Thioether sulfur-bound [Cu2] complexes showing catechol oxidase activity and DNA cleaving behaviour

Article abstract of DOI:10.1039/c8dt04183k

Rational ligand design approaches allowed {Cu₂(μ-OH/OMe)} cores to be accommodated within μ-phenoxido bis(tetradentate) and μ-phenoxido bis(tridentate) ligands having thioether donors. The complexes [Cu₂(μ-H₂L1)(μ-OH)](ClO₄)₂·2H₂O (1), [Cu₂(μ-L2)(μ-OH)(OH₂)](ClO₄)₂ (2a) and [Cu₂(μ-L2)(μ-OCH₃)(OH₂)](ClO₄)₂ (2b) were obtained from an N₂O₃S₂ donor set bearing the H₃L1 ligand (2,6-bis-[{2-(2-hydroxyethylthio)ethylimino}methyl]-4-methylphenol) and N₂OS₂ donor set containing the HL2 ligand (4-methyl-2,6-bis-[{2-(methylthio)phenylimino}methyl]phenol) without showing double phenoxido bridging or any type of preformed inter-fragment aggregation. Previously, we showed that H₃L (2,6-bis[((2-(2-hydroxyethoxy)ethyl)imino)methyl]-4-methylphenol), the ether analogue of H₃L1, in the presence of carboxylate anions, was responsible for the self-aggregation of preformed {Cu₂} fragments and gave two types of [Cu₄] complexes comprising [Cu₄O] and [Cu₄(OH)₂] cores (T. S. Mahapatra, A. Bauzá, D. Dutta, S. Mishra, A. Frontera and D. Ray, ChemistrySelect, 2016, 1, 64-74). The molecular structures of 1, 2a and 2b were determined via single crystal X-ray diffraction and solution studies, which indicated the presence of [Cu₂] species. This was further confirmed via UV-vis spectroscopy and HRMS analysis. The synthesized complexes were screened for their potential as catalysts for the catalytic oxidation of 3,5-di-tert-butylcatechol (3,5-DTBCH₂). A change in the mechanism of catalytic oxidation was observed with a change in the ligand backbone. All three complexes also showed DNA binding properties, which were further substantiated via molecular docking studies. Their DNA binding affinities were quantitatively ascertained using their intrinsic binding constant, K_b, values which were found to be 4.2 × 10⁴, 5.6 × 10⁴ and 4.8 × 10⁴ M^-1, respectively. Furthermore, the complexes displayed efficient DNA cleavage behaviour with pBR322 and the oxidative path was established in presence of ROS, singlet oxygen, ¹O₂, and the superoxide anion, O₂·^-.

Full text of DOI:10.1039/c8dt04183k

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Thioether sulfur-bound [Cu2] complexes showing
catechol oxidase activity and DNA cleaving
behaviour†
Cite this: DOI: 10.1039/c8dt04183k
Manisha Das,a Zeenat Afsan, b Dipmalya Basak,a Farukh Arjmand b and
a
Debashis Ray
*
Rational ligand design approaches allowed {Cu2(μ-OH/OMe)} cores to be accommodated within
μ-phenoxido bis(tetradentate) and μ-phenoxido bis(tridentate) ligands having thioether donors. The com-
plexes [Cu2(μ-H2L1)(μ-OH)](ClO4)2·2H2O (1), [Cu2(μ-L2)(μ-OH)(OH2)](ClO4)2 (2a) and [Cu2(μ-L2)(μ-OCH3)
(OH2)](ClO4)2 (2b) were obtained from an N2O3S2 donor set bearing the H3L1 ligand (2,6-bis-[{2-(2-
hydroxyethylthio)ethylimino}methyl]-4-methylphenol) and N2OS2 donor set containing the HL2 ligand
(4-methyl-2,6-bis-[{2-(methylthio)phenylimino}methyl]phenol) without showing double phenoxido brid-
ging or any type of preformed inter-fragment aggregation. Previously, we showed that H3L (2,6-bis[((2-(2-
hydroxyethoxy)ethyl)imino)methyl]-4-methylphenol), the ether analogue of H3L1, in the presence of car-
boxylate anions, was responsible for the self-aggregation of preformed {Cu2} fragments and gave two
types of [Cu4] complexes comprising [Cu4O] and [Cu4(OH)2] cores (T. S. Mahapatra, A. Bauzá, D. Dutta,
S. Mishra, A. Frontera and D. Ray, ChemistrySelect, 2016, 1, 64–74). The molecular structures of 1, 2a and
2b were determined via single crystal X-ray diffraction and solution studies, which indicated the presence
of [Cu2] species. This was further confirmed via UV-vis spectroscopy and HRMS analysis. The synthesized
complexes were screened for their potential as catalysts for the catalytic oxidation of 3,5-di-tert-butyl-
catechol (3,5-DTBCH2). A change in the mechanism of catalytic oxidation was observed with a change in
the ligand backbone. All three complexes also showed DNA binding properties, which were further substan-
tiated via molecular docking studies. Their DNA binding affinities were quantitatively ascertained using their
intrinsic binding constant, Kb, values which were found to be 4.2 × 104, 5.6 × 104 and 4.8 × 104 M−1, respect-
ively. Furthermore, the complexes displayed efficient DNA cleavage behaviour with pBR322 and the oxi-
dative path was established in presence of ROS, singlet oxygen, 1O2, and the superoxide anion, O2·−.
Received 19th October 2018,
Accepted 13th December 2018
DOI: 10.1039/c8dt04183k
rsc.li/dalton
and flexibility of the side arms.6 The availability of a central
phenol donor group may result in a [Cu2L(OH/OR)]-type frag-
Introduction
The design, synthesis and characterization of binucleating ment or complex in the presence of a hydroxide/alkoxido
ligand systems and their [Cu2] complexes have received bridge, while double-phenoxido bridges lead to [Cu2L2]-type
increasing interest in recent years due to their involvement in species. In [Cu2(OH/OR)]-type complexes or fragments, the
magnetic-exchange interactions,2 DNA binding and cleavage,3 opening up of the OH/OR bridge through protonation is easier
catecholase mimetic action,4 and as building units for the con- and their core is suitable for substrate binding followed by bi-
struction of tetra- and higher order coordination aggregates.5 metallic catalysis. Recent studies have shown that [Cu2] com-
Phenol-based ligands having two metal ion pockets can plexes can function as useful model systems for catalytic cate-
provide [Cu2L]- or [Cu2L2]-type species based on the availability chol oxidation.7 A Cu⋯Cu separation in the range of 2.9–3.2 Å
is ideal for binding the catechol molecule by two metal ion
centres.8 It has been demonstrated that occasionally double-
aDepartment of Chemistry, Indian Institute of Technology, Kharagpur 721 302, India. phenoxido- or -hydroxido-bridged [Cu2] complexes give cataly-
tically inactive species.9 Nature uses several metalloenzymes to
catalyse the precise and selective oxidation of organic sub-
E-mail: dray@chem.iitkgp.ac.in; Fax: (+91) 3222-82252; Tel: (+91) 3222-283324
bDepartment of Chemistry, Aligarh Muslim University, Aligarh 202002, UP, India
†Electronic supplementary information (ESI) available: X-ray crystallographic
strates for useful transformations. The surrounding coordi-
data in CIF format, Schemes S1–S4, Fig. S1–S20, Tables S1–S2.† CCDC
nation environment, resulting coordination geometry, brid-
ging groups and three-dimensional structural features of
1857614–1857616 for complexes 1–2b. For ESI and crystallographic data in CIF
or other electronic format see DOI: 10.1039/c8dt04183k
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different bimetallic copper-based enzyme active sites are tion of 2,6-diformyl-4-methylphenol and 2-(methylthio)aniline
diverse in nature. The balance between these parameters and in the same molar ratio led to 4-methyl-2,6-bis-[{2-(methylthio)
their optimization result in the observed functions of different phenylimino}methyl]phenol (HL2) (Scheme S2†). Subsequently,
copper proteins and copper enzymes. Type-3 copper enzymes the reactions of H3L1 and HL2 with Cu(ClO4)2·6H2O in the
such as tyrosinase (Tyr) and catechol oxidase (CO) are charac- presence of NEt3 was examined, as summarized in Scheme 1.
terized by copper centres with similar first coordination In MeOH, the room temperature reaction with H3L1 resulted a
spheres but varying secondary coordination spheres.10 green solution, from which green single crystals of 1 were
Cationic bimetallic systems are known to act as catalytic obtained in 72% yield (eqn (1a)). In search of [Cu4] tetra-
mimics and receptors for the binding of anionic DNA.11 DNA- hedron- or dicubane-type complexes, we repeated the reaction,
based asymmetric catalysis is based on a hybrid system gener- without any success, in the presence of AcO− ions suitable for
ated in situ by placing a catalytically active CuII complex in inter-dimeric aggregation (Scheme S3†). The available AcO−
close proximity to DNA through non-covalent interactions.12 ions were not suitable for this endeavour because Cu–S were
Also, there has been an upsurge in the development of copper- bonds formed immediately, thus inhibiting the aggregation
based antitumor therapeutic agents, which target DNA and reaction.
mediate cleavage reactions effectively via ROS, causing overall
H3L1 þ 2CuðClO4Þ2 ꢀ 6H2O þ 2NEt3
damage to the DNA helix and ultimately resulting in cell
CꢁH!3OH ½Cu2ðμ‐H2L1Þðμ‐OHÞꢂðClO4Þ2 ꢀ 2H2O
death.13,14 Synthetic binuclear complexes show a myriad of
ð1aÞ
binding modes to DNA, including intercalation, groove
binding, electrostatic and van der Waals interactions.
þ 2fðNHEt3ÞðClO4Þg þ 9H2O
While trying to isolate, crystallize and study the reaction of
H3L1 þ 2CuðClO4Þ2 ꢀ 6H2O þ 2CH3COONa
Cu(ClO4)2·6H2O with
2,6-bis-[{2-(2-hydroxyethylthio)ethyl-
CꢁH!3OH ½Cu2ðμ‐H2L1Þðμ‐OHÞꢂðClO Þ ꢀ 2H2O
ð1bÞ
4
2
imino}methyl]-4-methylphenol (H3L1) and (4-methyl-2,6-bis-
[{2-(methylthio)phenylimino}methyl]phenol (HL2) (Chart 1),
we isolated three products suitable for exploring biomimetic
catalytic and biophysical behaviour. Earlier Oshio et al. used
H3L (Chart 1, left) for the synthesis of 3d–4f aggregate
[Mn4Ln2], in which the ether oxygen side-arms remained dan-
gling.15 We were also unable to show any coordination from
the ether oxygen donors of the side-arms, which is the prere-
quisite for two types of [Cu4] aggregates.1 Thus, the binding
capacity of thioether-containing side-arms versus ether-bearing
side-arms can be compared for the competitive stabilization of
[Cu2] complexes in contrast to the immediate self-assembly of
[Cu4] aggregates.1 The reaction of HL2 with Cu(ClO4)2 was
briefly reported without any crystal structure.16
þ 2CH3COOH þ 2NaClO4 þ 9H2O
The elemental analysis, solution molar conductivity
measurement and single crystal X-ray diffraction results estab-
lished 1 as [Cu2(μ-H2L1)(μ-OH)](ClO4)2·2H2O. The reaction of
HL2 with Cu(ClO4)2·6H2O and NEt3 in a 1 : 2 : 2 molar ratio in
MeOH–H2O (1 : 1) mixed solvent medium gave 2a as a green
precipitate (eqn (2)). Recrystallization from saturated MeCN
Results and discussion
Synthetic methodology
Condensation of 2,6-diformyl-4-methylphenol and 2-(2-amino-
ethylthio)ethanol in a 1 : 2 molar ratio at room temperature in
solution provided 2,6-bis-[{2-(2-hydroxyethylthio)ethylimino}
methyl]-4-methylphenol (H3L1) (Scheme S1†). Solution reac-
Chart 1 H3L, H3L1 and HL2.
Scheme 1 Synthetic routes for 1–2b.
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solution provided green crystals of 2a in 64% yield suitable for L mol−1 cm−1) and 311 nm (ε = 35 300 L mol−1 cm−1) for 1, 2a
X-ray diffraction analysis. The same reaction performed in and 2b, respectively, which correspond to the CuII-bound
pure MeOH gave a green microcrystalline precipitate, which on intra-ligand n → π* and π → π* transitions originating from
recrystallization from MeCN, resulted in green crystals of 2b in the CuII-bound CvN groups.
62% yield (eqn (3)).
HL2 þ 2CuðClO4Þ2 ꢀ 6H2O þ 2NEt3
ꢁ! ½Cu2ðμ‐L2Þðμ‐OHÞðOH2ÞꢂðClO4Þ2
þ 2fðNHEt3ÞðClO4Þg þ 9H2O
Descriptions of the crystal structures
MeOHꢁH2O
ð2Þ
ð3Þ
[Cu2(μ-H2L1)(μ-OH)](ClO4)2·2H2O (1). Complex 1 crystallizes
in the monoclinic Cc space group with Z = 4. The cationic part
of its molecular structure is presented in Fig. 1 and its impor-
tant metric parameters are given in Table S1.† Its structure
consists of one [Cu2(µ-H2L1)(µ-OH)]2+ unit, two perchlorate
anions and two lattice water molecules. The two copper
centres are in different coordination geometries, where Cu1 is
square planar, while Cu2 is square pyramidal. The two copper
centres are doubly bridged by phenoxido O (O1) and hydroxido
O (O2) atoms. Imine nitrogen (N1/N2) and thioether sulfur
(S1/S2) atoms provide an NSOO donor set to each copper,
while one of the ethanol side arms provides apical coordi-
nation to the Cu2 centre. Around Cu1, the side arm remains
dangling and shows slight deviation from the ideal square
planar geometry (τ4 = 0.12).19 In the case of Cu2, the deviation
from ideal square pyramidal geometry was estimated from the
τ5 value, which is 0.01.20 The deviations mainly originated
from the displacement of the CuII centers from the NSOO
basal planes by 0.078 Å and 0.125 Å for Cu1 and Cu2, respect-
ively. The calculated solid angles (sum of the three angles)
around the tetrahedral S donors are 301.75° and 295.6° for S1
and S2, respectively, indicating their contribution for cuboidal
distortion, making apical coordination feasible from one side
of the alcohol arm. The Cu1–S1 and Cu2–S2 distances are
close at 2.3206(19) and 2.3468(19) Å, respectively. The phenox-
ido and hydroxide bridgehead angles within the Cu2O2 core
(Cu1–O1–Cu2 and Cu1–O2–Cu2) are 96.49(19)° and 103.3(2)°,
respectively, which brought the Cu⋯Cu separation below 3 Å
to 2.9465(10) Å. The in-plane Cu–OOH bond lengths of 1.878(5) Å
and 1.880(5) Å are shorter compared to similar reported
bonds.21 The Cu–OPh distances were slightly longer at 1.972(4) Å
and 1.978(4) Å. As a result of the flexible ligand backbone,
HL2 þ 2CuðClO4Þ2 ꢀ 6H2O þ 2NEt3
MeOH
ꢁ! ½Cu2ðμ‐L2Þðμ‐OCH3ÞðOH2ÞꢂðClO4Þ2
þ 2fðNHEt3ÞðClO4Þg þ 10H2O
The elemental analysis and solution molar conductivity
data initially indicated the above formulas for the obtained
solids of 2a and 2b, and the single crystal X-ray structure deter-
mination ultimately confirmed their molecular structures.
FT-IR characterization in the solid state
The spectra of the solid powder samples were recorded with
KBr pellets. The stretching frequency for the Cu2+ bound
–CvN group, which is characteristic for a Schiff base,
appeared at 1637 cm−1 for 1, while for 2a and 2b the same was
slightly shifted to a lower wavenumber at 1618 cm−1. The phe-
noxido-bridge responsible for the formation of the [Cu2] entity
resulted in the characteristic νC–O stretching frequencies at
1554 cm−1, 1534 cm−1 and 1535 cm−1 for 1, 2a and 2b, respect-
ively. Also for 1, the νO–H stretching frequency, which is charac-
teristic for CuII-bound ligand alcohol –OH arms, bridging
hydroxido groups and lattice water molecules, was detected as
a broad band at 3422 cm−1. For 2a and 2b, the νO–H stretching
frequency for the coordinated water molecule and bridging
hydroxide group appeared at 3468 and 3469 cm−1, respectively.
−
For the crystal lattice trapped ClO4 anions in the overall Td
symmetry, the ν3(T2)(νClO) band appeared as a single band at
∼1088 cm−1 and the ν4(T2)(δdOClO) mode at ∼625 cm−1 for 1, 2a
and 2b.17 The CuII-bound C–S stretching frequency (νC–S) from
the aliphatic and aromatic thioether donors appeared as weak
peaks at 769 cm−1, 759 cm−1 and 759 cm−1 for 1, 2a and 2b,
respectively (Fig. S1†).
Electronic spectral transitions in solution
MeCN solutions of 1–2b showed several representative bands
in the 800–200 nm spectral range (Fig. S2†). The band due to
low intensity d–d transitions appeared at 608 nm (ε = 193
L mol−1 cm−1), 606 nm (ε = 184 L mol−1 cm−1), and 607 nm
(ε = 212 L mol−1 cm−1) for 1, 2a and 2b, respectively. The
intense CuII-bound ligand-based absorptions were found at
around 400 nm. In the bound form, the phenoxido O to CuII
charge transfer (LMCT) transition was detected at 371 nm (ε =
11 900 L mol−1 cm−1) for 1, 404 nm (ε = 14 500 L mol−1 cm−1
)
for 2a, and 403 nm (εmax = 17 200 L mol−1 cm−1) for 2b.18 The
other transitions originating from the ligand were observed at
Fig. 1 Perspective view of the cationic part of 1. Color scheme: Cu,
259 nm (ε = 29 600 L mol−1 cm−1), 307 nm (ε = 29 400 green; O, red; N, blue; C, black; and S, yellow.
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the Cu2O2 core is distorted by 11.50° compared to an ideal presented in Fig. 3 and their bond parameters are tabulated in
plane.
Table S1.† The ligand anion L2− bridges two CuII ions through
The hydrogen atom of the bridging hydroxide (O2) group the central phenoxido part and two side arms bearing imine N
−
served as a hydrogen bond donor to O8 of the lattice ClO4
and thioether S donors. The two-solvent system provided solvent-
anion, leading to its deviation from the Cu2O2 plane by 42.44°. derived HO− and MeO− bridges holding the two CuII centres.
The other oxygen (O6) atom of ClO4− remains hydrogen bonded
The apical coordination of a single H2O molecule by one of
to the pendent imine alcohol arm O3, and thus is not available the CuII centres provided two different coordination geome-
for coordination to the Cu1 centre. Thus, the hydrogen-bonded tries. Similarly to the previous case, we were able to generate
−
inter link established through the ClO4 anions result in a 1D [Cu2] complexes having coordination asymmetry. The calcu-
chain structure (Fig. 2). The coordinated hydroxyl (O4) group lated τ4 values for Cu1 (0.14 for 2a and 0.17 for 2b) depict
remains hydrogen bonded to one lattice water oxygen (O1W) slight distortion, but sufficiently rule out any apical coordi-
atom, which is again hydrogen bonded to O3. A second lattice nation from the H2O molecule. For Cu2 the centres, the
water molecule (O2W) shows hydrogen bonding interactions Addison parameter (τ5) values (0.06 for 2a and 0.04 for 2b)
−
with O1 W and the oxygen (O9) atom of the second ClO4
.
dictate almost regular square pyramidal geometries. In 2b, the
[Cu2(μ-L2)(μ-OH)(OH2)](ClO4)2 (2a) and [Cu2(μ-L2)(μ-OCH3) Cu–OMe distances (1.895(5) Å and 1.900(4) Å) are slightly
ˉ
(OH2)](ClO4)2 (2b). Both 2a and 2b crystallize in the triclinic P1 shorter than the Cu–OOH distances (1.9147(19)
Å and
space group with Z = 2. Perspective views of both molecules are 1.9268(19) Å) in 2a, respectively. Whereas, the Cu–Oph dis-
tances (1.957(4) Å and 1.957(4) Å) in 2b are marginally longer
than that in 2a (1.9420(17) Å and 1.9391(18) Å) by 0.017 Å,
respectively. The Cu–N bond distances are close in both the
complexes (1.946(2) Å and 1.949(2) Å in 2a and 1.945(5)
and 1.944(5) Å in 2b); whereas, the Cu–S separations in
the hydroxido-bridged 2a are smaller (2.2740(9)
Å and
2.2676(11) Å) than the methoxido-bridged situation in 2b
(2.293(2) and 2.280(3) Å), respectively. The Cu⋯Cu distance in
2a (2.9535(12) Å) is smaller than that in 2b (2.982(2) Å). The
coordinated water molecule is synergistically stabilized by
hydrogen bonding interactions with lattice perchlorate anions
in both 2a and 2b (Fig. 4). In 2a, the hydroxido bridge shows
hydrogen bonding interaction with one of the perchlorate
anions, leading to deviation of the hydroxido proton away
Fig. 2 View of the hydrogen-bonded 1D chain through trapping of
anions and solvents in 1.
Fig. 3 Perspective views of 2a and 2b with partial atom numbering scheme. Anions are omitted for clarity. Color scheme: Cu, green; O, red;
N, blue; C, black; and S, yellow.
Fig. 4 Perspective view of the hydrogen bonding of 2a and 2b with a perchlorate counter anion.
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from the Cu2O2 plane by 51.33° (Fig. 4). Around the tetrahedral
For 1, the value of the coupling constant, J, was found to be
S donors in 2a and 2b, the calculated solid angles (sum of the −40.56 cm−1 with the g value of 2.15 and χTIP value of 2.0 ×
three adjacent angles) are 310.03 and 303.21° and 309.64° and 10−2 cm3 mol−1
. For 2a, a similar procedure yielded
306.19°, respectively, indicating distortion from tetrahedral J = −52.84 cm−1, g = 2.15 and χTIP = 2.2 × 10−2 cm3 mol−1. On
geometry toward cuboidal structures. On the other hand, the other hand, for 2b, the magnitude of J was found to be
around the bridging methoxido O atom in 2b, the calculated much higher at −268.92 cm−1 with g = 2.15 and χTIP = 1.9 ×
solid angle (sum of the three adjacent angles) is 359.57°, con- 10−2 cm3 mol−1. For the hydroxido and alkoxido-bridged
firming the planar structure clipping two CuII centres.
dinuclear CuII complexes, a linear correlation was found
Solid state magnetic exchange behavior. The temperature between the coupling constant, J, and the size of the Cu–O–Cu
dependence of the molar magnetic susceptibility, χM, values angle, with larger angles leading to a stronger antiferro-
for 1–2b was determined under a magnetic field of 0.1 T over magnetic exchange interaction.23,24 However, this exchange
the temperature range of 300–10 K to find any advantage for interaction was also found to be dependent on the extent of
the presence of two CuII centres in a single entity and their the displacement of the hydrogen atom of the hydroxido
exchange interactions. The plots of χMT versus T for 1, 2a and bridges away from the Cu2O2 plane and folding distortion of
2b are shown in Fig. 5. At 300 K, the values of χMT for 1 and 2a the plane itself. Large displacements and distortions lead to a
are 0.82 cm3 K mol−1 and 0.81 cm3 K mol−1, respectively, much weaker antiferromagnetic exchange or even ferro-
which are consistent with the expected values for two CuII sites magnetic exchange interactions.24–26 In 1, 2a and 2b, the
(0.75 cm3 K mol−1 for g = 2.0). The value of χMT for 2b at Cu–O–Cu angles are 103.26°, 100.50° and 103.56°, respectively,
0.52 cm3 K mol−1 is much lower than that expected for two non- and strong antiferromagnetic exchange interactions are
interacting and isolated CuII centres. In the case of 1 and 2a, expected. However both 1 and 2a show very weak interactions,
with a decrease in temperature, χMT gradually decreased until as exemplified by their low coupling constant, J. This is due to
100 K and showed a sharp drop at lower temperatures, reaching the displacement of the hydrogen atom of the hydroxido-
a minimum value of 0.010 cm3 K mol−1 and 0.002 cm3 K mol−1
,
bridge away from the Cu2O2 plane by 42.44° and 51.33° for 1
respectively, at 10 K. For 2b, a continual decrease in χMT was and 2a, respectively, as a result of the hydrogen bonding inter-
observed upon a decrease in temperature, with the data enter- actions with perchlorate counter anions. The molecular struc-
ing a plateau at low temperature values. The χMT value was very ture of 1 also shows distortion of the Cu2O2 core by 11.50°
close to zero below 50 K, reaching 0.001 cm3 K mol−1 at 10 K. with respect to the planar disposition of the atoms owing to
These observations indicate a predominant antiferromagnetic the more flexible ligand backbone, which further lowers the
exchange interaction between the two CuII ions in 1–2b. The coupling constant value. Hence, 1 has the weakest antiferro-
data were simulated using the FIT-MART22 program to deter- magnetic exchange interaction between the CuII centres. On
mine the magnitude of the exchange interaction. The value of g the contrary, 2b having a methoxido bridge and a planar
was allowed to vary freely and the temperature-independent Cu2O2 core exhibited the highest J value in accordance with
paramagnetism (TIP) was also considered. The Hamiltonian the Cu–O–Cu angle.
used for the simulation procedure is given below in eqn (4) and
the resulting curves are shown in Fig. 5.
Functional activity for catechol oxidation. The catalytic oxi-
dation of 3,5-di-tert-butylcatechol (3,5-DTBCH2), as a model
substrate, by the synthesized complexes was examined system-
atically under aerobic condition. The active sites of different
forms of naturally occurring catechol oxidases have been deter-
mined by X-ray crystallography; however, the mechanism of
catalytic action involving several intermediates has not been
elucidated unequivocally. The catalytic oxidation was exam-
ined spectrophotometrically by monitoring the increase in the
quinone (DTBQ) absorption band at around 400 nm as a
function of time. Solutions of complexes 1, 2a and 2b (∼1 ×
2
X
ꢀ
ꢁ
ˆ
ˆ
ˆ
Si
ˆ
H ¼ ꢁ2J1 SCu1SCu2 þ gμBH
ð4Þ
i¼1
10−5 mol L−1) were treated with 100 equiv. (∼1 × 10−3 mol L−1
)
of 3,5-DTBCH2 under aerobic conditions and time-dependent
UV-vis spectra were recorded up to 1 h. The experiments were
performed in two different solvents, MeOH and MeCN, produ-
cing distinct interpretations. The control experiments were
performed with Cu(ClO4)2·6H2O and 100 equiv. of DTBCH2,
where no change was observed in absorption intensity even
after 1 day.
Oxidation in MeCN. In pure MeCN medium, complex 1
showed an absorption band at 371 nm. Upon the addition of
3,5-DTBCH2, the original peak vanished with the appearance
of another peak at 434 nm. The spectrum remained
Fig. 5 Temperature dependence of χMT at 0.1 T for 1, 2a and 2b.
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unchanged after 30 min of mixing. When the reaction and new peaks developed at 385 nm and 387 nm for 2a and
medium was made slightly alkaline to pH 7.2 using diluted 2b, respectively. The addition of diluted Et3N to take the pH to
Et3N in MeCN, the peak at 434 nm vanished and a new band 7.2, led to the generation of a peak at 400 nm in both cases,
appeared at 404 nm, showing a time-dependent increase in showing a time-dependent increase in intensity. Similar obser-
intensity. Similar observations were made in MeCN–H2O (1 : 1) vations were made in MeCN–H2O (1 : 1) at pH 7.0. Thus, the
medium at pH 7.0 maintained by Tris-HCl buffer solution. formation of a stable catalyst–substrate adduct was predicted.
These observations lead us to suspect the formation of a stable 3 mL solution of 2a and 2b each in MeCN–H2O (1 : 1) with a
catalyst–substrate adduct in MeCN or MeCN–H2O (pH ∼ 7) concentration of 1.0 × 10−5 M was treated sequentially with 1 ×
solution. A small increase in the pH of the medium led to the 6–15 × 6 µL of 10−3 M solution of DTBCH2 in MeCN–H2O
collapse of this intermediate forming DTBQ. To prove this (1 : 1) at pH 7.0. Fig. 7 depicts the spectral changes for 2a and
model, 3 mL 10−5 M solution of 1 in MeCN–H2O (1 : 1) was 2b upon the addition of increasing amount of substrate. With
treated sequentially with 1 × 6–13 × 6 µL of 10−3 M solution of the sequential addition of substrate, the peaks at 315 nm and
DTBCH2 in MeCN–H2O (1 : 1) at pH 7.0. Fig. 6a depicts the 312 nm for 2a and 2b disappeared providing new absorption
spectral change with increase in the concentration of the sub- bands at 254 nm and 267 nm, respectively. The lack of iso-
strate. The spectra show isosbestic points at 395 nm and sbestic points in the case of 2a and 2b indicate changes in
246 nm, clearly suggesting the formation of a complex (cata- the structure of the species following the coordination of
lyst)–catechol (substrate) adduct in solution. To determine the DTBCH2. The plot of the absorbance values at 385 nm
stoichiometry of this adduct, the changes in absorbance at against the mole fraction [DTBCH2]/[complex 2a] (Fig. 8, left)
434 nm and 371 nm were plotted against the mole fraction for 2a clearly shows the stepwise formation of a 1 : 1 and
[DTBCH2]/[complex 1] (Fig. 6b). The plot clearly indicates the finally 1 : 2 catalyst–substrate adduct. On the other hand, in
formation of 1 : 1-type of adducts.
the case of 2b, the plot of absorbance at 387 nm vs. mole frac-
Complexes 2a and 2b exhibit bands at 409 nm and 407 nm, tion of [DTBCH2]/[complex 2b] (Fig. 8, right) showed no clear
respectively, in MeCN. Upon the addition of 100 equivalents of formation of a 1 : 1 adduct prior to the formation of the 1 : 2
3,5-DTBCH2, the peaks at 409 nm and 407 nm disappeared adduct.
Fig. 6 (a) Changes in the spectral features of 1 with an increase in DTBCH2 concentration in MeCN–H2O (1 : 1) at pH 7.0 and (b) plot of absorbance
of 1 and its adduct with DTBCH2 against [DTBCH2]/[Complex 1].
Fig. 7 Variations in the spectral features of (a) 2a and (b) 2b with an increase in DTBCH2 concentration in MeCN–H2O (1 : 1) at pH 7.0.
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Fig. 8 Plot of absorbance against mole fraction of [DTBCH2]/[2a] (left) and mole fraction of [DTBCH2]/[2b] (right).
Oxidation in 1 : 1 MeCN–H2O at pH 7.2 to 7.8. To probe the at 447 nm on increasing the pH of the medium. Earlier, we
effect of pH on the catalytic oxidation of DTBCH2 stemming assigned the 371 band for the phenoxido O to the CuII charge
from the collapse of the catalyst–substrate adduct, time-depen- transfer (LMCT) transition. With an increase in pH up to 7.8,
dent spectroscopic measurements were carried out on mix- there is a concomitant increase in the intensity of the band at
tures of 1, 2a and 2b with 100 equivalents of 3,5-DTBCH2 in 447 nm. Presumably, the coordinated alcohol arms of the
MeCN–H2O (1 : 1) at four pH values of 7.2, 7.4, 7.6 and ligand become deprotonated in alkaline medium, leading to the
7.8 maintained by 50 mM Tris-HCl buffer solution (Fig. S5†). generation of the band at 447 nm. It is possible for the deproto-
In all cases, a peak at around 404 nm was observed, which nated alcohol arms lying in close proximity to the bound cate-
exhibited a time-dependent increase in intensity. This indi- chol molecule to assist in the deprotonation of the latter. 2a
cates the catalytic oxidation of 3,5-DTBCH2 to 3,5-DTBQ by all and 2b have no alcohol arms, and hence, exhibit no change in
the complexes at a pH above 7.0, where their activity increased spectral features with an increase in the pH of the medium.
on going from pH 7.2 to 7.8. In the literature, such increase is
Oxidation in MeOH. Complex 1 was found to be catalytically
often ascribed to the deprotonation of catechol, forming a inactive in MeOH. On the other hand, both 2a and 2b readily
stronger catecholate–catalyst adduct. However, in the case of catalysed the oxidation of 3,5-DTBCH2 in MeOH under aerobic
1–2b, a stable catalyst–substrate adduct was already formed at conditions. Fig. S6† shows the change in spectral behaviour of
pH 7.0. Thus, deprotonation of the catechol after it coordi- these two complexes upon the addition of 100 equivalents of
nates to the CuII centre seems to be more important rather 3,5-DTBCH2 up to 1 h from the time of mixing. In the absence
than adduct formation. To investigate the enhanced catalytic of substrate, both complexes exhibited a broad absorption
activity of 1 with an increase in the pH of the medium, first we band at ∼404 nm in MeOH. Upon the addition of DTBCH2,
tried to identify the different species of 1 present in solution this band was blue shifted to 395 nm for 2a and 397 nm for
within the pH range of 7.0 to 7.8. Fig. 9 shows the spectral 2b. As the reaction proceeded, the band was stabilised at
changes of 1 within this pH window with an isosbestic point at 401 nm, showing a time dependent-increase in intensity and
400 nm. At pH 7.0, complex 1 showed a peak at 371 nm, which signifying the quick formation of quinone.
gradually diminished in intensity, while a new band appeared
Kinetic study. The kinetic evaluation of the oxidation of 3,5-
DTBCH2 in the presence of 1, 2a and 2b was performed in
MeOH and MeCN–H2O (1 : 1) at pH 7.2, 7.4, 7.6 and 7.8 main-
tained by the appropriate amount of 50 mM Tris-HCl buffer
solution. A fixed concentration (∼1 × 10−5 mol L−1) of 1, 2a
and 2b in appropriate medium was treated with varying con-
centrations of 3,5-DTBCH2 (10 to 100 equivalents) and the for-
mation of DTBQ was monitored by UV-vis spectrophotometry
by recording the change in absorbance at 401 nm in MeOH
and at 409 nm in MeCN–H2O within the first 5 min of mixing.
The reaction rates were calculated via the initial rate method
and analysed using eqn (5) (Fig. 10–12 and Fig. S6†).
n
½Sꢂ
Rate ¼ Vmax
ð5Þ
n
n
½KAꢂ þ ½Sꢂ
Herein, n denotes the number of cooperative binding sites
for the substrate. When n = 1, there is no substrate cooperativ-
Fig. 9 UV-vis spectral changes of 1 from pH 7.0 to 7.8.
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Fig. 10 Kinetic plots for 1 at four different pH values in MeCN–H2O (1 : 1).
Fig. 11 Kinetic plots for 2a at four different pH values in MeCN–H2O (1 : 1).
ity and the kinetics for catalysis follows the Michaelis–Menten the maximum reaction rate (Vmax), binding constant (KM),
model, giving KA (association constant) = KM (Michaelis– association constant (KA) and turnover number (kcat) for the
Menten constant). The obtained kinetic parameters including oxidation reaction are listed in Table 1. The rate constant
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Fig. 12 Kinetic plots for 2b at four different pH values in MeCN–H2O (1 : 1).
(knon) for the uncatalyzed oxidation of 3,5-DTBCH2 in 1 : 1 model of enzyme kinetics is followed by 2a and 2b only at
MeCN–H2O at pH 7.2, 7.4, 7.6 and 7.8 was also determined. higher pH values. In MeOH medium, both 2a and 2b displayed
The comparison of the kcat values with knon showed a manifold substrate cooperativity having n ≈ 2. The value of n ≈ 2 in
increase in reaction rate in the presence of 1, 2a and 2b MeOH and in MeCN–H2O (1 : 1) at a lower pH value is consist-
(Table 2).
ent with the formation of 1 : 2 catalyst–substrate adducts by 2a
Analysis of the above data clearly revealed that while 1 and 2b.
follows Michaelis–Menten kinetics at all pH values, both 2a
Cyclic voltammetry. To assess the pH dependence of the
and 2b showed an enhancement in reaction rate through sub- electron transfer reactions from the ligand-bound CuII centres,
strate cooperativity at a lower pH value, as exemplified by the cyclic voltammetry was performed on 1–2b in 1 : 1 MeCN–H2O
value of n, which is greater than unity. The Michaelis–Menten at pH 7.0, 7.2, 7.4, 7.6 and 7.8 (Fig. 13 and Fig. S7†). For 1, a
small change in reduction potential was noted on increasing
the pH value from 7.0 to 7.8 (Fig. 13). At pH 7.0, the reductive
responses were observed at −0.161 V and −0.432 V vs. Ag/AgCl
Table 1 Kinetic parameters for 1, 2a and 2b
for the two electron transfer steps CuIICuII → CuICuI and
Solvent
n
Vmax (M s−1
)
KM or KA (M) kcat (h−1
)
CuICuI → Cu0Cu0, respectively. The stripping of Cu0 from the
working electrode surface as CuII occurred at −0.168 V vs. Ag/
AgCl during the reverse scan. Increasing the pH value to 7.8
shifted the reduction peaks to more negative potentials of
−0.212 V and −0.492 V, respectively. This is probably due to
the deprotonation of the coordinated alcohol arm at higher
pH but cannot account for the increase in rate of catalytic oxi-
dation. Both 2a and 2b gave very similar cyclic voltammograms
with the CuIICuII → CuICuI transformation taking place at
−0.479 V and −0.485 V, respectively, vs. Ag/AgCl (Fig. S7†). No
shift in peak position was observed with an increase in pH of
the medium.
1
MeCN–H2O pH 7.2
MeCN–H2O pH 7.4
MeCN–H2O pH 7.6
MeCN–H2O pH 7.8
MeOH
1
1
1
1
6.565 × 10−8
6.829 × 10−4
23.634
64.886
77.500
135.136
—
18.024 × 10−8 3.696 × 10−4
21.528 × 10−8 2.524 × 10−4
37.538 × 10−8 5.021 × 10−4
—
—
—
2a
MeCN–H2O pH 7.2 2.017 5.449 × 10−8
MeCN–H2O pH 7.4 1.684 7.892 × 10−8
5.631 × 10−4
3.790 × 10−4
19.619
28.411
46.483
MeCN–H2O pH 7.6 1.162 12.912 × 10−8 3.520 × 10−4
MeCN–H2O pH 7.8 0.958 31.050 × 10−8 5.891 × 10−4
111.780
118.656
MeOH
1.920 32.960 × 10−8 2.417 × 10−4
2b
MeCN–H2O pH 7.2 2.032 5.276 × 10−8
MeCN–H2O pH 7.4 1.443 8.746 × 10−8
5.123 × 10−4
4.464 × 10−4
18.993
31.485
73.904
173.322
83.664
In MeOH solution, the cyclic voltammogram of 1 shows two
closely spaced reduction peaks at −0.411 V and −0.578 V vs.
Ag/AgCl due to the CuIICuII → CuIICuI and CuIICuI → CuICuI
MeCN–H2O pH 7.6 1.105 20.529 × 10−8 6.499 × 10−4
MeCN–H2O pH 7.8 0.923 48.145 × 10−8 8.648 × 10−4
MeOH
2.137 23.240 × 10−8 1.415 × 10−4
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Table 2 Comparison between knon and kcat for 1, 2a and 2b
kcat (h−1
)
kcat/knon
pH
knon (h−1
)
1
2a
19.619
28.411
46.483
2b
18.993
31.485
73.904
1
2a
2b
7.2
7.4
7.6
7.8
0.077
0.104
0.189
0.429
23.634
64.886
77.500
135.136
306.935
623.904
410.053
315.002
254.792
273.182
245.941
260.559
246.662
302.740
391.026
404.014
111.780
173.322
with the fact that the flexible backbone of H3L1 allowed the
metal ion centre to adopt a tetrahedral or T-shaped geometry
in the CuI state. On the other hand, HL2 is planar and rigid
due to the extensive delocalisation of the π electron cloud over
the entire ligand backbone. Accordingly, it was expected that 1
would show higher catechol oxidase activity. On the contrary,
the experimental data showed that 1 is inactive in MeOH
medium, while 2a and 2b exhibited moderate catalytic activity.
In MeCN−H2O (1 : 1), the catalytic oxidation occurred only at
pH values greater than 7.0. Hence, the role of the pH of the
medium is very important in deciding the catalytic potency of
these systems.
EPR responses in solution. To identify the formation of
organic radical-bound intermediates during the catalytic oxi-
dation process, solutions of 1–2b in MeCN−H2O at pH 7.4
were treated with 100 equivalents of 3,5-DTBCH2 and the EPR
spectra of the reaction mixtures were recorded at room tem-
perature (Fig. 15). As expected, for the antiferromagnetically
coupled CuI2I-based complexes the spectra showed a character-
istic peak at ∼1620 G (g ≈ 4.25) for the forbidden ΔMS = 2 tran-
sition and a broad peak with very weak intensity at ∼3385 G
(g ≈ 2.03). For 2a and 2b, an intense sharp signal at g = 2.01
was observed due to the formation of semiquinone radical
bound species (Fig. 15b and c, respectively). On closer obser-
vation, it was found that the signal splits into two, indicating
the presence of a lone electron on C4 of the benzene ring
bearing a single proton. Similar observations were made in the
EPR spectra of 2a and 2b in MeOH in the presence of
100 equivalents of 3,5-DTBCH2 (Fig. S8†). Interestingly, no
such signal was found in the spectrum of 1 in the presence of
Fig. 13 Cyclic voltammograms for 1 at pH 7.0, 7.2, 7.4, 7.6 and 7.8
(maintained by appropriate amount of 50 mM Tris-HCl buffer solution)
in MeCN–H2O (1 : 1) under an N2 atmosphere at the Pt disc working
electrode, Ag/AgCl reference electrode and Pt wire counter electrode.
transformations, respectively (Fig. 14, left). The small peak at
0.669 V vs. Ag/AgCl recorded during the anodic sweep is poss-
ibly due to the ligand-centred oxidation process. Both 2a and
2b registered a single reduction peak at −0.696 V vs. Ag/AgCl
for the two-electron reduction step CuIICuII → CuICuI (Fig. 14,
middle and right, respectively). The peaks at 0.626 V and 0.621
V vs. Ag/AgCl for 2a and 2b, respectively, are associated with
ligand-centred oxidation processes. The peaks corresponding
to the reduction of the CuII centres appeared at a less negative
potential for 1 compared to 2a and 2b, which is consistent
Fig. 14 Cyclic voltammograms for 1–2b in MeOH in the presence of TBAP recorded under an N2 atmosphere at the Pt disc working electrode,
Ag/AgCl reference electrode and Pt wire counter electrode.
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Fig. 15 EPR spectra at 298 K for (a) 1 + 100 equivalent 3,5-DTBCH2, (b) 2a + 100 equivalent 3,5-DTBCH2 and (c) 2b + 100 equivalent 3,5-DTBCH2
in MeCN/H2O (1 : 1) at pH 7.4.
3,5-DTBCH2 (Fig. 15a). This observation rules out the for- DTBCH)2}•+ (C52H66Cu2N2O6S2; Calcd 1004.2949) (Fig. S13 and
mation of the semiquinone radical in the case of 1. Hence, the S15†), respectively. In MeOH solution and in the presence
mechanistic pathway of catechol oxidation by 1 is different of 3,5-DTBCH2, the spectra of 2a and 2b exhibited peaks
from that by 2a and 2b.
at m/z = 1055.6642 and 1055.6709 from the adducts
HRMS analysis. To determine the stability of the complexes {Cu2(L2)(OH)(3,5-DTBCH)2(CH3OH)2-H}+ (C53H73Cu2N2O8S2;
in solution and also to identify the nature of the initial cata- Calcd 1055.3395) and {Cu2(L2)(OCH3)(3,5-DTBCH)2(H2O)
lyst–substrate adduct formed, HRMS analysis of solutions of (CH3OH)-H}+ (C53H73Cu2N2O8S2; Calcd 1055.3395) (Fig. S17
1–2b alone and in the presence of 3,5-DTBCH2 in MeOH and and S19†), respectively. Hence, as evidenced from the HRMS
MeCN–H2O (1 : 1) was performed. In MeOH and MeCN–H2O analysis, 1 forms an initial 1 : 1 adduct with the substrate,
(1 : 1) media, 1 displayed peaks at m/z = 495.0662 and while both 2a and 2b form 1 : 2 catalyst–substrate adducts.
495.0062, respectively, due to the presence of {Cu2(H2L1)}+ This is in accordance with the results obtained from the UV-vis
(C17H25Cu2N2O3S2; Calcd 494.9899) formed under the ESI con- titration experiments.
dition, leading to the reduction of the CuII centres and
Detection and estimation of H2O2. The amount of H2O2 pro-
removal of the hydroxido bridge. In MeOH and MeCN–H2O duced in each case during the catalytic oxidation of 3,5-
(1 : 1) media, another set of peaks at m/z = 530.0086 and DTBCH2 by 1–2b was estimated using a modified iodometric
530.0126 arose from the species {Cu2(H2L1)(OH)(H2O)}+ procedure at intervals of 2, 4, 6, 10, 20 and 30 min after the
(C17H28Cu2N2O5S2; Calcd 530.0032) (Fig. S9 and S10†), respect- addition of the substrate in MeCN–H2O (1 : 1) at pH 7.4
ively. The mass spectrum of 1 in MeCN–H2O (1 : 1) in the pres- (Fig. 16) and MeOH (Fig. S20†). For both 2a and 2b, the initial
ence of 100 equivalents of 3,5-DTBCH2 displayed a peak at m/z concentration of H2O2 formed was equivalent to the quinone
= 924.2861, corresponding to the species {[Cu2(H2L1)(OH)(3,5- concentration, but as the reaction proceeded, the quinone con-
DTBCH2)]ClO4·(H2O)5-H}+
(C31H59ClCu2N2O15S2;
Calcd centration exceeded that of H2O2. This implies the reduction
924.1632) (Fig. S11†). Both 2a and 2b displayed peaks at of O2 to H2O together with H2O2 in the latter stages of the reac-
m/z = 530.9732 in MeOH and m/z = 530.9694 in MeCN–H2O tion. Interestingly, in the case of 1, formation of H2O2 was not
(1 : 1), indicating the presence of the species {Cu2L2}+ detected in MeCN–H2O (1 : 1) at pH 7.4.
(C23H21Cu2N2OS2; Calcd 530.9687) in solution. Once again
Catalytic pathways and proposed mechanism of oxidation.
these were formed under the ESI conditions, leading to the Based on the above observations, we sought an explanation for
reduction of the CuII centers and removal of the hydroxido the biomimetic catechol oxidase (CO)-type catalytic activity
and methoxido bridges. In MeOH and MeCN–H2O (1 : 1), 2a shown by 1–2b. The ‘classic’ mechanism proposed by Krebs
exhibited another peak corresponding to the species {Cu2(L2) et al.,27 suggests an initial stoichiometric reaction between the
(OH)}2+ (C23H22Cu2N2O2S2; Calcd 273.9852) at m/z = 273.9848 CuI2I species and catechol, leading to the formation of a
(Fig. S16 and S12,† respectively). Likewise, the spectra for 2b in reduced CuI2 intermediate and a molecule of quinone. The
MeOH and MeCN–H2O (1 : 1) also showed a peak at m/z = coordination of triplet O2 to this intermediate produces the
753.1281 and 753.1168 from the species {[Cu2(L2)(OMe)(H2O)] peroxo-CuI2I adduct, which is responsible for the oxidation of a
ClO4·(H2O)4}+ (C24H36ClCu2N2O11S2; Calcd 753.0041) (Fig. S18 second equivalent of substrate. This probably requires the
and S14†), respectively. In the presence of 100 equivalents of coordination of the catechol or catecholate ion to both CuII
3,5-DTBCH2, the spectra of 2a and 2b in MeCN–H2O (1 : 1) dis- centres in the bridging mode, as suggested by Solomon et al.28
played peaks at m/z = 990.811 and 1104.4846 arising from the On the other hand, Reedijk et al.29 reported a mechanism
catalyst
substrate-adducts
{Cu2(L2)(OH)(3,5-DTBCH)2-H}+ based on the monodentate coordination of the catechol or
(C51H65Cu2N2O6S2; Calcd 991.2871) and {Cu2(L2)(OMe)(3,5- catecholate ion to a single CuII centre. This mechanism pro-
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Fig. 16 Estimation of H2O2 after 2, 4, 6, 10, 20 and 30 minutes of mixing with 3,5-DTBCH2 in MeCN–H2O2 (1 : 1) at pH 7.4 for 2a and 2b.
poses the possibility for one-electron transfer from the sub-
In 1, further assistance for the abstraction of a proton from
strate molecule to CuII, forming a semiquinonate-bound the catechol-OH is provided by the deprotonated alcohol arm
mixed valence CuICuII species. Coordination of O2 to the CuI of the ligand lying in close proximity to the substrate. This is
centre results in the formation of a CuII–superoxo species, fol- exemplified by the slightly higher values of kcat for 1 at various
lowed by the one-electron oxidation of semiquinone, releasing pH compared to that of 2a and 2b. Once deprotonated, the
one molecule of quinone and H2O2. In the crystal structure of uncoordinated catechol-OH function binds to the second CuII
CO from Ipomoea batatas, the presence of a phenylalanine side centre by displacing the ligand alcohol arm and the catecho-
chain over one of the CuII sites prevents the bidentate coordi- late can now bind both the metal centres in a bidentate brid-
nation of the catecholate ion to both metal ion centres.27 Our ging mode. Thus, the catalytic pathway follows the Krebs’ pro-
complexes form stable catalyst–substrate adducts at neutral pH posed ‘classic’ mechanism, which is in accordance with the
in MeCN–H2O (1 : 1) medium and show activity only at pH absence of a radical signal in the EPR experiment and lack of
greater than 7.0. This initiation of the catalytic activity in basic formation of H2O2. In the case of 2a and 2b, the kinetic experi-
medium can be explained considering the monodentate ments show the cooperative participation of the substrate in
coordination of 3,5-DTBCH to one CuII site, as predicted by enhancing the catalytic reaction rate. Both 2a and 2b form a
the HRMS study, and the necessity of the abstraction of the 1 : 2 adduct in MeCN–H2O (1 : 1) at pH 7.0. Most likely, the
proton from the uncoordinated phenolic-OH for the 2e− oxi-
dation of the catechol molecule. The EPR studies of 2a and 2b
in solution in the presence of 3,5-DTBCH2 confirmed the for-
mation of semiquinone radical species, which were not
detected in the case of 1. The deprotonation seems essential
for 2a and 2b for the formation and binding of the semiquino-
nate to the metal centre in the mixed valence CuICuII species.
Considering that the intermetallic distances are around 2.9 Å,
the semiquinonate has the option of binding to either the CuI
or CuII centre. When coordinated to the CuI centre, the reac-
tion should proceed according to the mechanism proposed by
Reedijk et al. (Schemes 3 and S4,† Path a), while coordination
to the CuII site leads to the reaction following a modified
‘classic’ mechanism by Krebs et al. (Schemes 3 and S4,† Path
b†). Thus, both H2O2 and H2O are expected to be formed. The
determination of the amount of H2O2 produced in the
medium indicated that the concentration of H2O2 was lower
than 3,5-DTBQ at the latter stages of the reaction. Thus, the
reaction proceeds according to the mechanism proposed by
Reedijk, involving a semiquinonate radical with a significant
contribution from the modified ‘classic’ mechanism.
Interestingly, the HRMS analysis of 1–2b in both MeOH and
MeCN media indicates the formation of [CuICuI(H2L1)]+ and
[CuICuI(L2)]+ under ESI conditions. These species are analo-
gous to the deoxy form of the natural enzyme, which indicates
their formation during the catalytic cycle occurs through a
mechanism similar to that proposed by Krebs et al.
Scheme 2 Catalytic pathway for 1.
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Scheme 3 Two different catalytic pathways ‘a’ and ‘b’ for 2a.
apical monodentate coordination of 3,5-DTBCH to the CuII mism is associated with the presence of synergistic non-covalent
centres occur from opposite ends and both catechol-OH interactions, namely, electrostatic binding, groove binding
groups show hydrogen bonding interaction with the bridging (major or minor) along the outside of the DNA helix, hydrogen
hydroxido and methoxido groups. For 2a, the bridging hydro- bonding and van der Waals interactions. On the other hand,
xido group acts as a hydrogen bond acceptor towards one cate- hypochromism is due to the intercalative mode involving strong
chol-OH, while acting as a hydrogen bond donor towards the π–π stacking interactions between the aromatic chromophore of
second catechol-OH, and thus activating the O–H bond of the complexes and the base pairs of DNA.
latter for proton abstraction. In the case of 2b, the hydrogen
The complexes 1, 2a and 2b exhibited intense absorption
bonding interaction of one catechol-OH with the bridging bands at 260–290 nm and 400–460 nm, which are attributed to
methoxido group weakens the hydrogen bonding interaction the intra-ligand charge transfer band corresponding to the
of the second catechol-OH, facilitating proton abstraction by a π–π* transition and ligand to metal ion LMCT bands, respect-
base. At higher pH values, this cooperative effect becomes less ively (Fig. 17). Upon incremental addition of ct-DNA (0–4.0 ×
important and the value of n moves closer to unity. MeOH 10−4 M) to fixed concentrations of 1, 2a and 2b (2 × 10−4 M), a
being more basic than MeCN and H2O molecules in the reac- hyperchromic effect with a slight blue-shift in the bands in
tion medium is able to abstract a proton from the substrate 265–290 nm region was observed. Meanwhile, the LMCT band
attached to 2a and 2b in the presence of substrate cooperativ- centred at 400–460 nm exhibited a hypochromic effect with a
ity. In 1, MeOH is unable to deprotonate the coordinated red-shift, which was accompanied by the appearance of distinct
alcohol arm of the ligand to assist the proton abstraction from isosbestic points at 305 nm and 398 nm for complexes 2a and
catechol, resulting in its inactivity. The proposed catalytic 2b and complex 1, respectively, indicating equilibrium between
pathways for 1–2b are depicted in Schemes 2, 3 and S4,† the free and DNA-bound probe in the ground state. The topo-
respectively.
logy of the ligands and incorporation of two copper centres in
Studies for in vitro ct-DNA binding. We selected several bio- the complexes resulted in a characteristic pattern in UV-vis titra-
physical methods to evaluate the ability of complexes 1–2b to tions, showing simultaneous “hyperchromic” and “hypochro-
interact with DNA via a non-covalent mode or cause DNA mic” changes. The hyperchromic effect can be attributed to
damage via an oxidative and/or cleavage mechanism, and strong electrostatic interactions due to the presence of posi-
thus, initiate the processes of programmed cell death or apop- tively charged synergic dinuclear metal centers with negatively
tosis. Electronic spectral measurements were performed to charged oxygen atoms of the DNA double helix, leading to
determine the interaction of 1, 2a and 2b with ct-DNA and to overall damage of DNA. On the other hand, the hypochromic
probe their possible binding modes. In general, hyperchro- effect arises due to the partial intercalation mediated via π–π*
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Fig. 17 Absorption spectra of (a) 1, (b) 2a and (c) 2b (2 × 10−4 M) in the presence of increasing concentrations of ct-DNA (0–4.0 × 10−4 M) in
Tris-HCl buffer (pH 7.2).
stacking interactions by the ligand scaffold of the complexes vides more reliable results for the mode of binding. Complexes
between the base pairs of the DNA double helix.30,31
1–2b displayed intense luminescence at ca. 320–350 nm (λem)
To ascertain the extent of binding for complexes 1–2b, the when excited at 265 nm in the absence of ct-DNA in 0.01 Tris-
intrinsic binding constant, Kb, values were calculated using HCl/50 mM NaCl buffer at room temperature. Upon the conco-
the following Wolfe–Shimmer equation (eqn (6)):32
mitant addition of ct-DNA (0–4.00 × 10−5 M) to the complexes,
a decrease in florescence intensity was observed without any
apparent shift in the emission maximum (Fig. 18). The
observed quenching demonstrated that the binding of com-
plexes 1–2b with ct-DNA is due to the change in the microenvi-
½DNAꢂ ½DNAꢂ
1
¼
þ
ð6Þ
ðεa ꢁ εf Þ ðεb ꢁ εf Þ kbðεb ꢁ εf Þ
where, [DNA] is the ct-DNA concentration, and εa, εf and εb are ronment around the fluorophore residue inside the DNA helix.
the apparent (Aabs/[CuII complex]), free and bound complex This suggests an intercalative mode of binding involving π–π
extinction coefficients, respectively. In the plot of [DNA]/ stacking interactions between the aromatic ligand and the
(εa − εf) vs. [DNA] with a slope of 1/(εb − εf) and intercept of base pairs of DNA.33,34 The quantitative assessment of the ct-
1/[Kb(εb − εf)], the Kb values were obtained using the ratio of DNA binding strength of complexes 1–2b was ascertained from
the slope to the intercept. The intrinsic binding constant, Kb, the values of the binding constants, K, calculated using the
values of complexes 1–2b with ct-DNA were found to be 4.2 × Scatchard equation,35 which were found to be 3.73 × 104 M−1
,
104 M−1, 5.6 × 104 M−1 and 4.8 × 104 M−1, respectively, which 6.81 × 104 M−1 and 4.72 × 104 M−1, respectively, well consistent
followed the order 2a > 2b > 1. This suggests 2a exhibits the with the results of the UV-vis spectral titrations with ct-DNA.
highest binding propensity, which can be attributed to the
Competitive binding experiments with ethidium bromide
additional stabilization by strong π–π stacking interactions due (EB) were also carried out to validate the intercalative binding
to the presence of more phenyl rings in the ligand backbone mode of the complexes. The EB planar cationic dye is a classi-
compared to 1 and structural differences between 2a and 2b cal intercalator that emits intense fluorescence in the presence
due to the presence of hydroxy and methoxy functional appen- of DNA at ca. 595 nm (λem) due to its ability to intercalate
dages, respectively. Complex 2a, which possess an apical between the adjacent base pairs of DNA helices. A substantial
hydroxy functionality, is a more active species as it can engage decrease in the fluorescence intensity of EB-DNA was observed
readily in hydrogen bonding and cleavage reactions, resulting upon increasing the concentration of the copper complexes
in a stronger binding strength.
1–2b, where [complex]/[DNA] = 1 : 1 to 6 : 1, indicating competi-
The binding affinity of the complexes with DNA was also tive binding between EB and the complexes towards ct-DNA
validated by employing fluorescence spectroscopy, which is a binding (Fig. 19). This decrease can be attributed to the displa-
more sensitive method than absorbance spectroscopy and pro- cement of the EB molecule from the EB-DNA complexes,
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Fig. 18 Emission spectra of complexes (a) 1, (b) 2a and (c) 2b in Tris-HCl buffer (pH 7.2) with and without ct-DNA at room temperature. The arrow
shows a change in intensity with increasing concentration of DNA.
which establishes the intercalative mode of binding. The mag- mobility of the plasmid DNA changed. In complexes 1–2b, the
nitude of the quenching efficiency was evaluated using the supercoiled form, SC, Form I, gradually diminished on increas-
Stern–Volmer constant, Ksv, values for complexes 1–2b, which ing the concentration (5–30 µM); whereas, the nicked circular
were found to be 3.08 × 104, 4.8 × 104 and 4.12 × 104 M−1
,
form, NC, Form II, increased (Lanes 2–7). However, at 30 µM
respectively. The Ksv values and plots reveal that complex 2a is concentration, the complexes exhibited the substantial conver-
the best quencher among the CuII complexes.
sion of Form I into Form II (Fig. 20a, 21a and 22a, respectively),
Circular dichroic studies. The CD spectra of complexes 1–2b suggesting significant cleavage activity. Furthermore, there was
upon incubation with ct-DNA revealed a decrease in the inten- no evidence of a slow moving linear circular form LC (Form III),
sity of both the positive and negative bands at 275 nm (due to which migrates between the SC and NC forms and is typical for
base stacking) and 246 nm (due to right handed helicity), double-strand scission. Therefore, these changes in the pattern
respectively (Fig. 20) with a slight red-shift in the positive band support the single-strand cleavage mediated by the complexes
at 275 nm. These changes in the CD spectra of 1–2b can be similar to many other CuII complexes previously synthesised.39,40
attributed to the partial intercalation mode of the Cu(II) com- Furthermore, many copper complexes have been proven to
plexes with ct-DNA, which could unwind the helix of ct-DNA, cleave DNA more efficiently in the presence of exogenous agents,
leading to the loss of helicity.36 Moreover, the red-shift such as ascorbate (Asc), glutathione (GSH), H2O2 and 3-mercap-
observed in the positive bands revealed that the complexes topropionic acid (MPA). Therefore, it was imperative to explore
could induce changes in the helicity and base stacking signa- the cleavage activity of 1–2b in the presence of these activators,
tures, which may result in a B → A conformational change in and it was observed that the activity was enhanced significantly,
the DNA double helix.37,38 Furthermore, the changes in intensity as depicted in Fig. 21b, 22b and 23b, respectively, in their pres-
followed the order 2a > 2b > 1, which is in good agreement with ence, which followed the order H2O2 > MPA ≈ GSH > Asc.41
the results obtained by UV-vis and fluorescence spectroscopy.
To predict the mechanism of the cleavage reactions by the
ct-DNA cleaving experiments. Since DNA cleaving activity is complexes, 1–2b (30 µM) were allowed to react with EtOH and
considered as direct evidence of metal complex-DNA inter- DMSO (hydroxyl radical scavengers), NaN3 (singlet oxygen sca-
actions, gel electrophoretic assays of complexes 1–2b were per- venger, O2) and SOD (superoxide anion radical, O2•−). It was
1
formed using supercoiled pBR322 plasmid DNA as the sub- observed that DMSO and EtOH did not show significant
strate in a medium of 5 mM Tris-HCl/50 mM NaCl buffer at quenching efficiency of the DNA cleavage (Fig. 21b, 22b and
pH 7.20. On varying the concentration of the complexes with 23b, respectively) ruling out the possibility of the involvement
the plasmid DNA (complex-DNA ratio), the electrophoretic of diffusible hydroxyl radicals in the cleavage reaction.
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Fig. 19 Emission spectra of EB-ct-DNA in the absence and presence of complex (a) 1, (b) 2a and (c) 2b in Tris-HCl buffer at pH 7.2. [Complex 1–2b] =
[EB] = [DNA] = 1.11 × 10−4 M. Arrow shows change in intensity with increasing concentration of complexes 1–2b.
The mechanistic pathway for the proposed oxidative clea-
vage involves initially the interaction of the CuII-complex with
DNA in the outer sphere mode, which leads to the reduction of
the CuII species to CuI species in the presence of the reducing
·−
agent. O2 is generated upon the reaction of the Cu(I) com-
plexes with dioxygen. The superoxide (O2−) dismutates to
Fig. 20 CD spectra of ct-DNA alone (red), ct-DNA + 1 (blue), ct-DNA +
2a (green) and ct-DNA + 2b (brown) in Tris-HCl buffer (pH = 7.2).
[Complex] = 10−4 M, [DNA] = 10−4 M.
However, in the presence of NaN3 and SOD, significant inhi-
bition occurred. These results indicate the oxidative cleavage
pathway of complexes 1–2b and suggest that ROS, singlet Fig. 21 Agarose gel electrophoretic patterns showing the cleavage of
pBR322 plasmid DNA (300 ng) by the complex under different con-
ditions for an incubation time of 45 min at 37 °C. (a) Lane 1, DNA
control; lane 2, DNA + 1 (5 µM); lane 3, DNA + 1 (10 µM); lane 4, DNA +
1 (15 µM); lane 5, DNA + 1 (20 µM); lane 6, DNA + 1 (25 µM); and lane 7
oxygen and the superoxide anion radical are responsible for
their cleavage activity.42,43 The cleavage studies were per-
formed in the presence of DAPI and methyl green to explore
the preference of the potential binding sites at the minor and
DNA + 1 (30 µM). (b) Lane 1, DNA control; lane 2, DNA + 1 (30 µM) + Asc
major grooves with the DNA helix. The addition of DAPI, (0.4 mM); lane 3, DNA + 1 (30 µM) + GSH (0.4 mM); lane 4, DNA + 1
(30 µM) + H2O2 (0.4 mM); lane 5, DNA + 1 (30 µM) + MPA (0.4 mM); lane
6, DNA + 1 (30 µM) + DMSO (5 µL); lane 7, DNA + 1 (30 µM) + EtOH
(5 µL); lane 8, DNA + 1 (30 µM) + NaN3 (20 mM); lane 9, DNA + 1
(30 µM) + SOD (5 units); lane 10, DNA + 1 (30 µM) + DAPI (8 mM); and
lane 11, DNA + 1 (30 µM) + methyl green (2.5 µL of a 0.01 mg mL−1
solution).
which is a minor groove binding agent, induced significant
cleavage inhibition activity, while methyl green (a major groove
binding agent) could not quench DNA cleavage (Fig. 21b, 22b
and 23b, lanes 10 and 11, respectively), thereby implicating the
clear preference of the complexes for DNA minor grooves.
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Fig. 22 Agarose gel electrophoretic pattern showing the cleavage of
pBR322 plasmid DNA (300 ng) by the complexes under different conditions
for an incubation time of 45 min at 37 °C. (a) Lane 1, DNA control; lane 2,
DNA + 2a (5 µM); lane 3, DNA + 2a (10 µM); lane 4, DNA + 2a (15 µM); lane
5, DNA + 2a (20 µM); lane 6, DNA + 2a (25 µM); and lane 7 DNA + 2a
(30 µM). (b) Lane 1, DNA control; lane 2, DNA + 2a (30 µM) + Asc (0.4 mM);
lane 3, DNA + 2a (30 µM) + GSH (0.4 mM); lane 4, DNA + 2a (30 µM) +
H2O2 (0.4 mM); lane 5, DNA + 2a (30 µM) + MPA (0.4 mM); lane 6, DNA + 1
(30 µM) + DMSO (5 µL); lane 7, DNA + 2a (30 µM) + EtOH (5 µL); lane 8,
DNA + 2a (30 µM) + NaN3 (20 mM); lane 9, DNA + 2a (30 µM) + SOD
(5 units); lane 10, DNA + 2a (30 µM) + DAPI (8 mM); and lane 11, DNA + 2a
(30 µM) + methyl green (2.5 µL of a 0.01 mg mL−1 solution).
Fig. 24 Molecular docked structures of (a) 1, (b) 2a, (c) and 2b in the
minor grooves of the DNA dodecamer duplex of sequence
d(CGCGAATTCGCG)2 (PDB ID: 1BNA).
the DNA double helix. For small molecules, the minor groove
is found to be the preferential target since the closer contiguity
of the strands allows more close contact in surface area and
tightly binds with the best fitting.45 The efficient docking of
complexes 1–2b revealed that these complexes partially interca-
late at the DNA intercalative sites or fit snugly into the curved
contour of the targeted DNA in the narrow and slightly deeper
guanine–cytosine (G–C) region of the minor groove (Fig. 24).46
The relative binding energy of the docked structures was found
to be −253.49, −306.00 and −294.07 kJ mol−1 for complexes
1–2b, respectively, where 2a exhibits the highest binding
affinity. This is attributed to the planarity of the 2a complex,
which possess an additional aromatic phenyl ring in its ligand
scaffold bound to CuII centres.
Experimental
Reagents and materials
Fig. 23 Agarose gel electrophoretic patterns showing the cleavage of
pBR322 plasmid DNA (300 ng) by the complexes under different con-
ditions for an incubation time of 45 min at 37 °C. (a) Lane 1, DNA
control; lane 2, DNA + 2b (5 µM); lane 3, DNA + 2b (10 µM); lane 4,
DNA + 2b (15 µM); lane 5, DNA + 2b (20 µM); lane 6, DNA + 2b (25 µM);
and lane 7 DNA + 2b (30 µM). (b) Lane 1, DNA control; lane 2, DNA + 2b
(30 µM) + Asc (0.4 mM); lane 3, DNA + 2b (30 µM) + GSH (0.4 mM); lane
4, DNA + 2b (30 µM) + H2O2 (0.4 mM); lane 5, DNA + 2b (30 µM) + MPA
(0.4 mM); lane 6, DNA + 2b (30 µM) + DMSO (5 µL); lane 7, DNA + 2b
(30 µM) + EtOH (5 µL); lane 8, DNA + 2b (30 µM) + NaN3 (20 mM); lane
9, DNA + 2b (30 µM) + SOD (5 units); lane 10, DNA + 2b (30 µM) + DAPI
(8 mM); and lane 11, DNA + 2b (30 µM) + methyl green (2.5 µL of a
0.01 mg mL−1 solution).
The chemicals used were obtained from the following sources:
copper hydroxide carbonate and triethylamine from Merck,
India, and 2-mercaptoethanol, 2-chloroethylamine hydro-
chloride and 2-(methylthio)aniline from Alfa Aesar. Copper(II)
perchlorate hexahydrate was freshly prepared by treating
hydrated copper(II) carbonate (22.12 g, 0.1 mol) with 1 : 1
aqueous perchloric acid solution. 2,6-Diformyl-4-methylphenol
was prepared following a modified literature procedure.47 All
other chemicals and solvents used were reagent-grade
materials and used as received without further purification.
Synthesis of ligands
H2O2, which generates ROS species responsible for DNA
strand scission via the oxidative pathway.44
[2-(2-Aminoethylthio)ethanol] (AET). 2-Marcaptoethanol
(0.780 g, 10 mmol) was added dropwise to a freshly prepared
aqueous (30%) NaOH solution (15 mL) under stirring for
30 min and then the solution was heated to 50 °C.
Subsequently, an aqueous solution of 2-chloroethylamine
hydrochloride (1.159 g, 10 mmol) was added dropwise to the
above mixture for 2 h. Finally the mixture was stirred for a
further 2 h at 50 °C to give a pale yellow solution. The resulting
solution was concentrated under reduced pressure and then
•ꢁ
CuðiÞ þ O2 ! CuðiiÞ þ O2
2O2•ꢁ þ 2Hþ ! O2 þ H2O2
ð7Þ
ð8Þ
ð9Þ
•
ROS species þ DNA ! DNA cleavage
Molecular docking study
The molecular docking study was performed on complexes dissolved in absolute ethanol and filtered to remove the pre-
1–2b with DNA to envisage their preferential binding sites in cipitate. Then the filtrate was concentrated under reduced
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pressure to afford AET as a pale yellow liquid. 1H NMR strong, br = broad, s = strong, m = medium, w = weak): 3422
(400 MHz, (CD3)2SO, δ ppm): 2.47–2.53 (4H, –CH2), 2.63 (2H, (br), 1637 (s), 1554 (m), 1088 (vs), 769 (w), 625 (m). UV-vis
methylene CH2 attached with N atom), 3.48 (2H, methylene spectra [λmax, nm (ε, L mol−1 cm−1)]: (MeCN solution) 608
CH2 attached with O atom). 13C NMR (100 MHz, (CD3)2SO, (193), 371 (11 900), 259 (29 600). Molar conductance, ΛM:
δ ppm): 34.31, 35.99 (methylene C attached with S), 42.18 (MeCN solution) 230 Ω−1 cm2 mol−1
.
(methylene C attached with N atom), 61.43 (methylene C
[Cu2(μ-L2)(μ-OH)(OH2)](ClO4)2 (2a). Complex 2a was pre-
attached with O atom).
pared in a similar manner as reported with slight modification
H3L1 (2,6-bis-[{2-(2-hydroxyethylthio)ethylimino}methyl]-4- for improved yield.16 To a stirred solution of HL2 (approx.
methylphenol). H3L1 was prepared by reacting 4-methyl-2,6- 0.41 g, 1 mmol) in MeOH (20 mL), an H2O solution (10 mL) of
diformylphenol (0.164 g, 1 mmol) with 2-(2-aminoethylthio) Cu(ClO4)2·6H2O (0.745 g, 2 mmol) was added. To this reaction
ethanol (0.242 g, 2 mmol) in MeOH at room temperature with mixture, NEt3 (0.202 g, 2 mmol) was added and the resulting
stirring for 1 h followed by refluxing for 2 h. Removal of the green solution was stirred for 2 h. Next the solution was fil-
solvent under vacuum yielded a yellow oily mass, which was tered, and the green precipitate was dissolved in MeCN and
characterized by FTIR and NMR spectroscopy. The as-obtained left for slow evaporation in air. Green block-shaped single crys-
oily product was used directly for the reactions with metal ion tals suitable for X-ray analysis were obtained after 1 week.
salts without further purification. FT-IR (cm−1, KBr pellet): Yield: 0.490 g (64%). Microanalytical data is consistent with
3368 (br), 2918 (m), 1637 (s), 1600 (w), 1458 (m), 1219 (w), the formula [Cu2(μ-L2)(μ-OH)(OH2)](ClO4)2. Anal. calcd for
1045 (w), 1010 (w), 871 (w), 772 (s). 1H NMR (400 MHz, C23H24Cu2N2O11S2Cl2 (766.56 g mol−1): C, 36.04; H, 3.16; N,
(CD3)2SO, δ ppm): 8.58 (2H, imine–H), 7.49 (2H, Ar–H), 3.65. Found: C, 36.32; H, 3.12; N, 3.68. Selected FT-IR bands:
2.81–3.74 (8H, methylene CH2 attached with S atom), 3.52 (4H, (KBr, cm−1, vs = very strong, br = broad, s = strong, m =
methylene CH2 attached with O atom), 3.74 (4H, methylene medium, w = weak) 3468 (br), 1618 (m), 1534 (s) 1089 (vs), 759
CH2 attached with N atom), 2.23 (3H, methyl–H). 13C NMR (w), 624 (m). UV-vis spectra [λmax, nm (ε, L mol−1 cm−1)]:
(100 MHz, (CD3)2SO, δ ppm): 161.94 (imine C), 159.98–121.27 (MeCN solution) 606 (184), 404 (14 500), 307 (29 400). Molar
(Ar C), 61.45 (methylene C attached to imine N), 59.64 (methyl- conductance, ΛM: (MeCN solution) 250 Ω−1 cm2 mol−1
.
ene C attached to OH group), 34.50–32.97 (methylene C
attached to S), 20.35 (methyl C).
[Cu2(μ-L2)(μ-OCH3)(OH2)](ClO4)2 (2b). 2b was synthesized
following the same procedure described for 2a using pure
HL2 (4-methyl-2,6-bis-[{2-(methylthio)phenylimino}methyl] MeOH solvent in place of the MeOH-H2O mixture. The green
phenol). 4-Methyl-2,6-diformylphenol (0.820 g, 5 mmol) and 2- microcrystalline precipitate obtained from the reaction
(methylthio)aniline (1.39 g, 10 mmol) were mixed in MeOH mixture was dissolved in MeCN and kept for slow evaporation
(20 mL) under stirring followed by refluxing for 2 h. The in air. Green block-shaped single crystals suitable for X-ray
solvent was removed under vacuum to obtain a yellow solid analysis were obtained after 1 week. Yield: 0.484 g (62%).
product, which was characterized via FT-IR and NMR spec- Microanalytical data is consistent with the formula [Cu2(μ-L2)
troscopy. Yield: 0.382 g (94%). FT-IR (cm−1, KBr pellet): 3436 (μ-OCH3)(OH2)](ClO4)2. Anal. calcd for C24H26Cu2N2O11S2Cl2
1
(br), 2916 (m), 1591 (vs), 1438 (s), 975 (m), 743 (m). H NMR (780.59 g mol−1): C, 36.93; H, 3.36; N, 3.59. Found: C, 37.01; H,
(400 MHz, (CD3)2SO, δ ppm): 8.58 (2H, imine–H), 7.49 (2H, Ar– 3.35; N, 3.58. Selected FT-IR bands: (KBr, cm−1, vs = very
H), 2.81–3.74 (8H, methylene CH2 attached with S atom), 3.52 strong, br = broad, s = strong, m = medium, w = weak) 3469
(4H, methylene CH2 attached with O atom), 3.74 (4H, methyl- (br), 1618 (m), 1535 (s) 1088 (vs), 759 (w), 624 (m). UV-vis
ene CH2 attached with N atom), 2.23 (3H, methyl–H). 13C NMR spectra [λmax, nm (ε, L mol−1 cm−1)]: (MeCN solution) 607
(100 MHz, (CD3)2SO, δ ppm): 161.94 (imine C), 118.76–147.12 (212), 402 (17 200), 311 (35 300). Molar conductance, ΛM:
(Ar C), 20.34 (methyl C), 14.86 (methyl C attached to S atom).
(MeCN solution) 250 Ω−1 cm2 mol−1
.
Caution! Complexes of metal ions containing organic ligands
together with perchlorate counter anions are explosive in nature.
Synthesis of complexes
[Cu2(μ-H2L1)(μ-OH)](ClO4)2·2H2O (1). The as-prepared solu- Although we did not face any problems while dealing with the title
tion of H3L1 (25 mL) containing 1 mmol of H3L1 (approx. compounds, it is safe to prepare a small amount of sample and
0.37 g) was diluted to 30 mL with MeOH. To this solution, an handle it with care.
MeOH solution (15 mL) of Cu(ClO4)2·6H2O (0.744 g, 2 mmol)
Physical measurements. Elemental analyses (C, H and N) of
was added slowly and the resulting green solution was stirred the compounds were performed with a PerkinElmer model
for ca. 15 min. Subsequently, an MeOH solution (10 mL) of 240C elemental analyser. A PerkinElmer RX1 spectrometer was
NEt3 (0.202 g, 2 mmol) was added dropwise to the above solu- used to record the FTIR spectra. A CHI 1120A electrochemical
tion obtain a dark green solution and stirring was continued analyzer was used for cyclic voltammetry measurements.
for 1 h. The solution was filtered and kept in air at room tem- Electrochemical measurements were performed at 25 °C in
perature for slow evaporation. Green block-shaped single crys- MeCN–H2O (1 : 1) at pH 7.0–7.8 (maintained by 50 mM Tris-
tals suitable for X-ray analysis were obtained after 12 days. HCl buffer solutions) and MeOH in a one-compartment cell
Yield: 0.54 g (72%). Anal. calcd for C17H30Cu2N2O14S2Cl2 with platinum as the working electrode, Pt wire as the counter
(748.52 g mol−1): C, 27.28; H, 4.04; N, 3.74. Found: C, 27.45; H, electrode and Ag-AgCl as the reference electrode. For the
4.02; N, 3.76. Selected FT-IR bands: (KBr, cm−1, vs = very electrochemical measurements in MeOH, TBAP was used as
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the external electrolyte while in MeCN–H2O (1 : 1) Tris-HCl a color code of red-white-blue, where red spots indicate shorter
buffer solution acted as the electrolyte. Electron paramagnetic contacts, white regions highlight contacts around the van der
resonance (EPR) spectra were recorded at 9.13 GHz (X-band) in Waals distance, and the blue areas are for longer contacts. Two
continuous-wave mode with a Bruker ELEXSYS 580 X-band more coloured properties, shape index and curvedness, were
EPR spectrometer equipped with a standard accessory for also specified based on the shape and the local curvature of
room temperature operation (298 K). Direct current (dc) mag- the surfaces. The Hirshfeld surface analysis of 1–2b is pre-
netic measurements were performed using polycrystalline sented in the ESI.†
samples of compounds 1, 2a and 2b using a Quantum Design
Kinetics for catalytic oxidation of 3,5-DTBCH2. The catecho-
MPMS SQUID VSM magnetometer in the temperature range of lase activity of the complexes were studied in MeOH and
300–10 K under an applied field of 0.1 T. Electronic spectra MeCN–H2O (1 : 1) at pH 7.2–7.8 (maintained by appropriate
were recorded on a PerkinElmer Lambda 25 using cuvettes 50 mM Tris-HCl buffer) separately using 3,5-di-tert-butylcate-
with a 1 cm path length and data were reported in λmax/nm. chol (3,5-DTBCH2) as the model substrate. The experiments
Emission spectra were recorded on a Shimadzu RF-5301PC were monitored on a Shimadzu UV 3100 UV-Vis-NIR spectro-
spectrofluorometer using slit widths of 10/10 nm. CD spectra photometer and were repeated thrice. With the progress of the
were measured on a Jasco J-815-CD spectropolarimeter at oxidation reaction, i.e., oxidation of 3,5-DTBCH2 to 3,5-DTBQ,
room temperature. All experiments involving the interaction of
a characteristic increase in the absorption intensity at
the complexes with ct-DNA were carried out in aerated buffer ∼400 nm was observed. The complex solution of constant con-
(5.0 mM Tris-HCl, 50.0 mM NaCl, pH = 7.3). The concentration centration (∼1 × 10−5 M) in MeCN–H2O (1 : 1) at pH 7.2–7.8
per base pair for DNA was determined spectrophotometrically and MeOH were reacted separately with increasing amounts of
by assuming the ε260 nm value to be 6600 M−1 cm−1 48
.
3,5-DTBCH2 (10 to 100 equivalents) at room temperature
Cleavage experiments were performed via Axygen electrophor- under aerobic conditions to study the kinetics of the oxidation
esis supported by a Genei power supply in the potential range reaction, and then from the quinone band maximum, the
of 50–500 V and visualized and photographed using a Vilber– change in absorbance was monitored. A 10 min reaction time
INFINITY gel documentation system.
scan for all the complex-substrate combinations was run and
Crystal data collection and refinement. Single crystal X-ray the data was treated using the initial rate method. The slope of
diffraction data for 1, 2a and 2b were collected on a Bruker the absorbance versus time plot indicates the rate of the reac-
SMART APEX-II CCD X-ray diffractometer furnished with a tion and based on eqn (5), a kinetic analysis was performed.
graphite-monochromated Mo Kα (λ = 0.71073 Å) radiation Experiments were also performed in the absence of the com-
source using the ω scan (width of 0.3° frame−1) method at plexes in MeCN–H2O (1 : 1) at pH 7.2–7.8 to determine the
293 K at a scan rate of 4 s per frame. The SAINT and XPREP auto-oxidation rate (knon) of 3,5-DTBCH2.
software49 were used for data processing and space group
Detection of H2O2 during catechol oxidation. Hydrogen per-
determination. Direct methods of SHELXS-201450 were used to oxide generation during the catalytic oxidation of 3,5-DTBCH2
solve the structure, which were then refined with full-matrix was determined using spectrophotometry following a modified
least squares using the SHELXL (2014/7)51 program package iodometric method.56 The reaction mixtures were prepared in
included in the WINGX system Version 2014.1.52 Data were a similar way to that for the kinetic experiments. The reaction
corrected for Lorentz and polarization effects, where an mixture was acidified with H2SO4 (1 × 10−3 M) to pH 2 after 1 h
empirical absorption correction was applied using SADABS.53 to completely quench the reaction. The oxidized product
The locations of the heaviest atoms (Cu) were determined quinone was extracted thrice with CH2Cl2 and then 1 mL of
easily. The O, N, and C atoms were subsequently determined 10% aqueous KI solution was added to the aqueous layer.
from the difference Fourier maps. These atoms were refined Ammonium molybdate (3%) solution was added in a catalytic
−
anisotropically. The H atoms on the hydroxido bridges were amount (3 drops) to accelerate the formation of triiodide (I3
)
fixed from the Fourier map, while the other H atoms were in the presence of H2O2.56 The reaction occurring in the
incorporated at calculated positions and refined with a fixed medium was H2O2 + 2I− + 2H+ → 2H2O + I2, and the I2 liber-
geometry and riding thermal parameters with respect to their ated from the reaction combined with excess iodide ions
−
carrier atoms. Crystallographic diagrams were presented using present in the reaction mixture to form I3 ions according to
the DIAMOND software.54 A summary of the crystal data and the reaction I2 (aq) + I− → I3−. The H2O2 liberated from the
relevant refinement parameters is summarized in Table 3. reaction mixture was detected spectrophotometrically by moni-
Crystallographic data (including structure factors) have been toring the band at 353 nm (ε = 26 000 L mol−1 cm−1), which is
deposited with the Cambridge Crystallographic Data Centre as the characteristic band of I3−. A blank experiment (without
supplementary publications CCDC 1857614, 1857615 and 3,5-DTBCH2 or without catalyst) was also carried out because
1857616,† respectively.
atmospheric O2 can oxidize I−.
Hirshfeld surface analysis. The surface characteristics of the
In vitro DNA binding and cleavage experiments. DNA
molecules in the crystals were mapped using Crystal Explorer binding experiments were performed in Tris-HCl/NaCl
(version3.1) using the crystal structure coordinates of the .cif (5 : 50 mM) buffer at pH 7.20, which included absorption spec-
files.55 Graphical plots of the molecular Hirshfeld surfaces tral traces, emission spectroscopy and CD.57,58 While measur-
were mapped with a normalized contact distance (dnorm) using ing the absorption spectra, an equal amount of ct-DNA was
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Table 3 Crystal data and structure refinement details for 1, 2a and 2b
Parameter
1
2a
2b
Formula
C17H30Cl2Cu2N4O14S2
748.55
C23H24Cl2Cu2N2O11S2
766.56
C24H26Cl2Cu2N2O11S2
780.59
F.W. (g mol−1
)
Crystal system
Space group
Monoclinic
Cc
Triclinic
P1
Triclinic
P1
ˉ
ˉ
Crystal color
Green
Green
Green
Crystal size/mm3
0.35 × 0.27 × 0.20
8.6838(8)
33.813(3)
9.7383(9)
90.00
104.186(5)
90.00
2772.2(4)
4
1.793
0.40 × 0.35 × 0.30
8.637(3)
12.523(4)
13.642(6)
77.99(2)
89.17(3)
78.25(2)
1412.4(9)
2
1.802
1.905
776
301(2)
0.41 × 0.37 × 0.33
8.566(7)
12.777(12)
13.867(7)
99.18(6)
91.21(6
104.27(8)
1449(2)
2
1.789
1.859
792
299(2)
a/Å
b/Å
c/Å
α/°
β/°
γ/°
V/Å3
Z
Dc/g cm−3
μ (mm−1
F(000)
T/K
)
1.946
1528
297(2)
Total reflns
R(int)
14 501
0.0497
16 409
0.0374
15 786
0.0849
Unique reflns
Observed reflns
Parameters
5061
4434
344
0.0461, 0.1315
1.043
0.814, −0.678
1857614
4994
4337
413
0.0276, 0.0805
1.079
0.351, −0.449
1857615
5159
3195
419
0.0550, 0.1585
1.068
0.946, −0.440
1857616
R1; wR2 (I > 2σ(I))
GOF (F2)
Largest diff peak and hole (e Å−3
CCDC no.
)
added to the reference solution to eliminate the absorbance of [Cu2] complexes into any high nuclearity [Cu4] or [Cu6] struc-
ct-DNA itself, and the absorbance of the Tris buffer was sub- tures. The larger size of the –S– atoms attached to the ethanol
tracted through baseline correction. Concentrated stock solu- groups in H3L1 gave rise to flexibility in adjacent arms, enfor-
tions of the complexes were prepared by dissolving the cing twisting around the –S– atom for apical alcohol –OH
complex in DMSO and diluting appropriately with the corres- coordination to only one CuII centre. In situ generation of OH−
ponding buffer to the required concentrations for all the bridges from water molecules present in the reaction medium
experiments.
clipped two CuII centres in the µ1,1 mode in 1. The reaction of
DNA cleavage experiments of supercoiled pBR322 (300 ng) HL2 in MeOH–H2O medium resulted in a µ-HO− bridge for
with 1–2b in Tris-HCl/NaCl (5 : 50 mM) buffer at pH 7.20 were 2a, while pure MeOH resulted a µ-MeO− bridge for 2b.
carried out using agarose gel electrophoresis, as described Justification for the ligand design is apparent from the cata-
earlier.59,60
lytic oxidation of 3,5-DTBCH2 and DNA binding propensity as
Molecular docking studies. Molecular docking studies were well as cleavage reactions for plasmid-type DNA. In pure MeCN
performed using the HEX 8.0 software,61,62 which is an interac- or MeCN–H2O at pH 7, complex 1 was responsible for a 1 : 1
tive molecular graphics program for calculating and displaying catalyst-catecholate adduct, while both 2a and 2b resulted 1 : 2
feasible docking modes of a molecule and DNA. The crystal adducts for the oxidation reactions to take place. The absence
structures of the B-DNA dodecamer d(CGCGAATTCGCG)2 (PDB of ligand arm coordination at the apical site favoured the
ID: 1BNA) was downloaded from the protein data bank (http:// coordination of a second molecule of 3,5-DTBCH. At higher
www.rcsb.org./pdb). Visualization of the docked pose was done pH values in MeCN–H2O medium, the coordinated alcohol
using the Discovery Studio molecular graphics program.
arm becomes deprotonated to assist proton abstraction from
the bound DTBCH molecule to bridge two CuII centres. The
catalytic oxidation takes place through a superoxido-Cu2
species, which finally provides H2O. This course of reaction is
supported from the absence of both a radical signal in the
Conclusion
Thioether donor-bearing N2O3S2 donor H3L1 and N2OS2 donor EPR spectrum and H2O2 in the reaction product. In the case of
HL2 ligands were used to synthesize a family of symmetric 2a and 2b, the detection of the semiquinone radical and very
[Cu2] complexes showing coordination asymmetry as potential low H2O2 concentration compared to quinone concentration
antitumor chemotherapeutic agents. In the case of H3L1, indicated two pathways, where one involves a peroxido-Cu2
coordination from one dangling alcohol arm from one side of species and the other a modified ‘classical’ mechanism. In
the ligand resulted in coordination asymmetry. The chosen MeOH at lower pH values, the observed substrate cooperativity
ligand systems were not favourable for the aggregation of the can be explained from the assistance of a second DTBCH
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molecule through hydrogen bonding interactions. The impor-
tance of proton abstraction from the bound catecholate
species is further supported by the mismatch in the kinetic
data and the reduction potential values. The binding studies
of the complexes with ct-DNA employing different biophysical
techniques revealed a partial intercalative mode with electro-
static interactions. The binding constant, Kb, values for the
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The authors declare no competing financial interest.
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Acknowledgements
M. D. and D. B. are grateful to IIT Kharagpur for their research
fellowship. We are also thankful to DST, New Delhi, for
providing the Single Crystal X-ray Diffractometer facility in
the Department of Chemistry, IIT Kharagpur under FIST
program. EPR facility by CRF, IIT Kharagpur is duly
acknowledged. M. D. and D. B. are thankful to Mr Sudipto
Khamrui for his help with EPR measurements. Financial
support to the Department of Chemistry, Aligarh Muslim
University from the DST-PURSE programme and DRS-1 (SAP)
from UGC, New Delhi is gratefully acknowledged. Z. A.
expresses her gratitude to the University Grants Commission
(UGC), New Delhi, for providing Fellowship under DST under
PURSE phase II programme.
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