Coordination geometry-induced optical imaging of L-cysteine in cancer cells using imidazopyridine-based copper(ii) complexes

Article abstract of DOI:10.1039/c8dt04634d

Overexpression of cysteine cathepsins proteases has been documented in a wide variety of cancers, and enhances the l-cysteine concentration in tumor cells. We report the synthesis and characterization of copper(ii) complexes [Cu(L1)₂(H₂

Full text of DOI:10.1039/c8dt04634d

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Coordination geometry-induced optical imaging
of L-cysteine in cancer cells using
Cite this: DOI: 10.1039/c8dt04634d
imidazopyridine-based copper(II) complexes†
Selvarasu Priyanga, a Themmila Khamrang,b Marappan Velusamy,b
Sellamuthu Karthi,c Balasubramaniem Ashokkumar c and
a
Ramasamy Mayilmurugan
*
Overexpression of cysteine cathepsins proteases has been documented in a wide variety of cancers, and
enhances the L-cysteine concentration in tumor cells. We report the synthesis and characterization of
copper(II) complexes [Cu(L1)2(H2O)](SO3CF3)2, 1, L1 = 3-phenyl-1-(pyridin-2-yl)imidazo[1,5-a]pyridine,
[Cu(L2)2(SO3CF3)]SO3CF3, 2, L2 = 3-(4-methoxyphenyl)-1-pyridin-2-yl-imidazo[1,5-a]pyridine, [Cu(L3)2(H2O)]
(SO3CF3)2, 3, L3
= 3-(3,4-dimethoxy-phenyl)-1-pyridin-2-yl-imidazo[1,5-a]pyridine and [Cu(L4)2(H2O)]
(SO3CF3)2, 4, L4 = dimethyl-[4-(1-pyridin-2-yl-imidazo[1,5-a]pyridin-3-yl)phenyl]amine as ‘turn-on’ optical
imaging probes for L-cysteine in cancer cells. The molecular structure of complexes adopted distorted tri-
gonal pyramidal geometry (τ, 0.68–0.87). Cu–Npy bonds (1.964–1.989 Å) were shorter than Cu–Nimi bonds
(2.024–2.074 Å) for all complexes. Geometrical distortion was strongly revealed in EPR spectra, showing g
k
(2.26–2.28) and A values (139–163 × 10−4 cm−1) at 70 K. The d–d transitions appeared around 680–741
k
and 882–932 nm in HEPES, which supported the existence of five-coordinate geometry in solution. The
Cu(II)/Cu(I) redox potential of 1 (0.221 V vs. NHE) was almost identical to that of 2 and 3 but lower than that
of 4 (0.525 V vs. NHE) in HEPES buffer. The complexes were almost non-emissive in nature, but became
emissive by the interaction of L-cysteine in 100% HEPES at pH 7.34 via reduction of Cu(II) to Cu(I). Among
the probes, probe 2 showed selective and efficient turn-on fluorescence behavior towards L-cysteine over
natural amino acids with a limit of detection of 9.9 × 10−8 M and binding constant of 2.3 × 105 M−1. The
selectivity of 2 may have originated from a nearly perfect trigonal plane adopted around a copper(II) center
(∼120.70°), which required minimum structural change during the reduction of Cu(II) to Cu(I) while imaging
Cys. The other complexes, with their distorted trigonal planes, required more reorganizational energy,
which resulted in poor selectivity. Probe 2 was employed for optical imaging of L-cysteine in HeLa cells and
macrophages. It exhibited brighter fluorescent images by visualizing Cys at pH 7.34 and 37 °C. It showed
relatively less toxicity for these cell lines as ascertained by the MTT assay.
Received 22nd November 2018,
Accepted 21st December 2018
DOI: 10.1039/c8dt04634d
rsc.li/dalton
particular, Cys has a pivotal role as an intracellular redox
buffer that influences detoxification and critical metabolic
Introduction
Biomolecules containing a thiol group such as L-cysteine (Cys), functions; it is also a potential neurotoxin, a biomarker for
homocysteine (Hcy) and glutathione (GSH) have crucial roles various medical diagnoses and active-site protein in several
in cellular function, including proliferation, antioxidant metalloenzymes in body fluids.2–5 Thus, a disproportionate
defense, signaling and maintaining redox homeostasis.1 In level of Cys causes various adverse effects in human
metabolism.6–11 Specifically, protease enzymes known as
“cysteine cathepsins” are located in the intracellular region,
aBioinorganic Chemistry Laboratory/Physical Chemistry, School of Chemistry,
Madurai Kamaraj University, Madurai, 625021, India.
and are involved in protein degradation and processing in
normal cell formation.12 In tumor-cell formation, they are
translocated to the cell surface and facilitate degradation of
the extracellular matrix (ECM), which produces an abnormal
amount of Cys by breaking disulfur linkages. The over-
expression of cysteine cathepsins has been reported in a wide
variety of cancers.13 Hence, it is essential to study the pathway
E-mail: mayilmurugan.chem@mkuniversity.org
bDepartment of Chemistry, North-Eastern Hill University, Shillong, 793022, India
cSchool of Biotechnology, Madurai Kamaraj University, Madurai, 625 021, India
†Electronic supplementary information (ESI) available. CCDC 1833674–1833677.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c8dt04634d
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and activity of Cys to understand the key biological roles of oxyphenyl)-1-pyridin-2-yl-imidazo[1,5-a]pyridine (L2), 3-(3,4-
cysteine cathepsins in the formation and proliferation of dimethoxy-phenyl)-1-pyridin-2-yl-imidazo[1,5-a]pyridine (L3)
tumor cells. In addition, detection and imaging of thiol-con- and
taining molecules in cancer biology is very important to phenyl]amine (L4) were synthesized according to a method-
ensure early diagnosis and treatment of disease. ology published previously with slight modifications
dimethyl-[4-(1-pyridin-2-yl-imidazo[1,5-a]pyridin-3-yl)
Some scholars have detected thiol-containing biomolecules (Scheme 1).28 The reaction of substituted benzaldehyde with
that mostly utilize biologically unviable metals such as Ir, Cd, di-pyridin-2-yl-methanone in the presence of NH4OAc and
Hg and Ru.14,15 Boron dipyrromethane (BODIPY), cyanine and acetic acid yielded the respective ligands, which were charac-
coumarin fluorescent units have been reported as chemodosi- terized by nuclear magnetic resonance (NMR) spectroscopy.
metric sensors for the detection of thiol-containing compounds Copper(II) complexes were isolated as green solids by the reac-
(Cys, Hcy, GSH) without selectivity or with poor selectivity.16–22 tion of one equivalent of Cu(SO3CF3)2 with two equivalents of
Recently, Cu(II)-based probes have received much attention as the corresponding ligands in acetonitrile at room temperature.
‘optical probes’ for imaging smaller metabolites and other bio- The formation of the complexes was confirmed by high-resolu-
chemical reactions produced during the formation and prolifer- tion-mass spectrometry (HR-MS) (Fig. S1–S4†) and they were
ation of tumor cells.24 As well explored in the literature, it is formulated as [Cu(L1)2(H2O)](SO3CF3)2 1, [Cu(L2)2(SO3CF3)]
challenging to generate CuII–thiolate bonds due to the thermo- SO3CF3 2, [Cu(L3)2(H2O)](SO3CF3)2
3 and [Cu(L4)2(H2O)]
dynamically favorable redox reaction (2RS− + 2CuII → RS-SR + (SO3CF3)2 4. The formulations were further supported by
2CuI).25 In general, copper(II) is reduced by thiols and sub- single-crystal X-ray structures. The suitable single crystals for
sequently coordinated to cuprous ions for generation of Cu(I)– X-ray diffraction were grown in acetonitrile by slow evaporation
thiol complexes.26 Only a few copper(II) complexes containing and diffusion methods.
anthracene, coumarin, fluorescein, and zwitterionic bipyridine
derivatives have been reported for the detection of thiols (Cys,
Molecular structure of copper(II) complexes
Hcy, GSH) and histidine with poor selectivity, which were
The single-crystal X-ray structures of 1–4 are shown in Fig. 1
and their selected bond lengths, bond angles are summarized
in Table 1. The complexes 1–3 were crystallized in a triclinic
studied without any chemical-biology perspective.23
Very recently, we developed copper(II)-probes based on terpyr-
idine and pyridine-bis-benzimidazole ligands for imaging of pre-
ˉ
system with a space group of P1, but the complex 4 was crystal-
incubated Cys in cancer cells, which showed excellent selectivity
lized in a monoclinic system with a space group of C2/c. The
and limit of detection.11,27 The Cys-probing mechanism is pro-
molecular structure of 1–4 exhibited a distorted trigonal–bipyr-
posed to operate via reduction of copper(II) centers into copper(I)
amidal coordination geometry as predicted by the Addison
followed by displacement, which leads to regeneration of the
structural index τ, 0.68–0.87 [τ = (β − α)/60; for perfect square
pyramidal and trigonal–pyramidal geometries, τ = 0 and 1,
fluorescent intensity of the original ligand at brighter visible-
light emission regions. The preferable tetrahedral coordination
respectively].29 The distorted trigonal bipyramidal geometry of
geometry of copper(I) cannot be adopted by rigid terpyridine
copper(II) centers were constituted by the coordination of four
and pyridine-bis-benzimidazole ligand systems.11,27
nitrogens of the ligand units and one oxygen of water or tri-
In this report, we designed and synthesized new copper(II)
flate anion molecules. The complex 2 exhibited a higher τ
value, 0.87, than that of 3 (τ, 0.75), which was lowered further
complexes of bidentate imidazopyridine-based ligands and
characterized them as optical probes for imaging of L-cysteine
to 0.68 for 1 and 4. The electron-releasing p-methoxy substitu-
via a ‘turn on’ fluorescence mechanism. These ligands are
ent on the phenyl rings of 2 enabled exhibition of a higher τ
value and adoption of more trigonal–bipyramidal coordination
geometry (or less square pyramidal). In fact, N2, N5 and O1
donors constituted nearly a perfect trigonal plane around
expected to provide the required geometrical plasticity for the
coordination of Cu(II) and Cu(I) ions. The ligand architecture
was modified by substituting methoxy and –NMe2 groups, and
we expected to tune the structural properties and imaging
copper(II) centers, as evidenced by bond angles of 120.70° for
mechanism. The p-substituted methoxy probe exhibited selec-
N2–Cu–O1; 119.21° for N2–Cu–N5, and 120.06° for N5–Cu–O1.
tive and efficient turn-on fluorescence behavior on interaction
These bond angles deviated completely from 120° for 3 (111.3,
with Cys, and imaged Cys in cancer cells via reduction of
copper(II) to copper(I) without ligand displacement. The
probing mechanism was established by electronic spectral,
redox and density functional theory (DFT) studies and live cell
imaging by laser scanning fluorescence microscopy.
Results and discussion
Synthesis and characterization
The
phenyl-substituted
imidazopyridine-based
ligands
3-phenyl-1-pyridin-2-yl-imidazo[1,5-a]pyridine (L1), 3-(4-meth- Scheme 1 Structure of ligands L1–L4.
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Fig. 1 Molecular structure of [Cu(L1)2(H2O)](SO3CF3)2 1 (A), [Cu(L2)2(SO3CF3)]-(SO3CF3) 2 (B), [Cu(L3)2(H2O)](SO3CF3)2 3 (C) and [Cu(L4)2(H2O)]
(SO3CF3)2 4 (D). Thermal ellipsoids are drawn at the 40% probability level. Hydrogen atoms and SO3CF3− ions have been omitted for clarity.
Table 1 Selected bond lengthsa [Å] and bond anglesa [°] for 1–4
129.6; N5–Cu–O1, 104.70°) and exhibited less trigonal–bipyra-
midal coordination geometry than that of 2 and 3. These data
1
2
3
4
clearly indicated that p-methoxy substituents on phenyl rings
enforced a nearly perfect trigonal plane and that additional
m-methoxy groups offered steric influence to deviate from the
trigonal plane around copper(II) centers. The bond angles N1–
Cu1–N4 (170.44–175.28°) of 1–4 deviated from 180°, indicating
a distortion in the axial coordination. The Cu–Owater bond dis-
tance of 1 (2.093 Å) was slightly shorter than those of 3
(2.196 Å), 4 (2.161 Å) and the Cu–Otriflate bond of 2 (2.174 Å.
The Cu–Npy bonds (1.964–1.989 Å) of the complexes were
shorter than the Cu–Nimi bonds (2.024–2.074 Å). The strong
overlapping of the p-orbital of pyridine nitrogen and d-orbital
of the Cu(II) center led to shorter Cu–N bonds than those of
imidazole nitrogen atoms. However, these Cu–N bond dis-
tances were longer than those of our previously reported
square pyramidal complexes of terpyridine (1.970–2.069 Å)11
and pyridine-bis-benzimidazole (1.958–2.000 Å) ligands.27
τ-Value
0.68
0.87
1.964(3)
1.980(3)
2.050(3)
2.048(3)
2.174(3)
173.11(13)
81.12(13)
103.10(12)
101.51(12)
81.31(12)
119.21(12)
85.72(12)
87.43(12)
120.70(12)
120.06(12)
0.75
0.68
Cu(1)–N(1)
Cu(1)–N(4)
Cu(1)–N(2)
Cu(1)–N(5)
Cu(1)–O(1)
N(1)–Cu(1)–N(4) 175.28(12)
N(1)–Cu(1)–N(2) 81.15(11)
N(4)–Cu(1)–N(2) 100.88(11)
N(1)–Cu(1)–N(5) 101.61(11)
N(4)–Cu(1)–N(5) 81.30(10)
N(2)–Cu(1)–N(5) 117.67(11)
N(1)–Cu(1)–O(1) 87.49(11)
N(4)–Cu(1)–O(1) 88.06(11)
N(2)–Cu(1)–O(1) 133.95(11)
N(5)–Cu(1)–O(1) 108.30(11)
1.978(3)
1.985(3)
2.053(3)
2.074(3)
2.093(3)
1.989(4)
1.980(4)
2.043(4)
2.049(4)
2.196(4)
173.58(17) 170.43(13)
81.65(16)
99.94(16)
103.09(16) 102.98(13)
80.90(17) 81.76(13)
128.12(16) 129.61(13)
86.86(17)
86.77(17)
111.3(2)
120.5(2)
1.982(3)
1.976(3)
2.024(3)
2.054(3)
2.167(3)
81.43(14)
102.08(14)
85.25(13)
85.50(13)
125.66(15)
104.70(15)
a Standard deviation in parenthesis; [τ = (β − α)/60; β(N1–Cu1–N4) =
175.28° and α(N2–Cu1–O1) = 133.95° for 1. β(N1–Cu1–N4) = 173.11°
and α(N2–Cu1–O1) = 120.70° for 2. β(N1–Cu1–N4) = 173.64° and α(N2–
Cu1–N5) = 128.11° for 3. β(N1–Cu1–N4) = 170.44° and α(N2–Cu1–N5) =
129.62° for 4; where by, for perfect square pyramidal and trigonal–
bipyramidal geometries, τ = 0 and 1, respectively.
Electronic spectral studies
The electron paramagnetic resonance (EPR) spectra of the
copper(II) complexes 1–4 were recorded in a methanol/DMF
mixture at 70 K and found to be axial. The EPR spectra were
128.12, 120.5°) due to additional electron-releasing methoxy simulated and their Hamiltonian parameters calculated,
groups at the meta-position, and resulted in significant distor- which were almost similar to that of the experimental data
tion in the trigonal plane as compared with 2. Conversely, the (Fig. S5,† Table 2). The hyperfine features resolved in the paral-
more electron-releasing –NMe2 group in 4 offered more distor- lel (g ) region and showed identical g values at 2.26–2.28 with
k
k
tion in the trigonal plane (N2–Cu–O1, 125.66; N2–Cu–N5, g⊥ values of 2.04–2.05 for all complexes (Fig. 2, Table 2). We
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Table 2 Electronic spectral parameters of copper(II) complexes
EPR parametersb
Electronic spectraa
Complex
λmax,nm (ε, M−1 cm−1
)
g
g⊥
A (cm−1
)
A⊥
f (cm)
α2
β2
γ2
K
k
K⊥
K
k
k
1
222 (28 800)
305 (7800)
374 (11 800)
680 (32)b
2.27c
2.26d
2.04c
2.04
154c
148d
9.5
147
0.7519
0.8821
0.7686
0.6750
0.6631
0.6894
0.6591
0.5781
0.6370
887 (20)
2
3
4
224 (31 800)
300 (15 000)
375 (31 800)
710 (34)
2.26c
2.25d
2.05c
2.05d
139c
150d
9.5
9.5
9.5
162
140
148
0.7041
0.7908
0.7489
0.9416
0.8718
0.8799
0.9032
0.7971
0.7397
0.6360
0.6303
0.5540
0.5530
0.6774
0.6344
885 (25)
222 (45 600)
294 (17 000)
377 (20 200)
725 (32)
2.28c
2.28d
2.05c
2.04d
163c
168d
882 (23)
222 (51 000)
306 (32 600)
384 (16 600)
741 (46)
2.27
2.04
153
932 (34)
a Concentration 1 × 10−2 M in acetonitrile/HEPES buffer pH 7.34 at 25 °C. b Measured at 70 K in methonal : DMF(8 : 2); A and A⊥ 10−4 cm−1. f = (g /A ),
k
k
k
α
dx2
2 = A /0.036 + (g − 2.0023) + 3/7 (g⊥ − 2.0023) + 0.04. K = α2β2 and K⊥ = α2γ2, K 2 = (g − 2.0023) ΔE (dxy − dx2 y2)/8λ0), K⊥2 = (g⊥ − 2.0023) ΔE(dxz,yz
− −
k
k
k
k
−
y2)/2λ0), K = α2β2, K⊥ = α2γ2, β2 = K /α2, γ2 = K⊥/α2, K = Aiso/Pβ2 + (gav − 2.0023)/β2. c kExperimental value. d Calculated value from simulated spectra.
k
k
Fig. 2 EPR spectra of 1–4 in a methanol/DMF mixture at 70 K. Microwave frequency, 9.647 GHz; microwave power, 1.0 mW; modulation amplitude,
10 G; modulation frequency, 100 kHz; time constant, 40.96.
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found g > g⊥ > 2.0023, suggesting an unpaired electron on the plane π-bonding in all complexes, whereas K = K⊥ for pure σ-
k
k
1
dx2−y2 orbital with the B2g ground state.30 Even though the bonding and K < K⊥ in-plane π-bonding have been reported.40
k
highest-energy singly occupied d-orbital was dx2−y2 its lobes The Fermi contact hyperfine interaction term (K) was calcu-
may not have been pointing directly at the ligands, which lated as 0.553 for 2, indicating an almost identical degree of
resulted in a smaller repulsive or antibonding interaction with covalent bonding character in Cu–N as predicted from α2
the ligand field as compared with perfect square-based geome- values.41 The K value is also a measure of the s-electron contri-
try.31 All the complexes exhibited f-values (g /A ) of bution to the hyperfine interaction.42 The K value of 2 was
k
k
140–162 cm−1, which were higher than those of square-based lower than those of 1, 3 and 4.
geometries (105–135 cm−1).32,33 The higher f-values revealed
The d–d transition appeared at 680 nm (ε, 32 M−1 cm−1) for
larger distortion in coordination geometry, suggesting strong 1 in acetonitrile : HEPES [4-(2-hydroxyethyl)-1-piperazineetha-
deviation from planarity.33 The line from minor g-values at nesulfonic acid] buffer at pH, 7.34 (1 × 10−2 M) and its ligand-
2.43, 2.31 for 1, 2.42, 2.29, 2.16 for 3, and 2.46, 2.32 for 4 corre- based transitions were merged (Table 2). The position of the
sponded to an additional minor species that may have origi- d–d transition was slightly varied for 2 (710 nm, ε, 34 M−1
nated from solvation and exchange of coordinated water mole- cm−1), 3 (725 nm, ε, 32 M−1 cm−1) and 4 (741 nm, ε, 46 M−1
cules with anions34 or from geometrical interconversion.35 cm−1). The energy order of d–d transitions was 1 > 2 > 3 > 4.
However, these minor species could not be discriminated by The energies of d–d transitions supported the existence of five-
the electronic absorption spectra in acetonitrile, methanol, coordinate geometry with dx2−y2 ground state in all com-
DMF or in a methanol–DMF mixture (Fig. S6†). Electrospray plexes.40,43 Also, low-energy shoulders observed around
ionization-mass spectrometry (ESI-MS) of these solutions 882–932 nm for 1–4 could be attributed to d–d transitions
revealed mass signatures only for the core [Cu(L)2]2+. A spectral characteristic of distorted square-pyramidal geometry,44 which
pattern for the 1 : 1 stoichiometry of ligand and copper(II) ions is due to geometrical interconversion in solution.
was not observed. Conversely, a minor component at 2.071 at a Interestingly, the solid-state absorption spectra of 1–4 showed
lower field was observed only for 2 because M(I) = −3/2.36 The d–d transitions around 735–787 nm and their positions were
values of g (2.26–2.28) and A (139–163 × 10−4 cm−1) further slightly shifted from the respective solution spectra (Fig. S7†).
k
k
supported the notion of geometrical distortion with a very The energies of d–d transitions were not significantly affected
weak axial interaction in solution. The distortion in the square while complexes interacted with selected amino acids such as
plane may have been caused by axial interaction by increasing alanine, arginine, glycine, histidine, leucine, proline, serine,
g values and decreasing A values. The lower values of hyper- threonine, tryptophan, and tyrosine in acetonitrile : HEPES
k
k
fine coupling A may have been contributed from strong Fermi buffer at pH, 7.34. Interestingly, while interacting, the com-
k
contact, spin dipoles and orbital dipoles.37 Normally, the plexes with Cys caused an immediate disappearance or huge
square-based CuN4 chromophore is expected to show g and A
decrease in intensity of d–d transitions due to the instan-
k
k
values of 2.200 and 200 × 10−4 cm−1, respectively.38 The devi- taneous reduction of Cu(II) into Cu(I) (Fig. S8†). In particular,
ation of square-based geometries agreed well with solid-state the d–d transition of 2 was decreased gradually by the
structural parameters calculated from single-crystal X-ray ana- measured addition of Cys and completely quenched by 5
lysis, whereby the non-zero τ-values correspond to strong dis- equivalents (Fig. 3). The solution turned yellow and after while
tortion in coordination geometry.38 In fact, the lower A value
k
of 2 (139 × 10−4 cm−1) was in good agreement with larger a
geometrical distortion parameter τ (0.87) in the solid state.
The lower A value of 2 was due to the larger distortion in a
k
square plane around copper(II) centers.39 Conversely, other
complexes exhibited a relatively higher A value (153–163 ×
k
10−4 cm−1) and relatively smaller geometrical distortion para-
meter τ (0.68–0.75) than 2 in the solid state.
The EPR parameters and energy of d–d transitions were
used to estimate bonding parameters such as covalency of in-
plane σ-bonds (α2), in-plane π-bonds (β2) and out-plane
π-bonds (γ2).40 The complex 2 exhibited lower α2 (0.7041) and
higher β2 (0.9416) and γ2 (0.9032) than those of 1, 3 and 4
(Table 2). These data suggested a slightly higher degree of
covalent bonding character in Cu–N bonds of 2 than that in
other complexes; in general, α2 values would be close to unity
for ionic bonding and decrease with increasing covalency.41
The orbital reduction factors K = α2β2 and K⊥ = α2γ2 were cal-
k
culated as 0.6631 and 0.636 for 2 and were almost identical to
Fig. 3 Electronic spectral changes upon addition Cys to 2 (5 × 10−6 M)
in HEPES buffer (pH 7.34) at 25 °C. Inset: Depletion of a d–d band of 2
those of 1, 3 and 4. All the complexes showed K > K⊥, thereby
k
revealing the persistence of a significant amount of out-of- by addition of Cys (10−2 M) in DMF HEPES buffer.
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caused a colorless precipitate corresponding to Cys-bound 360 nm (ε, 22 860 M−1 cm−1) in HEPES buffer at pH, 7.34. This
Cu(I) complexes (Fig. S9†), which was characterized by HR-MS, was accompanied by the simultaneous appearance of new
1H NMR, infrared (IR) and elemental analysis. HR-MS spectra bands around 224 nm (ε, 31 800 M−1 cm−1), 300 (ε, 15 000 M−1
showed a molecular ion peak at m/z, 785.3256 corresponding cm−1) and 375 nm (ε, 31 800 M−1 cm−1) corresponding to for-
to [CuI(L)2(Cys)] (Fig. S10†). The element composition was mation of 2 (Fig. S15†). The formation of 2 was further con-
measured by elemental analysis, and found to be, for firmed by HR-MS, whereby a molecular ion peak appeared at
CuC41H36N7O4S, the following: C, 62.60; H, 4.60; N, 12.44. This m/z = 814.12347, corresponding to [Cu(L2)2(SO3CF3)]+. The
was similar to the calculated elemental composition (C, 62.62; stability constant was calculated as 9.04 by pH-metric titration
H, 4.61; N, 12.47). This notion was further supported by 1H (Fig. S16†). The interaction of 2 (5 × 10−6 M) with Cys as a func-
NMR, whereby the formation of Cu(I) was accompanied with a tion of concentration was studied by following changes in
shift in the α-CH and β-CH2 resonances of Cys towards a ligand field transitions. The bands at 300 (ε, 15 000 M−1 cm−1
)
higher field along with ligand spectral signatures. α-CH and and 375 nm (ε, 31 800 M−1 cm−1) were decreased gradually
β-CH2 protons appeared at 3.99 and 3 ppm for free Cys, over the calculated addition of Cys, and saturated at 5 equiva-
respectively.45 One equivalent of Cys with 2 showed a broaden- lents. This observation clearly showed that only Cys could alter
ing of 1H NMR peaks, possibly due to a mixture Cu(II) and the absorption pattern of 2 via reduction of Cu(II) into Cu(I),
Cu(I) species. However, addition of 5 equivalents exhibited and led to the formation of [CuI(L)2(Cys)] (cf. above). After this
well-resolved 1H NMR peaks corresponding to [CuI(L)2(Cys)] formation, coordinated Cys possibly oxidized to attain for-
(Fig. S11†). The observed chemical-shift values of Cys were mation of the Cys-S-S-Cys bond, and then dissolved oxygen
similar to those of previously reported Cu(I)-Cys complexes.45 likely to facilitate the re-oxidation of Cu(I) for the binding of
The IR stretching frequencies (ν) appeared at 2915, 1577, 1482 next Cys.47 The addition of other biomolecules, such as homo-
¯
and 1401 cm−1, which showed considerable shifting from free cysteine, glutathione, and methionine, showed relatively negli-
Cys, and matched with
ν
¯
st
of CuI-(Cys) (Fig. S12†).46 gible changes in the absorption spectrum of 2. Also, the
Interestingly, treatment of [CuI(L)2(Cys)] with one equivalent of absorption spectral pattern of 2 was unaltered by the addition
H2O2 (30%) resulted in regeneration of the original d–d band of 10 equivalents of biologically relevant cations (K+, Ca2+,
of 2 at 693 nm (ε, 26 M−1 cm−1) and further addition of Cys Mg2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, and Zn2+) and anions such
led to disappearance of the d–d band again. This result clearly as sulfate and phosphate, at pH = 7.34 (Fig. S17†). Thus, probe
suggested that the interaction of Cys proceeded through the 2 was stable enough for imaging under biological conditions
reduction of Cu(II) to Cu(I) without ligand displacement without transmetalation or interferences by endogenous metal
(Fig. S13†). Conversely, our previously reported rigid Cu(II)- ions or anions at this pH.
complexes could not yield Cys-bound Cu(I) complexes, whereas
Redox studies
ligand displacement was noticed after reduction of the Cu(II)
center.11,27
The redox behavior of complexes was investigated by cyclic vol-
The ligand-based π–π* transitions of 1 were resolved at tammetry (CV) using a three-electrode cell configuration and
lower concentration (5 × 10−6 M) and showed three major NaCl as the supporting electrolyte in HEPES buffer solution at
absorption bands around 222 nm (ε, 28 800 M−1 cm−1), pH 7.34. A platinum sphere, platinum wire, and Ag/Ag+ were
305 nm (ε, 7800 M−1 cm−1) and 374 nm (ε, 11 800 M−1 cm−1
)
used as the working, auxiliary, and reference electrode,
in HEPES buffer at pH 7.34. The energy of these transitions respectively. The measured redox potentials were converted
was not significantly affected by ligand architecture and into a normal hydrogen electrode (NHE) by adding +0.205 V.27
almost identical spectral signatures were observed for 2–4 The Cu(II)/Cu(I) redox potential of probe 1 (0.221 V vs. NHE)
(Fig. S14†). However, their intensities were notably increased was lower than that for 4 (0.525 V vs. NHE) but almost identi-
by introducing electron-releasing methoxy and dimethylamine cal to that of 2 (0.237 V) and 3 (0.218 V) (Table 3, Fig. S18†).
groups on the phenyl rings of 1 to obtain 2 and 4, respectively, The Cu(II)/Cu(I) redox potentials of 1–4 were higher than the
and caused redshift. The interaction of selected amino acids biological redox potential ranges. In healthy differentiated
with 1, 3 and 4 showed almost no change or slight shift or cells, the glutathione/glutathione disulfide (GSH/GSSG) redox
change in the intensity of transitions at this lower concen- couple was approximately −0.22 V vs. NHE, but became more
tration. This may have been due to weaker coordination or reducing in proliferating cells (−0.26 V), which corresponded
coordination without major geometrical rearrangements and to a higher GSH : GSSG ratio. The extracellular redox potential
the oxidation state (cf. below) of the copper(II) center. of the cysteine/cysteine disulfide (CySH/CySSCy) couple is typi-
Interestingly, the interaction of Cys with 2 showed significant cally −0.080 V, whereas pathologic cells demonstrate CySH/
spectral changes. Nevertheless, the other amino acids men- CySSCy potentials ranging from −0.062 to −0.02 V in the extra-
tioned above almost unaltered the absorption spectral signa- cellular space.48 The treatment of Cys with 1, 3 and 4 displayed
tures of 2. The stability and formation of 2 were examined by only slight changes in Cu(II)/Cu(I) redox potentials.
monitoring absorption spectral changes at this biologically Nevertheless, the Cu(II)/Cu(I) redox potential of 2 shifted to a
relevant lower concentration (5 × 10−6 M). The gradual more negative potential by the reaction of Cys (E1/2, 0.190 V vs.
addition of Cu2+ to L2 resulted in the disappearance of the NHE; ΔE, 89 mV). It was studied further by electrochemical
ligand-based transitions at 317 (ε, 28 500 M−1 cm−1) and titration, whereby the gradual addition of Cys to 2 (1 × 10−3 M)
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Table 3 Optical and redox data for 1–4
Redox dataa
Emission data
Complex
Epa (V)
Epc (V)
ΔE (mV)
E1/2 (V)
E1/2 (V, vs. NHE)
λemie (nm)
Binding constantf
1 × 105
Detection limitf
1.9 × 10−7
1
0.062
0.339
0.070
0.003
0.019
0.08
0.057
0.040
0.356
0.365
−0.030
0.26
92
79
77
89
76
104
89
72
72
115
0.016
0.299b
0.032
0.221
0.504
0.237
0.190
0.186
0.217
0.218
0.209
0.525
0.512
490
2
−0.007
−0.059
−0.057
−0.024
−0.032
−0.032
0.284
467
2.3 × 105
9.9 × 10−8
−0.015b
−0.019c
0.028d
0.0125
0.004b
0.320
3
4
460
465
1.3 × 104
1.6 × 104
1 × 10−7
1.5 × 10−7
0.250
0.307c
a Concentration 1 × 10−3 M in HEPES buffer pH 7.34 at 25 °C (reference: saturated Ag/Ag+; supporting electrolyte: 0.1 M NaCl solution; scan rate =
50 mV s−1). To convert E1/2 vs. NHE add +0.205. b Complex + Cys. c Complex + His. d Complex + GSH. e Corresponding to respective ligands.
f Calculated in HEPES buffer pH 7.34 at 25 °C.
showed a shift in the Cu(II)/Cu(I) redox potential, along with His (Fig. S21a†). This result was supported further by the per-
increases of current values up to one equivalent (Fig. S19a and sistence of the d–d band after the interaction of 2 with His
b†). Further addition of Cys resulted in a gradual shift in redox (Fig. S22†). The binding constant (K) for the interaction of 2
potential with a decrease in current values. These changes and His was calculated to be 6.7 × 104 M−1 (Fig. S23†). Also, an
were linear up to seven equivalents of Cys and then started pre- non-significant change in Cu(II)/Cu(I) redox potentials was
cipitating a colorless solid (Fig. 4). The solid was characterized observed for the addition of five equivalents of GSH to 2, but
as [CuI(L2)2(Cys)] by HRMS and IR spectroscopies (cf. above). the current value decreased (Fig. S21b†). The other amino
The formation of Cys-bound reduced species [CuI(L2)2(Cys)] acids could not produce such electrochemical changes under
was interesting and different from our previous observation, identical conditions.
whereas the Cu(I) ion was displaced from the coordination
sphere and released free ligands.11,27 A completely different
redox behavior was noted for the reaction of Cu(SO3CF3)2 with
Cys under an identical condition, whereby the Cu(II)/Cu(I)
redox couple disappeared readily (Fig. S20†). Conversely,
Optical spectral studies
The ligands L1–L4 showed strong fluorescence in the range
460–490 nm in HEPES buffer at pH 7.34 (5 × 10−6 M) and the
energy of their emission wavelengths were strongly influenced
electrochemical titration of 2 with histidine (His) exhibited
by electronic substituents. The fluorescent quantum yields of
only a slight shift in Cu(II)/Cu(I) redox potential with signifi-
the ligands were calculated to be 4%, 3.8%, 7.6% and 1.1% for
cant decreases in current values without concomitant
L1, L2, L3 and L4, respectively (Table S1†). However, their
reduction, which may have been due to the coordination of
copper(II) complexes were found to be non-fluorescent. This
‘turn off’ behavior emerged due to the paramagnetic Cu(II)
center.11,27 Specifically, the fluorescence intensity of the ligand
L2 was completely quenched by the coordination of Cu2+. The
binding constant for Cu2+ and L2 was calculated as 5.3 × 106
(Fig. S24†). This finding was supported further by the highest
occupied molecular orbital/lowest unoccupied molecular
orbital (HOMO–LUMO) electron density map of 2.
Computational calculations were carried out using B3LYP, 6-
31G (for C, H, N, and O) and TD-DFT using LANL2DZ (for Cu)
basis sets in the Gaussian 09 program49 (Fig. S25, Table S2†).
The frontier molecular orbital analysis of L2 revealed the
HOMO to be localized on the entire ligand moiety, and LUMO
to be localized only on the imidazopyridine moiety. However,
for 2, the HOMO localized around the imidazopyridine moiety
and LUMO was localized around the pyridine moiety and
copper center. This scenario was due to the collapse of the
electronic conjugation of the ligand by the Cu2+ center
through internal charge transfer. That is, the electron density
localized on the ligand moiety was transferred to the copper(II)
center. After the interaction of Cys with 2, the HOMO localized
Fig. 4 Electrochemical titration of [Cu(L2)2(CF3SO3)2] 2 (1 × 10−3 M)
with various amounts of Cys using NaCl as the supporting electrolyte in
HEPES buffer at pH 7.34.
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around copper and Cys, and the LUMO localized around the
imidazopyridine moiety. A larger HOMO–LUMO energy gap for
2 (3.125 (2) eV) referred to higher kinetic stability and lower
chemical reactivity.50 Single-excitation time-dependent DFT
(TDDFT) was undertaken to explain the electronic structural
properties of the ground and excited states of 2. The vertical
transitions calculated by TDDFT were comparable with the
experimentally observed electronic spectra of 2, whereby the
vertical π → π* transition at ∼370 nm was almost closer to the
experimentally observed spectra at ∼375 nm.51 They corre-
sponded to the pure HOMO → LUMO+1 excitation. The
LUMO+1 localized around imidazopyridine and the copper
center. However, after the interaction of Cys with 2, the LUMO
localized around the imidazopyridine moiety and the HOMO
was transferred to copper and cysteine. The experimentally
absorbed π → π* transition spectra at 375 nm for 2 + Cys
corresponded to the HOMO−1 → LUMO excitation.
The emission property of 1, 3 and 4 was altered by the
interaction of selected amino acids (Fig. S26†) and turned on
their fluorescent intensity without selectivity. In particular,
the interaction of Cys and His with 1, 3 and 4 showed huge
fluorescent enhancement compared with that for other listed
amino acids. Amazingly, the probe 2 exhibited turned-on
fluorescent behavior only by the interaction of Cys, however,
it remained non-fluorescent with all other amino acids. The
intensity raised by 2 with Cys was 103-fold higher than its orig-
inal intensity (Fig. 5a). The fluorescence intensity increased
linearly upon gradual addition of Cys (Fig. S27†) and this
intensity enhancement was saturated at 12 equivalents. Then,
the intensity remained unchanged up to 17 equivalents
(Fig. S28†). The binding constant (K) for the interaction of 2
and Cys was calculated as 2.3 × 105 M−1 from the linear plot
Fig. 5 (a) Fluorescence spectra of 2 (5 × 10−6 M) with various amino
acids (5 × 10−5 M) in HEPES buffer pH, 7.34 at 25 °C. Inset: Fluorescence
spectra of L2, 2, 2 + Cys. (b) Bar diagram for the selectivity of various
amino acids and biologically relevant ions. Inset: Fluorescence spectra
of 2, 2 with HCy, His, GSH (5 × 10−5 M, λex = 367 nm).
obtained using the Benesi–Hildebrand expression, Io/I − Io
=
b/(a − b){1/K[M] + 1}; where Io and I are the intensities in the
absence and presence of the analyte, respectively, [M] is the
concentration of the analyte, and ‘a’ and ‘b’ are the intercept
and slope of the plot, respectively. The linear plot was obtained
by fitting (Io/I − Io) versus 1/[M].52 The limit of detection was fluorescence intensity immediately (Fig. 6). This finding suggested
calculated as 9.9 × 10−8 M by employing the formula [3× (stan- that 2 was highly sensitive toward Cys and able to be visualized
dard deviation/slope)] to the linear plot. However, other com- even at lower concentrations. There was no concomitant
plexes exhibited a lower binding constant with Cys (1.3 × 104 change in the fluorescence intensity of 2 over the pH range
to 1 × 105) as compared with 2 (Table 3). A Job’s plot was 4.5–9.5. However, 2 with Cys showed gradual increment in
obtained by fluorescence titration of 2 with Cys and revealed intensity on increasing pH up to 9.5 (Fig. S30†). The fluo-
one equivalent binding of Cys (Fig. S29†). The biological redox rescence intensity of 2 was not enhanced by biological cations
buffer glutathione (GSH) and homocysteine (Hcys) could not (K+, Ca2+, Mg2+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, and Zn2+) at pH
produce similar fluorescent enhancement even though they 7.34 (Fig. S31†). This clearly ruled out the possibility of trans-
contain a thiol group. Their enhancement of fluorescent inten- metalation in 2 by these biologically relevant cations.55 Also,
sity was very much lower even with 10 equivalents, which was the fluorescence intensity of 2 was not turned on by adding
negligible as compared with the intensity raised by Cys under biological anions such as SH−, PO43−, SO42− or H2S (Fig. S32†).
identical conditions (Fig. 5b). This disparity was due to the Similar to absorption spectral studies, addition of one equi-
higher pKa values of GSH (9.2), Hcys (10.0), than L-cysteine valent H2O2 (30%) to the solution of [CuI(L2)2(Cys)] led to
(8.3),53 and they are expected to show weaker coordination immediate quenching of fluorescence intensity due to the oxi-
with 2 than Cys and, hence, the reduction of Cu(II) to Cu(I).54 dation of Cu(I) to Cu(II) (Fig. S33†) (Scheme 2) and it was regen-
In fact, the fluorescence intensity of 2 was not raised even with erated by addition of another equivalent of Cys. The enhance-
10 equivalents of the other listed amino acids. However, the ment of fluorescence intensity by Cys may have occurred via
addition of one equivalent Cys to the same solution raised the two consecutive steps. First, the copper(II) center was reduced
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distorted trigonal plane adopted in 1, 3 and 4 may require
higher reorganization energy for electron transfer and, hence,
reduction of Cu(II) to Cu(I) by Cys58 and possibly led to poor
selectivity.
Imaging of living cells
The optical imaging probe 2 could visualize Cys selectively in
an isolated chemical environment as established by spectral
and redox methods. Thus, it provided suitable excitation/emis-
sion wavelengths and brightness for Cys imaging in living
cells. First, MTT [3-(4,5-dimethylthiozol-2-yl)-2,5-diphenyltetra-
zolium bromide] assays were carried out for Henrietta Lacks
cervical cancer (HeLa) cells and macrophage cells, and calcu-
lated as 86.26% and 75.26%, respectively. They exhibited low
cytotoxicity limits with cell viability up to 75 μM concentration
(Fig. 7). This finding suggested that probe 2 could be explored
as a biomarker for imaging Cys in living cells without affecting
their viability. Therefore, we investigated the Cys imaging
ability of probe 2 in HeLa cells and macrophages via a turn-on
fluorescent mechanism under physiological pH (7.34). HeLa
Fig. 6 Competitive binding experiments of 2 (5 × 10−6 M) with various
amino acids (5 × 10−5 M) in the absence (black) and presence of one
equivalent Cys (red) in HEPES buffer pH, 7.34 at 25 °C.
to copper(I) by Cys, as expected. Once reduced, the copper(I)
ion apparently requires trigonal-based coordination geome-
try.56 The reduction presumably involves significant geometri-
cal reorganization through internal changes in the bond
lengths and angles. In the Cys ligand, the sulfur atom has
three valence 3p orbitals: one of the p-orbitals is used for a
C–S bond and the remaining two degenerate p-orbitals are per-
pendicular to the C–S bond. The two degenerate p-orbitals can
interact with the copper center and split in energy depending
on the C–S–Cu angle. By changing the C–S–Cu angle via the
interaction between the S-pπ-orbital and Cu-dx2−y2 orbital, a
tetrahedral/tetragonal similar to the Cu–S interaction of blue
Cu-proteins can be adopted.57 While the interaction is weaken-
ing, the maximum absorption peak is shifted to higher energy
and vice versa. The required geometrical plasticity for the
coordination of Cu(II) and Cu(I) ions may be better stabilized
by the p-substituted methoxy group of 2. A nearly-perfect trigo-
nal plane of 2 (predicted by a lower A in EPR and molecular
k
structure) enforced faster electron transfer and selectivity as
compared with other complexes. In fact, it showed a higher
binding constant with Cys among the other complexes. The
Fig. 7 MTT assay in HeLa cells. Mean standard error from the tested
triplicate samples is represented as mean error bars. HeLa cells (red) and
macrophages (blue) were incubated with 0–75 μM of 2 for 24 h.
Scheme 2 The reaction of 2 with Cys in HEPES buffer at pH 7.34.
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and macrophage cells were incubated with 2 (5 μM) for 30 min showed almost similar fluorescence images (Fig. 8 and S34†)
at 37 °C. Monolayers of two cells were grown on cover-glass before and after exogenous treatment with Cys. These imaging
Petri dishes for the measurement of fluorescence and bright- studies on living cells revealed that 2 could enter cancer cells
field images. Then, laser scanning fluorescence microscopy and turn on fluorescence intensity by detecting Cys.
was done at an excitation wavelength of 367 nm using green Interestingly, probe 2 showed no significant changes in cell
filter. Acridine orange was used as a fluorescent intercalating structure or morphology under our experimental conditions.
agent. HeLa cells showed reasonable brighter fluorescence
images, possibly corresponding to the amount of Cys in living
cells. HeLa cells were pre-treated with 100 µM of Cys over
30 min at 37 °C followed by incubation with 2 (5 µM) for an
Summary
additional 30 min. The imaging of HeLa cells by laser scan-
ning fluorescence microscopy exhibited brighter fluorescence
images as compared with images from untreated cells. These
results clearly suggested that probe 2 could interact directly
with Cys in a transient manner through cell membranes at bio-
logical pH (7.34) and reduce the copper(II) center into copper
(I). However, pre-treatment of a thiol-blocking agent (200 µM
of N-ethylmaleimide (NEM)) with HeLa cells before addition of
2 (5 µM) showed only a very faint fluorescence image. Before
treatment with 2, the cells showed almost no fluorescence in a
similar region. The Cys imaging experiment was also examined
for macrophage cell lines to enable comparison of the
efficiency and specificity of probe 2. Macrophage cell lines also
The copper(II) complexes of imidazopyridine-based ligands
were synthesized as an optical imaging probe for visualizing
Cys in cancer cells. The molecular structure of the complexes
adopted a distorted trigonal pyramidal around copper(II)
centers in the solid state. This five-coordinate geometry also
persisted in solution, as supported by electronic spectral data.
Interestingly, the complex with a p-methoxy substituent on
phenyl rings adopted a nearly perfect trigonal plane around
the copper(II) center, which facilitated faster reduction of Cu(II)
to Cu(I) selectively by Cys. Other complexes exhibited strong
distortion around the trigonal plane (which may require
higher reorganization energy for electron transfer and
reduction of Cu(II) to Cu(I)) and resulted in poor selectivity.
Fig. 8 Fluorescence and bright-field images of HeLa cells: (a) cells in the absence of 2, (b) cells incubated with 2 (5 μM) for 30 min, (c) cells pre-
treated with 100 μM Cys and incubated with 2 (5 μM) for 30 min, and (d) cells pre-treated with 200 μM NEM and incubated with 2 (5 μM) for 30 min.
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The Cu(II)/Cu(I) redox potential of the complexes varied slightly Heraeus Vario Elemental automatic analyzer. Fourier trans-
by ligand electronics, whereby methoxy substituents on phenyl form-infrared (FT-IR) measurements were carried out using a
rings showed a lower redox potential than that of –NMe2 sub- Nicolet 6700 system. Absorption spectra were undertaken
stituents. All ligands exhibited strong fluorescence, but their using Agilent Technologies diode array 8453 spectrometers at
copper(II) complexes were non-fluorescent. The emission be- 298 K, and emission spectra were measured on an Agilent
havior of complexes varied significantly upon interactions of Technologies Cary Eclipse spectrofluorometer at 298 K.
amino acids, and turned on their fluorescent intensity without Electrochemical data were recorded in a Biologic SP-150
selectivity. Interestingly, one of the probes with p-methoxy sub- Electrochemical Workstation using saturated Ag/Ag+ and Pt as
stituents showed a selective turn-on fluorescence property a reference electrode and working electrodes, respectively; 0.1
towards Cys over other amino acids. It showed an excellent M NaCl was used as a supporting electrolyte. The electrodes
binding constant of 2.3 × 105 M−1 and a limit of detection of are calibrated by K4[Fe(CN)6] prior to measurements.
9.9 × 10−8 M at pH 7.34. The probing mechanism of Cys likely
Determination of X-ray structure
operated via reduction of copper(II) to copper(I) without ligand
displacement. After reduction, the electronic conjugation was These experiments were carried out on an Agilent
regenerated and fluorescence intensity enhanced at the bright Technologies Supernova-E CCD diffractometer. Single crystals
visible region. Furthermore, this probe exhibited significantly of suitable size were selected from the ‘mother’ liquor and
brighter fluorescence images for living cells such as HeLa and immersed in paraffin oil, then mounted on the tip of a glass
macrophage cell lines. The intensity of the images was fiber. A Mo radiation source and microfocus tube multilayer
enhanced by exogenous addition of Cys to living cells. Cell- mirror optics (λ = 0.71073 Å) were used for data collection. The
imaging experiments strongly suggested that our probe might structures were solved by direct methods using the program
cross membrane barriers readily to permeate into living cells SHELXS-2013. Refinement and all further calculations were
rapidly for imaging intracellular Cys with low cytotoxicity.
carried out using SHELXS 2013. H-atoms were included in cal-
culated positions and treated as ‘riding’ atoms using SHELX
default parameters. Non-H atoms were refined anisotropically
using weighted full-matrix least-square on F2. CCDC 1833674,
1833675, 1833676 and CCDC 1833677† contained the sup-
plementary crystallographic data for our study. Structural
refinement parameters 1–4 are summarized in Table 4.
Experimental section
Materials
Unless indicated otherwise, common reagents or materials
were obtained from a commercial source and used without
further purification. All solvents were used after appropriate
distillation or purification. The chemicals 3,4-dimethoxyben-
zaldehyde, di-pyridin-2-yl-methanone, copper(II) chloride,
copper(II) triflate, zinc(II) chloride, cobalt(II) chloride, iron(II)
chloride, iron(III) chloride, magnesium(II) chloride, manganese
(II) chloride, calcium chloride, sodium chloride, potassium
chloride, L-alanine, L-arginine, L-cysteine, L-glycine, L-histidine,
L-leucine, L-proline, L-serine, L-threonine, L-tryptophan,
L-tyrosine, L-methionine, DL-homocysteine, L-glutathione,
sodium phosphate and methyl mercaptoacetate were pur-
chased from Sigma-Aldrich. NH4OAc was obtained from
Merck.
DFT methods
Geometry optimizations were undertaken using DFT methods.
For metal ions, LANL2DZ basis sets with the Becke3–Lee–
Yang–Parr hybrid functional (B3LYP) were used. For elements
other than metals, the 6-311G (d) basis sets were employed. All
DFT and TD-DFT calculations were carried out using the
Gaussian 09 program package.59
Measurement of the stability constant
The pH of solutions was measured with an ELICO instrument
(Model LI120) equipped with a combined glass electrode
assembly. This instrument has a built-in internal electronic
voltage supply with a temperature compensator covering the
range from 0 °C to 100 °C. The instrument was calibrated with
Physical measurements
All reactions were carried out under an atmosphere of dry buffer solutions of acidic and basic pH before start the pH
nitrogen. Glassware was oven-dried prior to use. Unless indi- titrations. Specific solutions were used to calculate the metal–
cated otherwise, common reagents or materials were obtained ligand stability constant over a pH range of 0–14 by 0.1 M of
from a commercial source and used without further purifi- NaOH. The total volume (10 mL) of solution contained 0.2 mL
cation. All solvents were used after appropriate distillation or of 0.1 M HNO3, 2.8 mL of distilled water, 6 mL of dioxane and
purification. EPR spectra were recorded at 77 K using a JEOL 1 mL of 1 M NaNO3. For ligand and metal–ligand titrations,
X-band spectrometer (JES-FA100). The spectra simulation was 0.5 mL of 0.1 M ligand solution and 0.1 mL of 0.1 M metal
carried out using JEOL Anisotropic Simulation software solution were used subsequently.
(AniSim/FA). NMR spectra were recorded at 400 MHz using a
Bruker spectrometer. Chemicals-shift values and coupling con-
MTT assay using 2 for HeLa and macrophage cell lines
stants are given in ppm and Hz, respectively. HR-MS spectra The MTT assay is a simple colorimetric assay to measure cyto-
were measured on a Thermo Scientific Exactive Plus EMR toxicity. Metabolically active cells can convert this tetrazolium
instrument. Elemental analyses were carried out using a salt into a water-insoluble dark-blue formazan. The resultant
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Table 4 Crystal data and structure refinement for 1–4
1
2
3
4
Empirical formula
C40H31CuF6N7O7S2
963.38
Triclinic
ˉ
P1
11.0766(9)
14.1661(10)
14.7052(10)
69.381(6)
75.972(6)
72.748(7)
2037.7(3)
293(2)
C41H34CuF6N6O10S2
1012.40
Triclinic
ˉ
P1
11.4660(8)
11.5674(8)
17.1754(11)
88.282(5)
71.269(6)
81.010(6)
2130.4(3)
293(2)
C42H36CuF6N6O11S2
1042.43
Triclinic
ˉ
P1
10.1295(7)
15.0236(9)
16.3207(10)
101.942(5)
104.135(6)
98.175(6)
2307.3(3)
293(2)
C43H42CuF6N8O8S2
1040.50
Monoclinic
C2/c
27.6006(14)
15.8879(8)
21.9963(12)
90
108.440(6)
90
9150.5(9)
293(2)
Formula weight
Crystal system
Space group
a [Å]
b [Å]
c [Å]
α [°]
β [°]
γ [°]
V [Å3]
T [K]
Density [Mg m−3
]
1.570
1.578
1.500
1.511
Z
2
2
2
8
µ [mm−1
F (000)
]
0.726
982
0.703
1034
0.653
1066
0.655
4280
No. of reflections collected
12 975
1.023
0.0825
0.1460
14 752
1.047
0.0951
0.2115
10 939
1.029
0.1093
0.1938
29 689
1.041
0.0992
0.1995
Goodness of fit on F2
a
R1
b
wR2
a R1 = ∑||Fo| − |Fc||/∑|Fo|. b wR2 = [∑w(Fo2 − Fc2)2/∑w(Fo2)2]1/2
.
value is related to the number of living cells. To determine 367 nm line from an argon ion laser, and emitted fluorescence
cytotoxicity/viability, HeLa and macrophage cells were plated was monitored at 440 2 nm.
at 1 × 104 cells per well in a 96-well plate at 37 °C in an atmo-
General procedure for ligand synthesis
sphere of 5% CO2. Cells were maintained in Dulbecco’s modi-
fied Eagle’s medium (DMEM) supplemented with 10% fetal
bovine serum (FBS) and 100 units of penicillin–streptomycin.
After cell attachment, a fresh medium containing probe 2 of
varying concentrations was added. After 24 h, probe 2 was
removed and the medium was added, along with MTT dye
solution (0.5 mg ml−1), to cells. After 4 h of incubation at
37 °C in 5% CO2, the medium was removed and formazan crys-
tals were solubilized in 100 µL of dimethyl sulfoxide.
Absorbance was read on a microplate reader at 575 nm. The
relative cell viability (%) compared with control cells was calcu-
lated by [A]test/[A]control × 100. Remarkably, the MTT assay of
probe 2 towards HeLa and macrophage cell lines showed
viable cells accurately at 86.26% and 75.26%, respectively, up
to 75 μM of probe 2.
To a flask containing a mixture of substituted benzaldehyde
(1.25 g, 7.5 mmol), di-pyridin-2-yl-methanone (0.92 g,
5.0 mmol), and NH4OAc (1.93 g, 25 mmol) were added to
25 mL of dry acetic acid. The solution mixture was slowly
heated to reflux, stirred for 16 h, cooled, and 2 mL of water
was added. The solution was pumped dry, and the residue was
extracted with dichloromethane/water. The organic layer was
dried over anhydrous sodium sulfate, filtered, and dried. The
residue was chromatographed through silica gel (ethylacetate :
hexane = 1 : 3) to give a yellow powder.
3-Phenyl-1-pyridin-2-yl-imidazo[1,5-a]pyridine (L1). Ligand
L1 was prepared by the reaction of benzaldehyde, di-pyridin-2-
yl-methanone (0.92 g, 5.0 mmol), and NH4OAc (1.93 g,
25 mmol) with addition to 25 mL of dry acetic acid (1.25 g,
7.5 mmol). Yellow solid; yield: 56%. Melting range, 92–96 °C;
NMR (400 MHz, CDCl3), δ 8.65–8.62 (d, 1H, py-CH, J = 12 Hz),
Laser scanning fluorescence microscopy
The human-derived cervical cancer HeLa cell line was grown 8.57–8.56 (d, 1H, imz-CH, J = 4 Hz), 8.20–8.17 (d, 2H, py-CH, J
and maintained in DMEM containing 10% FBS, glutamine = 6 Hz), 7.78–7.77 (d, 2H, Ar-CH, J = 4 Hz), 7.67–7.63 (t, 1H,
(0.29 g L−1), sodium bicarbonate (2.2 g L−1), penicillin (100 000 imz-CH, J = 8 Hz), 7.50–7.46 (t, 2H, py-CH, Ar-CH, J = 8 Hz),
U L−1), and streptomycin (10 mg L−1) in an atmosphere of 5% 7.42–7.38 (t, 1H, Ar-CH, J = 8 Hz), 7.04–7.02 (t, 1H, Ar-CH, J = 4
CO2–95% air at 37 °C. A monolayer of HeLa cells was grown on Hz), 6.86–6.84 (t, 1H, imz-CH, J = 8 Hz), 6.60–6.57 (t, 1H, imz-
cover glass Petri dishes for fluorescence and bright-field CH, J = 6 Hz); ESI-MS for C18H14N3: 272.11 [M + H]+, found:
images. Briefly, HeLa cells grown in DMEM were washed with 272.1182 [M
+
H]+. Elemental analysis: calculated for
phosphate-buffered saline and treated with cysteine (100 μM). C18H13N3: C, 79.68; H, 4.83; N, 15.49%. Found: C, 79.67; H,
Then, complex (5 μM) was added and incubation allowed to 4.82; N, 15.48%.
proceed for 30 min. Subsequently, NEM (200 μM) and complex
3-(4-Methoxyphenyl)-1-pyridin-2-yl-imidazo[1,5-a]pyridine
(5 μM) were incubated for 30 min. Cells were fixed using 4% (L2). Ligand L2 was prepared by the reaction of 4-methoxyben-
paraformaldehyde, incubated at 37 °C for 20 min and zaldehyde (1.25 g, 7.5 mmol), di-pyridin-2-yl-methanone
mounted with DPX for imaging using a Carl Zeiss laser scan- (0.92 g, 5.0 mmol), and NH4OAc (1.93 g, 25 mmol) upon
ning microscope (LSM 710). Fluorophores were excited using addition to 25 mL of dry acetic acid. Yellow solid; yield: 62%;
Dalton Trans.
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melting range 105–110 °C; 1H NMR (400 MHz, CDCl3)
[Cu(L2)2(SO3CF3)]SO3CF3 (2). Yield: 0.39 g (60%). HR-ESI
δ 8.72–8.70 (d, 1H, py-CH, J = 8 Hz), 8.65–8.64 (d, 1H, py-CH, mass for [C39H30CuF3N6O5S]+ (m/z), calculated: 814.12465;
J = 4 Hz), 8.27–8.25 (d, 1H, py-CH, J = 8 Hz), 8.2–8.19 (d, 1H, found: 814.12347.
imz-CH, J = 8 Hz), 7.78–7.73 (t, 3H, imz-CH, py-CH, Ar-CH, J =
[Cu(L3)2(H2O)](SO3CF3)2 (3). Yield: 0.47 g (70%). HR-ESI
10 Hz), 7.13–7.07 (m, 3H, Ar-CH, J = 8 Hz), 6.96–6.92 (t, 1H, mass for [C40H34CuN6O4]2+ (m/z), calculated: 725.28176;
imz-CH, J = 8 Hz), 6.67–6.64 (t, 1H, imz-CH, J = 6 Hz), 3.9 (s, found: 725.19281.
aryl-OCH3, 3H); ESI-MS calculated for C19H15N3O: 301.12 (M+),
[Cu(L4)2(H2O)](SO3CF3)2 (4). Yield: 0.38 g (58%). HR-MS
found: 301.10 (M+). Elemental analysis: calculated for mass for [C40H36CuN8]2+ (m/z), calculated: 691.23589; found:
C19H15N3O: C, 75.73; H, 5.02; N, 13.94%. Found: C, 75.72; H, 691.23590.
5.50; N, 13.91%.
Characterization of [Cu(L2)2(Cys)]. 1H NMR (400 MHz,
3-(3,4-Dimethoxy-phenyl)-1-pyridin-2-yl-imidazo[1,5-a]pyridine DMSO and D2O mixture) δ 8.49–8.48 (d, 1H, J = 4.5 Hz, CHAr),
(L3). Ligand L3 was prepared by the reaction of 3,4-dimethoxy- 8.39–8.37 (d, 1H, J = 6.7 Hz, CHAr), 8.24–8.22 (d, 1H, J = 9.1 Hz,
benzaldehyde (1.25 g, 7.5 mmol), di-pyridin-2-yl-methanone CHAr), 8.15 (dd, 2H, J = 15.5, 7.6 Hz, CHAr), 7.74–7.72 (2, 2H, J
(0.92 g, 5.0 mmol), and NH4OAc (1.93 g, 25 mmol) upon = 7.4 Hz, CHAr), 7.41 (s, 1H, CHAr), 7.24–7.17 (m, 1H, CHAr),
addition to 25 mL of dry acetic acid. Yellow solid; yield: 68%; 7.13–7.11 (d, 2H, J = 7.5 Hz, CHAr), 6.92–6.88 (t, 1H, J = 6.5 Hz,
melting range, 138–142 °C; 1H NMR (400 MHz, CDCl3) δ CHAr). 3.79 (S, 4H, OCH3, α-CH), 2.91(s, 2H, β-CH2). HR-MS, m/
8.72–8.70 (d, 1H, py-CH, J = 8 Hz), 8.65–8.64 (d, 1H, imz-CH, z, 785.3256 (calculated for 785.18455). FT-IR stretching fre-
J = 4 Hz), 8.28–8.23 (t, 2H, py-CH, J = 10 Hz), 7.76–7.72 (t, 1H, quencies (ν) 2915, 1577, 1482 and 1401 cm−1 and were well-
¯
imz-CH, J = 8 Hz), 7.38–7.35 (d, 2H, Ar-CH, J = 12 Hz), shifted from free Cys and matched with ν of CuI-(Cys).
¯
st
7.13–7.10 (t, 1H, py-CH, J = 6 Hz), 7.04–7.02 (d, 1H, Ar-CH, J = Elemental analysis: calculated for CuC41H36N7O4S: C, 62.62; H,
8 Hz), 6.96–6.92 (t, 1H, imz-CH, J = 8 Hz), 8.68–8.65 (t, 1H, 4.61; N, 12.47. Found as C, 62.60; H, 4.60; N, 12.44.
imz-CH, J = 6 Hz), 3.99–3.97 (d, aryl-OCH3, 6H, J = 8 Hz);
ESI-MS calculated for C20H17N3O2: 331.13 (M+), found: 331.11
(M+). Elemental analysis: calculated for C20H17N3O2: C, 72.49; Conflicts of interest
H, 5.17; N, 12.68%. Found: C, 72.50; H, 5.16; N, 13.66%.
The authors declare no conflicts of interest.
Dimethyl-[4-(1-pyridin-2-yl-imidazo[1,5-a]pyridin-3-yl)phenyl]
amine (L4). Ligand L4 was prepared by the reaction of 4-(di-
methylamino)benzaldehyde (1.25 g, 7.5 mmol), di-pyridin-2-yl-
methanone (0.92 g, 5.0 mmol), and NH4OAc (1.93 g, 25 mmol)
Acknowledgements
upon addition to 25 mL of dry acetic acid. Yellow solid; yield:
We acknowledge the Science and Engineering Research Board
(SERB), New Delhi, Board of Research in Nuclear Sciences
(BRNS), Mumbai and Department of Biotechnology (DBT),
New Delhi, India for funding.
71%; melting range, 176–180 °C; 1H NMR (400 MHz, CDCl3)
δ 8.7–8.65 (d, 2H, py-CH, imz-CH, J = 10 Hz), 8.3–8.28 (d, 1H,
py-CH, J = 8 Hz), 8.23–8.21 (d, 1H, py-CH, J = 8 Hz), 7.75–7.69
(m, Ar-CH, imz-CH, 3H), 7.12–7.10 (d, 1H, py-CH, J = 8 Hz),
6.93–6.85 (m, imz-CH, Ar-CH, 3H), 6.64–6.61 (t, 1H, imz-CH J =
6 Hz), 3.05 (s, aryl-N(CH3)2, 6H); ESI-MS calculated for
References
C20H19N4: 315.16 [M + H]+, found: 315.16 [M + H]+. Elemental
analysis: calculated for C20H18N4: C, 76.41; H, 5.77; N, 17.82%.
Found: C, 76.39; H, 5.75; N, 17.80%.
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These complexes were prepared by following the same pro-
cedure. An illustrative example is provided below for 1.
[Cu(L1)2(H2O)](SO3CF3)2 (1). A acetonitrile (5 mL) solution
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