Rhenium(I) polypyridine diamine complexes as intracellular phosphorogenic sensors: Synthesis, characterization, emissive behavior, biological properties, and nitric oxide sensing

Article abstract of DOI:10.1002/chem.201402502

We report the development of a series of rhenium(I) polypyridine complexes appended with an electron-rich diaminoaromatic moiety as phosphorogenic sensors for nitric oxide (NO). The diamine complexes [Re(N^N)(CO)₃(py-DA)] [PF₆] (py-DA=3-(N-(2-amino-5-methoxyphenyl)aminomethyl)pyridine; N^N=1,10-phenanthroline (phen) (1a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me₄-phen) (2a), 4,7-diphenyl-1,10-phenanthroline (Ph ₂-phen) (3a)) have been synthesized and characterized. In contrast to common rhenium(I) diimines, these diamine complexes were very weakly emissive due to quenching of the triplet metal-to-ligand charge-transfer ( ³MLCT) emission by the diaminoaromatic moiety through photoinduced electron transfer (PET). Upon treatment with NO, the complexes were converted into the triazole derivatives [Re(N^N)(CO)₃(py-triazole)][PF ₆] (py-triazole=3-((6-methoxybenzotriazol-1-yl)methyl)pyridine; N^N=phen (1b), Me₄-phen (2b), Ph₂-phen (3b)), resulting in significant emission enhancement (I/I₀≈60). The diamine complexes exhibited high reaction selectivity to NO, and their emission intensity was found to be independent on pH. Also, these complexes were effectively internalized by HeLa cells and RAW264.7 macrophages with negligible cytotoxicity. Additionally, the use of complex 3a as an intracellular phosphorogenic sensor for NO has been demonstrated. Emission turned ON for NO: A series of rhenium(I) polypyridine complexes functionalized with an electron-rich diaminoaromatic moiety has been developed as a new class of phosphorogenic sensors for NO. Upon treatment with NO, the weakly emissive complexes were converted into the strongly emissive triazole derivatives, resulting in significant emission enhancement (I/I₀≈60; see figure). Experiments showed that the diamine complexes can sense NO that is 1) generated exogenously by NOC-7 in HeLa cells and 2) produced endogenously in RAW264.7 macrophages.

Full text of DOI:10.1002/chem.201402502

DOI: 10.1002/chem.201402502
Full Paper
&
Sensors
Rhenium(I) Polypyridine Diamine Complexes as Intracellular
Phosphorogenic Sensors: Synthesis, Characterization, Emissive
Behavior, Biological Properties, and Nitric Oxide Sensing
Alex Wing-Tat Choi, Vicki Man-Wai Yim, Hua-Wei Liu, and Kenneth Kam-Wing Lo*[a]
Abstract: We report the development of a series of rheniu-
m(I) polypyridine complexes appended with an electron-rich
diaminoaromatic moiety as phosphorogenic sensors for
nitric oxide (NO). The diamine complexes [Re(N^N)(CO)3(py-
moiety through photoinduced electron transfer (PET). Upon
treatment with NO, the complexes were converted into the
triazole derivatives [Re(N^N)(CO)3(py-triazole)][PF6] (py-tria-
zole=3-((6-methoxybenzotriazol-1-yl)methyl)pyridine;
DA)][PF6]
(py-DA=3-(N-(2-amino-5-methoxyphenyl)amino-
N^N=phen (1b), Me4-phen (2b), Ph2-phen (3b)), resulting
in significant emission enhancement (I/I0 ꢀ60). The diamine
complexes exhibited high reaction selectivity to NO, and
their emission intensity was found to be independent on
pH. Also, these complexes were effectively internalized by
HeLa cells and RAW264.7 macrophages with negligible cyto-
toxicity. Additionally, the use of complex 3a as an intracellu-
lar phosphorogenic sensor for NO has been demonstrated.
methyl)pyridine; N^N=1,10-phenanthroline (phen) (1a),
3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-phen) (2a), 4,7-
diphenyl-1,10-phenanthroline (Ph2-phen) (3a)) have been
synthesized and characterized. In contrast to common rhe-
nium(I) diimines, these diamine complexes were very weakly
emissive due to quenching of the triplet metal-to-ligand
charge-transfer (3MLCT) emission by the diaminoaromatic
Introduction
sion, and accumulation of NO, thereby to further understand
its roles in different biological systems.
Nitric oxide is an important cellular signaling molecule that is
associated with a variety of biological processes such as neuro-
transmission, muscle relaxation, and blood pressure and
immune response regulation.[1] It is biosynthesized in mamma-
lian cells by three distinct nitric oxide synthase (NOS) isoforms
including neuronal NOS (nNOS), endothelial NOS (eNOS), and
inducible NOS (iNOS).[2] The intracellular NO concentration
ranges from submicromolar to micromolar levels depending
on the types of NOS that are present in the cells.[3] A high in-
tracellular concentration of NO, which triggers the formation
of reactive nitrogen species (RNS), has been implicated in carci-
nogenesis and several neurodegenerative disorders such as
Alzheimer’s disease, Parkinson’s disease, multiple sclerosis, and
Huntington’s disease.[4] To date, a number of detection meth-
ods including colorimetry, chemiluminescence, electron para-
magnetic resonance, electrochemistry, and fluorescence have
been utilized to detect this important biological messenger.[5]
Among these methods, fluorescence detection allows imaging
intercellular NO with very high spatiotemporal resolution.[6]
Thus, it is an attractive method to study the production, diffu-
Thus far, two types of fluorescent molecular sensors for NO
have been developed.[7] The first type involves the use of a par-
amagnetic Cu2+ ion as a quencher to suppress the emission of
a fluorophore.[8] After the metal ion is reduced by NO, the
emission of the fluorophore is recovered. In the second type,
an electron-rich diaminoaromatic moiety is exploited to recog-
nize NO and to quench the emission of a fluorophore through
photoinduced electron transfer (PET).[6a] The diaminoaromatic
moiety is converted into a triazole derivative by NO under aer-
ated conditions and the emission of the fluorophore such as
fluorescein,[9] rhodamine,[10] BODIPY,[11] cyanine,[12] and naphtha-
limide[13] is thus reverted. Despite the development of these re-
agents, the possibility of using phosphorescent transition-
metal diamine complexes as intracellular NO sensors has not
been fully explored.[14,15] In the past few years, there has been
a fast-growing interest in the cellular-uptake properties of
phosphorescent rhenium(I) complexes with a focus on their
potential as cellular imaging and therapeutic reagents.[16–21]
Previously, we have synthesized a series of rhenium(I) and
iridium(III) diaminoaromatic complexes as sensors for NO.[15] Al-
though the rhenium(I) complexes display interesting phos-
phorogenic properties toward NO, they are unable to react ef-
ficiently with NO in living cells, probably due to insufficient re-
activity.
[a] A. W.-T. Choi, V. M.-W. Yim, Dr. H.-W. Liu, Prof. K. K.-W. Lo
Department of Biology and Chemistry, City University of Hong Kong
Tat Chee Avenue, Kowloon, Hong Kong (P.R. China)
Fax: (852)3442-0522
Herein, we report the synthesis, characterization, and photo-
physical and electrochemical properties of three phosphores-
cent rhenium(I) polypyridine diamine complexes [Re-
(N^N)(CO)3(py-DA)][PF6] (py-DA=3-(N-(2-amino-5-methoxyphe-
E-mail: bhkenlo@cityu.edu.hk
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201402502.
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such as [Re(phen)(CO)3(py-CC-DAPh)][CF3SO3] (py-CC-DAPh=3-
(2-(3,4-diaminophenyl)ethynyl)pyridine)
DAPh)(CO)3(pyridine)][CF3SO3] (bpy-CH2-DAPh=4-(N-(2-amino-
phenyl)aminomethyl)-4’-methyl-2,2’-bipyridine) (S2), and
(S1),
[Re(bpy-CH2-
[Re(Ph2-phen)(CO)3(py-CH2-DAPh)][CF3SO3] (py-CH2-DAPh=3-
(N-(2-aminophenyl)aminomethyl)pyridine) (S3) have also been
prepared (the Supporting Information, Scheme S1). However,
none of these complexes displayed satisfactory intracellular
NO-sensing properties due to slow reaction kinetics. Since the
reactivity of the complexes toward NO is strongly dependent
on the electron density of the diaminoaromatic moiety,[11a] we
believe that for a complex to function as a phosphorogenic
sensor for intracellular NO, a highly electron-rich diaminoaro-
matic ligand should be used. Thus, in this study, an electron-
donating methoxy group was added to the diaminoaromatic
ligand to increase its electron density. Additionally, the diami-
noaromatic moiety was attached to the complexes through an
insulating spacer-arm to reduce the electron-withdrawing
effect caused by the rhenium(I) center. By this modification, we
anticipate that the complexes will have an increased reactivity
toward NO and can function as phosphorogenic sensors in
living cells.
Regarding the synthesis of the complexes, 2-(tert-butoxycar-
bonylamino)-5-methoxyaniline was treated with 3-pyridinecar-
boxaldehyde, and the intermediate was reduced by NaBH4 to
Scheme 1. Structures of the rhenium(I) diamine complexes 1a–3a and the
triazole complexes 1b–3b.
afford
3-(N-(2-(tert-butoxycarbonylamino)-5-methoxyphenyl)-
aminomethyl)pyridine (py-DA-tBoc) (the Supporting Informa-
tion, Scheme S2). The diamine complexes 1a–3a were ob-
tained from the reaction of [Re(N^N)(CO)3(CH3CN)][CF3SO3]
with py-DA-tBoc, followed by deprotection of the tBoc group,
anion exchange with KPF6, and column chromatographic pu-
rification. The triazole complexes 1b–3b were prepared by
bubbling purified NO gas into a solution of the diamine com-
plexes 1a–3a in CH2Cl2 and subsequently purified by recrystal-
lization from CH2Cl2/diethyl ether. All the complexes were
stable in air and soluble in most organic solvents such as
CH2Cl2, CH3CN, and alcohols. They were characterized by using
1H NMR spectroscopy, positive-ion ESI-MS, and IR spectroscopy
and gave satisfactory microanalysis.
nyl)aminomethyl)pyridine; N^N=1,10-phenanthroline (phen)
(1a), 3,4,7,8-tetramethyl-1,10-phenanthroline (Me4-phen) (2a),
4,7-diphenyl-1,10-phenanthroline (Ph2-phen) (3a)) and their tri-
azole counterparts [Re(N^N)(CO)3(py-triazole)][PF6] (py-tria-
zole=3-((6-methoxybenzotriazol-1-yl)methyl)pyridine; N^N=
phen (1b), Me4-phen (2b), Ph2-phen (3b)) (Scheme 1). The NO-
sensing properties of the diamine complexes and the effects of
pH on the emission intensity of the phen complexes 1a and
1b have been studied. We have also investigated the cytotox-
icity and cellular-uptake properties of the diamine complexes
by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bro-
mide (MTT) assay and ICP-MS, respectively. Furthermore, the
NO-sensing properties of complex 3a in living cells have been
examined.
Photophysical and electrochemical properties
The electronic absorption spectral data of the complexes are
summarized in Table S1 (the Supporting Information). Similar
to other rhenium(I) diimines,[15a,16–27] all the complexes dis-
played intense absorption bands at 250–345 nm and weaker
absorption shoulders at 370–393 nm, which have been as-
signed to spin-allowed intraligand (1IL) (p!p*) (N^N and pyri-
dine ligands) and spin-allowed metal-to-ligand charge-transfer
(1MLCT)(dp(Re)!p*(N^N)) transitions, respectively. It is note-
worthy that the diamine and triazole complexes with the same
diimine ligand displayed very similar electronic absorption
spectra, indicating that the conversion of diamine complexes
into their triazole counterparts did not significantly alter their
electronic absorption properties.
Results and Discussion
Design and synthesis of complexes
Previously, we have shown that rhenium(I) diimine complexes
coordinated with the ligand 3,4-diaminopyridine are interest-
ing phosphorogenic sensors for NO.[15a] However, their poten-
tial to function as intracellular NO sensors is limited since these
complexes are unable to be converted into their triazole deriv-
atives inside living cells. Owing to the short intracellular life-
time of NO (k1/2 ꢀ5 s),[1a,4] the reactivity of the complexes must
be sufficiently high to compete with other intracellular NO re-
action pathways. In addition to 3,4-diaminopyridine, rhenium(I)
complexes coordinated with other diaminoaromatic ligands
Upon irradiation, all the complexes exhibited long-lived trip-
let metal-to-ligand charge-transfer 3MLCT (dp(Re)!p*(N^N))
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Table 1. Photophysical data of the rhenium(I) polypyridine complexes.
Table 2. Self-quenching constants and emission lifetimes at infinite dilu-
tion of the rhenium(I) diamine complexes 1a–3a in degassed CH3CN at
298 K.
[a]
Complex
Medium
lem
to
Fem
(T [K])
[nm]
[ms]
Complex
ksq
ti.d.
1a
CH2Cl2 (298)
CH3CN (298)
glass (77)[b]
CH2Cl2 (298)
CH3CN (298)
glass (77)[b]
CH2Cl2 (298)
CH3CN (298)
glass (77)[b]
CH2Cl2 (298)
CH3CN (298)
glass (77)[b]
CH2Cl2 (298)
CH3CN (298)
glass (77)[b]
CH2Cl2 (298)
CH3CN (298)
glass (77)[b]
529
530
477 sh, 501
494 sh, 524
494 sh, 526
1.36
2.03
10.6
3.70
2.36
37.9
6.52
5.30
0.009
0.004
[dm3 molꢁ1 sꢁ1
]
[ms]
1a
2a
3a
2.90ꢁ109
2.64ꢁ109
3.92ꢁ109
2.43
9.99
7.42
2a
3a
1b
2b
3b
0.015
0.006
467 (max), 499, 532 sh
544
545
522
527
544
0.012
0.007
21.7
3.28
1.82
their triazole counterparts (Table 1), reflecting that the emission
quenching of the diamine complexes is basically intermolecu-
lar in nature.
0.38
0.18
464 sh, 489
489 sh, 510
489 sh, 512
465 (max), 498, 534 sh
540
12.7
12.19
9.30
51.7
9.58
4.33
29.3
0.38
0.33
To gain further insights into the emission quenching behav-
ior, the electrochemical properties of the complexes have been
investigated by cyclic voltammetry, and the results are sum-
marized in Table 3. With reference to previous electrochemical
studies of related rhenium(I) polypyridine complexes,[22,23] the
quasi-reversible couple at +1.69 to +1.76 V and the first re-
duction wave at ꢁ1.16 to ꢁ1.41 V (all potentials are versus the
saturated calomel electrode (SCE)) have been assigned to the
oxidation of the rhenium(I) center and the reduction of the di-
imine ligands, respectively. Compared with the triazole ana-
logues, the diamine complexes displayed an additional irrever-
sible band at +0.40 to +0.42 V and a quasi-reversible couple
at +1.09 to +1.15 V. Since similar oxidation waves were also
observed for the uncoordinated diaminoaromatic ligand py-DA
(+0.38 and +1.13 V), these potentials have been assigned to
diaminoaromatic-based oxidation. The occurrence of these oxi-
dation waves at significantly less positive potentials than those
of o-diaminobenzene (+0.54 and +1.78 V) and o-diaminopyri-
dine (+0.60 and +1.59 V) indicates that the addition of a me-
thoxy group and the alkylation of the primary amine has sub-
stantially increased the electron density of the diaminoaromat-
ic unit.
0.42
0.17
556
506, 528 sh
[a] lex =355 nm, [Re]=10 mm. [b] EtOH/MeOH (4:1, v/v).
Figure 1. Emission spectra of complexes 1b (c), 2b (a), and 3b (g)
in CH2Cl2 at 298 K.
From the potentials of the diimine-based reduction of the
diamine complexes (ꢁ1.16 to ꢁ1.41 V, Table 3) and their low-
temperature emission energy (E00 =2.37–2.65 eV, Table 1), the
excited-state reduction potentials (E8[Re+*/0]) of the rhenium(I)
complexes have been determined to be +1.21 to +1.30 V. On
the basis of these potentials and the first oxidation potential
of the diamine complexes, reductive quenching of the excited
emission. The photophysical data are summarized in Table 1,
and the emission spectra of the triazole complexes 1b–3b in
CH2Cl2 at 298 K are shown in Figure 1 as examples. The struc-
tural features and long emission lifetimes of the Me4-phen
3
complexes suggest the involvement of IL (p!p*) (Me4-phen)
character in their emissive states. It is noteworthy
that the diamine complexes displayed much lower
Table 3. Electrochemical data of the rhenium(I) polypyridine complexes.[a]
emission quantum yields and shorter emission life-
times compared with their triazole counterparts. Ad-
ditionally, unlike the triazole analogues, the diamine
complexes exhibited concentration-dependent emis-
sion lifetimes, suggestive of the operation of a self-
quenching process in which the emission of the com-
plexes is quenched by the diaminoaromatic moiety.
The self-quenching rate constants (ksq) have been de-
termined to be on the order of 109 dm3 molꢁ1 sꢁ1
(Table 2), indicative of a very efficient quenching
pathway. Note that the emission lifetimes at infinite
dilution (ti.d.) (Table 2) are comparable to those of
Complex
Oxidation
Reduction
E1/2 or Ea [V]
E1/2 or Ec [V]
1a
2a
3a
1b
2b
3b
+0.42,[b] +1.15,[c] +1.76[c]
+0.42,[b] +1.11,[c] +1.69[c]
+0.40,[b] +1.09,[c] +1.72[c]
+1.76[c]
ꢁ1.17, ꢁ1.55, ꢁ1.75,[c] ꢁ2.10[b]
ꢁ1.41,[c] ꢁ1.63,[c] ꢁ1.83,[b] ꢁ2.06[b]
ꢁ1.16, ꢁ1.38,[b] ꢁ1.47,[c] ꢁ1.65,[c] ꢁ2.07[c]
ꢁ1.16, ꢁ1.45,[b] ꢁ1.77,[c] ꢁ2.11[b]
ꢁ1.39,[c] ꢁ1.62,[c] ꢁ1.82,[b] ꢁ2.07[b]
ꢁ1.14, ꢁ1.37,[b] ꢁ1.46,[c] ꢁ1.63,[c] ꢁ2.05[c]
+1.69[c]
+1.74[c]
[a] In CH3CN (0.1 moldmꢁ3 TBAP) at 298 K, glassy carbon electrode, sweep rate
100 mVsꢁ1, all potentials versus SCE. [b] Irreversible waves. [c] Quasi-reversible cou-
ples.
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complexes is favored by 0.82 to 0.88 eV. This reflects that the
emission quenching of the diamine complexes is an electron
transfer in nature. Interestingly, the reductive quenching pro-
cess from the oxidized ligand to the excited complexes was
also thermodynamically favorable (by 0.13 to 0.15 eV), which
further supports the very efficient emission quenching of the
complex by the diaminoaromatic moiety.
NO-Sensing properties
The NO-sensing ability of the diamine complexes has been ex-
amined by emission titration experiments using a NO-releasing
reagent 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 spontaneously releases two equivalents of NO
under physiological conditions (t1/2 =10 min, PBS buffer at
pH 7.4).[28] Upon addition of NOC-7 to a solution of the diamine
complex 1a, substantial emission enhancement (I/I0 =64) was
Figure 3. Absorption spectral traces of 1a (10 mm) in aerated potassium
phosphate buffer (50 mm, pH 7.4)/DMSO (99:1, v/v) at 298 K in the presence
of NOC-7 (0 to 40 mm).
&
*
Figure 4. pH titration curves for complexes 1a ( ) and 1b ( ) in aerated
100 mm KCl(aq)/DMSO (99:1, v/v) at 298 K.
Figure 2. Emission spectral traces of complex 1a (10 mm) in aerated potassi-
um phosphate buffer (50 mm, pH 7.4)/DMSO (99:1, v/v) at 298 K in the pres-
ence of NOC-7 (0 to 40 mm). The inset shows the emission titration curve of
complex 1a with NOC-7. The emission intensity was monitored at 550 nm.
Excitation wavelength=355 nm.
phorogenic sensors for NO in biological systems. The inde-
pendence of emission intensity of the diamine complex on pH
is most likely due to the fact that it is very difficult to proton-
ate both amines of the diaminoaromatic moiety (for example,
pKa1 and pKa2 of o-diaminobenzene are 4.6 and 0.8, respective-
ly).[29] Thus, electron transfer from the diaminoaromatic moiety
to the excited rhenium(I) complex still occurs very efficiently
even in an acidic medium. Similar observations have been re-
ported in other electron-rich diaminoaromatic compounds.[30]
Efficient in vivo NO sensors should have high reaction selec-
tivity toward NO over other reactive species such as reactive
oxygen species (ROS) and RNS that are present in biological
systems. Thus, the reaction selectivity of the diamine complex
observed (Figure 2). This gain in emission intensity has been
attributed to the conversion of the diamine complex into its
triazole counterpart, which has been subsequently confirmed
by ESI-MS (Figure S1, the Supporting Information). The emis-
sion intensity of the diamine complex showed good linearity
with [NOC-7] ranging from 0 to 15 mm (R2 =0.9867) with a de-
tection limit of ꢀ7.3 nm, which is considerably two orders of
magnitude lower than that of the previously reported rheni-
um(I) diaminoaromatic complex [Re(Ph2-phen)(CO)3(3,4-
diaminopyridine)](CF3SO3) (ꢀ300 nm).[15a]
A clear isosbestic
point (l=302 nm) was observed in the absorption spectral
traces of complex 1a in the presence of various amounts of
NOC-7 (Figure 3), which confirms that the complex was con-
verted into its triazole counterpart 1b as the sole product.
The effects of pH on the emission intensity of the diamine
complex 1a and the triazole analogue 1b have been investi-
gated, and the results are shown in Figure 4. The emission in-
tensity of both complexes was independent on pH between 2
and 11, and that complex 1a emitted with a much weaker
emission intensity than complex 1b in the same pH range,
which allows the diamine complexes to function as phos-
1a toward NO has been investigated using biologically rele-
ꢁ
vant species including HO , H2O2, O2ꢁ, OClꢁ, ONOOꢁ, NO2
,
C
and NO3ꢁ. Upon addition of these species to a solution of the
diamine complex, no significant emission changes were ob-
served in most cases. Although there was small emission en-
hancement upon treatment with ONOOꢁ (I/I0 =9), it was signif-
icantly smaller than that caused by NO (I/I0 =64; Figure 5).
These results suggest that the diamine complexes have high
reaction selectivity toward NO. It is important to point out that
modification of the complexes with different diimine ligands
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Table 4. Lipophilicity and cellular uptake of the rhenium(I) diamine com-
plexes 1a–3a.
Complex
Log
Po/w
No. of
mole [fmol][a]
1a
2a
3a
+0.80
+2.29
+3.91
0.35ꢂ0.02
2.81ꢂ0.07
11.91ꢂ0.29
[a] Numbers of moles of rhenium(I) associated with a typical HeLa cell
upon incubation at 378C for 1 h ([Re]=10 mm in the incubation medium).
assays, the viability of HeLa cells incubated with complex 3a
(10 mm) for 1 h was (92.6ꢂ5.6)%, indicating that the complex
did not impose substantial cytotoxicity toward the cells. De-
spite the high cellular-uptake efficiency (Table 4), no significant
intracellular emission was observed upon incubation of the
cells with the complex for 1 h. In contrast, after the cells were
further treated with NOC-7 for 1 h, the intracellular emission
intensity increased substantially (Figure 6). The microscopic
images revealed that the intracellular intensity of HeLa cells in-
creased steadily upon addition of NOC-7 and reached >3-fold
emission enhancement in 1 h (Figure 6). The absence of similar
changes in the control experiment (Figure 6) points out that
the observed emission enhancement is due to the intracellular
conversion of the diamine complex into its triazole derivative.
Interestingly, the emission enhancement was only observed in
the cytoplasmic region of the cells. In contrast, HeLa cells incu-
bated with the commercially available NO sensor 4-amino-5-
methylamino-2’,7’-difluorofluorescein-3’,6’-diacetate (DAF-FM
diacetate) showed non-specific staining and it was evenly dis-
tributed in the whole cell (Figure S6, the Supporting Informa-
tion). Thus, the intracellular localization properties of the dia-
mine complexes will enable them to monitor the changes of
intracellular NO levels at specific locations. Since the previously
reported rhenium(I) complexes [Re(N^N)(CO)3(3,4-diaminopyri-
dine)]+ are unable to react with intracellular NO,[15a] it becomes
apparent that rich electron density of the diaminoaromatic
ligand is crucial to the intracellular NO-sensing capability of
the complexes. The photostability of the triazole complex 3b
and the triazole form of DAF-FM diacetate, 2,7-difluoro-9-(4-
carboxy-1-methyl(benzotriazol-5-yl))xanthene-3,6-diacetate
(DAF-FM T) has been studied. The dependence of emission in-
tensity on the irradiation time and confocal microscopy images
of HeLa cells treated with complex 3b (10 mm, 5 min, lex =
405 nm, 25 mW) and DAF-FM T (10 mm, 5 min, lex =405 nm,
25 mW), respectively, are shown in Figures S7 and S8 (the Sup-
porting Information). After continuous exposure to 405 nm ir-
radiation (25 mW) for 150 seconds, the emission intensity of
complex 3b was maintained at 70% of its initial value, where-
as the fluorescence intensity of DAF-FM T decreased to only
30%. These results demonstrated the high photostability of
these rhenium(I) complexes, which renders them excellent can-
didates for time-lapse cellular imaging applications.
Figure 5. Selectivity of complex 1a (10 mm) to NO over other ROS and RNS
(200 mm) in aerated potassium phosphate buffer (50 mm, pH 7.4)/DMSO
(99:1, v/v) at 298 K. The emission intensity was monitored at 550 nm. Excita-
tion wavelength=355 nm.
did not affect their reaction sensitivity and selectivity (Figur-
es S2–S5, the Supporting Information). Thus, by using different
diimine ligands, these rhenium(I) complexes can function as se-
lective sensors for NO with multicolor emission properties.
We have investigated the possible effects of different biolog-
ical molecules including bovine serum albumin (BSA), human
serum albumin (HSA), and vesicles composed of 1,2-distearoyl-
sn-glycero-3-phosphocholine (DSPC) on the emission intensity
of complex 1a. Emission titration experiments revealed no sig-
nificant emission enhancement upon addition of the two
serum albumins to the diamine complex ([Re]=10 mm, [serum
albumin]=100 mm, I/I0 <1.1). Although we observed 4-fold
emission enhancement upon the incorporation of the complex
into DSPC vesicles and 1.4-fold enhancement when a solution
of the free complex was deaerated, these enhancement factors
were substantially lower than that caused by NO (I/I0 =64). In
other words, under all the conditions we have examined, the
emission intensity of the triazole complex 1b was significantly
higher than that of its diamine counterpart complex 1a, sup-
porting the argument that the diamine complexes in this work
are excellent phosphorogenic sensors for biological NO.
Cellular-uptake properties and live-cell confocal imaging
To investigate the possibility of using the diamine complexes
1a–3a as phosphorogenic sensors for intracellular NO, we
have first examined the cellular-uptake properties of the com-
plexes by ICP-MS. Upon incubation of HeLa cells with the com-
plexes ([Re]=10 mm) at 378C for 1 h, an average cell was
found to contain 0.35 to 11.91 fmol of rhenium (Table 4). The
cellular uptake of the complexes followed the order: 1a<
2a<3a, which is in accordance with the lipophilicity of the
complexes (Table 4). Similar results have also been observed in
related rhenium(I) complexes.[15a,21b,d] Since the Ph2-phen com-
plex 3a exhibited the highest cellular-uptake efficiency, its in-
tracellular imaging properties have been studied in more
detail.
After confirming that the diamine complex 3a can be used
to visualize NO generated exogenously by NOC-7, we have ap-
plied this complex to cultured murine macrophage RAW264.7
Complex 3a was first used to sense NO that was exogenous-
ly generated by NOC-7 in HeLa cells. As revealed by the MTT
Chem. Eur. J. 2014, 20, 9633 – 9642
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ꢀ5 mm)[32] and RAW264.7 cells
upon
stimulation
by
LPS
([NO]intracellular ꢀ2 mm).[31] Thus, our
complexes can detect intracellu-
lar NO at least in the micromolar
concentration. We believe that
the design of more sophisticated
systems, for example, dual emis-
sive complexes for ratiometric
sensing, would allow quantita-
tive measurements of NO con-
centrations in an intracellular
environment.
Conclusion
In this work, a series of rheni-
um(I) polypyridine complexes
functionalized with an electron-
rich diaminoaromatic moiety has
been developed as a new class
of phosphorogenic sensors for
NO. Owing to the efficient PET
from
the
diaminoaromatic
moiety to the excited rhenium(I)
complexes, all the diamine com-
plexes were very weakly emis-
sive. Upon treatment with NO,
the diamine complexes were
converted into the strongly
emissive triazole derivatives with
the emission intensity enhanced
by ꢀ60 fold. Also, the diamine
complexes exhibited high detec-
Figure 6. Brightfield and fluorescence images of HeLa cells incubated with complex 3a (10 mm) at 378C for 1 h.
The cells were post-incubated with a) 200 mm and b) 0 mm of NOC-7 in PBS at 378C for 1 h. c) The changes of
*
average fluorescence intensity of complex 3a-loaded HeLa cells (nꢃ10) upon addition of 200 mm ( ) and 0 mm
&
( ) of NOC-7 in PBS at 378C for 1 h.
cells, which are known to generate high levels of NO (in micro-
molar concentrations) upon stimulation by endotoxins or cyto-
kines.[31] Similar to the case of HeLa cells, treatment of
RAW264.7 cells with complex 3a gave only very weak emission
upon excitation. However, after stimulation of the cells by en-
dotoxin lipopolysaccharide (LPS) (1 mgmLꢁ1, 18 h), intense in-
tracellular emission was observed (Figure 7). The microscopic
images revealed that the RAW264.7 cells exhibited a 3.9-fold
increase of intracellular emission intensity (nꢃ10) upon stimu-
lation by LPS. It is important to note that the increase of emis-
sion intensity was suppressed by the addition of the NO syn-
thase inhibitor NG-nitro-l-arginine (LNNA) (Figure 7), indicating
that the augmentation of emission intensity was due to the in-
tracellular formation of NO. Thus, the NO generation process
can be readily traced by monitoring the emission intensity of
the cells.
tion sensitivity and reaction selectivity toward NO, and their
emission intensity was independent of the pH of the solution.
The cellular studies showed that the complexes can not only
detect NO produced exogenously in HeLa cells by NOC-7, but
also NO that is generated endogenously by murine macro-
phage RAW264.7 cells. The favorable phosphorogenic proper-
ties, pH-independent emission intensity, high detection sensi-
tivity, and reaction selectivity of these complexes render them
attractive reagents to visualize NO in living cells. The electron
density of the diaminoaromatic moiety of the current com-
plexes is significantly higher than that of the previous 3,4-dia-
minopyridine complexes[15a] due to the additional methoxy
substituent. As a result, greater enhanced reactivity and suc-
cessful intracellular sensing of NO have been accomplished.
Compared with their iridium(III) counterparts,[15b] the current
rhenium(I) complexes displayed higher emission enhancement
factors upon reaction with NO, which has been ascribed to the
efficient emission quenching of the rhenium(I) polypyridine
core by the diaminoaromatic moiety. Also, the emissive state
Although it is difficult to measure NO concentrations in
living cells because the emission intensity of the resulting tria-
zole complexes may be affected by different parameters such
as concentration of the complex and rigidity and polarity of
their local environment, the confocal microscopy study clearly
indicated that the emission of the complexes was turned on
by NO upon treatment of HeLa cells with NOC-7 ([NO]intracellular
3
of rhenium(I) polypyridine complexes is mainly MLCT in char-
acter, which allows tuning of emission energy using different
diimine ligands. The design of related phosphorescent transi-
tion-metal complexes as sensors for NO is underway.
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4-Methoxy-2-nitroaniline: A solu-
tion of 4-hydroxy-2-nitroaniline
(1.0 g, 6.5 mmol) in DMF (10 mL)
was cooled in an ice bath, and
sodium hydride (60% dispersion in
mineral oil, 260 mg, 6.5 mmol) was
added slowly. After the mixture
was stirred for 30 min, methyl
iodide
was
added
(82 mL,
6.5 mmol), and the suspension was
stirred at room temperature for
12 h. The mixture was evaporated
to dryness to afford a yellow solid,
which was purified by column
chromatography on silica gel. The
desired product was eluted with n-
hexane/ethyl acetate (4:1, v/v). The
solvent was removed under
vacuum to afford the product as
an orange solid. Yield: 970 mg
(89%). 1H NMR (300 MHz, CDCl3,
298 K): d=7.52 (s, 1H; H3), 7.06 (d,
1H, J=9.3 Hz; H5), 6.77 (d, 1H, J=
9.0 Hz; H6), 5.94 (br, 2H; NH2), 3.78
(s, 3H; CH3); MS (ESI, positive-ion
mode): m/z: 169 [M+H+]+.
Figure 7. Sensing of NO produced endogenously in RAW264.7 cells using complex 3a. Fluorescence images (top)
and brightfield images (bottom) of RAW264.7 cells upon incubation with complex 3a (10 mm) at 378C for 1 h
without pretreatment (left), pretreated with LPS (1 mgmLꢁ1, 18 h) (middle), and pretreated with both LPS
(1 mgmLꢁ1, 18 h) and LNNA (1 mm, 18 h) (right).
1-(tert-Butoxycarbonylamino)-4-
methoxy-2-nitrobenzene: 4-Me-
thoxy-2-nitro-aniline
(890 mg,
5.3 mmol) was added to a suspension of sodium hydride (60% dis-
persion in minerals oil, 1.5 g, 37 mmol) in THF (20 mL). After the
suspension was stirred for 10 min, a solution of di-tert-butyl car-
bonate (1.4 g, 6.4 mmol) in THF (20 mL) was added slowly, and the
mixture was stirred at room temperature for 24 h. Water (50 mL)
was added to quench the reaction, and the product was extracted
with ethyl acetate (50 mLꢁ3). The combined organic layer was
dried over MgSO4 and evaporated to dryness yielding a yellow
solid, which was purified by column chromatography on silica gel.
The desired product was eluted with n-hexane/ethyl acetate (6:1,
v/v). The solvent was removed under vacuum to afford the product
as a yellow solid. Yield: 1.3 g (89%); 1H NMR (300 MHz, CDCl3,
298 K): d=9.44 (s, 1H; NHtBoc), 8.44 (d, 1H, J=9.6 Hz; H6), 7.64 (d,
1H, J=3.0 Hz; H3), 7.21 (dd, 1H, J=9.3, 3.0 Hz; H5), 3.85 (s, 3H;
CH3), 1.54 (s, 9H; CH3 of tBoc); MS (ESI, positive-ion mode): m/z:
291 [M+Na+]+.
Experimental Section
Materials and synthesis
All solvents were of analytical reagent grade and purified accord-
ing to standard procedures.[33] Di-tert-butyl carbonate, 4-hydroxy-2-
nitroaniline, sodium hydride, 3-pyridinecarboxaldehyde, NaBH4,
Ph2-phen, phen, Me4-phen, silver(I) trifluoromethanesulfonate,
[Re2(CO)10], trifluoroacetic acid, KPF6, sodium nitrite, sodium nitrate,
sodium hypochlorite, potassium superoxide, H2O2, [(NH4)2Fe-
(SO4)2]·6H2O, 4,4’-dimethyl-2,2’-bipyridine, selenium oxide, 2-
amino-4-bromoaniline, o-diaminobenzene, and copper iodide were
purchased from Acros. Sodium peroxynitrite, methyl iodide, HSA,
BSA, and NOC-7 were obtained from Calbiochem. Ethynyltrimethyl-
silane, tetrakis(triphenylphosphine)palladium(0), tetra-n-butylam-
monium fluoride, and 5% palladium on carbon were purchased
from Aldrich. Purified NO gas was obtained from Scientific Gas En-
gineering Co., Ltd. MTT and DSPC were obtained from Sigma. All
these chemicals were used without further purification. [Re-
(N^N)(CO)3(CH3CN)][CF3SO3] was prepared as described previous-
ly.[34] The ligands 4-formyl-4’-methyl-2,2-bipyridine[35] and 3-ethynyl-
pyridine[36] were prepared according to reported procedures. All
buffer components were of biological grade and used as received.
Autoclaved Milli-Q water was used for the preparation of aqueous
solutions. HeLa and RAW264.7 cells were obtained from American
Type Culture Collection. Dulbecco’s modified Eagle’s medium
(DMEM), fetal bovine serum (FBS), phosphate buffered saline (PBS),
trypsin/ethylenediaminetetraacetic acid (EDTA), penicillin/strepto-
mycin, and DAF-FM diacetate were purchased from Invitrogen. The
growth medium for cell culture contained DMEM with 10% FBS
and 1% penicillin/streptomycin. The triazole compound DAF-FM T
was prepared by bubbling purified NO gas into a solution of DAF-
FM diacetate in PBS. The synthesis and characterization data of
complexes S1–S3 are included in the Supporting Information.
2-(tert-Butoxycarbonylamino)-5-methoxyaniline: A mixture of 5%
palladium on carbon (130 mg) and 1-(tert-butoxycarbonylamino)-4-
methoxy-2-nitrobenzene (1.3 g, 4.9 mmol) in MeOH (30 mL) was
stirred under a hydrogen atmosphere at room temperature for
12 h. The solid residue was removed by filtration, and the filtrate
was evaporated to dryness yielding a pale-brown solid, which was
purified by column chromatography on silica gel. The desired
product was eluted with n-hexane/ethyl acetate (2:1, v/v). The sol-
vent was removed under vacuum to afford the product as a pale-
grey solid. Yield: 900 mg (78%). 1H NMR (300 MHz, CDCl3, 298 K):
d=7.05 (d, 1H, J=8.4 Hz; H3), 6.36–6.33 (m, 2H; H4 and H6), 6.01
(br, 1H; NHtBoc), 3.98–3.74 (m, 5H; NH2 and OCH3), 1.50 ppm (s,
9H; CH3 of tBoc); MS (ESI, positive-ion mode): m/z: 239 [M+H+]+.
3-(N-(2-(tert-Butoxycarbonylamino)-5-methoxyphen-1-yl)amino-
methyl)pyridine (py-DA-tBoc): A mixture of 2-(tert-butoxycarbony-
lamino)-5-methoxyaniline (533 mg, 2.2 mmol), 3-pyridinecarboxal-
dehyde (210 mL, 2.2 mmol), and acetic acid (133 mL, 4.5 mmol) in
MeOH (10 mL) was heated at reflux under an inert atmosphere of
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nitrogen for 12 h. After the solution was cooled to room tempera-
ture, NaBH4 (424 mg, 11.2 mmol) was added slowly, and the mix-
ture was stirred at room temperature for 12 h. Water (10 mL) was
added to quench the reaction, and the product was extracted with
CH2Cl2 (30 mLꢁ3). The combined organic layer was dried over
MgSO4 and evaporated to dryness yielding a yellow solid, which
was purified by column chromatography on silica gel. The desired
product was eluted with CH2Cl2/MeOH (50:1, v/v). The solvent was
removed under vacuum to afford the product as a white solid.
(s, 2H; CH2NH), 3.33 ppm (s, 3H; OCH3); IR (KBr) n˜ =3421 (br, NꢁH),
2027 (s, CꢄO), 1919 (s, CꢄO), 841 cmꢁ1 (s, PF6ꢁ); MS (ESI, positive-
ion mode): m/z: 832 [MꢁPF6ꢁ]+; elemental analysis calcd (%) for
ReC40H31N5O4PF6: C 49.19, H 3.20, N 7.17; found: C 49.21, H 3.47, N
6.99.
[Re(N^N)(CO)3(py-triazole)][PF6] (N^N=phen (1b), Me4-phen
(2b), Ph2-phen (3b)): Purified NO was bubbled slowly into a solu-
tion of [Re(N^N)(CO)3(py-DA)][PF6] (0.04 mmol) in CH2Cl2 (10 mL)
for 10 min. The solution was evaporated to dryness to afford
a yellow solid. Recrystallization of the product from CH2Cl2/diethyl
ether afforded the complex as yellow crystals. Complex 1b: Yield:
24 mg (72%); 1H NMR (300 MHz, CD3OD, 298 K): d=9.50 (d, 2H,
J=4.8 Hz; H2 and H9 of phen), 8.81 (d, 2H, J=8.4 Hz; H4 and H7
of phen), 8.58 (d, 1H, J=5.1 Hz; H6 of pyridine), 8.13 (s, 2H; H5
and H6 of phen), 8.02 (t, 2H, J=5.4 Hz; H3 and H8 of phen), 7.96–
7.93 (m, 2H; H4 of pyridine and H4 of benzotriazole), 7.96 (s, 1H;
H2 of pyridine), 7.36 (t, 1H, J=6.0 Hz; H5 of pyridine), 7.13 (d, 1H,
J=8.7 Hz; H5 of benzotriazole), 6.72 (s, 1H; H7 of benzotriazole),
˜
1
Yield: 469 mg (64%). H NMR (300 MHz, CDCl3, 298 K): d=8.62 (s,
1H; H2 of pyridine), 8.51 (d, 1H, J=3.9 Hz; H6 of pyridine), 7.70 (d,
1H, J=7.8 Hz; H4 of pyridine), 7.26 (t, 1H, J=6.9 Hz; H5 of pyri-
dine), 7.07 (d, 1H, J=8.4 Hz; H3 of phenyl ring), 6.26 (d, 1H, J=
8.1 Hz; H4 of phenyl ring), 5.98 (br, 1H; CH2NH), 4.54 (br, 1H;
NHtBoc), 4.34 (s, 2H; CH2NH), 3.71 (s, 3H; OCH3), 1.49 (s, 9H; CH3
of tBoc); MS (ESI, positive-ion mode): m/z: 330 [M+H+]+.
[Re(N^N)(CO)3(py-DA)][PF6] (N^N=phen (1a), Me4-phen (2a),
Ph2-phen (3a)):
A mixture of [Re(N^N)(CO)3(CH3CN)][CF3SO3]
5.74 (s, 2H; CH NH), 3.73 (s, 3H; OCH ); IR (KBr): n=2035 (s, CꢄO),
2
3
1910 (s, CꢄO), 842 cmꢁ1 (s, PF6ꢁ); MS (ESI, positive-ion mode): m/z:
691 [MꢁPF6ꢁ]+; elemental analysis calcd (%) for ReC28H20N6O4PF6: C
40.25, H 2.41, N 10.06; found: C 40.05, H 2.63, N 9.88. Complex 2b:
(0.21 mmol) and py-DA-tBoc (0.21 mmol) in THF (30 mL) was
heated at reflux under an inert atmosphere of nitrogen for 12 h.
The mixture was evaporated to dryness to afford a yellow solid,
which was purified by column chromatography on silica gel. The
desired product was eluted with a mixture of CH2Cl2 and MeOH.
The solvent was removed by evaporation to afford a yellow solid,
which was then redissolved in CH2Cl2 (10 mL). Trifluoroacetic acid
(1 mL) was added to the solution and the mixture was stirred at
room temperature under an inert atmosphere of nitrogen for 3 h.
The solution was then evaporated to dryness to afford a brown
solid. The complex was converted into the hexafluorophosphate
salt by anion exchange with KPF6 in MeOH. Recrystallization of the
product from CH2Cl2/diethyl ether afforded the complex as brown
crystals. Complex 1a: Yield: 90 mg (52%); 1H NMR (400 MHz,
CD3OD, 298 K): d=9.58 (dd, 2H, J=5.2, 1.2 Hz; H2 and H9 of
phen), 8.69 (dd, 2H, J=8.0, 1.2 Hz; H4 and H7 of phen), 8.57 (d,
1H, J=5.6 Hz; H6 of pyridine), 8.08 (s, 2H; H5 and H6 of phen),
7.96–7.92 (m, 3H; H2 of pyridine and H3 and H8 of phen), 7.81 (d,
1H, J=8.0 Hz; H4 of pyridine), 7.30 (t, 1H, J=5.6 Hz; H5 of pyri-
dine), 6.77 (d, 1H, J=8.4 Hz; H3 of phenyl ring), 6.21 (d, 1H, J=
8.4 Hz; H4 of phenyl ring), 4.76 (s, 1H; H6 of phenyl ring), 4.19 (s,
2H; CH2NH), 3.32 ppm (s, 3H; OCH3); IR (KBr): n˜ =3423 (br, NꢁH),
2031 (s, CꢄO), 1919 (s, CꢄO), 846 cmꢁ1 (s, PF6ꢁ); MS (ESI, positive-
ion mode): m/z: 680 [MꢁPF6ꢁ]+; elemental analysis calcd (%) for
ReC28H23N5O4PF6·0.5Et2O: C 41.82, H 3.28, N 8.13; found: C 42.18, H
1
Yield: 20 mg (56%); H NMR (300 MHz, CD3OD, 298 K): d=9.22 (s,
2H; H2 and H9 of Me4-phen), 8.65 (d, 2H, J=6.0 Hz; H6 of pyri-
dine), 8.27 (s, 2H; H5 and H6 of Me4-phen), 7.97 (d, 1H, J=8.7 Hz;
H4 of pyridine), 7.86 (d, 1H, J=9.0 Hz; H4 of benzotriazole), 7.72 (s,
1H; H2 of pyridine), 7.37 (t, 1H, J=5.7 Hz; H5 of pyridine), 7.04 (d,
1H, J=9.0 Hz; H5 of benzotriazole), 6.69 (s, 1H; H7 of benzotria-
zole), 5.69 (s, 2H; CH2NH), 3.70 (s, 3H; OCH3), 2.86 (s, 6H; CH3 at C4
and C7 of Me4-phen), 2.67 ppm (s, 6H; CH3 at C3 and C8 of Me4-
phen); IR (KBr): n˜ =2031 (s, CꢄO), 1930 (s, CꢄO), 846 cmꢁ1: (s, PF6ꢁ);
MS (ESI, positive-ion mode): m/z: 747 [MꢁPF6ꢁ]+; elemental analy-
sis calcd (%) for ReC32H28N6O4PF6·H2O: C 42.21, H 3.32, N 9.24;
found: C 42.21, H 3.05, N 9.47. Complex 3b: Yield: 31 mg (78%);
1H NMR (300 MHz, CD3OD, 298 K): d=9.54 (d, 2H, J=5.4 Hz; H2
and H9 of Ph2-phen), 8.63 (d, 1H, J=5.4 Hz; H6 of pyridine), 8.14
(s, 2H; H5 and H6 of Ph2-phen), 8.02–8.00 (m, 4H; H2 of pyridine,
H4 of benzotriazole, and H3 and H8 of Ph2-phen), 7.71–7.66 (m,
11H; H4 of pyridine and C6H5 of Ph2-phen), 7.42 (t, 1H, J=5.7 Hz;
H5 of pyridine), 6.99 (s, 1H; H7 of benzotriazole), 6.94 (d, 1H, J=
9.0 Hz; H5 of benzotriazole), 5.80 (s, 2H; CH2NH), 3.78 ppm (s, 3H;
OCH ); IR (KBr): n=2027 (s, CꢄO), 1919 (s, CꢄO), 842 cmꢁ1 (s, PF6ꢁ);
˜
3
MS (ESI, positive-ion mode): m/z: 843 [MꢁPF6ꢁ]+; elemental analy-
sis calcd (%) for ReC32H31N5O4PF6·0.5H2O: C 48.20, H 2.93, N 8.43;
found: C 48.31, H 3.01, N 8.65.
1
3.13, N 8.28. Complex 2a: Yield: 135 mg (73%); H NMR (400 MHz,
CD3OD, 298 K): d=9.27 (s, 2H; H2 and H9 of Me4-phen), 8.71 (d,
2H, J=5.6 Hz; H6 of pyridine), 8.28 (s, 2H; H5 and H6 of Me4-
phen), 7.91 (s, 1H; H2 of pyridine), 7.82 (d, 1H, J=8.0 Hz; H4 of
pyridine), 7.35 (t, 1H, J=5.6 Hz; H5 of pyridine), 6.65 (d, 1H, J=
8.4 Hz; H3 of phenyl ring), 6.10 (d, 1H, J=8.4 Hz; H4 of phenyl
ring), 4.88 (s, 1H; H6 of phenyl ring), 4.17 (s, 2H; CH2NH), 3.23 (s,
3H; OCH3), 2.82 (s, 6H; CH3 at C3 and C8 of Me4-phen), 2.64 (s, 6H;
CH3 at C4 and C7 of Me4-phen); IR (KBr): n˜ =: 3422 (br, NꢁH), 2029
(s, CꢄO), 1912 (s, CꢄO), 845 cmꢁ1 (s, PF6ꢁ); MS (ESI, positive-ion
mode): m/z: 736 [MꢁPF6ꢁ]+; elemental analysis calcd (%) for Re-
C32H31N5O4PF6: C 43.65, H 3.55, N 7.95; found: C 43.70, H 3.78, N
Instrumentation and methods
1H NMR spectra were recorded on a Varian Mercury 300 MHz NMR
spectrometer or a Bruker 400 MHz NMR spectrometer at 298 K.
Positive-ion ESI mass spectra were recorded on a PerkinElmer Sciex
API 365 mass spectrometer. IR spectra were recorded on a Perkin-
Elmer 1600 series FTIR spectrophotometer. Elemental analyses
were carried out on a Vario EL III CHN elemental analyzer. Electron-
ic absorption and steady-state emission spectra were recorded on
a
Hewlett–Packard 8453 diode array spectrophotometer and
1
7.66. Complex 3a: Yield: 122 mg (59%); H NMR (400 MHz, CD3OD,
a SPEX FluoroLog 3-TCSPC spectrophotometer equipped with a Ha-
mamatsu R928 PMT detector, respectively. Emission lifetimes were
measured in the Fast MCS or the MCS lifetime mode with
a NanoLED N-375 as the excitation source. All the solutions for
photophysical studies were degassed with at least four successive
freeze-pump-thaw cycles and stored in a 10 cm3 round-bottomed
flask equipped with a sidearm 1 cm fluorescence cuvette and
sealed from the atmosphere by a Rotaflo HP6/6 quick-release
298 K): d=9.62 (d, 2H, J=5.2 Hz; H2 and H9 of Ph2-phen), 8.51 (d,
1H, J=5.2 Hz; H6 of pyridine), 8.42 (s, 1H; H2 of pyridine), 8.17 (s,
2H; H5 and H6 of Ph2-phen), 8.04 (d, 2H, J=5.6 Hz; H3 and H8 of
Ph2-phen), 7.87 (d, 1H, J=8.4 Hz; H4 of pyridine), 7.71–7.64 (m,
10H; C6H5 at C4 and C7 of Ph2-phen), 7.35 (t, 1H, J=7.6 Hz; H5 of
pyridine), 6.60 (d, 1H, J=8.4 Hz; H3 of phenyl ring), 5.98 (d, 1H,
J=8.0 Hz; H4 of phenyl ring), 5.51 (s, 1H; H6 of phenyl ring), 4.30
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Teflon stopper. Luminescence quantum yields were measured by
the optically dilute method[37] using a degassed CH3CN solution of
[Re(phen)(CO)3(pyridine)](CF3SO3) (Fem =0.18, lex =355 nm) as the
standard solution.[38] Cyclic voltammetric measurements were car-
ried out using a CH Instruments Electrochemical Workstation CHI
750 A. Details on MTT assays, ICP-MS, and the preparation of DSPC
vesicles have been reported previously.[21a,39] The lipophilicity of the
complexes was determined from the log k’w values (k’w =chroma-
tographic capacity factor at 100% aqueous solution). The log k’w
values were determined by reversed-phase HPLC on a C-18
column according to the method described by Minick.[40]
(10 mm). After incubation for 1 h, the medium was removed and
the cell layer was washed gently with PBS (1 mL ꢁ 3). The coverslip
was mounted onto a sterilized glass slide and then imaged using
a Leica TCS SPE confocal microscope. In the NO imaging experi-
ments, the cells were treated with complex 3a and incubated with
NOC-7 (200 mm) in PBS for 1 h, and finally washed with PBS (1 mLꢁ
3).
NO Imaging in stimulated RAW264.7 cells
RAW264.7 cells in growth medium were seeded on a sterilized cov-
erslip in a 60 mm tissue culture dish and grown at 378C under
a 5% CO2 atmosphere for 24 h. The medium was then removed
and replaced with medium containing LPS (1 mgmLꢁ1) to induce
the production of NO. After incubation for 18 h, the medium was
removed and the cell layer was washed gently with PBS (1 mLꢁ3).
The stimulated RAW264.7 cells were then treated with complex 3a
(10 mm) in medium/DMSO (99:1, v/v) for 1 h and finally washed
with PBS (1 mLꢁ3). The coverslip was mounted onto a sterilized
glass slide and then imaged using a Leica TCS SPE confocal micro-
scope. The control experiments were performed using a similar
procedure except that medium containing LPS (1 mgmLꢁ1) was re-
placed with 1) medium only and 2) medium containing LPS
(1 mgmLꢁ1) and LNNA (1 mm).
Emission-quenching studies
The self-quenching rate constants (ksq) and emission lifetimes at in-
finite dilution (ti.d.) of the diamine complexes were determined
using the equation 1/t=1/ti.d. +ksq[Re], in which t is the emission
lifetime of the complex at concentration [Re] in degassed solutions.
The slope and y-intercept of the linear fit of a plot of 1/t versus
[Re] gave ksq and 1/ti.d., respectively.
pH-Dependent emission studies
A stock solution (20 mL) of complexes 1a and 1b (50 mm), respec-
tively, in a mixture of 10 mm HCl and 100 mm KCl(aq)/DMSO (99:1,
v/v) was prepared. The pH of the stock solution was increased
gradually by addition of an appropriate amount of 1, 0.5 0.25, 0.13,
0.07, 0.04, or 0.02 n KOH (aq). After a desired pH value was ob-
tained, 2 mL of the stock solution was transferred to a quartz cuv-
ette and the emission spectrum was measured under aerated con-
ditions. The solution was then returned to the stock, and the pH
was further adjusted. The overall increase in the volume of the
stock solution due to pH adjustment was kept below 2% (i.e.,
400 mL).
Acknowledgements
We thank the City University of Hong Kong (Project No.
7004020) and the Hong Kong Research Grants Councils (Proj-
ect No. CityU 102311) for financial support. A.W.-T.C. acknowl-
edges the receipt of a Postgraduate Studentship, a Research
Tuition Scholarship, and a Student Research Excellence Award
administered by City University of Hong Kong.
NO Titration and selectivity assays
A series of solutions containing different concentrations of NOC-7
(0–40 mm) and the diamine complexes (10 mm) in potassium phos-
phate buffer (50 mm, pH 7.4)/DMSO (99:1, v/v) were prepared. The
solutions were gently stirred at room temperature for 1 h, and the
absorption and emission spectra of each solution were then mea-
sured under aerated conditions. In the selectivity assays, different
ROS and RNS (200 mm) were added into a solution of the diamine
complex (10 mm) in potassium phosphate buffer (50 mm, pH 7.4)/
DMSO (99:1, v/v). The solutions were gently stirred at room tem-
perature for 1 h and the emission spectrum of each solution was
then measured under aerated conditions. Sodium nitrite, sodium
nitrate, sodium peroxynitrite, sodium hypochlorite, and potassium
Keywords: imaging agents · luminescence · nitric oxide ·
rhenium · sensors
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Received: March 6, 2014
Published online on July 1, 2014
Chem. Eur. J. 2014, 20, 9633 – 9642
www.chemeurj.org
9642
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim