A Highly Selective and Efficient Copper(II) – “Turn-On” Fluorescence Imaging Probe for l-Cysteine

Article abstract of DOI:10.1002/ejic.201601229

A simple copper(II) complex of the anthracenyl-appended terpyridine ligand 4′-(anthracen-9-yl)-2,2′:6′,2′′-terpyridine (L), [Cu(L)Cl₂] (1, ESI-MS, m/z = 507.08), is reported as a highly selective “turn-on” optical imaging probe for l-cysteine. Probe 1 shows a Cu^II/Cu^Iredox potential [E_1/2= –0.194 V versus normal hydrogen electrode (NHE)] within a biologically viable range. The copper center in 1 adopts a square-pyramidal geometry (τ = 0.0851), and the Cu–N_pybond (1.970 ?) of the middle pyridine (py) ring is shorter than the other two Cu–N_pybonds (2.069 and 2.040 ?). The Cu–Cl bonds (2.444 and 2.027 ?) are labile enough to be replaced by solvent molecules. The square-based geometry is further supported by the A_∥value of 156.8 × 10^–4cm^–1determined by electron paramagnetic resonance (EPR) spectroscopy at 70 K. The d–d transition of 1 in acetonitrile/4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (acetonitrile/HEPES) buffer at pH 7.34 appears at λ =760 nm (ε = 198 m^–1cm^–1), and the ligand-based transitions are observed at λ = 327–384 nm. The ligand shows strong fluorescence at λ = 430 nm with a quantum yield of 11 %, but its emission intensity is completely quenched on coordination with Cu²⁺ions, as in 1. Probe 1 detects l-cysteine selectively through turn-on fluorescence intensity at λ_em= 431 nm with a limit of detection of 1.9 × 10^–8m at pH 7.34. Interestingly, probe 1 is able to visualize exogenously added l-cysteine in Henrietta Lack cervical cancer (HeLa) cells, human liver hepatocellular carcinoma (HepG2) cells, and human embryonic kidney 293 (HEK293) cells under identical conditions at pH 7.4 in confocal microscopy imaging.

Full text of DOI:10.1002/ejic.201601229

A Journal of
Accepted Article
Title: Highly Selective and Efficient Copper(II)-'Turn-on' Fluorescence
Imaging Probe for L-Cysteine
Authors: Mayil Murugan Ramasamy, Maheshwaran Duraiyarasu,
Nagendraraj Thavasilingam, Manimaran Paramasivam,
Ashokkumar Balasubramaniem, and Mukesh Kumar
This manuscript has been accepted after peer review and appears as an
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content of this Accepted Article.
To be cited as: Eur. J. Inorg. Chem. 10.1002/ejic.201601229
Link to VoR: http://dx.doi.org/10.1002/ejic.201601229

10.1002/ejic.201601229
European Journal of Inorganic Chemistry
FULL PAPER
Highly Selective and Efficient Copper(II)-‘Turn-on’ Fluorescence
Imaging Probe for L-Cysteine
Duraiyarasu Maheshwaran,[a] Thavasilingam Nagendraraj,[a] Paramasivam Manimaran,[b]
Balasubramaniem Ashokkumar,[b] Mukesh Kumar[c] and Ramasamy Mayilmurugan*, [a]
Abstract: A simple anthracenyl appended terpyridine copper(II)
complex [Cu(L)Cl2] 1 (ESI-MS, m/z = 507.08) [L= 4'-(anthracen-9-yl)-
2,2':6',2''-terpyridine] is reported as highly selective “turn-on” optical
imaging probe for L-cysteine. The probe 1 shows Cu(II)/Cu(I) redox
potential (E1/2, -0.194 V vs NHE) within a biologically viable range.
The molecular structure of 1 adopts square pyramidal geometry ( =
0.0851) and its Cu-Npy bond (1.970 Å) of middle pyridine is shorter
than other two Cu-Npy bonds (Cu-N, 2.069, 2.040 Å). The Cu-Cl bonds
(2.444, 2.027 Å) are labile enough to replace by solvent molecules.
The square based geometry is further supported by A|| value of 156.8
× 10-4 cm-1 in EPR at 70 K. The d-d transition of 1 appears at 760 nm
(, 198 M-1cm-1) along with the ligand-based transitions around 327-
384 nm in acetonitrile: HEPES buffer at pH 7.34. The ligand shows
strong fluorescence at 430 nm with a quantum yield of 11% but its
emission intensity is completely quenched on coordination with Cu2+
as in 1. The probe 1 detects L-cysteine (Cys) selectively by turn on
fluorescence intensity at em, 431 nm with a limit of detection 1.9 ×
formation.[4] In tumors cell formation, they are translocated into
the cell surface and involving degradation of extracellular matrix
(ECM) leading to malignant cell growth.[5] Recent attentive reports
revealed that cysteine cathepsins, which are overexpressed in a
wide variety of cancers, that produces Cys by breaking disulfur
linkage.[5] So, it is essential to track and locate Cys and its activity
to understand the key role in biological disorders. In addition to
that detection and discrimination of thiol-containing molecules in
cancer biology, is high importance to ensure early stage diagnosis
and treatment of disease.
Among available techniques[6] to detect these thiols,
fluorescence technique receives much attention from researchers
by its simplicity, high sensitivity, and good spatiotemporal
resolution properties.[7-9] Despite, limited reports are available in
the literature based on BODIPY (boron dipyrromethene), cyanine
and other fluorescent units as chemodosimetric sensors for
detecting thiol containing compounds (Cys, Hcy, GSH) without
selectivity.[10,11] On the other hand, only a few reports are available
10-8
M at pH 7.34. Interestingly, probe 1 is able to visualize
exogenously added Cys in Henrietta Lack cervical cancer (HeLa) cells, on Cu2+-based probes, which are appended with anthracene,
Human liver hepatocellular carcinoma (HepG2) cells and Human
Embryonic Kidney 293 (HEK293) cells under identical conditions at
pH 7.4 in confocal microscopic imaging.
coumarin, fluorescein and zwitterionic bipyridine derivative for
detecting amino acids (Histidine, Cys, Hcy) and GSH but none of
them were detecting Cys selectively.[12] There is a little knowledge
concerning the Cu2+-probes for selective Cys detection.
Nevertheless, Cu2+ based probes have received much attention
as ‘turn-on’ fluorescence probes owing to its tunable ‘Off-On’
Introduction
12f]
signaling property.[12a-d,
Due to its intrinsic paramagnetic
properties, Cu2+ (d9) tend to quench the fluorescence of ligand to
“off state” on complexation.[13] The soft S-donor nucleophile in Cys
has preferable tendency to coordinate with Cu2+ and subsequently
reduced into Cu+.[12a-d, 12f] Adopting these chemical and biological
concepts, we have synthesized a new Cu2+-probe based on 4'-
(anthracen-9-yl)-2,2':6',2''-terpyridine as fluorescence sensor for
Cys, which possesses terpyridine as coordination and anthracene
as fluorescence reporter units. Further, we were stimulated to use
this as ‘turn-on’ fluorescent molecular probe for visualizing
upregulated Cys expressions in the cancer cell. Previously, the
metal complexes of this ligand have been explored for the DNA
interaction, antitumor activity, and Cd-based sensor studies.[14] To
our knowledge, our report is the first example of well-defined Cu-
based ‘turn-on’ fluorescent probe for Cys imaging with high
selectivity and no such efforts have been reported in the literature.
Biologically active compounds containing thiol groups such as the
amino acid L-Cysteine, homocysteine (Hcy) and glutathione
(GSH) are playing several important roles in cellular functions
including cell proliferation, antioxidant defense, cell signaling and
maintaining redox homeostasis etc.[1] In particularly Cys plays a
vital role to maintain critical metabolic functions and detoxification
process by acting as intracellular redox buffer.[2] Change in the
level of Cys causes oxidative damage, hematopoiesis, psoriasis,
leukocyte loss, metabolic disorder, cardiovascular and
Alzheimer's disease.[3] Moreover, cysteine cathepsins are
protease family enzymes and located in the intracellular region
and involved in protein degradation and processing in normal cell
[a]
D. Maheswaran, T. Nagendraraj, Prof. Dr. R. Mayilmurugan
Bioinorganic Chemistry Laboratory/Physical Chemistry
School of Chemistry, Madurai Kamaraj University, Madurai 625 021,
Tamil Nadu, India.
Results and Discussion
E-mail: mayilmurugan.chem@mkuniversity.org/
ramamayil@gmail.com
[b]
[c]
P. Manimaran, Prof. Dr. B. Ashokkumar
School of Biotechnology, Madurai Kamaraj University,
Madurai 625 021, Tamil Nadu, India
Prof. Dr. Mukesh Kumar
Solid State Physics Division, Physics Group, Bhabha Atomic
Research Center, Mumbai, Maharashtra, India.
Synthesis and characterization: The ligand L [4'-(anthracen-9-
yl)-2,2':6',2''-terpyridine] was synthesized as published
previously[15] by reaction of 2-acetyl pyridine with 9-anthracene
aldehyde in the presence of NH4OH and KOH in ethanol (scheme
1) and characterized by NMR and ESI-MS (m/z, 410.17). The
copper(II) complex [Cu(L)Cl2] 1 has been isolated as green solid
by reaction of ligand and CuCl2 in methanol/dichloromethane (1:1
Supporting information for this article is given via a link at the end of
the document.
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10.1002/ejic.201601229
European Journal of Inorganic Chemistry
FULL PAPER
v/v) at 50 °C, the yield is 67%. The formation of the complex is
confirmed by ESI-MS (m/z, 507.08) and elemental analysis. The
suitable single crystals of 1 for X-ray diffraction were grown in
acetonitrile by slow evaporation.
Scheme 1. Synthesis of ligand and its copper complex [Cu(L)Cl2] 1.
Molecular structure and EPR spectra: The molecular structure
of 1 was determined by single-crystal X-ray crystallography
(Figure 1a) and crystallographic data together with selected bond
lengths and angles are summarized in Table 1. This complex
crystallizes in orthorhombic system with space group P 21 21 21.
The coordination environment around the copper(II) center is
described as square pyramidal with the trigonality index  of
0.0851 [ = (-)/60, where , N3-Cu1-N1 156.35 and , N2-
Cu1-Cl2 151.24; for perfect square pyramidal and trigonal
bipyramidal geometries the
 values are zero and unity
respectively].[16] The square pyramidal coordination of copper(II)
center is comprised of three nitrogen donors from anthracenyl
terpyridine unit and two chloride ions. All three pyridine nitrogens
and one chloride constitute square of the plan and an axial portion
is occupied by remaining chlorine ion. The Cu-Npy bond (1.970 Å)
of middle pyridine is shorter than other two Cu-Npy bonds (Cu-N,
2.069, 2.040 Å). The axial Cu-Cl1 bond distance (2.444 Å) is
slightly longer than the equatorial Cu-Cl2 bond distance (2.270 Å).
These Cu-Cl bond distances are similar to average Cu-Cl
distances (~2.4 Å) in previously reported copper complexes,
which has been ascertained as weaker.[17] Thus, the Cl- ions in 1
are labile enough to replace by solvent molecules followed by
analyte during optical imaging (cf. below). The sum of bond
angles N3-Cu1-N2 (79.07), N2-Cu1-N1 (78.35), N1-Cu1-Cl2
(98.13) and Cl2-Cu1-N3 (98.24) is 353.79 and closer to 360
suggesting that N1, N2, N3 and Cl2 are nearly coplanar. The N1-
Cu-N3 (156.35) and N2-Cu-Cl2 (151.24) bond angles are
deviated from the 180, indicating distortion in the square plane.
The square based geometry of 1 is further supported by EPR
spectral signatures (Figure 1b). The frozen solution spectra of 1
in methanol/DMF mixture at 70 K was found to be anisotropic and
its hyperfine features due to copper have been resolved in the
parallel region (g|| = 2.25, g⊥ = 2.05) (Table 2). The EPR spectrum
comprises of g values in the order of g|| > g⊥ > 2.0023 which
corresponds to normal EPR spectrum for copper(II) center with
Figure 1. The molecular structure of 1 (50% probability factor for thermal
ellipsoids) and hydrogen atoms are omitted for clarity (a). X-band EPR spectra
of 1 ethanol/DMF mixture at 70 K (b).
Table 1. Crystal data and structure refinement and selected bond length (Å)
and bond angles () of 1
empirical formula
fw
crystal system
space group
a (Å)
b (Å)
c (Å)
 (deg)
C29H19Cl2CuN3
543.91
Bond lengths and Bond anglesc
Cu(1)-N(1)
Cu(1)-N(2)
Cu(1)-N(3)
Cu(1)-Cl(1)
Cu(1)-Cl(2)
2.069(5)
1.970(4)
2.040(5)
2.4443(16)
Orthorhombic
P 21 21 21
11.2901(4)
13.9876(7)
16.3642(4)
90
2.2700(16)
90
90
N(2)-Cu(1)-N(3)
N(2)-Cu(1)-N(1)
N(3)-Cu(1)-N(1)
79.07(18)
78.35(19)
156.35(19)
 (deg)
 (deg)
3
2584.26(17)
V (Å )
T (K)
Cu K λ, (Å)
293(2)
1.54184
1.398
N(2)-Cu(1)-Cl(2) 151.24(13)
N(3)-Cu(1)-Cl(2) 98.24(14)
N(1)-Cu(1)-Cl(2) 98.13(15)
3
density (Mg/m )
Z
4
N(2)-Cu(1)-Cl(1) 105.37(13)
N(3)-Cu(1)-Cl(1) 97.95(14)
N(1)-Cu(1)-Cl(1) 94.74(14)
Cl(2)-Cu(1)-Cl(1) 103.36(7)
 (mm-1)
F(000)
3.264
1108
5838
no. of reflections
square based geometry. Further, the
A||
value of
collected
156.8 × 10-4 cm-1 suggested that complex 1 belongs to square
pyramidal geometry (A||= 154-162 × 10-4 cm-1 for square based
geometry[18]).
2
1.040
GOF on F
R1a
0.0505
0.1384
wR2b
aR1 = Σ||Fo|-|Fc||/Σ|Fo|; bwR2 = [Σw(Fo2-Fc2)2/Σw(Fo2)2]1/2
cStandard deviations in parenthesis.
.
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10.1002/ejic.201601229
European Journal of Inorganic Chemistry
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Electronic absorption spectral studies: The complex 1 exhibits
d-d transition at 760 nm (ε, 198 M-1cm-1) along with merged ligand
based transitions at the concentration of 1 × 10-3 M in acetonitrile:
HEPES buffer at pH 7.34 (Table 2, Figure S4). However ligand
based transitions such as π-π* (334 nm; ε, 54,280 M-1cm-1) of
terpyridine and π-π* transitions of anthracene moiety at 348 nm
(ε, 44,740 M-1cm-1), 366 nm (ε, 58,620 M-1cm-1) and 385 nm (ε,
54,160 M-1cm-1) are well resolved only at lower concentration (5 ×
10-6 M).[19] The formation and stability of 1 under the biological
relevant concentration of Cu2+ ion (1.06 ± 30 µg/mL in healthy
adults[20]) was monitored by UV-Vis spectral method. Upon
addition of Cu2+ ion to ligand (5 × 10-6 M) in acetonitrile: HEPES
buffer at pH 7.34, the ligand-based transitions at 334 nm
disappeared and new bands at 329 nm and 342 nm appeared.
However, the d-d transition is not perceived at this biologically
relevant lower concentration.
biological concentration of the Cu2+ ion. This formation is further
confirmed by ESI-MS analysis and shows a molecular ion peak
m/z = 507.08 corresponds to [Cu(L)Cl]+. On addition of various
amino acids to 1, only Cys reproduces the absorption pattern of L
and all other amino acids show no changes under identical
conditions. The interaction of Cys to
1 as a function of
concentration was studied by adding several portions of Cys and
reveal that the bands at 329 and 342 nm were decreased
gradually to regenerate the band at 334 nm with a clear isosbestic
point at 317 nm (Figure 2b). This observation clearly showed that
only Cys is able to regenerate absorption pattern of ligand
possibly by coordination with copper(II) center followed by
reduction into Cu(I) and subsequently released from coordination
sphere. Further, the addition of GSH to 1 slightly enhances the
intensity of bands around 366 and 385 nm and this is possibly due
to the interaction of GSH with copper(II) without releasing ligand
(S5a).[21] Nevertheless, none of the amino acids altered the
absorption spectral pattern of 1 even under conditions of 20-fold
excess. In addition to that, the absorption spectrum of 1 is not
altered by the addition of twenty equivalents of biologically
2-
relevant anions (PO4 , SH-) and cations (Na+, K+, Ca2+, Mg2+
,
Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Zn2+) at pH, 7.34. Thus, probe 1 is
stable enough for imaging without any transmetalation by
endogenous metal ions at this pH (Figure S5b).
Fluorescence spectral studies: The ligand shows strong
fluorescence at 430 nm with a quantum yield of 11% in
acetonitrile:HEPES buffer (7:3, v/v) solution at pH, 7.34 (5 × 10-6
M). Interestingly, this fluorescence intensity was quenched on the
coordination of ligand with copper(II) center to obtain 1 and it
became non-fluorescent. This is further confirmed by the
immediate quenching of fluorescence intensity of ligand while
adding one equivalent Cu2+ ion (Figure S6a). It is believed that
this ‘turn-off’ behavior arises due to the paramagnetic nature of
copper(II) center strongly disturbed the electronic resonance in
the ligand, which results in lowering of the fluorescence intensity.
It is supported by HOMO-LUMO electron density map obtained
by DFT method using B3LYP 6-31G (for C, H, and N)/LANL2DZ
(for Cu) basis set in Gaussian 09 program (Table 2, Figure 3).[22]
The frontier molecular orbitals for ligand and complex were
obtained from the optimized geometries, for the ligand, HOMO,
and LUMO are localized on the anthracenyl moiety. However, for
1, HOMO is localized on copper and chlorides. But, LUMO is
localized on terpyridine unit, copper, and chlorides. These results
clearly indicate that electronic resonance of anthracenyl moiety is
disrupted by Cu2+ center via internal charge transfer, which results
in quenching of fluorescence and change in absorption spectral
pattern of ligand. Thus, the electron density is localized only on
anthracenyl moiety for the free ligand and shifted around
copper(II) center for 1 due to its paramagnetic nature.
Figure 2. UV-Visible spectral changes over addition of Cu2+ ion into L (5 × 10-6
M) inset: plot of [Cu2+] Vs Absorbance (5 × 10-6 M) in acetonitrile:HEPES buffer
(pH 7.34) at 25C (a), addition Cys into 1 (5 × 10-6 M) inset: plot of [Cys] Vs
Absorbance at 334, 348, 366 and 385 nm (b).
The fluorescence behavior of 1 with various amino acids
was studied by adding 20 equivalents of them in separate
experiments. Interestingly, non-fluorescent 1 is ‘turned on’ its
fluorescence intensity only upon the addition of Cys, whereas it
remains non-fluorescent with all other amino acids. However,
glutathione, homocysteine, and L-histidine show very less
fluorescence enhancement with 20 equivalents and are negligible
Further, upon gradual addition of Cu2+ ion to the ligand, the
new bands at 329 and 342 nm gradually raised and the bands at
366 and 386 nm were decreased gradually with an isosbestic
point at 312 nm (Figure 2a), indicates the formation of 1 under the
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10.1002/ejic.201601229
European Journal of Inorganic Chemistry
FULL PAPER
compared to intensity raised by Cys (Figure 4, S7) under identical
conditions. Interestingly, glutathione and homocysteine were not
able to raise the fluorescent intensity as comparable with Cys
even though they possess thiol group. This is possibly due to the
differences in the pKa values of GSH (9.2), Hcy (10.0) and Cys
(8.3) [22] and the higher pKa value of GSH and Hcy is expected to
encourage weaker coordination with 1 than Cys.[24] Further, no
enhancement of fluorescence intensity is observed on adding
biological anions such as SH-, phosphate to 1. Also, the
Cys solution to 1, the fluorescence intensity is raised gradually
and it is found as linear (Figure 5b).
fluorescence intensity is not enhanced by treating
1 with
biologically relevant cations at pH, 7.34 (Figure 4b). This clearly
indicates that no transmetalation is possible in probe 1 by
biologically relevant cations.[25] The turn-on fluorescence behavior
of 1 with Cys found as 182 times higher than that of 1 and
quantum yield is 6%. This indicates that the probe 1 is able to
detect Cys at a concentration of 5 × 10-6 M, as evident from the
enhancement in fluorescence intensity. This enhancement is
saturated at seven equivalents of Cys and further addition of Cys
remains unchanged in intensity up to 10 equivalents (Figure S6b).
Figure 4. Fluorescence spectra of 1 (5 × 10-6 M) at upon addition of various
amino acids (1 ×10-4 M) in acetonitrile:HEPES buffer (7:3, v/v), pH, 7.34 at 25C
(a). Inset: Fluorescence spectra of L, 1 and 1 + Cys. Bar diagram for the
selectivity of various amino acids and biologically relevant ions (1 × 10-4 M ) (ex
= 366nm) (b).
The binding constant K was calculated from the linear plot as 7.7
× 104 M-1 by using Benesi-Hildebrand expression, I0/I-I0 = b/(a –b)
{1/(K[M]) + 1} where, I0 and I are intensities in the absence and
presence of analyte respectively, [M] is the concentration of
analyte, a and b are intercept and slope of the plot respectively.
The linear plot was obtained by fitting (I0/I-I0) vs 1/[M] (Figure
S9).[12d] The limit of detection was calculated as 1.9 × 10-8 M by
employing the formula [3 × (Standard deviation/slope)] from the
linear plot. These fluorescence studies revealed that 1 is highly
sensitive towards Cys and it can detect Cys even at lower level.
The time-dependent fluorescence spectrum of 1 with Cys was
obtained by monitoring fluorescence changes at 431 nm over the
period of 70 minutes. The decrease in fluorescence intensity at
431 nm is not rapid (Figure S11) and sustains its fluorescence
intensity over this period. Further, the stability of 1 was studied
Figure 3. Frontier orbitals of L, HOMO (a) and LUMO (b); 1, HOMO (c) and
LUMO (d) are calculated using B3LYP 6-31G/LANL2DZ level.
The competitive experiments with 1 and 20 equivalents of
various amino acids and biological anions SH-, phosphate are
performed and found that fluorescence intensity is not elevated.
However, it is readily raised by the addition of one equivalent of
Cys (Figure 5a) to this solution. Thus, the fluorescence intensity
of probe 1 is ‘turned-on’ selectively by Cys and other amino acids
remain silent. Also, both essential and non-essential amino acids
and biological anions such as SH-, phosphate are not interfering
with the detection of Cys. Upon addition of subsequent aliquots of
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10.1002/ejic.201601229
European Journal of Inorganic Chemistry
FULL PAPER
with 20 equivalents of well-known exogenous bidentate
(bipyridine and 1,10-phenonthroline) and polydentate (EDTA =
ethylenediaminetetraacetic acid) ligands by absorption and
emission spectral methods (Figure S5d and S8b). The 20
equivalents of bipyridine and 1,10-phenonthroline showed slight
changes in absorption and emission spectral patterns of 1 but
EDTA (20 equivalents) exhibited a noticeable enhancement in
fluorescent intensity under identical conditions. However, one
equivalent of EDTA exhibits only slight changes in absorption and
emission spectral patterns, which is indicative of probe 1 is stable
enough for imaging without significant displacement of copper
from the coordination sphere. There are no significant changes in
emission spectra of 1 at pH range 4.5-9.5. On the other hand, the
addition of Cys to 1 at pH 6.5 shows a drastic decrease in ‘turn
on’ emission intensity and further decreased on lowering pH to
5.5 and 4.5. The emission intensity has been slightly increased
on raising pH to 8.5 and 9.5. (Figure S10). Thus, the ‘turn on’
emission intensity is efficient around pH 7.34 for the detection of
Cys by 1.
Electrochemical studies and mechanism: The redox behavior
of 1 was investigated by cyclic voltammetry (CV) and differential
pulse voltammetry (DPV) using
a
three-electrode cell
configuration in acetonitrile:HEPES buffer (7:3, v/v) solution at
pH, .34 and NaCl was used as supporting electrolyte. A platinum
sphere, a platinum wire, and Ag/Ag+ were used as working,
auxiliary and reference electrodes respectively. The obtained
redox potential was converted into NHE (normal hydrogen
electrode) by adding +0.205 V. The complex 1 shows irreversible
Cu(II)/Cu(I) redox couple at E1/2, -0.194
V vs NHE. The
Cu(II)/Cu(I) redox potential of 1 is well within biological ranges and
hence presumably causes no oxidative damage at intra-
/extracellular space. In healthy differentiated cells, the
glutathione/glutathione disulfide (GSH/GSSG) redox couple is
approximately at -0.22 V vs NHE but becomes more reducing in
proliferating cells (-0.26 V), which corresponds to a higher GSH
to GSSG ratio. The extracellular redox potential of the
cysteine/cysteine (CySH/CySSCy) couple is typically at -0.080 V
whereas pathologic cells demonstrate CySH/CySSCy potentials
ranging from -0.062 to -0.02
V
in extracellular space.[26]
Interestingly, Cu(II)/Cu(I) redox couple of 1 is readily disappeared
on the addition of four equivalents Cys and new redox couple
appeared around E1/2, 0.031V vs NHE (Table 2, Figure 6, S12).
This is possibly due to the reduction of Cu(II) to Cu(I) by Cys and
this redox process is presumed to involve
a significant
geometrical reorganization from the internal changes in bond
lengths and angles of the complex.[27] The above redox potential
is identical to that of observed for in situ generated species by
treating Cu2+ with Cys (Figure 11c).
The solution containing mixture of 1 with one equivalent of
Cys was analyzed by ESI-MS and shows the molecular ion peak
at 410.10 corresponds to ligand and other fragment patterns at
m/z value of 507.02, 840.82 and 880.84 are corresponding to
[Cu(L1)Cl], [Na(L1)2] and [Cu(L1)2] respectively (Figure S18a).
Figure 6. Electrochemical titration of 1 with Cys (0 to 4 equivalents) (5 × 10-6 M)
in acetonitrile:HEPES buffer (pH 7.34) at 25C [reference: saturated Ag/Ag+;
supporting electrolyte: 0.1M NaCl solution; scan rate: = 50 mV s-1].
Figure 5. Competitive binding experiments of 1 (5 × 10-6 M) with various amino
acids (1 × 10-4 M ) in the absence (black) and presence of Cys (1 eq.) (a),
Changes in fluorescence spectra of 1 by varying concentration of Cys (5 × 10-7
M to 5 × 10-6 M) at 25C, pH 7.34 in acetonitrile:HEPES buffer (7:3) v/v. [ex
=
366nm, slits: 5nm/5nm]. Inset: linear fit for the addition (b).
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10.1002/ejic.201601229
European Journal of Inorganic Chemistry
FULL PAPER
occurs via two consecutive steps, firstly the copper(II) center is
reduced to copper(I) by Cys as expected. Once reduced, the
copper(I) ion is apparently released from coordination sphere,
owing to its preference for tetrahedral coordination geometry[27]
but the rigid terpyridine ligand system is unable to adopt that
preference (Scheme 2). Then the electron density of L is possibly
regenerated around the fluorophore for emission. Further, the
Cu(II)/Cu(I) redox potential of 1 is much lower than a reported
redox barrier for adopting tetrahedral geometry as known in the
blue copper proteins (E1/2, 0.276 - 0.307 V).[29]
Table 2. Electronic spectral, redox and computational data
1
1 + Cys
L
334
(54,280)
348
(44,740)
366
(58,620)
385
327 (58,840)
341 (63,300)
364 (29,780)
384 (25,240)
760 (198)*
332 (28,240)
349 (29,900)
366 (39,800)
385 (37,040)
Absorption spectraa
λmax (nm), (ε, M-1 cm-1)
(54,160)
Electrochemical datab
Epa (V)
Epc (V)
-0.5203
-0.2785
0.116
0.178
CV
E1/2(V)
-0.399
0.147
DPV
-0.2273
240
0.1519
62
∆E (mV)
EPR spectra
g||
2.25
g value
g⊥
2.05
A|| (cm-1)
Computational detailsd
(eV)
156.8 × 10-4
Scheme 2. Proposed mechanism for Cys detection by 1.
Optimized energy
-3.5304 × 104
-5.2513
-4.1001 × 104
-5.4069 (α spin)
-5.363 (β spin)
-2.717 (α spin)
-2.981 (β spin)
2.689 (α spin)
2.2.38 (β spin)
HOMO
LUMO
Cell imaging studies: The optical imaging probe 1 detects Cys
selectively over all amino acids in the isolated chemical
environment. Therefore, we further envisioned to use this
molecular probe as ‘turn-on’ fluorescent tool for Cys in cancer
cells in vitro at biological pH, 7.4. The probe 1 is containing a
fluorescent unit that provides suitable excitation and emission
wavelengths as well as brightness for Cys imaging. Laser
scanning confocal microscopy experiment was performed at
excitation wavelength 365 nm to elucidate the Cys imaging
capability of 1 in living cells (Figure 7). The Cys was visualized in
the two cancer and one tumorigenic cell line of different origin
such as Henrietta Lack cervical cancer (HeLa), Human liver
hepatocellular carcinoma (HepG2) and Human Embryonic Kidney
293 (HEK-293) cells under identical conditions. The monolayer of
all three cells was grown on cover glass Petri dishes for
fluorescence and bright field images. The probe 1 (5 µM) was
treated with HeLa cells and it was able to visualize Cys with
fluorescence images of less intensity (Figure 7). Further, HeLa
cells were pre-treated with 100 µM of Cys for 30 minutes and
incubated with 1 (5 µM) for 30 minutes and exhibit bright
fluorescence images (Figure 7). However, pre-treatment of a
known thiol blocking agent (200 µM) N-ethylmaleimide (NEM) to
HeLa cells prior to the addition of 1 (5 µM) showed no significant
fluorescence behavior (Figure 7). Further, Cys imaging of other
cell lines such as HepG2 and HEK-293 were also examined in
order to compare efficiency and specificity of 1. Thus, HepG2 and
HEK-293 cells were treated with 1 under identical conditions as
described previously. Both the cell lines showed fluorescence
nature but the brightness of images are less compared to that of
obtained from HeLa cells (Figure S19). Meanwhile, treatment of
200 M of NEM and 5 M of 1 to HepG2 and HEK-293 cells were
showed the fluorescence images with negligible intensity. All
these cell imaging studies revealed that 1 can enter into the
cancer cell simultaneously and “turn-on” fluorescence intensity by
detecting Cys. The probe 1 directly interacts with Cys by transitory
through cell membranes under at biological pH, 7.4 and its
-1.7209
3.5304
Energy gap
TD-DFT
Optimized energy
HOMO
-4.1001 × 104
-5.398 (α spin)
-5.363 (β spin)
-2.68 (α spin)
-2.981 (β spin)
2.68 (α spin)
LUMO
Energy gap
2.382 (β spin)
aConcentration of 5 × 10-6 M in acetonitrile:HEPES buffer (pH 7.34) at 25C.
*Concentration of 1 × 10-2 M in acetonitrile:HEPES buffer (pH 7.34) at 25C.
bConcentration of 5 × 10-6 M in acetonitrile:HEPES buffer (pH 7.34) at 25C.
[reference: saturated Ag/Ag+; supporting electrolyte: 0.1M NaCl solution;
scan rate: = 50 mV s-1]. To convert E1/2 vs NHE add +0.205 V.
cMethanol/DMF mixture at 70 K .
dDFT method using B3LYP 6-31G (for C, H and N)/LANL2DZ (for Cu)
basis set in Gaussian 09 program.
The FT-IR analysis of above solution exhibits stretching
frequencies (ῡ), at 3030, 1583, 1487 and 1408 cm-1 and are
matched with stretching frequencies of in situ generated species
from the reaction of Cu2+ and Cys (Figure S18b).[28] The ESI-MS
and FT-IR studies reveal that presumably copper displaced from
coordination sphere and releases the free ligand as noticed in the
electronic spectra and electrochemistry. Furthermore, the in situ
generated copper(I) complex [Cu(L)]+ 2 (formation was confirmed
by ESI-MS analysis, m/z, 475. 06 and 1HNMR) exhibited a
fluorescence spectrum at same energy with 31% decreased
intensity than the ligand (Figure S17).
The 1HMR studies for the reactions of 1 and 2 with Cys
shows no significant changes in the spectral pattern (Figure S13,
S14, and S15) of complexes and Cys. Also, there are no new
1HMR signals noticed for the oxidized product of Cys. The
enhancement in fluorescence intensity of 1 with Cys possible
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10.1002/ejic.201601229
European Journal of Inorganic Chemistry
FULL PAPER
copper(II) center reduced to copper(I). Finally, copper(I) is
decoordinated and releases fluorescent ligand, which presumed
to shows substantial turn-on fluorescence intensity (Figure 7,
S13). This mechanism is supported by detailed electronic
spectroscopic, redox and mass spectrometric measurements (cf.
above). So, the probe 1 is readily response to Cys at pH 7.4 in
HEPES-buffered solutions indicates that imaging is direct and
efficient towards Cys in cancer cells. Interestingly, probe 1 (5 M)
shows no significant changes in cell structure and morphology.
turned-on. Furthermore, The Cys has been visualized in the two
cancer and one tumorigenic cell lines of different origin such as
HeLa, HepG2 and HEK-293 cells under identical conditions by
confocal microscopy.
Experimental Section
Materials: Unless otherwise indicated, common reagents or materials
were obtained from the commercial source and used without further
purification. All the solvents were used after appropriate distillation or
purification.
The
chemicals
such
as
2-acetylpyridine,
9-
anthracenecarboxyaldehyde, copper(II) chloride, 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 purchased from Sigma-Aldrich. Sodium
hydroxide and ammonium hydroxide were purchased from Merck.
Physical Measurements: All reactions were carried out under an
atmosphere of dry nitrogen. Glassware was oven dried prior to use. Unless
otherwise indicated, common reagents or materials were obtained from
the commercial source and used without further purification. All the
solvents were used after appropriate distillation or purification. NMR
spectra were recorded at 300 MHz on a Bruker spectrometer. Chemicals
shifts values and coupling constants are given in ppm and Hz respectively.
ESI-MS spectra were measured on
a Thermo LC-MS instrument.
Absorption spectra were carried out using Agilent diode array 8453
spectrometer at 298 K and Emission spectra were measured on Agilent
Cary Eclipse Spectrofluorometric at 298 K. Electrochemical data were
recorded in Biologic SP-150 Electrochemical workstation using saturated
Figure 7. Confocal fluorescence and bright field images of HeLa cells: Cells in
the absence of 1 (a); Cells incubated with 1 (5 M) for 30 minutes without
treatment of Cys (b); Cells pretreated with 100 M Cys and incubated with 1 (5
M) for 30 minutes (c); Cells pretreated with 200 M NEM and incubated with 1
(5 M) for 30 minutes (d).
Ag/Ag+ and Pt as
respectively. The 0.1 M NaCl is used as supporting electrolyte. The
electrodes are calibrated by K4[Fe(CN)6] prior to measurements.
a reference electrode and working electrodes
Elemental analyses were carried out using a Heraeus Vario Elemental
automatic analyzer. IR spectra were taken in Shimadzu FT-IR instrument.
Electron paramagnetic resonance (EPR) measurements were recorded
using Bruker EMX Plus EPR Spectrometer. Confocal microscopic
experiments were carried out in CARL ZEISS (CLSM 710) instrument.
Conclusions
X-ray Structure Determination: Single crystal 1 of suitable size was
picked from the mother liquor, coated with paraffin oil and mounted on a
fiber loop for X-ray diffraction data collection on Agilent Supernova system
(Cu-Kα radiation) equipped with Titan CCD detector. Data processing,
including face based absorption correction, was carried out using
CrysAlisPro software suites. The structure was solved by the direct method
using Shelxs and refined using Shelxl.[30] All the non-hydrogen atoms were
refined anisotropically, whereas the hydrogen atoms were added at the
riding positions and refined isotropically. CCDC-1440688 contain the
supplementary crystallographic data for this paper. This data can be
obtained free of charge from The Cambridge Crystallographic Data Center
via ww.ccdc.cam.ac.uk/data request/cif.
A simple copper(II) complex of anthracenyl appended terpyridine
ligand has been synthesized as a probe for Cys imaging in cancer
cells. The molecular structure of probe adopts square pyramidal
geometry around copper(II) center and it is further supported by
EPR spectral data. The Cu(II)/Cu(I) redox potential of the probe (-
0.194 V vs NHE) is well within biologically range. The d-d and
ligand-based transitions are appeared at 760 and 327-384 nm
respectively in acetonitrile: HEPES buffer at pH 7.34. The probe
showed a selective and efficient “turn-on” fluorescence behavior
towards Cys over other amino acids at pH, 7.4 with the binding
constant of 7.7 × 104 M-1 and limit of detection is 1.9 × 10-8 M. The
redox, ESI-Mass, IR and 1HNMR studies revealed that the probe
showed bright visible-light emission on reacts with Cys by
reduction of copper(II) center to copper(I) and subsequent
displacement of copper. Because, the rigid terpyridine ligand
system unable to adopt preferable tetrahedral coordination
geometry of copper(I) which leads to collapsing of coordination
sphere, subsequently original fluorescence intensity of ligand is
DFT Methods: The geometry optimizations were performed by using
density functional theory (DFT) methods. For the metal ion involving
systems using LANL2DZ basis sets with the Becke3–Lee–Yang–Parr
hybrid functional (B3LYP) and the 6−311G (d) basis sets used for
elements other than metals. All DFT calculations were carried out by using
the Gaussian 09 program package.[31]
This article is protected by copyright. All rights reserved.

10.1002/ejic.201601229
European Journal of Inorganic Chemistry
FULL PAPER
Confocal Laser Scanning Microscopy (CLSM): The human-derived
cervical cancer HeLa cell line, Human liver hepatocellular carcinoma
(HepG2) cell line and Human Embryonic Kidney 293 (HEK-293) cell line
were grown and maintained in Dulbecco's modified Eagle's medium
(DMEM) containing 10% FBS, glutamine (0.29 g/L), sodium bicarbonate
(2.2 g/L), penicillin (100,000 U/L), and streptomycin (10 mg/L) in an
atmosphere of 5% CO2-95% air at 37 °C. For confocal imaging, a
monolayer of HeLa cells, HepG2 cells HEK-293 were grown on cover glass
Petri dishes for fluorescence and bright field images. Briefly, all three cells
grown in DMEM were washed with PBS solution and treated with cysteine
(100 M), complex (5 M) was added incubated for 30 minutes. For N-
ethylmaleimide (NEM) treatment, NEM (200 M) and complex (5 M) were
incubated for 30 minutes subsequently. The cells were fixed by using 4%
paraformaldehyde incubated at 37 °C for 20 minutes and mount with DPX
for imaging using a Confocal Laser Scanning Microscopy (Carl Zeiss LSM
710, Germany). Fluorophores were excited using 365 nm line from an
argon ion laser, and emitted fluorescence was monitored at 440±2 nm.
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Acknowledgements
We acknowledge Department of Science and Technology (DST)
and Department of Biotechnology (DBT), New Delhi for funding.
DM thanks to University Grants Commission (UGC), New Delhi
for fellowship. We also thank DST-PURSE program at the
department of microbiology, Bharathidasan University,
Tiruchirappalli, Tamil Nadu, India for providing the confocal
microscopic imaging facility.
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Keywords: Copper(II) probe • Cysteine imaging • Cell imaging
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10.1002/ejic.201601229
European Journal of Inorganic Chemistry
FULL PAPER
Entry for the Table of Contents
FULL PAPER
D. Maheshwaran, T. Nagendraraj, P.
Manimaran, B. Ashokkumar, Mukesh
Kumar, R. Mayilmurugan*
Page No. – Page No.
Title
Highly selective “turn-on” optical imaging copper(II) probe based on anthracenyl
terpyridine is reported. This detects L-cysteine (Cys) selectively at 431 nm with a limit
of detection 1.9 × 10-8 M at pH 7.34. The Cys in HeLa, HepG2, and HEK293 cells is
efficiently visualized by confocal microscopic imaging using this probe.
This article is protected by copyright. All rights reserved.