficients (log Do/w) determined
by the “shake-flask” method using three technical replicates. A saturated solution of 1-octanol with
phosphate buffer (pH 7.4) was prepared by mixing 16 mL of 1-octanol (99.9%) with 4 mL of 10 mM
phosphate buffer (4:1). A saturated solution of phosphate buffer with 1-octanol was prepared by mixing
16 mL of 10 mM phosphate buffer with 4 mL of 1-octanol. The saturated solutions were shaken for 24
hours at room temperature using a vortex mixer at 300 rpm. For determining log Do/w, [Ru(phen)3](Cl)2,
Ru-0T, Ru-1T, and Ru-2T were dissolved in 500 μL of the saturated phosphate buffer and diluted with
an equal volume of saturated 1-octanol to achieve a final volume of 1 mL and concentration of 25 µM;
Ru-3T and Ru-4T were dissolved in 500 μL of the saturated 1-octanol and diluted with an equal volume
of saturated phosphate buffer to achieve a final volume of 1 mL and concentration of 25 µM. The solutions
were inverted 200 times by hand and allowed to stand at ambient temperature for 24 h to give time for
the complex to disburse between the two layers. The 1-octanol and phosphate buffer layers were then
carefully separated using a syringe and transferred to a 96-well microtiter plate for absorbance to be read
at wavelengths corresponding to the absorption maxima of the complexes using a SpectraMax M2e
microplate reader. The concentration of metal complex in each layer was determined from standard
curves prepared with the saturated solution used for the initial dilution and then used to calculated log
Do/w as the ratio of the concentrations of compound detected in the 1-octanol layer to phosphate buffer.
The averages of the three technical replicates are reported.
Spectroscopy
Unless described otherwise, spectroscopy was performed on dilute (5−20 μM) solutions of the PF6− salts
of the complexes in spectroscopy grade acetonitrile that had distilled over calcium hydride and stored
under N2. Solutions were deoxygenated by five freeze-pump-thaw cycles in custom Schlenk-style
cuvettes. Emission spectra were internally corrected for nonlinear lamp output and detector response.
UV−Visible spectroscopy
Ultraviolet−visible (UV-vis) absorption spectra were collected with a Jasco V730 spectrometer in 5 mm
quartz cuvettes. Extinction coefficients were determined by regressing absorption versus concentration
for five dilutions at room temperature.
62
Emission Spectroscopy at Room Temperature
Steady-state emission spectra were acquired on deoxygenated solutions by sparging 30 min with argon
using a PTI Quantamaster spectrofluorometer equipped with a K170B PMT (maximum useable
wavelength ≈ 800 nm). Wavelength-dependent nonlinearities in lamp output and detector sensitivities
were corrected by the software. Generally, the most intense and longest-wavelength peak in the
excitation spectrum was chosen for λex. Appropriate optical filters were installed to reduce artifacts from
scatter and harmonics. Solutions were deoxygenated by argon-sparging in septum-capped 10 mm quartz
cuvettes.
Emission Spectroscopy at 77 K
The complexes were immobilized as glasses of 10 μM solutions of the PF6− salts in 4:1 ethanol:methanol,
contained in 5 mm NMR tubes, and frozen in liquid nitrogen. The solvent was not deaerated. Otherwise,
spectra were acquired as described above in section 2.2.3.
Singlet Oxygen Quantum Yield
The quantum yields for singlet oxygen production (ΦΔ) were measured from dilute (≈5 μM) solutions in
spectroscopic grade acetonitrile (not deaerated) at room temperature, and calculated from the intensity
of the 1O2 emission centered near 1276 nm using the relative actinometric method depicted in Equation
S1, where I is the integrated intensity of the emission, A is the absorbance of the solution at the excitation
wavelength, and η is the refractive index of the solvent. The subscript S denotes the standard solution,
in this case [Ru(bpy)3](PF6)2, for which ΦΔ,s=0.56,1 and (η2/ηS2)=1 since MeCN was used for both. These
experiments used the PF6− salts in air-saturated MeCN because water and other protic solvents quench
the 1O2 emission.
The emission was measured on a PTI Quantamaster spectrofluorometer with a Hamamatsu R5509-42
PMT that was cooled to −80 °C. The emission spectrum was measured over 1200–1350 nm with a 1000
nm long pass filter in place and integrated with baseline correction. The excitation wavelength was
chosen as the wavelength in the MLCT absorption region that produced the most intense signal in the
excitation spectrum.
𝐼 𝐴𝑠 𝜂 2
𝛷∆ = 𝛷∆,𝑠 ( ) ( ) ( 2 )
𝐼𝑠 𝐴 𝜂𝑠
Equation S1
63
Transient Absorption Spectroscopy
Dilute (≈5 μM) solutions of the complexes in spectroscopy grade acetonitrile were degassed by five
freeze-pump-thaw cycles in custom Schlenk-style cuvettes. Transient absorption (TA) lifetimes and
differential excited-state absorption (ESA) spectra were measured with an Edinburgh Instruments LP980 spectrometer equipped with the PMT-LP detector. 355 nm excitation pulses (0.1 Hz, ∼5 ns pulse
width, ∼7−9 mJ per pulse) were generated by the third harmonic of a Continuum Minilite Nd:YAG laser.
The ESA spectra were measured at 10 nm steps and corrected for luminescence, and single-wavelength
TA lifetime measurements were optimized for detector response. This system was also used to measure
the phosphorescence lifetimes (without a probe beam).
Triplet Energy Determination by Stern-Volmer Quenching
The energy of the 3ILCT states was determined by Stern-Volmer quenching of the excited state lifetime
as observed at the TA maximum of the PS signal in the near infrared. The transient lifetime of a dilute
(≈5 μM) solution of PS in CH2Cl2 was measured at varying (0 – ≈100 μM) concentrations of π-expansive
quenchers with known triplet energies,2 in accordance with Equation S2 (where τ and τ0 are the TA
lifetimes in presence and absence of quencher Q, respectively, and kq is the quenching constant). The
532 nm second harmonic of a Continuum Minilite Nd:YAG laser (0.1 Hz, ∼5 ns pulse width, ∼7−9 mJ per
pulse) was used to excite the edge of the PS 1MLCT band, while avoiding direct excitation of the
quencher.
1 1
= + 𝑘𝑞 [Q]
𝜏 𝜏0
Equation S2
Ru(II) compound solutions
For Ru-4T, stock solutions were prepared 25 mM in 100% DMSO. For the rest of the compounds, stock
solutions were prepared at 5 mM in 10% v/v DMSO:water. Dilutions were prepared in serial with 1x
Dulbecco’s Phosphate-Buffered Saline (DPBS) without Ca2+ or Mg2+ that was diluted from 10x DPBS
(Corning 20-031-CV). Less than 1.2 % v/v DMSO was applied at the highest concentration (300 µM)
Glass vials with PTFE-lined caps were used for stock solutions. The vials were wrapped with aluminum
foil to protect from light and stored at −20°C when not in use. When not in use, all stock solutions were
stored at −20°C wrapped in foil.
64
Cell culture
Non-pigmented male human melanoma (SK-MEL-28, ATCC HTB-72) cells were cultured and maintained
in EMEM (BioWhittaker, 12-125Q) media, which was further enriched with 10% FB essence (VWR,
10803-034) and 1% glutagro (L-alanyl-L-glutamine; VWR 45001-086). Cells were kept at 37°C under ≥
90% humidity and 5% USP-grade CO2 in a water-jacketed incubator (ThermoFisher, Thermo Scientific
4110). Split ratios between 1:2–1:5 were used to achieve 150,000–400,000 cells mL−2 at the start of each
passage. Cells were used within 15 passages from being purchased. Unless otherwise stated, a seeding
density of 3000 cells well-1 was used in each (photo)cytotoxicity screen in 384-well plates.
Cellular assays
The photobiological efficacy of each complex was evaluated using dose-response cell viability assays on
384-well plates in concentrations that range from 1×10−3 to 300 µM for all and from 1×10−12 to 300 µM for
Ru-4T due to its greater phototoxicity. Under all circumstances, well plates were only stacked 2-plates
high in the incubator to facilitate rapid heat exchange. To evaluate inter-assay reproducibility, Ru-3T and
Ru-4T were evaluated in several additional assays (Table S7–S10).
Following our recent examples,3,4 the compounds were screened for (photo)cytotoxicity via a doseresponse resazurin assay in a 384-well plate format. Into 384-well plates (Greiner Bio-One, 781182),
DPBS was added into the outmost two wells (144 well count) at 75 µL well−1 as a perimeter in the
biosafety cabinet. An electronic multichannel pipettor was used for the experimental set-up. There was
40 µL well−1 in total for all the inner wells including sample wells, positive and negative control wells.
Sample wells were composed of 10 µL well−1 complete media, 20 µL well−1 cell slurry (3000 well−1
SKMEL28 cells) and 10 µL well−1 compound dilutions in DPBS. Positive control wells (12 count) were
composed of 10 µL well−1 complete media, 20 µL well−1 cell slurry (3000 cells well−1) and 10 µL well−1
DPBS. Negative control wells (12 count) consisted of 30 µL well−1 complete media and 10 µL well−1
DPBS. Before cells were seeded in log-phase growth with >90% viability, the well plates were
preincubated (37°C, 5% CO2, ≥90% RH) with dispensed media. After cell seeding, plates were mixed
(up, down, left, right tilting) and then placed in the incubator and incubated 2–3 h to facilitate cell
attachment.
During the incubation, serial dilutions were prepared across 9 concentrations from 1×10−3 to 300 µM for
all compounds and additional 9 concentrations from 1×10−12 to 1×10−3 µM for Ru-4T in sterile 384-well
plates using DPBS as solvent. The lights in biosafety cabinet were kept off to minimize exposure to light
preventing premature activation in cells. The 384-well plates were incubated with lids for 2–3 h before
65
the compound dilutions were dispensed at 10 µL well−1. Replicates (triplicates) were dispensed row-wise
and spaced every 4 rows.
The 384-well plates were incubated overnight (13–20 h drug-to-light interval, DLI), followed by light
treatments. Light treatment was approximately 100 J cm−2 delivered at 18–24 mW cm−2 with cool white
Visible (400–700 nm), Blue (Prizmatix LED
453 nm), Green (Prizmatix LED, 523 nm), and Red (Prizmatix
LED, 633 nm). After illumination, plates were further incubated for 1 day before final viability
measurements. Edge effects were noted on the 384-well plate; therefore, the post-PDT period was set
for one day (20–23 h) to equilibrate cell viability instead of 48 h. Prewarmed 0.3 mM sterifiltered resazurin
in 0.2 M phosphate buffer (pH = 7.4) was dispensed across all well plates at 10 µL well−1. Resazurin dyed
plates were incubated for 4 h before reading fluorometrically on a Molecular Devices M2e (30 s shake,
bottom-read, λexc 530 nm, long-pass 570 nm, λem 620 nm).
Hypoxia protocol
The compounds were screened in parallel under normoxia (~18.5% O2) and hypoxia (1% O2). For
hypoxic-treated plates, the cells were incubated 1–2 h under normoxia (37°C, 5% CO2, ≥90% RH) to
allow time for cells to adhere. Then the plates were incubated at 1% O 2 (37°C, 5% CO2, ≥90% RH) in a
Biospherix Xvivo X3 chamber for 2–3 h before compound dilutions were dispensed in the biosafety
cabinet. Plates were then incubated in Biospherix chamber for 17–19 h (DLI). Before light treatment
(while still inside the Biospherix chamber), the dissolved oxygen concentration was measured in one
randomly chosen well in each plate using an immersive optical probe (Ohio Lumex, PyroScience
OXSOLV). The optical probe was calibrated using deaerated, deionized water before each experiment.
For measurements, the probe was carefully positioned in the center of the chosen well at a depth of
approximately 3 mm. We found that 7–9 µM O2 was optimal under a 1% O2 atmosphere, where the
dissolved O2 decreases with more depth (in normoxia-treated cells, the dissolved O2 concentration is
approximately 100 µM O2). The hypoxia-treated plates to be light-treated were then sealed inside the
Biospherix chamber using low-gas permeable and high transparency qPCR films (VWR, 89134-428) to
maintain hypoxic conditions during light treatment, which is the most critical point for testing oxygendependence of a PS. After light treatment, the films were removed in the biosafety cabinet and all hypoxictreated plates were moved to the normoxic incubator (37°C, 5% CO2, ≥90% RH). Alongside the normoxictreated plates, the hypoxic-treated plates were incubated for 20–23 h before cell viability determination.
66
Biological replicates
To verify reproducibility across assays for the more active Ru-3T and Ru-4T complexes, biological
replicates were performed as longitudinal studies following our previously reported examples.3,4
Biological replicates were assigned randomized and unique plate maps, and different pipet tips were
used for repeat #1 (Sartorius 790352), #2–3 (VWR 83007-352), and #4–5 (low retention Sartorius LHL790352). To minimize any possible contribution from stray light, the overhead lights in the lab space
were turned off for repeat #5. All cells used in these assays were between 10–15 passages, and all
assays were performed within one month of each other.
Light devices and protocols
Unless otherwise noted, each biological assay used a fluence of 100 J cm-2 and an irradiance of 18-22
mW cm-2. Three different light sources were used for visible, green, and red light treatments: a cool white
LED panel (SOLLA-CREE, 400–700 nm, maxima ~450 nm) and two UHP-LEDs (Prizmatix, 523 and 633
nm). Their spectral outputs are shown in Figure S33.
Data analysis and statistics
Data from the resazurin cell viability assay were corrected for background by subtracting the signal from
wells that contained only media and DPBS (no cells) and normalized relative to untreated cells. Because
the absorbance and emission of the metal complexes can interfere with the resazurin fluorescence signal,
wells treated with the highest concentrations of metal complex were also observed under a microscope.
If no cells were detected, these wells were assigned a value of zero. A more detailed discussion of assay
limitations for this class of complexes is provided in our 2019 review.5
Data points obtained from resazurin fluorescence were fit to a three-parameter log-logistic (Equation S3)
and logistic model (Equation S4) using GraphPad Prism 8.4.0. We use Equation S3 for summary
log(EC50) plots (Figure 12a) and in the dose-response curves shown in Figure 13, but we use Equation
S4 for data in log(PI) plots (Figure 12b) as well as the tabulated EC50 and PI values (Table S6–S10).
𝑌 = Bottom +
(𝑇𝑜𝑝 − 𝐵𝑜𝑡𝑡𝑜𝑚)
Equation S3
(1 + (10𝐿𝑜𝑔(𝐸𝐶50 −𝑋)×𝐻𝑖𝑙𝑙𝑠𝑙𝑜𝑝𝑒 )
𝑌 = Bottom +
(𝑇𝑜𝑝 − 𝐵𝑜𝑡𝑡𝑜𝑚)
𝐻𝑖𝑙𝑙𝑠𝑙𝑜𝑝𝑒
(1 + (𝐸𝐶50 ⁄𝑋)
)
Equation S4
67
Experiments were completed in triplicate and replicated data points are always plotted with error bars
denoting the standard deviation (SD). All EC50 values are reported alongside the standard error of the
mean (SEM). In cases where the hill slope was too steep to calculate a representative SEM, the SEM
was labelled as not determined (n.d.). Phototherapeutic indices (PI) are reported as the ratio of dark to
light EC50 values and serve as a phototherapeutic efficacy benchmark. Any summary plots showing Log
EC50 and Log PI values of the entire series of complexes are plotted with SEMs from log-logistic fits.
Synthetic Characterization
Description of NMR Assignments. The proton signals of tris-homoleptic compound [Ru(phen)3](Cl)2 are
characterized in detail by Pazderski et al.,6 and were used to establish the location of the phenanthroline
proton signals in compounds Ru-0T‒Ru-4T. In compounds Ru-0T‒Ru-4T, protons 2 and 9 are
electromagnetically distinct from one another. It is expected that the protons 2 would be more deshielded
than protons 9 due to their relative proximity to the imidazole-phenanthroline (IP) π-system. In
compounds Ru-0T‒Ru-4T, the furthest downfield signal is assigned as c due to its proximity to the
imidazole’s nitrogens. This signal often appears as a broad singlet due to prototropic tautomerization of
the neighboring imidazole. In compounds Ru-0T‒Ru-4T, protons 3, 8, and b are similar in their offsets
and splitting characteristics and were assigned using 1H-1H COSY correlations with their neighboring
protons (2/4, 7/9, a/c, respectively). In the case of compounds Ru-3T and Ru-4T, protons 4 and 7 are
distinguishable from one another, which allows for protons 3 and 8 to also be identified more accurately
through 1H-1H COSY correlations. In compound Ru-1T, the thiophene proton d is relatively deshielded
due to through-space induction with the imidazole’s nitrogens, and because of proton e’s distinct splitting
proton f’s location is determined by process of elimination and observed 1H-1H COSY correlations. In
compounds Ru-1T‒Ru-4T, the proton signals corresponding to the thiophene closest to the imidazole
were all assigned by locating the chemically distinct d signal, then assigning the other proton(s) in the
spin system using 1H-1H COSY correlations. The terminal thiophene’s protons in compounds Ru-2T‒Ru4T may be assigned using the doublet of doublets observed in monosubstituted thienyl 1H NMR spectra.
Using that signal as a starting point, the other two proton signals may be found through 1H-1H COSY
correlations, with the proton next to sulfur appearing more downfield. After that, for Ru-3T and Ru-4T,
the only remaining signals are those which correspond to the “internal” thiophenes, which are surrounded
by other thiophenes. This is somewhat straightforward for compound Ru-3T, which only contains one
internal thiophene, but determining which proton is f and which one is g remains a challenge without 13C
-1H HSQC and 13C -1H HMBC. For compound Ru-4T, the internal thiophene signals are more difficult to
assign. Here, the two spin systems (f‒g and h‒i) were first established using 1H-1H COSY correlations.
68
Then, 13C -1H HSQC was used to identify which 13C peaks correspond to the thiophene protons d‒k.
Then, 13C -1H HMBC was used to establish neighboring proton peaks. For example, e and f both correlate
to two of the same 13C peaks (141.46 ppm and 136.24 ppm), which are assumed to be carbons 12 and
13. Proton e correlates more strongly to the peak at 141.46 ppm, and proton f correlates more strongly
to the 136.24 ppm peak, which allowed for carbon 12 and carbon 13 to also be assigned as signals at
141.46 and 136.24, respectively. Because proton f was identified, proton g was also identified by process
of elimination. Proton g and proton h both correlate to the 13C peaks at 138.30 ppm and 136.36 ppm,
which are designated as carbons 14 and 15. Again, h correlated more strongly to 15 and g correlated
more strongly to 14. In addition, i and j both correlate to 13C peaks 137.78 ppm and 138.13 ppm, with k
weakly correlating to the 137.78 peak. Using these correlations, carbons 16 and 17 were assigned to the
138.13 and 137.78 peaks, respectively. This provided a working assignment of all of compound Ru-4T’s
protons and some key carbons. The remaining quaternary carbons (10-11, 19-24) were assigned using
13
C -1H HMBC correlations and process of elimination. Carbon 10 shows a weak 3-bond correlation to
proton d and is relatively downfield at 150.01 ppm. Carbon 11 was assigned based on the strong 2-bond
HMBC correlation with proton d. Carbons 19 and 20 were assigned based on 2-bond and 3-bond
correlations shared with protons 5/6 and 3/8, respectively. There are correlations between carbons 21‒
22 with protons 2, 9, 4, 7, 5, and 6, (but not 3 or 8) which allowed for carbons 21 and 22 to be identified.
Carbon 23 was identified using a strong correlation with proton a. Because proton c is decoupled, and
no 2-bond or 3-bond correlations are observed from that proton signal, the location of the signals
corresponding to carbons 24 and 25 remains unsolved; it is assumed that they overlap with other carbon
signals. An artefact appears near 138 ppm in the 13C NMR spectrum, which is attributed to the
surrounding area’s various radio stations.
69
NMR spectra
(b)
Figure S1. (a) Aromatic 1H NMR (700MHz, MeOD-d3, 298 K) spectrum and assignments for [Ru(phen)3](Cl)2. (b)
Aromatic 1H-1H COSY NMR (700MHz, MeOD-d3, 289 K) spectrum and assignments for [Ru(phen)3](Cl)2.
70
(b)
Figure S2. (a) Aromatic 1H NMR (700MHz, MeOD-d3, 298 K) spectra and assignments for Ru-0T. (b) Aromatic
71
1H-1H COSY NMR (700MHz, MeOD-d , 298 K) assignments for Ru-0T.
3
(b)
Figure S3. (a) Aromatic 1H NMR (700MHz, MeOD-d3, 298 K) spectra and assignments for Ru-1T. (b) Aromatic
72
1H-1H COSY NMR (700MHz, MeOD-d , 298 K) assignments for Ru-1T.
3
(b)
Figure S4. (a) Aromatic 1H NMR (700MHz, MeOD-d3, 298 K) spectra and assignments for compound Ru-2T. (b)
73
Aromatic 1H-1H COSY NMR (700MHz, MeOD-d3, 298 K) assignments for compound Ru-2T.
(b)
Figure S5. (a) Aromatic 1H NMR (700MHz, MeOD-d3, 298 K) spectra and assignments for Ru-3T. (b) Aromatic
74
1H-1H COSY NMR (700MHz, MeOD-d , 298 K) assignments for Ru-3T.
3
Figure S6. Aromatic 1H NMR (700MHz, MeOD-d3, 298 K) spectra and assignments for Ru-4T.
75
Figure S7. (a) Zoom of 13C NMR (700 MHz, MeOD-d3, 298 K) spectrum from 124-155 ppm with assignments for
Ru-4T. (b) Zoom of 13C NMR (700 MHz, MeOD-d3, 298 76
K) spectrum from 124.5-133.5 ppm for Ru-4T.
Figure S8. Aromatic 1H-1H COSY NMR (700 MHz, MeOD-d3, 298 K) spectrum and assignments for Ru-4T.
77
5,6
(b)
2
e
h
b,8
9 a
d
3
e l f j hg I k
j
i
g
l
b
3,8
k
5,6
f
d
Figure S9. (a) 13C-1H HSQC NMR (700 MHz, MeOD-d3, 298 K) spectrum of Ru-4T with assignments. (b) Zoom of
78
13C -1H HSQC NMR (700 MHz, MeOD-d , 298 K) spectrum for Ru-4T with assignments.
3
(a)
47
5,6
2 9 a
c
b,8
d 3
e l f j hgi k
e-i,l
3,8,b
5,6,d,k
c,11
19,20
15,13
16,17
4,7,14
12
22
20,21
10
a
2,9
Figure S10. (a) 13C-1H HMBC NMR (700 MHz, MeOD-d3, 298 K) of Ru-4T with assignments. (b) Zoom of 13C -1H
79
HMBC NMR (700 MHz, MeOD-d3, 298 K) spectrum for Ru-4T with assignments.
HRMS spectra
(a)
321.0547
(b)
321.0547
320.5554
322.0546
320.0552
321.5552
319.5560
322.5574
318.0571
318.5570
319.0538
323.0592
Figure S11. (a) High resolution ESI+-MS spectrum for [Ru(phen)3](Cl)2. (b) Zoom of 321.0547 m/z showing
isotopic distribution.
80
(a)
341.0582
681.1110
(b)
341.0582
340.5590
342.0575
340.0568
341.5585
339.5593
342.5599
338.0508
338.5615
339.0587
343.0587
(c)
681.1110
680.1114
683.1099
679.1074
678.1122
682.1127
684.1093
675.1134
676.1152
677.1060
685.1177
Figure S12. (a) High resolution ESI+-MS spectrum for Ru-0T. (b) Zoom of 341.0582 m/z showing isotopic
81
distribution. (c) Zoom of 681.1110 m/z showing isotopic distribution.
(a)
382.0523
763.0974
(b)
382.0523
381.5534
383.0530
381.0527
382.5522
380.5530
383.5520
379.0541
379.5565 380.0515
384.0548 384.5501
(c)
763.0974
762.0949
765.1003
764.1019
761.0944
760.0958
766.1007
757.0962
758.1046
759.0993
762.5959
763.5994 764.5969
767.0951
768.1075
Figure S13. (a) High resolution ESI+-MS spectrum for Ru-1T. (b) Zoom of 382.0523 m/z showing isotopic
82
distribution. (c) Zoom of 763.0974 m/z showing isotopic distribution.
(a)
423.0445
845.0851
(b)
423.0446
422.5454
424.0457
423.5480
422.0470
421.5463
424.5476
420.0462
420.5475 421.0464
425.0471
425.5375
(c)
845.0851
844.0836
847.0842
845.0884
843.0839
842.0859
848.0910
839.0843
840.0953
841.0805
842.5847
843.5835
844.5841
845.5865
846.5907
847.5874
849.0903
848.5858
850.0821
Figure S14. (a) High resolution ESI+-MS spectrum for Ru-2T. (b) Zoom of 423.0445 m/z showing isotopic
83
distribution. (c) Zoom of 845.0851 m/z showing isotopic distribution.
(a)
464.0405
927.0769
(b)
464.0405
465.0391
463.5386
464.5394
463.0413
465.5397
462.5411
461.0414
466.0410
461.5393 462.0416
466.5355 467.0385
(c)
927.0769
926.0753
929.0744
928.0802
925.0753
924.0768
930.0809
921.0807
926.5759
922.0743 923.0695
923.5782
924.5758
927.5784
925.5644
931.0784
928.5718
929.5668
932.0667
933.0780
Figure S15. (a) High resolution ESI+-MS spectrum for Ru-3T. (b) Zoom of 464.0405 m/z showing isotopic
84
distribution. (c) Zoom of 927.0769 m/z showing isotopic distribution.
(a)
505.0312
1009.0553
(b)
505.0312
506.0317
504.5341
505.5332
504.0336
506.5353
503.5337
507.0310
502.0345
502.5363 503.0304
507.5317
508.0332
(c)
1009.0663
1008.0619
1011.0553
1010.0600
1007.0590
1012.0641
1006.0576
1003.0624
1004.05931005.0577
1005.5635 1006.5521
1013.0623
1007.5663
1008.5578
1009.5630 1010.5575
1011.5534
1014.0498
1012.5630
1015.0631
Figure S16. (a) High resolution ESI+-MS spectrum for Ru-4T. (b) Zoom of 505.0312 m/z showing isotopic
85
distribution. (c) Zoom of 1009.0663 m/z showing isotopic distribution.
HPLC chromatograms
9.268
DAD1 A, Sig=285,8 Ref=850,20 (LIUBOV\LL000112.D)
mAU
800
600
400
200
0
15
20
15
25
30
35
min
20
25
30
35
min
15
20
25
30
35
min
15
20
25
30
35
min
9.268
5
10
DAD1 B, Sig=440,8 Ref=850,20 (LIUBOV\LL000112.D)
mAU
600
500
400
300
24.305
200
100
0
9.268
5
10
DAD1 C, Sig=490,8 Ref=850,20 (LIUBOV\LL000112.D)
mAU
140
120
100
80
60
40
20
0
9.268
5
10
DAD1 D, Sig=400,8 Ref=850,20 (LIUBOV\LL000112.D)
mAU
500
400
300
200
100
0
5
10
Figure S17. HPLC chromatogram for [Ru(phen)3](Cl)2 collected at the following wavelengths: 285, 400, 440, and
490 nm (99.5% purity by peak area).
86
9.069
DAD1 A, Sig=285,8 Ref=850,20 (RACHEL\RH000051.D)
mAU
1600
1400
1200
1000
800
600
9.417
9.727
9.807
400
200
0
15
20
25
30
35
min
15
20
25
30
35
min
15
20
25
30
35
min
15
20
25
30
35
min
9.068
5
10
DAD1 B, Sig=440,8 Ref=850,20 (RACHEL\RH000051.D)
mAU
700
600
500
400
300
9.417
200
100
0
9.068
5
10
DAD1 C, Sig=490,8 Ref=850,20 (RACHEL\RH000051.D)
mAU
175
150
125
100
75
50
25
0
9.069
5
10
DAD1 D, Sig=400,8 Ref=850,20 (RACHEL\RH000051.D)
mAU
500
400
300
9.417
200
100
0
5
10
Figure S18. HPLC chromatogram for Ru-0T collected at the following wavelengths: 285, 400, 440, and 490 nm.
(98% purity by peak area).
87
9.069
DAD1 A, Sig=285,8 Ref=850,20 (RACHEL\RH000051.D)
mAU
1600
1400
1200
1000
800
600
9.417
9.727
9.807
400
200
0
15
20
25
30
35
min
15
20
25
30
35
min
15
20
25
30
35
min
15
20
25
30
35
min
9.068
5
10
DAD1 B, Sig=440,8 Ref=850,20 (RACHEL\RH000051.D)
mAU
700
600
500
400
300
9.417
200
100
0
9.068
5
10
DAD1 C, Sig=490,8 Ref=850,20 (RACHEL\RH000051.D)
mAU
175
150
125
100
75
50
25
0
9.069
5
10
DAD1 D, Sig=400,8 Ref=850,20 (RACHEL\RH000051.D)
mAU
500
400
300
9.417
200
100
0
5
10
Figure S19. HPLC chromatogram for Ru-1T collected at the following wavelengths: 285, 400, 440, and 490 nm
(99.5% purity by peak area).
88
21.194
DAD1 A, Sig=285,8 Ref=850,20 (RACHEL\RH000047.D)
mAU
400
300
200
100
0
15
20
5
10
DAD1 B, Sig=440,8 Ref=850,20 (RACHEL\RH000047.D)
15
20
5
10
DAD1 C, Sig=490,8 Ref=850,20 (RACHEL\RH000047.D)
15
20
15
20
25
30
35
min
25
30
35
min
25
30
35
min
25
30
35
min
21.194
5
10
DAD1 D, Sig=400,8 Ref=850,20 (RACHEL\RH000047.D)
mAU
400
300
200
100
21.194
0
mAU
250
200
150
100
50
21.194
0
mAU
100
80
60
40
20
0
5
10
Figure S20. HPLC chromatogram for Ru-2T collected at the following wavelengths: 285, 400, 440, and 490 nm
(99.5% purity by peak area).
89
22.748
DAD1 A, Sig=285,8 Ref=850,20 (HOUSTON\HC000167.D)
mAU
800
700
600
500
400
10.817
200
22.213
22.452
23.067
23.172
23.477
300
100
0
15
20
5
10
DAD1 B, Sig=440,8 Ref=850,20 (HOUSTON\HC000167.D)
15
20
5
10
DAD1 C, Sig=490,8 Ref=850,20 (HOUSTON\HC000167.D)
15
20
15
20
25
30
35
min
25
30
35
min
25
30
35
min
25
30
35
min
22.748
5
10
DAD1 D, Sig=400,8 Ref=850,20 (HOUSTON\HC000167.D)
mAU
1000
800
600
22.213
22.452
23.066
23.173
23.477
400
200
22.748
0
mAU
800
600
22.213
22.452
23.066
23.173
23.477
400
200
22.748
0
mAU
200
150
22.212
100
50
0
5
10
Figure S21. (a) HPLC chromatogram for Ru-3T collected at the following wavelengths: 285, 400, 440, and 490
nm (96% purity by peak area).
90
24.114
DAD1 A, Sig=285,8 Ref=850,20 (HOUSTON\HC000219.D)
mAU
800
600
23.899
23.975
24.491
24.704
24.796
400
200
0
15
20
5
10
DAD1 C, Sig=490,8 Ref=850,20 (HOUSTON\HC000219.D)
15
20
5
10
DAD1 D, Sig=400,8 Ref=850,20 (HOUSTON\HC000219.D)
15
20
15
20
25
30
35
min
25
30
35
min
25
30
35
min
30
35
min
24.114
5
10
DAD1 B, Sig=440,8 Ref=850,20 (HOUSTON\HC000219.D)
mAU
1200
1000
800
600
23.898
23.976
24.491
24.700
24.796
400
200
24.114
0
mAU
400
300
23.898
23.976
24.488
200
100
24.114
0
mAU
1000
800
600
23.899
23.975
24.492
24.705
24.798
400
200
0
5
10
25
Figure S22. HPLC chromatogram for Ru-4T collected at the following wavelengths: 400, 285, 440, and 490 nm
(99.5% purity by peak area).
91
Lipophilicity measurements
Table S1. Log distribution coefficient (log Do/w) of 50 μM [Ru(phen)3](Cl)2 and Ru-nT (n=0–4) in 1-octanol and 10
mM phosphate buffer (pH = 7.4) using the shake-flask method.
alog D
Compound
log (Do/w ± SD)
[Ru(phen)3](Cl)2
– 1.853 ± 0.076
Ru-0T
– 1.524 ± 0.017
Ru-1T
– 0.810 ± 0.023
Ru-2T
– 0.212 ± 0.028
Ru-3T
+ 0.772 ± 0.188
Ru-4T
n.d.a
o/w for Ru-4T was undefined due to precipitation at the octanol:phosphate
measurable amount of Ru-4T in the phosphate buffer phase.
92
buffer interface leading to no
Computational Studies
Table S2. (a) Dihedral angles φ1–φ4 (degree) and Ru-N distances (Å) values obtained for singlet ( 1GS) and triplet
(T1) optimized [Ru(phen)3]2+ and Ru-nT geometries in a water environment at the PBE0/6-31+G**/SDD/level of
theory. (b) Ru-N bond labels and dihedral angles defined for the table.
(a)
[Ru(phen)3]2+
Ru-0T
Ru-1T
Ru-2T
1GS
T1
1GS
T1
1GS
T1
ij
/
/
/
/
177.08
ij
/
/
/
/
/
ij
/
/
/
/
ij
/
/
/
Ru-N1
2.08
2.05
Ru-N2
2.08
2.05
Ru-N3
2.08
Ru-N4
Ru-3T
1GS
T1
-176.24
176.36
/
-163.28
/
/
/
/
2.07
2.05
2.07
2.05
2.08
2.08
2.08
2.10
Ru-N5
2.08
Ru-N6
2.08
Ru-4T
1GS
T1
1GS
T1
-179.67
177.15
-179.75
-177.59
-179.74
179.94
-166.40
179.91
-166.59
179.87
/
/
162.62
-179.94
164.80
-179.92
/
/
/
/
/
-161.41
179.82
2.07
2.05
2.07
2.07
2.07
2.07
2.07
2.07
2.07
2.05
2.07
2.07
2.07
2.07
2.07
2.07
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.10
2.08
2.10
2.08
2.08
2.08
2.08
2.08
2.08
2.10
2.08
2.10
2.08
2.10
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
2.08
93
Table S3. Calculated percent contribution of the Ru d-orbital and the orbitals involving phen, IP, and nT chain to the
frontier orbitals (HOMO-1, HOMO, LUMO, LUMO+1) for [Ru(phen)3]2+ and Ru-nT in the singlet ground state (1GS).
HOMO-1
HOMO
LUMO
LUMO+1
%
Ru
phen
IP
nT
Ru
phen
IP
nT
Ru
phen
IP
nT
Ru
phen
IP
nT
[Ru(phen)3]2+
47
53
/
/
61
39
/
/
2
98
/
/
1
99
/
/
Ru-0T
46
29
25
/
58
26
16
/
2
57
41
/
2
69
29
/
Ru-1T
18
42
36
4
55
26
19
/
2
57
41
2
72
25
1
Ru-2T
55
25
19
1
1
39
35
26
2
56
39
3
2
68
25
5
Ru-3T
55
26
19
0
0
36
18
46
3
55
40
2
3
64
23
10
Ru-4T
55
27
18
0
0
27
13
61
3
52
38
7
3
40
21
36
94
Table S4. Computed absorption transitions >400 nm (λ and λexp), oscillator strength (f), theoretical peak assignment,
and predominant configuration.
Cmpd
ȜQP
Ȝexp / nm
f
[Ru(phen)3]2+
432
444
0.112
434
450
0.149
Assignment
Ru-0T
433
Ru-1T
438
0.119
457
434
Ru-2T
Ru-3T
455
0.207
0.247
0.247
434
0.118
460
1.034
0.266
449
H-1→L+1;
H-2→L+2
H-1→L+2;
H-2→L+1
H-1→L+1;
H-1→L+2;
H-2→L+1
H-3→L
H→L
443
466
H-1→L+2
H-2→L+2
0.118
457
H-2→L+1
H-3→L+1
H→L+1
H-1→L+1;
H-2→L+2
H→L
H→L+1;
H-3→L
438
0.771
H→L+3
Main Configuration
1MLCT (65%)
1MLCT (62%)
1MLCT (62%)
1MLCT (55%)
1MLCT (65%)
1MLCT (52%)/1LLCT (30%)
1MLCT (47%)/1LLCT (30%)
1MLCT (62%)
1ILCT/1IL (58%)/1LLCT(20%)
1MLCT (47%)/1LLCT (35%)
1ILCT/1IL (58%) /1LLCT (20%)
H→L+4;
410
413
0.110
H-1→L+3;
1MLCT (42%)/1LLCT(40%)
H-3→L+3
Ru-4T
488
465
462
435
2.231
0.185
436
0.110
95
H→L+1
H→L;
H→L+3
H-3→L+1;
H-2→L+2
1ILCT/1IL (77%)
1ILCT/1IL (53%)/1LLCT (30%)
1MLCT (56%)
96
97
Figure S23. Plots of HOMO-1 (H-1), HOMO (H), LUMO (L) and LUMO+1 (L+1) molecular orbitals for [Ru(phen)3]2+
and Ru-nT computed in water at the M06/6-31+G(d,p)/SDD/ level of theory.
98
99
100
101
Figure S24. Occupied and virtual NTOs and predominant configuration of the computed absorption wavelengths
>400 nm reported in Table S4.
102
Figure S25. Optimized T1 geometries for [Ru(phen)3]2+ and Ru-nT in a water environment at the PBE0/631+G(d,p)/SDD/ level of theory.
103
Figure S26. Occupied and Virtual Natural Transition Orbitals (NTOs) of the lowest energy triplet excited states (T 1):
(a) 3MLCT T1 states of [Ru(phen)3]2+ and Ru-nT (n=0,1). (b) Mixed ligand-based 3ILCT/3LLCT T1 states of Ru-2T,
Ru-3T, and Ru-4T, in a water environment at the M06/6-31+G**/SDD level of theory.
104
Table S5. Adiabatic labels and vertical energies (ΔEvert) of the lowest 3MLCT, 3ILCT, and 3MC states, along with the
corresponding adiabatic 3MLCT and 3ILCT energies (ΔEadia) computed with respect to the S0 minima, and emission
energies (ΔEem) for the 3MLCT states for [Ru(phen)3]2+ and Ru-nT (n=0–4) in a water environment at the TD-M06/631+G**/SDD level of theory. The experimental 3MLCT emission energies at 77 and 298 K and the energies
estimated for 3ILCT states from Stern-Volmer quenching rates are also provided for comparison.
3MLCT (eV)
3ILCT (eV)
ȴ
em, 77 K, 298 K
Compound
State
ȴ
vert
ȴ
adia
ȴ
em
[Ru(phen)3]2+
T1
2.43
2.21
2.01
2.19, 2.06
Ru-0T
T1
2.40
2.18
1.99
Ru-1T
T1
2.40
2.18
Ru-2T
T2
2.40
Ru-3T
T2
Ru-4T
T2
ȴ
vert
n.d.
T21
3.28
2.18, 2.01
n.d.
T18
3.07
1.98
2.16, 2.05
n.d.
T19
3.06
2.17
1.98
2.15, 2.03
T1
2.23
1.82
n.d.
T19
3.06
2.40
2.16*
1.99*
2.16, 2.02
T1
1.97
1.57
~1.5
T22
3.06
2.37
2.12
1.99
2.16, 2.02
T1
1.84
1.44
~1.5
T25
3.06
*Not fully converged due to degeneracy with the 3ILCT state; n.d.=not determined.
105
ȴ
adia
E0-0
State
Expt
State
ȴ
vert
3MC (eV)
Expt
Spectroscopic Characterization
Figure S27: Emission decays measured as ≈5 μM solutions in degassed MeCN at RT, with a 355 nm
excitation.
106
Figure S28. Excited state absorption profiles of the series, measured as ≈5 μM solutions in degassed MeCN at
RT, with a 355 nm excitation.
107
Figure S29: Transient absorption decays measured as ≈5 μM solutions in degassed MeCN at RT, with a
355 nm excitation.
108
Figure S30. ESA profile of Ru-1T at a longer time slice, showing a bleach near 460 nm and an
absorption at longer wavelengths, typical of a 3MLCT state.
Electrochemical Characterization
Figure S31. CDPV for the oxidation of (a) [Ru(phen)3]2+, (b) Ru-0T, (c) Ru-1T, (d) Ru-2T, (e) Ru3T, and (f) Ru-4T in DMF containing 0.1 M tetrabutylammonium hexafluorophosphate and
ferrocene as an internal standard.
109
Figure S32. CDPV for the reduction of (a) [Ru(phen)3]2+, (b) Ru-0T, (c) Ru-1T, (d) Ru-2T, (e) Ru3T, and (f) Ru-4T in DMF containing 0.1 M tetrabutylammonium hexafluorophosphate and
ferrocene as an internal standard.
110
Biological and Photobiological Characterization
Figure S33. Spectral outputs of light sources used in photobiological experiments.
111
Table S6: Cytotoxicity and photocytotoxicity of [Ru(phen)3](Cl)2 and Ru-0T–Ru-4T in normoxic (~18.5% O2, top) or
hypoxic (1% O2, bottom) treated SK-MEL-28 melanoma cells. Values correspond to the biological replicate shown
Resazurin-Cell Viability
EC50 ± SEM (μM)
PId
Complex
Oxygen%
Dark
Visiblea
Greenb
Redc
Visiblea
Greenb
Redc
[Ru(phen)3](Cl)2
~18.5
>300
22.2 ± 5.4
>300
>300
~14
~1
~1
Ru-0T
~18.5
>300
6.80 ± 2.49
>300
>300
~44
~1
~1
Ru-1T
~18.5
138 ± 6
5.94 ± 0.17
144 ± 4
220
23
1
Ru-2T
~18.5
113 ± 5
116 ± 6
194
211
1
Ru-3T
~18.5
66.4 ± 3.3
59.5 ± 1.7
1165
269
1
Ru-4T
~18.5
84.0 ± 2.5
16.3 ± 1.6
113731
1364
5
[Ru(phen)3](Cl)2
1
Ru-0T
0.626 ±
0.198
0.583 ± n.d.
0.536 ±
0.144
0.057 ±
0.247 ±
0.037
0.015
(7.39 ±
0.062 ±
n.d.)*10-4
0.0076
>300
>300
>300
>300
~1
~1
~1
1
>300
>300
>300
>300
~1
~1
~1
Ru-1T
1
147 ± 5
172 ± 6
147 ± 4
156 ± 5
1
1
1
Ru-2T
1
120 ± 5
137 ± 9
128 ± 7
122 ± 5
1
1
1
Ru-3T
1
52.5 ± 1.7
75.3 ± 5.8
64.1 ± 4.7
54.0 ± 2.1
1
1
1
Ru-4T
1
86.2 ± 2.1
2.05 ± 0.38
1.34 ± 0.74
91.9 ± 3.0
42
64
1
Light treatments were approximately 100 J cm −2 delivered at 18–22 mW cm−2 with acool white visible (400–700 nm), b green
523 nm, cred 633 nm, and d PI = phototherapeutic index. Hypoxia and normoxia experiments were ran in parallel. PI not
determined when dark EC50 exceeded 300 μM. SEM not determined when hill slope was too steep.
in Figure 13.
112
Table S7. Inter-assay performance: cytotoxicity and photocytotoxicity of Ru-3T in normoxic-treated (~18.5% O2)
SK-MEL-28 melanoma cells.
Resazurin-Normoxia (18.5% O2) Ru-3T Repeats
EC50 ± SEM (μM)
Greenb
Redc
Visiblea
Greenb
Redc
0.247 ± 0.015
59.5 ± 1.7
1165
269
1
0.395 ± 0.251
3.29 ± 0.34
1591
309
37
0.0689 ± n.d.
0.219 ± 0.037
3.77 ± 0.05
1800
566
33
92.1 ± 2.0
0.0786 ± n.d.
0.638 ± n.d.
3.37 ± 0.09
1172
144
27
4
82.9 ± 2.3
0.0715 ± n.d.
0.164 ± n.d.
50.3 ± n.d.
1159
505
2
5
110 ± 3
0.0642 ± n.d.
0.264 ± 0.032
34.8 ± 2.9
1713
417
3
6
70.4 ± 3.1
0.186 ± 0.038
69.4 ± 3.4
2542
378
1
0.302 ± 0.166
32.1 ± 28.7
1592 ± 501
370 ± 144
14.9 ± 16.6
Repeat
Dark
0
66.4 ± 3.3
1
122 ± 5
2
124 ± 4
3
Mean ±
SDe
Visiblea
PId
0.0570 ±
0.0368
0.0767 ±
0.0075
95.4 ± 23.7
0.0277 ±
0.0033
0.0635 ±
0.0174
min
66.4
0.057
0.164
3.29
1159
144
1
max
124
0.0786
0.638
69.4
2542
566
37
Light treatments were approximately 100 J cm −2 delivered at 18–22 mW cm−2. acool white visible (400–700 nm), b green 523
nm, cred 633 nm, d PI = phototherapeutic index (dark EC50 / light EC50), and n.d. = SEM not determined due to overly steep
hill slope. e Did not test for outliers or run meta-analysis, use with caution.
Repeats used different plate maps (all), different tips (Sartorius 790352 repeat #0, VWR 83007-352 repeats #1–2, low
retention Sartorius LH-L790352 repeats #3–4), changed cell parent seed stock for repeats 3–6, and overhead lights were
off in #4–6. Serum and consumable lots were identical for repeats 0–6. Cell passage numbers were within 15. Run in
parallel with normoxic repeats.
113
Table S8. Inter-assay performance: cytotoxicity and photocytotoxicity of Ru-3T in hypoxic-treated (1% O2) SK-MEL28 melanoma cells.
Resazurin-Hypoxia (1% O2) Ru-3T Repeats
EC50 ± SEM (μM)
PId
Repeat
Dark
Visiblea
Greenb
Redc
Visiblea
Greenb
Redc
0
52.5 ± 1.7
75.3 ± 5.8
64.1 ± 4.7
54.0 ± 2.1
1
1
1
1
120 ± 4
112 ± n.d.
115 ± 5
1
1
1
120 ± 68
0.352 ±
2
118 ± 3
1.00 ± 0.07
0.018
68.8 ± 3.2
118
335
2
3
98.5 ± 2.7
1.15 ± n.d.
1.04 ± 0.73
93.6 ± 3.3
86
95
1
4
78.6 ± 1.6
1.14 ± n.d.
1.11 ± n.d.
77.9 ± 1.2
69
71
1
5
109 ± 2
0.215 ±
0.420 ±
0.061
0.056
111 ± 4
507
260
1
72.1 ± 3.3
0.447 ±
0.813 ±
6
0.067
0.310
62.5 ± 3.5
161
89
~1
92.7 ± 25.6
27.3 ± 46.5
26.8 ± 47.4
83.3 ± 23.8
135 ± 174
122 ± 128
1.14 ± 0.38
min
52.5
0.215
0.352
54.0
1
1
1
max
120
112
120
115
507
335
2
Mean ±
SDe
Light treatments were approximately 100 J cm −2 delivered at 18–22 mW cm−2. acool white visible (400–700 nm), b green 523
nm, cred 633 nm, d PI = phototherapeutic index (dark EC50 / light EC50), and n.d. = SEM not determined due to overly steep
hill slope. e Did not test for outliers or run meta-analysis, use with caution.
Repeats used different plate maps (all), different tips (Sartorius 790352 repeat #0, VWR 83007-352 repeats #1–2, low
retention Sartorius LH-L790352 repeats #3–4), changed cell parent seed stock for repeats 3–6, and overhead lights were
off in #4–6. Serum and consumable lots were identical for repeats 0–6. Cell passage numbers were within 15. Run in
parallel with normoxic repeats.
114
Table S9. Inter-assay performance: cytotoxicity and photocytotoxicity of Ru-4T in normoxic-treated (~18.5% O2)
SK-MEL-28 melanoma cells.
Resazurin-Normoxia (18.5% O2) Ru-4T Repeats
EC50 ± SEM (μM)
PId
Repeat
Dark
Visiblea
Greenb
Redc
Visiblea
Greenb
Redc
0
84.0 ± 2.5
(7.39 ± n.d.)x10-4
1
106 ± 3
(4.01 ± 0.51)x10-8
0.0616 ±
0.0076
0.147 ±
0.019
16.3 ± 1.6
1.14x105
1364
5
1.38 ±
n.d.
2.64x109
721
77
2
101 ± 2
(3.22 ± 0.19)x10-7
0.0306 ±
0.004
1.88 ±
0.11
3.14x108
3301
54
3
92.0 ± 2.3
(8.04 ± 0.55)x10-4
0.257 ±
0.015
1.44 ±
0.68
1.14x105
358
64
4
90.9 ± 1.9
(8.58 ± n.d.)x10-3
0.0792 ±
n.d.
11.4 ±
0.5
1.06x104
1148
8
5
85.5 ± 1.3
(6.38 ± 3.86)x10-4
0.0321 ±
0.0020
7.69 ±
0.16
1.34x105
2664
11
6
101 ± 2
(4.85 ± 0.44)x10-3
0.0593 ±
0.0074
50.6 ± n.d.
2.08x104
1703
2
Mean ±
SDe
94.3 ± 8.4
(2.23 ± 3.26)x10-3
0.095 ±
0.081
12.96 ±
17.55
(4.22 ± 9.86)x108
1608 ±
1050
31.6 ± 32.1
min
84
4.01x10-8
0.0306
1.38
1.06x104
358
2
max
106
8.58x10-3
0.257
50.6
2.64x109
3301
77
Light treatments were approximately 100 J cm −2 delivered at 18–22 mW cm−2. acool white visible (400–700 nm), b green 523 nm,
cred 633 nm, d PI = phototherapeutic index (dark EC
50 / light EC50), n.d. = SEM not determined due to overly steep hill slope.
e Did
not test for outliers or run meta-analysis, use with caution.
Repeats used different plate maps (all), different tips (Sartorius 790352 repeat #0, VWR 83007-352 repeats #1–2, low retention
Sartorius LH-L790352 repeats #3–4), changed cell parent seed stock for repeats 3–6, and overhead lights were off in #4–6. Serum
and consumable lots were identical for repeats 0–6. Cell passage numbers were within 15. Run in parallel with normoxic repeats.
115
Table S10. Inter-assay performance: cytotoxicity and photocytotoxicity of Ru-4T in hypoxic-treated (1% O2) SKMEL-28 melanoma cells.
Resazurin-Hypoxia (1% O2) Ru-4T Repeats
EC50 ± SEM (μM)
PId
Repeat
Dark
Visiblea
Greenb
Redc
Visiblea
Greenb
Redc
0
86.2 ± 2.1
2.05 ± 0.38
1.34 ± 0.74
91.9 ± 3.0
42
64
1
6.44 ± 8.22
19.2 ± 15.6
22.0 ± 15.4
15
5
4
98.3 ± 2.3
0.0323 ±
0.0039
0.199 ±
0.012
6.19 ± 0.25
3043
494
16
87.4 ± 2.1
0.299 ± 0.172
0.809 ±
0.451
4.55 ± 1.84
292
108
19
81.8 ± 1.2
0.252 ± 0.060
0.562 ±
0.052
85.7 ± 2.3
325
146
1
5
92.5 ± 2.7
0.0537 ±
0.0040
0.229 ±
0.011
97.0 ± 4.2
1723
404
1
6
102 ± 2
0.0701 ± n.d.
0.211 ±
0.040
106 ± 2
1455
483
1
Mean ±
SDe
92.0 ± 7.2
1.31 ± 2.37
3.22 ± 7.06
59.0 ± 45.8
985 ± 1136
243 ± 209
6.14 ± 7.88
min
81.8
0.0323
0.199
4.55
15
5
1
max
102
6.44
1.34
106
3043
494
19
1
2
3
4
95.5 ± 2.4
Light treatments were approximately 100 J cm −2 delivered at 18–22 mW cm−2. acool white visible (400–700 nm), b green 523
nm, cred 633 nm, d PI = phototherapeutic index (dark EC50 / light EC50), n.d. = SEM not determined due to overly steep hill
slope. e Did not test for outliers or run meta-analysis, use with caution.
Repeats used different plate maps (all), different tips (Sartorius 790352 repeat #0, VWR 83007-352 repeats #1–2, low
retention Sartorius LH-L790352 repeats #3–4), changed cell parent seed stock for repeats 3–6, and overhead lights were
off in #4–6. Serum and consumable lots were identical for repeats 0–6. Cell passage numbers were within 15. Run in
parallel with normoxic repeats.
116
Figure S34. Radar plot of [Ru(phen)3](Cl)2 and Ru-0T–Ru-4T concerning relevant (photo)physical,
electrochemical, and (photo)biological values. Upper bounds are labelled for respective values and lower bounds
converge on the origin. Log(PI) values and EC50’s are derived from the data shown in Table S6, and the lower
bounds are 0. The lower bounds of the other values were chosen to emphasize trends among the series, and are
as follows: ФΔ=0.25, Log(D)= -2, TTA=0, Eoxidation (1st)=550 mV, Ereduction (1st)= -1850 mV, Log(EC50, dark-normoxia)=0.
Photophysical values are derived from Table 2, electrochemical values from Table 4, and Log(D) values are from
This
Tableradar
S1. plot was generated using R (version 4.3.1) 7 with the fsmb package.8 The text was formatted using
Inkscape.9
117
References
(1)
DeRosa, M. C.; Crutchley, R. J. Photosensitized Singlet Oxygen and Its Applications. Coord. Chem.
Rev. 2002, 233–234, 351–371. https://doi.org/10.1016/S0010-8545(02)00034-6.
(2)
Montalti, M.; Credi, A.; Prodi, L.; Gandolfi, M. T. Handbook of Photochemistry; CRC Press, 2006.
https://doi.org/10.1201/9781420015195.
(3)
Cole, H. D.; Roque, J. A.; Shi, G.; Lifshits, L. M.; Ramasamy, E.; Barrett, P. C.; Hodges, R. O.;
Cameron, C. G.; McFarland, S. A. Anticancer Agent with Inexplicable Potency in Extreme Hypoxia:
Characterizing a Light-Triggered Ruthenium Ubertoxin. J. Am. Chem. Soc. 2022, 144 (22), 9543–
9547. https://doi.org/10.1021/jacs.1c09010.
(4)
Roque III, J. A.; Cole, H. D.; Barrett, P. C.; Lifshits, L. M.; Hodges, R. O.; Kim, S.; Deep, G.; FrancésMonerris, A.; Alberto, M. E.; Cameron, C. G.; McFarland, S. A. Intraligand Excited States Turn a
Ruthenium Oligothiophene Complex into a Light-Triggered Ubertoxin with Anticancer Effects in
Extreme
Hypoxia.
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8317–8336.
https://doi.org/10.1021/jacs.2c02475.
(5)
Monro, S.; Colón, K. L.; Yin, H.; Roque, J.; Konda, P.; Gujar, S.; Thummel, R. P.; Lilge, L.; Cameron,
C. G.; McFarland, S. A. Transition Metal Complexes and Photodynamic Therapy from a TumorCentered Approach: Challenges, Opportunities, and Highlights from the Development of TLD1433.
Chem. Rev. 2019, 119 (2), 797–828. https://doi.org/10.1021/acs.chemrev.8b00211.
(6)
Pazderski, L.; Pawlak, T.; Sitkowski, J.; Kozerski, L.; Szłyk, E. 1H NMR Assignment Corrections and
1
H, 13C, 15N NMR Coordination Shifts Structural Correlations in Fe(II), Ru(II) and Os(II) Cationic
Complexes with 2,2′-Bipyridine and 1,10-Phenanthroline. Magnetic Resonance in Chemistry 2010,
48 (6), 450–457. https://doi.org/10.1002/mrc.2600.
(7)
R Core Team. R: A Language and Environment for Statistical Computing; R Foundation for
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Nakazawa, M. Fmsb: Functions for Medical Statistics Book with Some Demographic Data; 2019.
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118
CHAPTER 3. RU(II) TRIFLUOROMETHYL BIPYRIDINE-BASED OLIGOTHIENYL
COMPLEXES FOR PHOTODYNAMIC THERAPY
Houston D. Cole,a Abbas Vali,a John A. Roque III,a,b, Ge Shi,a Alisher Talgatov,a Gurleen
Kaur,a Wesley McDonald,a Elamparuthi Ramasamy,a Colin G. Cameron,a* Sherri A.
McFarlanda*
a Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, Texas, 76019-0065 United States
b Department of Chemistry and Biochemistry, The University of North Carolina at Greensboro, Greensboro, North Carolina
27402, USA
c Department of Chemistry, University of Houston, 112 Fleming Building, Houston, Texas, 77204-5003, USA
*Corresponding authors: C.G.C. <colin.cameron@uta.edu> ORCID 0000-0003-0978-0894, S.A.M.
<sherri.mcfarland@uta.edu> ORCID 0000-0002-8028-5055
3.1 ABSTRACT
In our recent investigations, we have developed and extensively studied a new series of Ru(II)
polypyridyl complexes featuring two 4,4'-bis(trifluoromethyl)-2,2'-bipyridine (4,4'-btfmb) ligands
and one imidazo[4,5-f][1,10]phenanthroline (IP) tethered to thiophene rings (nT) as secondary
ligands. The [Ru(4,4'-btfmb)2(IP-nT)]2+ scaffold represents a unique class of photosensitizers in
the field of photodynamic therapy (PDT) bearing several electron-deficient trifluoromethyl
groups (-CF3). Electrochemically, these complexes demonstrated significant shifts in the
oxidation potentials of the nT moiety, ranging between +580 mV and +1020 mV. Reductions in
the nT units were particularly notable, recorded from −2230 mV in Ru-3T to as low as −2960
mV in Ru-4T. The series exhibited a range of singlet oxygen quantum yields from 0.13 to 0.66,
with Ru-3T showing the highest efficiency. From a spectroscopic perspective, the lower
thiophene-substituted complexes (Ru-0T–2T) predominantly exhibited metal-to-ligand charge
transfer (3MLCT) states with lifetimes around 0.61–1.5 µs. In contrast, the higher thiophenesubstituted variants (Ru-3T–4T) displayed longer-lived intraligand charge transfer (3ILCT)
states, lasting ~20 µ. The photobiological efficacy of these complexes against melanoma cells
(SK-MEL-28) demonstrated a dependence on the number of thiophene rings. While Ru-0T
was minimally active, a notable increase in cytotoxicity was observed with increasing
119
thiophene content, culminating in the exceptional potency of Ru-4T (EC50=10 nM, PI=10,000).
The complexes in this series stand out as being highly reproducible in their photobiological
efficacy, with all EC50 values falling within one order of magnitude of one another over five
biological replicates. The activity was also influenced by the wavelength of light, with significant
persistence under hypoxic conditions, especially noted in Ru-3T and Ru-4T. These results
highlight the significance of the extended 3ILCT states in the higher thiophene-substituted
complexes, suggesting their vital role in enhancing photocytotoxicity. This research not only
contributes to our understanding of the structure-activity relationships in Ru(II) polypyridyl
complexes but also underscores their potential as robust and effective photosensitizers in
PDT.
Keywords: Ruthenium, metal-to-ligand charge transfer (MLCT), intraligand (IL), melanoma,
photodynamic therapy
3.2 INTRODUCTION
Cancer continues to be a leading cause of mortality worldwide, second only to heart diseases. 1
Despite substantial progress in available medical treatments,2–6 there remains a critical need
for innovative therapeutic strategies and supplementary treatments to enhance the efficacy of
traditional methods like surgery, radiotherapy, and chemotherapy. In this regard, photodynamic
therapy (PDT) is a distinct and promising method for targeted cancer therapy. It employs a
non-toxic photosensitizer (PS), harmless light, and molecular oxygen to produce cytotoxic
reactive oxygen species (ROS) that target tumor cells. PDT is advantageous due to its
localized approach and minimal invasiveness, resulting in reduced side effects and improved
patient quality of life.7,8 PDT's precision comes from the selective accumulation of PSs in tumor
tissues and the controlled activation by light. Consequently, phototoxic effects are restricted to
areas where the PS, light, and oxygen coexist in time and space. Optimizing the light protocol,
including wavelength, fluence, irradiance, and dosimetry, as well as the drug-to-light interval
(DLI), is key to maximizing PDT's effect. The oxygen-dependent nature of PDT is challenging
when addressing hypoxic tumors. Moreover, PDT can exacerbate hypoxia by depleting oxygen
during irradiation, limiting ROS production and thus reducing the efficacy against cancer cells.
120
Therefore, there is a drive to develop light-activated compounds that function through oxygenindependent mechanisms. Metal complexes, particularly Ru(II) polypyridyl systems, are of
significant interest in this regard. The strategic selection of ligand-metal combinations allows
for a range of excited state configurations with unique photophysical and reactive properties.
Approaches have included the photorelease of bulky ligands to expose phototoxic metals
and/or ligands,9–17 photocaging of chemotherapeutics and enzyme inhibitors,14,18–39 photoredox
reactions,40,41 and enhancing ROS production even under low oxygen conditions. 13,16,42
Our research group has a strong focus on metal complexes as PSs for their diverse
mechanisms of action. Their modular design and relatively straightforward synthesis allow for
rapid alteration of their physicochemical, photophysical, and biological characteristics, and
aligns with our tumor-specific approach to PS development. We advocate that no singular ideal
PS exists; rather, PS design should be tailored to the intended application. Our Ru(II)
polypyridyl complex, TLD1433, is a prime example, currently in Phase II clinical trials for
treating non-muscle invasive bladder cancer (NMIBC) with PDT.7,43 It exhibits significant
phototoxicity towards cancer cells while maintaining low dark toxicity. Clinically, it is activated
using green light to prevent harm to underlying muscles.
To expand our understanding of oligothiophene-based metal complexes and to develop new
PSs, we are exploring variations in metal ions, coligands, thienyl groups, counter ions, and
coordination structures.7,10,13,16,42,44,45 Our long-term aim is to establish structure-activity
relationships (SARs) for photoactive metal complexes, considering their physicochemical,
photophysical, electrochemical, and biological properties. Part of our SAR study involves
replacing certain hydrogens with fluorine in order to study the effects of electronegativity on the
properties of metal-centered PSs. In this study we introduce a series of Ru(II) PSs, each
featuring two 4,4'-bis(trifluoromethyl)-2,2'-bipyridine (4,4'-btfmb) coligands and a imidazo[4,5f][1,10]phenanthroline (IP) ligand with n=0–4 thiophene rings (nT). The syntheses and
structural characterization of complexes Ru-3T and Ru-4T were previously published,46,47 and
in this study the photocytotoxic effects of the five [Ru(4,4'-btfmb)2(IP-nT)]2+ variants and the
reference compound [Ru(4,4'-btfmb)3]2+ were examined on melanoma cells under various light
conditions and oxygen levels. Their lipophilicities, ground state absorption and emission
121
characteristics, excited state configurations and lifetimes, and redox properties have been
systematically studied.
The remarkable activities of these complexes, particularly those with longer thienyl chains, are
of considerable interest to us. This research offers a comprehensive insight into the
photophysical and biological behaviors of these complexes, providing a robust platform for
future biological studies into the underlying dynamics of PDT efficacy in a range of
oligothiophene-containing metal complexes. Furthermore, it introduces two new hypoxia-active
PSs, presenting potential avenues for further exploration and development.
3.3 MATERIALS AND METHODS
The complexes in this series were characterized by 1H NMR, HPLC, and ESI+ MS. They were
evaluated for lipophilicity, ground and excited state characteristics using absorption and
emission spectroscopy, electrochemistry, and (photo)cytotoxicity. Further details regarding
procedures and characterization data are available in the Supplementary Information.
Instrumentation
A CEM Discover microwave reactor was used to perform microwave reactions. Flash column
chromatography was carried out on the Teledyne ISCO EZ Prep UV model of CombiFlash® EZ
Prep using SILICYCLE SiliaSepTM 25 g prepacked silica cartridges. Size-exclusion
chromatography was performed using a gravity column packed with Sephadex® LH-20. The
NMR spectra were collected on JEOL 500 MHz spectrometers (University of North Carolina at
Greensboro, University of Texas at Arlington) operating at 500 MHz for 1H experiments, and
on an Agilent 700 MHz Magnet spectrometer (The Joint School of Nanoscience and
Nanoengineering at Greensboro) operating at 700 MHz for 1H experiments. The chemical
shifts are reported in parts per million (ppm) and were referenced to the residual solvent
peaks. High resolution ESI+ mass spectra were obtained using a Thermo Fisher Scientific LTQ
Orbitrap XL instrument (Triad Mass Spectrometry Laboratory at University of North Carolina at
Greensboro) and Shimadzu IT-TOF instrument (Shimadzu Center for Advanced Analytical
Chemistry at University of Texas at Arlington). HPLC analyses were carried out on an
Agilent/Hewlett Packard 1100 series instrument in 100 µM solutions in methanol using a
122
Hypersil GOLD C18 reversed-phase column with an A→B gradient (98% → 5% A; A=0.1%
formic acid in H2O, B=0.1% formic acid in MeCN). Reported retention times are accurate to
within ±0.1 min.
Synthesis and Characterization
We previously published the synthetic methods and structural characterization of Ru-3T and
Ru-4T,46,47 and Ru(4,4ʹ-btfmb)3]2+ has been reported by others.48–51 To the best of our
knowledge, all other complexes presented in this study have not been reported. All solvents
and reagents were purchased from commercial sources and used without further purification.
Water used for all biological experiments was deionized to a resistivity ≥ 18.2 MΩ using either
a Barnstead or Milli-Q® filtration system. Methanol was purchased from Fisher Scientific (ACS
grade for synthesis, HPLC grade for LC eluent, OptimaTM grade for HPLC and MS sample
preparation). Deuterated solvents for NMR were purchased from Cambridge Isotope
Laboratories. Ruthenium(III) trichloride trihydrate was purchased from Ark Pharm and Acros
Organics. Ru(4,4ʹ-btfmb)2Cl2•2H2O52 and IP-based ligands53 were prepared according to
adapted literature procedures. The synthesis of IP-based ligands follows that described below
for IP-4T. [2,2′:5′,2″:5″,2‴-quaterthiophene]-5-carbaldehyde (4T-CHO) was prepared as
previously described.54,55 Final products are synthetically characterized in Figure S1‒Figure
S21 via 1H NMR, 1H–1H COSY NMR, HPLC, and ESI+–MS. The Cl− salts of final complex
products were obtained via anion metathesis on HCl-treated Amberlite IRA-410 resin with
methanol as eluent and isolated in vacuo. Final complexes are a mixture of Δ/Λ isomers.
[Ru(4,4ʹ-btfmb)3](Cl)2 Ru(Cl)3∙xH2O (58 mg, 0.2 mmol) and 4,4ʹ-btfmb (175 mg, 0.6 mmol) was
added to a microwave vessel containing argon-purged ethylene glycol (3 mL), then the mixture
was subject to microwave irradiation at 180°C for 45 min with stirring. The resulting dark red
solution was then transferred to a separatory funnel with deionized water (25 mL) and CH2Cl2
(25 mL). After gentle agitation, the CH2Cl2 was drained and the remaining aqueous layer was
washed with CH2Cl2 (25 mL) until the CH2Cl2 layer was colorless. Then, CH2Cl2 (25 mL) and
saturated aqueous KPF6 (5 mL) was added, and the mixture was shaken gently. The CH2Cl2
layer was drained and the product was further extracted from the aqueous layer with CH 2Cl2
(25 mL) until the aqueous layer was colorless. The CH2Cl2 extracts were then combined and
123
concentrated under reduced pressure. The crude product was then purified using silica gel
flash column chromatography with a gradient of MeCN to 10% water in MeCN, followed by
7.5% water in MeCN with 0.5% KNO3. The product-containing fractions were then combined
and concentrated under vacuum, then transferred to a separatory funnel with CH 2Cl2 (25 mL),
deionized water (25 mL), and saturated aqueous KPF6 (1 mL). The resulting mixture was
gently agitated and the CH2Cl2 layer was drained. Additional CH2Cl2 (25 mL) was used to
extract the remaining product until the aqueous layer was colorless. The CH 2Cl2 layers were
then combined and dried under vacuum. This was then converted to the corresponding Cl - salt
in quantitative yield using Amberlite IRA-410 with MeOH as the eluent, then purifying further
using Sephadex LH-20 with MeOH as the eluent, affording a dark red solid (50 mg, 20%). 1H
NMR (400MHz, MeOD-d3, ppm): δ 9.41 (d, J=1.9 Hz, 6H), 8.15 (d, J=5.9 Hz, 6H), 7.86
(dd,J=6.0, 1.8 Hz, 6H). HRMS (ESI+) m/z for [M-2Cl-]2+ calcd: 489.0169. Found: 488.9529.
HPLC retention time 22.41 min (99.5% purity by peak area).
[Ru(4,4ʹ-btfmb)2(phen)](Cl)2 (Ru-phen) Ru(4,4ʹ-btfmb)2Cl2∙2H2O (91 mg, 0.12 mmol) and phen
(22 mg, 0.1 mmol) were added to a microwave vessel containing argon-purged ethylene glycol
(4 mL) and subjected to microwave irradiation at 180°C for 15 min. The resulting dark red
mixture was then isolated and purified in the same manner as [Ru(4,4ʹ-btfmb)3](Cl)2, yielding
the desired product as a dark red solid (43 mg, 45%). 1H NMR (400MHz, MeOD-d3, ppm): δ
9.38 (d, J=14.1 Hz, 4H), 8.81 (d, J=8.0 Hz, 2H), 8.36 (s, 2H), 8.28 (d, J=5.9 Hz, 2H), 8.23 (d,
J=5.3 Hz, 2H), 7.94 – 7.84 (m, 6H), 7.67 (dd, J=6.0, 1.8 Hz, 2H). HRMS (ESI+) m/z for [M-2Cl]2+ calcd: 433.0295. Found: 432.9739. HPLC retention time 22.41 min (99.5% purity by peak
area).
[Ru(4,4ʹ-btfmb)2(IP)](Cl)2 (Ru-0T) Ru(4,4ʹ-btfmb)2Cl2∙2H2O (91 mg, 0.12 mmol) and IP (22 mg,
0.1 mmol) were added to a microwave vessel containing argon-purged ethylene glycol (4 mL)
and subjected to microwave irradiation at 180°C for 15 min. The resulting dark red mixture was
then isolated and purified in the same manner as [Ru(4,4ʹ-btfmb)3](Cl)2, yielding the desired
product as a dark red solid (81.3 mg, 51%). 1H NMR (400MHz, MeOD-d3, ppm): δ 9.38 (dd,
J=16.6, 1.9 Hz, 4H), 9.10 (d, J=8.4 Hz, 2H), 8.68 (s, 1H), 8.29 (d, J=5.9 Hz, 2H), 8.15 (dd,
J=5.3, 1.3 Hz, 2H), 7.95 (d, J=6.0 Hz, 2H), 7.92 (dd, J=8.3, 5.3 Hz, 2H), 7.87 (dd, J=6.0, 1.9
124
Hz, 2H), 7.66 (dd, J=6.0, 1.9 Hz, 2H). HRMS (ESI+) m/z for [M-2Cl-]2+ calcd: 453.0326. Found:
452.9735. HPLC retention time 20.33 min (>98% purity by peak area).
[Ru(4,4ʹ-btfmb)2(IP-1T)](Cl)2 (Ru-1T). Ru(4,4ʹ-btfmb)2Cl2∙2H2O (91 mg, 0.12 mmol) and IP-1T
(30 mg, 0.1 mmol) were added to a microwave vessel containing ethylene glycol (4 mL) and
subjected to microwave irradiation at 180°C for 15 min. The dark red solution was transferred
to a separatory funnel with H2O (25 mL) and CH2Cl2 (25 mL). CH2Cl2 layer was used to wash
the aqueous layer. CH2Cl2 (25 mL) and saturated aqueous KPF6 (5 mL) were used to extract
the product from the aqueous layer. The product was then purified using silica gel flash column
chromatography. The product-containing fractions were transferred to a separatory funnel with
CH2Cl2 (25 mL), H2O (25 mL), and saturated aqueous KPF6 (1 mL) and the [Ru(4,4ʹ-btfmb)2(IP1T)](PF6)2 product was isolated via extraction. The PF6− salt was then converted to the
corresponding Cl− salt in quantitative yield using Amberlite IRA-410 with MeOH as the eluent,
followed by further purification using Sephadex LH-20 with MeOH as the eluent, yielding
[Ru(4,4ʹ-btfmb)2(IP-1T)](Cl)2 as a dark red solid (43 mg, 25%). 1H NMR (400MHz, MeOD-d3,
ppm): δ 9.40 (d, J=1.9 Hz, 2H), 9.36 (d, J=1.8 Hz, 2H), 9.19 (s, 2H), 8.29 (d, J=5.8 Hz, 2H),
8.12 (d, J=5.2 Hz, 2H), 8.01 (d, J=3.6 Hz, 1H), 7.97 (d, J=6.1 Hz, 2H), 7.91 (dd, J=8.3, 5.2 Hz,
2H), 7.88 (dd, J=6.0, 1.9 Hz, 2H), 7.74 (d, J=5.0 Hz, 1H), 7.68 (dd, J=6.1, 1.8 Hz, 2H), 7.31
(dd, J=5.0, 3.7 Hz, 1H). HRMS (ESI+) m/z for [M-2Cl]2+ calcd: 494.0265. Found: 494.0261, m/z
for [M-2Cl-H]+ calcd: 987.0456. Found: 987.0474. HPLC retention time 21.75 min (99% purity
by peak area).
[Ru(4,4ʹ-btfmb)2(IP-2T)](Cl)2 (Ru-2T). Ru(4,4ʹ-btfmb)2Cl2∙2H2O (91 mg, 0.12 mmol) and IP-2T
(38 mg, 0.1 mmol) were added to a microwave vessel containing argon-purged ethylene glycol
(4 mL) and subjected to microwave irradiation at 180°C for 15 min. The resulting dark red
mixture was then isolated and purified in the same manner as Ru-1T, yielding the desired
product as a dark solid (33 mg, 29%). 1H NMR (400MHz, MeOD-d3, ppm): δ 9.40 (d, J=1.9 Hz,
2H), 9.37 (d, J=1.9 Hz, 2H), 9.17 (s, 2H), 8.29 (d, J=5.8 Hz, 2H), 8.13 (dd, J=5.2, 1.2 Hz, 2H),
7.99 (d, J=6.1 Hz, 2H), 7.94 (d, J=3.8 Hz, 1H), 7.91 (dd, J=8.3, 5.2 Hz, 2H), 7.88 (dd, J=5.9,
1.9 Hz, 2H), 7.71 – 7.67 (m, 2H), 7.47 (dd, J=5.1, 1.1 Hz, 1H), 7.43 – 7.38 (m, 2H), 7.13 (dd,
J=5.1, 3.6 Hz, 1H). HRMS (ESI+) m/z for [M-2Cl-]2+ calcd: 535.0203. Found: 535.0201, m/z for
125
[M-2Cl--H]+ calcd: 1069.0333. Found: 1069.0353. HPLC retention time 22.86 min (95.2%
purity by peak area).
[Ru(4,4ʹ-btfmb)2(IP-3T)](Cl)2 (Ru-3T). Ru(4,4ʹ-btfmb)2Cl2∙2H2O (151 mg, 0.2 mmol) and IP-3T
(76 mg, 0.164 mmol) were added to a microwave vessel containing argon-purged ethylene
glycol (4 mL) and subjected to microwave irradiation at 180°C for 15 min. The resulting dark
red mixture was then isolated and purified in the same manner as Ru-1T, yielding the desired
product as a dark red solid (55 mg, 27%). 1H NMR (700MHz, MeOD-d3, ppm): δ 9.40 (d, J=2.0
Hz, 2H), 9.37 (d, J=1.9 Hz, 2H), 9.26 (s, 1H), 9.11 (s, 1H), 8.29 (d, J=5.8 Hz, 2H), 8.14 (dd,
J=5.2, 1.3 Hz, 2H), 7.99 (s, 2H), 7.95 – 7.90 (m, 3H), 7.88 (dd, J=6.0, 1.9 Hz, 2H), 7.69 (dd,
J=6.1, 1.9 Hz, 2H), 7.40 (d, J=3.8 Hz, 1H), 7.35 (d, J=3.8 Hz, 1H), 7.31 (dd, J=3.5, 1.1 Hz, 1H),
7.23 (d, J=3.8 Hz, 1H), 7.09 (dd, J=5.1, 3.6 Hz, 1H). 13C NMR (700MHz, MeOD-d3, ppm): δ
163.28, 163.08, 162.89, 159.60, 159.43, 155.07, 154.53, 150.34, 141.88, 140.97, 140.81,
140.77, 140.60, 139.10, 137.67, 135.97, 131.57, 129.82, 129.20, 126.94, 126.39, 125.84,
125.75, 125.40, 125.33, 125.23, 124.45, 124.34, 123.11, 123.02, 122.89, 122.79, 119.88.
HRMS (ESI+) m/z for [M-2Cl-]2+ calcd: 576.0142. Found: 576.0141, m/z for [M-2Cl--H]+ calcd:
1151.0211. Found: 1151.0232. HPLC retention time 23.80 min (95.4% purity by peak area).
[Ru(4,4ʹ-btfmb)2(IP-4T)](Cl)2 (Ru-4T). Ru(4,4ʹ-btfmb)2Cl2∙2H2O (114 mg, 0.2 mmol) and IP-4T
(90 mg, 0.164 mmol) were added to a microwave vessel containing argon-purged ethylene
glycol (4 mL) and subjected to microwave irradiation at 180°C for 15 min. The reaction mixture
was transferred to a 100mL beaker and diluted with ~30 mL H2O, then treated with 3 mL of
saturated aqueous KPF6 and stirred for 5 minutes. At this time, a red precipitate formed, and
was collected using a Büchner filtration apparatus. The product was then purified following the
same procedure as described for Ru-1T, yielding [Ru(4,4ʹ-btfmb)2(IP-4T)](Cl)2 as a dark red
solid (77 mg, 35%). 1H NMR (700 MHz, MeOD-d3, ppm): δ 9.40 (d, J=2.0 Hz, 2H), 9.37 (d,
J=1.9 Hz, 2H), 9.26 (s, 1H), 9.11 (s, 1H), 8.29 (d, J=5.8 Hz, 2H), 8.14 (dd, J=5.2, 1.3 Hz, 2H),
7.99 (s, 2H), 7.95 – 7.90 (m, 3H), 7.88 (dd, J=6.0, 1.9 Hz, 2H), 7.69 (dd, J=6.1, 1.9 Hz, 2H),
7.40 (d, J=3.8 Hz, 1H), 7.35 (d, J=3.8 Hz, 1H), 7.31 (dd, J=3.5, 1.1 Hz, 1H), 7.23 (d, J=3.8 Hz,
1H), 7.09 (dd, J=5.1, 3.6 Hz, 1H). 13C NMR (175 MHz, MeOD-d3, ppm): δ 163.28, 163.08,
162.89, 159.60, 159.43, 155.07, 154.53, 150.34, 141.88, 140.97, 140.81, 140.77, 140.60,
126
139.10, 137.67, 135.97, 131.57, 129.82, 129.20, 126.94, 126.39, 125.84, 125.75, 125.40,
125.33, 125.23, 124.45, 124.34, 123.11, 123.02, 122.89, 122.79, 119.88. HRMS (ESI+) m/z:
[M–2Cl]2+ calcd for C49H26F12N8RuS3 576.0142; Found: 576.0141. [M–2Cl–H]+ calcd for
C49H25F12N8RuS3 1151.0211; Found: 1151.0232. HPLC retention time 23.80 min (99% purity
by peak area).
Electrochemistry
Voltammetry was performed in dimethylformamide (DMF, Fisher HPLC grade) that had been
dried and deoxygenated with an Inert PureSolv MD7 solvent purification system, with 100 mM
tetrabutylammonium hexafluorophosphate (TBAPF6) (Fisher) as the supporting electrolyte, in a
two-compartment low volume cell with the three-electrode configuration under argon. A 3 mm
glassy carbon disc was used as the working electrode with a platinum wire counter electrode
and a Ag/AgCl/4M KCl reference electrode. Ferrocene (Fc) was used as an internal standard.
The complex solutions were approximately 4 mM for oxidation sweeps and 0.25 mM for
reduction sweeps.
Measurements were conducted at room temperature using a WaveNow potentiostat (Pine
Research Company) with Aftermath software. Cyclic differential-pulse voltammetry (CDPV)
measurements used a sweep rate of 2 mV∙s-1 with a modulation amplitude varying from 12.5 to
100 mV. For reversible processes, the formal redox potential E°′ was taken as the average of
Epa (anodic peak potential) and Epc (cathodic peak potential). For quasi-reversible processes,
only Epa or Epc is reported.
3.4 RESULTS AND DISCUSSION
Synthesis and Characterization
[Ru(4,4'-btfmb)3]+2 and Ru-nT were synthesized utilizing our established method for related
Ru(II) 4,4'-bis(trifluoromethyl)-2,2'-bipyridine-based complexes.13 The complexes were initially
isolated as PF6− salts and subsequently purified using flash chromatography on silica. These
PF6− salts were then efficiently converted to their corresponding Cl− salts through anion
metathesis with Amberlite IRA-410, followed by further purification using size-exclusion
127
chromatography on Sephadex LH-20. The final yields were approximately 60% for [Ru(4,4'btfmb)3]+2, Ru-0T, Ru-1T, and Ru-3T, around 40% for Ru-2T, and close to 30% for Ru-4T.
These complexes underwent thorough characterization by 1D and 2D 1H NMR spectroscopy
(Figure 1, Figure S1–Figure S7), with signal assignments for [Ru(4,4'-btfmb)3]+2 and Ru-0T–
Ru-4T conducted using 1H–1H COSY NMR. The assignments aligned with those of our
previously reported, related compounds.13,16,42 Additionally, these complexes were
characterized by high-resolution ESI+ mass spectrometry (Figure S8–Figure S14). HPLC
analyses confirmed that the complexes were ≥95% pure, as determined by integration (Figure
S15–Figure S21).
The lipophilicities of the chloride salts of complexes were determined by measuring their
distribution between 1-octanol and 10 mM phosphate buffer (pH 7.4) following the “shakeflask” method as we described previously.44 The log Do/w values for this series span four orders
of magnitude, with [Ru(4,4ʹ-btfmb)3]2+ being the most hydrophilic at about -2 and Ru-4T most
lipophilic near +2 (Figure 2). The three reference compounds lacking thiophene rings
([Ru(4,4ʹ-btfmb)3]2+, Ru-phen, and Ru-0T) as well as Ru-1T show a preference for the
aqueous buffer and accordingly have negative log Do/w values. In contrast, complexes with two
to four thiophenes (Ru-2T, Ru-3T, Ru-4T) have positive log Do/w values and increase on going
from n=2 to 4. The addition of the trifluoromethyl group qualitatively improved the overall
aqueous solubility of the complexes with positive log Do/w values compared to analogous Ru(II)
and Os(II) complexes with other coligands where 4T often leads to precipitation at the
octanol/buffer interface.42,44
128
Figure 1. Labelled structures and 1H NMR spectra showing aromatic region for [Ru(4,4ʹ-btfmb)3](Cl)2 and Ru-nT
(n=0−4) in MeOD-d3 (Cl− salts; 298 K). All spectra were collected at 500 MHz, except for Ru-4T, which was
collected at 700 MHz.
129
Figure 2. Lipophilicity of [Ru(4,4ʹ-btfmb)3](Cl)2, Ru-phen, and Ru-nT (n=0–4) in 1-octanol and phosphate buffer
using the in-house “shake-flask” method.
Spectroscopy
Electronic Absorption
Figure 3: UV-vis spectra of [Ru(4,4ʹ-btfmb)3]2+, Ru-phen, and Ru-nT (n=0–4) normalized to the π‒π* peak near
295 nm.
The UV-vis absorption spectra of the series are overlaid in Figure 3, and the corresponding
molar extinction coefficients for various peak maxima are summarized in Table 1. The sharp
peaks near 295 nm and below are due to π‒π* transitions involving the 4,4ʹ-btfmb ligand,49 as
well as phen and/or IP in the cases of Ru-phen and Ru-nT. The energies of these transitions
are not affected by the length of the thiophene chain appended to the IP ligand. The broader
130
and less intense peak between 400 and 500 nm with a local maximum at 456 nm in [Ru(4,4ʹbtfmb)3]+2 is due to Ru2+(dπ)→L(π*) 1MLCT transitions. The substitution of one 4,4ʹ-btfmb
ligand for phen or IP causes the lowest energy 1MLCT transitions to red-shift by 10‒20 nm,
suggesting more electron density on the metal center as would be expected for these
heteroleptic complexes. Assuming that the 4,4'-btfmb π* orbital remains the acceptor orbital for
the lowest energy 1MLCT transitions in all cases, the effect of phen or IP is primarily on the dπ
orbital energy. This effect is most evident for Ru-phen, Ru-0T, and Ru-1T. For complexes
containing the IP-nT ligand, additional ligand-localized transitions overlap the 1MLCT
transitions. These isolated transitions can be seen in the absorption spectra of the analogous
uncomplexed IP-nT ligands and free oligothiophenes.56 Ru-2T through Ru-4T have
contributions from 1LLCT (nTĺIP) and 1ILCT (nTĺnT) transitions that shift to longer
wavelengths and increase in 1ILCT character with increasing n. The lowest energy singletsinglet transitions tend to be mixed 1MLCT/1LLCT states for Ru-2T and mixed 1ILCT/1LLCT for
Ru-3T and Ru-4T, based on what has been observed previously in related compounds. 7,13,42,43
Table 1: Molar Extinction coefficients at Selected Wavelengths for the Complexes of This Study.
cmpd
λabs (nm) (log (ε / M−1 cm−1))
[Ru(4,4ʹ-btfmb)3]+2
244 (4.34), 294 (4.84), 427 (4.01), 456 (4.12),
Ru-phen
260 (4.69), 296 (4.80), 353 (4.05), 432 (4.09), 471 (4.12)
Ru-0T
248 (4.68), 296 (4.84), 341 (4.13), 435 (4.12), 470 (4.13)
Ru-1T
245 (4.57), 294 (4.92), 335 (4.51), 462 (4.20)
Ru-2T
250 (4.68), 295 (4.89), 370 (4.69), 462 (4.35)
Ru-3T
251 (4.58), 294 (4.76), 410 (4.57)
Ru-4T
246 (4.57), 297 (4.81), 351 (4.33), 441 (4.70)
Singlet Oxygen Sensitization
The singlet oxygen quantum yields (ΦΔ) of the complexes were calculated by measuring the
intensity of O2 phosphorescence (1∆g→3Σg) centered at 1260 nm against [Ru(bpy)3]2+ as the
standard (ΦΔ=0.56).57 These are reported in Table 2. [Ru(4,4ʹ-btfmb)3]+2, Ru-phen, and Ru-0T
are moderately efficient 1O2 generators (ΦΔ=0.47‒0.64) and similar to [Ru(bpy)3]2+. However,
the thiophene-containing complexes Ru-1T‒Ru-4T exhibit larger differences (ΦΔ=0.13‒0.66)
131
and are less efficient than what has been observed for related compounds. 7,43 The largest 1O2
quantum yield was measured for Ru-3T at 66%. While all of the complexes show a
wavelength-dependence for ΦΔ, Ru-4T exhibits a notable concentration dependence for the
1O
2 quantum yield (Figure S22 and Table S1). These differences in ΦΔ appear to be unrelated
to differences in other photophysical parameters such as emission wavelengths and triplet
lifetimes (vide infra).
Emission
Figure 4: Normalized emission spectra [Ru(4,4ʹ-btfmb)3]2+ and the Ru-nT series as PF6‒ salts at RT and at 77K.
The RT emission was measured in MeCN degassed by freeze-pump-thaw (5 cycles). The 77K emission was
measured in a 4:1 EtOH:MeOH glass. Excitation wavelengths are noted in parentheses.
All of the complexes in the series exhibited a broad, featureless red emission band at room
temperature (Figure 4a, Table 2). This emission was centered around 636 nm (τem =1.5 µs) for
the parent [Ru(4,4ʹ-btfmb)3]2+ complex in MeCN at room temperature (RT) and shifted to
shorter wavelengths with vibronic intervals of around 1350 cm -1 at 77 K (Figure 4b).48–51 Such
behavior is consistent with emissive 3MLCT states in Ru(II) complexes with polypyridyl
ligands.58 The thermally induced Stokes shift (ΔES) of around 830 cm-1 is slightly smaller than
the related model complex [Ru(bpy)3]2+ (ΔES= 1127 cm−1).59 Similar to what was observed for
the 1MLCT absorption bands, the introduction of a phen or an IP ligand results in bathochromic
shifts of up to 30 nm but otherwise similar spectra and lifetimes (τem =0.8‒0.9 µs). The 3MLCT
emission energies and lifetimes (τem =0.6‒0.8 µs) also do not depend of the number of
132
thiophenes in the IP-nT ligand, suggesting that the π* acceptor orbitals in the Ru(dπ)→L(π*)
transitions are localized to the phen/IP portion of the IP-nT ligands. However, the emission
quantum yield (Φem) decreases from 16% for [Ru(4,4ʹ-btfmb)3]2+ to 9% for Ru-phen and Ru0T. Addition of one thiophene drops the value of Φem to around 2%, and additional thienyl rings
further decrease the emission output to <1%. Although Ru-4T produces weak but detectable
emission, a value for Φem was not calculated due to the extremely low signal-to-noise ratio.
The values for Φem increase up to ten-fold at 77 K but Ru-4T is still only about 0.14%. While
the nature of the emissive 3MLCT state does not appear to change across the series, the lower
emissive quantum yields with increasing n point toward the involvement of additional excited
states.
Table 2: Spectroscopic data for compounds [Ru(4,4ʹ-btfmb)3](PF6)2, Ru-phen, and Ru-0T±Ru-4T as PF6− salts.
Excitation wavelengths are indicated in parenthesis. Emission lifetimes were measured following a <5 ns 355 nm
laser pulse.
RT emission
cmpd
λem. (λex)
Φem
τem / μs
636 (458)
1.6×10−1
1.5
Ru-phen
660 (470)
9.0×10−2
0.89
Ru-0T
662 (470)
9.1×10−2
0.83
Ru-1T
661 (460)
1.8×10−2
0.79
Ru-2T
659 (457)
4.1×10−3
0.62
Ru-3T
664 (462)
1.3×10−3
0.79
Ru-4T
650 (435)
v. wk.
0.64
/ nm
[Ru(4,4ʹbtfmb)3]2+
τTA / μs)
77K emission
λem. (λex)
/ nm
604, 653
(460)
619, 668
(472)
619, 669
(470)
620, 670
(469)
634, 687
(463)
623, 673
(470)
625, 672
(468)
133
Φem,77K
ΦΔ (λex / nm)
λex=355
λex=532
nm
nm
2.5×10−1
0.47 (462)
1.5
1.5
2.4×10−1
0.64 (470)
0.89–0.94
0.77–0.92
1.9×10−1
0.50 (469)
0.78–0.86
0.78–0.87
2.0×10−1
0.13 (462)
0.81–0.93
0.71–0.87
4.3×10−2
0.28 (466)
0.61–0.72
0.63–0.71
9.7×10−3
0.66 (470)
20–21
22–24
1.4×10−3
0.40 (467)
19–20
20–21
Transient Absorption and Excited State Pathways
Figure 5: Transient absorption (TA) spectra of [Ru(4,4ʹ-btfmb)3](PF6)2, Ru-phen, and Ru-nT as PF6− salts in
degassed MeCN at RT (λex=355 nm). The profiles for λex=532 nm are similar (Figure S24). The dotted line
indicates ΔO.D.=0.
The triplet excited states were investigated using nanosecond transient absorption (TA)
spectroscopy with excitation from a 355 nm or 532 nm laser of ≤5 ns pulse width. The
responses with the two different excitation wavelengths are similar so only the shorter
wavelength excitation is shown in Figure 5. The differential excited state absorption (ESA)
spectra were collected using solutions of the PF6− salts of the compounds in degassed (5x
freeze-pump-thaw) MeCN. Early time slices are presented in Figure 5 and the full relaxation
spectra are collected in Figure S23 and Figure S24. Transient lifetimes were measured at the
ESA maxima and bleach minima and are listed in Table 2.
The ESA profiles of the [Ru(4,4ʹ-btfmb)3](PF6)2, Ru-phen, Ru-0T, and Ru-1T are similar,
consisting primarily of a strong 1MLCT ground state bleach near 450 nm superimposed with
new ESA characteristic of the 3MLCT state. The stronger ESA near 375 nm involves bftmb−
transitions, and the extremely weak and broad absorption past 525 nm is due to 4,4'-btfmb− or
LMCT transitions involving Ru(II). With TA lifetimes match the emission lifetimes and support
lowest lying 3MLCT states for these complexes.
The ESA profile of Ru-2T exhibits features consistent with an IP-nT ligand-localized triplet
excited state, as we have previously reported.7,13,45,60–62 This broad and rather intense ESA
134
near 450‒700 nm is superimposed on the bleach with its minimum near 370 nm due to a
strong ground state absorption contributed by the IP-2T ligand (Figure 3). Ru-2T has the
signature of an 3IL-based excited state, but its TA lifetime matches the 3MLCT emissive lifetime
at all wavelengths. This suggests an equilibrium between 3MLCT and 3IL-based states that are
close in energy and decay with a common lifetime.63
The ESA spectra of Ru-3T and Ru-4T are characteristic of oligothiophene-based 3ILCT states,
with longer TA lifetimes. The excited state relaxation pathways are proposed in Scheme 1. Ru3T shows a bleach corresponding to the ground state ππ* transition near 410 nm and a broad
ESA with its maximum near 610 nm. For Ru-4T these were shifted to around 430 nm and 655
nm, respectively. These ESA features are similar to those of the free IP-3T and IP-4T
ligands.42,44 The decays are monoexponential, with τ=~20 μs for both complexes, indicating
that the long-lived 3ILCT is decoupled from the shorter-lived emissive 3MLCT, and further
supported by the very low emission quantum yields for Ru-3T and Ru-4T.
The TA spectra for each compound was also collected with 532 nm excitation, and there is
little difference between the two wavelengths (compare Figure S24 and Figure S25). This
suggests that the excited state dynamics on the ns to µs timescales are independent of the
excitation wavelength.
135
Scheme 1: Jablonski diagram illustrating the excited state relaxation pathways of complexes Ru-3T and Ru-4T.
Energies are not to scale.
Electrochemistry
Figure 6: Redox potentials (vs. Fc) of [Ru(4,4ʹ-btfmb)3]2+, Ru-phen, and Ru-nT as PF6− salts in degassed MeCN
at RT and their tentative assignments.
The electrochemistry of Ru(II) polypyridyl complexes is typified by single electron processes
involving one-electron oxidation of the metal center and three sequential reductions on each of
the ligands.64 Oxidation of the Ru2+ center (+0.98 V versus Fc, MeCN) tends to be
electrochemically reversible, and the ensuing low spin 4d5 complex is chemically stable. In
complexes like [Ru(bpy)3]2+, the first reduction (‒1.72 V versus Fc, MeCN) involves the lowestenergy ligand π* orbital. Since the low spin 4d6 configuration is thereby unaffected, the
136
complex remains substitutionally inert, and the process is also reversible. The added electron
is localized on one ligand, and thus [Ru(bpy)3]2+ exhibits three sequential one-electron
reductions under straightforward electrochemical conditions. In a potential window widened by
low temperature cyclic voltammetry, [Ru(bpy)3]2+ has been shown to participate in a total of six
one-electron reductions.65,66
There are additional redox processes for some members of the present series due to the
presence of the electrochemically active oligothiophene67 unit in complexes Ru-2T‒Ru-4T.
The formal redox potentials of the series were measured by cyclic differential pulse
voltammetry (CDPV) to enhance the signal, with ferrocene (Fc) as an internal reference (E1/2
(Fc/Fc+)=0.380 V vs SCE68). The potentials are tabulated in Table 3 and plotted in Figure 6
with tentative assignments.
The metal oxidation of [Ru(4,4′-btfmb)3]2+ and the three one-electron reductions agree well with
published data49 and are around 0.3‒0.5 V more positive than the corresponding processes in
[Ru(bpy)3]2+, a consequence of the electron-withdrawing nature of the 4,4′-btfmb ligands. The
shift of the Ru2+→ Ru3+ oxidation is slightly attenuated when one 4,4′-btfmb ligand is replaced
with phen or IP-nT, which mirrors the shift of the MLCT absorption energy (Table 1). The
oxidation of nT becomes more favorable with increasing n, consistent with the behavior of free
oligothiophenes.69 The oxidation potential of the thienyl group ranges from +1.02 V for Ru-1T
to +0.58 for Ru-4T and is less positive than the metal center in all cases, indicating that nT is
more easily oxidized than Ru(II) regardless of the number of thiophenes. The oxidation of nT
does shift the Ru2+→ Ru3+ oxidation slightly more positive, consistent with a decrease in
electron density on the metal.
[Ru(4,4′-btfmb)3]2+ and the other complexes without thiophenes (Ru-phen, Ru-0T) as well as
Ru-1T exhibit five sequential reductions spanning −1.24 to −2.81 V (Figure 6). For [Ru(4,4′btfmb)3]2+ this involves sequential one-electron reductions on each of the three 4,4′-btfmb
ligands followed by second reductions on two of those ligands (within the experimental
potential window). We believe this study is the first to report the second reductions of the two
4,4′-btfmb ligands occurring between −2.2 and −2.9 V for [Ru(4,4′-btfmb)3]2+.
137
When one of the 4,4′-btfmb ligands is replaced by phen, IP-0T, or IP-1T, the third reduction
involves phen or IP and is less favorable by around 0.4‒0.5 V (owing to their lack of electronwithdrawing ‒CF3 substituents). Ru-2T to Ru-4T exhibit similar reductions involving the 4,4′btfmb coligands and IP, but they accommodate additional reductions on the oligothienyl
groups. For Ru-2T, the 2T group accepts only one electron and this sixth reduction is the least
favorable and occurs near −2.81 V. The 3T group of Ru-3T can be doubly reduced (−2.54 and
−2.96 V), where the first nT reduction is easier than the last 4,4′-btfmb reduction. Continuing
this trend, the 4T group of Ru-4T accommodates three electrons (−2.23, −2.55, and −2.93 V),
with the first nT reduction occurring near that of IP at −2.13 V. The effect of the thiophene
chain length is dramatic. The nT reduction potential shifts positive by more than 0.5 V on going
from two to four thiophenes, and the Ru-4T complex can accommodate at least eight extra
electrons in its ground state at room temperature!
Table 3. Formal redox potentials for the hexafluorophosphate salts of the complexes measured using CDPV at
approximately 1.0 mM in MeCN containing TBAPF6. The potentials are referenced in volts (V) against ferrocene
as the internal standard. The working and reference electrodes were glassy carbon and Ag/AgCl/4M KCl,
respectively. Overlapping waves were deconvoluted mathematically (error approximately ±0.02 V).
LL2−→LL22−
LL1−→LL12−
LL3→LL3−
LL2→LL2−
LL1→LL1−
Ru2+→Ru3+
[Ru(4,4'-btfmb)3]2+
−2.75
−2.21
−1.61
−1.39
−1.24
+1.34
Ru-phen
−2.70
−2.30
−2.01
−1.49
−1.29
+1.18
Ru-0T
−2.81
−2.29
−2.14
−1.54
−1.33
+1.18
Ru-1T
−2.75
−2.30
−2.12
−1.51
−1.32
+1.22
+1.02
−2.81
−2.71
−2.29
−2.12
−1.51
−1.31
+1.26
+0.89
−2.96
−2.54
−2.86
−2.32
−2.15
−1.54
−1.32
+1.29
+0.69
−2.55
−2.23
−2.83
−2.33
−2.13
−1.52
−1.32
+1.27
+0.58
Compound
nT2−→nT3−
nT−→nT2−
Ru-2T
Ru-3T
Ru-4T
−2.93
nT→nT−
138
nT→nT+
In Vitro Photobiological Activity
The compounds in this series were assessed for their cytotoxicity in the absence of light (dark)
as well as their light-triggered cytotoxicity against human skin melanoma cells (SK-MEL-28)
and lung carcinoma cells (A549) cultured as 2D monolayers (Figure 7). Details can be found in
our previously published work7,13,16 as well as in the SI.
Normoxia.
Figure 7. In vitro cytotoxicity and photocytotoxicity log (EC50 ± SEM) values (left) and PI values (right) obtained
from dose−response curves in the SK-MEL-28 melanoma cell line (a) and A549 cell line (b) with [Ru(4,4ʹbtfmb)3](Cl)2 and Ru-phen−Ru-4T. Treatments include dark (black circles) or light delivered at a fluence of 100 J
cm−2 and irradiance of ~20 mW cm-2. The light wavelengths were broadband visible (400−700 nm, blue squares),
523 nm (green inverted triangles), or 633 nm (red triangles. Data collected under normoxic (∼18.5% O2) and
hypoxic (1% O2) conditions is represented with closed symbols and open symbols, respectively.
For each photobiological assay in normoxia, SK-MEL-28 or A549 cells growing in log phase
were seeded into 384-well plates, with one set for dark cytotoxicity evaluation and another set
for photocytotoxicity assessment. After allowing the cells to adhere for 3−5 hours at 37°C, they
were treated with varying concentrations of the compounds (1 nM to 300 µM for all
compounds, 1 aM to 300 µM for Ru-4T). After a 13−20 h drug-to-light interval (DLI), the light
plates were exposed to specific light treatments, while the dark plates remained in the
139
incubator. The light treatment used a fluence of 100 J cm-2 emitted from broadband visible
(400−700 nm, 21 mW cm-2) or monochromatic (±2.5 nm) green (523 nm, 18 mW cm-2) or red
(633 nm, 18 mW cm-2) LEDs. After light treatment, the plates were allowed to incubate in
normoxia at 37°C for 24 h. Cell viability was then assessed indirectly using a resazurin-based
cell viability assay. Sigmoidal fits of the dose-response curves were used to calculate the
effective concentrations required to reduce cell viability by 50% (EC50 values) for both
treatment conditions. Phototherapeutic indices (PIs), representing the amplification of cytotoxic
effects upon light exposure, were calculated as ratios of dark-to-light EC50 values.
In the absence of light activation, the complexes in this series were relatively nontoxic to SKMEL-28 and A549 cells (Figure 7, Table S4−Table S5). [Ru(4,4ʹ-btmfb)3]2+, Ru-phen, and Ru0T exhibited dark EC50 values that exceeded the highest concentration tested in the assay
(>300 µM), indicating a lack of toxicity. Consequently, the phototherapeutic indices (PIs) for
these compounds were technically undefined but were reported as a lower limit using 300 µM
as the dark cytotoxicity threshold. Ru-1T−Ru-3T were considered nontoxic toward both cell
lines, with dark EC50 values >100 µM. Ru-4T had the lowest dark EC50 values, which were still
relatively high at 99 µM and 120 µM in in SK-MEL-28 and A549 cells, respectively.
SKMEL28 cells. [Ru(4,4ʹ-btmfb)3]2+, Ru-phen, and Ru-0T were inactive against both cell lines
under any light condition. Incorporation of thiophene rings (n=1−4 thienyl groups) resulted in
progressively higher potency with visible light, spanning from 22 µM (PI=11) for the least active
thienyl compound Ru-1T to as low as 10 nM (PI=10,000) for the most active compound Ru-4T
in SK-MEL-28. Appending two thiophenes (Ru-2T) improved the potency 37-fold, shifting the
EC50 values into the sub-micromolar regime near 0.61 µM (PI=290). Another 20-fold
enhancement in photocytotoxicity was accomplished on going to three thiophene rings (Ru-3T;
SK-MEL-28: EC50=0.21 µM, PI=920), and yet another 20-fold improvement occurred with four
thiophenes (Ru-4T; SK-MEL-28: EC50=10 nM, PI=10,000).
The activity of the series was mostly diminished with longer-wavelength green or red light. With
green light, Ru-1T lost most of its activity (EC50=103 µM, PI=2). The activity Ru-2T dropped by
a factor of 3 but was still single-digit micromolar (EC50=1.9 µM, PI=91), and Ru-4T dropped by
140
10-fold but remained sub-micromolar (EC50=0.12 µM, PI=815). Ru-3T, on the other hand,
maintained its activity (EC50=0.27 µM, PI=731). With red light, Ru-3T (EC50=12.1 µM; PI=329)
and Ru-4T (EC50=5.64 µM; PI=461) showed modest activity. This outcome aligns with the
anticipated behavior of compounds that exhibit minimal absorption of red light.70
A549 cells. The A549 cell line proved more resistant to the light-triggered compounds than SKMEL-28 under similar conditions, but the overall trend in activity remained the same (Figure 7,
Table S4). The cytotoxicity was minimal for all complexes, with EC50 values ranging from 120–
300 µM. Compounds [Ru(4,4ʹ-btfmb)3](Cl)2, Ru-phen, and Ru-0T were completely inactive
under all light conditions, and Ru-1T (visible EC50=29 µM, PI=7) was the least active thienyl
complex, followed by Ru-2T (visible EC50=1.37 µM, PI=127), Ru-3T (visible EC50=0.70 µM,
PI=280), and finally Ru-4T (EC50=0.077 µM, PI=1500). Treatments with red and green light
similarly attenuated the overall activity of the series.
Hypoxia.
The assays under hypoxic conditions mirrored the procedure followed for normoxia, with a
notable exception: post-cell adhesion, the plates—both for dark and light conditions—were
transferred to a hypoxia chamber with 1% O2 atmosphere for a duration of 2–3 h prior to
introducing the compounds. After a DLI of 18 h in the hypoxia chamber, the concentration of
dissolved O2 was verified in the assay wells using an immersive optical probe before sealing
the plates to be light treated with transparent qPCR film that has a low gas permeability. Light
was delivered outside the hypoxia chamber for approximately 1.5 h, while the dark plates were
kept inside an incubator. The films were removed from the light plates at the end of the
illumination period. Both dark and light plates were then incubated in normoxia (37°C, 5% CO2,
with relative humidity above 90%) for 20–23 h, after which time the cell viability was assessed.
Hypoxic conditions broadly attenuated the activity of the series in both cell lines, where A549
cells were completely resistant to all treatments and SK-MEL-28 cells were moderately
sensitive under certain conditions. Regardless of light-treatment, [Ru(4,4ʹ-btfmb)3](Cl)2 and
compounds Ru-0T–Ru-2T exhibited no photocytotoxic effect under hypoxic conditions toward
SK-MEL-28 cells. On the other hand, Ru-3T showed an EC50 value of ~1 µM with visible and
141
green light treatments. With red light, Ru-4T was less active (EC50=26 µM, PI=4), but
otherwise the series was inactive. The dramatic decline in the activity of Ru-3T and Ru-4T,
coupled with the complete inactivity of the other compounds in the series under hypoxic
conditions, suggests that oxygen plays a pivotal role in their photocytotoxic mechanisms. This
observation solidifies the hypothesis that the primary driver behind the observed
photocytotoxicity of these compounds in normoxic conditions stems from oxygen-dependent
photophysical processes.
Biological replicates.
Figure 7 and Table S5 show representative activity against SK-MEL-28 for one biological
replicate based on the mean values from three technical replicates with minimal standard
deviation. Since a greater degree of variation is expected over biological replicates, we
assessed the activity of the most potent compounds (Ru-3T and Ru-4T) over five biological
replicates (each performed in triplicate) with SK-MEL-28 cells (Table S6−Table S9). Repeat 0
corresponds to the data in Figure 7 and Table S5. The subsequent biological replicates are
labeled Repeats 1–5. SK-MEL-28 cells were selected for these studies because this is the cell
line we have historically used to rank potency across all compounds made in our lab. 13
Without light both Ru-3T and Ru-4T were completely nontoxic over all biological replicates in
both normoxia and hypoxia, with mean EC50 of 197 µM for Ru-3T and 100 µM for Ru-4T. The
visible EC50 values for Ru-3T in normoxia ranged from 70 to 700 nM (mean=440 nM); the
corresponding visible PIs ranged from 305–2700 (PIavg=1400). The EC50 values for Ru-4T
under the same conditions ranged from 9–59 nM with a mean of 17 nM (PI=1900–12000,
mean=6500). All 6 replicates were within an order of magnitude of each other.
Using green light, the EC50 values for Ru-3T fluctuated between 0.22–0.66 µM (PIavg=0.29
µM), with PIs between 300–1000 (PIavg=548). The mean values were determined to be 0.29
µM and 548, respectively. For Ru-4T, the results were slightly more variable, with green EC50
values from 54 to 320 nM, and corresponding PIs extending from 800–3000 range. Even
though Ru-4T was generally more active than Ru-3T, the difference in their activities was only
around five-fold.
142
Under red light illumination in normoxia, the EC50 values of both Ru-3T and Ru-4T were
reproducibly in the single digit micromolar regime. Ru-3T exhibited red EC50 values between 2
and 7 µM (mean=2.2 µM) and PIs ranging from 23 to 101 (PIavg=52). The activity of Ru-4T was
slightly more consistent than Ru-3T, with red EC50 values falling between 1.3 to 3.3 µM
(mean=2.2 µM) with PIs between 21 and 91 (PIavg=53). The red-derived activity of Ru-3T and
Ru-4T is notable as both complexes exhibit vanishingly low molar extinction coefficients at 633
nm. This characteristic has been reported previously,70 and tends to require lowest-lying 3ππ*
triplets with prolonged lifetimes such as those observed for Ru-3T and Ru-4T.
The activity of Ru-3T in hypoxia was mostly consistent but repeats 3 and 4 were outliers that
should be ignored: repeat 3 displayed unusually high activity compared to the other replicates,
and no activity at all was observed in repeat 4 (see Table S16). In repeats 1, 2, and 5, the
visible and green activity of Ru-3T improved when compared to our initial measurements, but
all values were still within one order of magnitude. With visible light, the EC 50 value was 1.2 µM
(PI=170) in repeat 0 but improved to a range of 410–870 nM (PI=230–700, PIavg=300) in
repeats 1, 2, and 5. Similarly, the initial green EC50 value for Ru-3T was 1.1 µM (PI=180),
which lowered to 0.3–1.0 µM (PI=200–700, PIavg=270) in subsequent assays. The observation
of two distinct outliers, and the fact that the activity of Ru-3T was actually greater in
subsequent biological replicates, underscores the importance of performing biological
replicates when evaluating photobiological efficacy.
The activity of Ru-4T varied slightly more in hypoxia than it did in normoxia, but all EC50 values
were still within roughly one order of magnitude. Using visible light treatment, repeats 0 and 2–
5 fell between 0.13–0.54 µM (PI=200–400), but repeat 1 presented a slightly lower EC50 of 35
nM (PI=2900). With green and red light treatments, Ru-4T proved to be consistently more
active than what was originally observed in repeat 0, with initial EC50 values of 1.0 µM (PI=90)
and 26 µM (PI=4), respectively, which improved to a range of 200–900 nM (PI=100–500,
PIavg=210) and 6–9 µM (PI=7–30, PIavg=9) during repeats 1–5. The average hypoxic red
activity (<10 µM) of Ru-4T is particularly notable for this class of PS, and is comparable to the
“ubertoxin” ML19C01 (which presents sub-nanomolar normoxic phototoxicity).13,16
143
In conclusion, across six biological replicates, Ru-4T has consistently displayed superior
activity compared to Ru-3T, particularly when activated with visible light under normoxic
conditions. Under these conditions, Ru-4T had an average EC50 of 13 nM (PIavg=9300).
Notably, the values obtained in all biological replicates were within one order of magnitude,
making Ru-4T more consistent than ML19C01, which varies by up to 6 orders of
magnitude.13,16 This marked improvement in consistency may be related to the improved
aqueous solubility observed in this family of complexes, and the effects of fluorination on
biological reproducibility is being investigated further.
3.5 CONCLUSIONS
Herein we synthesized a series of Ru(II) polypyridyl complexes featuring two 4,4'-btfmb
coligands and the IP-nT ligand (n=1–4) and compared these systems to the reference
compounds [Ru(4,4'-btfmb)3]+2, Ru-phen, and Ru-0T. This study stands as part of our larger
initiative to better understand the impact of structural variations on the photophysical,
photobiological, and electrochemical profiles of oligothienyl-containing metal complexes.
The emission quantum yields were moderate for [Ru(4,4'-btfmb)3]+2 and become very low as
the number of thiophenes increased in the IP-nT ligand. Ru-3T and Ru-4T displayed the
longest T1 lifetimes in the series at approximately 20 μs. The 1O2 quantum yields ranged from
poor to moderate, with Ru-3T and Ru-4T having the higher quantum yields among the thienyl
complexes.
Electrochemically, the Ru(II/III) oxidation processes were typical for this family of complexes.
Additional thiophene oxidations (nT0/+) were observed at +1.02 V for Ru-1T to as low as +0.58
V for Ru-4T. All complexes underwent at least five reductions: two for each 4,4'-btfmb ligand
and one for the phen or IP ligands. Complexes Ru-2T–Ru-4T underwent additional reductions
involving their nT groups, occurring at potentials ranging from −2.23 V to −2.96 V. Specifically,
Ru-4T could accommodate 8 additional electrons, highlighting its potential for potential
photoredox applications.
144
Over five biological replicates, Ru-4T demonstrated EC50 values averaging 13 nM (PIavg=9300)
in normoxia under visible light. Notably, all values fell within one order of magnitude of each
other, making Ru-4T significantly more consistent than some previously studied IP-4T
complexes. We attribute this consistency in part to the improved aqueous solubility for these
complexes. It is common for IP-4T complexes to precipitate in aqueous solution, but Ru-4T
dissolves very well.
In summary, this fluorinated family of Ru(II) polypyridyl complexes stands out for its notable
water solubility, tight consistency in photobiological assays, and compelling electrochemical
properties. These findings highlight Ru-4T as an excellent candidate for further applications.
3.6 ASSOCIATED CONTENT
Synthetic characterization (1D and 2D NMR, HPLC, HRMS) and (photo)biological data are
included in the Supporting Information. This material is available free of charge via the Internet
at https://www.acs.com.
3.7 ACKNOWLEDGEMENTS
S.A.M. and C.G.C. thank the National Cancer Institute (NCI) of the National Institutes of Health
(NIH) (Award R01CA222227) and the National Science Foundation (NSF) (Award NSF
2102459) for support. The content in this work is solely the responsibility of the authors and does
not necessarily represent the official views of the National Institutes of Health. S.A.M. also thanks
Dr. Daniel Todd as UNCG’s Triad Mass Spectrometry Facility manager and his assistants
Jennifer Simpson and Diane Wallace. S.A.M. likewise thanks Dr. Franklin Moy (UNCG) and Dr.
Brian Edwards (UTA) for their experimental support and instrument maintenance as NMR facility
managers.
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3.9 SUPPORTING INFORMATION
Synthesis and Characterization
Preparation of Target Complexes
Chart S1. Structures of [Ru(btfmb)3](Cl)2, Ru-phen, and Ru-0T–Ru-4T.
Ru(4,4ʹ-btfmb)3 was synthesized using an adapted literature procedure that describes the
microwave-assisted synthesis of tris-homoleptic Ru(II) polypyridyl complexes.1 To a solution of
Ar-purged ethylene glycol, Ru(Cl)3xH2O was combined with 3 equivalents of 4,4′-btfmb ligand
and subjected to microwave irradiation for 45 minutes at 180 °C. The crude reaction mixture is
then isolated and purified using the same method described above.
Complexes Ru-phen and Ru-0T–Ru-3T were prepared by adapting previously described
synthetic methods.2,3 Briefly, Ru(4,4ʹ-btfmb)2(Cl)2•2H2O was combined in 20% excess with the
corresponding phen or IP-based ligand in a solution of argon-purged ethylene glycol and was
subjected to microwave irradiation (hold temperature: 180 °C) for 15 minutes. The complexes
were isolated as their PF6− salts by transferring the crude reaction mixture to a separatory
funnel containing H2O. The products were then washed with dichloromethane (DCM) several
times, which removes any unreacted Ru(4,4ʹ-btfmb)2(Cl)2•2H2O precursor. An aqueous
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solution of saturated KPF6 is then added to the separatory funnel, and then the products were
extracted using DCM. The complexes are then purified using flash silica gel chromatography
(100% MeCN → 10% water in MeCN → 7.5% H2O, 0.5% KNO3, in MeCN) and then converted
to their Cl salts via elution through a column of amberlite resin. Finally, the complexes are
purified using size-exclusion chromatography, which affords the separation of other Ru(II)
species such as [Ru(4,4ʹ-btfmb)3](Cl)2 that may elute at similar times when using (normal
phase) silica chromatography.
The isolation of Ru-4T was modified due to its poor solubility in DCM. To circumnavigate this,
the crude reaction mixture was diluted with water in a beaker and treated with saturated
aqueous KPF6, and then the solid [Ru(4,4ʹ-btfmb)2(IP-4T)](Cl)2 was isolated via a Büchner
filtration apparatus and was subsequently washed with 100 mL of H2O and a few drops of
ether. Thereafter, the complex was purified using the same methods described above.
1H NMR and 1H–1H COSY NMR characterization
The Ru(II) polypyridyl complexes with α-oligothiophene chains were characterized in-depth
using 1H NMR spectroscopy. Our strategy for assigning the 1H signals across all the
complexes was centered around identifying each spin system present on the ligands: btfmb,
phen, or IP-nT. This began with [Ru(4,4ʹ-btfmb)3](Cl)2, where three diagnostic signals were
observed. These signals enabled the rapid assignment of btfmb signals in all other complexes
in the series. The symmetry and electronic properties of the Ru-phen complex were altered by
replacing one btfmb ligand with phen, resulting in unique phenanthroline-based signals. Their
distinct Newtonian characteristics and the through-bond correlations observed in 1H -1H COSY
aided in their identification.
The assignment of Ru-0T–Ru-3T was largely straightforward. Specifically, Ru-0T and Ru-1T
were the easiest to assign due to the electromagnetic and chemical distinctions between the
imidazole or thiophene peaks from the other pyridyl 1H signals. However, with each additional
thiophene ring on the alpha-oligothiophene chain, the complexity of the assignment increased.
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A crucial observation was the distinct chemical shift of the thiophene ring closest to the
imidazole unit, attributed to its spatial proximity to nearby nitrogen atoms. This allowed us to
distinguish the first ring of the thiophene chain from all other signals in the spectra for Ru-1T–
Ru-4T. In the case of Ru-2T, the assignment process was simplified due to the presence of
only two thiophene rings: the first positioned next to the imidazole and the second
characterized by three signals. The assignment of Ru-3T was also straightforward, with
thiophene rings being easily discernable.
However, the Ru-4T complex posed a unique challenge due to the presence of two
electromagnetically similar thiophene rings in the middle of the alpha-oligothiophene chain. To
overcome this, we first utilized 1H-13C HSQC NMR to associate the 13C signals with the 1H
signals confirmed via 1H-1H COSY NMR. Subsequently, we used 13C-1H HMBC NMR to
identify correlations shared between unidentified thienyl ring 13C atoms and previously
identified thienyl protons, which were then used to assign the remaining thiophene ring protons
in the Ru-4T complex.
Characterization with HPLC and ESI+ MS
Each complex was evaluated using HPLC and MS to further confirm their purity. The HPLC
method utilized a reverse-phase silica column and a gradient of 98% → 5% A; A = 0.1% formic
acid in H2O, B = 0.1% formic acid in MeCN, and was adopted from previously reported
examples,2–4 In general, the complexes eluted between 20–24 minutes. The purity of the
complexes was determined to be at least 98% by integration.
The complexes were evaluated by ESI+-MS via direct injection (Figure S8–Figure S14. In
every case, the observed ions shown signature isotopic distributions corresponding to a +2
charged Ru(II) species, where the Cl− counteranions have dissociated, but otherwise the
complex was left intact. Ru-1T–Ru-4T shown a +1 charged peak, which is due to loss of a
proton from a nitrogen on the imidazole unit.
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Lipophilicity Determination
To determine the lipophilicity (or distribution coefficient, D) of the compounds, an in-house
modified "shake-flask" method was employed at pH 7.4.5 Saturated 1-octanol solution was
prepared by mixing 1-octanol with 10 mM phosphate buffer (pH 7.4) in a ratio of 4:1. Similarly,
a saturated phosphate buffer solution was prepared by mixing 10 mM phosphate buffer with 1octanol in the same ratio. These solutions were vigorously shaken at 300 rpm for 24 hours at
room temperature using a VWR vortex mixer to achieve saturation. Excess solvent from
saturation was removed from both solutions using pipette before further use. For the
lipophilicity determination, a 50 μM solution of chloride salt of each compound was prepared in
saturated 1-octanol or saturated phosphate buffer (500 μL), then an equal volume (500 μL) of
the other saturated solvent was added to the solution, resulting in a final volume of 1 mL. The
mixture was then shaken 200 times then centrifuged at 11,000 rpm (~10,000x g) for 2 minutes
using a BioRad Model 16K Microcentrifuge, which allowed for separation of the 1-octanol and
phosphate buffer layers. The layers were then separated using a syringe, and the amount of
the compound in each layer was measured by utilizing the standard curve specific to the
compound and solvent. Absorbance measurements were taken at the characteristic longest
wavelength peak for each compound using a SpectraMax M2e plate reader.
Spectroscopy
Spectroscopic characterization was undertaken with dilute (5–20 μM) solutions of the
complexes as PF6− salts in spectroscopic grade MeCN.
UV-visible spectroscopy
Steady state ultraviolet visible near-infrared absorption spectra were measured at room
temperature on a Jasco v730 dual beam spectrophotometer with 5 mm pathlength cuvettes.
Molar extinction coefficients (ε) were determined at selected peak maxima by regressing the
absorption at five different concentrations.
Singlet oxygen quantum yield
The quantum yields of singlet oxygen sensitization (ΦΔ) were calculated from the steady state
intensity of the 1O2 emission band near 1276 nm. This signal was measured on a PTI
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Quantamaster emission spectrometer with a 1000 nm long pass filter and a Hamamatsu
R5509-42 near-infrared photomultiplier tube detector that was cooled to −80 °C. The
instrument internally corrected for any wavelength-dependent nonlinearity in the lamp and
detector. The ΦΔ values were calculated using a relative actinometric formula (Equation S1),
where ‘𝐼’ denotes the integrated emission intensity, ‘A’ is the absorbance of the UV-vis
spectrum at the excitation wavelength, ‘𝜂’ is the refractive index of the solvent. The symbols
‘𝐴𝑆 ’ and ‘𝐼𝑆 ’ represent the absorbance and refractive index corresponding with the standard,
which was [Ru(bpy)3](PF6)2 (Φ∆=0.56 in aerated MeCN).6 All measurements were conducted
using solutions of the PF6− salts of the complexes in air-saturated MeCN.
𝐼 𝐴𝑆 𝜂2
ΦΔ = ΦΔ,𝑆 ( ) ( ) ( 2 )
𝐼𝑆 𝐴 𝜂𝑆
Equation S1
Steady-state emission
Steady-state emission spectra were measured on a PTI Quantamaster spectrometer with a
K170B PMT (max ≈800 nm). Any wavelength-dependent nonlinearity in lamp output and
detector sensitivity was corrected internally by the instrument. Suitable long pass filters were
employed to minimize artifacts caused by harmonics and scatter. The most intense and
longest-wavelength peak in the excitation spectrum was selected for the excitation wavelength
(λex). Room temperature emission of the new complexes was measured from a solution of
spectroscopy-grade MeCN that had been degassed with five free-pump-thaw cycles in a
custom Schlenk-style cuvette. Emission at 77K was measured in a 4:1 EtOH:MeOH glass held
in a 5 mm NMR tube immersed in liquid nitrogen in a custom apparatus. The quantum yields
for emission (Φem) were calculated using a relative actinometric formula (Equation S2), where
‘𝐼’ denotes the integrated emission intensity, ‘A’ is the absorbance of the UV-vis spectrum at
the excitation wavelength, ‘𝜂’ is the refractive index of the solvent. The symbols ‘𝐴𝑆 ’ and ‘𝐼𝑆 ’
represent the absorbance and refractive index corresponding with the standard, which was
[Ru(bpy)3](PF6)2 (Φem=0.018 in MeCN left in air,6 0.4 in EtOH:MeOH7 glass).
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𝐼 𝐴𝑆 𝜂2
Φem = Φem,𝑆 ( ) ( ) ( 2 )
𝐼𝑆 𝐴 𝜂𝑆
Equation S2
Transient Absorption spectroscopy
Transient absorption (TA) lifetimes and differential excited state absorption (ESA) spectra were
measured on an Edinburgh Instruments LP-980 spectrometer equipped with the PMT-LP
detector. A Continuum Minilite Nd:YAG laser generated 355 or 532 nm excitation pulses (≈5 ns
pulse width, ≈7–9 mJ per pulse). Sample solutions were prepared in a custom Schlenk-style
cuvettes in spectroscopy-grade MeCN that had been degassed with five free-pump-thaw
cycles. The ESA spectra were recorded at 10 nm intervals, and single wavelength TA lifetime
measurements were optimized for maximum detector response. Emission lifetimes were also
measured with this configuration, less the probe beam.
Photobiology
Following our previous work,2–4 resazurin-based assays were used to determine the in vitro
cytotoxicity and photocytoxicity of the complexes in this series against normoxic and hypoxic
male human melanoma cells (SK-MEL-28, ATCC HTB-72).
Cell culture
The nonpigmented male human melanoma cells (SK-MEL-28, ATCC HTB-72) were cultivated
and maintained in EMEM (BioWhittaker, 12-125Q) was used as the basal medium, which was
supplemented with 10% FB essence (VWR, 10803-034), 1% glutagro (L-alanyl-L-glutamine;
VWR 45001-086), 1% sodium pyruvate (ThermoFisher, 11360070) and 1% NEAA
(ThermoFisher, 11140050). The cells were incubated using a water-jacketed incubator
(ThermoFisher, Thermo Scientific 4110) at a temperature of 37°C, with humidity levels above
or equal to 90%, and a CO2 concentration of 5%. To initiate each passage, split ratios from
1:2–1:5 were employed to achieve a cell density of 150,000–400,000 cells per milliliter. The
cells were utilized within 15 passages from the time of purchase. For photobiological assays,
the SK-MEL-28 cells were seeded into 384-well plates with a seeding density of 3000 cells per
well.
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For the cultivation and screening of male lung carcinoma cells (A549, ATCC CCL-185), EMEM
(BioWhittaker, 12-125Q) was used as the basal medium, which was supplemented with 10%
FB essence (VWR, 10803-034) and 1% glutagro (L-alanyl-L-glutamine; VWR 45001-086). The
cells were incubated using a water-jacketed incubator (ThermoFisher, Thermo Scientific 4110)
at a temperature of 37°C, ≥90% humidity, and 5% CO2. To initiate each passage, split ratios
from 1:4–1:6 were employed to achieve a cell density of 150,000–400,000 cells mL-1. The cells
were utilized within 15 passages from the time of purchase. The A549 cells were seeded into
384-well plates for cytotoxicity screening with a seeding density of 4,500 cells per well.
Cellular assays
We assessed the photobiological effectiveness of each compound by conducting doseresponse cell viability assays on 384-well plates. Concentrations ranging from 1×10−3 to 300
µM were used for all compounds, while a broader concentration range of 1×10−12 to 300 µM
was utilized for Ru-4T due to its higher phototoxicity. To ensure quick heat exchange, the well
plates were stacked only two plates high in the incubator, irrespective of the experimental
conditions. To evaluate the reproducibility between assays, we performed additional
assessments of Ru-3T and Ru-4T.
Ru(II) compound solutions
Stock solutions of Ru-4T were prepared at concentrations of 21 mM and 25 mM in 100%
DMSO. As for the other compounds, stock solutions were prepared at a concentration of 5 mM
in a 10% v/v DMSO:water mixture. Dilutions were then prepared in a serial manner using 1x
Dulbecco's Phosphate-Buffered Saline (DPBS) without Ca2+ or Mg2+, which was obtained by
diluting 10x DPBS (Corning 20-031-CV). The highest concentration (300 µM) of the diluted
solutions contained less than 1.2% v/v DMSO. Glass vials with PTFE-lined caps were utilized
to store the stock solutions, and these vials were wrapped in aluminum foil to shield them from
light. When not in use, all stock solutions were kept at −20°C while wrapped in foil.
Cytotoxicity and photocytotoxicity
In accordance with our recent examples, the compounds were subjected to (photo)cytotoxicity
screening using a resazurin assay in a 384-well plate format. Greiner Bio-One 384-well plates
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(781182) were used for this purpose. In the biosafety cabinet, DPBS was added to the
outermost two wells (a total of 144 wells) at a volume of 75 µL per well, forming a perimeter.
An electronic multichannel pipettor was employed for the experimental setup. The total volume
for all inner wells, including sample wells, positive control wells, and negative control wells,
was 40 µL per well. The sample wells consisted of 10 µL of complete media, 20 µL of cell
slurry (3000 SK-MEL-28 cells per well, 4500 A549 cells per well), and 10 µL of compound
dilutions in DPBS. The positive control wells (12 in total) were prepared with 10 µL of complete
media, 20 µL of cell slurry (3000 cells per well), and 10 µL of DPBS. The negative control wells
(12 in total) consisted of 30 µL of complete media and 10 µL of DPBS. Before seeding the
cells, the well plates were preincubated with dispensed media at a temperature of 37°C, 5%
CO2, and a relative humidity of at least 90%. After cell seeding, the plates were gently mixed
by tilting them up, down, left, and right, and then placed in the incubator for 2–3 hours to
facilitate cell attachment.
During the incubation period, we prepared serial dilutions of the compounds in sterile 384-well
plates using DPBS as the solvent. For all compounds, dilutions were prepared across nine
concentrations ranging from 1×10−3 to 300 µM. In addition, for Ru-4T, an extra set of nine
concentrations ranging from 1×10−12 to 1×10−3 µM was included. To minimize exposure to light
and prevent premature activation in cells, the lights in the biosafety cabinet were kept off
during the dilution preparation. The 384-well plates, along with their lids, were incubated for 2–
3 hours before the compound dilutions were dispensed at a volume of 10 µL per well.
Replicates (triplicates) were dispensed in a row-wise manner with a spacing of every four
rows.
The 384-well plates were incubated overnight, with a drug-to-light interval (DLI) ranging from
13 to 20 hours. Following the incubation, light treatments were administered. The light
treatment consisted of delivering approximately 100 J cm −2 of light at an intensity of 18–24 mW
cm−2. The light used included cool white Visible light (400–700 nm), Blue light (Prizmatix LED,
453 nm), Green light (Prizmatix LED, 523 nm), and red light (Prizmatix LED, 633 nm). After the
light treatments, the plates were further incubated for 1 day before the final viability
measurements were taken. It is worth noting that edge effects were observed on the 384-well
162
plate, which led to the adjustment of the post-photodynamic therapy (PDT) period to one day
(20–23 hours) to allow for cell viability equilibration, instead of the standard 48-hour period.
Prewarmed sterifiltered resazurin in 0.2 M phosphate buffer (pH = 7.4) at a concentration of
0.3 mM was dispensed into all wells of the plates, with a volume of 10 µL per well. The
resazurin-dyed plates were then incubated for 4 hours before fluorometric measurements were
taken using a Molecular Devices M2e plate reader. The measurements were conducted with a
30-second shaking step, a bottom-read configuration, an excitation wavelength of 530 nm, a
long-pass filter at 570 nm, and an emission wavelength of 620 nm.
Hypoxia cytotoxicity and photocytotoxicity
The screening of the compounds was conducted simultaneously under two different oxygen
conditions: normoxia (approximately 18.5% O2) and hypoxia (1% O2). For the plates treated
with hypoxia, the cells were allowed to incubate for 1–2 hours at 37°C, 5% CO2, and a relative
humidity of at least 90% under normoxia to promote adhesion after seeding. Subsequently, the
plates were transferred to a Biospherix Xvivo X3 chamber and incubated for an additional 2–3
hours at 1% O2, 37°C, 5% CO2, and a relative humidity of at least 90% before the compound
dilutions were dispensed in the biosafety cabinet. The plates were then placed back into the
Biospherix chamber and incubated for a DLI of 17–19 hours. To confirm the hypoxic condition,
the dissolved oxygen level inside the Biospherix chamber was measured using an immersive
optical probe, with a range of 5–7 µM. Following confirmation, the hypoxic-treated plates
designated for light treatment were sealed inside the Biospherix chamber using low-gas
permeable and highly transparent qPCR films (VWR, 89134-428) to maintain the hypoxic
conditions during the light treatment. This step is crucial to evaluate the oxygen-dependence of
the photosensitizer (PS). After the light treatment, the films were removed in a biosafety
cabinet, and all the hypoxic-treated plates were transferred to a normoxia incubator (37°C, 5%
CO2, and a relative humidity of at least 90%). Like the normoxic-treated plates, the hypoxictreated plates were incubated for an additional 20–23 hours before the final viability
measurements were taken.
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Dosimetry Studies
To assess the impact of irradiance on the photoactivity of Ru-4T, specialized dosimetry
experiments were conducted. The Modulight ML8500 platform was utilized for precise well-bywell illumination, employing lasers with center wavelengths of 525 nm, 630 nm, and 753 nm.
Various irradiances ranging from 10 to 100 mW cm −2 were applied while maintaining a
constant fluence of 100 J cm−2. The spectral output was captured using a Luzchem SPR fiber
optic detector in conjunction with an Ocean Optics USB4000 spectrophotometer and an Ocean
Optics UV-Vis XSR fiber optic with a diameter of 230 μm (see Figure S26). Irradiance levels
were measured using a Thorlabs Optical Power Meter PM100D along with their corresponding
thermal power sensor S310C.
Longitudinal studies
The reproducibility of the more active Ru-3T and Ru-4T complexes was tested across multiple
assays using SK-MEL-28. The results of the five repeat assays can be found in Figure S29
and Table S5–S10. For each repeat assay, randomized and unique plate maps were assigned.
Different types of pipet tips were utilized for each repeat: Sartorius 790352 for repeat #1, VWR
83007-352 for repeat #2–3, and low retention Sartorius LH-L790352 for repeat #4–5. This
approach aimed to minimize any potential contribution from stray light. During repeat #5, the
overhead lights in the laboratory space were turned off to further reduce the impact of stray
light on the assay results. All cells utilized in these experiments were within the range of 10–15
passages, and all assays were performed within a one-month timeframe to maintain
consistency and minimize potential variations.
Light devices and protocols.
For all biological assays, unless stated otherwise, a consistent fluence of 100 J cm -2 and an
irradiance ranging between 18–22 mW cm-2 were employed. The light treatments utilized three
different light sources for visible, green, and red light. These sources included a cool white
LED panel from SOLLA-CREE, covering a spectral range of 400–700 nm with a maximum
absorption around 450 nm. Additionally, two UHP-LEDs from Prizmatix were used, emitting
light at 523 nm (green) and 633 nm (red) respectively. The spectral outputs of these light
sources can be referenced in Figure S26.
164
Data manipulation and statistics
Data from the resazurin cell viability assay were corrected for background by subtracting the
signal from wells that contained only media and DPBS (no cells) and normalized relative to
untreated cells. Because the absorbance and emission of the metal complexes can interfere
with the resazurin fluorescence signal, wells treated with the highest concentrations of metal
complex were also observed under a microscope. If no cells were detected, these wells were
assigned a value of zero. A more detailed discussion of assay limitations for this class of
complexes is provided in our 2019 review.4
Data points obtained from resazurin fluorescence were fit to a three-parameter log-logistic
(Equation S3) and logistic model (Equation S4) using GraphPad Prism 8.4.0. We use Equation
S3 for summary log(EC50) plots (Figure 12a) and in the dose-response curves shown in Figure
13, but we use Equation S4 for data in log(PI) plots (Figure 12b) as well as the tabulated EC50
and PI values (Table S5–S10).
𝑌 = Bottom +
(𝑇𝑜𝑝 − 𝐵𝑜𝑡𝑡𝑜𝑚)
Equation S3
(1 + (10𝐿𝑜𝑔(𝐸𝐶50 −𝑋)×𝐻𝑖𝑙𝑙𝑠𝑙𝑜𝑝𝑒 )
𝑌 = Bottom +
(𝑇𝑜𝑝 − 𝐵𝑜𝑡𝑡𝑜𝑚)
(1 + (𝐸𝐶50 ⁄𝑋)
𝐻𝑖𝑙𝑙𝑠𝑙𝑜𝑝𝑒
)
Equation S4
Experiments were completed in triplicate and replicated data points are always plotted with
error bars denoting the standard deviation (SD). All EC50 values are reported alongside the
standard error of the mean (SEM). In cases where the hill slope was too steep to calculate a
representative SEM, the SEM was labelled as not determined (n.d.). Phototherapeutic indices
(PI) are reported as the ratio of dark to light EC50 values and serve as a phototherapeutic
efficacy benchmark. Any summary plots showing Log EC50 and Log PI values of the entire
series of complexes are plotted with SEMs from log-logistic fits.
165
Maximum tolerated dose in mice
To determine the maximum tolerated dose (MTD) of Ru-3T and Ru-4T, a cohort of 8-week-old
female C57BL/6J mice weighing approximately 20 g each was used. The MTD evaluation was
carried out via two different injection routes: intraperitoneal (IP) and intravenous (IV). For IP
injections, the mice were dosed with varying concentrations of 25–200 mg kg−1, with a total
volume of 200 µL injected into the lower right abdominal quadrant. For IV injections,
concentrations of 12.5–50 mg kg−1 were administered, with a total volume of 100 µL injected
into the tail vein. The vehicle used for both injection routes consisted of a 10% DMSO solution
in 0.9% saline. Prior to injection, compound solutions were prepared immediately and
sonicated to ensure complete dissolution. Injections were delivered slowly after visually
confirming compound dissolution. Following the injections, the mice were continuously
monitored for a period of 2 hours, with additional frequent monitoring over 6 hours, and
periodic monitoring over a span of 2 weeks. If a combination of moderately severe signs of
clinical toxicity appeared, or a single severe sign appeared, or if 2 weeks had passed since the
injection, the mice were euthanized. The MTD was determined as the dose that induced
moderate signs of clinical toxicity.
166
Synthetic Characterization
NMR Spectra
Figure S1. (a) Structure labelling and aromatic 1H NMR (700MHz, 798 K) spectrum of [Ru(4,4ʹ-btfmb)3](Cl)2 with
assignments. (b) Aromatic 1H-1H COSY NMR (700MHz, 798 K) spectrum of [Ru(4,4ʹ-btfmb)3](Cl)2.
167
Figure S2. (a) Structure labelling and aromatic 1H NMR (700MHz, 798 K) spectrum of Ru-phen with assignments.
(b) Aromatic 1H-1H COSY NMR (700MHz, 798 K) assignments for Ru-phen.
168
Figure S3. (a) Structure labelling and aromatic 1H NMR (700MHz, 289 K) spectrum of Ru-0T with assignments. (b)
Aromatic 1H-1H COSY NMR (700MHz, 798 K) assignments for Ru-0T.
169
Figure S4. (a) Structure labelling and aromatic 1H NMR (700MHz, 798 K) spectrum of Ru-1T with assignments. (b)
Aromatic 1H-1H COSY NMR (700MHz, 798 K) assignments for Ru-1T.
170
Figure S5. (a) Structure labelling and aromatic 1H NMR (700MHz, 798 K) spectrum of Ru-2T with assignments. (b)
171 for Ru-2T.
Aromatic 1H-1H COSY NMR (700MHz, 798 K) assignments
Figure S6. (a) Structure labelling and aromatic 1H NMR (700MHz, 798 K) spectrum of Ru-3T with assignments.
(b) Aromatic 1H-1H COSY NMR (700MHz, 798 K) assignments for Ru-3T.
172
Figure S7. (a) Structure labelling and aromatic 1H NMR (700MHz, 798 K) spectrum of Ru-4T with assignments.
(b) Aromatic 1H-1H COSY NMR (700MHz, 798 K) assignments for Ru-4T.
173
High-Resolution Mass Spectra (HRMS-ESI+)
Figure S8. (a) High resolution ESI+-MS spectrum for [Ru(4,4ʹ-btfmb)3](Cl)2 (b) Zoom of 488.9529 m/z showing
isotopic distribution.
174
Figure S9. (a) High resolution ESI+-MS spectrum for Ru-phen (b) Zoom of 432.9739 m/z showing isotopic
distribution.
175
Figure S10. (a) High resolution ESI+-MS spectrum for Ru-0T (b) Zoom of 488.9529 m/z showing isotopic
distribution.
176
Figure S11. (a) High resolution ESI+-MS spectrum for Ru-1T. (b) Zoom of 432.9739 m/z showing isotopic
distribution. (c) Zoom of 987.0456 m/z showing isotopic distribution.
177
Figure S12. (a) High resolution ESI+-MS spectrum for Ru-2T (b) Zoom of 464.0405 m/z showing isotopic
distribution. (c) Zoom of 927.0769 m/z showing isotopic distribution.
178
Figure S13. (a) High resolution ESI+-MS spectrum for Ru-3T (b) Zoom of 576.0128 m/z showing isotopic
distribution. (c) Zoom of 1151.0299 m/z showing isotopic distribution.
179
Figure S14. (a) High resolution ESI+-MS spectrum for Ru-4T (b) Zoom of 617.0052 m/z showing isotopic
distribution. (c) Zoom of 1232.0035 m/z showing isotopic distribution.
180
HPLC Chromatographs
22.412
DAD1 A, Sig=285,8 Ref=850,20 (LIUBOV\LL000114.D)
mAU
500
400
300
200
100
0
15
20
5
10
DAD1 C, Sig=490,8 Ref=850,20 (LIUBOV\LL000114.D)
15
20
5
10
DAD1 D, Sig=400,8 Ref=850,20 (LIUBOV\LL000114.D)
15
20
5
15
20
25
30
35
min
25
30
35
min
25
30
35
min
25
30
35
min
22.412
5
10
DAD1 B, Sig=440,8 Ref=850,20 (LIUBOV\LL000114.D)
mAU
140
120
100
80
60
40
20
22.412
0
mAU
60
50
40
30
20
10
22.412
0
mAU
60
50
40
30
20
10
0
10
Figure S15. HPLC chromatogram for [Ru(4,4ʹ-dtfmb)3](Cl)2 collected at the following wavelengths: 285,
400, 440, and 490 nm.
181
22.412
DAD1 A, Sig=285,8 Ref=850,20 (LIUBOV\LL000114.D)
mAU
500
400
300
200
100
0
15
20
5
10
DAD1 C, Sig=490,8 Ref=850,20 (LIUBOV\LL000114.D)
15
20
5
10
DAD1 D, Sig=400,8 Ref=850,20 (LIUBOV\LL000114.D)
15
20
5
15
20
25
30
35
min
25
30
35
min
25
30
35
min
25
30
35
min
22.412
5
10
DAD1 B, Sig=440,8 Ref=850,20 (LIUBOV\LL000114.D)
mAU
140
120
100
80
60
40
20
22.412
0
mAU
60
50
40
30
20
10
22.412
0
mAU
60
50
40
30
20
10
0
10
Figure S16. HPLC chromatogram for Ru-phen collected at the following wavelengths: 285, 400, 440, and
490 nm.
182
20.335
DAD1 A, Sig=285,8 Ref=850,20 (HOUSTON\HC000180.D)
mAU
300
250
200
150
21.357
100
50
0
15
20
5
10
DAD1 C, Sig=490,8 Ref=850,20 (HOUSTON\HC000180.D)
15
20
5
10
DAD1 D, Sig=400,8 Ref=850,20 (HOUSTON\HC000180.D)
15
20
15
20
25
30
35
min
25
30
35
min
25
30
35
min
25
30
35
min
20.335
5
10
DAD1 B, Sig=440,8 Ref=850,20 (HOUSTON\HC000180.D)
mAU
80
60
40
20
20.335
0
mAU
50
40
30
20
10
20.335
0
mAU
40
30
20
10
0
5
10
Figure S17. HPLC chromatogram for Ru-0T collected at the following wavelengths: 285, 400, 440, and
490 nm.
183
21.754
DAD1 A, Sig=285,8 Ref=850,20 (HOUSTON\HC000178.D)
mAU
1750
1500
1250
1000
20.208
500
250
0
15
20
5
10
DAD1 C, Sig=490,8 Ref=850,20 (HOUSTON\HC000178.D)
15
20
5
10
DAD1 D, Sig=400,8 Ref=850,20 (HOUSTON\HC000178.D)
15
20
15
20
25
30
35
min
25
30
35
min
25
30
35
min
25
30
35
min
21.753
5
10
DAD1 B, Sig=440,8 Ref=850,20 (HOUSTON\HC000178.D)
21.210
21.297
21.472
21.543
22.145
22.317
22.522
750
mAU
400
300
100
22.317
22.522
21.209
21.298
21.472
21.543
200
21.753
0
mAU
250
200
21.209
21.297
21.472
21.543
100
50
22.317
22.522
150
21.753
0
mAU
200
150
50
22.318
22.523
21.210
21.297
21.472
21.543
100
0
5
10
Figure S18. HPLC chromatogram for Ru-1T collected at the following wavelengths: 285, 400, 440, and
490 nm.
184
21.754
DAD1 A, Sig=285,8 Ref=850,20 (HOUSTON\HC000178.D)
mAU
1750
1500
1250
1000
0
20.5
21
19.5
20
DAD1 C, Sig=490,8 Ref=850,20 (HOUSTON\HC000178.D)
20.5
21
19.5
20
DAD1 D, Sig=400,8 Ref=850,20 (HOUSTON\HC000178.D)
20.5
21
20.5
21
21.5
22.522
22.317
22
22.5
23
min
23
min
23
min
23
min
21.753
19.5
20
DAD1 B, Sig=440,8 Ref=850,20 (HOUSTON\HC000178.D)
22.145
250
21.472
21.543
21.210
20.208
500
21.297
750
mAU
400
300
22.522
22.317
21.472
21.543
21.209
100
21.298
200
0
21.5
22
22.5
21.753
mAU
250
200
150
22.522
22.317
21.472
21.543
21.209
50
21.297
100
0
21.5
22
21.753
22.5
mAU
200
150
22.523
22.318
21.472
21.543
21.210
50
21.297
100
0
19.5
20
21.5
22
22.5
Figure S19. Zoom of HPLC chromatogram for Ru-2T collected at the following wavelengths: 285, 400,
440, and 490 nm.
185
Figure S20. (top) HPLC chromatogram for Ru-3T collected at the following wavelengths: 285 nm. (middle) Zoom
of HPLC chromatogram for Ru-3T collected at 285 nm. (bottom) Overlay of UV-Vis absorption spectra of HPLC
186
Chromatogram peaks of Ru-3T at 285 nm.
Figure S21. (a) HPLC chromatogram for Ru-4T collected at the following wavelengths: 285 nm. (b) Zoom of
187
HPLC chromatogram for Ru-4T collected at the following
wavelengths: 285 nm. (c) Overlay of UV-Vis absorption
spectra of HPLC Chromatogram peaks of Ru-4T at 285 nm.
Table S1. Analytical HPLC method using the Hypersil GOLD C18 Column.
time (min)
%MeCN
%Water
Pre-run
20
2
98
Run
0
2
98
2
5
95
5
30
70
15
30
70
20
60
40
30
95
5
35
2
98
40
2
98
Post-run
10
2
98
Flow rate (mL min−1)
1
Both eluents contain 0.1% optima grade formic acid with runs involving a 20 μL injection at 50–200 µM of metal
complex dissolved in optima grade MeOH.
188
LIPOPHILICITY
Table S2. Log distribution coefficient (log Do/w) of chloride salts of [Ru(btfmb)3], Ru-phen and Ru-nT
(n=0–4) in 1-octanol and 10mM phosphate buffer (pH = 7.4) using the shake-flask method.
Compound
log (Do/w ± SD)
[Ru(4,4ʹ-btfmb)3](Cl)2
– 2.184 ± 0.071
Ru-phen
– 1.862 ± 0.080
Ru-0T
– 1.349 ± 0.026
Ru-1T
– 0.336 ± 0.037
Ru-2T
+ 1.182 ± 0.042
Ru-3T
+ 1.580 ± 0.052
Ru-4T
+ 2.172 ± 0.132
189
Spectroscopic Characterization
Singlet Oxygen Sensitization
Ru-4T concentration (µM)
Figure S22: The dependence of 1O2 quantum yield on the concentration of Ru-4T in acetonitrile
Table S3: Singlet oxygen sensitization quantum yield and varying concentration and excitation wavelength.
Concentration (μM)
ΦΔ @ λex=440 nm
ΦΔ @ λex=440 nm
20
0.22
0.29
15
0.26
0.34
11
0.29
0.37
8.4
0.31
0.39
6.3
0.32
0.40
190
Transient Absorption
Figure S23: Differential excited state absorption profiles for the series in degassed MeCN at RT, λex=532 nm.
191
Figure S24: Time-sliced differential excited state absorption spectra, excitation pulse 355 nm.
192
Figure S25: Time-sliced differential excited state absorption spectra, excitation pulse 532 nm.
193
Photobiological Evaluation
Light Sources and Absorbed Photons
Relative Emission
(a)
100
Relative Emission
633 nm Prizmatix
75
523 nm Prizmatix
453 nm Prizmatix
50
Cool White
Visible CREE
25
0
400
(b)
Light Source
500
600
700
Wavelength (nm)
800
100
Light Source
633 nm Prizmatix
75
523 nm Prizmatix
453 nm Prizmatix
50
Cool White
Visible CREE
25
0
400
500
600
700
Wavelength (nm)
800
Figure S26. Relative spectral emissions or output of the light sources applied in photobiological studies
manufacturer of the LED chip and/or light device is indicated (Prizmatix or CREE). Two color schemes are shown
with (a) being colorblind-friendly and (b) matching colors used in reported biological plots (approximately matching
apparent colors).
194
(a)
vis. &
453 nm 523 nm 633 nm
(104 M-1 cm-1)
Norm. Spectral Emission
8
6
4
2
0
300
400
500
600
btfmb
phen
0T
1T
700
2T
Wavelength (nm)
3T
(b)
vis. &
453 nm 523 nm 633 nm
4T
Norm. Spectral Emission
0.15
(104 M-1 cm-1)
Cmpd
0.10
0.05
Light Source
Visible
453 nm
523 nm
633 nm
0.00
300
400
500
600
700
Wavelength (nm)
Figure S27. Overlay of [Ru(4,4ʹ-btfmb)3](Cl)2, Ru-phen, and Ru-0T–Ru-4T for their molar extinction coefficients in
MeCN (single extrapolated concentration, 20 μM only) and normalized spectral outputs of light sources applied in
photobiological studies. Top (a) shows full range and bottom (b) shows the zoom (bottom 1/50 th of top y-axis).
Color fill was only used in the top plot of the PSs for clarity. The dotted line in (b) indicates the threshold used for
the absorbed photon flux estimate (0.27% norm. spectral emission).
195
Table S4. Approximate photon flux density (mol m−2 s−1) absorbed by 20 μM of [Ru(4,4ʹ-btfmb)3](Cl)2, Ru-phen,
and Ru-0T–Ru-4T in MeCN (5 mm pathlength). Does not correct for scatter or reflection.
cmpd
633 nma
523 nmb
453 nmc
Visibled
V:Re
V:Gf
B:Vg
G:Rh
[Ru(btfmb)3](Cl)2
8.70 × 10−8
2.47 × 10−5
1.62 × 10−4
4.68 × 10−5
538
2
3
284
Ru-phen
8.44 × 10−7
4.09 × 10−5
1.75 × 10−4
5.67 × 10−5
67
1
3
49
Ru-0T
9.97 × 10−7
4.63 × 10−5
1.81 × 10−4
5.99 × 10−5
60
1
3
46
Ru-1T
2.02 × 10−6
6.64 × 10−5
2.03 × 10−4
7.27 × 10−5
36
1
3
33
Ru-2T
2.05 × 10−6
8.15 × 10−5
2.79 × 10−4
9.73 × 10−5
48
1
3
40
Ru-3T
6.42 × 10−6
8.84 × 10−5
3.36 × 10−4
1.18 × 10−4
18
1
3
14
Ru-4T
7.60 × 10−6
1.24 × 10−4
4.47 × 10−4
1.58 × 10−4
21
1
3
16
ared 633 nm, bgreen 523 nm, cblue 453 nm, dcool white visible (400–700 nm), eratio of visible to red absorbed
photon flux, fratio of visible to green absorbed photon flux, gratio of blue to visible absorbed photon flux, and hratio
of green to red absorbed photon flux. PS and light source overlay in Table S2.
Note that the values above were calculated from the absorption values reported in the spectroscopy section (5
mm pathlength, 20 µM PF6− salts in MeCN) and use an equivalent irradiance (20.5 mW cm−2) to facilitate
comparison.
196
Hy
p.
JR4-141, SKMEL28,
Activity: log (EC50)
No
rm
.
Hy
p.
JR4-141, A549,
Activity: log (EC50)
No
rm
.
Cytotoxicity and Photocytotoxicity
4T
Dark
4T
Dark
3T
633 nm
3T
633 nm
2T
523 nm
2T
523 nm
1T
Vis
1T
Vis
0T
453 nm
0T
453 nm
phen
phen
btfmb
btfmb
-1
0
1
2
-3
3
-2
-1
0
1
2
3
Log (EC50 / M)
Log (EC50 / M)
JR4-141, A549,
Activity: PI
p.
Hy
JR4-141, SKMEL28,
Activity: PI
.
rm
No
No
rm
.
-2
Hy
p.
-3
633 nm
4T
633 nm
3T
523 nm
3T
523 nm
2T
Vis
2T
Vis
1T
453 nm
1T
453 nm
PI
JR4-141, SKMEL28,
Activity: log (PI)
No
rm
.
Hy
p.
JR4-141, A549,
Activity: log (PI)
1
20
40
60
8
1 00
0
20
0
50
0
80
0
1 20 40 60 80 00 00 00 00 00 00 00 00
1 2 5 8 10 20 30 40
PI
No
rm
.
btfmb
Hy
p.
btfmb
30
0T
phen
10
0T
phen
00
0
00
0
50
00
0
4T
4T
633 nm
4T
633 nm
3T
523 nm
3T
523 nm
2T
Vis
2T
Vis
1T
453 nm
1T
453 nm
0T
0T
phen
phen
btfmb
btfmb
0
1
2
3
4
5
0
Log (PI)
1
2
3
4
5
Log (PI)
Figure S28. Summary activity plots of Ru complexes [Ru(4,4ʹ-btfmb)3](Cl)2, Ru-phen, and Ru-0T–Ru-4T against
A549 cells (left) and SK-MEL-28 (right). Treatments in either hypoxic (1% O2) or normoxic (18.5–21% O2)
conditions are denoted by symbols for dark (sham, 0 J cm−2) and light treatments (100 J cm−2, ~20 mW cm−2) with
red 633 nm, green 523 nm, cool white visible, and blue 453 nm.
197
Table S5. Comparison of (photo)cytotoxicities of [Ru(4,4ʹ-btfmb)3](Cl)2, Ru-phen, and Ru-0T–Ru-4T in hypoxic
(1% O2) and normoxic (18.5–21% O2) treated male lung carcinoma A549 cells.
EC50 ± SEM (µM)a
633
523
453
633
Visibled,e
d
d
d
d
nm
nm
nm
nm
523
nmd
453
nmd
Visibled,e
PIb
cmpd
O2%
[Ru(btfmb)3](Cl)2
1
>300
>300
>300
>300
>300
1
1
1
1
Ru-phen
1
>300
>300
>300
>300
>300
1
1
1
1
Ru-0T
1
>300
>300
>300
>300
>300
1
1
1
1
Ru-1T
1
211 ±
7
208 ±
5
213 ±
5
212 ±
6
212 ± 8
1
1
1
1
Ru-2T
1
182 ±
7
170 ±
4
178 ±
5
176 ±
5
190 ± 5
1
1
1
1
Ru-3T
1
175 ±
12
166 ±
6
187 ±
7
215 ±
9
207 ± 9
1
1
1
1
Ru-4T
1
120 ±
4
96.7
± 6.5
126 ±
5
126 ±
4
127 ± 6
1
1
1
1
[Ru(btfmb)3](Cl)2
18.5
>300
>300
>300
>300
>300
1
1
3
3
Ru-phen
18.5
>300
>300
>300
>300
>300
1
1
2
1
Ru-0T
18.5
>300
>300
>300
>300
>300
1
2
2
2
Ru-1T
18.5
199 ±
7
189 ±
5
12.3 ±
1.9
28.6 ±
3.2
1
2
16
7
Ru-2T
18.5
174 ±
4
152 ±
6
0.50 ±
0.056
1.37 ±
0.35
1
55
348
127
Ru-3T
18.5
195 ±
10
12.1
± n.d.
16
329
491
280
18.5
119 ±
4
5.64
± n.d.
0.40 ±
0.08
0.033
±
0.0023
0.70 ±
n.d.
Ru-4T
132 ±
11
3.19
±
1.19
0.59
± n.d.
0.26
±
0.081
0.077 ±
n.d.
21
461
3617
1541
Darkc
aEffective concentration to reduce relative cell viability to 50% (EC
50) and standard error of the mean (SEM),
bphototherapeutic index (PI) provides the ratio of dark (sham) to light EC
50 values,
cdark treatment or absence of
light during treatment, dlight treatment uses 100 J cm−2 at ~20 mW cm−2, and ecool white visible (400–700 nm).
*n.d. = SEM not determined to a steep hill slope.
198
Table S6. Comparison of (photo)cytotoxicities [Ru(4,4ʹ-btfmb)3](Cl)2, Ru-phen, and Ru-0T–Ru-4T in hypoxic (1%
O2) and normoxic (18.5% O2) SK-MEL-28 cells.
EC50 ± SEM (µM)a
Darkc
633
nmd
523 nmd
453 nmd
Visibled,e
633
nmd
523
nmd
PIb
453
nmd
1
>300
>300
>300
>300
>300
1
1
1
1
Ru-phen
1
>300
>300
>300
>300
>300
1
1
1
1
Ru-0T
1
>300
>300
>300
>300
>300
1
1
1
1
Ru-1T
1
254 ±
8
251 ±
5
240 ± 5
271 ± 7
264 ± 9
1
1
1
1
Ru-2T
1
160 ±
4
160 ±
3
163 ± 5
179 ± 5
174 ± 5
1
1
1
1
Ru-3T
1
195 ±
7
198 ±
8
1.08 ±
n.d.
1.24 ±
n.d.
1.18 ±
n.d.
1
181
157
165
Ru-4T
1
99.2
± 3.6
25.9
± 3.2
1.09 ±
0.29
0.12 ±
0.015
0.13 ±
0.036
4
91
863
769
[Ru(btfmb)3](Cl)2
18.5
>300
>300
>300
71.8 ±
9.8
>300
1
1
4
1
Ru-phen
18.5
>300
>300
>300
281 ±
n.d.
300 ±
n.d.
1
1
~1
1
Ru-0T
18.5
>300
>300
>300
145 ± 21
290 ±
n.d.
1
1
2
~1
Ru-1T
18.5
238 ±
9
228 ±
5
103 ± 4
10.1 ±
n.d.
22.4 ±
2.4
1
2
24
11
Ru-2T
18.5
176 ±
5
110 ±
3
1.93 ±
0.15
0.64 ±
n.d.
0.61 ±
0.1
2
91
277
290
Ru-3T
18.5
196 ±
6
0.27 ±
0.021
0.12 ±
0.009
0.21 ±
0.032
28
731
1675
920
Ru-4T
18.5
101 ±
3
7.02
± 0.1
2.74
±
0.41
0.12 ±
0.01
0.0024 ±
0.00018
0.010 ±
0.001
37
815
41564
10100
compound
% O2
[Ru(btfmb)3](Cl)2
aEffective concentration to reduce relative cell viability to 50% (EC
Visibled,e
50) and standard error of the mean (SEM),
bphototherapeutic index (PI) provides the ratio of dark (sham) to light EC
50 values,
cdark treatment or absence of
light during treatment, dlight treatment uses 100 J cm−2 at ~20 mW cm−2, and ecool white visible (400–700 nm).
*n.d. = SEM not determined to a steep hill slope.
199
(b)
5
Dark EC50
5
633 nm PI
4
633 nm EC50
4
523 nm PI
3
523 nm EC50
2
Vis EC50
Repeat #
Repeat #
(a)
2
1
1
0
0
-3
-2
-1
0
1
2
Vis PI
3
0
3
1
Log (EC50 ± SEM / μM)
3
4
5
Log (PI)
(c)
(d)
5
Dark EC50
5
633 nm PI
4
633 nm EC50
4
523 nm PI
3
523 nm EC50
2
Vis EC50
Repeat #
Repeat #
2
2
1
1
0
0
-3
-2
-1
0
1
2
Vis PI
3
0
3
Log (EC50 ± SEM / μM)
1
2
3
4
5
Log (PI)
Figure S29. Interassay performance with various factors changed across each repeat as described in the
experimental section. Cytotoxicity and photocytotoxicity of Ru-3T (top, a+b) and Ru-4T (bottom, c+d) in
normoxic- (filled symbols, solid lines; ~18.5% O2) and hypoxic-treated (open symbols, dashed lines; 1% O2) SKMEL-28 melanoma cells. Log (EC50 ± SEM) values (left) and PI values (right). Treatments included dark (0 J
cm−2) and 100 J cm−2 doses of 633 nm, 523 nm, and visible (400–700 nm) light.
“Repeat 0” is the data reported in the compound family’s dose-response in SK-MEL-28 about
one year after repeats 1–5 (completed over one month).
200
Table S7. Interassay performance: cytotoxicity and photocytotoxicity of Ru-3T in hypoxic-treated (1% O2) male
SK-MEL-28 melanoma cells.
Resazurin-Hypoxia (1% O2) Ru-3T Repeats
EC50 ± SEM (μM)
PId
Repeat
Dark
Reda
Greenb
Visc
Reda
Greenb
Visc
0*
195 ± 7
198 ± 8
1.08 ± n.d.
1.18 ± n.d.
1
181
165
1
123 ± 6
109 ± 12
0.59 ± 0.04
1
209
304
2
238 ± 11
160 ± 24
0.84 ± 0.22
1
283
377
3
210 ± 13
7.54 ± 0.29
28
724
707
4
170 ± 3
5
0.41 ±
0.037
0.63 ±
0.068
0.29 ±
0.30 ±
0.024
0.059
128 ± 31
219 ± 69
190 ± 41
1
1
1
199 ± 8
165 ± 19
1.01 ± 0.08
0.87 ± 0.98
1
197
230
189 ± 39
128 ± 67
37.1 ± 89.1
13.9 ± 30.8
6 ± 11
266 ± 243
297 ± 238
min
123
7.54
0.290
0.297
1
1
1
max
238
198
219
190
28
724
707
Mean ±
SDe
Light treatments were approximately 100 J cm−2 delivered at 18–22 mW cm−2.a red 633 nm, b green 523 nm, c
cool white visible (400–700 nm), d PI = phototherapeutic index (dark EC50 / light EC50), *original run in repeat 0
from Table S4, and n.d. = SEM not determined due to overly steep hill slope. e Did not test for outliers or run
meta-analysis, use with caution.
Repeats used different plate maps (all), different tips (Sartorius 790352 repeat #1, VWR 83007-352 repeats
#2–3, low retention Sartorius LH-L790352 repeats #4–5), changed cell parent seed stock for repeats 4–5, and
overhead lights were off in #5. Serum and consumable lots were identical for repeats 1–5. Cell passage
numbers were equal. Run in parallel with normoxic repeats.
201
Table S8. Interassay performance: cytotoxicity and photocytotoxicity of Ru-3T in normoxic-treated (~18.5% O2)
male SK-MEL-28 melanoma cells.
Resazurin-Normoxia (~18.5% O2) Ru-3T Repeats
EC50 ± SEM (μM)
Repeat
Dark
Reda
0*
196 ± 6
7.02 ± 0.10
1
123 ± 6
5.44 ± 0.11
2
230 ± 12
4.70 ± n.d.
3
225 ± 10
3.36 ± 0.9
4
195 ± 9
1.94 ± 0.06
5
213 ± 10
PId
Reda
Greenb
Visc
28
731
920
0.070 ± n.d.
23
515
1767
0.70 ± n.d.
49
364
331
0.084 ± n.d.
67
1023
2691
0.65 ± n.d.
0.64 ± n.d.
101
299
305
4.90 ± n.d.
0.60 ± n.d.
0.10 ± n.d.
43
358
2158
197 ± 39
4.56 ± 1.75
0.29 ± 0.41
0.44 ± 0.21
52 ± 29
548 ± 280
1362 ± 994
min
123
1.94
0.22
0.070
23
299
305
max
230
7.02
0.66
0.70
101
1023
2691
Mean ±
SDe
Greenb
Visc
0.27 ±
0.21 ±
0.021
0.032
0.24 ±
0.023
0.63 ± n.d.
0.22 ±
0.037
Light treatments were approximately 100 J cm−2 delivered at 18–22 mW cm−2. a red 633 nm, b green 523 nm, c
cool white visible (400–700 nm), d PI = phototherapeutic index (dark EC50 / light EC50), *original run in repeat 0
from Table S4, and n.d. = SEM not determined due to overly steep hill slope. e Did not test for outliers or run
meta-analysis, use with caution.
Repeats used different plate maps (all), different tips (Sartorius 790352 repeat #1, VWR 83007-352 repeats
#2–3, low retention Sartorius LH-L790352 repeats #4–5), changed cell parent seed stock for repeats 4–5, and
overhead lights were off in #5. Serum and consumable lots were identical for repeats 1–5. Cell passage
numbers were equal. Run in parallel with normoxic repeats.
202
Table S9. Interassay performance: cytotoxicity and photocytotoxicity of Ru-4T in hypoxic-treated (1% O2) male
SK-MEL-28 melanoma cells.
Resazurin-Hypoxia (1% O2) Ru-4T Repeats
EC50 ± SEM (μM)
PId
Repeat
Dark
Reda
Greenb
Visc
0*
99.2 ± 3.6
25.9 ± 3.2
1.09 ± 0.29
1
102 ± 3
5.71 ± 0.15
2
113 ± 5
4.30 ± 0.06
3
114 ± 4
3.48 ± 0.13
4
97.8 ± 4.2
5
0.13 ±
0.036
Reda
Greenb
Visc
4
91
769
18
464
2948
26
224
411
33
278
221
21
111
183
7
95
369
0.22 ±
0.035 ±
0.013
0.0026
0.51 ±
0.28 ±
0.043
0.028
0.41 ±
0.52 ±
0.041
0.032
4.59 ± 0.70
0.88 ± 2.24
0.54 ± 0.21
65.3 ± 1.5
9.32 ± 0.90
0.68 ± 0.22
98.6 ± 17.7
8.88 ± 8.58
0.63 ± 0.32
0.47 ± 0.87
18 ± 11
211 ± 146
817 ± 1065
min
65.3
3.48
0.220
0.0346
4
91
183
max
114
25.9
1.09
0.535
33
464
2948
Mean ±
SDe
0.18 ±
0.069
Light treatments were approximately 100 J cm−2 delivered at 18–22 mW cm−2. ared 633 nm, b green 523 nm, c
cool white visible (400–700 nm), d PI = phototherapeutic index (dark EC50 / light EC50), *original run in repeat 0
from Table S4, and n.d. = SEM not determined due to overly steep hill slope. e Did not test for outliers or run
meta-analysis, use with caution.
Repeats used different plate maps (all), different tips (Sartorius 790352 repeat #1, VWR 83007-352 repeats
#2–3, low retention Sartorius LH-L790352 repeats #4–5), changed cell parent seed stock for repeats 4–5, and
overhead lights were off in #5. Serum and consumable lots were identical for repeats 1–5. Cell passage
numbers were equal. Run in parallel with normoxic repeats.
203
Table S10. Interassay performance: cytotoxicity and photocytotoxicity of Ru-4T in normoxic-treated (~18.5% O2)
male SK-MEL-28 melanoma cells.
Resazurin-Normoxia (~18.5% O2) Ru-4T Repeats
EC50 ± SEM (μM)
Repeat
Dark
Reda
Greenb
0*
101 ± 3
2.74 ± 0.41
0.12 ± 0.010
1
106 ± 3
2.36 ± 0.10
2
115 ± 4
1.27 ± n.d.
0.24 ± 0.037
3
114 ± 4
1.64 ± 0.83
0.19 ± 0.029
1.84 ± 0.08
0.32 ± 0.038
3.39 ± n.d.
0.095 ± n.d.
100 ± 17
2.21 ± 0.78
min
69.9
max
115
95.2 ±
4
3.0
69.9 ±
5
1.1
Mean ±
SD
e
PId
Visc
Reda
Greenb
Visc
37
815
10402
45
1949
11964
91
479
4406
70
597
1949
52
296
2957
0.0093 ± n.d.
21
738
7500
0.36 ± 0.35
0.017 ± 0.010
53 ± 25
812 ± 587
1.27
0.054
0.0089
21
296
1949
3.39
0.32
0.059
91
1949
11964
0.0097 ±
0.00068
0.054 ±
0.0088 ±
0.0030
0.0012
0.026 ±
0.0083
0.059 ± n.d.
0.032 ±
0.0028
6530 ±
4092
Light treatments were approximately 100 J cm−2 delivered at 18–22 mW cm−2 ared 633 nm, b green 523 nm, c
cool white visible (400–700 nm), d PI = phototherapeutic index (dark EC50 / light EC50), *original run in repeat 0
from Table S4, and n.d. = SEM not determined due to overly steep hill slope. e Did not test for outliers or run
meta-analysis, use with caution.
Repeats used different plate maps (all), different tips (Sartorius 790352 repeat #1, VWR 83007-352 repeats
#2–3, low retention Sartorius LH-L790352 repeats #4–5), changed cell parent seed stock for repeats 4–5, and
overhead lights were off in #5. Serum and consumable lots were identical for repeats 1–5. Cell passage
numbers were equal. Run in parallel with normoxic repeats.
204
CHAPTER 4. CHIRALITY MATTERS: ENANTIOMERICALLY RESOLVED RU(II)
OLIGOTHIENYL COMPLEXES FOR PHOTODYNAMIC THERAPY
Houston D. Cole,a Troy T. Handlovic,a Saba Aslani,a John A. Roque III,a,b, Ge Shi,a
Elamparuthi Ramasamy,a Colin G. Cameron,a* Daniel W. Armstrong,a Sherri A. McFarlanda*
a Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, Texas, 76019-0065 United States
*Corresponding authors: C.G.C. <colin.cameron@uta.edu> ORCID 0000-0003-0978-0894, S.A.M.
<sherri.mcfarland@uta.edu> ORCID 0000-0002-8028-5055
4.1 ABSTRACT
Keywords: Ruthenium, enantiomers, cancer, metal-to-ligand charge transfer (MLCT),
intraligand (IL), melanoma, photodynamic therapy
4.2 INTRODUCTION
The chiral configurations of bis-heteroleptic and tris-homoleptic Ruthenium polypyridyl
complexes are defined as being either the delta (Δ) or lambda (Λ) enantiomer (Scheme 1).
The resolution of these chiral complexes may be performed synthetically1 or through highpressure liquid chromatography (HPLC) using a chiral stationary phase.2–4 In collaboration with
the Armstrong group, we have previously reported the HPLC-driven separation of various
ruthenium and osmium oligothienyl complexes using R-napthylenecarbamate cyclofructan 6
(CF6-RN) column matrixes.5,6 The absolute configurations of these enantiomers may be
confirmed using vibrational circular dichroism (VCD).6
205
Scheme 1. Structure of lambda (Λ) and delta (Δ) enantiomers of [Ru(bpy)3]+2.
The racemates of certain Ru(II) polypyridyl complexes bearing dipyridophenazine (dppz) or
benzodipyridophenazine (dppn) type ligands are known for their DNA binding properties7–12 as
well as their capacity for DNA binding-induced luminescence.13 A number of studies have
expanded upon this by evaluating the binding properties of the delta (Δ) and lambda (Λ)
enantiomers with DNA, where they typically found the delta enantiomer to bind more strongly
to DNA than the lambda enantiomer. Consequently, the delta enantiomer is also observed to
be more cytotoxic than the delta enantiomer and racemate in certain cancerous cell lines,12,14–20
with studies suggesting that the primary source of this cytotoxicity stems from binding-induced
DNA damage or disruption of the mitochondrial membrane.
In the context of photodynamic therapy (PDT), the effect of chirality on the photobiological
activity of Ru(II) polypyridyl complexes is far less understood. A study that was published this
year (2023)12 observed that, at the same concentration (30 µM), ∆-[Ru(DIP)2(dppz)]+2 (DIP=
4,7-diphenylphenanthroline) was more phototoxic than the corresponding lambda enantiomer
in HeLa cells when exposed to visible light. We seek to expand upon the PDT community’s
understanding of the effects of chirality on photocytotoxicity by comparing the dose-response
activity of enantiomerically pure (∆/Λ) photosensitizers with their corresponding racemates. In
this study, we investigate two previously published Ru(II) oligothienyl complexes bearing 4,4ʹbtfmb ligands and an imidazo[4,5-f][1,10]phenanthroline ligand tethered to n=3–4 thiophene
rings (IP-nT). We selected these complexes because of their highly consistent photobiological
activity, which allows us to effectively distinguish between the effects of chirality from other
factors that contribute to the photobiological variance observed in certain oligothienyl systems.
206
4.3 RESULTS AND DISCUSSION
Chart 1. Structures of ¨/ȁ-Ru-3T and ¨/ȁ-Ru-4T. The racemates are mixtures of both
enantiomers.
Resolution of enantiomers via HPLC
A previously described chiral chromatographic technique was used for Ru-3T and Ru-4T.
Briefly, racemates were eluted through a CF6-RN column matrix using an eluent consisting of
MeOH/TFA/TEA. Three batches were prepared in total: A, B, and C. Batch A and B were
prepared using the same procedure. Batch B shown evidence of TEA and TFA salts by 1H
NMR and 19F NMR and was visibly less soluble than expected, another batch was prepared
without the use of triethylamine (TEA).
In Vitro Photobiological Activity
The complexes rac/¨/ȁ-Ru-3T and rac/¨/ȁ-Ru-4T were thoroughly evaluated for their
cytotoxic and photocytotoxic properties against normoxic-treated SKMEL28 cells over several
resazurin-based photobiological assays in which we determine the effective concentration to
207
eliminate 50% of cells (EC50) in the dark and under green (523 nm) and cool white visible
(400–700 nm) light as well as the phototherapeutic index (PI) of each compound under each
light condition (Table 1, Table S3–Table S8). The PI is defined as the ratio of dark to light EC50
values; in cases where the dark EC50 value exceeds the upper concentration limit of 300 µM,
the PI is technically undefined but is estimated using 300 µM as the dark EC50 value. Details
regarding the cell culture conditions, solution preparation, and photobiological assay
procedures may be found in our previous work8,9 as well as the Supplemental Information.
Unless otherwise specified, the racemates of Ru-3T and Ru-4T were tested alongside their
enantiomerically resolved counterparts.
Compound rac-Ru-3T exhibits minimal activity towards melanoma cells in the dark but
reached sub-micromolar EC50 values when exposed to visible or green light (visible EC50=16
nM, green EC50=220 nM). Our initial investigations showed that the enantiopure samples were
less active overall than the racemate both in the dark and light, but all values were within one
order of magnitude of one another. This was further confirmed in two follow-up assays where
we tested rac/¨/ȁ-Ru-3T alongside one another under the same conditions as the original run
(Figure S10, Table S3–Table S5).
Our investigations comparing rac/¨/ȁ-Ru-4T shown that the enantiomerically pure
compounds shared similar toxicity in the dark. However, in the presence of green or visible
light, rac-Ru-4T (visible EC50= 6.4 nM) and ¨-Ru-4T (visible EC50= 14 nM) were significantly
less photocytotoxic than ȁ-Ru-4T (visible EC50= 0.21 nM), where ȁ-Ru-4T out-performs racRu-4T by a factor of 35 (visible PI= 16,000 vs. 560,000)!
208
Table 1. Cytotoxicity and photocytotoxicity of rac/¨/ȁ-Ru-3T and rac/¨/ȁ-Ru-4T against normoxic-treated
SKMEL28 cells (~18.5% O2).
Resazurin-Cell Viability
Compound
PIc
EC50 ± SEM
Dark
Visiblea
Greenb
Visiblea
Greenb
ǻȁ
-Ru-3T 192 ± 8 μM
16 ± n.d. nM
0.22 ± 0.027 μM 12,000
870
ǻ-Ru-3T
210 ± 7 μM
97 ± n.d. nM
0.32 ± 0.031 μM
2200
670
ȁ-Ru-3T
>300 μM
79 ± n.d. nM
0.25 ± 0.029 μM
3800
1200
ǻȁ
-Ru-4T 110 ± 3 μM
6.4 ± 0.6 nM
97 ± 5.8 nM
16,000
1100
ǻ-Ru-4T
127 ± 4 μM
14 ± n.d. nM
180 ± n.d. nM
900
700
ȁ-Ru-4T
117 ± 3 μM
0.21 ± 0.016 nM
0.85 ± n.d. nM
560,000
14,000
Light treatments were approximately 100 J cm−2 delivered at 18–24 mW cm−2 with a cool white Visible
(400–700 nm), b Green (523 nm), and c PI = phototherapeutic index. Hypoxia and normoxia experiments
were ran in parallel. *n.d. = SEM not determined due to steep hill slope.
To confirm our findings in the initial assay described in Table 1, we tested the compounds
against one another an additional 17 times (18 total assays), where we probed the effects of:
1) inter-assay variability, 2) batch-to-batch variability (of the compounds), and 3) the potential
impact/presence of impurities. In order to isolate the effects of inter-assay variability, we kept
all conditions exactly the same for 10 additional assays using the same batch of material used
in the original run, which are coded as A1–A12 in Table S6–Table S8. In each experiment, we
observed that 1) the racemate was typically less toxic than either isolated enantiomer in the
dark (by up to a factor of two) and 2) the trend in light-triggered photocytotoxicity was that ȁRu-4T was the most phototoxic by a margin of roughly one order of magnitude, with ¨-Ru-4T
being the least phototoxic among the three and rac-Ru-4T being somewhere between the two
in terms of activity. Notably, the EC50 values for each complex under every light condition
209
during all 12 assays were consistently within 1 order of magnitude of one another, which aligns
with our previous observations of rac-Ru-3T and rac-Ru-4T.
The batch-to-batch variability of rac/¨/ȁ-Ru-4T was tested by preparing the racemate two
additional times then resolving each new batch of racemate, once using the exact same
procedure (Table S6–Table S8, B1–B4) and another time using a modified purification method
(Table S6–Table S8, C1–C2). The dark toxicity and photobiological efficacies of rac-Ru-4T
and ¨-Ru-4T from batch B were the same as the complexes from batch A, with EC50 values
within one order of magnitude of the original results. Conversely, ȁ-Ru-4T was less toxic in the
dark and with light treatments during all four repeat assays. The dark toxicity of ȁ-Ru-4T from
batch B exceeded the upper concentration limit of 300 µM, and the visible EC50 values
decreased by a factor of two in two examples (from ~0.5 nM to ~3 nM), and in the other two
examples their EC50 values dropped by over an order of magnitude (from ~0.5 nM to ~100
nM). The photocytotoxic trends were similar under green light, where the overall activity
decreased from 1–10 nM to roughly 300 nM during the four runs.
This sudden decrease in activity was particularly concerning because batches A and B were
identical in their HPLC traces. However, further analysis of their stock solutions revealed that
the lambda enantiomer in batch B was visibly less water soluble than previous batches (Figure
S12), and 1H NMR analysis confirmed the presence of cationic ammonium salts. These kinds
of salts are difficult to remove via simple work up procedures because of their similar solubility
properties to our complexes as well as their exceedingly high boiling points. We believe the
overall attenuation in activity was from the added mass of these salts causing less ȁ-Ru-4T to
be added to cells. To circumvent this issue, we were able to prepare batch C by removing TEA
from the eluent, thus removing the possibility of ammonium salts. Thankfully, the complexes
from batch C have displayed the same overall trends in activity that were observed in batch A
in the two biological replicates so far (Table S6–Table S8, C1–C2). Our observations here
further emphasize the importance of biological replication as well as the importance of fully
characterizing enantiomerically resolved samples after isolation.
210
4.4 SUMMARY AND FUTURE DIRECTIONS
In summary, the dark toxicity and photobiological activity of rac-Ru-3T, ¨-Ru-3T, and ȁ-Ru-3T
were, under these conditions, the same against SKMEL28 cells. However, expanding the IPnT ligand to have n = 4 thiophenes caused two major effects: 1) the dark toxicity of the isolated
¨-Ru-4T and ȁ-Ru-4T complexes was lower than rac-Ru-4T by roughly a factor of two; 2) ¨Ru-4T is the least active with light treatments and ȁ-Ru-4T was the most active under light
treatments, with rac-Ru-4T being in between. This enantioselectivity exceeds an order of
magnitude, and our best results displayed an improvement in activity by a factor of 35. To the
best of our knowledge, this is the first study to compare the EC50 values of racemic PSs with
their delta and lambda enantiomers. The source of this specificity is still unknown and is under
active investigation.
4.5 ASSOCIATED CONTENT
Synthetic characterization (1D and 2D NMR, HPLC, HRMS) of the racemates and
(photo)biological data are included in the Supporting Information.
Acknowledgements
S.A.M. and C.G.C. thank the National Cancer Institute (NCI) of the National Institutes of Health
(NIH) (Award R01CA222227) as well as the National Science Foundation (NSF) (Award NSF
2102459) for support. The content in this work is solely the responsibility of the authors and
does not necessarily represent the official views of the National Institutes of Health. S.A.M.
also thanks Dr. Daniel Todd as UNCG’s Triad Mass Spectrometry Facility manager and his
assistants Jennifer Simpson and Diane Wallace. S.A.M. likewise thanks Dr. Franklin Moy
(UNCG) and Dr. Brian Edwards (UTA) for their experimental support and instrument
maintenance as NMR facility managers.
211
4.6 REFERENCES
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(2)
Sun, P.; Krishnan, A.; Yadav, A.; MacDonnell, F. M.; Armstrong, D. W.
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75–80. https://doi.org/10.1016/j.molstruc.2008.02.030.
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Sun, P.; Wang, C.; Breitbach, Z. S.; Zhang, Y.; Armstrong, D. W. Development of New
HPLC Chiral Stationary Phases Based on Native and Derivatized Cyclofructans. Anal. Chem.
2009, 81 (24), 10215–10226. https://doi.org/10.1021/ac902257a.
(4)
Sun, P.; Wang, C.; Padivitage, N. L. T.; Nanayakkara, Y. S.; Perera, S.; Qiu, H.; Zhang,
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4.7 SUPPORTING INFORMATION
Materials and Methods
Synthesis and Characterization of Racemates
The parent complexes rac-Ru-3T and rac-Ru-4T was prepared as previously described.1 The
characterization data pertaining to the racemates may be found in Figure S1–S8.
Enantiomeric resolution of rac-Ru-3T and rac-Ru-4T
Parent complexes rac-Ru-3T and rac-Ru-4T were resolved and assigned as being the Λ or ∆
enantiomer using our previously described techniques.1 All preparative and analytical
separations were conducted using a JASCO (Tokyo, Japan) semi-prep supercritical fluid
chromatograph (SFC-2000-7). The instrument allows for flow rates up to 20 mL/min and a
pressure limit of 500 bar. The system contains two HPLC pumps (PU-2086), an autosampler
(AS-2059-SFC) with a 100 µL loop, a 6-column selector (HV-2080-06), a column oven (CO2060), a UV detector (UV-2075), and a back pressure regulator (BP-2080). The carbon dioxide
pump was held at a constant -10 °C by a Julabo (Seelbach, Germany) chiller to ensure the
CO2 stays liquefied, and the column oven was held at a constant 40 °C. Separations were
done on the Larihc CF6-RN phase which contains R-(-)-1-(1-Napthyl)ethyl (RN) isocyanate
functionalized cyclofructan-6 (CF-6) bonded to 5 µm fully porous silica particles. Columns were
synthesized and packed by AZYP, LLC (Arlington, Tx) in the dimensions of 250 x 4.6 (i.d.) for
analytical separations and 250 x 7.8 (i.d.) for preparative separations. For preparative
separations, solutions were made at 10 mg/mL in methanol, introduced to the system every ~7
minutes in 100 µL full loop injections (~1 mg of racemate), signal was monitored at 500 nm,
and fractions were collected using the SCF-Vch-Bp automated valve unit. The SFC system
and the valve unit were both controlled by JASCO’s Chrom Nav (ver. 2). The mobile phase
was 50/50 (v/v) CO2/modifier where the modifier is 100/0.25/0.25 (v/v/v) MeOH/TFA/TEA. For
the 4.6 (i.d.) column a flow rate of 4 mL/min was used and for the 7.8 (i.d.) column a flow rate
of 12 mL/min was used to approximately match the linear velocity between the columns. The
collected fractions were concentrated under reduced pressure, resuspended in water, then
extracted with DCM several times to remove any residual ions from the eluent. The DCM
215
fractions were then combined, concentrated under reduced pressure, and passed through a
12-inch column of HCl-treated Amberlite IRA-420 using MeOH as the eluent. The collected
fractions were concentrated under reduced pressure and used for subsequent photobiological
assays. To evaluate the integrity of each of the enantiomerically resolved samples we
evaluated each batch of ¨ȁ
-Ru-4T via 1H NMR and 19F NMR to confirm that all TFA and TEA
had been removed from the sample. The final yields of the enantiomerically resolved
complexes are as follows: 50 mg of rac-Ru-3T yielded 13 mg of ¨-Ru-3T (99.1% ee), 8 mg of
ȁ-Ru-3T (>99.8% ee by HPLC) (42% rec); 70 mg of rac-Ru-4T yielded 20 mg of ¨-Ru-4T
(98.4% ee by HPLC), 20 mg of ȁ-Ru-4T (98.8% ee by HPLC) (67% rec).
Two additional batches of ¨ȁ
-Ru-4T were prepared in order to evaluate the batch-to-batch
variability of the enantiomerically resolved complexes, which are grouped as “A”, “B”, or “C” in
the photobiological assays in Figure S11 and Table S3–S7. Batch B was prepared following
the exact procedure described above. Batch C was resolved using a mobile phase of 50/50
(v/v) CO2/modifier where the modifier is 100/0.25 (v/v) MeOH/TFA, and was then concentrated
under reduced pressure, redissolved in minimal MeOH, and passed through a 12-inch column
of HCl-treated Amberlite IRA-420 using MeOH as the eluent.
Cell culture
Following previously described techniques2,3 resazurin-based assays were used to assess the
in vitro (photo)cytotoxicity of the compounds in this series against both normoxic and hypoxic
male human melanoma cells (SK-MEL-28, ATCC HTB-72).
The nonpigmented male human melanoma cells (SK-MEL-28, ATCC HTB-72) were cultured
and maintained using EMEM (BioWhittaker, 12-125Q) media, which was further enriched with
10% FB essence (VWR, 10803-034) and 1% Gluta Grow (L-alanyl-L-glutamine; VWR 45001086). These cells were incubated in a water-jacketed incubator (ThermoFisher Thermo
Scientific 4110), which was set at 37°C. The humidity was maintained at or above 90%, and
the CO2 was kept at a 5% concentration. Split ratios ranging from 1:2 to 1:5 were used to
achieve a cellular density between 150,000–400,000 cells·ml-1. These cells were used within
216
15 passages of their procurement. Photobiological assays were conducted on SK-MEL-28
cells after being dispensed into 384-well plates at a seeding density of 3000 cells per well.
Cellular assays
We evaluated the photobiological potency of each compound by executing dose-response cell
viability assays on 384-well plates. We applied a concentration range from 1×10 −3 to 300 µM
for rac/¨/ȁ-Ru-3T and a range of 1×10−12 to 300 µM for rac/¨/ȁ-Ru-4T. To facilitate rapid
thermal interchange, irrespective of the experimental setup, we limited the stacking of well
plates in the incubator to only two high. Additional tests on Ru-3T and Ru-4T were performed
to assess the consistency between different assays, as reflected in Figure S10–Figure S11.
Ru(II) compound solutions
Stock solutions for Ru-3T and Ru-4T were formulated at 21 mM and 25 mM concentrations
respectively, using 100% DMSO. The remaining compounds had their stock solutions
prepared at a concentration of 5 mM in a mixture of 10% v/v DMSO and water. Dilutions were
created using 1x Dulbecco's Phosphate-Buffered Saline (DPBS) devoid of Ca2+ or Mg2+, which
was obtained by diluting 10x DPBS (Corning 20-031-CV). The highest concentration (300 µM)
of the diluted solutions contained less than 1.2% v/v DMSO. Stock solutions were stored in
glass vials with PTFE-lined caps and these vials were wrapped in aluminum foil to prevent light
exposure. All stock solutions were kept at −20°C, wrapped in foil, when not in use.
Cytotoxicity and photocytotoxicity
Following our recent methodology,2,3 the compounds were put through (photo)cytotoxicity
screening using a resazurin assay in a 384-well plate configuration. We used Greiner Bio-One
384-well plates (781182) for this procedure. Inside the biosafety cabinet, DPBS was dispensed
into the outermost two wells (144 wells in total), each receiving a volume of 75 µL, creating a
boundary. For the experimental arrangement, we used an electronic multichannel pipettor. All
inner wells, encompassing sample wells, positive control wells, and negative control wells, had
a total volume of 40 µL per well. The sample wells contained 10 µL of complete media, 20 µL
of cell slurry (3000 SK-MEL-28 cells per well), and 10 µL of compound dilutions in DPBS. The
positive control wells (12 in total) contained 10 µL of complete media, 20 µL of cell slurry (3000
217
cells per well), and 10 µL of DPBS. The negative control wells (12 in total) contained 30 µL of
complete media and 10 µL of DPBS. Prior to cell seeding, the well plates were preincubated
with the dispensed media at 37°C, 5% CO2, and a relative humidity of at least 90%. Post cell
seeding, the plates were gently agitated by tilting in all directions and subsequently placed in
the incubator for 2-3 hours to promote cell attachment.
During the incubation stage, we arranged serial dilutions of the compounds in sterile 384-well
plates, utilizing DPBS as the solvent. For all compounds, dilutions were arrayed over nine
concentrations from 1×10−3 to 300 µM. Additionally, for Ru-4T, we included an extra nine
concentrations spanning from 1×10−12 to 1×10−3 µM. To safeguard against premature cellular
activation and limit exposure to light, we ensured the lights in the biosafety cabinet were
switched off while preparing the dilutions. The 384-well plates, along with their lids, underwent
a pre-incubation period of 2–3 hours before we dispensed the compound dilutions at a volume
of 10 µL per well. Experiments were performed in triplicate, and the sample wells were
arranged row-wise, separated by every four rows.
The 384-well plates were subjected to overnight incubation, with a drug-to-light interval (DLI)
oscillating between 13 to 20 hours. Post-incubation, the plates received light treatments which
involved the delivery of approximately 100 J cm−2 of light at an intensity ranging from 18–24
mW cm−2. The spectrum of light encompassed cool white visible light (400–700 nm), blue light
(Prizmatix LED, 453 nm), green light (Prizmatix LED, 523 nm), and red light (Prizmatix LED,
633 nm). After light treatments, the plates experienced another day of incubation prior to the
execution of final viability measurements. It is significant to highlight that edge effects surfaced
on the 384-well plate, influencing the adjustment of the post-photodynamic therapy (PDT)
period to one day (20–23 hours) for achieving cell viability equilibration, diverging from the
conventional 48-hour period. We introduced prewarmed sterifiltered resazurin in a 0.2 M
phosphate buffer (pH = 7.4) with a concentration of 0.3 mM into all wells of the plates, allotting
a volume of 10 µL per well. The resazurin-stained plates then underwent a 4-hour incubation
before the fluorometric measurements were conducted using a Molecular Devices M2e plate
reader. These measurements included a 30-second shaking step, a bottom-read setup, an
218
excitation wavelength of 530 nm, a long-pass filter at 570 nm, and an emission wavelength of
620 nm.
Hypoxia cytotoxicity and photocytotoxicity
Compounds were scrutinized under two distinct oxygen settings: normoxia (~18.5% O 2) and
hypoxia (1% O2). During the hypoxia treatment phase, the cells, after seeding, were allowed a
1–2-hour incubation at 37°C, 5% CO2, and a minimum relative humidity of 90% under
normoxic conditions, ensuring adequate cell adhesion. Following this, the plates made a
transition to the specialized environment of a Biospherix Xvivo X3 chamber. Here, they were
further incubated for 2–3 hours under settings of 1% O2, 37°C, 5% CO2, and ≥90% relative
humidity. Post this phase, the compound dilutions were introduced in a biosafety cabinet.
The plates then resumed their residence in the Biospherix chamber for a DLI spanning 17–19
hours. To validate the hypoxic state, an immersive optical probe was deployed to measure the
dissolved oxygen content within the chamber, which consistently registered between 5–7 µM.
Once this hypoxic environment was confirmed, plates slated for light treatment were
hermetically sealed within the Biospherix chamber using qPCR films from VWR (89134-428),
notable for their low gas permeability and high transparency. This procedure was imperative
for the study of the oxygen reliance of the photosensitizer (PS).
Upon completion of the light treatment, the sealing films were removed under the sterile
confines of a biosafety cabinet. All the plates that underwent hypoxia treatment were then
migrated to an incubator with normoxic conditions set at 37°C, 5% CO 2, and a relative humidity
≥90%. In a manner akin to their normoxic counterparts, these plates were afforded a further
20–23 hours of incubation ahead of the ultimate viability assessment.
Longitudinal studies
The consistency and reliability of the performance of the rac/¨/ȁ-Ru-3T and rac/¨/ȁ-Ru-4T
complexes were rigorously examined over multiple assays using SK-MEL-28. The data from
these repeated tests can be consulted in Figure S10, Figure S11, and Table S2–Table S7.
219
Each repetition was characterized by a uniquely randomized plate map to negate any
unintended influences from stray light. Cells involved in these trials were between 10–15
passages.
Light devices and protocols.
For all biological assays, unless stated otherwise, a consistent fluence of 100 J cm -2 and an
irradiance ranging between 18–22 mW cm-2 were employed. The light treatments utilized three
different light sources for visible, green, and red light. These sources included a cool white
LED panel from SOLLA-CREE, covering a spectral range of 400–700 nm with a maxima
around 450 nm. Additionally, two UHP-LEDs from Prizmatix were used, emitting light at 523
nm (green) and 633 nm (red) respectively. The spectral outputs of these light sources can be
referenced in Figure S9.
Data manipulation and statistics
Data from the resazurin cell viability assay were corrected for background by subtracting the
signal from wells that contained only media and DPBS (no cells) and normalized relative to
untreated cells. Because the absorbance and emission of the metal complexes can interfere
with the resazurin fluorescence signal, wells treated with the highest concentrations of metal
complex were also observed under a microscope. If no cells were detected, these wells were
assigned a value of zero. A more detailed discussion of assay limitations for this class of
complexes is provided in our 2019 review.4
Data points obtained from resazurin fluorescence were fit to a three-parameter log-logistic
(Equation S1) and logistic model (Equation S2) using GraphPad Prism 8.4.0. We use Equation
S1 for summary log(EC50) plots (Figure 12a) and in the dose-response curves shown in Figure
13, but we use Equation S2 for data in log(PI) plots (Figure 12b) as well as the tabulated EC50
and PI values (Table 1 and Table S2–Table S7).
𝑌 = Bottom +
(𝑇𝑜𝑝 − 𝐵𝑜𝑡𝑡𝑜𝑚)
Equation S1
(1 + (10𝐿𝑜𝑔(𝐸𝐶50 −𝑋)×𝐻𝑖𝑙𝑙𝑠𝑙𝑜𝑝𝑒 )
220
𝑌 = Bottom +
(𝑇𝑜𝑝 − 𝐵𝑜𝑡𝑡𝑜𝑚)
(1 + (𝐸𝐶50 ⁄𝑋)
𝐻𝑖𝑙𝑙𝑠𝑙𝑜𝑝𝑒
)
Equation S2
Experiments were completed in triplicate and replicated data points are always plotted with
error bars denoting the standard deviation (SD). All EC50 values are reported alongside the
standard error of the mean (SEM). In cases where the hill slope was too steep to calculate a
representative SEM, the SEM was labelled as not determined (n.d.). Phototherapeutic indices
(PI) are reported as the ratio of dark to light EC50 values and serve as a phototherapeutic
efficacy benchmark. Any summary plots showing Log EC50 and Log PI values of the entire
series of complexes are plotted with SEMs from log-logistic fits.
221
Synthetic Characterization
(a)
H2O
MeOH
ϯϯ͛
ϲ͛ a
d,b
ϱ͛
5
e,j
f
g
h
6
i
c
Figure S1. 1H NMR (700 MHz) spectra of rac-Ru-3T in MeOD-d3 at 298 K with structure labelling and 1H NMR
assignments. Top: Full spectrum. Bottom: Zoom of aromatic region from 6.9–9.6 ppm.
222
ϯ͛ϯ
ϲ͛ a
c
d,b
ϱ͛ 5
6
e,j f
h g
i
i
g
h
e,jf
5
ϱ͛
d,b
6
a
ϲ͛
c
3
ϯ͛
e,j
f
g
h
i
i
g
h
f
e,j
Figure S2. 1H-1H COSY (700 MHz) NMR Spectra of rac-Ru-3T in MeOD-d3 at 298 K with structure labelling and
1H NMR assignments. Top: Zoom of aromatic region. Bottom: zoom of aromatic thiophene signals.
223
(a)
(b)
ϯ͛ϯ
ϲ͛ a
6 ϱ͛ 5
db
f,l
e
jhgi
k
c
Figure S3. 1H NMR (700 MHz) spectra of rac-Ru-4T in MeOD-d3 at 298 K with structure labelling and 1H NMR
assignments. Top: Full spectrum. Bottom: Zoom of aromatic region from 6.8—9.6 ppm.
224
ϯ͛ϯ
ϲ͛ a
c
d,b
6 ϱ͛ 5
e
f,l j,h,g
i
k
k
i
j,h,g
f,l
e
5
ϱ͛
6
a
d,b
ϲ͛
c
3
ϯ͛
e
f,l
g
j h
i
k
k
i
g
h
j
f,l
e
Figure S4. 1H-1H COSY NMR (700 MHz) spectra of rac-Ru-4T in MeOD-d3 at 298 K with structure labelling and
1H NMR assignments, aromatic region. Top: Zoom of entire aromatic region. Bottom: Zoom of aromatic region
highlighting key thiophene peaks.
225
Figure S5. (top) HPLC chromatogram for rac-Ru-3T collected at the following wavelengths: 285 nm. (middle)
Zoom of HPLC chromatogram for rac-Ru-3T collected at 285 nm. (bottom) Overlay of UV-Vis absorption spectra
226
of HPLC Chromatogram peaks of rac-Ru-3T at 285 nm.
Figure S6. (a) HPLC chromatogram for rac-Ru-4T collected at the following wavelengths: 285 nm. (b) Zoom of
227 wavelengths: 285 nm. (c) Overlay of UV-Vis
HPLC chromatogram for rac-Ru-4T collected at the following
absorption spectra of HPLC Chromatogram peaks of rac-Ru-4T at 285 nm.
Figure S7. (a) High resolution ESI+-MS spectrum for rac-Ru-3T (b) Zoom of 576.0128 m/z showing isotopic
distribution. (c) Zoom of 1151.0299 m/z showing isotopic distribution.
228
Figure S8. (a) High resolution ESI+-MS spectrum for rac-Ru-4T (b) Zoom of 617.0052 m/z showing isotopic
distribution. (c) Zoom of 1232.0035 m/z showing isotopic distribution.
229
Table S1. Analytical HPLC method using the Hypersil GOLD C18 Column.
time (min)
%MeCN
%Water
Pre-run
20
2
98
Run
0
2
98
2
5
95
5
30
70
15
30
70
20
60
40
30
95
5
35
2
98
40
2
98
Post-run
10
2
98
Flow rate (mL min−1)
1
Both eluents contain 0.1% optima grade formic acid with runs involving a 20 μL injection at 50–200 µM of metal
complex dissolved in optima grade MeOH.
230
Photobiological Evaluation
Relative Emission
(a)
100
Relative Emission
633 nm Prizmatix
75
523 nm Prizmatix
453 nm Prizmatix
50
Cool White
Visible CREE
25
0
400
(b)
Light Source
500
600
700
Wavelength (nm)
800
100
Light Source
633 nm Prizmatix
75
523 nm Prizmatix
453 nm Prizmatix
50
Cool White
Visible CREE
25
0
400
500
600
700
Wavelength (nm)
800
Figure S9. Relative spectral emissions or output of the light sources applied in photobiological studies
manufacturer of the LED chip and/or light device is indicated (Prizmatix or CREE). Two color schemes are shown
with (a) being colorblind-friendly and (b) matching colors used in reported biological plots (approximately matching
apparent colors).
231
Figure S10. Interassay cytotoxicity and photocytotoxicity of rac-Ru-3T (top), ¨-Ru-3T (middle), and ȁ-Ru-3T
(bottom) in normoxic-treated SK-MEL-28 melanoma cells. (EC50 ± SEM) values (left) and PI values (right).
Treatments included dark (0 J cm−2) and 100 J cm−2 doses of 523 nm and 453 nm light.
232
Figure S11. Interassay cytotoxicity and photocytotoxicity of rac-Ru-4T (top), ¨-Ru-4T (middle), and ȁ-Ru-4T
(bottom) in normoxic-treated SK-MEL-28 melanoma cells. (EC50 ± SEM) values (left) and PI values (right).
Treatments included dark (0 J cm−2) and 100 J cm−2 doses of 523 nm and 453 nm light. The experiment codes
are on the y-axis and they indicate the batch of material that was used as well as the total number of runs for
each batch. The total number of assays ran is 12.
233
Table S2. Interassay performance: cytotoxicity and photocytotoxicity of rac-Ru-3T in normoxic-treated (~18.5%
O2) male SK-MEL-28 melanoma cells.
Resazurin-Normoxia (~18.5% O2) rac-Ru-3T Repeats
EC50 ± SEM (μM)
PId
Assay
Repeat
Dark
Bluea
Greenb
Bluea
Greenb
1*
192 ± 8
0.22 ± 0.027
0.016 ± n.d.
870
12000
GS9-104
2
160 ± 7
0.043 ± 0.0044
0.57 ± n.d.
3700
280
GS9-108
3
200 ± 14
0.058 ± n.d.
0.12 ± n.d.
3400
1600
GS9-110
Light treatments were approximately 100 J cm−2 delivered at 18–22 mW cm−2. a blue 453
nm, b green 523 nm, c, d PI = phototherapeutic index (dark EC50 / light EC50), e cool white
visible light (400-700 nm) used in lieu of blue light *original run in Table 1.
234
Batchcode
Table S3. Interassay performance: cytotoxicity and photocytotoxicity of ¨-Ru-3T in normoxic-treated (~18.5% O2)
male SK-MEL-28 melanoma cells.
Resazurin-Normoxia (~18.5% O2) ¨-Ru-3T Repeats
EC50 ± SEM (μM)
PId
Assay
Repeat
Dark
Bluea
Greenb
Bluea
Greenb
1
170 ± 9
0.098 ± n.d.
0.19 ± n.d.
1745
900
GS9-94
2*
210 ± 7
0.097 ± n.d.
0.32 ± 0.031
2165
656
GS9-104
3
230 ± 8
0.30 ± 0.034
1.1 ± 0.03
760
207
GS9-108
4
210 ± 13
0.059 ± 0.014
0.29 ±0.078
3610
734
GS9-110
Light treatments were approximately 100 J cm−2 delivered at 18–22 mW cm−2. a blue 453
nm, b green 523 nm, c, d PI = phototherapeutic index (dark EC50 / light EC50), e cool white
visible light (400-700 nm) used in lieu of blue light *original run in Table 1.
235
Batchcode
Table S4. Interassay performance: cytotoxicity and photocytotoxicity of ȁ-Ru-3T in normoxic-treated (~18.5% O2)
male SK-MEL-28 melanoma cells.
Resazurin-Normoxia (~18.5% O2) ȁ-Ru-3T Repeats
EC50 ± SEM (μM)
PId
Assay
Repeat
Dark
Bluea
Greenb
Bluea
Greenb
1
250 ± 17
0.074 ± n.d.
0.18 ± n.d.
3400
1400
GS9-94
2*
>300
0.079 ± n.d.
0.25 ± 0.029
3800
1200
GS9-104
3
240 ± 8
0.061 ± 0.0059
0.34 ± 0.10
4000
710
GS9-108
4
250 ± 15
0.026 ± 0.0021
0.22 ± 0.07
9800
1200
GS9-110
Light treatments were approximately 100 J cm−2 delivered at 18–22 mW cm−2. a blue 453
nm, b green 523 nm, c, d PI = phototherapeutic index (dark EC50 / light EC50), e cool white
visible light (400-700 nm) used in lieu of blue light. *original run in Table 1.
236
Batchcode
Table S5. Interassay performance: cytotoxicity and photocytotoxicity of rac-Ru-4T in normoxic-treated (~18.5%
O2) male SK-MEL-28 melanoma cells. The “Run” column indicates the total number of assays on one complex,
and the “Experimental Code” indicates which batch was used as well as the total number of assays with that
batch.
Resazurin-Normoxia (~18.5% O2) rac-Ru-4T Repeats
EC50 ± SEM (μM)
Run
Experiment
Code
PId
Dark
Visiblec
Greenb
Visiblec
Greenb
Assay Batchcode
1*
A1
105 ± 3
(6.4 ± 0.64)*10-3 e
0.097 ± 0.0058
16000e
1100
GS9-104
2
A2
104 ± 3
(15.0 ± 2.1)*10-3 e
0.24 ± 0.049
6900e
430
GS9-108
3
A3
99 ± 3.8
(4.0 ± 0.27)*10-3 e
0.086 ± n.d.
25000e
1200
GS9-110
4
A4
68 ± 1.3
(0.89 ± n.d.)*10-3
0.070 ± 0.0027
77000
970
GS9-150
0.14 ± 0.012
30000
450
GS9-157
5
A5
63 ± 0.7
(2.1 ± 0.15)*10-3
6
A6
64 ± 1.2
(6.2 ± 0.31)*10-3
0.14 ± 0.019
10000
460
GS9-160
7
A7
79 ± 1.5
(1.7 ± 0.13)*10-3
0.043 ± 0.0033
45000
1800
GS9-168
8
A8
65 ± 1.5
(1.1 ± n.d.)*10-3
0.032 ± 0.0028
62000
2000
GS9-172
9
A9
90 ± 1.1
(2.7 ± 0.35)*10-3
0.14 ± 0.017
33000
640
GS9-175
10
A10
67 ± 1.3
(5.3 ± 0.43)*10-3
0.055 ± 0.0037
13000
12000
GS9-178
11
A11
52 ± n.d.
(1.3 ± 0.10)*10-3
0.048 ± 0.0037
40000
1000
GS9-185
12
B1
57 ± 2.0
(2.4 ± 0.11)*10-3
0.033 ± 0.0021
24000
1700
GS10-60
0.024 ± 0.0075
26000
2700
GS10-64
13
B2
67 ± 2.4
(2.6 ± 0.18)*10-3
14
B3
51 ± n.d.
(0.89 ± n.d.)*10-3
0.046 ± 0.0034
5700
1100
GS10-65
15
B4
27 ± 1.2
(0.74 ± 0.17)*10-3
0.039 ± 0.0035
3600
680
GS10-67
16
C1
64 ± 1.3
(6.4 ± 0.55)*10-3
0.11 ± n.d.
10000
600
GS10-74
17
C2
67 ± 1.1
(2.9 ± 0.41)*10-3
0.054 ± 0.0804
23000
1200
GS10-81
Light treatments were approximately 100 J cm−2 delivered at 18–22 mW cm−2 ablue 453 nm, b green 523 nm, c cool white visible (400–700 nm), d PI =
phototherapeutic index (dark EC50 / light EC50),. e blue 453 nm used, *original run in repeat 0 from Table 1.
237
Table S6. Interassay performance: cytotoxicity and photocytotoxicity of ¨-Ru-4T in normoxic-treated (~18.5% O2)
male SK-MEL-28 melanoma cells.
Resazurin-Normoxia (~18.5% O2) ¨-Ru-4T Repeats
EC50 ± SEM (μM)
Run
Experiment
Code
PId
Dark
Visiblec
Greenb
Visiblec
Greenb
Assay
Batchcode
1*
A1
127 ± 5
0.014 ± n.d.
0.18 ± n.d.
9000
706
GS9-94
2
A2
123 ± 4
(1.1 ± 0.070)*10-3 e
0.14 ± 0.010
110000
879
GS9-104
3
A3
129 ± 4
(19.0 ± 1.1)*10-3 e
0.22 ± 0.038
6800
586
GS9-108
4
A4
126 ± 7
(0.50 ± 0.037)*10-3 e
0.14 ± n.d.
252000
900
GS9-110
5
A5
121 ± 4
(0.62 ± n.d.)*10-3
0.11 ± 0.017
200000
1100
GS9-150
(7.55 ± 1.10)*10-3
280000
15000
GS9-157
6
A6
110 ± 3
(0.39 ± 0.074)*10-3
7
A7
122 ± 4
(0.021 ± 0.002)*10-3
0.16 ± 0.030
5800
760
GS9-160
8
A8
123 ± 4
(1.9 ± 0.17)*10-3
0.16 ± 0.046
66000
770
GS9-168
9
A9
116 ± 3
(4.1 ± 0.31)*10-3
0.15 ± 0.012
28000
770
GS9-172
10
A10
120 ± 4
(2.2 ± 0.60)*10-3
0.046 ± 0.0018
55000
2600
GS9-175
11
A11
118 ± 3
(7.8 ± 0.63)*10-3
0.12 ± 0.028
15000
1000
GS9-178
12
A12
113 ± 3
(2.2 ± 0.30)*10-3
0.055 ± 0.0025
52000
2100
GS9-185
13
B1
108 ± 3
(0.8 ± 0.16)*10-3
0.0140 ± 0.0064
134000
7700
GS10-60
0.013 ± n.d.
114000
8500
GS10-64
14
B2
115 ± 5
(1.0 ± n.d.)*10-3
15
B3
104 ± 3
(0.85 ± n.d.)*10-3
0.017 ± 0.0157
123000
6100
GS10-65
16
B4
97 ± 3
(0.84 ± n.d. )*10-3
0.017 ± 0.0112
11500
5700
GS10-67
17
C1
118 ± 3
(3.4 ± 0.28)*10-3
0.065 ± n.d.
35000
1800
GS10-74
18
C2
122 ± 4
(0.78 ± n.d.)*10-3
0.012 ± 0.0007
160000
10000
GS10-81
Light treatments were approximately 100 J cm−2 delivered at 18–22 mW cm−2 ablue 453 nm, b green 523 nm, c cool white visible (400–700 nm), d PI =
phototherapeutic index (dark EC50 / light EC50),. e blue 453 nm used, *original run in repeat 0 from Table 1.
238
Table S7. Interassay performance: cytotoxicity and photocytotoxicity of ȁ-Ru-4T in normoxic-treated (~18.5% O2)
male SK-MEL-28 melanoma cells.
Resazurin-Normoxia (~18.5% O2) ȁ-Ru-4T Repeats
EC50 ± SEM (μM)
Run
Experiment
Code
PId
Assay
Dark
Visiblec
Greenb
Visiblec
Greenb
(0.85 ± n.d.)*10-3
560000
14000
GS9-94
Batchcode
1*
A1
117 ± 3
(0.21 ± 0.016)*10-3
2
A2
113 ± 3
(0.61 ± n.d.)*10-3 e
(20.0 ± 1.0)*10-3
190000
5700
GS9-104
3
A3
103 ± 3
(0.63 ± 0.072)*10-3 e
(33.0 ± 1.8)*10-3
160000
3100
GS9-108
4
A4
134 ± 7
(0.33 ± 0.016)*10-3 e
(20.0 ± 1.7)*10-3
410000
6700
GS9-110
5
A5
105 ± 3
(0.29 ± 0.042)*10-3
(0.80 ± n.d.)*10-3
360000
13000
GS9-150
(3.7 ± 0.19)*10-3
340000
21000
GS9-157
6
A6
96 ± 2.3
(0.28 ± 0.030)*10-3
7
A7
98 ± 2.6
(0.32 ± 0.034)*10-3
(4.5 ± 0.19)*10-3
300000
22000
GS9-160
8
A8
105 ± 3
(0.62 ± n.d.)*10-3
(12.2 ± 0.80)*10-3
170000
8600
GS9-168
9
A9
95 ± 2.9
(0.63 ± n.d.)*10-3
(2.8 ± 0.15)*10-3
150000
34000
GS9-172
10
A10
101 ± 3
(0.64 ± n.d.)*10-3
(6.3 ± 0.35)*10-3
160000
16000
GS9-175
11
A11
99 ± 2.4
(0.32 ± 0.060)*10-3
(7.4 ± 1.10)*10-3
310000
13000
GS9-178
12
A12
93 ± 2.7
(0.30 ± 0.054)*10-3
(7.2 ± 0.56)*10-3
310000
130000
GS9-185
13
B1
>300
(3.2 ± 0.27)*10-3
0.23 ± 0.016
94000
1300
GS10-60
0.15 ± n.d.
96000
2100
GS10-64
14
B2
>300
(3.1 ± 0.20)*10-3
15
B3
>300
(97.0 ± n.d. )*10-3
0.31 ± 0.030
3100
970
GS10-65
16
B4
>300
(96.0 ± n.d.)*10-3
0.31 ± 0.026
3100
960
GS10-67
17
C1
82 ± 1.5
(1.5 ± 0.28)*10-3
0.022 ± 0.0034
56000
3800
GS10-74
18
C2
90 ± 1.1
(0.59 ± n.d.)*10-3
(3.49 ± 0.22)*10-3
150000
26000
GS10-81
Light treatments were approximately 100 J cm−2 delivered at 18–22 mW cm−2 ablue 453 nm, b green 523 nm, c cool white visible (400–700 nm), d PI =
phototherapeutic index (dark EC50 / light EC50),. e blue 453 nm used, *original run in repeat 0 from Table 1.
239
Figure S12. 300 µM stock solutions of ȁ-Ru-4T used in photobiological assays reported in Tables S2 through S7.
Left: batch A. Center: batch B. Right: batch C. The stock solution of batch A was prepared several months prior to
the other two batches, and so some compound has precipitated on the walls.
240
CHAPTER 5. CONCLUSIONS AND PERSPECTIVES
We set out to evaluate the various properties that lead to photocytotoxicity in Ru(II) polypyridyl
systems bearing the IP-nT ligand, where we tested the effects of the coligand (Chapters 2 and
3) as well as the chiral configuration of the photosensitizers (PSs) (Chapter 4). Our investigations
in Chapter 2 led to the identification of [Ru(phen)2(IP-4T)](Cl)2 as being another “ubertoxin”, with
phototoxicity occurring at as concentrations as low as ~10 fM and phototherapeutic indices (PIs)
(the ratios of dark to light EC50 values) reaching over 1 trillion! This extremely high degree of
activity has, so far, only been observed in the 2,9-dimethyl phenanthroline analog published
previously,1,2 but is otherwise unprecedented in the literature. Alongside this activity is the very
high degree of photobiological variability, sometimes spanning 5 or 6 orders of magnitude. In
response, we performed extensive biological replication on both complexes and documented
our findings. We do not have a clear explanation for this variation, but we suspect that it is related
to relatively poor solubility observed in certain IP-4T complexes.
In Chapter 3, we combined our IP-nT ligands with two 4,4ʹ-bistrifluoromethyl-2,2ʹ-bipyridine (4,4ʹbtfmb) coligands to evaluate the effects of fluorination on the photobiological, photophysical and
electrochemical properties of our complexes. The complexes were somewhat less active in
comparison to our phenanthryl complexes (10 nM vs 0.7 nM), but their activity was extremely
consistent, with all EC50 values being within one order of magnitude of one another. We
speculate this is related to the complexes being much easier to dissolve in aqueous media, but
we are uncertain what is specifically causing this behavior. The electrochemical properties of
these complexes are also of note. Specifically, the fluorinated 4,4ʹ-btfmb ligands facilitate the
acceptance of additional electrons while in the ground state (compared to the standard three
reductions in [Ru(bpy)3]+2 type complexes, one for each ligand), with [Ru(4,4ʹ-btfmb)2(IP4T)](Cl)2 being able to tolerate a staggering 8 electrons! This observation is likely contributing to
the overall photobiological activity, but the specific mechanisms related to this are still under
active investigation.
241
In Chapter 4, we probe the effects of chirality on the photobiological activity of our complexes.
In order to minimize interference from biological variation, we chose to focus on the
aforementioned [Ru(4,4ʹ-btfmb)2(IP-nT](Cl)2 complexes, and we utilized chiral HPLC to resolve
the complexes in collaboration with the Armstrong group. Our photobiological assays did not
show any differences in the dark toxicity of any of the complexes. Furthermore, rac/Δ/Λ-[Ru(4,4ʹbtfmb)2(IP-3T](Cl)2 all shared similar light-derived activity. However, the activity of rac/Δ/Λ[Ru(4,4ʹ-btfmb)2(IP-4T](Cl)2 is significantly different when tested alongside one another.
Specifically, the lambda enantiomer was the most active, followed by the racemate, with the
delta enantiomer being the least active among the three. The difference in activity was roughly
one order of magnitude when comparing the lambda enantiomer with either the racemate or
delta enantiomer, with Λ-[Ru(4,4ʹ-btfmb)2(IP-4T](Cl)2 leading in terms of activity by up to a factor
of 30 (rac-Ru-4T visible EC50=6.4 µM, ȁ-Ru-4T EC50=0.21 µM)!
In summary, this dissertation identifies several new and interesting photoactive complexes for
photodynamic therapy. These studies add a layer of complexity to our larger initiative of
correlating structural variations with photobiological activities across different families of
coordination complexes, where we are considering the metal ion, coligands, thienyl-appended
ligands, number of thiophenes, counter ions, ionizable groups, protonation states, and
coordination number and geometry. In the future, we seek to further expand upon these studies
by combining other phenanthroline-based and trifluoromethylated ligands with our IP-nT system,
and we plan to study the effects of chirality on other IP-nT complexes against SKMEL28 as well
as other cancer cell lines.
5.1 REFERENCES
(1) Roque III, J. A.; Cole, H. D.; Barrett, P. C.; Lifshits, L. M.; Hodges, R. O.; Kim, S.; Deep,
G.; Francés-Monerris, A.; Alberto, M. E.; Cameron, C. G.; McFarland, S. A. Intraligand
Excited States Turn a Ruthenium Oligothiophene Complex into a Light-Triggered
Ubertoxin with Anticancer Effects in Extreme Hypoxia. J. Am. Chem. Soc. 2022, 144 (18),
8317–8336. https://doi.org/10.1021/jacs.2c02475.
242
(2) Cole, H. D.; Roque, J. A.; Shi, G.; Lifshits, L. M.; Ramasamy, E.; Barrett, P. C.; Hodges,
R. O.; Cameron, C. G.; McFarland, S. A. Anticancer Agent with Inexplicable Potency in
Extreme Hypoxia: Characterizing a Light-Triggered Ruthenium Ubertoxin. J. Am. Chem.
Soc. 2022, 144 (22), 9543–9547. https://doi.org/10.1021/jacs.1c09010.
243