New ruthenium(II) complexes functionalized with coumarin derivatives: Synthesis, energy-transfer-based sensing of esterase, cytotoxicity, and imaging studies

Article abstract of DOI:10.1002/chem.201103025

Two new bichromophoric ruthenium(II) complexes, [Ru(bpy) ₂(bpy-CM)](PF₆)₂ and [Ru(bpy) ₂(bpy-CM343)](PF₆)₂ (bpy=2,2'-bipyridine, CM=coumarin) with appended coumarin ligands have been designed and synthesized. The energy-transfer-based sensing of esterase by the complexes has been studied by using UV/Vis and luminescence spectroscopic methods. The cytotoxicity and the cellular uptake of one of the complexes have also been investigated. Woodruff-not only tasty! Two new bichromophoric ruthenium(II) complexes with appended coumarin ligands have been synthesized. The energy-transfer-based sensing of esterase by the complexes was studied by using UV/Vis and luminescence spectroscopy. The Ru^II complex containing a coumarin 343 derivative was found to exhibit only a slight cytotoxicity towards HepG2 cells, and it was easily taken up in the living cells to show evident response to the esterase (see figure). Copyright

Full text of DOI:10.1002/chem.201103025

FULL PAPER
DOI: 10.1002/chem.201103025
New Ruthenium(II) Complexes Functionalized with Coumarin Derivatives:
Synthesis, Energy-Transfer-Based Sensing of Esterase, Cytotoxicity, and
Imaging Studies
[
a, b]
[a]
[c]
[a]
Mei-Jin Li,
Keith Man-Chung Wong, Changqing Yi, and Vivian Wing-Wah Yam*
Dedicated to Prof. Hubert Le Bozec on the occasion of his 60th birthday
Abstract: Two new bichromophoric ruthenium(II) complexes, [Ru
ACHTUNGTRENNUNG( bpy) ACHTUNGTNERNUNG( bpy-
2
CM)](PF ) and [Ru(bpy) (bpy-CM343)](PF ) (bpy=2,2’-bipyridine, CM=cou-
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
A
H
N
T
E
N
G
ACHTUNGTRENNUNG
6
2
2
6 2
marin) with appended coumarin ligands have been designed and synthesized. The
energy-transfer-based sensing of esterase by the complexes has been studied by
using UV/Vis and luminescence spectroscopic methods. The cytotoxicity and the
cellular uptake of one of the complexes have also been investigated.
Keywords: coumarins
transfer · luminescence · ruthenium
·
energy
[2b]
Introduction
coworkers. Large emission spectral changes upon hydroly-
sis of the phosphodiester group by the enzyme were ob-
served. Due to the cleavage of the spacer by the enzyme,
the FRET efficiency is decreased and such spectral changes
are studied for ratiometric measurements. Another new
FRET system, involving the ruthenium(II) bathophenan-
throline complex acting as the acceptor, and the carbostyril
derivative acting as the donor, was synthesized by Bann-
Recently, fluorescent sensors based on fluorescence reso-
nance energy transfer (FRET) have attracted significant in-
terest and are currently being exploited for various studies
and applications in molecular and supramolecular photophy-
[1]
[2]
[3]
sics, biology, and molecular devices. Systems of particu-
lar interest are bichromophoric molecules in which a donor
and an acceptor are linked by a spacer and the excited-state
energy from the donor is transferred to the acceptor. Be-
cause this process is distance- and orientation-dependent,
the efficiency and the rate of energy transfer can be modi-
fied by variation of the mutual distance and the orientation
of the donor and acceptor moieties, which can be achieved
by external perturbation on the spacer group. These kinds
of systems usually show large Stokes shifts and are poten-
tially useful for chemosensing or biosensing applications. In
recent years, some ratiometric fluorescent sensors based on
[4a]
warth and coworkers. The donor and acceptor could be
easily introduced into the peptide moieties and such system
could be employed for a protease assay. Although there
have been a number of reports on the study of rutheniu-
[5]
m(II) polypyridyl complexes, it was only until quite recent-
ly that the highly luminescent ruthenium(II) complexes of
[6]
polypyridyl ligands have been utilized in bioimaging de-
spite the widespread interest in their study as oxygen sen-
[7]
sors and DNA intercalators.
Our efforts have been to study the FRET processes of
transition-metal complexes and to utilize the concept of
FRET for the design of FRET-based sensors, which are rela-
tively scarce in inorganic and organometallic systems, as
most of the luminescent chemosensors are based on photo-
induced electron transfer (PET) and photoinduced charge
transfer (PCT) processes. As an extension of our previous
work on the FRET studies of coumarin-appended pyridyltri-
carbonylrhenium(I) 2,2’-bipyridyl complexes with oligoether
[2b,4]
FRET have been developed for protease sensing.
An
enzyme-cleavable sensor based on FRET for phosphodies-
terase activity was synthesized and reported by Nagano and
[
a] Dr. M.-J. Li, Dr. K. M.-C. Wong, Prof. V. W.-W. Yam
Department of Chemistry, The University of Hong Kong
Pokfulam Road, Hong Kong SAR (P.R. China)
E-mail: wwyam@hku.hk
[8]
spacers, herein we report the synthesis, photophysics, elec-
trochemistry, energy-transfer-based sensing of esterase, cyto-
toxicity, and bioimaging studies of two ruthenium(II) di-
[
b] Dr. M.-J. Li
Key Laboratory of Analysis and
Detection Technology for Food Safety
(
Ministry of Education and Fujian Province)
A
T
N
T
E
U
G
a coumarin moiety
Department of Chemistry, Fuzhou University
Fuzhou 350108 (P.R. China)
(
are found to give high FRET efficiency from the coumarin
unit to the ruthenium(II) complex unit. Drastic electronic
absorption and emission spectral changes have been ob-
served, resulting from the modulation of the energy-transfer
process upon cleavage of the ester linkage by the enzymatic
[
c] Dr. C. Yi
School of Engineering, Sun Yat-Sen University
Guangzhou 510275 (P.R. China)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201103025.
Chem. Eur. J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
1
&
ÞÞ
These are not the final page numbers!

(
2
t, J=6.2 Hz, 2H; coumarin 343), 2.75 (t, J=6.2 Hz, 2H; coumarin 343),
.44 (s, 3H; CH on bpy), 1.97 ppm (m, 4H; coumarin 343) MS (EI+):
3
+
m/z: 467 [M] .
Synthesis of [Ru
according to a modification of a literature procedure for the related ruth-
2 6 2
AHCTUNGTERNNGNU( bpy) ACHTUNGTNERUNG( bpy-CM)] ACHUTGNENRUN(G PF ) : The complex was synthesized
[
9]
enium(II) polypyridine complexes. Bpy-CM (41 mg, 0.11 mmol) was
added to a solution of cis-[Ru(bpy) Cl ]·2H O (52 mg, 0.1 mmol) in abso-
lute ethanol (50 mL) and the mixture was heated to reflux under N for
A
H
U
G
R
N
N
2
2
2
2
2
+
5 H during which the purple-black solution turned red-orange. After re-
moval of solvent under vacuum, the residue was dissolved in a minimum
amount of water (1 mL), and the complex was precipitated by addition
Scheme 1. Chemical structures of [Ru
ACHTUNGTRENNUNG( bpy)
2
A
H
U
T
E
N
N
2
A
H
U
G
E
N
N
and [Ru-
2
+
6
of a saturated aqueous KPF solution. The desired product was obtained
by filtration and subsequent recrystallization by slow diffusion of diethyl
ether vapor into an acetonitrile solution of the complex afforded the
product as red-orange crystals (64 mg, 60%). H NMR (400 MHz,
hydrolysis activity of esterase. The complex containing cou-
marin 343 has been found to exhibit only a slight cytotoxici-
ty towards HepG2 cells and show high sensitivity toward
sensing of esterase in the buffer solution and living cells,
rendering it an ideal candidate for the ratiometric protease
assay in a living cell.
1
3
CD CN): d=8.74 (s, 1H; aromatic proton), 8.70 (s, 1H; aromatic
proton), 8.48 (m, 4H; aromatic protons), 8.42 (s, 1H; aromatic proton),
8.05 ( m, 4H; aromatic protons), 7.72 (m, 7H; aromatic protons), 7.54 (d,
J=5.6 Hz, 1H; bpy), 7.48 (d, J=5.6 Hz, 1H; bpy), 7.40 (m, 6H; aromatic
protons), 7.25 (d, J=5.6 Hz, 1H; bpy), 5.55 (s, 2H; CH O), 2.54 ppm (s,
2
+
+
3
H; CH
analysis calcd (%) for C42
.20, N 7.62; found: C 45.90, H 3.21, N 7.52.
Synthesis of [Ru(bpy) (bpy-CM343)](PF ; The complex was prepared
6 2
by using a procedure similar to that for [Ru(bpy) (bpy-CM)](PF )
3
); MS (FAB+): m/z: 931
A
H
U
G
R
N
U
G
6
] , 786 [Mꢀ2PF
6
] ; elemental
H
32
N
6
O
4
2
F
12·1.5H O (1103): C 45.74, H
2
3
A
H
U
G
R
N
U
G
2
A
H
U
T
E
N
N
6 2
)
Experimental Section
A
H
U
G
R
N
U
G
2
A
H
U
T
E
N
N
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
except bpy-CM343 was used instead of bpy-CM. Subsequent recrystalli-
zation by slow diffusion of diethyl ether vapor (51.4 mg, 0.11 mmol) into
Materials and reagents: Ruthenium
ride, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
ACHTUNGTRENUNNG( III) chloride hydrate, thionyl chlo-
an acetonitrile solution of the complex afforded a red solid (76 mg,
(
(
MTT) were purchased from Aldrich. 3-Carboxylic acid coumarin and 4-
2-hydroxyethyl)piperazine-1-ethane sulfonic acid (HEPES) were of ana-
1
6
5%). H NMR (400 MHz, CD
3
CN): d=8.73 (d, J=4.9 Hz, 1H; aromat-
ic proton), 8.48 (m, 6H; aromatic protons), 8.05 (m, 4H; aromatic pro-
tons), 7.72 (m, 6H; aromatic protons), 7.54 (d, J=5.6 Hz, 1H; bpy), 7.48
lytical grade and were purchased from Lancaster Synthesis Ltd. A por-
cine liver esterase (PLE) suspension in (NH SO (3.2m, pH 8.0) was
purchased from Sigma–Aldrich and used as received. cis-[Ru-
4
)
2
4
(
5
(
1
d, J=5.6 Hz, 1H; bpy), 7.40 (m, 4H; aromatic protons), 7.25 (d, J=
.6 Hz, 1H; bpy), 7.03 (s, 1H; coumarin 343), 5.49 (s, 2H; CH O), 3.42
m, 4H; coumarin 343), 2.72 (m, 4H; coumarin 343), 2.55 (s, 3H; CH ),
[
9]
[10]
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(bpy)
2
Cl
2
]·2H
2
O,
4-methyl-4’-hydroxylmethyl-2,2’-bipyridine,
and
[
11]
3
+
coumarin 343 acid chloride
methods.
were synthesized according to literature
.94 ppm (m, 4H; coumarin 343); MS (FAB+): m/z: 1026 [MꢀPF
6
] ,
+
2+
8
81 [Mꢀ2PF
RuP
H 3.58, N 8.29.
6
] , 441 [Mꢀ2PF
6
] ; elemental analysis calcd for
Synthesis of coumarin-functionalized 4,4’-dimethyl-2,2’-bipyridine (bpy-
CM): Bpy-CM was prepared by modification of a literature method for
C
48
H
41
N
7
O
4
2 2
F12·H O (1189): C 48.49, H 3.64, N 8.25; found: C 48.56,
[
12]
the related compound. Coumarin-3-carboxylic acid (0.23 g, 1.2 mmol)
was heated to reflux with thionyl chloride (5 mL) for 2 h to give a clear
solution. The excess of thionyl chloride was removed by distillation and
the residue was dried in vacuum. The white solid was then dissolved in
Cell culture: HepG2 cells (American Type Culture Collection, Manassas,
VA, USA) were cultured in RMPI 1640 medium supplemented with
1
5
0% fetal bovine serum, 1% penicillin, and streptomycin in a humidified
% CO atmosphere.
2
dry CH
a
1
2
Cl
solution of 4-methyl-4’-hydroxylmethyl-2,2’-bipyridine (0.20 g,
.0 mmol) and Et N (0.2 mL) in dry CH Cl (10 mL) at 08C. The mixture
2
(5 mL), and the solution was added in a dropwise manner to
Cell viability assay: The MTT assay was used to determine the viability
2
+
2
of HepG2 cells upon treatment with [Ru ACTHUNGTRNEUNG( bpy) (bpy-CM343)] , as de-
3
2
2
[
13]
scribed in detail elsewhere. HepG2 cells were seeded in 96-well tissue
was heated to reflux for overnight after the addition was complete. The
mixture was then cooled to room temperature and the solvent was re-
moved under vacuum. Purification by chromatography on silica gel with
6
culture plates at the density of 4ꢁ10 cells per well and incubated for
2
+
2
three days. After treatment with [Ru ACHTNUGTRNEUN(G bpy) (bpy-CM343)] for 24 h, the
1
plates were washed twice with culture medium, then MTT was added and
the plates were incubated for another 4 h. Cells without treatment of
CHCl
300 MHz, CDCl
in), 8.54 (d, J=4.9 Hz, 1H; bpy), 8.49 (s, 1H; bpy), 8.25 (s, 1H; bpy),
.64 (m, 2H; coumarin), 7.49 (d, J=4.9 Hz, 1H; bpy), 7.36 (m, 2H; cou-
marin), 7.15 (d, J=4.9 Hz, 1H; bpy), 5.50 (s, 2H; CH O), 2.45 ppm (s,
3
as eluent gave bpy-CM as a white solid (0.18 g, 48%). H NMR
(
3
): d=8.71 (d, J=4.9 Hz, 1H; bpy), 8.63 (s, 1H; coumar-
2
+
[
2
Ru ACHTUNTGNERNUG( bpy) (bpy-CM343)] were used as control. The relative cytotoxicity
was expressed in percentage of [ODsampleꢀODblank]/[ODcontrolꢀODblank]ꢁ
7
1
00. Data were collected from three independent experiments and ex-
2
+
pressed as the mean ꢁ standard deviation (SD). The statistical differen-
ces were analyzed by a paired Studentꢂs t-test. P values less than 0.05
were considered to indicate statistical differences.
3
H; CH
Synthesis of coumarin 343-functionalized 4,4’-dimethyl-2,2’-bipyridine
bpy-CM343): Coumarin 343 acid chloride (155 mg, 0.51 mmol) was dis-
solved in dry CH Cl (10 mL) and the solution was added in a dropwise
manner to the solution of 4-methyl-4’-hydroxylmethyl-2,2’-bipyridine
0.10 g, 0.5 mmol) and Et N (0.75 mL) in dry CH Cl (15 mL) at 08C.
3
); MS (EI+): m/z: 372 [M] .
(
Live cell confocal imaging: HepG2 cells in growth medium (1ꢁ
2
2
5
ꢀ1
1
0 cellsmL ) were seeded in a 35 mm tissue culture dish and incubated
at 378C under a 5% CO atmosphere for 48 h. The culture medium was
removed and replaced with a mixture of medium/DMSO (99:1, v/v) con-
(
3
2
2
2
After addition, the mixture was stirred at 608C for 12 h. The mixture was
cooled to room temperature and the solvent was removed under vacuum.
2
+
2
taining [Ru ACHTUNGRTNEUNG( bpy) (bpy-CM343)] (50 mm). After incubation for 1 h, the
medium was removed, and the cell layer was washed gently with PBS.
The cell layer was then trypsinized and added up to a final volume of
Purification by chromatography on silica gel with CHCl
3
as eluent gave
bpy-CM343 as an orange-red solid (0.15 g, 64%). H NMR (400 MHz,
CDCl ): d=8.68 (d, J=4.9 Hz, 1H; bipyridyl proton at 6-position), 8.54
d, J=4.9 Hz, 1H; bipyridyl proton at 3-position), 8.41 (s, 1H; bipyridyl
1
3
mL with PBS. Imaging was performed by using a confocal microscope
3
(
Leica TCS SP5) with an excitation wavelength at l=432 nm. The emis-
(
sion was measured by using a long-pass filter at l=455 nm.
proton at 6’-position), 8.39 (s, 1H; coumarin 343), 8.23 (s, 1H; bipyridyl
proton at 3’-position), 7.53 (d, J=4.9 Hz, 1H; bipyridyl proton at 5-posi-
tion), 7.14 (d, J=4.9 Hz, 1H; bipyridyl at 5’-position), 6.95 (s, 1H; cou-
Michaelis–Menten kinetics of esterase hydrolysis: An aliquot of porcine
liver esterase (PLE, 6 mL) was added into a cuvette containing HEPES
buffer (2.4 mL, 0.05m, pH 7.5, 0.1m KCl) and various concentrations
2
marin 343), 5.44 (s, 2H; CH O on bpy), 3.34 (m, 4H; coumarin 343), 2.88
&
2
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New Ruthenium(II) Complexes Functionalized with Coumarin Derivatives
FULL PAPER
ꢀ
(
0.1–20 mm) of the complex. The cuvette was shaken vigorously and the
night. Subsequent metathesis to the PF6 salts was accom-
plished by addition of a saturated aqueous solution of KPF6
to the corresponding complex chlorides. The identity of the
emission intensity at l=488 nm (excitation at l=445 nm) was monitored
for 5 min at 258C. The rate of increase in emission intensity between 100
and 200 s was determined and converted into mmmin of coumarin 343
by using a standard curve. The rate of increase in concentration of cou-
marin 343 was plotted as a function of substrate concentration and a theo-
ꢀ
1
1
ligands was confirmed by satisfactory H NMR spectroscopy
and EI mass spectrometry, whereas those of the complexes
were confirmed by satisfactory elemental analyses, H NMR
spectroscopy, and FAB mass spectrometry.
1
retical fit according to
Eq. (1)].
a
Michaelis–Menten model was performed
[
V
max½Sꢂ
V ¼ K
ð1Þ
Photophysical properties: The electronic absorption spec-
trum of the complex [Ru ACTHUNGTRENN(UNG bpy) ACHUTNTGNERNU(G bpy-CM)] AHCTNGRUETNNU(G PF ) in CH CN
2 6 2 3
M
þ ½Sꢂ
In Equation (1) V is the rate of reaction, Vmax is the maximum reaction
rate, [S] is the concentration of the substrate, K is the Michaelis con-
stant, an indicator of the affinity that an enzyme has for a given sub-
strate.
or in HEPES buffer (0.05m, pH 7.5, 0.1m KCl) shows an in-
tense band at lꢃ290 nm and a less intense bands at approx-
imately lꢃ420–460 nm. The higher energy absorption band
M
is ascribed to the p!p* intra
ACTHUNGTRENNGNUl i ACHTUNGTRENNUNGg and (IL) transition of the bi-
pyridine ligands and coumarin, whereas the lower energy
Physical measurements and instrumentation: Electronic absorption spec-
tra were recorded on a Hewlett–Packard 8452A diode array spectropho-
tometer. Steady-state emission was recorded on a Spex Fluorolog-2
absorption band is tentatively assigned as the [dp(Ru)!p*-
1
Model F111 fluorescence spectrophotometer. H NMR spectra were re-
AHCTUNGTRENNUNG( bpy-CM)] metal-to-ligand charge transfer (MLCT) transi-
corded on a Bruker DPX-300 or Bruker DPX-400 Fourier transform
NMR spectrometer with chemical shifts were reported relative to tetra-
methylsilane. FAB+ and EI+ mass spectra were recorded on a Finnigan
MAT95 mass spectrometer. Elemental analysis of the complexes was per-
formed on a Carlo Erba 1106 elemental analyzer at the Institute of
Chemistry of the Chinese Academy of Sciences in Beijing.
tion. For [Ru ACHTUNTGNERNUG( bpy) (bpy-CM343)] ACHTUGNTERNNN(GU PF ) , the electronic ab-
2 6 2
sorption spectrum shows an intense absorption band at l
ꢃ280 nm and a less intense band at lꢃ440 nm. The higher
energy absorption band is assigned as IL p!p* transition of
the bipyridine ligands. In view of the fact that the free
ligand bpy-CM343 also shows an absorption band at l=
Luminescence quantum yields were measured by the optical dilute
[
15]
method reported by Demas and Crosby. A degassed aqueous solution
of [Ru(bpy) ]Cl (f=0.042, excitation wavelength at l=436 nm) was
4
40 nm, the lower energy absorption band with higher ex-
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
3
2
[
16]
used as the reference. All solutions for photophysical studies were de-
gassed on a high-vacuum line in a two-compartment cell consisting of
a 10 mL Pyrex bulb and a 1 cm path length quartz cuvette and sealed
from the atmosphere by a Bibby Rotaflo HP6 Teflon stopper. The solu-
tions were subject to no less than four freeze-pump-thaw cycles.
tinction coefficient (e=43590) is accordingly assigned as
a mixture of the IL transitions of coumarin 343 and the
[
dp(Ru)!p*
ACHTUNGTRENNUNG( bpy)] MLCT transition. Similar assignments
for the related ruthenium(II) diimine systems have also
been reported in the literature. The electronic absorption
data of the complexes are summarized in Table 1.
[5]
Energy transfer efficiency (hET) was determined by using the Equa-
tion (2), in which I
without the acceptor, respectively; and f
sion quantum yields with and without the acceptor, respectively.
D
and IDA are the donor emission intensities with and
D
and fDA are the donor emis-
Upon excitation at the absorption wavelength of coumar-
in and courmarin 343, l=300 and 430 nm, respectively, both
2
+
2+
h
ET ¼ 1ꢀðI =IDAÞ or 1ꢀðf
D
D
=fDA
Þ
ð2Þ
[Ru
A
H
U
G
R
N
U
G
2
A
H
U
G
R
N
U
G
and [Ru
A
H
U
T
E
N
N
(bpy)
(bpy-CM343)]
2
are
found to exhibit intense orange emission at lꢃ620 nm in
MeCN or in HEPES buffer (0.05m, pH 7.5, 0.1m KCl) at
Results and Discussion
room temperature. The origin of such an orange emission is
3
attributed to the typical triplet [dp(Ru)!p*
A
T
N
T
N
U
G
Synthesis and characterization: Conversion of coumarin 3-
carboxylic acid to coumarin 3-carboxylic chloride was ach-
ieved by the reaction of thionyl chloride under heating to
reflux conditions for 5 h. Reaction of 4-methyl-4’-hydroxyl-
excited state (Figure 1). The emission quantum yields of
2
+
[Ru
A
T
N
T
E
N
N
(bpy)
A
H
U
G
R
N
U
(bpy-CM)]
and [Ru
A
H
U
G
E
N
N
(bpy) (bpy-CM343)]
2
2
HEPES buffer (0.05m, pH 7.5, 0.1m KCl) at room tempera-
ꢀ
2
ꢀ2
ture are 0.96ꢁ10 and 1.53ꢁ10 , respectively. The excita-
[
10]
2+
methyl-2,2’-bipyridine
with coumarin 3-carboxylic chlo-
tion spectra of [Ru ACHTNUGTRENNUNG( bpy) ACHTUNGETRNNUNG( bpy-CM)] and [Ru AHCNUTGTRENNUN(G bpy) (bpy-
2 2
[11]
2+
ride or coumarin 343 acid chloride
afforded the ligands
CM343)] in HEPES buffer (0.05m, pH 7.5, 0.1m KCl) are
depicted in Figures S1 and S2 in the Supporting Informa-
tion, respectively. It is interesting to note that an additional
bpy-CM and bpy-CM343, respectively. The ruthenium(II)
complexes were synthesized by reacting the ligand bpy-CM
or bpy-CM343 with cis-[Ru
A
H
U
G
R
N
U
G
emission band at l=485 nm assignable to the intra
ACHTUNGTRENNUNGl i ACHTUNGTRENNUNGg and
2
2
2
anol under heating to reflux conditions under N for over-
emission from coumarin 343 is observed in [Ru(bpy) (bpy-
ACHTUNGTRENNUNG
2
2
II
Table 1. Electronic absorption and electrochemical data of the Ru complexes in CH
3
CN.
[
a]
[a]
Complex
l
abs [nm]
Oxidation
E
1/2 [V]
Reduction
E1/2 [V]
3
ꢀ1
ꢀ1
[b]
[c]
[b]
[c]
(
e [dm mol cm ])
versus SCE (DE
p
[mV])
p
versus SCE (DE [mV])
[
d]
[
Ru
Ru
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(bpy)
2
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(bpy-CM)]
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(PF
6
)
2
246 (22350), 255 (20560), 288 (80190),
30 (14310), 420 (9440), 454 (12730)
246 (29660), 254 (28020), 287 (80720),
25 (12910), 445 (43590)
+1.23 (59)
ꢀ1.20, ꢀ1.39 (59), ꢀ1.59 (60), ꢀ1.85 (110)
ꢀ1.41 (60), ꢀ1.61 (80), ꢀ1.85 (83)
3
[
A( bpy)
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(bpy-CM343)]
A( PF
C
H
T
U
N
G
T
R
E
N
N
U
N
G
6
)
2
+0.91 (65), +1.22 (57)
3
ꢀ
1
[
a] In acetonitrile (0.1m nBu
4
NClO
4
) at 298 K, working electrode, glassy carbon; scan rate, 100 mVs . [b] E1/2 =(Epa+Epc)/2; Epa and Epc are peak anodic
=EpaꢀEpc. [d] Irreversible wave.
and peak cathodic potentials, respectively. [c] DE
p
Chem. Eur. J. 2012, 00, 0 – 0
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V. W.-W. Yam et al.
free ligand bpy-CM. On the other hand, the reduction cou-
ples at about ꢀ1.60 and ꢀ1.85 V are attributed to the succes-
sive reductions of the two bipyridine ligands, similar to that
[17]
observed in the related ruthenium(II) diimine complexes.
Similarly, the first reduction couple at ꢀ1.40 V is assigned to
ligand-centered reductions of bpy-CM343, whereas the
second and the third reduction couples at about ꢀ1.60 and
ꢀ
1.85 V are ascribed to the successive reductions of the two
2
+
bipyridine ligands in [Ru
A
H
U
G
E
N
N
(bpy) (bpy-CM343)] . The occur-
2
rence of the ligand-centered reduction of bpy-CM and bpy-
CM343 at a less negative potential is probably due to the
presence of an electron-withdrawing ester group on the bi-
pyridine, resulting in the stabilization of the p* orbital of
the bpy-CM and bpy-CM343 ligands.
Figure 1. Emission spectra of [Ru
(bpy) (bpy-CM343)](PF (a) with excitation wavelength at l=300
and 430 nm, respectively, in HEPES buffer (0.05m, pH 7.5, 0.1m KCl) at
98 K.
2 6 2
ACHTUNGTRENNGNU( bpy) ACHTUNGTNERNU(G bpy-CM)] ACHUTNGERNNU(G PF ) (c) and [Ru-
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
6 2
)
2
Enzyme-cleavable sensing for esterase activity: The sensing
behaviors of the ruthenium(II) complexes for esterase activ-
ity have been investigated by electronic absorption and
emission spectroscopic methods. The electronic absorption
CM343)]
weak emission from the organic coumarin fluorophore is
found for [Ru(bpy) (bpy-CM)](PF ) (Figure 1), even with
ACHTUNGTRENNUNG( PF ) . On the other hand, no observable or very
6 2
2
+
spectral changes of [Ru
A
H
U
G
R
N
U
G
after treat-
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
A
H
U
G
R
N
N
A
H
U
G
R
N
U
G
ment of PLE are studied in HEPES buffer (0.05m, pH 7.5,
0.1m KCl). The low-energy absorption band is found to shift
to the blue slightly from l=455 to approximately 435 nm,
corresponding to the absorption band of free coumarin 343.
The absorbance at l=455 nm is decreased by approximately
16% upon treatment with PLE over one hour. Similar ob-
2
6 2
the excitation wavelength at the coumarin absorption band
at l=300 nm. The absence of the coumarin emission is sug-
gestive of an efficient energy transfer from the coumarin
donor to the ruthenium(II) complex acceptor in [Ru
(bpy-CM)](PF ) . Although a quantitative energy-transfer
efficiency from the coumarin to the ruthenium(II) moiety in
ACHTUNGTNRENUNG( bpy) -
2
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
ACHTUNGTRENNUNG
6
2
servations have also been reported in other related sys-
2
+
[4e]
[Ru
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(bpy)
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(bpy-CM)] cannot be determined due to the ob-
tems.
Two well-defined isosbestic points at lꢃ330 and
2
servation of very weak to negligible emission from the cou-
marin fluorophore, the energy-transfer efficiency of the
unity is expected from this observation. On the contrary, the
observation of the characteristic emission of coumarin 343
430 nm are observed. Time-dependent studies on the elec-
tronic absorption spectral changes for [Ru(bpy) (bpy-
CM343)] over one hour upon treatment with PLE have
been performed (Figure 2) and the change in the absorbance
as a function of time reveals an exponential decay trace
AHCTUNGTRENNUNG
2
2
+
in [Ru ACHTUNGTRENNUNG( bpy) (bpy-CM343)] ACTHUNGTERNN(GNU PF ) is attributed to the less ef-
2 6 2
ficient energy transfer from coumarin 343 to the rutheniu-
m(II) moiety, with an energy-transfer efficiency of about
(inset of Figure 2). It is noteworthy that [Ru ACHTUNGETRNNUNG( bpy) (bpy-
2
2
+
CM343)] has been hydrolyzed by porcine liver esterase in
HEPES buffer, which leads to the large changes in the spec-
troscopic properties. In contrast, no hydrolysis by PLE is ob-
9
5%, probably due to the less effective overlap between the
1
coumarin 343 emission and the MLCT absorption band of
2
+
the ruthenium(II) complex.
served in the complex [Ru
ACHTUNGTNERUNNG( bpy) ACTHUNGTRENNU(GN bpy-CM)] .
2
As the observation of emission at l=485 and 620 nm in
2
+
Electrochemical properties: The ruthenium(II) complexes
[Ru ACHTUNGERTNNUNG( bpy) (bpy-CM343)] is suggestive for energy transfer
2
2
+
2+
[Ru
ACHTUNGTRENNUNG( bpy) ACHTUNGTRENNUNG( bpy-CM)] and [Ru ACHUNTGTRENNUNG( bpy) (bpy-CM343)] show
2 2
typical oxidation couples at approximately +1.2 V versus
II
III
SCE (Table 1), which are assigned as the Ru to Ru oxida-
tion, on the basis of similar oxidation reported in other re-
[17]
lated ruthenium(II) diimine complexes.
An additional
quasi-reversible couple at approximately +0.9 V versus SCE
2
+
is observed in [Ru ACHTUNGTRENNUNG( bpy) (bpy-CM343)] . Such a quasi-re-
2
versible couple is tentatively assigned as the oxidation of the
amino group on coumarin 343. Both complexes show three
quasi-reversible reduction couples at about ꢀ1.40, ꢀ1.60,
and ꢀ1.85 V versus SCE, and an additional irreversible
cathodic wave at ꢀ1.20 V versus SCE is observed in [Ru-
2
+
2+
ACHTUNGTRENNUNG( bpy) ACHTUNGTRENNUNG( bpy-CM)] . For [Ru AHCTUNGTRENNGUN( bpy) ACHTNURTGENNNU(G bpy-CM)] , the cathodic
2 2
wave at ꢀ1.20 V and the reduction couple at ꢀ1.40 V are
tentatively assigned to the reductions of coumarin and bipyr-
idine ligand on the bpy-CM ligand in view of the observa-
tion of similar reductions in the cyclic voltammogram of the
Figure 2. Electronic absorption spectral changes of [Ru
CM343)](PF upon treatment of PLE over one hour. The inset shows
the change in absorbance at l=455 nm as a function of time.
2
ACHTUNGTNERNUNG( bpy) (bpy-
A
H
U
G
E
N
N
6 2
)
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New Ruthenium(II) Complexes Functionalized with Coumarin Derivatives
FULL PAPER
from the coumarin 343 donor to the ruthenium(II) acceptor,
such an energy-transfer process is anticipated to be modulat-
ed upon the cleavage of the ester group by the hydrolysis of
PLE activity. Upon addition of PLE to a solution of [Ru-
tum yield of the former is even larger than that of the later.
This may probably be due to the rather large difference in
their structures that leads to a difference in the lumines-
2
+
cence properties of [Ru
A
H
U
G
R
N
N
(bpy) (bpy-CM343)]
and [Ru-
2
2
+
2+
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(bpy) (bpy-CM343)] in HEPES buffer, the emission spec-
ACHTUNGTREN(NGNU bpy) (bpy-CH OH)] . In view of the fact that the energy-
2
2
2
trum with the excitation at the isosbestic wavelength of
transfer efficiency is strongly dependent on the distance be-
tween the donor and the acceptor, hydrolysis of the ester
430 nm exhibits a drastic emission enhancement at l=
4
85 nm, whereas the characteristic emission of the rutheniu-
group on the bpy-CM343 ligand of [Ru ACHTUNGERTUNNNG( bpy) (bpy-
2
2
+
m(II) luminophore at l=620 nm is relatively weak, as
shown in Figure 3. The principle of operation of the porcine
CM343)] by PLE would separate the donor and the ac-
ceptor far apart and hence lead to the blocking of the
energy-transfer process. As a result, the emission is mainly
attributed to the fluorescence of coumarin 343 at l=
4
85 nm, whereas the emission from the ruthenium(II) lumi-
nophore at l=620 nm is relatively weak. As the excitation
was made at the isosbestic wavelength of the electronic ab-
sorption spectral changes (Figure 2), the change of emission
intensity due to a change in the absorbance could be mini-
mized and the increase of the intensity for coumarin 343 is
due only to the suppression of the energy-transfer process.
Although a decrease in the emission intensity of the ruthe-
nium(II) luminophore at l=620 nm is anticipated due to
the suppression of the energy-transfer process, the observa-
tion of an apparently less-affected emission intensity of the
ruthenium(II) luminophore at l=620 nm may not be unrea-
sonable given the influence of the emission tail from the
very intense coumarin 343 emission that would mask the in-
tensity of the 620 nm band as well as the higher lumines-
cence quantum yield of the hydrolyzed product, [Ru-
2 6 2
Figure 3. Emission spectral changes of [Ru ACHUTNRGNENUG( bpy) (bpy-CM343)] ACHTUNGETRNNUN(G PF )
upon treatment with PLE over one hour. The inset shows the change in
emission intensity at l=485 nm as a function of time.
2
+
ꢀ2
liver esterase sensor is shown in Scheme 2. Upon addition of
A
H
U
G
R
N
N
(bpy) (bpy-CH OH)]
(3.77ꢁ10 ), than that of [Ru-
2
2
2
+
2+
ꢀ2
PLE, [Ru
the corresponding components [Ru
and CM343-COOH. The luminescence quantum yields of
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(bpy) (bpy-CM343)] would be hydrolyzed into
A( bpy) (bpy-CM343)] (1.53ꢁ10 ). Although photoinduced
H
U
G
R
N
U
G
2
2
2
+
A
H
U
G
R
N
N
(bpy) (bpy-CH OH)]
electron transfer may be thermodynamically allowed based
on the electrochemical data, such a process is not favored in
view of the presence of the insulating methylene group be-
tween the bipyridine and the coumarin 343 moieties. Time-
dependent studies on the emission spectral changes at l=
485 nm as a function of time after treatment with PLE over
2
2
2
+
the two units, [Ru
A
H
U
G
E
N
N
(bpy) (bpy-CH OH)]
and CM343-
2
2
COOH, in HEPES buffer have been determined to be
ꢀ
2
3
.77ꢁ10 and 0.2848, respectively. No direct conclusion
could be inferred from a comparison of the luminescence
2
+
quantum yield of [Ru
A
H
U
G
E
N
N
(bpy) (bpy-CH OH)] with that of
one hour have been performed (inset of Figure 3). [Ru-
2
2
2
+
2+
[Ru
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(bpy) (bpy-CM343)] , because the luminescence quan-
A
H
U
G
R
N
N
(bpy) (bpy-CM343)]
exhibits a large emission spectral
2
2
change, in which the main emission maximum is shifted
from 620 to 485 nm, together with a drastic emission color
change after the hydrolysis of the ester group by the enzyme
esterase. The kinetics of the PLE activity on [Ru ACHTUNGRETNN(UNG bpy) (bpy-
2
2
+
CM343)] has been probed spectroscopically. From the Mi-
chaelis–Menten plot shown in Figure 4, the Michaelis con-
stant (K ) and the maximum reaction rate (Vmax) are deter-
M
ꢀ
1
mined to be 1.70 mm and 0.037 mmmin , respectively.
Cytotoxicity studies: An MTT assay has been employed to
measure the metabolic activity of mitochondria in the cells
based on the principle that living cells are capable of reduc-
ing the lightly colored tetrazolium salt into an intense col-
[
13c]
ored formazan derivative.
Figure 5 shows the viability of
2
+
HepG2 cells upon treatment with [Ru
for 24 h. The results reveal that [Ru
A
H
U
G
R
N
N
(bpy) (bpy-CM343)]
2
2
+
A
H
U
G
R
N
U
(bpy) (bpy-CM343)]
2
exhibits only a slight cytotoxicity towards HepG2 cells. A
similar viability is also observed in four times higher concen-
tration of the ruthenium(II) complex for the imaging study.
Scheme 2. The principle of operation of the porcine liver esterase sensor
based on the FRET mechanism and photographs showing the emission
color change of the solution before and after addition of esterase.
Chem. Eur. J. 2012, 00, 0 – 0
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www.chemeurj.org
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V. W.-W. Yam et al.
2
+
Figure 4. Michaelis–Menten plot of [Ru
by treatment of PLE.
2
ACHTUNGTRENNUNG( bpy) (bpy-CM343)] hydrolysis
Figure 6. Images of confocal laser scanning microscopy of HepG2 cells in
2
+
2
the presence of [Ru CAHTUNGTNREUNG( bpy) (bpy-CM343)] : A) Merged image; B) bright-
field image; C and D) fluorescence images. Yellow arrows indicate the
dead cells, whereas red arrow indicates cell debris.
the dye into the living cells to be visualized by time-lapsed
microscopy.
2
Figure 5. Viability of HepG2 cells upon treatment with [Ru ACHTGNUTRNEUN(G bpy) (bpy-
2
+
CM343)] for 24 h.
Conclusion
Two new bichromophoric ruthenium(II) complexes with ap-
pended coumarin ligands have been designed and synthe-
sized. The energy-transfer process from the coumarin donor
to the ruthenium(II) acceptor has been observed. The cleav-
age of the ester linkage in the complexes by esterase togeth-
er with its associated spectroscopic changes has been stud-
ied. The electronic absorption and emission spectra exhibit-
ed a large spectral change, resulting from the reduced
FRET efficiency after the hydrolysis of the ester group by
Sensing of esterase in living cells: HepG2 cells are exposed
to [Ru ACHTUNGTRENNUNG( bpy) (bpy-CM343)] at a concentration of 50 mm for
2
2
+
one hour, in which time blue emission has been observed in
the living HepG2 cells (Figures 6A and C), whereas red
emission has been found in the dead cells or cell debris (Fig-
ures 6A and D). The bright-field morphology of these ex-
amined cells is shown in Figure 6B. The distribution of the
complex in the cytoplasm is not even, but localized in the
pronuclear region. However, the nuclei show much weaker
emission, indicative of negligible nuclear uptake of the com-
plex. From the image, it is likely that the complex binds to
the hydrophobic organelles such as the Golgi apparatus, the
II
the enzyme due to the cleavage of the spacer. The Ru com-
plex containing the coumarin 343 derivative is found to ex-
hibit only a slight cytotoxicity towards HepG2 cells, and it is
easily taken up in the living cells to show obvious response
to esterase as shown in the imaging study.
[18]
endoplasmic reticulum, and the mitochondria. All the in-
tracellular luminescence images are readily observable with-
out the need of prior washing to remove [Ru ACHTUNGTRENN(UNG bpy) (bpy-
2
2
+
CM343)] in the medium. In fact, the complex is rapidly
taken up and reacted in the living cells. It is believed that
the esterase in the living cells would react with [Ru-
Acknowledgements
V.W.-W.Y. acknowledges support from the University of Hong Kong
under the Distinguished Research Achievement Award Scheme. This
work has also been supported by a Collaborative Research Fund (CRF)
grant (HKUST2/CRF/10) and a General Research Fund (GRF) grant
(HKU 7064/11P) from the Research Grants Council of Hong Kong Spe-
cial Administrative Region, China. M.-J.L. acknowledges the National
Natural Science Foundation of China (NSFC No. 20801014).
2
+
A
C
H
T
U
N
G
T
R
E
N
N
U
N
G
(bpy) (bpy-CM343)] and cause a change in the lumines-
cence from red to blue. Therefore, the need for an extensive
washing step before imaging is not necessary due to the
unique property of this Ru complex. Moreover, the omis-
2
II
sion of the washing step allows the internalization process of
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New Ruthenium(II) Complexes Functionalized with Coumarin Derivatives
FULL PAPER
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Received: September 27, 2011
Published online: && &&, 0000
Chem. Eur. J. 2012, 00, 0 – 0
ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemeurj.org
&
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ÞÞ
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V. W.-W. Yam et al.
Woodruff—not only tasty! Two new
bichromophoric ruthenium(II) com-
plexes with appended coumarin ligands
have been synthesized. The energy-
transfer-based sensing of esterase by
the complexes was studied by using
UV/Vis and luminescence spectrosco-
Ruthenium Complexes
M.-J. Li, K. M.-C. Wong, C. Yi,
V. W.-W. Yam* ................ &&&& — &&&&
New Ruthenium(II) Complexes Func-
tionalized with Coumarin Derivatives:
Synthesis, Energy-Transfer-Based
Sensing of Esterase, Cytotoxicity, and
Imaging Studies
II
py. The Ru complex containing a cou-
marin 343 derivative was found to
exhibit only a slight cytotoxicity
towards HepG2 cells, and it was easily
taken up in the living cells to show evi-
dent response to the esterase (see
figure).
&
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ꢀ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Chem. Eur. J. 0000, 00, 0 – 0
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