Synthesis, Characterization and Photobiological Studies of Ru(II) Dyads Derived from α-Oligothiophene Derivatives of 1,10-Phenanthroline

Article abstract of DOI:10.1111/php.13012

Three new bis(2,2′-bipyridine)-heteroleptic Ru(II) dyads incorporating thienyl groups (n?=?1–3, compounds 1, 2 and 3, respectively) appended to 1,10-phenanthroline were synthesized and characterized to investigate the impact of n on the photophysical and photobiological properties within the series. All three complexes showed unstructured emission near 618?nm from a triplet metal-to-ligand charge transfer (³MLCT) state with a lifetime (τ_em) of approximately 1?μs. Transient absorption measurements revealed an additional excited state that was nonemissive and long-lived (τ_TA?=?43?μs for 2 and 27?μs for 3), assigned as a triplet intraligand (³IL) state that was accessible only in 2 and 3. All three complexes were strong singlet oxygen (¹O₂) sensitizers, with quantum yields (Φ_?) for 2 and 3 being the largest (74–78%), and all three were photocytotoxic to cancer cells with visible light activation in the order: 3?>?2?>?1. Cell-free DNA photodamage followed the same trend, where potency increased with decreasing ³IL energy. Compounds 2 and 3 also showed in?vitro photobiological effects with red light (625?nm), where their molar absorptivities were <100?m^?1?cm^?1. These findings highlight that Ru(II) dyads derived from α-oligothiophenes directly appended to 1,10-phenanthroline—namely 2 and 3—possess low-lying ³IL states that are highly photosensitizing, and they may therefore be of interest for photobiological applications such as photodynamic therapy (PDT).

Full text of DOI:10.1111/php.13012

DR. S. MCFARLAND (Orcid ID : 0000-0002-8028-5055)
Article type : Special Issue Research Article
Synthesis, Characterization, and Photobiological Studies of Ru(II) Dyads Derived from -
Oligothiophene Derivatives of 1,10-Phenanthroline†
Susan Monro,1 Colin G. Cameron,2 Xiaolin Zhu,3 Katsuya L. Colón,2 Huimin Yin,1 Tariq
Sainuiddin,1 Marc Hetu,1 Mitch Pinto,1 Anderson Fuller,1 Leah Bennett,1 John Roque III,2
Wenfang Sun,*3 Sherri A. McFarland*1,2
1Department of Chemistry, Acadia University, 6 University Avenue, Wolfville, Nova Sco-
tia B4P 2R6, Canada
2Department of Chemistry and Biochemistry, University of North Carolina at Greens-
boro, 301 McIver Street, Greensboro, North Carolina 27402-6170 USA
3Department of Chemistry and Biochemistry, North Dakota State University, Fargo,
North Dakota 58108-6050 USA
Corresponding authors’ emails: samcfarl@uncg.edu (Sherri McFarland);
wenfang.sun@ndsu.edu (Wenfang Sun)
†This article is part of a Special Issue celebrating Photochemistry and Photobiology's 55th Anniver-
sary.
This article has been accepted for publication and undergone full peer review but has not
been through the copyediting, typesetting, pagination and proofreading process, which may
lead to differences between this version and the Version of Record. Please cite this article as
doi: 10.1111/php.13012
This article is protected by copyright. All rights reserved.

ABSTRACT
Three new bis(2,2′-bipyridine)-heteroleptic Ru(II) dyads incorporating thienyl groups
(n=1-3, compounds 1, 2, and 3, respectively) appended to 1,10-phenanthroline were syn-
thesized and characterized to investigate the impact of n on the photophysical and
photobiological properties within the series. All three complexes showed unstructured
emission near 618 nm from a triplet metal-to-ligand charge transfer (3MLCT) state with
a lifetime (τem) of approximately 1 μs. Transient absorption measurements revealed an
additional excited state that was nonemissive and long-lived (τTA=43 μs for 2 and 27 μs
for 3), assigned as a triplet intraligand (3IL) state that was accessible only in 2 and 3. All
three complexes were strong singlet oxygen (1O2) sensitizers, with quantum yields (∆)
for 2 and 3 being the largest (74-78%), and all three were photocytotoxic to cancer cells
with visible light activation in the order: 3 > 2 > 1. Cell-free DNA photodamage followed
the same trend, where potency increased with decreasing 3IL energy. Compounds 2 and
3 also showed in vitro photobiological effects with red light (625 nm), where their molar
absorptivities were <100 M-1 cm-1. These findings highlight that Ru(II) dyads derived
from -oligothiophenes directly appended to 1,10-phenanthroline – namely 2 and 3 –
possess low-lying 3IL states that are highly photosensitizing, and they may therefore be
of interest for photobiological applications such as photodynamic therapy (PDT).
This article is protected by copyright. All rights reserved.

INTRODUCTION
Ru(II) complexes for photobiological applications
Coordination complexes and organometallic complexes have emerged as attractive al-
ternatives to organic compounds for light-based applications ranging from dye-
sensitized solar cells (DSSCs) to photodynamic therapy (PDT). Their modular architec-
tures lend well to systematic structural changes for tuning (photo)chemical, (pho-
to)biological, and (photo)physical properties. An important feature for their use as pho-
tosensitizers is the sheer number of excited states that are accessible with visible and
near-infrared light (1–4). Metals and ligands can be selected to produce lowest-energy
excited states with predictable reactivities that can be fine-tuned with further chemical
modification to one or more ligands. This concept has been demonstrated in the wide-
ly-studied Ru(II) polypyridyl class of complexes.
Briefly, the excited state dynamics of [Ru(bpy)3]2+ (bpy=2,2′-bipyridine), and many of
its derivatives, are governed by a lowest-lying triplet metal-to-ligand charge transfer
(3MLCT) excited state that exhibits an intrinsic lifetime of approximately 1 s in deoxy-
genated solution at room temperature (1). This state decays primarily through phospho-
rescence or intersystem crossing (ISC) in the absence of oxygen and other excited state
quenchers, but can sensitize singlet oxygen (1O2) in oxygenated solution. While the
metal-centered (MC) and intraligand (IL) triplet excited states of [Ru(bpy)3]2+ are high-
er in energy than the 3MLCT state and largely inaccessible with visible light, judicious
changes to the auxiliary ligands have resulted in systems with accessible 3MC or 3IL
states that play a defining role in the photophysical dynamics.
Notable strategies include the use of substituted ligands that cause steric clash in the
coordination sphere of the metal ion, to populate dissociative 3MC states upon
This article is protected by copyright. All rights reserved.

photoexcitation. Photoinduced ligand loss in these strained Ru(II) complexes has been
exploited for covalent modification of biomolecules such as DNA as a means of cytotox-
icity for photochemotherapy (PCT) (5,6), although PCT has not yet been developed
clinically. Alternatively, -extended ligands have been used to lower the energy of 3IL
states and prolong excited state lifetimes for improved 1O2 photosensitization (and to
afford additional cytotoxic mechanisms) in the design of better PDT agents (7–12). The
term metal-organic dyad has been used to describe these Ru(II) complexes that incor-
porate -expanded organic chromophores that are tethered to one of the chelating lig-
ands, and these systems were first studied by groups investigating triplet-triplet energy
transfer in bichromophoric systems (13). The predicted utility (at the time) of such
supramolecular systems was in the area of optoelectronic and luminescence-based
technologies, but more recently, their potential in photobiological applications such as
PDT/PCT has been recognized (7,14–20).
Ru(II) dyads with low-energy 3IL states and prolonged lifetimes
Ford and Rogers were the first to report on the attachment of organic chromophores
to diimine ligands to impart novel photophysical properties to Ru(II) polypyridyl com-
plexes in 1992 (21). In their example, a pyrenyl group appended to 1,10-phenanthroline
(phen), spatially isolated from Ru(II), led to an 3IL-3MLCT excited state equilibrium
where the 3IL state served as an energy reservoir to prolong the 3MLCT lifetime more
than ten-fold (from 0.8 s to 11.2 s). Harriman and Ziessel later replaced the flexible
hydrocarbon chain of the earlier Ru(II)-pyrenyl metal-organic dyad with an alkynyl
linker directly conjugated to bpy, which increased the forward and reverse rates of tri-
This article is protected by copyright. All rights reserved.

plet energy transfer and extended the 3MLCT lifetime to 42 s (22). Building on this
work, Castellano and Ziessel found that including two or three (rather than one) of the
pyrenylethynylene-substituted bpy ligands led to pure 3IL states with slightly longer
intrinsic lifetimes (57 and 65 s, respectively) (23).
In 2010 two groups reported separately on Ru(II) dyads incorporating one
pyrenylethnylene chromophore appended to phen (PEP), but differing in the substitu-
tion position (3-PEP versus 5-PEP) (14,24). These PEP-based Ru(II) dyads also possessed
lowest-lying, pure 3IL states with lifetimes of 140 and 240 s, respectively, that were ex-
tremely sensitive to trace oxygen (19). Zhao demonstrated the utility of such systems for
luminescent oxygen sensing (24) while McFarland and Thummel demonstrated their
potent in vitro PDT effects (19). While pure 3IL states (derived from 3* transitions
localized to the organic chromophore) are good producers of cytotoxic 1O2 through
Type II energy transfer processes, 3IL states with some amount of charge-transfer char-
acter (3ILCT) offer the opportunity to exploit Type I electron transfer reactions as well as
potential oxygen-independent pathways for PDT/PCT. Therefore, we had been simulta-
neously investigating -oligothienyl-based Ru(II) dyads with accessible 3IL and 3ILCT
states.
-Oligothiophene-based Ru(II) dyads
Thiophene rings linked through the 2,2′ positions form stable, conjugated chains of
oligomers and polymers. Oligo- and polythiophene derivatives are generally able to do-
nate and accept electrons at moderate energies without decomposition (25); the inter-
esting photophysical and electrochemical properties of conjugated oligo- and
This article is protected by copyright. All rights reserved.

polythiophenes arise from the expansive π-system that can stabilize excited states and
distribute positive and negative charges when the number of thiophene rings n is suffi-
ciently large. Optical absorption by the π-extended system leads to symmetry-
permitted excited states that can be driven to long-lived charge separation under the
influence of a suitable acceptor or donor (26).
The capacity for charge separation and transport is useful in the fields of solar energy
conversion and molecular electronics, but even oligomers of smaller n are well suited to
such applications. Triplet states are accessible from ISC of a singlet exciton, with an ex-
change energy of around 0.7 eV in conjugated polymers in general (27) and 1.72 eV for
2,2′:5′,2′′-terthiophene (3T) (28). The “natural” size of the triplet exciton is n=3‒4
thiophene rings (29), but the quantum yield of triplet formation decreases as the num-
ber of rings increases, leveling off at around n=5 (30). We were thus interested in
oligothiophenes of about n=3-5 for our Ru(II) dyads.
Early work by Scaiano and coworkers showed that relatively short thiophene chains
(such as 3T) act as good 1O2 generators (28,31), which led us to focus on 3T as the organ-
ic chromophore for certain Ru(II) dyads. The 3ππ* states of 3T have been estimated as
1.72 eV (28) to 1.93 eV (32), lower in energy than the 3MLCT state of most polypyridyl
Ru(II) complexes (33) making it possible to capture photons with 1MLCT states and de-
liver them effectively to spin-forbidden 3IL states on the organic chromophore. The slow
ISC rates characteristic of oligothienyl-based 3ππ* states are responsible for the extend-
ed triplet lifetimes observed for these Ru(II) dyads, especially those with 3IL states that
are sufficiently low in energy that they do not equilibrate with 3MLCT states. The impli-
cation is that long intrinsic lifetimes provide ample opportunity for bimolecular reac-
tions with oxygen and other excited state quenchers.
This article is protected by copyright. All rights reserved.

In addition, Ru(II) dyads having oligothienyl-substituted ligands with n as small as 2
exhibit long-lived charge separation via 3ILCT states formed by reductive quenching of
the initially populated MLCT state as demonstrated by Wolf and MacDonnell (33–35)
These 3ILCT states can participate in electron transfer reactions with oxygen and also
undergo autoionization steps to propagate chain reactions. This Type I/II dual reactivity
has been explored computationally for Ru(II) dyads containing the oligothienyl-
appended imidazo[4,5-f][1,10]phenanthroline (IP-nT) ligand, where n=1-4 (36), to ex-
plain the potent in vitro PDT effects observed with certain members of this family (7).
One example that serves to illustrate the potential of such systems in photobiological
applications is [Ru(dmb)2(IP-3T)]Cl2 (dmb=4,4′-dimethyl-2,2′-bipyridine), also known
as TLD1433, a Ru(II) dyad that we developed that is the first Ru(II)-based photosensitiz-
er to enter a human clinical trial and is being investigated for treating nonmuscle inva-
sive bladder cancer with PDT (ClinicalTrials.gov Identifier: NCT03053635).
In the present investigation, we explore a new family of Ru(II) dyads with thiophene
units (n=1-3) directly attached to phen at C5 (Chart 1). These structures are reminiscent
of the analogous pyrenyl-containing system first reported by Castellano that exhibited a
24 s lifetime (37). This study arose out of our ongoing interest in exploring the
photophysical and photobiological properties of oligothienyl-based chromophores in a
variety of Ru(II) dyad constructs.
[CHART 1]
This article is protected by copyright. All rights reserved.

MATERIALS AND METHODS
Synthesis and characterization. 1,10-phenanthroline monohydrate, ammonium ace-
tate, thiophene, n-butyllithium, tributyltinchloride, tetrakis(triphenylphosphine)-
palladium(0), thiophene (1T), 2,2′-bithiophene (2T), 2,2′:5′,2′′-terthiophene (3T), were
purchased from Sigma-Aldrich and used without further purification.
[Ru(bpy)2Cl2]∙2H20 was prepared by an established procedure (38). Microwave reactions
were performed in a CEM Discover microwave reactor. NMR spectra were collected us-
ing Bruker AVANCE 500 (Dalhousie University Nuclear Magnetic Resonance Research
Resource) or 300 (Acadia Centre for Microstructureal Analysis) MHz spectrometers,
and ESI mass spectra were obtained using a Bruker microTOF focus mass spectrometer
(Dalhousie University Mass Spectrometry Laboratory). HPLC analysis was carried out
using a Hypersil GOLD C18 reversed-phase column with an A-B gradient (98%  40%
A; A=0.1% formic acid in H2O, B=0.1% formic acid in MeOH). Reported retention times
are correct to within ± 0.1 min.
-
The metal complexes for this investigation were isolated and purified as PF6 salts and
subsequently subjected to anion metathesis on Amberlite IRA-410 with MeOH in order
to yield more water-soluble Cl- salts for biological analysis. 1H NMR and electrospray
-
ionization ESI (+ve) mass spectra were collected on PF6 salts in CD3CN and CH3CN,
respectively.
5-bromo-1,10-phenanthroline. 5-bromo-1,10-phenanthroline was prepared according to
an adapted literature procedure (39,40). 1,10-Phenanthroline monohydrate (4.02 g) was
dried in a vacuum oven (50 °C) for 3 days before use, resulting in a final weight of ap-
proximately 3.67 g. A dry heavy-walled pressure tube was charged with dry 1,10-
phenanthroline (3.6 g, 20 mmol) and then cooled in an ice bath. Oleum (sulphuric acid
This article is protected by copyright. All rights reserved.

fuming 20%, 12 mL) was added and the tube was stirred for 10 min, followed by
dropwise addition of bromine (0.6 mL, 11.6 mmol) dropwise using a dry 2.5 mL gastight
syringe. The pressure tube was tightly capped with a PTFE stopper, and heated slowly to
135 °C. Two days later, the color changed from a dark brown to a clear orange/red. The
reaction mixture was cooled, then slowly and carefully poured into a beaker containing
ice water and swirled, where the color changed to a brown/orange color. The mixture
was neutralized with stirring by adding NH4OH dropwise, changing the pH from 4 to
pH 10. The mixture became a yellow/orange cloudy color, with a dark red oil separated.
The mixture was transferred to a separatory funnel and two chloroform extractions (~15
mL) were performed. The chloroform layer, a clear, light yellow color, was introduced to
a beaker and a scoopful of activated charcoal was added and stirred for 15-20 min. The
mixture was then poured into an Erlenmeyer flask containing anhydrous MgSO4, then
gravity filtered through Whatman #1 filter paper into a round-bottom flask, and con-
centrated under vacuum. The light pink/purple solid was suspended in warm ether
(300-400 mL), resulting in a light purple solution with precipitate, and this was filtered
over a bed of Celite by applying light vacuum. The filtrate was then concentrated under
reduced pressure and dried to yield the final product as a colorless solid (2.448 g, 47%).
Rf = 0.38 (alumina, 5% MeOH/DCM). 1H NMR (300 MHz, CDCl3): δ 9.26 – 9.17 (m, 2H;
a, h), 8.67 (d, J = 6.6 Hz, 1H; f), 8.21 – 8.11 (m, 2H; c,d), 7.81 – 7.59 (m, 2H; g,f).
5-(2-thiophene)-1,10-phenanthroline (phen-1T). The starting material 2-
(tributylstannyl)thiophene (IT-SnBu3) was prepared according to a published method
with some exceptions (41). In summary, a 100 mL dry, argon purged, 3-hole round-
bottom flask with stir bar was charged with anhydrous tetrahydrofuran (30 mL).
Thiophene (320 L, 4 mmol) was added via a dry 1 mL gas-tight syringe and the flask
This article is protected by copyright. All rights reserved.

was immersed in a dry ice/acetone bath (approximately -80 °C) while slowly adding n-
BuLi (2.8 mL, 1.6 M, 4.4 mmol) dropwise via a dry 5 mL gas-tight syringe. This was
stirred for 30 min, then the reaction was quenched with a dropwise addition of
tributyltinchloride (1.3 mL, 4.8 mmol). The flask was stirred for 45 min more, then
brought slowly to room temp and stirred for 3 days. TLC revealed no spot for the
thiophene starting material (using various solvent conditions with silica or alumina
plates) but the reaction showed a new spot under short wavelength 254 nm. The mix-
ture was transferred to a 200 mL beaker and saturated NH4Cl was added (20 mL) while
stirring, producing an aqueous NH4Cl cloudy white bottom layer and a clear, pale yel-
low top layer. The mixture was transferred to a separatory funnel and an ether extrac-
tion (30-50 mL) was done, where the top ether layer was dried over anhydrous Na2SO4,
then filtered using a medium glass-sintered frit. The solvent was removed by rotary
evaporation and dried overnight to give a clear, pale yellow oil (1.4395 g, 95%). Rf = 0.87
(alumina, 100% hexanes).
To make the phen-1T ligand, a 50 mL dry Schlenk flask was charged with 5-bromo-
1,10-phenanthroline (518 mg, 2 mmol), 1T-SnBu3 (756 mg, 2 mmol),
tetrakis(triphenylphosphine)palladium(0) (69 mg, 0.06 mmol, 3 mol%), and dry DMF
(20 mL). The flask was argon purged and the reaction refluxed at 100 °C for 3 days. The
pale brown product was cooled then poured into a separatory flask holding saturated
NH4Cl (50 mL), followed by two DCM extractions and one water wash. The DCM layer
was dried over anhydrous Na2SO4, filtered, then concentrated under reduced pressure
to obtain a pale orange oil. Purification was performed using alumina column chroma-
tography, starting with 100% DCM and increasing polarity to 5% MeOH/DCM. This
resulted in a clear brown/red oil (185 mg, 35%). Rf = 0.31 (alumina, 5% MeOH/DCM). 1H
This article is protected by copyright. All rights reserved.

NMR (300 MHz, (CD3)2SO): δ 9.08 (s, 1H; g), 8.49 (dd, J = 48.3, 8.7 Hz, 2H; a,d), 7.93 –
7.66 (m, 3H; c,f,j), 7.64 – 7.46 (m, 4H; b,e,i,h).
5-(2,2′-bithiophene)-1,10-phenanthroline (phen-2T). 5-(tributylstannyl)-2,2′-
bithiophene (2T-SnBu3) was prepared according to a published method (41). Briefly, a
100 mL dry, argon purged, 3-neck round-bottom flask with stir bar was charged with
anhydrous THF (30 mL), then 2,2′-bithiophene (582 mg, 3.5 mmol) was added, and the
flask was immersed in a dry ice/acetone bath and cooled to -80 °C followed by dropwise
addition of n-BuLi (2.4 mL, 1.6 M, 3.9 mmol) dropwise via a dry 5 mL gas-tight syringe.
This was stirred for 30 min, then the reaction was quenched with a dropwise addition of
tributyltinchloride (1.14 mL, 4.2 mmol). The flask was stirred for 40 min more, then
brought slowly to room temp and stirred overnight. The colour changed from clear light
brown to clear light orange/brown. The mixture was transferred to a 200 mL beaker and
saturated NH4Cl (20 mL) was added while stirring, resulting in a bilayer of aqueous
NH4Cl cloudy white bottom layer and a clear amber/green top layer. The mixture was
transferred to a separatory funnel, an ether extraction (30-50 mL) was done, and the top
clear green ether layer was dried over anhydrous Na2SO4, then filtered using a medium
glass-sintered frit. The solvent was removed by rotary evaporation and dried overnight
to give a clear amber-green oil (1.14 g, 71%). Rf = 0.30 (Silica, 100% hexanes). The prod-
uct was used without further purification.
The ligand phen-2T was then synthesized by charging a dry 50 mL Schlenk flask with
5-bromo-1,10-phenanthroline (518 mg, 2 mmol), 2T-SnBu3 (756 mg, 2 mmol),
tetrakis(triphenylphosphine)palladium(0) (69 mg, 0.06 mmol, 3 mol%), and dry DMF
(20 mL). The flask was argon purged and the reaction refluxed at 100 °C for 3 days. The
product changed from a clear beige/brown to dark brown colour. It was cooled then
This article is protected by copyright. All rights reserved.

poured into a separatory flask containing saturated NH4Cl (75 mL) followed by two
DCM extractions and one water wash. The DCM layer was dried over anhydrous
Na2SO4, filtered, and then concentrated down resulting in a brown/orange oil (5.631 g).
Purification was performed using alumina column chromatography, starting with 100%
DCM and increasing polarity from 2% to 5% MeOH/DCM. This resulted in a clear
brown oil (263 mg, 58%). Rf = 0.5 (alumina, 5% MeOH/DCM). 1H NMR (300 MHz,
(CD3)2SO): δ 9.10 (ddd, J = 7.6, 4.3, 2.0 Hz, 2H; a,d), 8.67 (ddd, J = 14.2, 8.4, 1.7 Hz, 1H;
l), 8.52 (ddd, J = 17.2, 8.0, 1.8 Hz, 3H; c,f,g), 7.79 (ddd, J = 10.0, 8.1, 4.3 Hz, 2H; b,e), 7.57
(m, 1H; h), 7.48 (d, J = 3.7 Hz, 1H; i), 7.43 (dd, J = 3.7, 1.1 Hz, 1H; j), 7.14 (dd, J = 5.1, 3.6
Hz, 1H; k).
5-(2,2′:5′,2′′-terthiophene)-1,10-phenanthroline (phen-3T). The starting material 5-
(tributylstannyl)-5′-(2,2′:5′,2′′-terthiophene) was prepared according to a published
method with some exceptions (41). A 100 mL dry, argon purged, 3-hole round-bottom
flask with stir bar was charged with anhydrous THF (30 mL), then 2,2′:5′2:-terthiophene
(496 mg, 2 mmol) was added and the flask was immersed in a dry ice/acetone bath and
cooled to -80 °C while slowly adding n-BuLi (1.4 mL, 1.6 M, 2.2 mmol) dropwise via a
dry 5 mL gas-tight syringe. This was stirred for 30 min, then the reaction was quenched
with a dropwise addition of tributyltinchloride (651 L, 2.4 mmol). The flask was stirred
for 45 min more, then brought slowly to room temp and stirred for 3 days. The mixture
was transferred to a 200 mL beaker and saturated NH4Cl (30 mL) was added while stir-
ring, resulting in a bilayer of aqueous NH4Cl cloudy white bottom layer and a clear pale
green top layer. The mixture was transferred to a separatory funnel, an ether extraction
(30-50 mL) was done, and the top clear green ether layer was dried over anhydrous
Na2SO4, then filtered using a medium glass-sintered frit. The solvent was removed by
This article is protected by copyright. All rights reserved.

rotary evaporation and dried overnight to give a clear emerald green oil (977 mg, 91%).
Rf = 0.35 (silica, 100% hexanes) that was used without further purification.
The ligand phen-3T was then synthesized by charging a dry 50 mL Schlenk flask con-
taining 10 mL dry DMF with 5-bromophenanthroline (324 mg, 1.25 mmol) and 3T-
SnBu3 (672 mg, 1.25 mmol). After argon purging,
tetrakis(triphenylphosphine)palladium(0) (43 mg, 0.04 mmol, 3 mol%). The flask was
argon purged again and the reaction refluxed at 100 °C for 3 days. The product changed
from clear pale brown to a dark brown/red color. It was cooled and then poured into a
separatory funnel containing saturated NH4Cl (75 mL) followed by two DCM extrac-
tions and one water wash. The DCM layer was dried over anhydrous Na2SO4, filtered,
and then concentrated to give a dark brown/orange oil (2.42 g). Purification was per-
formed using alumina column chromatrography, starting with 100% DCM and increas-
ing polarity from 2% to 5% MeOH/DCM. This resulted in a clear orange oil (405 mg,
76%). Rf = 0.84 (alumina, 1% MeOH/DCM). 1H NMR (300 MHz, DMSO-d6) δ 9.15 (ddd,
J = 10.8, 4.4, 1.7 Hz, 2H; a,d), 8.66 (dd, J = 8.4, 1.6 Hz, 1H; c), 8.53 – 8.47 (m, 2H; g,f),
7.93 (dd, J = 8.4, 4.3 Hz, 1H; b), 7.81 (dd, J = 8.1, 4.3 Hz, 1H; e), 7.66 – 7.50 (m, 7H;
h,i,j,k,l,m,n).
Ru(bpy)2(phen-1T)PF6 (1). Ru(bpy)2Cl2 (364 mg, 0.71 mmol) and phen-1T (185 mg, 0.71
mmol) were added to a microwave vessel containing 99% ethanol (1.5 mL) and ethylene
glycol (1.5 mL). The mixture was subjected to microwave irradiation at 180 °C for 15 min.
TLC (silica, 7.5% H2O/MeCN + 0.5% KNO3) indicated that the starting materials were
exhausted, therefore the mixture was cooled and transferred (using dI water) to a 500
mL separatory funnel and slightly diluted with water. DCM extractions removed excess
Ru(bpy)2Cl2, then the aqueous layer containing product was introduced to saturated
This article is protected by copyright. All rights reserved.

KPF6 (approximately 2-3 mL). Further DCM extractions containing the desired product
were concentrated down to dry crude product (457.2 mg). The crude product was puri-
fied by silica flash chromatography, starting with 100% MeCN and the polarity was
gradually increased (up to 7.5% H2O/MeCN + 0.5% - 1% KNO3). This had to be repeated
three times to yield the purest possible product. Yield: 30.9 mg, 6.4%. Rf = 0.30 (10%
H2O in MeCN + 0.5% KNO3). 1H NMR (500 MHz, CD3CN): δ 8.84 (dd, J = 8.5, 1.2 Hz,
1H; c), 8.62 (dd, J = 8.3, 1.2 Hz, 1H; f), 8.56 (d, J = 7.8 Hz, 2H; 3’), 8.53 (d, J = 8.1 Hz, 2H;
3), 8.36 (s, 1H; g), 8.16 – 8.11 (m, 4H; 4’,a,d), 8.03 (td, J = 7.9, 1.4 Hz, 2H; 4), 7.88 (dd, J =
5.4, 1.3 Hz, 2H; 6’), 7.79 – 7.74 (m, 3H; b,e,j), 7.61 (dd, J = 9.7, 5.6 Hz, 2H; 6), 7.51 (dd, J =
3.6, 1.2 Hz, 1H; h), 7.48 (ddd, J = 7.7, 5.6, 1.4 Hz, 2H; 5′), 7.37 (dd, J = 5.1, 3.6 Hz, 1H; i),
7.30 – 7.25 (m, 2H; 5). MS (ESI+) m/z: 821.07 [M-(PF6)]+, 338.33 [M-2(PF6)]2+. HRMS
(ESI+) m/z for C36H26N6RuS: calc’d 338.0486, found: 338.0479. HPLC retention time:
16.15 min (97%).
Ru(bpy)2(phen-2T)PF6 (2). Ru(bpy)2Cl2 (151 mg, 0.29 mmol) and phen-2T (99.7 mg,
0.29 mmol) were added to a microwave vessel containing 99% ethanol (1.5 mL) and eth-
ylene glycol (1.5 mL). The mixture was subjected to microwave irradiation at 180 °C for
15 min. TLC (silica, 7.5% H2O/MeCN + 0.5% KNO3) indicated that the starting materi-
als were exhausted, therefore the mixture was cooled and transferred (using dI water) to
a 500 mL separatory funnel containing 100 mL dI water. DCM extractions removed ex-
cess Ru(bpy)2Cl2, then the aqueous layer containing product was introduced to saturat-
ed KPF6 (approximately 2-3 mL). Further DCM extractions removed the desired com-
pound which was concentrated down to dry crude product (148.9 mg). The crude prod-
uct was purified by silica flash chromatography, starting with 100% MeCN and the po-
larity was gradually increased (up to 7.5% H2O/MeCN + 0.5% to 1% KNO3). This was
This article is protected by copyright. All rights reserved.

repeated twice to afford the purest possible product. Yield: 18.1 mg, 6.0%. Rf = 0.34
1
(7.5% H2O in MeCN + 0.5% KNO3). H NMR (500 MHz, CD3CN): δ 8.94 (dd, J = 8.5, 1.2
Hz, 1H; c), 8.63 (dd, J = 8.2, 1.2 Hz, 1H; f), 8.57 (dd, J = 8.3, 1.0 Hz, 2H; 3’), 8.53 (dd, J =
8.2, 1.2 Hz, 2H; 3), 8.39 (s, 1H; g), 8.17 – 8.11 (m, 4H; 4’,a,d), 8.04 (td, J = 8.0, 1.5 Hz, 2H;
4), 7.88 (d, J = 5.2 Hz, 2H; 6’), 7.78 (ddd, J = 8.2, 5.2, 1.6 Hz, 2H; b,e), 7.61 (ddt, J = 9.4,
5.7, 1.2 Hz, 2H; 6), 7.51 – 7.47 (m, 4H; 5′,h,i), 7.45 (d, J = 3.8 Hz, 1H; l), 7.43 (dd, J = 3.6,
1.1 Hz, 1H; j), 7.28 (tdd, J = 5.9, 3.3, 1.6 Hz, 2H; 5), 7.17 (dd, J = 5.1, 3.6 Hz, 1H; k). MS
(ESI+) m/z: 903.00 [M-PF6]+, 379.33 [M-2(PF6)]2+. HRMS (ESI+) m/z for C40H28N6RuS:
calc’d 379.0425, found 379.0412. HPLC retention time: 23.36 min (96%).
Ru(bpy)2(phen-3T)PF6 (3). Ru(bpy)2Cl2 (210 mg, 0.4 mmol) and phen-3T (167 mg, 0.4
mmol) were added to a microwave vessel containing 99% ethanol (1.5 mL) and ethylene
glycol (1.5 mL). The mixture was subjected to microwave irradiation at 180 °C for 15
min. TLC (silica, 7.5% H2O/MeCN + 0.5% KNO3) indicated that the starting materials
were exhausted, therefore the mixture was cooled and transferred (using dI water) to a
500 mL separatory funnel containing 100 mL dI water. DCM extractions removed excess
Ru(bpy)2Cl2, then the aqueous layer containing product was introduced to saturated
KPF6 (approximately 2-3 mL). Further DCM extractions removed the desired compound
which was concentrated down to dry crude product (328 mg). The crude product was
purified by silica flash chromatography, starting with 100% MeCN and the polarity was
gradually increased (up to 7.5% H2O/MeCN + 0.5% to 1% KNO3). This was repeated
twice to yield the purest possible product. Yield: 20.4 mg, 4.5%. Rf = 0.33 (7.5% H2O in
MeCN + 0.5% KNO3). 1H NMR (500 MHz, CD3CN): δ 8.93 (dd, J = 8.6, 1.2 Hz, 1H; c),
8.63 (dd, J = 8.3, 1.2 Hz, 1H; f), 8.56 (d, J = 8.3 Hz, 2H; 3’), 8.52 (d, J = 8.3 Hz, 2H; 3), 8.39
(s, 1H; g), 8.16 – 8.10 (m, 4H; 4’,a,d), 8.03 (td, J = 7.9, 1.5 Hz, 2H; 4), 7.88 (d, J = 5.9 Hz,
This article is protected by copyright. All rights reserved.

2H; 6’), 7.77 (ddd, J = 8.3, 5.2, 2.4 Hz, 2H; b,e), 7.63 – 7.58 (m, 2H; 6), 7.50 – 7.45 (m,
4H; 5′,h,k), 7.43 (dd, J = 5.1, 1.1 Hz, 1H; n), 7.36 (d, J = 3.8 Hz, 1H; i), 7.35 (dd, J = 3.6, 1.1
Hz, 1H; l), 7.30 – 7.25 (m, 3H; 5,j), 7.13 (dd, J = 5.1, 3.6 Hz, 1H; m). MS (ESI+) m/z:
985.00 [M-PF6]+, 420.33 [M-2(PF6)]2+. HRMS (ESI+) m/z for C44H30N6RuS: calc’d
420.0363, found 420.0366. HPLC retention time: 25.51 min (100% purity).
Photophysical methods. Photophysical measurements were performed on dilute solu-
−
tions (5‒20 μM) of the metal complexes as their PF6 salts in spectroscopic grade MeCN.
Molar extinction coefficients (ε) were determined from the slopes of absorption versus
concentration plots for five concentrations, and measured in duplicate. Quantum yields
for emission (Φem) and singlet oxygen (ΦΔ) were measured relative to [Ru(bpy)3](PF6)2
as the standard (s) according to eq 1, where I, A, and η are the integrated emission in-
tensity, the absorbance at the excitation wavelength, and the refractive index of the sol-
vent, respectively. Reference quantum yields used for [Ru(bpy)3](PF6)2 were: Φem = 0.012
at 298 K in aerated MeCN (42), Φem = 0.095 at 298 K in deaerated MeCN (43), Φem
=
0.38 at 77 K in frozen 4:1 v/v EtOH:MeOH (18), and ΦΔ = 0.56 in aerated MeCN (44).
(1)
Solutions were degassed by argon sparging in a long-stemmed cuvette (Luzchem
QSC10S) for steady-state emission experiments. Samples for 77 K measurements were
prepared in 4:1 EtOH:MeOH solution in a 5 mm i.d. NMR tube, and then frozen in liq-
uid nitrogen in a quartz-tipped cold finger Dewar (Wilmad Labglass). UV-vis absorp-
tion spectra were recorded on a Jasco V-730 spectrometer, and steady-state emission
spectra were measured on a PTI Quantamaster equipped with a K170B PMT for measur-
ing visible emission from the metal complex and a Hamamatsu R5509-42 NIR PMT for
This article is protected by copyright. All rights reserved.

measuring NIR emission (<1400 nm) from 1O2 (centered around 1276 nm). Emission
and excitation spectra were corrected for the wavelength dependence of lamp output
and detector response.
Emission lifetimes (ex=457 nm, em=em max) were collected on degassed MeCN solu-
tions at room temperature or on 4:1 v/v EtOH:MeOH glass at 77 K using a PTI
LaserStrobe system, equipped with an R928 stroboscopic detector integrated with a GL-
3300 nitrogen/GL-301 dye laser (2−3 nm fwhm). The nanosecond transient difference
absorption (TA) spectra and decays were measured in degassed MeCN solutions on an
Edinburgh LP920 laser flash photolysis spectrometer. The third harmonic output (355
nm) of a Nd:YAG laser (Quantel Brilliant, pulse width = 4.1 ns, repetition rate = 1 Hz)
was used as the excitation source. Each sample was purged with argon for 45 min prior
to measurement.
Metal compound solutions for cellular assays. Stock solutions of the chloride salts of
the Ru(II) complexes were prepared at 5 mM in 10% DMSO in water and kept at −20 °C
prior to use. Working dilutions were made by diluting the aqueous stock with pH 7.4
Dulbecco’s phosphate buffered saline (DPBS). DPBS is a balanced salt solution of 1.47
mM potassium phosphate monobasic, 8.10 mM sodium phosphate dibasic, 2.68 mM
potassium chloride, and 0.137 M sodium chloride (no Ca2+ or Mg2+). DMSO in the assay
wells was under 0.1% at the highest complex concentration.
Cell culture reagents. For cellular assays, characterized FBS (VWR), RPMI 1640 media
(Corning Cellgro), EMEM (Corning Cellgro), and Dulbecco’s phosphate buffered saline
(DPBS 1×, Mediatech, 21-031-CV), were purchased from VWR. Trypsin-EDTA solution
This article is protected by copyright. All rights reserved.

(0.25% w/v Trypsin/0.53 mM EDTA, 30-2101), human promyelocytic leukemia cells (HL-
60) and human malignant melanoma cells (SK-MEL-28) were procured from ATCC.
Cell culture of HL-60 cells. HL-60 human promyelocytic leukemia cells (ATCC CCL-
240) were cultured at 37 °C under 5% CO2 in RPMI 1640 (Mediatech Media MT-10-040-
CV) supplemented with 20% FBS (PAA Laboratories, A15-701) and were passaged 3−4
times per week according to standard aseptic procedures. Cultures were started at
200,000 cells mL−1 in 25 cm2 tissue culture flasks and were subcultured when growth
reached 800,000 cells mL−1 to avoid senescence associated with prolonged high cell
density. Complete growth medium was prepared in 200 mL portions as needed by com-
bining RPMI 1640 (160 mL) and FBS (40 mL, prealiquoted and heat inactivated) in a
250 mL Millipore vacuum stericup (0.22 μm), and filtering.
Cell culture of SK-MEL-28. Adherent SK-MEL-28 malignant melanoma cells (ATCC
HTB-72) were cultured in Eagle’s Minimum Essential Medium (EMEM, Mediatech Me-
dia MT-10-009-CV) supplemented with 10% FBS. They were incubated at 37 °C under
5% CO2, and were passaged 2−3 times per week according to standard aseptic proce-
dures. SK-MEL-28 cells were started at 200,000 cells mL−1 in 75 cm2 tissue culture flasks
and were subcultured when growth reached 550,000 cells mL−1 by removing old culture
medium and rinsing the cell layer once with DPBS, followed by dissociation of the cell
monolayer with trypsin-EDTA solution. Complete growth medium was added to the cell
suspension to allow appropriate aliquots of cells to be transferred to new cell vessels.
Complete growth medium was prepared in 250 mL portions as needed by combining
EMEM (225 mL) and FBS (25 mL, prealiquoted and heat inactivated) in a 250 mL Milli-
pore vacuum stericup (0.22 μm) and filtering.
This article is protected by copyright. All rights reserved.

Cell culture of CCD-1064Sk cells. Adherent CCD-1064Sk normal skin fibroblast cells
(ATCC CRL-2076) were cultured in Iscove's Modified Dulbecco's Medium (IMDM,
ATCC 30-2005) supplemented with 10% FBS. They were incubated at 37 °C under 5%
CO2, and were passaged 2−3 times per week according to standard aseptic procedures.
CCD-1064Sk cells were started at approximately 200,000 cells mL−1 in 75 cm2 tissue cul-
ture flasks and were subcultured when growth reached confluency, approximately
500,000 cells mL−1 by removing old culture medium and rinsing the cell layer once with
DPBS, followed by dissociation of the cell monolayer with trypsin-EDTA solution.
Complete growth medium was added to the cell suspension to allow appropriate ali-
quots of cells to be transferred to new cell vessels. Complete growth medium was pre-
pared in 250 mL portions as needed by combining IMDM (225 mL) and FBS (25 mL,
prealiquoted and heat inactivated) in a 250 mL Millipore vacuum stericup (0.22 μm)
and filtering.
Cytotoxicity and photocytotoxicity assays. Cell viability experiments were performed
in triplicate in 96-well ultra-low attachment flat bottom microtiter plates (Corning Cos-
tar, Acton, MA), where outer wells along the periphery contained 200 μL of DPBS to
minimize evaporation from sample wells. Cells growing in log phase (HL-60 cells:
∼800,000 cells mL−1. SK-MEL-28 cells: ∼550,000 cells mL−1. CCD-1064Sk cells: ~500,000
cells mL−1) with at least 93% viability were transferred in 50 μL aliquots to inner wells
containing warm culture medium (25 μL) and placed in a 37 °C, 5% CO2 water-jacketed
incubator (Thermo Electron Corp., FormaSeries II, Model 3110, HEPA Class 100) for 3 h
to equilibrate (and allow for efficient cell attachment in the case of both the SK-MEL-28
and CCD-1064Sk adherent cells). Metals compounds were serially diluted with DPBS
and prewarmed at 37 °C before 25 μL aliquots of the appropriate dilutions were added to
This article is protected by copyright. All rights reserved.

cells. PS-treated microplates were incubated at 37 °C under 5% CO2 for 16 h drug-to-
light intervals. Control microplates not receiving a light treatment were kept in the dark
in an incubator, and light-treated microplates were irradiated under one of the follow-
ing conditions: visible light (400−700 nm, 44 mW∙cm−2) using a 190 W BenQ MS 510
overhead projector or red light (625 nm, 52.5 mW∙cm−2) from an LED array (PhotoDy-
namic Inc., Halifax, NS). Irradiation times using these two light sources were approxi-
mately 38 and 32 min, respectively, to yield total light doses of 100 J cm−2. Both untreat-
ed and light-treated microplates were incubated for another 48 h before 10 μL aliquots
of prewarmed, sterile filtered 0.6 mM resazurin reagent (Sigma Aldrich Canada), ac-
cording to a standard protocol (45), were added to all sample wells and subsequently
incubated for another 3 h. Cell viability was determined on the basis of the ability of the
resazurin redox indicator to be metabolically converted to a fluorescent dye by only live
cells. Fluorescence was quantified with a Cytofluor 4000 fluorescence microplate reader
with the excitation filter set at 530 ± 25 nm and emission filter set at 620 ± 40 nm. EC50
values for cytotoxicity (dark) and photocytotoxicity (light) were calculated from sig-
moidal fits of the dose-response curves using Graph Pad Prism 6.0 according to eq 2,
where yi and yf are the initial and final fluorescence signal intensities. For cells growing
in log phase and of the same passage number, EC50 values are generally reproducible to
within ±25% in the submicromolar regime, ±10% below 10 μM, and ±5% above 10 μM.
Phototherapeutic indices (PIs), a measure of the therapeutic window, were calculated
from the ratio of dark to light EC50 values obtained from the dose-response curves.
yi - yf
y = yi +
1+
10(logEC
-
x)´ (Hillslope)
50
(2)
This article is protected by copyright. All rights reserved.

DNA photocleavage assays. DNA photocleavage experiments were performed accord-
ing to general plasmid DNA gel mobility shift assay with 20 µL total sample volumes in
0.5 mL microcentrifuge tubes. Transformed pUC19 plasmid (1.3 µL, > 95% form I) was
added to 10 µL of 10 mM Tris-HCl buffer (pH 7.5). Dilutions of the Ru(II) compound(s)
were prepared in ddH2O and added in 5 µL aliquots to the appropriate tubes to yield
final Ru(II) concentrations ranging from 5 to 100 µM. Then, ddH2O (3.7 µL) was added
to bring the final assay volumes to 20 µL. Control samples with no metal complex re-
ceived 8.7 µL of water. Sample tubes were kept at 37 ˚C in the dark or irradiated with
broadband visible light (14 J cm-2 delivered from a Luzchem LZC-4X photo reactor over
the course of 30 min). After treatment, all samples (dark and light) were quenched by
the addition of 4 µL of gel loading buffer (0.025% bromophenol blue, 40% glycerol).
Samples volumes of 11.8 µL were loaded onto 1% agarose gels cast with 1x TAE (40 mM
Tris-acetate, 1 mM EDTA, pH 8.2) containing ethidium bromide (0.75 µg mL-1) and
electrophoresed for 30 min at 8 V cm-1 in 1x TAE. The bands were visualized using the
Gel Doc-It Imaging system (UVP) with Vision Works software.
RESULTS AND DISCUSSION
Synthesis and characterization
An overview of the synthetic steps for preparing the Ru(II) dyads is shown in Scheme
1. The previously unreported thienyl-appended 1,10-phenanthroline ligands phen-1T,
phen-2T, and phen-3T were synthesized via a Pd(0)-catalyzed Stille cross-coupling be-
tween the tributylstannane of thiophene (1T-SnBu3) or oligothiophene (2T-SnBu3 3T-
SnBu3) and 5-bromo-1,10-phenanthroline in 35-60% yield after purification on alumina.
This article is protected by copyright. All rights reserved.

The nT-SnBu3 starting materials were obtained in 70-95% yield by lithiation of the cor-
responding nT in THF followed by quenching with SnBu3Cl and were used without fur-
ther purification (41). 5-Bromo-1,10-phenanthroline was prepared in 47% yield from
freshly dried 1,10-phenanthroline and bromine in oleum (39).
The Ru(II) dyads were assembled from the corresponding phen-nT ligands and
[Ru(bpy)2Cl2]∙2H20 in 1:1 ethanol:ethylene glycol using microwave irradiation and iso-
–
lated as their PF6 salts. Purification was carried out on silica gel and was repeated 2-3
times to obtain very modest yields (5-6%) of the final complexes in >96% purity by
HPLC. Characterization by 1D 1H and 2D 1H-1H NMR (Figures S1-S6) as well as ESI-MS
(Figures S8-S13) confirmed the structures of the desired products. The hydrogen as-
signments in the 1H NMR spectra are compared for the three complexes in the Support-
–
ing Information (Figure S7). Photophysical characterization was done on the PF6 salts
in MeCN for comparison with published photophysical data for other Ru(II) com-
pounds and to be able to measure 1O2 quantum yields by emission at 1270 nm, which
would otherwise be quenched in aqueous solution. For biological experiments, the
compounds were converted to their water-soluble Cl– salts in quantitative yield using
anion-exchange chromatography.
[SCHEME 1]
[TABLE 1]
This article is protected by copyright. All rights reserved.

Photophysical properties
Absorption. The electronic spectra of complexes 1‒3 at room temperature are shown in
Figure 1a and tabulated in Table 1. Four key features appeared. The intense sharp peak
near 284 nm for all three Ru(II) dyads, also a feature observed in Ru(bpy)32+ (18), can be
attributed to the 1IL state (i.e., analogous to the ππ* transition) centered on the bpy lig-
ands. Similarly, the peak near 450 nm was assigned to MLCT transitions between the
Ru(II) center and the bpy ligands. The analogous transition between Ru(II) and the
more -extended ligands occurred around 420 nm and did not vary with the number of
appended thiophene rings, suggesting that this MLCT transition was primarily associ-
ated with the phen portion of the ligand. A broad peak of increasing intensity occurred
around 325 nm in 1, 380 nm in 2, and 400 nm in 3, and the decrease in energy with the
increasing n points to ππ* transitions localized on the appended thienyl ring(s) of the
phen ligands.
Emission. Complexes 1‒3 produced moderate (Φem ≈ 10-1–10-3, Table 1), broad and fea-
tureless emission near 618 nm at room temperature in Ar-sparged MeCN (Figure 1b). At
77 K (in 4:1 MeOH:EtOH glass), this emission shifted to shorter wavelengths (~580 nm),
increased in quantum yield, and developed typical 3MLCT vibronic structure (Figure
1c). The vibronic intervals were around 1350 cm-1, which is characteristic of diimine in-
volvement in the emissive state (40) and did not change significantly between the three
complexes. The ΔEs values of around 1060 cm-1 compare well with ΔEs=1127 cm−1 for
[Ru(bpy)3]2+ under the same conditions (15,23), and the ≈1 μs lifetime (em) of all three
approximates the 0.9 μs lifetime of [Ru(bpy)3]2+ (18), further supporting this assign-
ment of the 3MLCT state. The fact that the emission maximum did not change with n
(Figure 1b-c) indicates that the π* acceptor orbital of the 3MLCT state is the same in the
This article is protected by copyright. All rights reserved.

three complexes: the 3MLCT phosphorescence is thus ascribed to the
Ru(dπ) → bpy(π*) configuration.
[FIGURE 1]
Despite the similarities in the electronic configuration of the emissive states in the se-
ries, compound 1 exhibited much stronger 3MLCT phosphorescence (Table 1) at both
room temperature and at 77K, at least an order of magnitude greater than 2 or 3, with
no significant effect on the 3MLCT lifetime. This suggests that 2 and 3 may differ sub-
stantially from 1 in terms of their competing nonradiative 3MLCT relaxation pathways.
Transient absorption (TA). The triplet excited states of complexes 1–3 were also probed
using nanosecond transient absorption (TA) spectroscopy (Figure 2a and S14), and their
spectroscopic signatures were analyzed in the context of the well-established TA pro-
files of similar Ru(II) complexes. The TA spectra are differential measurements, consist-
ing of new excited-state absorption (ESA) bands (positive signals) that are superim-
posed on ground state transitions that are no longer available (negative signals). For
typical Ru(II) polypyridyl complexes, these differential spectra usually comprise a
prominent bleach in the mid-visible, due to the loss of the 1A1  1MLCT ground-state
absorption, plus a new sharp absorption in the UV and a broader one in the red-NIR,
both of which arise from the diimine ligand radical anion of the 3MLCT excited state
(46).
The TA signal of 1 (Figure 2a, S14a) exhibited these features, and the excited state life-
time (TA) measured at different wavelengths across the TA spectrum was similar to that
This article is protected by copyright. All rights reserved.

observed in the emission experiment, demonstrating that (i) the excited state of 1
probed by TA is the emitting state, and (ii) no additional states appear to contribute to
the excited state relaxation pathway for 1 on this timescale. Compounds 2‒3 had very
different profiles, characterized by a broad, and much longer-lived ESA in the red-NIR
that partly overlapped the MLCT bleach and affected its apparent position. This signa-
ture has been observed previously for other oligothiophene-containing Ru(II)
polypyridyl complexes and was attributed to 3IL states involving * transitions local-
ized on the thienyl groups (33). The lifetime of this new signal was 43 μs for 2 (Figure
S14b) and 27 μs for 3 (Figure 2b), pointing to a long-lived 3IL state that can be populated
only when n2 and that cannot (re)populate the 3MLCT state since its emissive lifetime
did not vary between the complexes. In other words, all of the Ru(II) dyads exhibited
3MLCT emission (em=1 μs), but only 2 and 3 displayed 3IL ESA with the prolonged life-
time.
[FIGURE 2]
Photophysical model. The 3MLCT emission from all three compounds occurred at
around 618 nm, or 2.1 eV. The triplet state energies ET(0‒0) of thiophene, bithiophene,
and terthiophene have been reported as 3.50, 2.23, and 1.93 eV, respectively (32). Using
these values as estimates for the 3IL states for the complexes, the major differences be-
tween the three complexes are the energies of their 3IL-based states. The 3IL state for 1
was inaccessible with ex=355 nm (3.49 eV), which presumably populates
nonthermalized 1IL and/or 1MLCT states that rapidly undergo internal conversion (IC)
This article is protected by copyright. All rights reserved.

to singlet states that can populate the 3MLCT state in all three complexes and the 3IL
state for only 2 and 3, as illustrated on the Jabłonski diagram in Scheme 2. The current
hypothesis is that the 3IL state for 1 is energetically uphill and inaccessible from the
thermalized 1MLCT and/or 1IL states.
The fact that the emissive lifetimes of the 3MLCT states for all three complexes were
2+
similar and monoexponential (≈1 μs, typical for unsubstituted Ru(bpy)3 -type com-
plexes) (4) suggests that the 3IL states did not act as “triplet reservoirs”, unlike some
other π-expansive Ru(II) dyads based on pyrene (33,47) The observation of 3MLCT life-
times for 2 and 3 that were not shorted or lengthened relative to the typical 1-μs lifetime
for Ru(II) polylpyridyls supported the notion that the 3IL states are not populated di-
rectly from 3MLCT states (which would shorten em, assuming the forward rate con-
stants for this process were faster than the rate constants for emission) and that the 3IL
states are not in thermal equilibrium with 3MLCT states (which would lengthen em).
The absence of a rise time (matching the decay of the 3MLCT state) for the formation of
the 3IL state for 2 and 3 also suggests that the 3IL states were not populated directly
from the 3MLCT manifold in these two systems. Nevertheless, the photophysical
scheme is tentative without further testing with different excitation wavelengths and on
shorter timescales.
Singlet oxygen sensitization. The quantum yields for singlet oxygen generation (ΦΔ)
were calculated for compounds 1-3 in air-saturated MeCN at room temperature using
2+
the integrated emission centered at 1268 nm (Table 1). Ru(bpy)3 , which is an efficient
1O2 sensitizer, was used as a standard (ΦΔ=0.56) (44). Compound 1 had a 1O2 quantum
yield of 58%, implying good conversion efficiency from the 3MLCT state, comparable to
the standard. Compounds 2 and 3 were more efficient still, 78% and 74%, respectively.
This article is protected by copyright. All rights reserved.

Since the spectroscopic data suggests that the thiophene-centered 3IL state dominates
in these two complexes and that it is not in equilibrium with the 3MLCT, one can con-
clude that the longer-lived 3IL state is more efficient at sensitizing 1O2 and that the 1O2
sensitization competes favorably with the nonradiative decay pathway from this state.
This is not surprising, since terthiophene itself is an excellent 1O2 sensitizer, with
ΦΔ=0.86 (31). The relatively large ΦΔ values in this series, especially for 2 and 3, suggest
that these Ru(II) dyads may be photobiologically active and could be exploited for pho-
todynamic applications such as PDT.
[SCHEME 2]
[TABLE 2]
In vitro photobiological activity
The cytotoxicities and photocytotoxicities of Ru(II) dyads 1-3 were evaluated in two
different cancer cell lines: SKMEL28 human melanoma cells as a representative cell line
that grows as adherent 2D monolayers, and HL60 human leukemia cells as one that
grows as a suspension. In addition, CCD-1064Sk human skin fibroblast cells were used
to determine whether the compounds showed any cytotoxicity toward noncancerous
cells. Briefly, cells growing in log phase were dosed with metal complex (1 nM-300 M)
in 96-well plates and incubated at for 16 h at 37 C in an atmosphere containing 5% CO2
prior to a light or sham (dark) treatment. The light treatments were 100 J cm-2 delivered
from visible (400-700 nm, 34 mw cm-2) or red (625 nm, 29 mW cm-2) LEDs (Figure S15);
This article is protected by copyright. All rights reserved.

the compounds showed no observable photobleaching at these light doses. The samples
were incubated for an additional 48 h (37 C, 5% CO2) followed by the addition of
Alamar Blue and its fluorescence quantification 16 h later. The dark and light
cytotoxicities were calculated as the effective concentrations required to reduce cell via-
bility to 50% (EC50) under a given condition. Photocytotoxicity indices (PIs) were de-
termined as the ratio of dark to light EC50 values. Selectivity factors (SF) were defined as
the ratio of the dark EC50 for CCD-1064Sk cells to the dark EC50 for a given cancerous
cell line. PIs and SFs greater than one signify light-amplified cytotoxic effects and selec-
tivity for cancer cells over normal cells, respectively. As long as the light EC50 value is
significantly less than the dark EC50 value for the noncancerous cell line, it is not neces-
sary to have selectivity for cancer cells over healthy cells because the selectivity can be
achieved by spatiotemporal control of the light delivery. The SFs were determined to
assess whether any of the compounds might be of interest as traditional chemothera-
peutics.
All three Ru(II) dyads were considered nontoxic to the CCD-1064Sk normal skin fibro-
blast cells (EC50>100 M) in the absence of a light trigger, and compound 1 was nontoxic
toward the two cancer cell lines as well (Table 2). Compounds 2 and 3 were slightly cyto-
toxic toward SKMEL28 cells (EC50=32 and 50 M, respectively), but nontoxic to HL60
cells (EC50>100 M). At this point, it is uncertain whether the four-fold selectivity of 3
and eight-fold selectivity of 2 toward melanoma cells (but no selectivity toward HL60
cells) stems from an inherent difference in the identities of these two cancerous cell
lines and their uptake of the compounds or whether it is simply due the difference in
the way these two cell lines grow (dendritic adherent monolayers versus spherical sus-
pension cells). It is also not clear why there is some selectivity for SKMEL28 cells over
This article is protected by copyright. All rights reserved.

noncancerous cells with regard to the two oligothienyl dyads and no preference when
n=1. Regardless, the cytotoxicities of these compounds are marginal, and thus they are
of little interest as traditional chemotherapeutics. However, this property makes them
potentially attractive as light-responsive prodrugs for photobiological applications such
as PDT.
[FIGURE 3]
The cytotoxicities of all the compounds toward both cancer cell lines were enhanced
upon visible light activation (Table 2, Figure 3), yielding EC50 values as low as 125 nM
(PI=400) for 3 toward SKMEL28 cells and PIs >800 (EC50=310 nM) for 2 toward HL60.
Compound 1, with one thienyl group, was the least potent with EC50 values in the low
micromolar range (1.4 and 6.8 M toward SKMEL28 and HL60, respectively). However,
the low dark cytotoxicity of 1 still led to PIs >210 in SKMEL28 and >44 in HL60. Com-
pounds 2 and 3 exhibited light EC50 values in the range of 125-310 nM with PIs between
150 and 835. The activity plot shown in Figure 4 clearly demonstrates that
phototoxicities toward both cell lines increase as the number of thienyl groups increas-
es: 1 < 2 < 3, with PIs generally being larger in the HL60 cell line (except for 1).
[FIGURE 4]
This article is protected by copyright. All rights reserved.

The visible light used for the photocytotoxicity experiments included higher-energy
blue wavelengths, which overlap well with the 1MLCT absorption band for all the Ru(II)
dyads (Figure 1a) and give rise to photocytotoxic effects. However, there is more focus
on using red and near-infrared wavelengths for in vivo photobiological applications,
especially when there is a need to penetrate tissue and minimize scatter. Therefore, we
also determined EC50 values and PIs using single-wavelength red LEDs emitting at 625
nm. Despite the fact that absorption of light for the series at this wavelength is minimal
(625 nm <<100 M-1 cm-1), we have previously shown that red PDT effects are possible even
when molar extinction coefficients are <100 M-1 cm-1 for a variety of Ru(II) dyads derived
from oligothienyls (7,36) and other -expansive systems (48). The criterion appears to
be a critical degree of -conjugation on one of the ligands that lowers the energy of the
3IL state below that of the 3MLCT state (<2.1 eV), making the former accessible.
Based on our previous studies, it was expected that compound 1 (with a single thienyl
group) would not give rise to photocytotoxic effects with red light in either cell line.
However, compounds 2 and 3 yielded EC50 values of approximately 14 and 2 M, respec-
tively, in both cancer cell lines. The PIs measured in SKMEL28 cells were less than those
in HL60 cells due to the higher dark cytotoxicity toward this cell line. Notably, the PIs
for compound 3 were 25 and and 76 with red light. Even when any dark cytotoxic con-
tribution to the observed photocytotoxicity is considered, the red photobiological activ-
ity increased with -expansion in this series. The theoretical basis for this phenome-
non, from computational methods on related Ru(II) dyads containing thienyl groups,
appears to be direct population of a low-lying 3IL state for n2, but the increased organ-
ic character of these states lead to small SOC values (on the order of a few tens of cm-1)
and thus very weak S-T absorption (36). The fact that any red photocytotoxicity is ob-
This article is protected by copyright. All rights reserved.