Synthesis and study of 2-acetyl amino-3-[4-(2-amino-5-sulfo-phenylazo)- phenyl]-propionic acid: A new class of inhibitor for hen egg white lysozyme amyloidogenesis

Article abstract of DOI:10.1039/c2md20236k

The compounds capable of blocking the aggregation of amyloidogenic proteins may have therapeutic potentials. We report on the synthesis of 2-acetyl amino-3-[4-(2-amino-5-sulfo-phenylazo)-phenyl]-propionic acid as an HEWL (hen egg white lysozyme) amyloid inhibitor starting from phenylalanine. The compounds arrest the monomers and exhibit anti-aggregating activity. Moreover, the acetyl derivative 1 served as a better inhibitor than the trifluoroacetyl derivative 2 against in vitro amyloid fibrillogenesis of the HEWL model system.

Full text of DOI:10.1039/c2md20236k

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ARTICLE TYPE
Synthesis and study of 2-Acetyl amino-3-[4-(2-amino-5-sulfo-phenylazo)-
phenyl]-propionic acid: A new class of inhibitor for Hen egg white
lysozyme amyloidogenesis
5
Sibaprasad Maity, Ravi Kumar, Suman Kumar Maity, Poulami Jana, Santu Bera and Debasish
Haldar*
Received (in XXX, XXX) Xth XXXXXXXXX 200X, Accepted Xth XXXXXXXXX 200X
DOI: 10.1039/b000000x
10 The compounds capable of blocking the aggregation of amyloidogenic proteins may have therapeutic
potentials. We report on the synthesis of 2ꢀAcetyl aminoꢀ3ꢀ[4ꢀ(2ꢀaminoꢀ5ꢀsulfoꢀphenylazo)ꢀphenyl]ꢀ
propionic acid as HEWL (Hen egg white lysozyme) amyloid inhibitor starting from phenylalanine. The
compound arrests the monomers and exhibits antiꢀaggregating activity. Moreover, the acetyl derivative 1
served as better inhibitor than the trifluoro acetyl derivative 2 against in vitro amyloid fibrillogenesis of
15 HEWL model system.
Hen eggꢀwhite lysozyme, an enzyme with 129 amino acids that
Introduction
lyses the cell walls of bacteria, has four disulfide bonds and
adopts mainly helical conformation (~30% αꢀhelix; ~6% βꢀ
50 sheet).13 Though there is only ~40% sequence similarity, hen egg
white lysozyme retain a high degree of structural homology with
the human lysozyme which has been found to form amyloid
fibrils that are responsible for hereditary nonꢀneuropathic
systemic amyloidosis.14
A
significant number of fatal human diseases including
Alzheimer’s systemic amyloidoses, Parkinson's disease,
Huntington's disease, type 2 diabetes, Creutzfeldt–Jakob disease
20 and prion disease are associated with the deposition of protein
fibrils in various organs.1ꢀ7 These amyloidogenic diseases
individually have their unique neuropathological, clinical and
biochemical characteristics. Up to now, more than 40 different
human proteins and peptides, including transthyretin, αꢀ
25 synuclein, β2ꢀmacroglobulin and βꢀamyloid have been isolated as
the misfolded fibrillar components of disease related amyloid
deposits.8ꢀ12 The corresponding amyloidogenic precursor proteins
associated with various diseases are not related to each other in
sequence or in structure, but the amyloid fibrils formed share
30 some common ultra structural and morphological characteristics.
Despite extensive investigations on amyloid fibril formation, the
details of aggregation process at atomic level are still unknown
due to insolubility and noncrystallinity of the amyloid plaque. So,
it is important to develop successful therapeutic strategies for
35 inhibiting amyloid formation to prevent amyloid related diseases.
55
The protein aggregation into soluble oligomers, protofibrils,
mature amyloid fibrils, or a combination of these is a requirement
for toxicity. Hence, inhibition of amyloid aggregation may
represent a possible therapeutic strategy for the prevention and
treatment of amyloid diseases. Goda et al. has reported that hen
60 egg white lysozyme forms amyloid fibrils in
a highly
concentrated ethanol solution.15 They have also reported that
addition of salt further promotes the association of the proteins. A
number of diverse small molecules including synthetic peptides,
other chemical compounds also have been synthesized to inhibit
65 or reduce the aggregation of various proteins, particularly in
relation to Aβ deposition, aggregation of lysozyme and
transthyretin.16ꢀ17 Ducruix et al. has reported that the cations and
anions have different effects on the protein–protein interactions
of HEWL.18
70
In this context, we have designed and synthesized 2ꢀAcetyl
aminoꢀ3ꢀ[4ꢀ(2ꢀaminoꢀ5ꢀsulfoꢀphenylazo)ꢀphenyl]ꢀpropionic acid
containing cationic and anionic parts in the same moity like
Congo red to inhibit or reduce the aggregation of various proteins
(Figure 1).19 In this report, utilizing hen egg white lysozymes as a
a Department of Chemical Sciences, Indian Institute of Science Education
and Research Kolkata, Mohanpur, West Bengal 741252, India, Fax:
40 +913325873020; Tel: +913325873119; E-mail: deba_h76@yahoo.com;
and deba_h76@iiserkol.ac.in
† Electronic Supplementary Information (ESI) available: Synthesis and
75 model system20, attempts were made to investigate the antiꢀ
amyloidogenic and antiꢀaggregating activities of modified amino
acids against the formation of aggregated species or amyloid
fibrils of lysozyme.20 Through UVꢀVisible, fluorescence and
1
characterization of compound 1 and 2, H NMR, 13C NMR, ESI Table 1,
Fugures ESI S1ꢀ2, Figure S1ꢀS20. other electronic format see
45 DOI: 10.1039/b000000x/
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50 on a fluorescence spectrometer (Perkin Elmer) in aqueous
medium ( pH= 2.0) at excitation wavelength 293 nm (slit 5/5). To
2 mL of HEWL solution (1×10ꢀ4 mg/mL) 20 ꢁL of both the
compounds (2×10ꢀ4 M) were added individually and spectra were
recorded. For reference (blank) 2 mL of HEWL solution (1x 10ꢀ4
55 mg/mL) at pH=2.0 has been used.
For ThT binding study to 2.5 mL of ThT solution (2.5×10ꢀ5 M) in
0.1 M sodium phosphate buffer pH= 7.4) 40ꢀL of each growing
solution at different time interval were added and incubated for
20 min and data were collected. Each time freshly prepared ThT
60 solution in sodium phosphate buffer has been used. Slit (5/10),
Excitation wavelength= 410 nm
TCSPC spectroscopy interactions of HEWL monomers with
compound 1 or 2 has been demonstrated. The anti amyloidogenic
behavior of both the compounds were confirmed by Congo red,
Thioflavin
T
binding assay, CD spectroscopy, FTꢀIR
5
spectroscopy, atomic force microscopy and crossꢀpolarized
optical microscopic studies. Moreover, compound 1 (acetyl
derivative) served as more effective inhibitor in comparison with
compound 2 (trifluroacetyl derivative) against amyloid fibril
formation of HEWL.
10
Circular Dichroic (CD) spectroscopy
CD study of hen egg white lysozyme fibrils with or without
modified amino acids in aqueous medium ( pH= 2.0) was carried
65 out on a JASCO Jꢀ815ꢀ150S instrument at a temperature of
25°C. 100 microlitre of each solution (15 days incubation) was
diluted to 500 microlitre with same acidic water (pH= 2.0) and
spectra were recorded.
Fig. 1 The schematic presentation of reported inhibitors 1 and 2.
Atomic force microscopy
70 The morphology of the reported compounds were investigated
by atomic force microscopy (AFM). A small amount of three
solution incubated at different time were diluted with acidic water
(pH= 2.0) and drop cashed on clean microscope cover glass and
then dried by slow evaporation. The materials were then allowed
75 to dry under vacuum at 30ºC for two days. Images were taken
with an NTMDT instrument, model no. APꢀ0100 in semicontactꢀ
mode.
Experimental section
15 Preparation of lysozyme solution
10 mL stock solution containing lysozyme at a concentration 2
mg/mL in double distilled water at pH 2.0 (by adding required
amount of HCl) was prepared. In each of the three vials 1.8 mL
of this stock solution was added. In the first vial 200 microlitre of
20 acidic solvent was added. In the second and third vial 200
microlitre of compound 1 and 2 solution (2×10ꢀ4 M in same
acidic water) were added seperately. These solution were
incubated at 65ºC for 15 days. These solutions were used for
different experiments.
Time Correlated Single Photon Counting (TCSPC)
spectroscopy
80 The fluorescence lifetimes were measured by the method of
timecorrelated single photon counting using
a picosecond
25 NMR experiments
spectrofluorometer from Horiba Jobin Yvon IBH. The instrument
was equipped with FluoroHub single photon counting controller,
Fluoro3PS precision photomultiplier power supply, and FCMCPꢀ
85 50SC MCPꢀPMT detection unit. A laser head or a nanoꢀ LED
pulsed diode powered by a pulsed diode controller (IBH) was
used as the excitation light source. The life time of free native
HEWL was recorded at concentration (1×10ꢀ4 mg/mL in acidic
water (pH=2.0). For complex 100ꢀL of both the compounds
90 (2×10ꢀ4 M) were added individually with free HEWL solution and
data were recorded. The excitation was done at 280 nm
wavelength and emission were recorded at 350 nm for all the
samples. The typical response time of this laser head was <100
ps. To calculate the lifetime, the fluorescence decay curves were
95 analyzed by an iterative fitting program provided by IBH. The
fluorescence decay curves were fitted with the equation:
All NMR studies were carried out on Jeol JNMꢀECS 400 MHz
spectrometer at 298 K. Compound concentrations were in the
range 1–10 mmol in CDCl3 and (CD3)2SO.
FT-IR spectroscopy
30 All reported solidꢀstate FTꢀIR spectra were obtained with a
Perkin Elmer Spectrum RX1 spectrophotometer. The solutions
were lyophilized and spectra were taken by KBr disk technique.
Mass spectrometry
Mass spectra were recorded on a QꢀTof Micro YA263 highꢀ
35 resolution (Waters Corporation) mass spectrometer by positiveꢀ
mode electro spray ionization.
UV/Vis spectroscopy
UV/Vis absorption spectra were recorded on
a UV/Vis
spectrophotometer (Hitachi). For binding of HEWL monomers
40 with compound 1 and 2 to 2 mL of HEWL solution (1x 10ꢀ4
mg/mL) in acidic water (pH=2.0) 20 ꢁL of both the compounds
(2×10ꢀ4 M) were added gradually and spectra were recorded. For
reference (blank) 2 mL of HEWL solution (1x 10ꢀ4 mg/mL) at
pH=2.0 has been used. For congo red binding assay 80ꢀL of each
45 growing solution were added to 2 mL of congo red solution
(2.15×10ꢀ5 M) in 0.1 M sodium phosphate buffer pH = 7.4 and
incubated for 20 minutes and data were collected.
Where Ai and τi are the amplitude and lifetime of the ith
100 fluorescence component respectively and n is the number of
fluorescence exponentials required for the best nonlinear least
squares (NLLS) fitting of the fluorescence decay curves.
Congo red assay
Fluorescence spectroscopy
For quenching experiments fluorescence spectrum was recorded
An alkaline saturated Congo red solution was prepared. The
2
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the absorption of lysozyme gradually increases with the increase
fibrils were stained by alkaline Congo red solution (80%
methanol/20% glass distilled water containing 10 ml of 1% 40 in concentration of the compounds 1 or 2. The absorption
NaOH) for 2 min and then the excess stain (Congo red) was
removed by rinsing the stained fibril with 80% methanol/20%
glass distilled water solution for several times. The stained fibrils
were dried under vacuum at room temperature for 24 h, then
visualized at 40× magnification and birefringence was observed
between crossed polarizers (Olympus optical microscope
equipped with polarizer and CCD camera).
maxima shifted from 277 nm to 258 nm upon addition of 100 ꢀL
of 2×10ꢀ4 M compound 1 where as the maxima shifted from 277
nm to 265 nm upon addition of same amount of compound 2
solution of same concentration. Moreover, there is a band at 380
45 nm (ESI Figure 1). This blue shift may be due to interaction of
compound 1 or 2 with Tryp62 or Tryp108 of HEWL through
various nonꢀcovalent interactions. There is a minor overlapping
of absorption bands that may influence the HEWL maximum
wavelength shift.
5
10 Results and Discussion
The compounds 1 and 2 were synthesized according to route
depicted in Scheme 1. In the first step methyl ester protected
phenylalanine 4 was obtained by treating phenylalanine with
SOCl2 and methanol. The amine group was protected by
15 acetylation. The nitration followed by the reduction developed
the amine 7. The compounds 1 and 2 were obtained by diazo
coupling reaction of 7 with sulfanilic acid. Considering the
binding propensities of Congo red with amyloid fibril, the
compounds were designed such that the compounds have some
20 structural and functional similarity with Congo Red. The
trifluoromethyl group has significant electronegativity and often
used a bioisostere to create derivatives by replacing a methyl
group. So, we have prepared a trifluoromethyl derivative 2 of
compound 1. The toxicity of the compounds 1 and 2 towards
25 neuronal cells were investigated by MTT assays and the
compounds failed to show any significant neuronal toxicity. The
antiꢀaggregating activities of both the compounds were studied
through different spectroscopic and microscopic techniques.
50
Fig. 2 UVꢀVisible spectra of lysozyme (1×10ꢀ4 mg/ mL) after addition of
different amount of (a) Compound 1 (b) Compound 2 at pH= 2.0
Concentration of both the compounds were 2×10ꢀ4 M in aqueous solution
at pH=2.0.
55
Fluorescence quenching measurements have been widely
used to study the interaction of various organic compounds with
hen egg white lysozyme and other proteins.21ꢀ22 From the
literature Tryp62 and Tryp108 are the most dominating
fluorophores and positioned at the active sites which mainly bind
60 with substrates. The Tryp28 and Tryp111 are present at the
hydrophobic matrix region, which stabilize the protein
structure.23 Hence most of the outer substrates bind with Tryp62
or Tryp108 that results the change in its spectroscopic
properties.24 The fluorescence experiments have performed on
65 hen egg lysozyme in presence of different amount of compound 1
or 2. Fig. 3 shows that the fluorescence intensity of hen egg
lysozyme gradually quenched by increasing amount of compound
1 or 2 with large red shift of fluorescence maxima. The maxima
shifted from 350 nm to 385 nm on addition of 100 ꢀL of 2x 10ꢀ4
70 M compound 1. But in presence of same amount of compound 2
the maxima of lysozyme shifted from 350 nm to 374 nm only.
The fluorescence quenching indicates strong interactions of
30
Scheme 1 Reagents and conditions: a) (i) SOCl2, MeOH, 0°C, (ii)
NaHCO3, 83%; b) for R = CH3, Ac2O, pyridine, rt, 84%; for R = CF3,
CF3COOEt, ET3N, MeOH, rt,72%; (c ) 1:1 conc. HNO3: conc. H2SO4,
35 41%; d) [H], 51%; e) NaNO2, dil. HCl, sulfanilic acid, 5°C, 90%.
Initially, the interaction of native hen egg white lysozyme with
the two synthetic compounds was investigated by UVꢀVis
spectroscopy. From Fig. 2, the blue shift of maxima exhibits that
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lysozyme with compound 1 and 2. From UVꢀVisible and
fluorescence spectra it may be conclude that compound 1 interact
with HEWL more efficiently than compound 2. We have also
calculated binding constant for both the compounds using the
equation
HEWL
HEWLꢀ
compound 1
HEWLꢀ
compound 2
1.43
1.44
1.32
0.53
0.72
0.70
3.41
5.29
3.72
0.46
0.27
0.29
1.09
1.18
1.15
5
35
Comparing the data listed in the table 1, the lifetime of one
component (τ1) is almost same for all the three cases. But lifetime
of other component (τ2) increased upon complexation of both the
compounds indicates formation of complex of HEWL with
log[F0/Fꢀ1] = log K + n log[Q]
Where F0 and F is the fluorescence intensity of fluorophore in the
10 absence and in the presence of quencher, respectively. K, n, [Q]
40 compound 1 and 2.
Fig. 4 Time dependent decay curves of hen egg white lysozyme (blue),
lysozymeꢀcompound
1 conjugate (red) and lysozymeꢀcompound 2
45 conjugate (green). Black curve indicates promt i.e. instrumental response
function (IRF).
To determine the inhibitory effect of these two compounds on
HEWL fibrilization, HEWL was incubated in absence and
presence of compound 1 or 2 at pH=2.0 at 650C for two weeks.
50 The inhibitory effects were tested by Congo red and ThioflavinꢀT
binding studies.26 Fig. 5 shows that absorption of Congo red
alone, in presence of HEWL, HEWLꢀCompound 1 and HEWLꢀ
Compound 2 complex after incubation for 2 weeks. From Fig. 5,
with HEWL the intensity of Congo red increased and maxima
55 shifted to higher wavelength with appearance of a new peak at
537 nm. For the HEWLꢀCompound 1 or 2 complexes, absorbance
increases less than that of control with almost no change of peak
position of Congo red. For HEWLꢀCompound 1 complex
intensity of Congo red increase less than that of HEWLꢀ
60 Compound 2 complex.
Fig. 3 Fluorescence spectra of HEWL (1×10ꢀ4 mg/ mL) after addition of
different amount of (a) Compound 1 (b) Compound 2 [pH= 2.0]
Concentration of both the compounds were 2×10ꢀ4 M in aqueous solution
15 at pH=2.0, λex= 293 nm.
are binding constant, number of binding site and concentration of
compound 1 or 2. From log[F0/Fꢀ1] vs log[Q] curve (ESI Fig. 2)
we have calculated binding constant for HEWLꢀcompound 1 and
HEWLꢀcompound
2 complex. The binding constant for
20 lysozymeꢀcompound 1 complex is 7.32×105 where as for
compound 2 it is 5.52 x 104 (ESI table 1). Hence binding affinity
of compound 1 is stronger than that of compound 2.
To confirm the intermolecular interaction of compound 1
and
2 with HEWL monomer time resolved fluorescence
25 spectroscopy were done for HEWL, HEWLꢀcompound 1 and
HEWLꢀcompound 2 complex using TCSPC (timeꢀcorrelated
single photon count) technique.25 Fig. 4 shows the decay curves
for HEWL monomer complex with both the compounds along
with lysozyme monomer. All of the decay curves fitted well with
30 biexponential decay pattern. The fitted decay parameters are
listed in table 1.
Table 1: Nonlinear least squares (NLLS) fitting parameters
obtained from fluorescence decay curves (Fig. 4)
Fig. 5 UVꢀVisible spectra of Congo red (black), Congo red in presence of
Sample
A1
A2
τ1 (ns)
τ2(ns)
χ2
HEWL (red), HEWLꢀCompound
1 complex (green) and HEWLꢀ
4
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Compound
2 complex (blue). [Sodium phosphate buffer pH=7.4,
Concentration of Congo red was 2.15 x 10ꢀ5 M, 80ꢀL stock solution was
added for each case].
The effect of these two compounds on the kinetics of HEWL
amyloid formation was also investigated through monitoring
maximal Thioflavin T emission intensity over the course of 4
days.26c Fig. 6 shows the relative intensity of Thioflavin T after
addition of same amount of three incubated solutions at different
time intervals. From Fig. 6, the fibrilization rate of HEWL
5
10 decreases in presence of compound 1 or 2. This bar diagram also
indicates that inhibitory effect of compound 1 is more than that of
compound 2.
Fig. 7 Far UVꢀCD spectra of HEWL (squere), HEWL in presence of
50 compound 1 (circle) and compound 2 (triangle) after incubation at
pH=2.0 at 650C for 15 days.
15
Furhter information on conformational preferences of hen egg
white lysozyme amyloid fibril at different condition was obtained
from FTIR studies.28ꢀ29 Fig. 8 shows the FTIR spectra of HEWL
20
55 alone, HEWLꢀcompound 1, HEWLꢀcompound
2 complex
incubated at pH=2.0 at 65°C for 15 days. FTIR spectra of
lysozyme amyloid fibril (Figure 8a) have amide I bands at 1633
cmꢀ1 and amide II band at 1503 cmꢀ1 indicating presence of β
sheet conformation in aggregated form.30 But for HEWLꢀ
60 compound 1 complex (Fig. 8b) and HEWLꢀcompound 2 (Fig.
8c) the amide I band occur at 1654 cmꢀ1 and 1675 cmꢀ1 and amide
II band occur at 1539 cmꢀ1 and 1540 cmꢀ1 respectively which
indicate αꢀhelix or random coil conformation in the complex.
25
Fig. 6 ThT binding assay of (a) HEWL (black), (b) in presence of
compound 1 (2 × 10ꢀ4 M) (red) and (c) in presence of compound 2 (2 × 10ꢀ
30 4 M) (green) at different time intervals. ThT concentration 2.5 x 10ꢀ5 M in
0.1 M sodium phosphate buffer pH= 7.4, λex= 410 nm.
CD is an excellent method of determining the backbone
structural transition of proteins by interaction with other
compounds. The far UVꢀcircular dichroism spectra of hen egg
35 white lysozyme amyloid fibril26 in absence and in presence of
compounds 1 or 2 after incubation at 65°C for 15 days is depicted
in Fig. 7. The HEWL solution incubated for 15 days in absence of
any inhibitors shows a net ellipticity transition from a random
organization with a characteristic peak at 195ꢀ200 nm (and a
40 positive transition about 230 nm) to an ordered betaꢀsheetꢀrich
structure with a negative peak at 217 nm and a positive peak at
195 nm, typical of amyloid behavior.27 But in presence of
compound 1 HEWL solution shows the negative peaks at 206
nm, 212 nm and a shoulder at 224 nm indicates αꢀhelical
45 conformation. Compound 2 also shows similar result. Hence,
both the compounds inhibit the βꢀsheet aggregation of HEWL.
65
Fig. 8 FTꢀIR spectrum of (a) hen egg white lysozyme amyloid fibril, (b)
lysozymeꢀcompound
conjugate.
1 conjugate and (c) lysozyme compound 2
To further investigate the morphological change during
70 fibrilization of hen egg white lysozyme in presence of compound
1 or 2, the AFM studies have been performed.31 To insight into
fibrilization process during amyloid formation of hen egg white
lysozyme , the AFM has been performed with sample having
different incubation time. Figure 9a, 9b and 9c show the AFM
75 images of HEWL amyloid obtained by incubation at 65°C at pH
2.0 for 2 days, 5 days and 15 days respectively in absence of
compound 1 or 2. From these micrographs, the monomer of
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HEWL is gradually selfꢀaggregated to form fiber like structure
with increasing of incubation time. Fibriller aggregates have
diameter range 50ꢀ80 nm. Fig. 9d and 9e show the morphology of
HEWL aggregrate in presence of compound 1 and compound 2
respectively, after incubation under same condition for 15 days.
HEWLꢀcompound 1 or 2 conjugate exhibit granular aggregate
with a diameter ca 100 nm (Figure 9d and 9e respectively).
The morphological resemblance between these hen egg
white lysozymeꢀcompound 1 or 2 conjugate and lysozyme
Fig. 10 (a) Congo red assay of hen egg white lysozyme fibriller
aggregates showing greenꢀgold birefringence under cross polarizer. (b)
30 and (c) Scattered greenꢀgold birefringence of (b) lysozymeꢀcompound 1
conjugate and (c) lysozymeꢀcompound 2 conjugate under cross polarizer.
5
Conclusions
In conclusion, the design, synthesis and amyloid inhibition
propensities
of
2ꢀAcetyl
aminoꢀ3ꢀ[4ꢀ(2ꢀaminoꢀ5ꢀsulfoꢀ
35 phenylazo)ꢀphenyl]ꢀpropionic acid has been reported. The
compounds have structural and functional similarity with Congo
Red and contain cation and anions. The compound arrests the
monomers and exhibits antiꢀaggregating activity against hen egg
white lysozyme in vitro. Moreover, acetyl derivative served as
40 more effective inhibitor in comparison with the trifluro acetyl
derivative. This result shed some light on the rational design of
effective therapeutic agent for amyloidogenic diseases and may
foster the in vivo studies.
10 amyloid stained by Congo red (CR) binding assay has been
performed.32ꢀ33 The aggregated fibrils obtained from the hen egg
white lysozyme at pH 2 after incubation for 15 days, were stained
by Congo red and were observed through cross polarizers. Figure
10a exhibits the greenꢀgold birefringence of Congo red bound
15 fibrils of lysozyme amyloid under cross polarizer.34ꢀ35 But
lysozymeꢀcompound 1 or 2 conjugate exhibits scattered greenꢀ
gold birefringence under cross polarizer (Figure 10b and 10c
respectively).
45 Acknowledgment
20
We acknowledge the DST, New Delhi, India, for financial
assistance Project No. (SR/FT/CSꢀ041/2009). S. Maity, P. Jana,
and S. K. Maity acknowledge C.S.I.R., New Delhi, India for
50 financial assistance. S. Bera thanks U. G. C., India for research
fellowship. We gratefully acknowledge the I.A.C.S., Jadavpur,
Kolkataꢀ700032, India for using the CD. We are thankful to Dr.
Jayasri Das Sarma, Department of Biological Chemistry, IISERꢀ
Kolkata, India for the neuronal toxicity experiments by MTT
55 assay.
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