Inhibition of copper-mediated aggregation of human γD-crystallin by Schiff bases

Article abstract of DOI:10.1007/s00775-016-1433-0

Abstract: Protein aggregation, due to the imbalance in the concentration of Cu²⁺ and Zn²⁺ ions is found to be allied with various physiological disorders. Copper is known to promote the oxidative damage of β/γ-crystallins in aged eye lens and causes their aggregation leading to cataract. Therefore, synthesis of a small-molecule ‘chelator’ for Cu²⁺ with complementary antioxidant effect will find potential applications against aggregation of β/γ-crystallins. In this paper, we have reported the synthesis of different Schiff bases and studied their Cu²⁺ complexation ability (using UV–Vis, FT-IR and ESI-MS) and antioxidant activity. Further based on their copper complexation efficiency, Schiff bases were used to inhibit Cu²⁺-mediated aggregation of recombinant human γD-crystallin (HGD) and β/γ-crystallins (isolated from cataractous human eye lens). Among these synthesized molecules, compound 8 at a concentration of 100 μM had shown ~95% inhibition of copper (100?μM)-induced aggregation. Compound 8 also showed a positive cooperative effect at a concentration of 5–15 μM on the inhibitory activity of human αA-crystallin (HAA) during Cu²⁺-induced aggregation of HGD. It eventually inhibited the aggregation process by additional ~20%. However, ~50% inhibition of copper-mediated aggregation of β/γ-crystallins (isolated from cataractous human eye lens) was recorded by compound 8 (100 μM). Although the reductive aminated products of the imines showed better antioxidant activity due to their lower copper complexing ability, they were found to be non-effective against Cu²⁺-mediated aggregation of HGD. Graphical Abstract: [Figure not available: see fulltext.]

Full text of DOI:10.1007/s00775-016-1433-0

J Biol Inorg Chem
DOI 10.1007/s00775-016-1433-0
ORIGINAL PAPER
Inhibition of copper‑mediated aggregation of human
γD‑crystallin by Schiff bases
Priyanka Chauhan1 · Sai Brinda Muralidharan2 · Anand Babu Velappan2 ·
Dhrubajyoti Datta3 · Sanjay Pratihar4 · Joy Debnath2 · Kalyan Sundar Ghosh1
Received: 30 August 2016 / Accepted: 13 December 2016
© SBIC 2017
Abstract Protein aggregation, due to the imbalance in the
concentration of Cu2+ and Zn2+ ions is found to be allied
with various physiological disorders. Copper is known to
promote the oxidative damage of β/γ-crystallins in aged eye
lens and causes their aggregation leading to cataract. There-
fore, synthesis of a small-molecule ‘chelator’ for Cu2+ with
complementary antioxidant effect will find potential appli-
cations against aggregation of β/γ-crystallins. In this paper,
we have reported the synthesis of different Schiff bases and
studied their Cu2+ complexation ability (using UV–Vis,
FT-IR and ESI-MS) and antioxidant activity. Further based
on their copper complexation efficiency, Schiff bases were
used to inhibit Cu2+-mediated aggregation of recombinant
human γD-crystallin (HGD) and β/γ-crystallins (isolated
from cataractous human eye lens). Among these synthe-
sized molecules, compound 8 at a concentration of 100
μM had shown ~95% inhibition of copper (100 μM)-
induced aggregation. Compound 8 also showed a positive
cooperative effect at a concentration of 5–15 μM on the
inhibitory activity of human αA-crystallin (HAA) during
Cu2+-induced aggregation of HGD. It eventually inhibited
the aggregation process by additional ~20%. However,
~50% inhibition of copper-mediated aggregation of β/γ-
crystallins (isolated from cataractous human eye lens) was
recorded by compound 8 (100 μM). Although the reduc-
tive aminated products of the imines showed better antioxi-
dant activity due to their lower copper complexing ability,
they were found to be non-effective against Cu2+-mediated
aggregation of HGD.
Graphical Abstract
Electronic supplementary material The online version of this
article (doi:10.1007/s00775-016-1433-0) contains supplementary
material, which is available to authorized users.
* Joy Debnath
joydebnath@scbt.sastra.edu
* Kalyan Sundar Ghosh
kalyan@nith.ac.in
1
Department of Chemistry, National Institute of Technology
Hamirpur, Hamirpur, Himachal Pradesh 177005, India
2
Department of Chemistry, School of Chemical
and Biotechnology, SASTRA University, Thanjavur,
Tamilnadu 613401, India
3
Department of Chemistry, Indian Institute of Science
Education and Research Pune, Pune, Maharashtra 411008,
India
4
Keywords Schiff base · Copper complex · γ-Crystallin ·
α-Crystallin · Cu2+-induced aggregation inhibition
Department of Chemical Sciences, Tezpur University,
Tezpur 784 028, India
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J Biol Inorg Chem
In the earlier reports on regulation of metal-induced
protein aggregation, it was found that 8-hydroxyquinoline
derivatives can significantly inhibit metal-mediated aggre-
gation of Aβ peptide [24, 25]. 8-Hydroxyquinoline conju-
gated with cyclodextrin showed anti-aggregation efficiency
against metal-induced aggregation of β-lactoglobulin [26].
Several bifunctional small molecules can also control the
metal-induced aggregation of Aβ peptide [27, 28]. It is also
reported that 8-hydroxyquinoline Schiff base compounds
can act as antioxidants and modulate Cu2+-mediated Aβ
peptide aggregation [29]. Some of these metal chelators
are in the process of clinical trials against neurodegenera-
tive disorders. Among them, clioquinol (chelating agent
of copper and zinc) can also dissolve the amyloid deposits
in vitro and in vivo, which were generated due to copper
and zinc dyshomeostasis [25]. This compound was also
used successfully against the Parkinson’s disease, where
it reduces the oxidation and amyloid burden [30]. Another
derivative of 8-hydroxyquinoline, e.g., (5, 7-dichloro-
2-[(dimethylamino)-methyl-8-hydroxyquinoline] (PBT2)
has also been successfully completed the phase II trial for
the Alzheimer’s (http://clinicaltrials.gov) and mouse model
for the Huntington’s disease [31].
In this study, we have designed and synthesized a series
of Schiff bases (metal chelator) to inhibit Cu2+-mediated
aggregation of human γD-crystallin (HGD). The major
reasons behind the selection of these Schiff bases are
their architectural flexibility, ease of their synthesis and
their ability to form stable complex with Cu2+. In fact,
the π-electron donating capability of the imine nitrogen
atom in these ligands would have a great contribution in
the formation of stable complexes with transition metals.
Additionally, the antioxidant activity of Schiff bases [29,
32, 33] may also protect the proteins from Cu2+-induced
oxidative damage. Therefore, the Schiff bases having suit-
able functional groups can be used against copper-induced
protein aggregation by virtue of their synergistic effects of
the metal complexation and antioxidant activity. To get a
further insight about the role of imine group in aggrega-
tion inhibition, the reduced analogues of these Schiff bases
were synthesized and tested.
Introduction
Some metal ions, especially Cu2+ and Zn2+ have been
found to be involved in the process of protein aggregation
and can eventually cause different neurodegenerative dis-
orders like Alzheimer’s, Parkinson’s disease, etc. [1–3].
Dyshomeostasis of these metal ions leads to the Aβ aggre-
gation and subsequent neurotoxicity in Alzheimer’s disease
along with the formation of reactive oxygen species (ROS)
[4–6]. Although the specific mechanism of metal-induced
aggregation is not very clear, the generation of oxidative
stress by the metal ions during aggregation has been well
documented in literature [7, 8]. Similarly, metal-catalyzed
oxidation of eye lens crystallin proteins causes their aggre-
gation and results in cataract [9–12], a leading cause of
blindness. Three major types of crystallins (α-, β- and γ-)
are present in the eye lens and there is no turnover of these
proteins in the aged fiber cells of the lens. Generally due to
the aggregation of β- and γ-crystallins, light scattering ele-
ments are produced and hence increase the opacity of the
lens. In addition to that, the aggregates once formed will
remain for rest of the life of an individual due to the lack
of methods to remove the aggregated proteins from lens.
At present, there is almost no other alternative to the cata-
ract treatment except eye surgery. Hence, the development
of pharmaceuticals that can inhibit or even slow down the
aggregation process will have remarkable impact and by
that a significant amount of medical cost could be saved.
Copper is present in a tightly bound form with the
crystallins in normal eye lens and this tight binding sup-
presses Cu2+-mediated ROS generation in the lens [13].
Alpha-crystallin (small heat shock protein in the lens) is
also involved in sequestration of Cu2+ ion present in lens to
exhibit its cytoprotective effects against oxidation [14]. In
addition, the binding of Cu2+ and Zn2+ with α-crystallins
causes an increase in their chaperone activity [15, 16]. But
with ageing of the lens, α-crystallins as molecular chaper-
one start to form stable complexes with their natural sub-
strate β/γ-crystallins and prevent their aggregation [17,
18]. It has also been reported that the binding of β- and
γ-crystallin to α-crystallin decreases its Cu2+-binding capa-
bility [19]. Therefore, with increase in the age of an indi-
vidual, the amount of free α-crystallin as well as its chap-
erone activity is significantly compromised. This should
indeed increase the concentration of free copper ion in the
cataractous lens [20–22] and can create oxidative insults
for β/γ-crystallins. A recent study has confirmed Cu2+ and
Zn2+ induced rapid aggregation of HGD leads to the forma-
tion of light scattering aggregates [23]. So the sequestration
of copper ions by small molecules would be a promising
strategy against this situation to prevent further oxidative
damage and aggregation of β/γ-crystallins.
Materials and methods
Synthesis of compounds
Two different sets of compounds have been prepared for
comparison of their complexing ability with Cu2+ and their
relative antioxidant activity. The first set contains the Schiff
bases and the second set of compounds was synthesized by
the reductive amination of the Schiff bases (Scheme 1).
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J Biol Inorg Chem
Scheme 1 Synthesis of ligand
molecules 3–11. Reagents and
reaction conditions: i crushing
in a mortar and pestle, rt, 5 min;
ii Na2S·H2O in EtOH, 70 °C,
30 min. iii NaBH4 in MeOH,
0 °C-rt, 3 h; iv acetic acid (cat.)
in methanol, rt-reflux, 1–3 h;
v NaBH3CN/AcOH (cat.) in
DCM, 0 °C-rt, overnight
Schiff bases
Reductive aminated pdts
NH2
NH2
NH2
OHC
R1
(i)
(iii)
+
N
H
NH2
N
R1
R1
3; R1 = OH
4; R1 = NO2
1; R1 = OH
2; R1 = NO2
6; R1 = OH
7; R1 = NO2
(ii)
5; R1 = NH2
OH
OH
OH
OHC
R2
(iv)
(v)
+
N
N
H
NH2
R1
R1
1; R2 = OH
2; R2 = NO2
8; R1 = OH
9; R1 = NO2
10; R1 = OH
11; R1 = NO2
Compound 3 The o-phenylenediamine (0.50 g, 4.6 mmol)
was crushed in a mortar pestle followed by the addition
of salicylaldehyde (0.5 ml, 4.6 mmol). These two com-
ponents were then mixed together at room temperature
for 5 min (TLC). The solid thus obtained was purified
over the silica gel column to yield compound 3 (0.903 g,
it was quenched with water, and the solid precipitate thus
obtained was filtered out and purified through silica gel
column to obtain compound 5 (0.13 g, 90%). Color: olive
green solid; mp: 118–120 °C; FT-IR (KBr): 3445 (s), 3369
(s), 3286 (s), 1615 (s), 1487, 744 (s). 1H NMR (300 MHz)
(CDCl3) δ 6.82–6.71 (m, 4H), 6.97 (dd, J = 1.5, 8.1 Hz,
1H), 7.09–6.95 (m, 1H), 7.25–7.19 (m, 1H), 7.36 (dd,
J = 1.5, 7.8 Hz, 1H), 8.56 (s, 1H). HRMS (ESI+): m/z
calcd for C13H14N3 [M + H]+: 212.1182; found: 212.1182.
Compound 6 The compound 3 (0.20 g, 0.94 mmol) was dis-
solved in 10 ml of MeOH and the temperature was brought
to 0 °C by using an ice bath. To this cold solution, a meas-
ured amount of sodium borohydride (0.11 g, 2.82 mmol)
was added and stirring was continued for another 3 h at
room temperature. After completion of the reaction (TLC),
it was quenched by adding water. The compound was then
filtered and reprecipitated from hexane and purified through
silica column to yield compound 6 (0.18 g, 88%). Color:
1
92%). Color: yellow solid; mp: 108–110 °C; H NMR
(300 MHz) (DMSO-d6) δ 6.93 (dt, J = 0.9, 9.0 Hz, 1H),
7.05 (d, J = 8.1 Hz, 1H), 7.26–7.21 (m, 2H), 7.40–7.32 (m,
4H), 8.64 (s, 1H). HRMS (ESI+): m/z calcd for C13H13N2O
[M + H]+: 213.1022; found: 213.1020.
Compound 4 The o-phenylenediamine (0.36 g, 3.30 mmol)
was crushed in a mortar pestle followed by the addition of
o-nitrobenzaldehyde (0.50 g, 3.30 mmol). These two com-
ponents were then mixed together at room temperature
for 5 min (TLC). The solid thus obtained at the end of the
reaction was purified over a silica gel column to yield com-
pound 4 (0.76 g, 95%). Color: yellow orange solid; mp:
89–91 °C; FT-IR (KBr): 3447, 3363, 1616 (s), 1518 (s),
1
pale brown solid; mp: 149–151 °C; H NMR (300 MHz)
1
1339 (s), 749 (s). H NMR (300 MHz) (DMSO-d6) δ 5.27
(CDCl3) δ 4.41 (s, 2H), 6.94–6.72 (m, 6H), 7.25–7.18 (m,
2H). HRMS (ESI+): m/z calcd for C13H15N2O [M + H]+:
215.1179; found: 215.1176.
(s, 2H, NH2), 6.55 (t, J = 1.5 Hz, 1H), 6.73 (d, J = 8.1 Hz,
1H), 7.02 (t, J = 7.2 Hz, 1H), 7.11 (d, J = 7.8 Hz, 1H),
7.78 (td, J = 7.8, 33.0 Hz, 1H), 8.05 (d, J = 8.1 Hz, 1H),
8.33 (d, J = 7.8 Hz, 1H), 8.86 (s, 1H). HRMS (ESI+):
m/z calcd for C13H12N3O2 [M + H]+: 242.0924; found:
242.0923.
Compound 5 It was prepared by selective reduction of the
nitro in presence of the imine with the help of Na2S·H2O
[34, 35]. The ethanolic solution (10 ml) of compound 4
(0.17 g, 0.68 mmol) was mixed with sodium sulphide
(Na2S·H2O) (0.40 g, 4.1 mmol) and then refluxed for
30 min at 70 °C. After completion of the reaction (TLC)
Compound 7 The compound 4 (0.20 g, 0.89 mmol) was dis-
solved in 10 ml of MeOH and the temperature was brought
to 0 °C by using an ice bath. To this cold solution, a meas-
ured amount of sodium borohydride (0.26 g, 4.14 mmol)
was added and stirring was continued for another 3 h at
room temperature. After completion of the reaction (TLC),
it was quenched by adding water. The compound was fil-
tered and reprecipitated from hexane and purified through
silica column to yield compound 7 (0.18 g, 90%). Color:
orange solid; mp-101–103 °C; FT-IR (KBr): 3392, 1595,
1 3

J Biol Inorg Chem
1
1508 (s), 1335 (s), 744 (s). H NMR (300 MHz) (CDCl3)
To this cold solution, a measured amount of NaBH3CN
(0.94 g, 24.3 mmol) was added and stirring was continued
for another 1 h at room temperature. After completion of
the reaction (TLC), it was quenched by adding water and
extracted by chloroform (2 times). The combined organic
layers were then concentrated under reduced pressure
and purified through silica gel column to afford comp 10
(0.17 g, 85%). Color: brown liquid; FT-IR (KBr): 3405,
δ 4.71 (s, 2H), 6.48 (dd, J = 5.4, 7.5 Hz, 1H), 6.68–6.78
(m, 3H), 7.02 (t, J = 7.2 Hz, 1H), 7.43 (t, J = 6.9 Hz,
1H), 7.53–7.62 (m, 2H), 8.05 (d, J = 6.9 Hz, 1H). HRMS
(ESI+): m/z calcd for C13H14N3O2 [M + H]+: 244.1081;
found: 244.1079.
Compound 8 Salicylaldehyde (0.52 g, 4.26 mmol) was dis-
solved in methanol (10 ml) and stirred at room temperature
for 5 min followed by the slow addition of 2-aminophenol
(0.46 g, 4.26 mmol). It was then stirred at room tempera-
ture for 1 h. Then the methanol was removed under reduced
pressure and the organic portion was extracted with ethyl
acetate (50 ml × 3). Finally, the organic layer was washed
with water and brine and dried over sodium sulphate fol-
lowed by the concentration under reduced pressure. The
crude residue thus obtained was purified by column chro-
matography to afford compound 8 (0.81 g, 89.3%). Color:
1
1611, 1521 (s), 1342 (s), 729 (s). H NMR (300 MHz)
(CDCl3) δ 4.74 (s, 2H), 6.46 (d, J = 7.8 Hz, 1H), 6.64
(t, J = 7.2 Hz, 1H), 6.76 (d, J = 7.2 Hz, 2H), 7.45–7.40
(m, 1H), 7.60–7.54 (m, 1H), 7.67–7.4 (m, 1H), 8.08
(dd, J = 1.2, 8.1 Hz, 1H). HRMS (ESI+): m/z calcd for
C13H13N2O3 [M + H]+: 245.0921; found: 245.0923.
Complexation studies of the compound 3–11 with Cu2+
1
dark orange solid; mp: 189–191 °C; H NMR (300 MHz)
For complexation studies, stock solution of the metal ion
was prepared by dissolving CuCl2 in water. All the metal–
ligand complexations were studied using spectroscopy
grade solvents at room temperature.
(CDCl3) δ 6.97–7.06 (m, 4H), 7.15 (d, J = 5.4 Hz, 1H),
7.21–7.26 (m, 1H), 7.40–7045 (m, 2H), 8.69 (s, 1H).
HRMS (ESI+): m/z calcd for C13H12NO2 [M + H]+:
214.0863; found: 214.0866.
Compound 9 2-Aminophenol (0.4 g, 3.67 mmol) was dis-
solved in methanol (10 ml) followed by the addition of
2-nitrobenzaldehyde (0.55 g, 3.67 mmol) and catalytic
amount of acetic acid. The reaction mixture was then
refluxed for 3 h. Thereafter, the methanol was removed
under reduced pressure and the crude thus obtained was
crystallized from ethylacetate to yield compound 9 (0.74 g,
83.8%). Color: greenish yellow colored solid; mp-109–
111 °C; FT-IR (KBr): 3378, 1519 (s), 1339 (s), 1228 (s),
752 (s). 1H NMR (300 MHz) (CDCl3) δ 6.95–6.92 (m, 1H),
7.04–7.02 (m, 1H), 7.36–7.24 (m, 2H), 7.64 (t, J = 5.7 Hz,
1H), 7.75 (t, J = 5.7 Hz, 1H), 8.06 (d, J = 6.0 Hz, 1H), 8.26
(d, J = 6.0 Hz, 1H), 9.17 (s, 1H). HRMS (ESI+): m/z calcd
for C13H11N2O3 [M + H]+: 243.0764; found: 243.0760.
Compound 10 The compound 8 (0.50 g, 2.3 mmol) was
dissolved in 5 ml of DCM and 0.5 ml of acetic acid and the
temperature was then brought to 0 °C by using an ice bath.
To this cold solution, a measured amount of NaBH3CN
(0.29 g, 4.2 mmol) was added and stirring was continued
for another 1 h at room temperature. After completion
of the reaction (TLC), it was quenched by adding water
and extracted with chloroform (2 times). The combined
organic layers were then concentrated under reduced pres-
sure and purified through silica gel column to afford comp
UV–Vis spectroscopy
The spectra were recorded using a UV–Vis spectrophotom-
eter (Lasany make). For each compound, absorption spectra
were collected on successive addition of CuCl2 solution to a
solution of that compound in 10 mM phosphate buffer, pH
7.0. The stoichiometry of the respective metal complexes
was calculated by mole-ratio method using the Job plot. The
absorbance (at a fixed wavelength) upon subsequent addi-
tion of Cu2+ was plotted against the mole fraction of Cu2+
.
The stoichiometry was obtained from the breakpoint in that
plot. The association constant (Ka) of the copper complex
was calculated using the Benesi–Hildebrand equation [36],
which was also used elsewhere previously to determine the
binding constants of metal–ligand complexes [37–40].
1
1
1
=
+
.
ꢀAsatKa[Cu2+
]
ꢀA
ꢀAsat
In the above expression ΔA is the absorbance difference
caused on addition of Cu2+ to the solution of the ligand at
a fixed wavelength and ΔAsat is the maximum absorbance
difference at that wavelength. The association constant (Ka)
of the copper complexes of the compounds was determined
from ratio of the intercept and slope obtained from the
plot between the reciprocal of ΔA against the reciprocal of
Cu2+ concentration.
1
10 (0.44 g, 88%). Color: brown solid; mp: 99–101 °C; H
NMR (300 MHz) (CDCl3) δ 4.41 (s, 2H), 6.91–6.72 (m,
6H), 7.25–7.15 (m, 2H). HRMS (ESI+): m/z calcd for
C13H14NO2 [M + H]+: 216.1019; found: 216.1020.
FT‑IR spectroscopy
Compound 11 The compound 9 (0.20 g, 8.1 mmol) was
dissolved in 5 ml of DCM and 0.5 ml of acetic acid and
the temperature was then brought to 0 °C using an ice bath.
FT-IR spectra were collected using a Perkin-Elmer spec-
trophotometer (Model Spectrum RX-IFTIR). The copper
1 3

J Biol Inorg Chem
complexes of compounds 8 and 9 were prepared by mix-
ing methanolic solutions of the metal ion and the respective
compound at a molar ratio of 1:1 and then the solution was
dried to remove methanol. Spectra were collected by mak-
ing a film of KBr with the metal complex.
Purification of proteins
Expression and purification of HGD
HGD-clone was kindly gifted by Prof. D. Balasubrama-
nian, L.V. Prasad Eye Hospital, Hyderabad, India. The
plasmid DNA was transformed into BL21(DE3) competent
E. coli cells (New England Biolabs). The bacterial cultures
were grown at 37 °C in LB Media to an absorbance of ~0.8
at 600 nm. Overexpression of HGD was induced by 1 mM
of isopropyl 1-thio-d-galactopyranoside (IPTG). Cultures
were then further grown for additional 5 h and bacterial
cells were collected by centrifugation. Purification of HGD
was done following the method of Pande et al. [43]. Briefly,
the cell pellet was lysed in 50 mM Tris–HCl buffer at pH
8.0 containing 25 mM NaCl, 2 mM EDTA, 1 mM each of
phenylmethylsulfonyl fluoride (PMSF) and benzamidine
hydrochloride in presence of lysozyme (250 μg/ml) using
rapid freeze–thaw technique. 50 μl of DNase (Himedia)
was then added and incubated for 1 h at room tempera-
ture followed by centrifugation. HGD was found mostly
in the supernatant, which was subjected to size exclusion
(Sephacryl S-200), followed by cation-exchange chroma-
tography (SP-Sepharose). Purity of the protein was checked
on an SDS-PAGE gel (SM Fig. S8A). The concentration of
HGD was determined by using an extinction coefficient of
2 mM−1 cm−1 at 280 nm [44].
Mass spectrometry
High-resolution mass spectrum (HRMS) of the copper
complex of compound 8 was recorded using Waters Micro-
mass Q-Tof Micro. The instrument is a hybrid quadrupole
time-of-flight mass spectrometer equipped with electro-
spray ionization (ESI) technique. Before recording the
ESI-MS, copper complex of compound 8 was prepared by
mixing methanolic solutions of compound 8 and CuCl2 at a
molar ratio of 1:1.
Theoretical calculations
All the calculations were performed using Gaussian 09
suite of program [41]. Geometries of the probable copper
complexes of compound 8 were optimized at B3LYP level
of theory employing effective core potential (ECP). The
valence basis sets LANL2DZ were used for copper and 6-
31G* basis set for other atoms. The formation energy of the
probable complexes was computed at same level of theory
with zero-point energies (ZPE) and thermal corrections at
298 K.
Expression and purification of HAA
Antioxidant assay
HAA clone was kindly gifted by Prof. K.P. Das, Bose Insti-
tute, Kolkata, India. The plasmid DNA was transformed
into BL21 (DE3) E. coli cells (New England Biolabs) and
the bacterial culture was grown at 37 °C till the absorbance
of the culture became ~0.8 at 600 nm. Overexpression of
HAA was further induced by 1 mM of IPTG and the cul-
ture was grown for an additional 5 h. Cells were pelleted by
centrifugation. Cell pellets were resuspended in lysis buffer
(50 mM Tris–HCl at pH 8.0 containing 25 mM NaCl,
2 mM EDTA, and 1 mM each of PMSF and benzamidine
hydrochloride and was lysed with lysozyme (250 µg/ml)
followed by five cycles of a rapid freeze and thraw. This
cell suspension was incubated with DNase (Himedia) for
an hour at room temperature and then it was centrifuged.
Both the supernatant and pellet were tested for the presence
of HAA using SDS-PAGE and HAA was found mostly in
The free-radical scavenging capacity of compounds 3–11
was determined by using 1,1-diphenyl-2-picrylhydrazyl
(DPPH) radical decolorization assay as described else-
where [42]. Briefly, the methanolic solution of the com-
pounds at different concentrations were added to the DPPH
solution (~120 µM) in methanol and incubated for 15 min.
Then the absorbance of the DPPH alone (control sample)
and the DPPH mixed different concentrations of the com-
pound (test samples) were measured at 517 nm. All the
solutions were protected from the light during mixing and
incubation. The DPPH free-radical scavenging activity was
calculated using the following equation. The IC50 values
for each compound as well as that for ascorbic acid (a com-
mon antioxidant) were calculated by the linear regression
analysis.
(Absorbance of control − absorbance of test sample)
%Radical scavenging activity =
×100
Absorbance of control
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J Biol Inorg Chem
the supernatant. The supernatant was then loaded to a size
exclusion column (Sephacryl S-300 HR) buffered with
0.1 M phosphate buffer of pH 7.0. Three iterations of size
exclusion were performed to get the final purified HAA
used for further studies. Protein purity was verified using
SDS gel electrophoresis (SM Fig. S8B). The concentration
of HAA was determined by using an extinction coefficient
of 0.83 mM−1 cm−1 at 280 nm [45].
using a spectrofluorimeter (Shimadzu RF 5301PC). The
excitation and emission wavelengths were set at 440 and
480 nm, respectively.
Results and discussion
To establish the aforementioned concept, Schiff bases
were designed in such a way that they can act as bidentate
ligand. The Cu2+ ion is known to form both five- and six-
member ring with a bidentate ligand [47]. Implementation
of this information had been carried out for further refine-
ment of the ligand structure, i.e., incorporation of suitable
functional groups at the ortho position of benzylidene and
benzyl moiety.
In this work, we have initially studied the complexa-
tion of a series of Schiff bases and their reduced aminated
products with Cu2+ using multispectroscopic techniques.
A major absorption band was found in the range of 350–
365 nm for the Schiff bases. This is due to the π → π*
transition in an extended conjugated system involving the
aromatic rings and the nitrogen atom of imine. For com-
pound 8, an additional band was also observed at 436 nm.
In the reduced aminated products, due to the loss of that
extended conjugation, the same transition was appeared at
lower wavelength (275–290 nm).
During complexation, new peaks were generated at 393
and 310 nm with a decrease in absorbance at 351 nm for
compound 3 (Fig. 1a). In case of compound 4, significant
development of new absorption band was not observed
upon addition of the metal ion (SM Fig. S1A). For com-
pound 5, generation of new peaks at 469 and 307 nm were
recorded along with decrease in absorbance at 364 nm
(Fig. 1b); whereas on addition of Cu2+, compound 8
showed an decrease in absorbance at 436 nm with a blue
shift and disappearance of the peak at 336 nm (Fig. 1c).
However, in case of compound 9, a new absorption band
was developed at 430 nm on addition of Cu2+ along with
the disappearance of the band at 347 nm (Fig. 1d). The
absorption bands of the copper complexes of various
ligands appeared at different wavelengths due to the pres-
ence of unlike functional groups in the ligands. However,
compound 6 (reduced imine) did not show any significant
change in the absorption spectra on addition of Cu2+ (SM
Fig. S1B). For compound 7, a tiny peak was developed at
394 nm (SM Fig. S1C). In case of compound 10, forma-
tion of a new peak was observed at 400 nm (Fig. 1e) in
its copper complex. For compound 11, only a small hump
was found to be developed at 438 nm upon the addition
of Cu2+ (SM Fig. S1D). The molar stoichiometry of the
copper complex of compounds 3, 5, 8, 9 and 10 was cal-
culated from the Job plot (SM Fig. S2) and mentioned in
Table 1.
Purification of β/γ‑crystallins from eye lens
Human eye lenses were collected from nearby civil hos-
pital and stored at −80 °C until required. The lenses were
homogenized in cold water and the insoluble part was
separated by centrifugation (18,000 rpm). The pH of the
supernatant was further reduced to 5.0. It was then again
centrifuged (18,000 rpm) and the precipitate was discarded
following the method of Spector [46]. The soluble β/γ-
crystallins fraction (SDS-PAGE shown in SM Fig. S8C)
was used for further studies.
Aggregation studies
A solution of HGD or β/γ-crystallins from cataractous lens
(~0.3 mg/ml) in 10 mM phosphate buffer (pH 7.0) was
mixed with 100 μM Cu2+ and then immediately placed in
the UV–Vis spectrophotometer. The solution turbidity was
measured as the apparent absorbance at 550 nm for an hour
time period. Similar set was also run in the presence of the
compound (at different concentrations) with the same pro-
tein solution under same condition. The cuvette tempera-
ture was maintained at 37 °C using a Peltier controller dur-
ing the experiment. Different concentrations (10–100 μM)
of compound 8 were used to study the inhibition of Cu2+
(100 μM)-induced aggregation of HGD. Inhibition studies
of copper (100 μM)-mediated aggregation of HGD were
also carried out individually in presence of other com-
pounds (3, 4, 5, 9 and 10) using 100 μM concentration of
each. Inhibition of copper-induced aggregation of HGD
was also carried out in presence of the different concentra-
tions of HAA alone, using a ratio of HAA:HGD from 0.1:1
to 2:1 under similar experimental conditions. Further, based
on our findings, we had used 6.16 μM of HAA and dif-
ferent concentrations of compound 8 (10–30 μM) against
Cu2+ (100 μM)-induced HGD aggregation to find out its
cooperative effect on HAA.
Thioflavin T assay
A solution of β/γ-crystallins (0.1 mg/ml) was mixed with
ThT (20 μM) in 10 mM phosphate buffer, pH 7.0 along
with 100 μM of Cu2+ in absence and presence of 100 μM
of compound 8. The fluorescence emission was recorded
1 3

J Biol Inorg Chem
A
B
C
D
E
Fig. 1 UV–Vis spectra of the compounds in 10 mM phosphate
buffer, pH 7.0 before and after addition of aqueous solution of CuCl2
for a compound 3; b compound 5; c compound 8; d compound 9; and
e compound 10. The UV–Vis spectra of the compound only is shown
in black, while the spectra after addition of one and three molar
equivalent of Cu2+ is shown in red and blue, respectively
The association constant (Ka) of the copper complexes
for compounds 3, 5, 8, 8 and 10 was determined using
Benesi–Hildebrand plot (SM Fig. S3) and mentioned in
Table 1. Comparison of the stability constants infers the
better complexing ability of phenolic –OH group, followed
by aromatic –NH2 and aromatic –NO2 groups. The poor
association constants of the copper complexes of reduced
amines as compared to their respective imines is due to
of their capability to form two favorable intramolecular
hydrogen bonding, which impeded them to form stable
Table 1 Molar stoichiometry and stability constants of the copper
complexes of the compounds
Compound
M:L molar stoichiometry
Ka (M−1
)
3
1:1
1:1
1:1
1:1
1:1
2.22 0.14 × 104
0.64 0.03 × 103
6.12 0.31 × 105
0.59 0.05 × 103
0.67 0.03 × 103
5
8
9
10
1 3

J Biol Inorg Chem
Fig. 2 Probable energy-opti-
mized structures of the Cu2+
complex of compound 8
Table 2 ESI-MS characterization of Cu2+-complex of compound 8
m/z found in ESI-MS Relative intensity (%) Assignment of peak
(ΔGf) between the product and the reactant with zero-point
energies (ZPE) and thermal corrections at 298 K. The ΔGf
was found to be more favorable in case of complex C-2,
where two copper atoms were bridged between two phe-
nolate groups of ligand and suggested to be more stable as
compared to complex C-1 (Fig. 2).
212.0
213.1
274.2
275.0
338.3
400.2
47
8
[bL–H]+
[L]+
[aML–2H]+
[ML–H]+
[M2L–H]+
[M3L–2H]+
26
100
8
Further, FT-IR spectroscopy was used to investigate the
involvement of various functional groups within the metal
coordination sphere. The presence of IR bands at 3375,
3304 and 3045–2844 cm−1 in compound 8 represents the
participation of phenolic –OH in intermolecular and intra-
molecular hydrogen bonding (SM Fig. S5A). However,
the IR spectra of its Cu2+ complex showed broadening of
the first band and disappearance of the second band. This
indicates a significant change of the phenolic–OH environ-
ment of the ligand in the copper complex (SM Fig. S5B).
Specifically the desertion of the band at 3045–2844 cm−1
suggested deprotonation of the phenolic –OH group to
form metal–oxygen bond. The migration of the C–O
stretching band from 1275–1222 to 1291 cm−1 also proved
the involvement of the phenolic –OH group in complexa-
tion. The coordination through the imine nitrogen was also
evident from the shift of imine band from 1632–1592 to
1611–1583 cm−1. The migration of this band to a lower fre-
quency region accounted for the decrease of electron den-
sity in the azomethine group due to its involvement in cop-
per complexation. Generation of new bands in the region
of 500–400 cm−1 also suggested the formation of metal–
oxygen and metal–nitrogen bonds in the copper complex. A
similar type of behavior was also found in the copper com-
plex of compound 9 (SM Fig. S5C and S5D).
42
a
M: copper
b
L: compound 8
complex with Cu2+. However, in case of imines, only one
weak intramolecular hydrogen bonding interaction is pos-
sible, which makes them better complexing agent for Cu2+
ion as compared to their reduced counterparts (SM Fig.
S4).
Among all these compounds, compound 8 was found to
form the most stable complex with Cu2+. The copper com-
plex of compound 8 was further characterized in detail. To
determine the probable structure of the Cu2+ complex of
compound 8, we opt for the energy-optimized calculation
after several attempts to crystallize that copper complex
were in vain. Based on the binding stoichiometry of 1:1
between copper and ligand (obtained from the Job plot),
two probable structures (C‑1 and C‑2) were optimized at
B3LYP level of theory using effective core potential (ECP)
along with valence basis sets LANL2DZ for copper and 6-
31G* basis set for other atoms. The energetically most sta-
ble structure was determined from free energy difference
1 3

J Biol Inorg Chem
Fig. 3 Antioxidant activity of a
compounds 3, 4, 5, 8 and 9 and
b compounds 6, 7, 10 and 11 as
determined by DPPH method
A 100
B
100
80
60
40
20
0
Comp 8
Comp 4
Comp 3
Comp 5
Comp 9
80
60
40
20
0
Comp 6
Comp 7
Comp 10
Comp 11
0
50
100
150
200
0
20
40
60
80
100
Concentration (µM)
Concentration (µM)
activity was evaluated. In this assay technique, DPPH can
accept an electron or hydrogen radical due to the pres-
ence of an odd electron in it. As hydrogen transfer occurs
from the antioxidant molecule to DPPH, the absorbance
of DPPH decreases at 517 nm. The antioxidant activities
of compounds 3–11 as a function of their concentrations
are shown in Fig. 3a, b. The corresponding IC50 values for
the free-radical scavenging capacities of compounds 3–11
are shown in Fig. 4. The IC50 for a known antioxidant like
ascorbic acid was also determined by the same assay for
comparison. Antioxidant potency of the reduced aminated
products was found to be same with that of ascorbic acid.
It is pertinent to note that the antioxidant activity of com-
pound 8 was the highest among all the imine compounds
and also comparable with ascorbic acid because of its two
phenolic –OH groups. Lowest antioxidant activity was
observed for compound 4 containing an electron-with-
drawing nitro group. Among all these compounds, we had
found that the compounds containing phenolic –OH group
showed better antioxidant activity in DPPH free-radical
scavenging (compound 8 vs. 3, and 9 vs. 4) due to lower
bond dissociation energy of O–H bond than that of N–H
bond in this kind of ortho-disubstituted aromatic com-
pounds [48, 49]. This is also supported by an earlier report
by Bendary et al. [50]. It is reported that the presence of
di-active groups in the ortho position of a compound is
very crucial for its antioxidant efficacy [48, 51, 52]. In this
kind of di-substituted structure, the hydrogen atoms (pref-
erentially attached with electronegative oxygen or nitrogen
atom) are abstracted followed by the stabilization of the
resulting phenoxy radical [52]. A similar kind of molecu-
lar scaffold of reduced aminated products explained their
higher free-radical scavenging activity compared to their
corresponding imine counterpart. This is observed for com-
pounds 6, 7 (N–H at the ortho position of NH2) and com-
pounds 10 and 11 (N–H at the ortho position of OH). The
plausible mechanism is shown in SM Fig. S7.
600
400
200
0
9
11 AA
3
8
10
6
4
7
5
Fig. 4 IC50 for the antioxidant activity of the compounds as obtained
from DPPH method. AA ascorbic acid
Appearance of more than one isosbestic points in the
UV–Vis titration spectra (for compound 8) provided an
indication about the presence of different complex species
in the solution, which was rigorously determined with the
help of ESI-MS spectrometry (SM Fig. S6; Table 2). The
ESI-MS findings were suggestive of copper complexa-
tion accompanied with the deprotonation of phenolic –OH
group. Along with the molecular ion peak of compound 8
(SM Fig. S6), few new peaks were observed due to singly
charged metal–ligand complex species like [ML–H]+ and
[ML–2H]+, [M2L–H]+ and [M3L–2H]+. A distinct molec-
ular ion peak (275.0; with 100% relative intensity) was
found for the metal–ligand complex with 1:1 stoichiometry
(ML), which unambiguously suggested that the major com-
plex species between Cu2+ and compound 8 had the molar
stoichiometry of 1:1 and which corroborated well with the
results obtained from the Job plot. In addition to this, some
metal–ligand complex species having stoichiometries other
than 1:1 also appeared. A typical isotopic pattern of the
copper complexes was found from the ESI-MS spectrum
and it confirmed the existence of metal–ligand complex
species like [ML–H]+ and [M3L–2H]+. The peak for [ML–
H]+ and [ML–2H]+ explicitly indicated the involvement of
deprotonated phenolic –OH groups of compound 8 in the
complexation process.
The inhibitory effect of these synthesized compounds
against copper-mediated aggregation of HGD was fur-
ther studied. Notably, compound 8 (20 μM) showed an
To find out the efficiency of the synthesized molecules
against Cu2+-mediated ROS generation, their antioxidant
1 3

J Biol Inorg Chem
Additive effect of Comp 8 & HAA
Actual inhibition
100
80
60
40
20
0
100
80
60
40
0
10
20
30
10
20
50
30
100
Concentration of Comp.8(µM)
Cocentration of Comp.8 (µM)
Fig. 6 The positive cooperative effect of compound 8 (at different
concentrations) on the inhibitory activity of HAA against copper-
mediated aggregation of HGD (~0.5 mg/ml) in 10 mM phosphate
buffer, pH 7.0. The concentrations of Cu2+ and HAA were 100 and
6.16 μM, respectively. The black line represents the additive effect
of individual inhibitions done by HAA and compound 8 (at different
concentrations) and the red one for the actual case
Fig. 5 Percentage inhibition of Cu2+ (100 μM)-induced aggregation
of HGD (0.5 mg/ml) in 10 mM phosphate buffer, pH 7.0 by different
concentrations of compound 8 (10–100 μM). Protein aggregation was
monitored by increase in the apparent absorbance at 550 nm due to
the increase in the turbidity of HGD solution on addition of Cu2+ in
absence and presence of the compound
aggregation inhibition of ~29% for HGD in presence of
Cu2+ (100 μM) and it reached the saturation level (~95%)
at 100 μM concentration of compound 8 (Fig. 5). Aggrega-
tion inhibition was determined by the apparent change in
absorbance at 550 nm. For compound 3, ~67% aggregation
inhibition was recorded at a concentration of 100 μM (SM
Fig. S9A). The lower protection efficiency of compound
3 as compared to 8 against copper-mediated aggregation
can be attributed to the lower stability constant of the cop-
per complex for compound 3 compared to compound 8,
although the other imines 4 and 9 (100 μM) did not show
any inhibition towards HGD aggregation in presence of 100
μM of Cu2+ (SM Fig. S9B and S9D). Similarly, we also
tested compound 10 (reduced aminated product) to check
its inhibitory potential, but it did not show any inhibition of
the aggregation process (SM Fig. S9E). For all these mol-
ecules, the inhibitory activity against Cu2+-mediated aggre-
gation matches well with the corresponding association
constants of their copper complexes. Higher the associa-
tion constant, lower will be the amount of free Cu2+ ions,
which can cause the aggregation of HGD. For compound
5, inhibition of aggregation of HGD was noticed only up to
~20 min in the temporal study (SM Fig. S9C). After that,
light scattering was enhanced significantly, which may be
due to the breakdown of the metal complex (lower stability)
and subsequent release of Cu2+ ions from its complex in
presence of HGD. Better inhibitory activity of compound 8
compared to 3, 4, 5 and 9 can be attributed to the presence
of two phenolic –OH groups and these groups remain non-
protonated unlike the –NH2 group with significantly high
pKa value. This experimental data clearly demonstrated that
in vitro inhibition of Cu2+-mediated aggregation is primar-
ily dependent on the copper complexing ability of the mol-
ecule and not on the antioxidant activity.
Protein
Protein+Cu2+
0.4
Protein+Cu2++ Comp 8
0.3
0.2
0.1
1000Time (sec)
0
2000
3000
Fig. 7 Time evolution of the turbidity of a solution of β/γ-crystallins
(0.25 mg/ml) and Cu2+ (100 μM) in absence (red) and presence
(blue) of 100 μM of compound 8
On the other hand, HAA is known for its chaperon
activity and is largely responsible for protection of β-
and γ-crystallins from their aggregation in lens. It is also
reported that human αB-crystallin can suppress HGD
aggregation promoted by Cu2+ [23]. Therefore, to find out
the effect of compound 8 on the aggregation inhibition by
HAA, we had studied the aggregation of HGD with vary-
ing concentrations of compound 8 in presence of a fixed
concentration of HAA (6.16 μM). At this particular con-
centration (6.16 μM) of HAA, only ~44% aggregation
inhibition was recorded. Nevertheless, the inhibition was
found to be increased by ~11% in the presence of com-
pound 8 (10 μM). However, at this particular concentration
(10 μM), compound 8 alone showed only ~1% aggregation
inhibition of HGD. Thus, the extended inhibitory activity
of HAA in presence of the compound 8 eloquently proved
its positive cooperative effect. This cooperative effect was
1 3

J Biol Inorg Chem
found only at the lower concentration of compound 8 and
diminished with the increasing concentration of the com-
pound 8 (Fig. 6). This finding implies that compound 8
is not only able to inhibit the protein aggregation process
through direct complexation with Cu2+, but also able to
enhance the chaperone activity of HAA leading to better
aggregation inhibition than HAA alone.
of Cu2+ ion with complementary antioxidant activity. This
study showed that the stability of the Cu–ligand complexes
were dependent on the various functional groups present
in their corresponding Schiff bases. The phenolic hydroxyl
group was found to play an important role for the formation
of stable Cu2+ complex. The change of IR absorption band
and the existence of the [ML–H]+ and [ML–2H]+ peaks
in ESI-MS spectra also authenticated the important role of
phenolic hydroxyl group during complexation. The Schiff
base (compound 8) with two phenolic hydroxyl groups
inhibited the aggregation of HGD up to 95% in presence
of Cu2+. However, other compounds 3, 4, 5 and 9 showed
moderate to no inhibitory activity against Cu2+-induced
aggregation of HGD. This finding clearly showed the prec-
edence of the complexing ability over the antioxidant activ-
ity. Furthermore, compound 8 also showed ~50% inhibition
against the aggregation of β/γ-crystallins purified and iso-
lated from cataractous human eye lens in the presence of
To ascertain the protective role of compound 8 on a real
system, the purified β/γ-crystallins from cataractous human
eye lens were used for the aggregation studies in presence
of Cu2+. The turbidity of the solutions was monitored at
550 nm as a function of time as mentioned previously. The
apparent absorbance (at 550 nm) of a solution containing
β/γ-crystallins was found to be increased in the presence
of 100 μM Cu2+ due to the aggregation and then plateaued
out. A reduction in turbidity was observed in presence of
the compound 8, which suggested its inhibitory effect
towards metal-induced aggregation of β/γ-crystallins.
In presence of the compound 8, ~50% aggregation inhi-
bition was recorded at 100 μM concentration (Fig. 7).
Compound 8 was found to be less protective against
Cu2+-induced aggregation of β/γ-crystallins as compared
to the recombinant HGD. This can be attributed to vari-
ous posttranslational modifications that already occurred
in the β/γ-crystallins (isolated from cataractous eye lens).
However, despite of the reduced activity, this experimental
data clearly showed the beneficial role of the Schiff bases
against Cu2+-induced aggregation of β/γ-crystallins at the
onset of the cataract formation without involving the HAA.
We had also investigated the fibrillogenic nature of
the β/γ-crystallins aggregate formed due to the addition
of Cu2+ using thioflavin T (ThT) fluorescence assay. The
fluorescence emission intensity of ThT at 480 nm will be
increased if it interacts with amyloids. Therefore, we meas-
ured the emission fluorescence intensity of ThT at 480 nm
in presence of the β/γ-crystallins (0.1 mg/ml) and 100 μM
of Cu2+ at pH 7.0. Very weak fluorescence emission inten-
sity of ThT at 480 nm suggested that the aggregate was
mostly amorphous rather than amyloid fibril type (SM Fig.
S10), which is in agreement with the observation reported
earlier [23], and it resembled the common aggregation pat-
tern frequently observed in the eye lens.
Cu2+
.
These new findings implemented the potential protective
role of the Schiff base against the aggregation process dur-
ing dyshomeostasis of the Cu2+ ion and also executed their
additional role to enhance the chaperon activity of HAA by
a positive cooperative effect. Therefore, it is expected that
the improvement of the complexing ability of Schiff bases
can make it a more potent inhibitor against Cu2+-medi-
ated aggregation of β- and γ-crystallins. This particular
approach can be further developed as a potential alternative
to conventional cataract treatment.
Acknowledgements KSG is grateful to Science and Engineering
Research Board (SERB) under Department of Science and Tech-
nology (DST), Govt. of India, for funding through its sanctioned
project (No. SB/FT/LS-277/2012). JD is grateful to DST (SB/FT/
CS-008/2013), New Delhi, India, for financial support. AB thanks
DST for a fellowship. JD is thankful to CARISM and CRF, SASTRA
University, for availing their 300 MHz NMR and UV–Vis spectropho-
tometer. PC and KSG are thankful to CMSE, NIT Hamirpur, for pro-
viding some of the instrumentation facilities. KSG is also grateful to
Prof. D. Balasubramanian, L.V. Prasad Eye Hospital, Hyderabad, and
Prof. K.P. Das, Bose Institute, Kolkata, for proving the clones of HGD
and HAA, respectively.
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