A thienoquinoxaline and a styryl-quinoxaline as new fluorescent probes for amyloid-β fibrils

Article abstract of DOI:10.1016/j.crci.2011.10.009

Simple and easy to prepare quinoxaline derivatives proved able to stain amyloid fibers such as aggregated lysozyme and Aβ(1-40)-peptide by a fluorescence "turn on" mechanism. Thienoquinoxaline 1 allowed the detection of lysozyme and Aβ(1-40) fibers at λ = 555 and 532 nm, respectively, with excitation at λ = 450 nm. Styryl-quinoxaline 2 stained lysozyme and Aβ(1-40) fibers with fluorescence at λ = 579 and 567 nm, respectively, upon excitation at λ = 453 nm. The apparent K _d values for Aβ(1-40) fibers were 77 and 294 nM for 1 and 2, respectively. The sensitivity of the aggregates detection assay with these new dyes was higher than that of thioflavin T. Considering their unique fluorescence properties compared to other dyes reported in the field, they can be considered as additional staining tools for the detection and studies of peptide/protein aggregation.

Full text of DOI:10.1016/j.crci.2011.10.009

C. R. Chimie 15 (2012) 79–85
Contents lists available at SciVerse ScienceDirect
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www.sciencedirect.com
´
Full paper/Memoire
A thienoquinoxaline and a styryl-quinoxaline as new fluorescent probes
for amyloid- fibrils
b
Hanane Benzeid a,b,c, Emmanuelle Mothes a,b, El Mokhtar Essassi c, Peter Faller a,b,
,
*
a, ,b
*
`
Genevieve Pratviel
a CNRS, laboratoire de chimie de coordination (LCC), 205, route de Narbonne, 31077 Toulouse, France
b Universite´ de Toulouse, UPS, INPT, LCC, 31077 Toulouse, France
c
´ ´
ˆ
´
´
´
Laboratoire de chimie organique heterocyclique, pole de competences pharmacochimie, universite Mohammed V-Agdal, faculte des sciences, avenue Ibn Battouta,
BP 1014, Rabat, Morroco
A R T I C L E I N F O
A B S T R A C T
Article history:
Simple and easy to prepare quinoxaline derivatives proved able to stain amyloid fibers such
Received 5 May 2011
as aggregated lysozyme and A
Thienoquinoxaline 1 allowed the detection of lysozyme and A
532 nm, respectively, with excitation at = 450 nm. Styryl-quinoxaline 2 stained lysozyme
and A (1-40) fibers with fluorescence at = 579 and 567 nm, respectively, upon excitation
at = 453 nm. The apparent Kd values for A (1-40) fibers were 77 and 294 nM for 1 and 2,
respectively. The sensitivity of the aggregates detection assay with these new dyes was
higher than that of thioflavin T. Considering their unique fluorescence properties compared
to other dyes reported in the field, they can be considered as additional staining tools for the
detection and studies of peptide/protein aggregation.
b
(1-40)-peptide by a fluorescence ‘‘turn on’’ mechanism.
Accepted after revision 20 October 2011
Available online 13 December 2011
b
(1-40) fibers at = 555 and
l
l
l
b
Keywords:
l
b
Ab-peptide
Fibrils
Fluorescent staining
Lysozyme
Quinoxaline
´
ß 2011 Academie des sciences. Published by Elsevier Masson SAS. All rights reserved.
1. Introduction
vivo imaging of these pathological hallmarks, are of great
importance. Moreover, in a more general context, a variety
The development of markers for fibrillar aggregates and
prefibrillar structures is an extremely active research field
due to the involvement of peptide or protein aggregation in
neurodegenerative diseases including prion, Parkinson’s,
Huntington’s and Alzheimer’s disease [1,2]. In each
disease, a different protein is implicated. Finding amyloid
imaging agents for different aggregation states as well as
different peptide/protein aggregates depending on the
nature of the peptide/protein is crucial in order to afford
tools for monitoring fibrils formation in vitro and in vivo.
They would provide the means for a better understanding
of neurodegenerative disorders, and would afford agents
for early diagnosis. Thus, molecular probes that specifically
target protein/peptide aggregates and allow in vitro and in
of convenient analytical tools for the (kinetic, structural. . .)
studies of protein misfolding processes leading to aggre-
gation and/or fibrillation would be useful [3].
Among the various imaging methodologies (MRI, PET,
radiolabel) [4–8], fluorescent dyes are attractive since they
are inexpensive and sensitive. They are extensively used
for in vitro and ex vivo staining of amyloids. In vivo near-
infrared imaging offers the advantage of a noninvasive
operation that does not require expensive instrumenta-
tion. Hence, the fluorescent staining of amyloids has
greatly evolved during the last decade.
The early dye Thioflavin T (ThT) [9–12] is still widely
used as detection agents for amyloids. ThT is a blue-
emission dye (lmax(em) ꢀ 480 nm). Non charged analogues
of ThT have been prepared for better blood–brain barrier
crossing yet are endowed with similar blue fluorescence
properties [10,13–15]. Congo Red analogue reached a
green fluorescence (lmax(em) ꢀ 550 nm) [16,17] as well as
* Corresponding authors.
E-mail addresses: peter.faller@lcc-toulouse.fr (P. Faller),
genevieve.pratviel@lcc-toulouse.fr (G. Pratviel).
pentameric thiophene derivatives
(lmax(em) = 550 nm)
1631-0748/$ – see front matter ß 2011 Acade´mie des sciences. Published by Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.crci.2011.10.009

80
H. Benzeid et al. / C. R. Chimie 15 (2012) 79–85
Fig. 1. Synthesis of quinoxalines.
Fig. 2. Fluorescence ‘‘turn on’’ of 1 induced by aggregates of lysozyme (A) and A
aggregated material (solid line), 6 M of non aggregated material (dashed line), and in the buffer (dotted line). Emission spectra were recorded in 20 mM
phosphate buffer pH = 7.4, NaCl 100 mM (DMSO 0.5% in A and 0.8% in B) with ex = 450 nm.
b(1-40) (B). Compound 1 (3 mM) was incubated in the presence of 6 mM of
m
l

H. Benzeid et al. / C. R. Chimie 15 (2012) 79–85
81
Fig. 3. Fluorescence ‘‘turn on’’ of 2 induced by aggregates of lysozyme (A) and A
aggregated material (solid line), 6
b
(1-40) (B). Compound 2 (3
m
M) was incubated in the presence of 6
m
M of
m
M of non aggregated material (dashed line), and in the buffer (dotted line). Emission spectra were recorded in 20 mM
phosphate buffer pH = 7.4, NaCl 100 mM (DMSO 0.5% in A and 0.8% in B) with
lex = 453 nm.
[12]. Polythiophenes [18], bis-thiophene derivative NIAD-4
In the present work, we report on convenient novel
[19]
(
l
max(em) = 600 nm) and styryl derivative 2C40
tools for amyloid staining consisting of quinoxaline
derivatives that are essentially nonfluorescent in
(
l
max(em) = 650 nm) exhibit a red fluorescence [20]. Further
red-shifting of spectral characteristics afforded potential
near-infrared fluorescent probes such as oxazine deriva-
tive AOI987 [21], and curcumin derivative CRANAD-2
aqueous buffers and exhibit
a yellow fluorescence
(
lmax(em) ꢀ 530–580 nm,
l
max(ex) = 468 nm) when bound
to amyloid fibers such as lysozyme and
peptide aggregates.
Ab(1-40)-
(
l
max(em) = 700 nm) compatible with in vivo use [22].
Recently, morphologically distinct fibrilar deposits
could be resolved with luminescent conjugated polythio-
phenes [18,23], or Nile red dye [24], and early fibrillation
states could be detected by fluorescence quenching effect
with TROL dye or by fluorescence enhancement with
2. Results and discussion
Quinoxaline derivatives 1–3 were prepared by simple
procedure. Condensation of o-phenylenediamine and ethyl
pyruvate led to quinoxaline 3, which was reacted with 4-
N,N’-dimethylbenzaldehyde to give styryl quinoxaline 2.
pentameric thiophene p-FTAA
[25,26].
(
lmax(em)
ꢀ
550 nm)

82
H. Benzeid et al. / C. R. Chimie 15 (2012) 79–85
Fig. 4. Emission spectra of 1 (3
m
M, dotted line.....), 2 (3
m
M, dashed line–––), and ThT (3
m
M, solid line) incubated separately with 6
mM aggregated
lysozyme (A) and Ab(1-40) (B) in 20 mM potassium phosphate buffer, NaCl 100 mM (DMSO 0.5%) pH 7.4. lex = 440 nm for ThT, and 468 nm for 1 and 2.
Emission bandpass = 10 nm, excitation bandpass = 10 nm.
Thienoquinoxaline 1 was obtained from 2 in the presence
of Lawesson’s reagent (Fig. 1).
compound 2 to aggregated lysozyme and A
accompanied by a dramatic enhancement of the fluores-
cence emission, max(em) = 579 and 567 nm, respectively
upon excitation of the dye at 453 nm. The ratio between
the fluorescence intensity in the presence and in the
b(1-40) is also
The two compounds were soluble in DMSO with molar
extinction coefficients of 30 900 MÀ1 cmÀ1 at 450 nm and
26 700 MÀ1 cmÀ1 at 453 nm for 1 and 2, respectively. They
exhibited bright fluorescence in DMSO while the aqueous
solutions only have trace emission. In DMSO, both dyes
l
absence of fibrils was ꢀ90 for both lysozyme and A
A residual fluorescence was noted when the dyes were
incubated with non aggregated A (1-40) (dashed line
b(1-40).
showed the same fluorescence emission
l
max(em) = 600 nm
b
and excitation maxima max(ex) = 468 nm (Stokes
l
Fig. 2B and dotted line Fig. 3B) due to partial aggregation of
the peptide. The control in the absence of peptide material
was fluorescence negative (Fig. 2 and Fig. 3).
shift = 132 nm). The spectra are provided as Supplementa-
ry Information (Appendix A: Figs. S1 and S2). The
compounds were stable under irradiation conditions used
in this study, i.e. less than 3 min.
Considering structural analogy with thioflavin T (ThT)
or its non charged benzothiazole analogues [10,14], and
CRANAD-2 [22], that can be used as probes for amyloid
fibrils, 1 and 2 were assessed for their suitability as new
probes for two types of fibrils, the aggregated states of
The fluorescence spectra of Figs.
2 and 3 were
obtained with excitation wavelength corresponding to
the absorbance maximum of each compound, namely
453 and 450 nm for 1 and 2, respectively. However,
fluorescence excitation spectra of both compounds in
interaction with both fibrillar targets show the same
l
max(ex) = 468 nm (not shown). Slightly higher fluores-
cence intensity is observed (ꢀ 25% increase) when
ex = 468 nm is used instead of ex = 450 (or 453) nm.
A
b
(1-40) peptide and lysozyme from chicken egg white.
When added to suspensions of A (1-40) and lysozyme
M) previously exposed to conditions causing aggrega-
tion, both 1 (Fig. 2) and 2 (Fig. 3) (3 M) exhibited
b
l
l
(6
m
The sensitivity of compounds 1 and 2 with respect to ThT
in amyloid fibers detection assay was compared (Fig. 4).
Compounds 1 and 2 were ca 6- and 3-fold more sensitive
m
fluorescence in 20 mM phosphate buffer pH 7.4 and NaCl
100 mM. Weak fluorescence response was observed if the
dye was alone in buffer or if the peptide was not in an
aggregated form (Fig. 2 for 1) (Fig. 3 for 2). In the presence
than ThT for the detection of lysozyme and A
peptide aggregate, respectively.
b(1-40)-
Fluorescence became more intense with increasing
concentrations of dyes as illustrated in Fig. 5 in the case of
of both aggregated lysozyme and A
cence of 1 is ‘‘turned on’’ with a > 100-fold fluorescence
intensity increase. The max(em) were observed at 555 and
532 nm for aggregated lysozyme and A (1-40), respec-
tively, upon irradiation at 450 nm. The binding of
b(1-40) the fluores-
interaction with A
tion experiments the apparent binding affinity constant
of the two compounds with aggregated A (1-40) peptide
were calculated (Appendix A: Fig. S3). For comparison
b(1-40) aggregates. From these titra-
l
b
b
Fig. 5. Emission spectra of increasing concentrations of 1: 0 to 2.5
mM (A), 2: 0 to 6.6
mM (B), and ThT: 0 to 10 mM (C) incubated with aggregated Ab(1-40)
peptide 6 M in 20 mM potassium phosphate buffer pH 7.4, NaCl 100 mM.
m
lex = 440 nm, bandpass = 10 nm and emission bandpass = 10 nm.

H. Benzeid et al. / C. R. Chimie 15 (2012) 79–85
83
Fig. 6. Emission spectra of ThT (3
mM) bound to aggregated lysozyme (6
mM) (dashed line) and in the presence of competitor 1 (3
mM) (A) and 2 (3
mM) (B)
(solid line). Emission bandpass = 10 nm.
lex = 440 nm, bandpass = 10 nm.
purpose ThT was also tested separately under the same
experimental conditions. The apparent dissociation
constant of 1 (Kd = 77 Æ 17 nM) and 2 (Kd = 294 Æ 90 nM)
was lower than that of ThT (Kd = 925 Æ 30 nM). The Kd of ThT
was in accordance with previous data [9,10]. The Kd is
slightly higher than the apparent Kd = 20 nM of the
fluorophore K114, which is used as a cell permeable
amyloid staining dye and works on the same principle,
i.e. it is not soluble in buffer but binds to amyloid fibrils [17].
Since, three classes of binding sites have been evidenced on
Ab(1-40) peptide [13,27], it would be interesting the know
whether the new compounds show competitive or inde-
pendent binding with respect to ThT. Unfortunately, it is not
possible to determine whether 1 and 2 are able to displace
ThT bound to Ab(1-40) through competition experiments
monitoring the decrease of the fluorescence of bound ThT
upon addition of increasing concentration of competitor
dye. The fluorescence spectra of 1 and ThT overlap in the
470–500 nm window of emission as well as in the excitation
window. The low fluorescence emission of 2 in the same
emission window also biased the assay. On the other hand,
equimolar quantity of 1 and/or 2 turned off the fluorescence
of lysozyme/ThT complex after 5 min with concomitant
exhibition of their own typical fluorescence (Fig. 6).
However, in this case as well, it is not possible to ascertain
the competitive binding of the small molecules to the same
site in fibrils since the fluorescence of bound ThT might be
quenched by 1 and 2 (FRET).
Interestingly, compound 1 and 2 turned on the
fluorescence upon interaction with fibrils as is the case
for ThT, but in contrast to ThT, 1 and 2 showed a blue shift
in emission (Appendix A: Figs. S1 and S2). As the origin of
the shift in ThT is still under discussion [11,28–31], the
behavior of 1 and 2 is more classical, and can be explained
by the lower stabilisation of the dipolar moment due to
the more apolar environment of 1 and 2 in the amyloid
fibrils compared to the solvent [32]. In a more apolar
environment, the electron-vibrational coupling between
the fluorophore and the solvent is reduced which leads to
a lower displacement parameter and hence the observed
blue shift for emission. Furthermore, the fluorescence of
the two dyes in complex with fibrils showed some
variations depending on the nature of the tested
aggregated model (Table 1). The maximum of fluores-
cence emission of the lysozyme staining appeared always
at higher wavelength compared to A
each dyes. As a consequence the blue shift is higher in
the (1-40) environment compared to lysozyme:
b(1-40) amyloid for
A
b
l
l
max = 532 nm (A
max = 567 nm (A
b
) vs 555 nm (lysozyme) for 1, and
b) vs 579 nm (lysozyme) for 2. The
larger blue shift of 1 and 2 bound to Ab(1-40) amyloid
can be explained by a more apolar binding site compared
to lysozyme. Additionally, the fluorescence blue shift
exhibited by 1 when bound to fibrils is always more
important than that of 2, which may be related to the
more rigid aromatic structure of 1. The reason is not
known, but could be explained by a more polar excited
state of 1 compared to 2.
Table 1
3. Conclusion
Fluorescence maximum
aggregated material.
lmax (nm) of free dyes and dyes in complex with
New quinoxaline derivatives (1 and 2) proved able to
DMSO
Buffer
A
b
(1-40) fibrils
Lysozyme
stain amyloid fibers such as aggregated lysozyme and A
40)-peptide with fluorescence detection at
ꢀ 530–580 nm
and excitation at
ꢀ 450 nm. The apparent Kd values for
aggregated A (1-40) were 77 nM and 294 nM for 1 and 2,
b(1-
1
2
l
Excitation
Emission
468
600
–
–
468
468
555
l
(495 and) 532
b
respectively. The main interest of these compounds in the
staining of peptide aggregates is (i) their fluorescence ‘‘turn
on’’ with a ca 100-fold increase of fluorescenceupon binding
to amyloids, which makes them sensitive tools, (ii) the large
difference between excitation and emission wavelength in
the complex (> 100 nm) associated with emission at high
Excitation
Emission
468
600
–
–
468
567
468
579
ThT
Excitation
Emission
385
445
450
482
450
482

84
H. Benzeid et al. / C. R. Chimie 15 (2012) 79–85
wavelength (> 530 nm), (iii) their high affinity for fibrils
and, (iv) in contrast to ThT, their different emission
properties when in complex with distinct types of fibrils.
In summary, since their fluorescent properties are different
from other available markers, compounds 1 and 2 afford
supplementary staining tools for amyloid fibers detection.
154.0, 151.6, 138.3, 133.1, 131.7, 129.7 (2xC), 129.3, 128.3,
124.1, 123.8, 116.6, 115.6, 112.6 (2xC), 40.2 (2xC).
Elemental analysis (%) calcd for C18H17N3OÁ0.4CH3
CH2OH: C 72.89, H 6.31, N 13.56; found: C 72.75, H
6.32,
N
14.13. Positive ESI-MS m/z (M + H)+ 292.4.
UV–vis (DMSO) lmax nm (
F(8C) 264.
e M
À1 cmÀ1) 453 (26 700).
Synthesis of N,N-dimethyl-4-(thieno[2,3-b]quinoxalin-
2-yl)aniline or quinoxaline (1). Compound (1 g,
4. Experimental
2
3.43 mmol) in toluene was refluxed with Lawesson’s
reagent (1.4 g, 3.43 mmol) for 30 min. The solvent was
evaporated. The crude solid was taken in hot ethanol,
filtered, washed with ethanol. The solid was dried under
vacuum. Yield: 0.73 g (2.4 mmol, 70%) brown solid.
4.1. Materials
Thioflavin T (C.I. 49005, basic yellow 1), o-phenylene-
diamine and Lawesson’s reagent were purchased from
Acros. Ethyl pyruvate and 4-(dimethylamino)benzaldehyde
were from Aldrich. All the solutions were prepared using
milliQ water. Dimethyl sulfoxide (analysis grade) was
purchased from Merck. Buffers and lysozyme from chicken
egg white (molecular weight 13 930 Da, molar extinction
coefficient 3.65 Â 104 MÀ1ÁcmÀ1) were purchased from
1H NMR (300 MHz, d6-DMSO)
(s, 1H, H), 7.83 (d, 2H, H NMe2, 3J = 9 Hz), 7.82 (m, 2H), 6.86
d 8.09-8.13 (m, 2H), 7.89
f
(d, 2H, H
,
3J = 9 Hz). 13C NMR (75.5 MHz, CDCl3)
d
NMe2
f
157.0, 153.7, 152.7, 151.8, 141.1, 139.7, 129.0, 128.8, 128.4,
128.3, 128.1(2xC), 120.7, 113.0, 112.0(2xC), 40.2(2xC).
Elemental analysis (%) calcd for C18H15N3Sꢁ0.8CH3CH2OH:
C 68.78, H 5.83, N 12.28; found: C 68.44, H 5.20, N 12.35.
Positive FAB (MNBA) m/z (M + H)+ 306. UV–vis (DMSO) lmax
Sigma-Aldrich. Synthetic A
b (1-40) peptide (sequence:
DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV)
was obtained from GeneCust (Dudelange, Luxembourg)
and was used without further purification. All experi-
ments were performed in 20 mM potassium phosphate
buffer pH 7.4, NaCl 100 mM, and conducted at room
temperature. Thienoquinoxaline (1) and styryl-quinoxa-
line (2) were not water-soluble. They were dissolved in
DMSO and added to the samples as concentrated
solutions (final DMSO 0.2 to 3%).
nm (e M
À1 cmÀ1) 450 (30 900), 312 (14 800). F(8C) 260.
4.3. Preparation of amyloid fibrils of lysozyme from chicken
egg white
Lysozyme (2 mg/mL, 143 mM) was incubated in
100 mM glycine buffer pH = 2.5, NaN3 0.02% at 57 8C upon
stirring (thermomixer) during 10 days. After aggregation
(evidenced by Thioflavin T fluorimetric assay) [33] the
aggregated lysozyme solution was stored at À20 8C for
several weeks. Before tests an aliquot was diluted in
20 mM phosphate buffer pH = 7.4, NaCl 100 mM and the
pH adjusted with concentrated NaOH solution. Final
4.2. Synthesis of quinoxaline 3
Condensation of o-phenylenediamine (1 g, 9.25 mmol)
and ethyl pyruvate (1.03 mL, 9.25 mmol) was performed in
10 mL of 4 N HCl solution during 15 min at ambient
temperature. After filtration of the reaction medium the
product was washed with distilled water until neutral
filtrate was obtained. The solid was dried under vacuum.
Yield: 1.18 g (7.4 mmol, 80%) white solid. 1H NMR
lysozyme concentration was 12
4.4. Preparation of A (1-40) fibrils
Peptide A (1-40) was dissolved in 25 mM sodium
mM.
b
(300 MHz, d6-DMSO)
Ar), 7.46 (m, 1H, HAr), 7.25 (m, 2H, HAr), 2.40 (s, 3H, CH3).
13C NMR (63 MHz, d6-DMSO)
159.7, 155.4, 132.4, 132.1
d
12.30 (s, 1H, NH), 7.68 (d, 1H,
b
H
hydroxide solution, pH = 12.0 (ꢀ 4 mg/mL). One volume of
this initial solution was diluted with 3 volumes of 10 mM
phosphate buffer pH = 7.4, NaCl 100 mM, NaN3 0.02%. The
pH was adjusted to 7.4 with concentrated HCl. The peptide
concentration was measured by UV–vis spectrometry
taking the molar extinction coefficient of tyrosine at
d
(Cq), 129.7, 128.3, 123.5, 115.7 (CAr), 21.0 (CH3). Elemental
analysis (%) calcd for C9H8N2O: C 67.49, H 5.03, N 17.49;
found: C 67.45, H 5.49, N 17.50. Positive FAB-MS (MNBA)
m/z (M + H)+ 161. UV–vis (DMSO) lmax nm ( À1 cmÀ1
e M )
340 (3 000), 332 (3 000), 280 (2 400).
276 nm (
was 273
e
m
= 1410 MÀ1 cmÀ1). Concentrations of A
M. Aggregation of A (1-40) was allowed to
b(1-40)
Synthesis of (E)-3-(4-(dimethylamino)styryl)quinoxa-
b
lin-2-one or styryl-quinoxaline (2). Compound
prepared by condensation of quinoxaline
2
3
was
(1 g,
proceed upon incubation at 30 8C for 21 days. After
aggregation (evidenced by Thioflavin T fluorimetric assay
[33] aggregated peptide solutions was stored at -20 8C for
several weeks.
6.25 mmol) with 4-N,N’-dimethylbenzaldehyde (1.8 g,
12.5 mmol) for 2 h at 200 8C. The reaction mixture was
allowed to cool down. The crude product was taken in
ethanol (50 mL), heated at 100 8C for 10 min, filtrated. The
solid was washed with ethanol and dried under vacuum.
Yield 1.02 g (5.3 mmol, 85%) yellow solid. 1H NMR
4.5. Non-aggregated material
Non-aggregated material consisted of freshly prepared
solutions. Lysozyme was dissolved in 20 mM phosphate
buffer pH = 7.4, NaCl 100 mM and Ab(1-40) in 25 mM
sodium hydroxide solution, pH = 12.0. Further dilutions
with 20 mM phosphate buffer pH = 7.4, NaCl 100 mM were
prepared and pH adjusted to 7.4 with HCl and used.
(250 MHz, d6-DMSO)
CHC=C
3J = 16 Hz), 7.73 (dd, 1H, HAr
(d, 2H, HAr
3J = 8.8 Hz), 7.43 (m, 2H, HAr), 7.38 (d, 1H,
CHC=C, 3J = 16 Hz), 7.29 (m, 2H, HAr), 6.79 (d, 2H, 3J = 8.7 Hz),
3.00 (s, 6H, 2CH3). 13C NMR (75.5 MHz, d6-DMSO)
155.4,
d
12.38 (s, 1H, NH), 7.99 (d, 1H,
,
,
3J = 8.7 Hz), 7.58
,
d

H. Benzeid et al. / C. R. Chimie 15 (2012) 79–85
85
4.6. Compounds preparation
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Safas FLX-Xenius fluorimeter (cuvette and plate reader).
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), diluted from the stock solution into 200 L (plate) or
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m
M
A
b
m
m
and compound 2 utilized
respectively, 10 nm bandpass. Emission were recorded at
em(max) = 480 nm (ThT), 535 nm (compound 1), and
lex = 440, 450, and 453 nm,
l
560 nm (compound 2), 10 nm bandpass. Excitation
wavelength is indicated in the Figure legends. Experi-
ments were conducted at room temperature. The Kd
binding curves were generated by software Kaleidagraph
with non linear one site binding regression. Measure-
ments were carried out after 5 min of incubation of dye
with aggregated material. Longer incubation times did not
result in higher fluorescence.
Acknowledgements
This work was partly supported by the International
Associated Moroccan-French Laboratory on Molecular
Chemistry (LIA, LCMMF). We would like to thank Dr. Neil
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Appendix A. Supplementary data
Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.crci.
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