Copper(II)-benzimidazole complexes as efficient fluorescent probes for l-cysteine in water

Article abstract of DOI:10.1039/c7dt01895a

Copper(ii) complexes [Cu(L1)(H₂O)₂](SO₃CF₃)₂1 and [Cu(L2)(H₂O)₂](SO₃CF₃)₂2 based on 2,6-bis(benzimidazolyl)pyridine were synthesized and are reported herein as highly selective "turn-on" optical probes for l-cysteine. The Cu(ii)/Cu(i) redox potential of probe 1 (0.14 V vs. NHE) was lower than that of 2 (0.233 V vs. NHE) in water. The molecular structure of 2 adopted a square pyramidal geometry (τ = 0.2545), with the Cu-N_py bond (1.958 ?) of its middle pyridine unit being shorter than the other two Cu-N_benzim bonds (Cu-N, 1.995, 2.000 ?). The axial Cu-O2 bond distance (2.247 ?) was slightly longer than the equatorial Cu-O1 bond distance (1.953 ?). The square-based geometry was further supported by the A_∥ value of 156 × 10^-4 cm^-1 in EPR at 70 K. The d-d and ligand-based transitions appeared at 662 and 314-356 nm for 1 and 651 and 313-360 nm for 2, respectively, in HEPES buffer at pH 7.34. These probes showed selective and efficient "turn-on" fluorescence behaviour towards Cys over other natural amino acids with a binding constant for 1 of 5.4 × 10⁴ and 1.30 × 10⁴ M^-1 for 2 and a limit of detection of 2.9 × 10^-8 M and 3.32 × 10^-8 M, respectively, for 1 and 2 at pH 7.34. The quantum yield for the detection of Cys by 1 (14.7%) was much lower than by 2 (23%). The fluorescence intensity of 1 and 2 were also slightly enhanced by histidine, but at a relatively lower level than that exhibited by Cys.

Full text of DOI:10.1039/c7dt01895a

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Submitted to : Dalton Trans. (Revised)
Manuscript ID: DT-ART-05-2017-001895.R1
Copper(II)-Benzimidazole Complexes as an Efficient Fluorescent
Probe for L-Cysteine in Water
Duraiyarasu Maheshwaran, Selvarasu Priyanga and Ramasamy Mayilmurugan*
Bioinorganic Chemistry Laboratory/Physical Chemistry, School of Chemistry, Madurai Kamaraj
University, Madurai 625 021, Tamil Nadu, India.
*Eꢀmail: mayilmurugan.chem@mkuniversity.org / ramamayil@gmail.com
Abstract:
The
copper(II)
complexes
[Cu(L1)(H O) ](SO CF )
1
and
2
2
3
3 2
[
Cu(L2)(H O) ](SO CF ) 2 based on 2,6ꢀbis(benzimidazolyl)pyridine have been synthesized
2
2
3
3 2
and reported as highly selective “turnꢀon” optical probe for Lꢀcysteine. The Cu(II)/Cu(I) redox
potential of the probe 1 (0.14 V vs NHE) is lower than of 2 (0.233 V vs NHE) in water. The
molecular structure of 2 adopts square pyramidal geometry (τ = 0.2545) and its CuꢀN bond
py
(1.958 Å) of middle pyridine is shorter than other two CuꢀNbenzim bonds (CuꢀN, 1.995, 2.000 Å).
The axial CuꢀO2 bond distance (2.247 Å) is slightly longer than the equatorial CuꢀO1 bond
ꢀ4
distance (1.953 Å). The square based geometry is further supported by A value of 156 × 10
||
ꢀ1
cm in EPR at 70 K. The dꢀd and ligandꢀbased transitions appear at 662 and 314ꢀ356 nm for 1
and 651 and 313ꢀ360 nm for 2 respectively in HEPES buffer at pH 7.34. These probes showed a
selective and efficient “turnꢀon” fluorescence behavior towards Cys over other natural amino
4
4
ꢀ8
acids with the binding constant (1, 5.4 × 10 ; 2, 1.30 × 10 ) with a limit of detection 2.9 × 10 M
ꢀ8
and 3.32 × 10 M respectively at pH, 7.34. The quantum yield for the detection of Cys by 1
14.7%) is much lower than by 2 (23%). The fluorescence intensity of 1 and 2 have been also
(
slightly enhanced by histidine but it is relatively lower than that of exhibited by Cys.
1

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Introduction
Amino acids play an important role in biological systems, among the twenty amino acids,
cysteine is acting as powerful detoxifiers and active site for several metalloenzymes in the body
1
fluids. Also, it involves in maintain critical metabolic functions by acting as intracellular redox
2
buffer. The change in the level of Cys causes oxidative damage, hematopoiesis, psoriasis,
3
leukocyte loss, metabolic disorder, cardiovascular and Alzheimer's disease. Moreover, the
protease family enzymes cysteine cathepsins are translocated into the cell surface and involving
4
degradation of extracellular matrix (ECM) leading to malignant cell growth. Thus, abnormal
level of this amino acids is indicating a variety of diseases such as chronic kidney disease,
hematopoiesis decrease, leucocyte loss, psoriasis, pulmonary diseases, stress, psychological
5
disorders, etc. The level of amino acid Cys has to be determined to avoid such complications.
So the detection of amino acid can be developed by various methods such as spectroscopic,
5
c,5d,6
chromatographic and electrochemical methods.
Several chemodosimetric sensors have been reported for detecting thiol containing
5
b, 7
compounds (Cys, Hcy, GSH) without selectivity.
However, only a limited reports are
2
+
available on Cu ꢀbased probes which include anthracene, coumarin, a zwitterionic
chromophore, fluorescein and other fluorophores for detecting amino acids (Histidine, Cys, Hcy)
8
and GSH without selectively. Very recently, we have developed a copper(II)ꢀbased probe for
9
2+
Cys detection with an excellent limit of detection and selective. These Cu ꢀbased probes have
received much attention as ‘turnꢀon’ fluorescence probes owing to its tunable ‘OffꢀOn’ signaling
8
2+
9
property. Due to its intrinsic paramagnetic property of Cu (d ) which tend to quench the
1
0
fluorescence intensity of ligand to “off state” on complexation. The soft Sꢀdonor nucleophile in
2
+
+ 8
Cys has preferable tendency for the coordination with Cu and subsequently reduced into Cu .
2
+
In this report, we have synthesized and studied Cu ꢀprobes based on 2,6ꢀ
bis(benzimidazolyl)pyridine as ‘turnꢀon’ fluorescent probe for LꢀCysteine. This probing
mechanism has established by spectral, redox and geometrical changes by DFT studies on
interaction with amino acid Cys. The ligand 2,6ꢀbis(benzimidazolyl)pyridine has a very similar
ligand architecture to our previously reported terpyridineꢀbased ligand and two benzimidazole
11
arms replaces pyridine arms. Previously, the Cu(II), Zn(II, Au(III) and lanthanides coordination
1
2ꢀ16
complexes of this tridentate ligand have been extensively studied
for DNA interaction and
2

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1
7
validation with cancer cells, detection of copper(II) ions and detection of pyrophosphate.
However none these studies focused on detection of biomaterials and metabolites.
Results and Discussion
Synthesis and characterization: The ligands L1 and L2 were synthesized according to
1
1
the previously published method by the reaction of a 2,6ꢀpyridineꢀdicarboxylic acid and 1,2ꢀ
diaminobenzene in the presence of polyphosphoric acid (PPA) at 230 °C under nitrogen. The
methylation of L1 using methyl iodide in the presence of KOH in acetone results the formation
of L2 and it is characterized by NMR and ESIꢀMS (m/z, 340.44). The copper(II) complexes have
been isolated as green solid by the reaction of Cu(SO CF ) with ligands L1 and L2 in methanol
3
3 2
at 55 °C. The formation of complexes was confirmed by HRꢀESIꢀMS and formulated as
Cu(L1)(H O) ](SO CF ) 1 and [Cu(L2)(H O) ](SO CF ) 2 (Figure S1). This is further
[
2
2
3
3 2
2
2
3
3 2
supported by the single crystal Xꢀray structure of 2. The suitable single crystal of 2 for Xꢀray
diffraction has been grown in the acetonitrileꢀwater mixture by slow evaporation at room
temperature.
Scheme 1. The structure of copper complexes 1- 3 used in the present study.
Molecular structure and EPR spectra
The single crystal Xꢀray structure of 2 shown in Figure 1 with selected atom labeling.
The complex has been crystallized in a triclinic system with a space group of Pꢀ1 (Table 1,
Figure 1a and Figure S2) and geometry around copper(II) centre is distorted square pyramidal.
This structural geometry revealed by the trigonality index τ of 0.2545 [τ = (β – α)/60; β (O1ꢀ
Cu1ꢀN3) = 173.18° and α (N1ꢀCu1ꢀN4) = 157.94°; for perfect square pyramidal and trigonalꢀ
1
8
bipyramidal geometries, τ = 0 and 1 respectively]. This square pyramidal coordination
3

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geometry of copper(II) centre is constituted by three nitrogen donors from the ligand and two
water molecules. All three nitrogen atoms and one water molecule constitute square plan and
remaining axial portion is occupied by a water molecule. The CuꢀN (1.958 Å) of middle
py
pyridine is shorter than other two CuꢀNBenzim bonds (CuꢀN, 1.995, 2.000 Å). The axial CuꢀO2
bond distance (2.247 Å) is slightly longer than the equatorial CuꢀO1 bond distance (1.953 Å).
The CuꢀN bond lengths of 2 are almost similar to those of previously reported
1
9
[
Cu(L1)(H O) ](NO ) (1.98 ꢀ 2.034 Å). However, these CuꢀN bond distances are shorter than
2 2 3 2
structurally similar 3 (Cu–N ,1.970 ꢀ 2.069 Å) (Figure 1b). The sum of bond angles N3ꢀCu1ꢀN4
py
(
79.95°), N3ꢀCu1ꢀN1 (79.95°), N1ꢀCu1ꢀO1 (101.37°) and O1ꢀCu1ꢀN4 (97.55°) is 358.42° and
closer to 360° suggesting that N1, N2, N3, and O1 are nearly coplanar in 2. The N1ꢀCuꢀN4
157.94°) and N3ꢀCuꢀO1 (173.18°) bond angles deviate from the 180°, indicating distortion in
the square plane.
The square based geometry of 2 is further supported by EPR spectrum (Figure 2). The
frozen solution spectra of 2 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 2.29, g 2.06)
(
|| =
⊥
=
(
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 square based geometry. Further,
ꢀ
4
ꢀ1
the A value of 156 × 10 cm suggested that 2 belongs to square pyramidal geometry (A = 154
||
||
ꢀ4
ꢀ1
20
ꢀ
162 × 10 cm for square based geometry ). This EPR spectral signature of 2 are similar to
ꢀ4
ꢀ1
that of 1 (g , 2.05; g , 2.29 ) but the geometrical parameter is higher than 1(A 124 × 10 cm ).
⊥
||
||
This reveals stronger geometrical distortion in 1. However, the A value of 2 is closer to that of 3
||
ꢀ4
ꢀ1
(
156.8 × 10 cm ) and g (2.05) and (g , 2.29) values are similar.
⊥ ||
Electronic absorption spectral studies
The complexes 1 and 2 exhibit dꢀd transitions at 662 (ε, 90 M cm ) and 651nm (ε, 84
ꢀ1
ꢀ1
ꢀ1
ꢀ1
ꢀ2
M cm ) respectively along with merged ligand based transitions in the concentration of 1 × 10
M in HEPES buffer at pH 7.34 (Table 2, Figure 3). However, πꢀπ* ligand transitions at 309 and
ꢀ1
ꢀ1
ꢀ1
ꢀ1
3
27 nm (ε, 11,480 and 13,020 M cm ) for L1 and 313 nm (ε, 10,360 M cm ) for L2 are well
ꢀ6
2+
ꢀ
resolved only at lower concentration (5 × 10 M). On coordination of a Cu ion with L1 (5 × 10
6
M) in HEPES buffer at pH 7.34, the ligandꢀbased transitions at 309 and 327 nm were
disappeared and two new bands were formed at 314 and 356 nm appeared (ε, 19,740 and 13,180
4

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ꢀ1
ꢀ1
2+
ꢀ
M cm ). On other hand, Cu ion coordination with L2 shows new bands at 360 nm (ε, 9560 M
1
ꢀ1
ꢀ1
ꢀ1
cm ) along with the ligandꢀbased transition at 313 nm (ε, 16,860 M cm ). However, the dꢀd
transition is not perceived at this lower concentration. The changes in the dꢀd bands of 1 and 2
ꢀ2
with various amino acids were studied at the concentration of 1 × 10 M. The dꢀd transition of 1
and 2 slightly lowered by addition of various natural amino acids (alanine, arginine, glycine,
histidine, leucine, proline, serine, threonine, tryptophan, and tyrosine) (Figure S3e and S3f).
However, the dꢀd band was readily disappeared on the addition of Cys (Figure S3c and S3d). On
other hand, the intensity of dꢀd transition has been decreased by the addition of His to 2 (Figure
ꢀ6
S5f). At the lower concentration (5 × 10 M), the addition of only one equivalent of Cys to 1 and
reproduces the absorption pattern of L1 and L2 respectively and all other amino acids exhibited
no concomitant changes under identical conditions (Figure S3a, S3b, and Figure S4). These
2
2
+
results reveal that the possibility of Cu displacement from coordination sphere of 1 and 2 on
interaction with Cys (Figure 3). We have reported very similar observation for 3 with Cys under
9
identical conditions. The addition of other biomolecules homocysteine (Hcy) and glutathione
(GSH) containing thiol exhibits slight changes in absorption bands of 1 and 2 (Figure S5a, and
Figure S5b).
Fluorescence Studies
The ligands L1 and L2 showed strong fluorescence at 406 and 392 nm with the quantum
ꢀ6
yield of 3.83 % and 11.3 % respectively in HEPES buffer at pH, 7.34 (5 × 10 M). Nevertheless,
the complexes 1 and 2 are found as nonꢀfluorescent. This is further confirmed by the immediate
2
+
fluorescence intensity quenching of ligand while adding one equivalent Cu ion (Figure S7).
This nonꢀfluorescent nature of 1 and 2 raised up by the paramagnetic nature of copper(II) centre,
which strongly disturbed the electronic resonance of the ligand and hence lowering in the
fluorescence intensity. This is further supported by HOMOꢀLUMO electron density map
obtained by DFT method using B3LYP 6ꢀ31G (for C, H, and N)/LANL2DZ (for Cu) basis set in
2
1
Gaussian 09 program (Table 2, Figure 4, Figure S6). The L1 and L2 show optimized energy, ꢀ
4
4
2
.7365× 10 and ꢀ2.9504 × 10 eV respectively, the frontier molecular orbitals HOMO and
LUMO are localized across the benzimidazole moiety. However, HOMO and LUMO are
localized around benzimidazole moiety and copper centre respectively for 1 and 2. The ground
4
4
state energies of complexes are lower (1, ꢀ6.3894 × 10 eV; 2, ꢀ6.6033× 10 eV) than that of
5

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ligands. However, these HOMO and LUMO of ligand L1 and L2 are different from our
previously reported ligand L3, where they are localized on fluorophore unit anthracenyl moiety.
The HOMO of 3 localized around the copper centre and LUMO was localized only on
terpyridine unit. These results are clearly indicating that electronic resonance of benzimidazole
2
+
moiety is strongly disrupted by Cu centre via internal charge transfer and hence quenching of
fluorescence intensity and change in absorption spectral pattern (cf. above).
The treatment of 1 and 2 with various amino acids (Figure 5 and 6) show no enhancement
ꢀ5
in the fluorescence intensity. However, the interaction of Cys (5 × 10 M) with 1 enhances the
fluorescence intensity almost 1.3 and 28 times higher than the original intensity of L1 and 1
respectively with a quantum yield of 14.7 % in aqueous solution. This increase in fluorescence
intensity has been saturated at 15 equivalents of Cys and no enhancement on further more
addition (Figure 7a). A huge fluorescence enhancement was observed for 2 with Cys and this
enhancement 143 times higher than that of 2 with a quantum yield of 23%. The increase in
intensity has been saturated with 13 equivalents of Cys and subsequent equivalents have shown
no further enhancement (Figure 7b).Thus, almost a similar enhancement in fluorescence intensity
has been noticed for 1 and 2 with the similar probing mechanism. Further, these fluorescence
responses of 1 and 2 with Cys are similar to that of our previously reported complex 3 with Cys
ꢀ6
9
and showed ‘turnꢀon’ mechanism with the quantum yield of 6% in 5 × 10 M at pH 7.34. The
fluorescence intensity was raised by the addition of His to 1 and 2 with a quantum yield of 5 and
11.3 % respectively. However, the increase in the emission intensity is 50% lower than that of
Cys and saturated with 15 and 13 equivalents of His for 1 and 2 respectively (Figure S8b and
S9b). On other hand, Hcy and NꢀacetylꢀLꢀcysteine (NAC) with 1 and 2 were shown significantly
lower enhanced of fluorescence intensity than Cys. However, the fluorescent intensity
enhancement by GSH with 1 and 2 is comparatively negligible (Figure S10).
The fluorescence titration of 1 and 2 with Cys and His have been followed the
HildebrandꢀBenesi equation, I /I-I = b/(a – b) {1/(K[M]) + 1} where, I and I are intensities in
0
0
0
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 (I /I-I ) vs
0
0
8d
1
/[M]. Upon addition of small aliquots of Cys to 1 and 2 raised the fluorescence intensity
gradually and they were found to be linear (Figure 8). The binding constant values were
6

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4
4
ꢀ1
calculated as 5.403 × 10 and 1.307 × 10 M for 1 and 2 respectively. The limit of detection was
calculated by employing the formula [3 × (Standard deviation/slope)] from the linear plot and 1
ꢀ8
ꢀ8
and 2, shows 2.9 × 10 M and 3.32 × 10 M respectively. These binding constant values are
4
–1
slightly lower than that of 3 (7.7×10 M ) but the limit of detection values are almost similar to
–
8
3
(1.9 × 10 M). The fluorescence studies revealed that 1 and 2 are highly selective towards Cys
and it can detect Cys even at lower concentration level. The competitive experiments of one
equivalent of Cys with 20 equivalents of other natural amino acids show that fluorescence
intensity readily raised only by Cys. Thus, the fluorescence intensity enhancement of 1 and 2
with Cys (Figure S12) is not affected by 20 equivalents of other natural amino acids. On other
5
4
hand, the binding constant values calculated for His with 1 and 2 as 1.4031 × 10 and 7.554 × 10
ꢀ1
M respectively are almost similar to that of Cys. However the limit of detection values 8.03 ×
ꢀ7
ꢀ7
1
0 M and 2.32 × 10 M for 1 and 2 respectively are lower than the values of Cys (Figure S11
and Figure S13).
Redox properties
The redox behavior of 1 and 2 has been investigated by cyclic voltammetry (CV) using a
threeꢀelectrode cell configuration in HEPES buffer solution at pH, 7.34 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 measured redox potential was converted into
NHE (normal hydrogen electrode) by adding +0.205 V. The irreversible Cu(II)/Cu(I) redox
couples at E , 0.014 and 0.233 V vs NHE have been observed for 1 and 2 respectively.
1
/2
Interestingly, this Cu(II)/Cu(I) redox couples were readily disappeared on the addition of four
equivalents Cys and new cathodic peak potential has been formed around 0.245 ꢀ 0.316 V vs
NHE (Table 2, Figure 9). The cathodic peak potential is identical to that of observed for in situ
2
+
generated Cu ꢀCys species. 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
6
k, 22
internal changes in bond lengths and angles of the complex.
The interaction of His with 1
and 2 shows a formation of new redox potentials at 0.1605 and 0.077 V vs NHE for 1 and 2
respectively. This indicates that the coordination of His with the complexes rather than reduction
of copper(II) centre and followed release as noticed Cys (Table 2, Figure S14).
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The solution of 1 and 2 with one equivalent of Cys were analyzed by ESIꢀMS and
showed the molecular ion peak at 311.98 and 340.03 corresponds to L1 and L2 respectively
(Figure S15). The FTꢀIR analysis exhibits stretching frequencies (ῡ) at 3034, 1581, 1485 for 1
ꢀ
ꢀ1
and 1406 cm and 3019, 1584, 1484 and 1405 cm for 2, which are matched with stretching
2
+
23
frequencies of in situ generated Cu ꢀCys species (Figure S17). The ESIꢀMS and FTꢀIR studies
reveal that presumably copper displacement from coordination sphere and release of the free
ligand as noticed by the electronic spectra and electrochemistry. Thus, the enhancement in
fluorescence intensity of 1 and 2 with Cys presumably proceeds via two consecutive steps, firstly
the copper(II) centre 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
6
k, 21
coordination geometry
but the rigid pyridineꢀbisꢀbenzimidazole ligand system is unable to
adopt this preference (Scheme 2). Then the electron density of ligand is possibly regenerated
around the fluorophore for the emission. Further, the Cu(II)/Cu(I) redox potential of 1 and 2 are
much lower than a reported redox barrier for adopting tetrahedral geometry as known in the blue
2
4
copper proteins (E , 0.276 ꢀ 0.307 V). On other hand , the amino acid His coordinates with 1
1
/2
and 2 and produces ‘turnꢀon’ fluorescence enhancement by transferring the electron density
towards benzimidazole units from copper centre. This is further supported by DFT studies,
revealing that HOMO and HOMOꢀ1 orbitals are localized on the histidine units and LUMO and
LUMO +1 are localized on benzimidazole units (Figure 10). This is consistent with ESIꢀMS
spectra of 1 and 2 with His, shows the species m/z, 684.10 and 684.33 corresponding to
+
+
[
(L1)Cu(His) ] and [(L2)Cu(His)(MeOH) ] respectively (Figure S16).
2 4
Scheme 2. Proposed mechanism for Cys and His detection by 1 and 2.
Conclusions
8

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The copper(II) complexes of 2,6ꢀbis(1Hꢀbenzo[d]imidazolꢀ2ꢀyl)pyridine ligands L1 and
L2 have been synthesized as a probe for Cys. The molecular structure of probe 2 adopts square
pyramidal geometry around copper(II) centre and it is further supported by EPR spectral data.
The probes showed a selective and efficient “turnꢀon” fluorescence behavior towards Cys over
other natural amino acids at pH, 7.34 with the excellent binding constants values with the limit of
ꢀ8
detection around 10 M at pH, 7.34. The redox, ESIꢀMass and IR studies revealed that the probe
showed bright visibleꢀlight emission on reacts with Cys by reduction of copper(II) centre to
copper(I) and subsequent displacement of copper. Because, the rigid pyridineꢀbisꢀbenzimidazole
ligand systems unable to adopt preferable tetrahedral coordination geometry of copper(I) which
leads to collapsing of coordination sphere, subsequently original fluorescence intensity of ligand
is turnedꢀon. On other hand, a relatively small fluorescence enhancement by probes with
histidine unit via the coordination of His rather than the displacement of copper, which produces
through releasing electron density to the benzimidazole units from the copper centre.
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 2,6ꢀpyridineꢀdicarboxylic acid, 1,2ꢀ
diaminobenzene, Polyphosphoric acid, methyl Iodide, ammonium hydroxide, potassium
hydroxide, copper triflate, amino acids like LꢀAlanine, LꢀCysteine, LꢀGlycine, LꢀHistidine, Lꢀ
Leucine, LꢀProline, LꢀSerine, LꢀThreonine, LꢀTryptophan were purchased from SigmaꢀAldrich
and used as such without further purification.
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. ESIꢀMS spectra were measured on a Thermo LCꢀMS 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.
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Electrochemical data were recorded in Biologic SPꢀ150 Electrochemical workstation using
+
saturated Ag/Ag and Pt as a reference electrode and working electrodes respectively. The 0.1 M
NaCl is used as supporting electrolyte. The electrodes are calibrated by K [Fe(CN) ] prior to
4
6
measurements. IR spectra were taken in Shimadzu FTꢀIR instrument. Electron paramagnetic
resonance (EPR) measurements were recorded using Bruker EMX Plus EPR Spectrometer.
X-ray Structure Determination: Single crystal 2 of suitable size was picked from the mother
liquor, coated with paraffin oil and mounted on a fiber loop for Xꢀray diffraction data collection
on Agilent Supernova system (CuꢀKα 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
25
Shelxl. All the nonꢀhydrogen atoms were refined anisotropically, whereas the hydrogen atoms
were added at the riding positions and refined isotropically. CCDCꢀ1551263 contain the
supplementary crystallographic data for this paper. This data can be obtained free of charge from
The Cambridge Crystallographic Data Centre via ww.ccdc.cam.ac.uk/data request/cif.
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
2
6
program package.
Synthesis of 2,6-bis(1H-benzo[d]imidazol-2-yl)pyridine (L1)
1
1 1
This ligand was synthesized and characterized by a previously reported method.
H
NMR (d6ꢀDMSO, 300 MHz): δ 8.31 (d, J = 7.8 Hz, 2H), 7.99 (t, J = 7.8 Hz, 1H), 7.73 (d, J = 7.3
+
5
Hz, 2H), 7.60 (d, J = 7.1 Hz, 2H), 7.25 (t, J = 7.8 Hz, 4H) ppm. ESIꢀMS (m/z): [C H N ],
19
14
312.84.
Synthesis of 2,6-bis(1-methyl-1H-benzo[d]imidazol-2-yl)pyridine (L2)
1
1 1
The ligand L2 was also synthesized and characterized as described previously. H NMR
(d6ꢀDMSO, 300 MHz): δ 8.30 (d, J = 9.6 Hz, 2H), 7.98 (t, J = 8.7 Hz, 1H), 7.69 (d, J = 7.5 Hz,
1
0

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2H), 7.41 (d, J = 7.8 Hz, 2H), 7.24 (dt, J = 13.3, 6.7 Hz, 4H), 4.18 (s, 6H) ppm. ESIꢀMS (m/z):
+
[
C H N ], 340.44.
2
1
18
5
Synthesis of [Cu(L1)(H O) ](SO CF ) 1
2
2
3
3 2
The ligand L1 (0.155 g, 0.5 mmol) was dissolved in 10 mL of hot methanol and
Cu(SO CF ) (0.5 mmol) in 5 mL of methanol was added dropwise and stirred for 30 minutes at
3
3 2
55°C. A green coloured product was filtered off and washed with cold methanol and diethyl
ether to remove excess ligand. Then it was dried under vacuum. Yield: 0.171 g (91.4%). HRꢀ
+
ESIꢀMS (m/z): [((L1)Cu(H O))/2]2MeOH, 226.0285; [(L1)Cu]+2H , 375.0383 and
2
+
[
(L1)Cu(H O) ]+H , 411.0143.
2
2
Synthesis of [Cu(L2)(H O) ](SO CF ) 2
2
2
3
3 2
The complex 2 was synthesized by using above procedure. The ligand L2 was used
+
instead of ligand L1. Yield: 0.083 g (72%). HRꢀESIꢀMS: (m/z): [((L2)Cu)/2] , 201.0388;
+
+
[
((L2)Cu (H O) )/2 + 3H] ; 240.0460, [(L2)Cu(H O) ] , 438.0898.
2 4 2 2
Acknowledgements
We acknowledge Science and Engineering Research Board (SERB), New Delhi, Board
of Research in Nuclear Sciences (BRNS), Mumbai and Department of Biotechnology (DBT),
New Delhi for funding. DM thanks to University Grants Commission (UGC), New Delhi for
fellowship.
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6.
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4

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Figures and Tables
(a)
(b)
Figure 1. Single crystal Xꢀray crystal structure of 2 (a) and its overlay with 3 (b). Thermal
ꢀ
ellipsoids are shown at the 50% probability level. Hydrogen atoms and SO CF are omitted for
3
3
clarity.
1
5

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1
2
3
Figure 2. Xꢀband EPR spectra of 1ꢀ3 in methanol/DMF mixture at 70 K.
1
6

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(a)
(b)
Figure 3. UVꢀVisible spectra of L1, 1 and 1 + Cys (a) and L1, 2 and 2 + Cys (b), pH 7.34 at
ꢀ6
ꢀ
2
5°C in HEPES buffer (5 × 10 M). Insets: dꢀd transitions of corresponding complexes at 1 × 10
M.
2
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7

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Figure 4. Energy profile diagram for L1, 1, L2 and 2; HOMO and LUMO are calculated by TDꢀ
DFT using B3LYP 6ꢀ31G/LANL2DZ level.
1
8

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(a)
(b)
Figure 5. Fluorescence spectra of 1 and addition of various amino acids (20 equivalents) in
ꢀ6
HEPES buffer pH, at 7.34 at 25°C (5 × 10 M), insert: Fluorescence spectra comparison of L1,
, 1 + Cys and 1 + His (a). Bar diagram for the selectivity of various amino acids (λ = 327 nm,
1
ex
slits: 5nm/5nm) (b).
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9

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(a)
(b)
Figure 6. Fluorescence spectra of 2 and addition of various amino acids (20 equivalents) in
ꢀ6
HEPES buffer pH, 7.34 at 25°C (5 × 10 M). Inset: Fluorescence comparison plot of L1, 2, 2+
Cys and 2+ His (a). Bar diagram for the selectivity of various amino acids (λ = 313 nm, slits:
ex
5nm/5nm) (b).
2
0

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(
a)
(b)
Figure 7. Fluorescence spectra of 1 (a) and 2 (b) and the addition of various equivalents of Cys
ꢀ6
in HEPES buffer solution pH, 7.34 at 25°C (5 × 10 M). Insets: Saturation plots.
2
1

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(a)
(b)
ꢀ6
ꢀ6
Figure 8. Fluorescence intensity changes of 1 (5 × 10 M ) (a) and 2 (5 × 10 M ) (b) on
addition of Cys (concentration) in pH 7.34.
2
2

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(a)
(b)
Figure 9. Cyclic voltamagrams of 1 (a), (concentration) and 2 (b) (concentration) with Cys (5 ×
ꢀ
6
+
1
0
0 M) in HEPES buffer (pH 7.34) at 25°C [reference: saturated Ag/Ag ; supporting electrolyte:
ꢀ1
.1M NaCl solution; scan rate: = 50 mV s ].
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3

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HOMO
HOMO -1
LUMO
LUMO +1
Figure 10. Frontier orbitals of 1 with His calculated using B3LYP 6ꢀ31G/LANL2DZ level.
2
4

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Dalton Transactions
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DOI: 10.1039/C7DT01895A
Table 1. Crystal data and structure refinement and selected bond length (Å) and bond angles (°) of 2
c
Empirical
formula
FW
Crystal system
space group
a (Å)
b (Å)
c (Å)
α (deg)
β (deg)
γ (deg)
Bond lengths and Bond angles
C23 H21 Cu F6 N5 O8 S2
737.11
Triclinic
P ꢀ1
12.7225(6)
15.1150(6)
17.1761(7)
85.831(3)
68.352(4)
Cu(1)ꢀO(1)
Cu(1)ꢀO(2)
Cu(1)ꢀN(4)
Cu(1)ꢀN(3)
Cu(1)ꢀN(1)
1.953(3)
2.247(3)
1.995(4)
1.958(4)
2.000(4)
O(1)ꢀCu(1)ꢀO(2)
O(1)ꢀCu(1)ꢀN(1)
O(1)ꢀCu(1)ꢀN(3)
O(1)ꢀCu(1)ꢀN(4)
N(1)ꢀCu(1)ꢀO(2)
N(1)ꢀCu(1)ꢀN(4)
N(3)ꢀCu(1)ꢀO(2)
N(3)ꢀCu(1)ꢀN(1)
N(3)ꢀCu(1)ꢀN(4)
N(4)ꢀCu(1)ꢀO(2)
91.82(14)
101.37(17)
173.18(15)
97.55(17)
99.71(16)
157.94(17)
94.55(14)
79.95(17)
79.95(17)
90.87(15)
7
1.152(4)
V (Å3)
T (K)
Cu Kα λ, (Å)
Density
2901.3(2)
100.15
1.54184
3
1
4
.687(Mg/m )
Z
ꢀ
ꢀ
1
(mm )
3.278
F(000)
1492
3
Crystal size
no.
0.07 × 0.05 × 0.05 mm
of
reflections
collected
10923
2
1.043
GOF on F
a
R1
0.0841
0.2377
b
wR2
2
2 2
2 2 1/2
(
a) R1 = Σ||Fo| – |Fc||/Σ|Fo|. (b) wR2 = [Σw(Fo – Fc ) /Σw(Fo ) ] . [c] Standard deviations in parentheses.
2
5

Dalton Transactions
Page 26 of 27
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DOI: 10.1039/C7DT01895A
Table 2. Electronic spectral, redox and computational data
L1
309
(11,480)
327
1
1 + Cys
1 + His
323
(13,980) 313
L2
2
2 + Cys
2 + His
313
(18,580)
a
Absorption spectra
314 (19,740)
356 (13,180)
662 (90)*
313 (16,860)
360 (9560)
651 (84)*
ꢀ
1
309 (16,660)
327 (18,860) 378
(1440)
313
λ
(nm), (ε, M
max
ꢀ
1
(10,360)
(18,760) 370
(2060)
cm )
(13,020)
Electrochemical
b
data
ꢀ
0
0.55
.007
ꢀ0.4891
ꢀ0.235
ꢀ0.3347
ꢀ0.02
ꢀ0.4119
ꢀ0.1275
154
Epa (V)
Epc (V)
E1/2(V)
ꢀ0.3129
ꢀ0.0696
ꢀ0.1908
243
ꢀ0.2189
ꢀ
ꢀ
0.3036
0.0445
ꢀ0.3181
0.1525
ꢀ
0
2
1
0.1113
0.0405
0.434
.0797
32
ꢀ0.1314
174
∆
E (mV)
46
215
c
EPR spectra
g||
2.29
2.05
124 × 10
2.29
2.06
156 × 10
g value
g
⊥
ꢀ
1
ꢀ4
ꢀ4
A (cm )
||
Computational
d
details (eV)
ꢀ
1
2.7365×
0
4
ꢀ2.9505
× 10
4
Optimized energy
HOMO
4
ꢀ6.3894 × 10
4
ꢀ6.6033× 10
ꢀ
7.011(α spin)
ꢀ6.831(α spin)
ꢀ6.817(β spin)
ꢀ3.166(α spin)
ꢀ3.895(β spin)
3.665 (α spin)
2.922 (β spin)
ꢀ5.783
ꢀ1.605
4.177
ꢀ5.835
ꢀ1.287
4.547
ꢀ
ꢀ
ꢀ
3
2
5.363 (β spin)
3.275 (α spin)
4.007 (β spin)
.735 (α spin)
.99 (β spin)
LUMO
Energy gap
TD-DFT
ꢀ
2.9504
ꢀ
1
2.7365×
0
4
4
4
Optimized energy
4
ꢀ6.3894 × 10
× 10
ꢀ6.6033× 10
ꢀ
ꢀ
7.011 (α spin)
6.997 (β spin)
ꢀ6.831 (α spin)
ꢀ6.817 (β spin)
ꢀ3.166 (α spin)
ꢀ3.895 (β spin)
3.665 (α spin)
2.922 (β spin)
HOMO
LUMO
ꢀ5.783
ꢀ1.605
ꢀ5.835
ꢀ1.287
4.547
ꢀ3.275 (α spin)
ꢀ
3
2
4.007 (β spin)
.735 (α spin)
.99 (β spin)
Energy gap
4.177
a
ꢀ6
Concentration of 5 × 10 M in HEPES buffer (pH 7.34) at 25°C.
Concentration of 1 × 10 M in HEPES buffer (pH 7.34) at 25°C.
Concentration of 5 × 10 M in HEPES buffer (pH 7.34) at 25°C.
reference: saturated Ag/Ag ; supporting electrolyte: 0.1M NaCl solution;
scan rate: = 50 mV s ]. To convert E vs NHE add +0.205 V.
Methanol/DMF mixture at 70 K .
DFT method using B3LYP 6ꢀ31G (for C, H and N)/LANL2DZ (for Cu)
basis set in Gaussian 09 program.
*
b
ꢀ2
ꢀ6
+
[
ꢀ1
1/2
c
d
2
6

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Dalton Transactions
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DOI: 10.1039/C7DT01895A
Table of content
The copper(II)ꢀbenzimidazole complexes detect Lꢀcysteine over other natural amino acids at pH,
.34 by ‘turnꢀon’ fluorescence mechanism via reduction of Cu(II) to Cu(I) followed by
displacement with excellent selectivity.
7
2
7