Rhenium(i) polypyridine complexes functionalized with a diaminoaromatic moiety as phosphorescent sensors for nitric oxide

Article abstract of DOI:10.1039/c3nj00033h

A series of rhenium(i) polypyridine complexes functionalized with a diaminoaromatic moiety has been developed as phosphorescent sensors for nitric oxide (NO). The diamine complexes [Re(N=N)(CO)₃(py-diamine)](CF ₃SO₃) [py-diamine = 3,4-diaminopyridine; N-N = 1,10-phenanthroline (phen) (1a), 2,9-dimethyl-1,10-phenanthroline (Me ₂-phen) (2a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me ₄-phen) (3a), 4,7-diphenyl-1,10-phenanthroline (Ph₂-phen) (4a)] were synthesized and characterized. These complexes were only weakly emissive due to the diaminoaromatic moiety that quenches the ³MLCT [dπ(Re) → π*(N=N)] emission by photoinduced electron transfer (PET). However, in the presence of NO, these diamine complexes were converted to the triazole derivatives [Re(N=N)(CO)₃(py-triazole)](CF ₃SO₃) [py-triazole = 1H-1,2,3-triazolo[4,5,c]pyridine; N=N = phen (1b), Me₂-phen (2b), Me₄-phen (3b), Ph ₂-phen (4b)], which revealed intense emission upon excitation. The emission of the complexes was independent to pH under neutral and basic conditions (pH > 6). The cytotoxicity and cellular uptake properties of these complexes were studied by the 3-(4,5-dimethyl-2-thiazolyl)-2,5- diphenyltetrazolium bromide (MTT) assay and ICP-MS, respectively. The potential application of these complexes as intracellular NO sensors was also investigated.

Full text of DOI:10.1039/c3nj00033h

New Journal of Chemistry
A journal for new directions in chemistry
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Volume 37 | Number 6 | June 2013 | Pages 1633–1844
Themed issue: 崛起的中国—Hello from China!
ISSN 1144-0546
PAPER
Kenneth Kam-Wing Lo et al.
Rhenium(I) polypyridine complexes
with a diaminoaromatic moiety as
phosphorescent sensors for nitric
oxide
1144-0546(2013)37:6;1-A

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Rhenium(I) polypyridine complexes functionalized
with a diaminoaromatic moiety as phosphorescent
sensors for nitric oxide†
Cite this: NewJ.Chem., 2013,
37, 1711
Alex Wing-Tat Choi, Che-Shan Poon, Hua-Wei Liu, Heung-Kiu Cheng and
Kenneth Kam-Wing Lo*
A series of rhenium(I) polypyridine complexes functionalized with a diaminoaromatic moiety has been
developed as phosphorescent sensors for nitric oxide (NO). The diamine complexes [Re(N^N)(CO)3-
(py-diamine)](CF3SO3) [py-diamine = 3,4-diaminopyridine; N^N = 1,10-phenanthroline (phen) (1a),
2,9-dimethyl-1,10-phenanthroline (Me2-phen) (2a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-phen)
(3a), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen) (4a)] were synthesized and characterized. These
complexes were only weakly emissive due to the diaminoaromatic moiety that quenches the 3MLCT
[dp(Re) - p*(N^N)] emission by photoinduced electron transfer (PET). However, in the presence of NO,
these diamine complexes were converted to the triazole derivatives [Re(N^N)(CO)3(py-triazole)](CF3SO3)
[py-triazole
= 1H-1,2,3-triazolo[4,5,c]pyridine; N^N = phen (1b), Me2-phen (2b), Me4-phen (3b),
Received (in Montpellier, France)
9th January 2013,
Ph2-phen (4b)], which revealed intense emission upon excitation. The emission of the complexes was
independent to pH under neutral and basic conditions (pH 4 6). The cytotoxicity and cellular uptake
properties of these complexes were studied by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
bromide (MTT) assay and ICP-MS, respectively. The potential application of these complexes as
intracellular NO sensors was also investigated.
Accepted 25th February 2013
DOI: 10.1039/c3nj00033h
www.rsc.org/njc
physiological lifetime (t1/2 = ca. 5 s) and rapidly diffuses
Introduction
through most cells and tissues,1a,4 it is a challenging task to
Nitric oxide (NO) is an important free radical that is involved in
a variety of biological processes such as neurotransmission,
muscle relaxation and blood pressure and immune response
regulation.1 It is endogenously produced in different mamma-
lian cells by nitric oxide synthase (NOS) which catalyzes the
conversion of L-arginine to NO and L-citrulline.2 Three isoforms
of NOS, namely neuronal NOS (nNOS), endothelial NOS (eNOS)
and inducible NOS (iNOS), are present in biological systems.
High intracellular concentrations of NO, which triggers the
formation of reactive nitrogen species (RNS), have been impli-
cated in carcinogenesis and several neurodegenerative disor-
ders such as Alzheimer’s disease, Parkinson’s disease, multiple
sclerosis and Huntington’s disease.3 Since NO has a short
monitor the formation and migration of NO in biological
systems. Among different detection methods, fluorescence
detection allows imaging of intra- and intercellular NO with
high spatiotemporal resolution.5 Thus, it is an ideal method to
study the production, diffusion processes and biological roles
of NO.
In the past decade, two distinct groups of fluorescent NO
sensors have emerged.6 The first involves the use of a para-
magnetic metal ion, such as Cu2+, as a quencher to suppress
the emission of a fluorophore.7 In the presence of NO, the
metal ion is reduced and displaced from the fluorophore,
resulting in emission enhancement. The second group exploits
an electron-rich diaminoaromatic moiety as both a recognition
unit for NO and a quencher for the emission of fluorophores
through photoinduced electron-transfer (PET).5a The diamino-
aromatic moiety has been covalently linked to different organic
fluorophores such as fluorescein,8 rhodamine,9 BODIPY10 and
cyanine11 to yield interesting NO sensors with various optical
properties. Despite the development of these reagents, the
possibility of using phosphorescent transition metal diamine
Institute of Molecular Functional Materials [Areas of Excellence Scheme, University
Grants Committee (Hong Kong)] and Department of Biology and Chemistry, City
University of Hong Kong, Tat Chee Avenue, Kowloon, Hong Kong, P. R. China.
E-mail: bhkenlo@cityu.edu.hk; Fax: +852 3442 0522; Tel: +852 3442 7231
† Electronic supplementary information (ESI) available: Electronic absorption
spectral data of the rhenium(I) polypyridine complexes. See DOI: 10.1039/
c3nj00033h
c
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complexes as intracellular NO sensors is scarce.12 The advan- diamine complex 4a were also investigated. The cytotoxicity and
tages of using phosphorescent transition metal complexes as cellular uptake properties of the diamine complexes were
biological probes include their intense and long-lived emission, studied by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium
large Stokes shifts and high photostability.13 In the past few bromide (MTT) assay and ICP-MS, respectively. The intracellular
years, there has been a fast-growing interest in the cellular NO-sensing properties of complex 4a were also investigated.
uptake properties of phosphorescent rhenium complexes with
a focus on their potential as cellular imaging and therapeutic
reagents.14–19 With our ongoing interest in phosphorescent
rhenium(I) polypyridine complexes as biomolecular and cellular
probes,19 we envisage that the modification of these complexes
with a diaminoaromatic moiety will generate a new class of
phosphorogenic sensors for NO.
Results and discussion
Synthesis of complexes
The diamine complexes 1a–4a were obtained from the reaction
of [Re(N^N)(CO)3(CH3CN)](CF3SO3) with py-diamine in refluxing
THF, followed by column chromatographic purification and
recrystallization from CH2Cl2/diethyl ether. The triazole com-
plexes 1b–4b were prepared by bubbling purified NO gas into a
solution of the diamine complexes 1a–4a in CH2Cl2 and sub-
sequently purified by recrystallization from CH2Cl2/diethyl
ether. All the complexes were stable in air and soluble in most
organic solvents such as CH2Cl2, CH3CN and alcohol. They
were characterized by 1H NMR, positive-ion ESI-MS and IR
spectroscopy and gave satisfactory microanalysis.
Herein, we report the synthesis, characterization and electro-
chemical and photophysical properties of four phosphorescent
rhenium(I) polypyridine diamine complexes [Re(N^N)(CO)3(py-
diamine)](CF3SO3) [py-diamine = 3,4-diaminopyridine; N^N =
1,10-phenanthroline (phen) (1a), 2,9-dimethyl-1,10-phenanthroline
(Me2-phen) (2a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-phen)
(3a), 4,7-diphenyl-1,10-phenanthroline (Ph2-phen) (4a)] and
their triazole counterparts [Re(N^N)(CO)3(py-triazole)](CF3SO3)
[py-triazole = 1H-1,2,3-triazolo[4,5,c]pyridine; N^N = phen (1b),
Me2-phen (2b), Me4-phen (3b), Ph2-phen (4b)] (Chart 1). The
effects of pH on the emission intensity of the Ph2-phen complexes
4a and 4b were examined. The NO-sensing properties of the
Electrochemical properties
The electrochemical properties of the complexes were studied
by cyclic voltammetry and the electrochemical data are
summarized in Table 1. The diamine complexes displayed
two irreversible waves at +0.94 to +1.14 V and +1.16 to +1.51 V
vs. SCE. Since these features were absent in the triazole
complexes 1b–4b, we assigned them to the oxidation of the
diaminoaromatic ligand py-diamine. With reference to the
previous electrochemical studies of related rhenium(I) polypyridine
complexes,20–22 the reversible or quasi-reversible couples
occurred at +1.57 to +1.76 V were ascribed to the rhenium(II/I)
oxidation. It is noteworthy that the rhenium(II/I) oxidation
couple of the diamine complexes generally occurred at less
positive potentials (+1.57 to +1.63 V) compared with their
triazole counterparts (+1.62 to +1.76 V), which is probably due
to the strong electron-donating effects of the diaminoaromatic
ligand. The first reduction couples and waves at ꢀ1.17 to
ꢀ1.48 V were assigned to the reduction of the diimine ligands.
This argument is supported by the observation that the
potential of the first reduction wave of the rhenium complexes
followed the order, Ph2-phen E phen 4 Me2-phen 4 Me4-phen,
Table 1 Electrochemical data of the rhenium(I) polypyridine complexesa
Complex
Oxidation, E1/2 or Ea/V
Reduction, E1/2 or Ec/V
1a
2a
3a
4a
1b
2b
3b
4b
+0.94,b +1.23,b +1.63b
+1.14,b +1.51,b +1.62b
+1.12,b +1.45,b +1.57b
+1.01,b +1.16,b +1.61b
+1.76b
ꢀ1.19, ꢀ1.60,b ꢀ1.88,b ꢀ2.10c
ꢀ1.27,b ꢀ1.65,b ꢀ1.93c
ꢀ1.46, ꢀ1.66,b ꢀ1.79,b ꢀ1.94b
ꢀ1.17, ꢀ1.44, ꢀ1.65,c ꢀ2.03b
ꢀ1.19,b ꢀ1.63,c ꢀ1.91,c ꢀ2.27c
ꢀ1.43,c ꢀ1.62,c ꢀ1.91c
+1.62b
+1.67b
ꢀ1.48,c ꢀ1.70,b ꢀ1.95,b ꢀ2.23b
ꢀ1.17,b ꢀ1.43,b ꢀ1.69,c ꢀ2.02b
+1.74b
a
In CH3CN (0.1 mol dmꢀ3 TBAP) at 298 K, glassy carbon electrode,
sweep rate 100 mV sꢀ1, all potentials versus SCE. Irreversible waves.
b
Chart 1 Structures of the rhenium(I) polypyridine diamine complexes 1a–4a
and the triazole analogues 1b–4b.
c
Quasi-reversible couples.
c
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which is in accordance with the stability of the p* orbitals of the
diimine ligands.
Electronic absorption and emission properties
The electronic absorption properties of all the complexes were
studied and the data are summarized in Table S1 (ESI†).
In general, both the diamine and triazole complexes displayed
similar electronic absorption spectra, indicating that the con-
version of the diamine complexes to their triazole counterparts
did not significantly affect the electronic absorption properties.
Similar to other rhenium(I) polypyridine complexes,19–27
all the complexes displayed intense absorption bands at
ca. 251–342 nm and weaker absorption shoulders at ca.
370–396 nm, which were assigned to spin-allowed intraligand
(1IL) (p-p*) (N^N and pyridine ligands) and spin-allowed
metal-to-ligand charge-transfer (1MLCT) [dp(Re) - p*(N^N)]
transitions, respectively.
Fig. 1 Emission spectra of the rhenium(I) triazole complexes in CH3CN at 298 K.
the Me4-phen complexes 2a and 2b in alcohol glass at 77 K
Upon irradiation, all the complexes exhibited long-lived suggest the involvement of 3IL (p - p*) (Me4-phen) character in
3MLCT [dp(Re) - p*(N^N)] emission. The photophysical data their emissive states. It is noteworthy that the diamine com-
are summarized in Table 2 and the emission spectra of the plexes showed significantly lower emission quantum yields and
triazole complexes 1b–4b in CH3CN at 298 K are shown in shorter emission lifetimes compared with their triazole coun-
Fig. 1. The structural features and long emission lifetimes of terparts (Table 2), which is most likely a consequence of
Table 2 Photophysical data of the rhenium(I) polypyridine complexesa
Complex
Medium (T/K)
lem (nm)
to (ms)
Fem
1ab
CH2Cl2 (298)
CH3CN (298)
Glassc (77)
556
554
513
0.18
0.15
10.85
0.010
0.00081
2ab
3ab
4ab
1b
CH2Cl2 (298)
CH3CN (298)
Glassc (77)
527
528
490
2.32
1.58
11.31
0.036
0.00096
CH2Cl2 (298)
CH3CN (298)
Glassc (77)
522
521
0.73
0.21
0.045
0.00038
470 (max), 501, 534 sh
29.02 (76%), 90.32 (24%)
CH2Cl2 (298)
CH3CN (298)
Glassc (77)
561
564
525, 556 sh
2.73
1.70
17.07
0.028
0.0023
CH2Cl2 (298)
CH3CN (298)
Bufferd (298)
Glassc (77)
533
549
550
497
2.95
1.34
0.49
11.03
0.28
0.097
0.0053
2b
3b
CH2Cl2 (298)
CH3CN (298)
Bufferd (298)
Glassc (77)
518
537
538
493
3.83
1.54
0.52
16.55
0.37
0.072
0.050
CH2Cl2 (298)
CH3CN (298)
Buffere (298)
Glassc (77)
515
514
518
10.01
10.89
8.00
0.62
0.48
0.42
466 (max), 499, 536 sh
34.19 (64%), 112.29 (36%)
4b
CH2Cl2 (298)
CH3CN (298)
Bufferd (298)
Glassc (77)
545
565
565
511, 536 sh
8.57
4.90
1.22
22.85
0.38
0.068
0.021
a
b
The concentrations of the complexes were adjusted so that the absorbance at the excitation wavelength (355 nm) was equal to 0.1. The emission
c
d
quantum yields of the diamine complexes 1a–4a in aqueous buffer were too low for accurate determination. EtOH/MeOH (4 : 1 v/v). 50 mM
potassium phosphate buffer pH 7.4/MeOH (9 : 1 v/v). 50 mM potassium phosphate buffer pH 7.4/MeOH (7 : 3 v/v).
e
c
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emission quenching due to the presence of the diaminoaro- the electron-withdrawing effects of the rhenium(I) center. Inter-
matic moiety. From the potentials of the diimine-based estingly, similar to the diamine complex, the triazole analogue
reduction of the diamine complexes 1a–4a (ꢀ1.17 to ꢀ1.46 V, complex 4b also displayed pH-dependent emission (Fig. 2).
Table 1) and their low-temperature emission energy (E00 = 2.36 However, the effect of pH on the emission intensity is
to 2.64 eV, Table 2), the excited-state reduction potentials less significant compared with that of the diamine complex
(Eo[Re+*/0]) of the rhenium complexes were calculated to be (IpH=2.5 : IpH=12 = 13 and 1.4 for complexes 4a and 4b, respec-
ca. +1.18 to +1.26 V vs. SCE. On the basis of these potentials and tively). The change of emission intensity in the acidic region fits
the first oxidation potential of the diamine complexes (+0.94 to an apparent pKa value of 4.73 ꢁ 0.05 for complex 4b. We
+1.14 V), reductive quenching of the excited complexes is tentatively assigned the weaker emission in the alkaline region
favored by ca. 0.06 to 0.29 eV. This indicates that the emission to the deprotonation of the triazole moiety, yielding the triazo-
quenching mechanism of the diamine complexes is most likely late form, which suppresses the emission of the complex. It is
electron transfer in nature.
noteworthy that the pKa value is lower than that of benzo-
triazole (pKa = 6.7),9 which is not unexpected since the
triazolate moiety is stabilized by the electron-withdrawing
pyridine ligand and the positively charged rhenium(I) center.
Interestingly, a blue shift of the emission maxima was observed
as the pH was decreased (Fig. 2), which originates from the
weaker electron-donating effects of the triazole ligand and
hence more stabilized dp(Re) orbitals.
pH dependent emission studies
The effects of pH on the emission intensity of the diamine
complex 4a and its triazole counterpart complex 4b were
investigated and the results are shown in Fig. 2. The emission
intensity of both complexes was relatively independent to pH
under neutral and basic conditions (pH 4 6). Upon decreasing
the pH of the solution, the diamine complex displayed emis-
sion enhancement, which was ascribed to the suppression of
PET quenching due to the protonation of the diaminoaromatic
moiety. Although the pKa value could not be determined
(Fig. 2), it should be much smaller than that of 1,2-diamino-
benzene (pKa = 4.52).28 This is reasonable because of the
reduced electron density on the amine nitrogen atoms due to
NO-sensing properties
The NO-sensing ability of the diamine complexes was investi-
gated by luminescence titrations using 3-(2-hydroxy-1-methyl-2-
nitrosohydrazino)-N-methyl-1-propanamine (NOC-7) as
a
titrant. NOC-7 is a stable NO-amine compound that sponta-
neously releases two equivalents of NO under physiological
conditions (t1/2 = 10 min, PBS buffer at pH 7.4).29 Upon addition
of NOC-7 to a solution of the diamine complex, significant
emission enhancement (I/Io = 31) was observed (Fig. 3). The
emission enhancement was attributed to the conversion of the
diamine complex to its triazole counterpart, which was subse-
quently confirmed by ESI-MS. The large emission enhancement
factor and low emission quantum yields of the diamine com-
plexes render them potential phosphorogenic sensors for NO.
The diamine complex 4a showed good signal linearity with
NOC-7 concentrations ranging from 0 to 50 mM (R2 = 0.99386).
The detection limit was determined to be ca. 300 nM, which is
comparable to
a
ruthenium(II)-based NO sensor.12 It is
Fig. 2 pH titration curves for complexes 4a (top) and 4b (bottom) (10 mM) in
aerated 100 mM KCl (aq)/MeOH (9 : 1 v/v) at 298 K. The emission intensity of
complexes 4a and 4b was monitored at 568 and 560 nm, respectively. The inset
shows the emission spectral traces of the complexes upon decreasing pH.
Fig. 3 Emission spectral traces of complex 4a (5 mM) in aerated potassium
phosphate buffer (50 mM, pH 7.4)/MeOH (9 : 1 v/v) at 298 K in the presence of 0
to 100 mM NOC-7. The inset shows the emission titration curve of complex 4a
with NOC-7. The emission intensity of complex 4a was monitored at 568 nm.
c
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noteworthy that the end point for the titration of the
diamine complex 4a with NO was not 1 : 1 (Fig. 3, inset),
which is commonly observed in other diaminoaromatic
compounds.5,6,12 This suggests that a complicated reaction
mechanism may be involved in the formation of the triazole
complex. In fact, diaminoaromatic compounds are known to
react with the oxidized products of NO such as nitrous anhydride
(N2O3) instead of NO itself.5a Nevertheless, since those oxidized
products can only be generated by NO under physiological
conditions, diaminoaromatic compounds are still widely
employed in the design of fluorescent NO sensors.5,6
Biological properties and NO-sensing in living cells
We investigated the possibility of using the diamine complexes
1a–4a as phosphorescent sensors for intracellular NO. First, the
cellular uptake properties of these complexes were examined by
ICP-MS. Upon incubation with the complexes ([Re] = 10 mM) at
37 1C for 1 h, an average HeLa cell (volume = 3.4 pL) contained
0.40 to 2.43 fmol of rhenium (Table 3). Due to their
more lipophilic diimine ligands, the Ph2-phen, Me2-phen and
Me4-phen complexes displayed more effective cellular uptake
than the phen complex. Similar results were also observed in
related rhenium(I) complexes.19h,j It is important to point out
that the intracellular rhenium concentrations of all four com-
plexes (0.12 to 0.72 mM) were much higher than that in the
medium before uptake (10 mM), indicative of the cellular
accumulation of the complexes. The cytotoxicity of the diamine
complexes 1a–4a toward HeLa cells over an incubation period
of 48 h was investigated by the MTT assay (Table 3). The
Ph2-phen, Me2-phen and Me4-phen complexes exhibited higher
cytotoxicity, which was in accordance with their higher uptake
efficiency.19h,j
The diamine complex 4a was used to visualize intracellular
NO that was exogenously generated by a donor (NOC-7) in HeLa
cells. Upon incubation with complex 4a for 1 h, weak intra-
cellular emission was observed in the cytoplasmic region of the
HeLa cells (Fig. 4). Owing to the highly emissive nature of the
triazole complexes, the intracellular conversion of the diamine
complexes to their triazole derivatives should lead to enhanced
intracellular luminescence. However, after the cells were
further incubated with NOC-7 (100 mM) for 30 min, the intra-
cellular emission of the HeLa cells displayed no significant
difference (Fig. 4). Similar results were also observed upon
further increasing the concentration of NOC-7 (500 mM) and
Fig. 4 Laser-scanning confocal microscopy images of HeLa cells incubated with
complex 4a (10 mM) at 37 1C for 1 h. The cells were post-incubated with (top row)
and without (bottom row) NOC-7 (100 mM) in PBS at 37 1C for 30 min.
incubation time (1 h) (data not shown). Under the same
conditions, significant emission enhancement was observed
for the HeLa cells which were incubated with a commercially
available NO sensor 4-amino-5-methylamino-20,70-difluoro-
fluorescein diacetate (DAF-FM diacetate). Also, we found that
the HeLa cells incubated with the triazole complex 4b displayed
strong intracellular luminescence (Fig. 5), despite the less
efficient uptake by HeLa cells (1.67 fmol per average cell) than
its diamine counterpart (2.43 fmol per average cell). All these
observations indicated that complex 4a could not be converted
to the triazole derivative under intracellular conditions. This is
probably related to the short intracellular lifetime of NO (t1/2
=
ca. 5 s)1a,4 and the rather low reactivity of the complex. Never-
theless, the cellular studies showed that the complex was able
to penetrate the cellular membrane and accumulate in the
HeLa cells. With appropriate modification of the molecular
structure, related rhenium(I) diamine complexes are expected
to function as phosphorescent sensors for NO in an intra-
cellular environment.
Table 3 Cellular uptake and cytotoxicity (IC50, 48 h) of the rhenium(I) diamine
complexes and cisplatin toward the HeLa cell line
Complex
No. of molea (fmol)
Concentration (mM)
IC50 (mM)
1a
2a
3a
4a
0.40 ꢁ 0.02
1.05 ꢁ 0.01
1.91 ꢁ 0.07
2.43 ꢁ 0.04
N.A.
0.12 ꢁ 0.01
0.31 ꢁ 0.01
0.56 ꢁ 0.02
0.72 ꢁ 0.04
N.A.
51.0 ꢁ 4.6
17.6 ꢁ 0.5
6.1 ꢁ 0.3
2.5 ꢁ 0.1
21.6 ꢁ 1.4
Cisplatin
a
Fig. 5 Laser-scanning confocal microscopy images of HeLa cells incubated with
complexes 4a (top row) and 4b (bottom row) (10 mM) at 37 1C for 1 h.
Number of moles of rhenium(I) associated with a typical HeLa cell
upon incubation with the complexes (10 mM) at 37 1C for 1 h.
c
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Conclusion
diethyl ether. Complex 1a was isolated as yellow crystals. Yield:
169 mg (80%). 1H NMR (300 MHz, acetone-d6, 298 K) d 9.81
(d, 2H, J = 5.1 Hz, H2 and H9 of phen), 9.08 (d, 2H, J = 8.4 Hz,
H4 and H7 of phen), 8.36–8.30 (m, 4H, H3, H5, H6 and H8 of
phen), 7.67 (s, 1H, H2 of pyridine), 7.38 (d, 1H, J = 6.0 Hz, H6 of
pyridine), 6.30 (d, 1H, J = 6.0 Hz, H5 of pyridine), 5.79 (br, 2H,
NH2 at C4 of pyridine), 4.44 (br, 2H, NH2 at C3 of pyridine). IR
(KBr) n/cmꢀ1: 3367 (br, NꢀH), 2026 (s, CRO), 1912 (s, CRO),
1165 (m, CF3SO3ꢀ), 1030 (m, CF3SO3ꢀ). Positive-ion ESI-MS ion
In this work, a new class of NO sensors have been developed
from phosphorescent rhenium(I) polypridine complexes. Upon
reaction with NO, the weakly emissive diamine complexes were
converted into the triazole derivatives, resulting in significant
emission enhancement. The emission of the complexes was
independent of pH under neutral and basic conditions. These
complexes were found to accumulate in HeLa cells with moderate
cytotoxicity. The large emission enhancement factors and low
emission quantum yields of the diamine complexes cause them
to function as phosphorogenic sensors for NO. Since the
reactivity of the complexes toward NO is determined by the
electron density of the diaminoaromatic moiety,10 complexes
with an attached electron-rich diaminoaromatic ligand are
anticipated to function as highly reactive NO sensors. To
achieve this, we believe that different electron-donating groups,
such as a methoxy moiety can be attached to the diamino-
aromatic ligands to increase their electron density. Additionally,
the diaminoaromatic moiety should be separated from the
electron-withdrawing rhenium(I) metal center through an
insulating spacer-arm. The design of related phosphorescent
transition metal diamine complexes as intracellular NO sensors
is under way.
clusters at m/z 559 {M
ꢀ
CF3SO3ꢀ}+. Anal. Calcd for
ReC21H15N5O6SF3ꢂH2O: C, 34.72; H, 2.36; N, 9.64. Found: C,
34.85; H, 2.69; N, 9.98. Complex 2a was isolated as yellow
1
crystals. Yield: 188 mg (85%). H NMR (300 MHz, acetone-d6,
298 K) d 8.82 (d, 2H, J = 8.1 Hz, H4 and H7 of Me2-phen), 8.21
(d, 2H, J = 8.4 Hz, H3 and H8 of Me2-phen), 8.13 (s, 2H, H5 and
H6 of Me2-phen), 7.16 (s, 1H, H2 of pyridine), 6.89 (d, 1H, J =
6.0 Hz, H6 of pyridine), 6.18 (d, 1H, J = 6.0 Hz, H5 of pyridine),
5.75 (br, 2H, NH2 at C4 of pyridine), 4.35 (br, 2H, NH2 at C3 of
pyridine), 3.45 (s, 6H, CH3 of Me2-phen). IR (KBr) n/cmꢀ1: 3368
(br, N–H), 2027 (s, CRO), 1911 (s, CRO), 1162 (m, CF3SO3ꢀ),
1028 (m, CF3SO3ꢀ). Positive-ion ESI-MS ion clusters at m/z 587
{M ꢀ CF3SO3ꢀ}+. Anal. Calcd for ReC23H19N5O6SF3: C, 37.50; H,
2.60; N, 9.51. Found: C, 37.80; H, 2.85; N, 9.57. Complex 3a was
isolated as yellow crystals. Yield: 205 mg (89%). 1H NMR (300 MHz,
acetone-d6, 298 K) d 9.53 (s, 2H, H2 and H9 of Me4-phen), 8.42
(s, 2H, H5 and H6 of Me4-phen), 7.71 (s, 1H, H2 of pyridine),
7.41 (d, 1H, J = 6.3 Hz, H6 of pyridine), 6.30 (d, 1H, J = 6.0 Hz,
H5 of pyridine), 5.75 (br, 2H, NH2 at C4 of pyridine), 4.39
(br, 2H, NH2 at C3 of pyridine), 2.91 (s, 6H, CH3 at C4 and C7 of
Me4-phen), 2.78 (s, 6H, CH3 at C3 and C8 of Me4-phen). IR (KBr)
n/cm1: 3369 (br, N–H), 2026 (s, CRO), 1910 (s, CRO), 1161
(m, CF3SO3ꢀ), 1031 (m, CF3SO3ꢀ). Positive-ion ESI-MS ion
Experimental
Materials and synthesis
All solvents were of analytical reagent grade and purified
according to standard procedures.30 Diimine ligands including
phen, Me2-phen, Me4-phen and Ph2-phen, AgCF3SO3 and
cisplatin were purchased from Acros. Re(CO)5Cl and py-diamine
were obtained from Aldrich. MTT was purchased from Sigma.
NOC-7 was obtained from Merck. Purified NO gas was obtained
from Scientific Gas Engineering Co., Ltd. All these chemicals were
used without further purification. [Re(N^N)(CO)3(CH3CN)](CF3SO3)
was prepared as described previously.31 Autoclaved Milli-Q
water was used for the preparation of aqueous solutions. All
buffer components were of biological grade and used as
received. HeLa cells were obtained from American Type Culture
Collection. Dulbecco’s modified Eagle’s medium (DMEM),
fetal bovine serum (FBS), phosphate buffered saline (PBS),
trypsin-EDTA, DAF-FM diacetate and penicillin/streptomycin
were purchased from Invitrogen. The growth medium for cell
culture contained DMEM with 10% FBS and 1% penicillin/
streptomycin.
clusters at m/z 615 {M
ꢀ
CF3SO3ꢀ}+. Anal. Calcd for
ReC25H23N5O6SF3: C, 39.27; H, 3.03; N, 9.16. Found: C, 39.34;
H, 3.18; N, 9.42. Complex 4a was isolated as orange crystals.
Yield: 188 mg (73%). 1H NMR (300 MHz, acetone-d6, 298 K)
d 9.87 (d, 2H, J = 5.7 Hz, H2 and H9 of Ph2-phen), 8.27–8.23
(m, 4H, H3, H5, H6 and H8 of Ph2-phen), 7.80–7.66 (m, 11H, H2
of pyridine and C6H5 of Ph2-phen), 7.49 (d, 1H, J = 6.3 Hz, H6 of
pyridine), 6.36 (d, 1H, J = 6.3 Hz, H5 of pyridine), 5.96 (br, 2H,
NH2 at C4 of pyridine), 4.66 (br, 2H, NH2 at C3 of pyridine). IR
(KBr) n/cmꢀ1: 3372 (br, N–H), 2023 (s, CRO), 1909 (s, CRO),
1158 (m, CF3SO3ꢀ), 1030 (m, CF3SO3ꢀ). Positive-ion ESI-MS ion
clusters at m/z 711 {M
ReC33H23N5O6SF3: C, 46.05; H, 2.69; N, 8.14. Found: C, 46.00;
H, 3.02; N, 8.25.
ꢀ
CF3SO3ꢀ}+. Anal. Calcd for
[Re(N^N)(CO)3(py-diamine)](CF3SO3) [N^N = phen (1a),
Me2-phen (2a), Me4-phen (3a), Ph2-phen (4a)]
[Re(N^N)(CO)3(py-triazole)](CF3SO3) [N^N = phen (1b),
Me2-phen (2b), Me4-phen (3b), Ph2-phen (4b)]
A mixture of [Re(N^N)(CO)3(CH3CN)](CF3SO3) (0.30 mmol) and Purified NO was bubbled slowly into
a
solution of
py-diamine (0.30 mmol) in THF (30 mL) was refluxed under an [Re(N^N)(CO)3(py-diamine)](CF3SO3) (0.070 mmol) in CH2Cl2
inert atmosphere of nitrogen for 12 h. The mixture was evapo- (10 mL) for 10 min. The solution was evaporated to dryness to
rated to dryness to give a yellow solid, which was purified by give a yellow solid. Recrystallization of the product from
column chromatography on alumina. The desired product was CH2Cl2/diethyl ether afforded the complex as yellow crystals.
1
eluted with a mixture of CH2Cl2 and methanol and it was Complex 1b. Yield: 42 mg (83%). H NMR (300 MHz, acetone-
subsequently recrystallized from a mixture of CH2Cl2 and d6, 298 K) d 10.05 (d, 2H, J = 5.4 Hz, H2 and H9 of phen), 9.51
c
1716 New J. Chem., 2013, 37, 1711--1719
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(s, 1H, H2 of pyridine), 9.09 (d, 2H, J = 8.1 Hz, H4 and H7 of HP6/6 quick-release Teflon stopper. Luminescence quantum
phen), 8.59 (d, 1H, J = 6.6 Hz, H6 of pyridine), 8.40–8.32 (m, 4H, yields were measured by the optically dilute method32 using a
H3, H5, H6 and H8 of phen), 7.82 (d, 1H, J = 6.6 Hz, H5 of degassed CH3CN solution of [Re(phen)(CO)3(pyridine)](CF3SO3)
pyridine). IR (KBr) n/cmꢀ1: 3448 (br, N–H), 2035 (s, CRO), 1906 (Fem = 0.18, lex = 355 nm) as the standard solution.33 Details of
(s, CRO), 1164 (m, CF3SO3ꢀ), 1029 (m, CF3SO3ꢀ). Positive-ion the MTT assays and ICP-MS have been reported previously.19g
ESI-MS ion clusters at m/z 570 {M ꢀ CF3SO3ꢀ}+. Anal. Calcd for
pH dependent emission studies
ReC21H12N6O6SF3ꢂ0.5C2H5OC2H5: C, 36.52; H, 2.27; N, 11.11.
Found: C, 36.52; H, 2.21; N, 11.08. Complex 2b. Yield: 42 mg
A 20 mL stock solution of complexes 4a and 4b (10 mM),
respectively, in a mixture of 10 mM KOH and 100 mM KCl(aq)/
MeOH (9: 1 v/v) was prepared. The pH of the stock solution was
increased gradually by addition of appropriate amounts of 1,
0.5 0.25, 0.13, 0.07, 0.04 or 0.02 N HCl(aq). After a desired pH
value was obtained, 2 mL of the stock solution was transferred to
a quartz cuvette for emission measurements. The solution was
then returned to the stock and the pH of the solution was further
adjusted. The overall increase in the volume of the stock solution
due to pH adjustment was kept below 2%.
1
(81%). H NMR (300 MHz, acetone-d6, 298 K) d 8.96 (s, 1H, H2
of pyridine), 8.81 (d, 2H, J = 8.1 Hz, H4 and H7 of Me2-phen),
8.28 (d, 2H, J = 8.7 Hz, H3 and H8 of Me2-phen), 8.11–8.06 (m,
3H, H6 of pyridine and H5 and H6 of Me2-phen), 7.71 (d, 1H, J =
6.6 Hz, H5 of pyridine), 3.56 (s, 6H, CH3 of Me2-phen). IR (KBr)
n/cmꢀ1: 3449 (br, N–H), 2033 (s, CRO), 1925 (s, CRO), 1155
(m, CF3SO3ꢀ), 1029 (m, CF3SO3ꢀ). Positive-ion ESI-MS ion
clusters at m/z 599 {M
ꢀ
PF6ꢀ}+. Anal. Calcd for
ReC23H16N6O6SF3ꢂ0.5H2O: C, 36.52; H, 2.27; N, 11.11. Found:
C, 36.54; H, 2.33; N, 11.28. Complex 3b. Yield: 47 mg (87%).
1H NMR (300 MHz, acetone-d6, 298 K) d 9.79 (s, 2H, H2 and H9
of Me4-phen), 9.53 (s, 1H, H2 of pyridine), 8.63 (d, 1H, J =
6.6 Hz, H6 of pyridine), 8.42 (s, 2H, H5 and H6 of Me4-phen),
7.78 (d, 1H, J = 6.6 Hz, H5 of pyridine), 2.91 (s, 6H, CH3 at C4
and C7 of Me4-phen), 2.81 (s, 6H, CH3 at C3 and C8 of
Me4-phen). IR (KBr) n/cmꢀ1: 3448 (br, N–H), 2032 (s, CRO),
1947 (s, CRO), 1159 (m, CF3SO3ꢀ), 1029 (m, CF3SO3ꢀ).
Positive-ion ESI-MS ion clusters at m/z 626 {M ꢀ CF3SO3ꢀ}+.
Anal. Calcd for ReC25H20N6O6SF3ꢂ1.5H2O: C, 37.41; H, 2.89; N,
10.47. Found: C, 37.47; H, 2.71; N, 10.72. Complex 4b. Yield:
44 mg (72%). 1H NMR (300 MHz, acetone-d6, 298 K) d 10.10
(d, 2H, J = 5.4 Hz, H2 and H9 of Ph2-phen), 9.58 (s, 1H, H2 of
pyridine), 8.70 (d, 1H, J = 6.9 Hz, H6 of pyridine), 8.32 (d, 2H,
J = 5.7 Hz, H3 and H8 of Ph2-phen), 8.20 (s, 2H, H5 and H6 of
Ph2-phen), 7.89 (d, 2H, J = 6.6 Hz, H5 of pyridine), 7.69–7.66
(m, 10H, C6H5 of Ph2-phen). IR (KBr) n/cmꢀ1: 3448 (br, N–H),
2027 (s, CRO), 1918 (s, CRO), 1159 (m, CF3SO3ꢀ), 1030
(m, CF3SO3ꢀ). Positive-ion ESI-MS ion clusters at m/z 722
{M ꢀ CF3SO3ꢀ}+. Anal. Calcd for ReC33H20N6O6SF3ꢂCH3CNꢂ2H2O:
C, 44.31; H, 2.87; N, 10.33. Found: C, 44.28; H, 2.77; N, 10.25.
Detection of NO in aqueous buffer solutions
A series of solutions containing different concentrations of NOC-
7 (0 to 100 mM) and the diamine complex 4a (5 mM) in potassium
phosphate buffer (50 mM, pH 7.4)/MeOH (9: 1 v/v) was prepared.
The solutions were gently stirred at room temperature for 2 h
and their emission spectra were then measured.
Live-cell confocal microscopy and imaging of intracellular NO
HeLa cells in growth medium were seeded on a sterilized
coverslip in a 60 mm tissue culture dish and grown at 37 1C
under a 5% CO2 atmosphere for 48 h. The culture medium was
then removed and replaced with medium/DMSO (99 : 1 v/v)
containing complex 4a (10 mM). After incubation for 1 h, the
medium was removed and the cell layer was washed with PBS
(1 mL ꢃ 3). The coverslip was mounted onto a sterilized glass
slide and then imaged using a Leica TCS SPE confocal micro-
scope. In the NO imaging experiment, after being treated with
complex 4a, the cells were incubated with NOC-7 (100 mM) in
PBS for 30 min and finally washed with PBS (1 mL ꢃ 3).
Instrumentation and methods
Acknowledgements
1H NMR spectra were recorded on a Varian Mercury 300 MHz
NMR spectrometer at 298 K. Positive-ion ESI mass spectra were
recorded on a Perkin-Elmer Sciex API 365 mass spectrometer.
IR spectra were recorded on a Perkin-Elmer 1600 series FT-IR
spectrophotometer. Elemental analyses were carried out on a
Vario EL III CHN elemental analyzer. Electronic absorption and
steady-state emission spectra were recorded on a Hewlett-
Packard 8453 diode array spectrophotometer and a SPEX
We thank the Hong Kong University Grants Committee (Areas
of Excellence Scheme AoE/P-03/08) and the Hong Kong
Research Grants Councils (project no. CityU 102311) for
financial support. A. W.-T. C. acknowledges the receipt of a
Postgraduate Studentship and a Research Tuition Scholarship
administered by City University of Hong Kong.
FluoroLog 3-TCSPC spectrophotometer equipped with
a
Notes and references
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four successive freeze-pump-thaw cycles and stored in a 10 cm3
round-bottomed flask equipped with a side-arm 1 cm fluores-
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