2-Quinolone and coumarin polymethines for the detection of proteins using fluorescence

Article abstract of DOI:10.1016/j.dyepig.2009.07.009

A series of novel, polymethine chalcone dye - 2-quinolone derivatives and their boron difluoride complexes were synthesized. The spectral-luminescence properties of a series of 2-quinolones and their coumarin analogues were characterized in the presence of a denaturing agent (sodium dodecyl sulfate), native bovine serum albumin as well as a combination of serum albumin and sodium dodecyl sulfate. A?study of the influence of the BF₂-ether group on the sensitivity of the polymethine dyes to proteins revealed that three of the dyes, namely two hydroxyquinoline dyes containing a 4-diethylamino-2-hydroxyphenyl substituent and a coumarine dye that contained an indolenine substituent, displayed high emission and bright fluorescence (quantum yield ≤ 0.27) and thus offer promise for use in protein detection.

Full text of DOI:10.1016/j.dyepig.2009.07.009

Dyes and Pigments 84 (2010) 159–164
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
2-Quinolone and coumarin polymethines for the detection of proteins
using fluorescence
,
a
b
a
Vladyslava B. Kovalska a , Kateryna D. Volkova , Alexander V. Manaev , Mykhaylo Yu. Losytskyy ,
*
Igor N. Okhrimenko b, Valery F. Traven b, Sergiy M. Yarmoluk a
a Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150 Zabolotnogo St., 03143 Kyiv, Ukraine
b Department of Organic Chemistry, Mendeleev University of Chemical Tehnology of Russia, Miusskaja Sq. 3, 125047 Moscow, Russian Federation
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel, polymethine chalcone dye – 2-quinolone derivatives and their boron difluoride
complexes were synthesized. The spectral-luminescence properties of a series of 2-quinolones and their
coumarin analogues were characterized in the presence of a denaturing agent (sodium dodecyl sulfate),
native bovine serum albumin as well as a combination of serum albumin and sodium dodecyl sulfate.
A study of the influence of the BF2-ether group on the sensitivity of the polymethine dyes to proteins
revealed that three of the dyes, namely two hydroxyquinoline dyes containing a 4-diethylamino-2-
hydroxyphenyl substituent and a coumarine dye that contained an indolenine substituent, displayed
high emission and bright fluorescence (quantum yield ꢀ 0.27) and thus offer promise for use in protein
detection.
Received 22 June 2009
Received in revised form
14 July 2009
Accepted 15 July 2009
Available online 22 July 2009
Keywords:
Polymethine dyes
Coumarin and 2-quinolone derivatives
Fluorescent detection
Proteins
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
synthesized 2-quinolones and their coumarin analogues were
evaluated as fluorescent dyes for the detection of native proteins
Optical markers for biomolecules are widely used in biochemical
and medical studies [1,2]. Detection based upon fluorescence has
received much attention and notable progress has been made in
both fluorescence instrumentation and the synthesis of novel flu-
orophores [2–4]. Polymethine dyes are well known to be sensitive
fluorescent probes for the non-covalent labeling of proteins [5–8].
Novel polymethines can be obtained from boron difluoride
complexes of hydroxyl(acyl)arenes and heteroarenes. The incor-
poration of the boron atom greatly increases the reactivity of the
acetyl function in ortho-hydroxyacetophenones, benzoylacetones
and acetylnaphthols [9–11]. The methyl group in such complexes
undergoes a facile condensation with carbonyl compounds and
equivalents. The recent research group recently developed a similar
procedure for the synthesis of polymethine – heteroarene deriva-
tives [12–15]. For example, complex 1 was found to be useful
starting substrate for the synthesis of 4-hydroxy-3-cinnamoylcou-
marins both as the BF2-complex 2 and the free base 3 (Fig. 1).
This paper concerns the synthesis of novel polymethine dyes
(Fig. 2) derived from the boron difluoride complex of 3-acetyl-
using bovine serum albumin (BSA) as model protein and as probes
for the nonspecific detection of proteins using a BSA/sodium
dodecyl sulfate (SDS) mixture. The influence of dye structure upon
selectivity towards certain protein was studied.
2. Experimental
The 1H NMR spectra (400 MHz) were recorded on a Bruker WP-
400 SY spectrometer in DMSO-d6 or CDCl3 with TMS as an internal
reference. The mass spectra were recorded on a MAT-112 spec-
trometer operating at 80 eV. M.p.’s (Pyrex capillary) are not
corrected.
2.1. Materials
Methanol, anhydrous dimethylformamide (DMF) distilled
under reduced pressure and 0.05 M Tris–HCl buffer (pH 8.0) were
used as solvents. Bovine serum albumin (BSA) and sodium dodecyl
sulfate (SDS) were purchased from ‘‘Sigma’’ (USA). Boron difluor-
ide complex 4 was obtained by the reaction of the 4-hydroxy-
3-acetylquinolon-2 with boron trifluoride etherate, as described
previously [6]. The dyes of coumarin series 2a [12], 2b, 2c, 3a, 3b,
3c [13], 2d, and 3d [14] were obtained according to the known
4-hydroxy-1-methyl-2-quinolone
4 (Fig. 1). In addition, the
* Corresponding author.
E-mail address: v.b.kovalska@imbg.org.ua (V.B. Kovalska).
0143-7208/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2009.07.009

160
V.B. Kovalska et al. / Dyes and Pigments 84 (2010) 159–164
F
F
B
F
F
F
F
O
O
B
B
O
O
O
O
OH
O
O
O
Me
R
Me
R
N
O
O
O
O
O
Me
4
1
2
3
Fig. 1. General structure of parent boron difluoride complexes and previously synthesized polymethine dyes.
procedures, their melting temperatures were in agreement with
the literature data.
2.5.2. 3-{(E)3-[2-hydroxy-4-(N,N-diethyaminol)-phenyl]prop-2-
enoyl}-4-difluoroboronoxy-1-methylquinone-2 (5b)
Yield 73%. M.p. 257–258 ꢃC. 1H NMR (CDCl3):
d 1.21 (m, 6H, Me);
3.45 (m, 4H, NCH2); 3.65 (s, 3H, MeN); 6.41 (s, 1H, Hi); 6.58 (d, 1H,
Hh, J ¼ 9.2 Hz); 7.24–7.32 (m, 2H, Hc, Hd); 7.68–7.81 (m, 2H, Hb, Hg);
8.37 (d, 3JH,H ¼ 8.2 Hz, 1H, Ha); 8.41 (d, 3JH,H ¼ 14.6 Hz, 1H, Hf); 8.50
(d, 3JH,H ¼ 14.6 Hz, 1H, He). Anal. calcd. for C23H23BF2N2O4: C, 62.75;
H, 5.27; N, 6.36. Found: C, 62.81; H, 5.29; N, 6.43.
2.2. Preparation of stock solutions
Dye stock solutions were prepared by dissolving the dyes
(2 ꢁ10ꢂ3 M) in DMF. Stock solutions of BSA (0.2 mg/ml) and SDS
(0.05%) as well as BSA/SDS were prepared by their dissolution in
0.05 M Tris–HCl buffer (pH 8.0).
2.5.3. 3-[(E)3-(5-piperidinyle-1-thien-2-yl)prop-2-enoyl]-4-
difluoroboronoxy-1-methylquinone-2 (5c)
Yield 75%. M.p. 296–297 ꢃC. 1H NMR (DMSO-d6):
d 1.71 (m, 6H,
2.3. Preparation of working solutions
3
Hj, Hk); 3.61 (s, 3H, MeN); 3.78 (m, 4H, Hi); 7.01 (d, JH,H ¼ 5.1 Hz,
3
All working solutions were prepared immediately before
experimentation. Working solutions of free dyes were prepared by
dilution of the dye stock solution in Tris–HCl buffer (pH 8.0).
Working solutions of the dyes in the presence of SDS, BSA and
BSA/SDS were prepared by dilution of the dye stock solution using
SDS, BSA or BSA/SDS stock solution, respectively. The concentra-
tions of dye, BSA and SDS in the working solutions were 5 ꢁ10ꢂ6 M,
0.2 mg/ml and 0.05%, respectively.
1H, Hh); 7.32 (t, 1H, Hc); 7.55 (d, JH,H ¼ 8.3 Hz, 1H, Hd); 7.67–7.77
3
(m, 2H, Hb, Hf); 8.04–8.11 (m, 2H, Ha, Hg); 8.31 (d, JH,H ¼ 12.9 Hz,
1H, He). Anal. calcd. for C22H21BF2N2O3S: C, 59.74; H, 4.79; N, 6.33.
Found: C, 59.81; H, 4.72; N, 6.40.
2.6 Dyes 6a–6c
A solution of 5a–c (3 mmol) in 60% ethanol (10 ml) was treated
with 1.59 g (15 mmol) Na2CO3. Then the mixture was boiled for
6–10 h. The decomposition of BF2-complex was controlled by TLC;
the resultant precipitate was filtered off, dried and crystallized from
2-propanol.
2.4. Spectroscopic measurements
Absorption spectra were recorded on Specord M-40 spectro-
photometer (Carl Zeiss, Germany). Fluorescence excitation and
emission spectra were registered on Cary Eclipse fluorescence
spectrophotometer (Varian, Australia). Fluorescence emission was
excited at the maximum of the fluorescence excitation spectrum.
Both excitation and emission slits had the width equal to 5 nm.
Fluorescence measurements were performed using the quartz cell
(1 ꢁ1 cm). The quantum yield value for 3a and 6b in presence of
BSA/SDS mixture was determined using Rhodamine 6G solution in
ethanol as the reference (quantum yield value 0.95). All the
measurements were performed at room temperature.
2.6.1. 4-Hydroxy-3-[(E)3-(1-ethyl-1,2,3,4-tetrahydroquinoline-6-
yl)prop-2-enoyl]-1-methylquinone-2 (6a)
Yield 75%. M.p. 198–199 ꢃC. 1H NMR (CDCl3):
d 1.18 (t, 3H, Me);
1.96 (m, 2H, Hk); 2.76 (t, 2H, Hj); 3.36 (m 4H, Hl, NCH2); 3.65 (s, 3H,
3
MeN); 6.55 (d, JH,H ¼ 8.7 Hz, 1H, Hi); 7.19–7.41 (m, 4H, Hc, Hd, Hg,
3
Hh); 7.63 (t, 1H, Hb); 8.04 (d, JH,H ¼ 15.4 Hz, 1H, Hf); 8.24 (d,
3JH,H ¼ 8.2 Hz, 1H, Ha); 8.48 (d, 3JH,H ¼ 15.4 Hz, 1H, He); 18.82 (s, 1H,
OH). Anal. calcd. for C24H24N2O3: C, 74.21; H, 6.23; N, 7.21. Found: C,
74.28; H, 6.22; N, 7.15.
2.5. Dyes 5a–5c
2.6.2. 4-Hydroxy-3-{(E)3-[2-hydroxy-4-(N,N-diethylamino)-
phenyl]prop-2-enoyl}-1-methylquinone-2 (6b)
A solution of 4 (1.06 g, 4 mmol) in acetic anhydride (10 ml) was
added to a solution of aldehyde (4 mmol) in acetic anhydride (2 ml)
at 60 ꢃC. The mixture was heated at 90 ꢃC for 30 min, and then
cooled. The resultant precipitate was filtered off and crystallized
from glacial acetic acid.
Yield 80%. M.p. 183–184 ꢃC. 1H NMR (CDCl3):
d 1.17 (m, 6H, Me);
3.34 (m, 4H, NCH2); 3.65 (s, 3H, MeN); 6.13–6.28 (m, 2H, Hi, Hh,);
7.20–7.31 (m, 2H, Hc, Hd); 7.50–7.69 (m, 2H, Hb, Hg); 8.24–8.33 (m,
2H, Ha, Hf); 8.62 (d, 3JH,H ¼ 15.4 Hz, 1H, He); 18.86 (s, 1H, OH). Anal.
calcd. for C23H24N2O4: C, 70.39; H, 6.16; N, 7.14. Found: C, 70.44; H,
6.19; N, 7.12.
2.5.1. 3-[(E)3-(1-ethyl-1,2,3,4-tetrahydroquinoline-6-yl)propyl-2-
enoyl]-4-difluoroboronoxy-1-methylquinone-2 (5a)
2.6.3. 4-Hydroxy-3-[(E)3-(5-piperidinyle-1-thiene-2-yl)prop-2-
Yield 82%. M.p. 294–295 ꢃC. 1H NMR (DMSO-d6):
d
1.20 (t, 3H,
enoyl]-1-methylquinone-2 (6c)
Me); 1.93 (m, 2H, Hk); 2.80 (t, 2H, Hj); 3.52 (m, 4H, Hl, NCH2); 3.66
Yield 86%. M.p. 202–203 ꢃC. 1H NMR (DMSO-d6):
d 1.65 (m, 6H,
3
3
(s, 3H, MeN); 6.87 (d, JH,H ¼ 9.0 Hz, 1H, Hi); 7.39 (t, 1H, Hc);
Hj, Hk); 3.61 (s, 3H, MeN); 3.77 (m, 4H, Hi); 6.37 (d, JH,H ¼ 4.2 Hz,
7.54–7.71 (m, 3H, Hd, Hg, Hh); 7.84 (t, 1H, Hb); 8.13 (d, 3JH,H ¼ 8.3 Hz,
1H, Ha,); 8.32 (d, 3JH,H ¼ 14.8 Hz, 1H, Hf); 8.41 (d, 3JH,H ¼ 14.8 Hz, 1H,
He). Anal. calcd. for C24H23BF2N2O3: C, 66.08; H, 5.31; N, 6.42.
Found: C, 66.04; H, 5.27; N, 6.49.
1H, Hh); 7.28 (t, 1H, Hc); 7.48–7.54 (m, 2H, Hd, Hg); 7.74 (t, 1H, Hb);
3
7.93 (d, JH,H ¼ 14.8 Hz, 1H, Hf); 8.08–8.16 (m, 2H, Ha, He). Anal.
calcd. for C22H22N2O3S: C, 66.98; H, 5.62; N, 7.10. Found: C, 67.02; H,
5.59; N, 7.14.

V.B. Kovalska et al. / Dyes and Pigments 84 (2010) 159–164
161
F
F
B
OH
O
O
O
N
O
N
O
O
O
3a
2a
F
O
F
O
OH
O
B
O
O
N
O
O
N
3b
2b
F
O
F
O
OH
O
O
B
NH
NH
O
O
O
3c
2c
F
F
OH
O
O
B
O
O
S
S
N
N
O
O
O
3d
2d
F
O
F
O
OH
O
B
N
N
O
N
N
O
6a
5a
F
OH
N
O
OH
F
B
O
OH
O
N
O
N
N
O
5b
6b
OH
N
O
O
F
F
B
O
O
S
N
S
N
N
O
6c
5c
Fig. 2. Structures of studied dyes.

162
V.B. Kovalska et al. / Dyes and Pigments 84 (2010) 159–164
3. Results and discussion
spectra, the short-wavelength band being more intensive than
long-wavelength one. Generally it should be noted that emission of
the dyes containing BF2-ether group is less intensive than that of
their unsubstituted analogues.
The synthesis of several dyes starting with boron difluoride
complex 4 is shown in Scheme 1. Condensations of 4 with alde-
hydes in acetic anhydride undergo with high yields and provide
boron difluoride complexes 5a–c of polymethines 6a–c. Reported
yields are given for analytically pure compounds, as shown by good
results of elemental analysis. The pure dyes were also characterized
by 1H NMR spectral method. The large values of the coupling
constants between protons of the methine chain (J ¼ 12–15 Hz) are
indicative of trans-configuration.
3.2. Characterization of fluorescent properties of chalcone dyes
in the presence of BSA
Characteristics of emission and excitation spectra of the dyes
and their boron difluoride complexes in presence of BSA are given
in Table 1. It should be noted that only for one hydroxyquinolone
dye (6c) positions of maxima of excitation and emission spectra
were close to these in buffer. In spectra of other dyes in BSA
complexes rather significant shifts took place or even new bands
appeared. Maxima of excitation spectra of studied hydrox-
yquinolone dyes in complexes with BSA are situated in the
495–596 nm region. In the spectra of these dyes single excitation
and emission bands were observed. Emission maxima of the dyes
are located between 569 and 626 nm. Stokes shifts values for these
dyes vary between small (10 nm for 5c) and large (85 nm for 6a)
ones. The increase in emission intensity for the studied hydrox-
yquinolone dyes in the presence of BSA (IBSA/I0) reaches 900 times
(5a). Particularly, for the most bright dye–BSA complexes (5b) the
high emission enhancement (in 470 times) is observed.
Interaction of coumarins 2a, 3a, 2b and 2c with BSA is accom-
panied by significant shift of the bands in excitation and/or emis-
sion spectra, or with appearing of the new ones, as compared to the
dyes spectra in buffer. For other coumarins corresponding maxima
positions were close to these observed in buffer. Thus in the exci-
tation/emission spectra of coumarins two bands were observed,
with exceptions of dyes 2a, 3b and 3d, having single band. Fluo-
rescence excitation maxima of studied dyes are located between
402 nm and 576 nm and emission maxima are situated in the range
480–614 nm. Observed Stokes shifts values are between 14 and
104 nm. All coumarins increase their fluorescence intensity in
presence of BSA but demonstrate from low to moderate intensity
level of fluorescence. The highest intensity increase and the
3.1. Characterization of fluorescent properties of free chalcone
dyes in buffer
Selected characteristics of fourteen polymethine dyes in buffer
are presented in Table 1. In fluorescence excitation spectra of
majority of hydroxyquinolone dyes single band, situated between
472 and 581 nm is observed, exception is the dye 6b, that has two
maxima on 451 nm and 549 nm. The same situation is in the
fluorescence emission spectra of hydroxyquinolone dyes, which
have single emission band with the maxima in the range
549–630 nm. In the case of hydroxyquinolone dye 6b, two emission
bands with maxima on 543 nm and 593 nm are observed. The dyes
demonstrate from medium to large values of the Stokes shifts,
which lay in the range 44–137 nm. Very low intensity of intrinsic
fluorescence of all dyes should be noted (0.6–4.4 a.u.).
In excitation spectra of all coumarin dyes two bands are pre-
sented, except these of 2a and 2c, where only long-wavelength
maxima are observed. The maxima of short-wavelength bands are
located between 404 and 470 nm, and these of long-wavelength
ones are between 470 and 585 nm. Emission maxima of the dyes
are situated in the range 462–556 nm for the short-wavelength
maxima and 522–712 nm for the long-wavelength bands. The
studied coumarins have from medium to large Stokes shifts values
from 26 nm to 134 nm. Low intrinsic fluorescence is observed for
the coumarins (0.2–41 a.u.), for these dyes having two bands in the
O
F
F
B
O
O
OH
O
O
j
f
g
i
a
d
N
b
k
Me
OH
e
l
h
N
c
N
O
N
N
Me
Me
Me
Me
5a
6a
O
F
F
B
OH
O
O
OH
O
O
OH
Me
N
i
Me
4
N
Me
N
g
N
Me
N
O
Me
h
Me
Me
Me
5b
6b
F
F
B
OH
O
O
N
O
O
S
j
j
i
O
S
S
N
k
N
N
N
O
g
i
h
Me
Me
6c
5c
Scheme 1. Scheme of dyes synthesis from boron difluoride complex 4.

V.B. Kovalska et al. / Dyes and Pigments 84 (2010) 159–164
163
Table 1
Characteristics of fluorescence excitation/emission spectra of studied chalcone dyes (5 ꢁ10ꢂ6 M) solutions in buffer, as well as in the presence of SDS (0.05%), BSA (0.2 mg/ml)
and BSA/SDS mixture (0.2 mg/ml and 0.05%, respectively).
Dye
In buffer
ex, nm
In SDS presence
ex, nm em, nm
In BSA presence
ex, nm em, nm
In BSA/SDS presence
ex, nm em, nm
l
l
em, nm
I, a.u.
0.6
l
l
I, a.u.
3
l
l
I, a.u.
l
l
I, a.u.
5a
472
549
504
586
507
578
505
576
588
629
587
620
145
81
770
174
585
495
578
626
580
614
537
171
2082
6a
5b
6b
479
546
451
616
594
543
1.2
4.4
2.5
490
544
595
599
23.7
9.6
505
573
27.4
507
569
675
506
569
1273
549
581
545
585
453
496
593
630
594
712
525
588
1.7
0.75
1.5
3.7
41
5c
6c
2a
3a
560
537
574
615
595
586
1.6
25
11.5
596
550
576
462
606
594
590
520
19
36
138
1173
606
540
572
618
591
588
34
414
192
19.5
540
400
564
458
100.5
6
523
432
574
429
555
524
606
533
545
2.3
67
225
540
405
540
562
467
680
3659
2b
3b
2c
3c
2d
3d
413
462
1.6
4
2
418
520
552
560
5.6
0.6
435
520
551
588
8
9.2
511
578
45
402
518
407
464
470
574
471
480
552
481
514
552
614
552
14.4
517
404
470
470
564
464
547
539
493
522
556
610
548
586
1.1
6
0.8
0.4
0.2
6
515
472
550
505
8.4
4
35
4
15
3
471
408
471
537
510
516
61.1
40
148
24.7
560
523
576
607
591
596
1.1
8.7
13.5
603
558
611
589
13
377
0.8
265
lex (lem) – maximum wavelength of fluorescence excitation (emission) spectrum, I – emission intensity, a.u. – arbitrary units.
brightest emission in BSA complex were observed respectively for
the unsubstituted dyes 3d (about 63 times) and 3a (1173 a.u.).
It is shown that for studied chalcone dyes pair (i.e. boron
difluoride complexes dye and non-substituted one) emission
intensity in BSA presence is higher for non-substituted compounds,
the exception being 5a–6a, and 5b–6b dyes pairs. The highest
emission increasing and fluorescence intensity in the BSA presence
are observed for the hydroxyquinoline dyes 5a, 5b, 6b and cou-
marine dyes 3a, 3d. These dyes will be further studied for their
sensitivity to various proteins.
Table 1. For majority of studied hydroxyquinoline dyes in the
presence of BSA/SDS mixture in excitation and emission spectra
single band is observed, while in corresponding spectra of the dye
5a two bands exist. Fluorescence excitation maxima for the
hydroxyquinoline dyes are located between 505 and 606 nm,
emission maxima for these dyes being situated between 569 and
629 nm. Only for the dye 6c positions of excitation/emission
maxima are similar to these in buffer. For all the dyes positions of
the bands in excitation/emission spectra in the presence of BSA/SDS
mixture are quite close to these in presence of BSA (only in the case
of 5a additional band appears). Stokes shifts values for all hydrox-
yquinoline dyes in BSA/SDS complexes are between 12 and 82 nm.
Fluorescence enhancement up to hundreds times upon addition of
BSA/SDS mixture is observed for these dyes, fluorescence intensity
reaching 1273 a.u. (Fig. 3) for the dye 6b (quantum yield for 6b in
presence of BSA/SDS mixture is about 0.23). The noticeable fluo-
rescence is also observed for dyes 6a and 6c.
Emission intensity of hydroxyquinolines in the presence of BSA/
SDS mixture is higher comparing with ‘‘pure’’ SDS presence in
16–50 times. Theexcitationand emissionbands of thedyes observed
in BSA/SDS mixture are close to these recorded in SDS presence,
except 5a where additional band appears and 5b where the 32 nm
excitation and 21 nm emission spectra shifts took place. It was
shown that emission intensity of unsubstituted hydroxyquinolines
in the presence of BSA/SDS mixture is higher than for corresponding
boron difluoride complexes analogues.
3.3. Characterization of fluorescent properties of chalcone dyes
in presence of SDS
Characteristics of excitation and emission spectra of studied
polymethine dyes in presence of SDS are given in Table 1. Despite
the used concentration of SDS (0.05%) was lower than its critical
micelle concentration (0.24%), spectral changes were observed for
all studied dyes upon addition of SDS. Thus from small to medium
increasing of emission (up to 31 times) was recorded for the pol-
ymethines, but because of low intrinsic fluorescence of studied
dyes, the fluorescence intensity of chalcones in SDS presence is
quite insignificant. In spectra of hydroxyquinoline dyes 5a, 6b and
coumarine dyes 2a, 3a and 3d in the presence of SDS the band
appeared which was not observed in spectra of free dyes in buffer.
For other dyes shift of excitation and emission bands on up to
28 nm occurred upon addition of SDS. For the coumarine dyes 3b
and 3d having two bands in intrinsic excitation and emission
spectra, considerable redistribution of the bands intensities was
noted in the presence of SDS.
In spectra of coumarin dyes 2b and 3c in BSA/SDS mixture
presence two bands in excitation and emission spectra are
observed, while other coumarin dyes have single bands in corre-
sponding spectra. Excitation maxima of coumarines are situated
between 405 and 603 nm, maxima of fluorescence emission
spectra being located in the range 467–680 nm. For the dyes 2a,
3a, 3c, and 2d in BSA/SDS mixture presence in emission spectra
the mostly pronounced bands have similar positions to those in
BSA presence. Stokes shifts values for coumarines are between 8
and 102 nm. The lowest increasing of the dye fluorescence upon
3.4. Characterization of fluorescent properties of chalcone dyes
in the presence of BSA/SDS mixture
Fluorescent properties of chalcone dyes and their boron
difluoride complexes in presence of BSA/SDS mixture are given in

164
V.B. Kovalska et al. / Dyes and Pigments 84 (2010) 159–164
high value of quantum yield and thus could be proposed for using
as probes for nonspecific detection of proteins.
550
575
600
625
650
675
700
a
250
200
150
100
50
250
200
150
100
50
5b in buffer, x 50 times
5b in BSA presence
5b in BSA-SDS presence
5b in SDS presence
4. Conclusions
A series of novel chalcones derived from the boron difluoride
complex of 3-acetyl-4-hydroxy-1-methyl-2-quinolone was
synthesized. The 2-quinolones, together with the previously
reported coumarin analogues, were studied in the presence of SDS,
native BSA and BSA/SDS mixture using fluorescence spectroscopy.
The dyes displayed low intrinsic fluorescence intensity. The emis-
sion intensity of the non-substituted chalcones in buffer, as well as
in the presence of SDS, BSA and BSA/SDS mixture was generally
higher than that of the corresponding boron difluoride complex.
The interaction of the dyes with BSA and BSA/SDS mixture was
accompanied by changes in excitation/emission spectra. The addi-
tion of SDS enhanced fluorescence intensity by 30 times. The
fluorescence response of the dyes in the presence of both BSA or
BSA/SDS mixture was more pronounced and the emission
enhancement was several hundred times.
The hydroxyquinoline dye 5b gave strong emission enhance-
ment (470 times) and provided the brightest complex with native
BSA. In the cases of the hydroxyquinoline 6b and coumarin 3a, very
high fluorescence intensity in the presence of BSA and BSA/SDS
mixture (quantum yields reaches 0.23 and 0.27 correspondingly)
together with noticeable emission enhancement (one hundreds
times) were observed. Thus the presence of diethylaminophenyl
and dimethyl indolenyl increased the affinity of the chalcones to
both native and denaturized proteins. Dyes 5b, 6b and 3a are
proposed for further study as fluorescent probes for protein
detection.
x 50 times
0
700
0
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575
600
625
650
675
Wavelength, nm
500
550
600
650
700
b
1400
1200
1000
800
600
400
200
0
1400
1200
1000
800
600
400
200
0
6b in buffer, x50 times
6b in BSA presence
6b in BSA-SDS presence
6b in SDS presence
x 50 times
500
References
550
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700
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Two of chalcone dyes, namely hydroxyquinoline 6b and cou-
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high emission increasing value, and also these dyes have rather