Synthesis of redox sensitive dyes based on a combination of long wavelength emitting fluorophores and nitroxides

Article abstract of DOI:10.1016/j.dyepig.2010.03.030

New, nitroxide-fluorophore acceptor-donor compounds were synthesized based on long wavelength (570-790 nm) emitting 9-diethylamino-5H-benzo[a]phenoxazin-5-one, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene and metal-ligand complex fluorophores. The fluorophores and nitroxides were linked via a robust C{double bond, long}C bond. The steady-state spectral properties of the new donor-acceptor compounds and their diamagnetic (sterically hindered amine) derivatives were studied. Titration of nitroxides with ascorbic acid sodium salt to diamagnetic N-hydroxy compounds resulted in fluorescence enhancement. The Ru-complex modified with nitroxide exhibited fluorescence increase and electron paramagnetic resonance band broadening upon B-deoxyribonucleic acid addition providing evidence of binding with B-deoxyribonucleic acid.

Full text of DOI:10.1016/j.dyepig.2010.03.030

Dyes and Pigments 87 (2010) 218e224
Contents lists available at ScienceDirect
Dyes and Pigments
journal homepage: www.elsevier.com/locate/dyepig
Synthesis of redox sensitive dyes based on a combination of long
wavelength emitting fluorophores and nitroxides
a
b
a
a,
*
}
Balázs Bognár , József Jeko , Tamás Kálai , Kálmán Hideg
a Institute of Organic and Medicinal Chemistry, University of Pécs, P.O. Box 99, H-7602 Pécs, Hungary
b Department of Chemistry, College of Nyíregyháza, H-4440 Nyíregyháza, Sóstói st. 31/B, Hungary
a r t i c l e i n f o
a b s t r a c t
Article history:
New, nitroxide-fluorophore acceptor-donor compounds were synthesized based on long wavelength
(570e790 nm) emitting 9-diethylamino-5H-benzo[a]phenoxazin-5-one, 4,4-difluoro-4-bora-3a,4a-
diaza-s-indacene and metal-ligand complex fluorophores. The fluorophores and nitroxides were linked
via a robust C]C bond. The steady-state spectral properties of the new donor-acceptor compounds and
their diamagnetic (sterically hindered amine) derivatives were studied. Titration of nitroxides with
ascorbic acid sodium salt to diamagnetic N-hydroxy compounds resulted in fluorescence enhancement.
The Ru-complex modified with nitroxide exhibited fluorescence increase and electron paramagnetic
resonance band broadening upon B-deoxyribonucleic acid addition providing evidence of binding with
B-deoxyribonucleic acid.
Received 3 December 2009
Received in revised form
28 March 2010
Accepted 29 March 2010
Available online 7 April 2010
Keywords:
BODIPY
B-DNA
Ó 2010 Elsevier Ltd. All rights reserved.
EPR
Fluorescence
Nitroxide radical
Nile Red
1. Introduction
nitroxide decreases. In other words, the nitroxide redox status can
be followed by both fluorescence and EPR spectroscopy. A further
Optical sensors for biomolecules and biochemical processes are
widely used in biochemical and medical studies [1,2]. Detection
based upon fluorescence has received much attention and signifi-
cant progress has been made in both fluorescence instrumentation
and in the synthesis of novel fluorophores [3,4]. The development
of the EPR technique [5] inspired researchers to synthesize new
spin labels and construct new double (EPR active and fluorescent)
sensors [6,7].
Fluorophore-nitroxide donor-acceptor compounds have been
utilized mainly for the detection of radicals or probing the redox
reactions in biological and chemical systems, including the detec-
tion of hydroxyl [8] or glutathionyl radical [9], Fe2þ or ascorbic acid
[10,11].
The fluorescence of nitroxide-fluorophore compounds is weak
owing to electron transfer from the fluorophore to nitroxide radical
or electron exchange between nitroxide and the excited singlet
state of the fluorophore [7]. When the nitroxide (“c-form”, Fig. 1)
function is reduced to N-hydroxylamine (“b-form”) the fluores-
cence intensity increases, while the intensity of the EPR signal of
extension of this idea, when the sterically hindered precursor
amine (“a-form”) instead of the nitroxide is attached to fluorophore
and its oxidation by reactive oxygen species (ROS) results in
a decrease in fluorescence with nitroxide formation [12]. In the past
decade a series of new donor-acceptor probes have been synthe-
sized varying both the nitroxide (nitronyl- [13], pyrrolidine- [14],
piperidine-nitroxide [15]) and the fluorophore (acridine [9],
umbelliferone [11], naphthyl [7], cyanine dye [16], polyaromatics
[14,17], naphthalimides [18], dansyl [6,15], fluorescamine [19] and
BODIPY [13,20]) moiety. However, these fluorophores emit mainly
below 600 nm and for biological and clinical application it is pref-
erable to apply long wavelength excitation and emission. At longer
wavelengths there is less sample absorbance, e.g. biological
samples are more transparent to red light, less autofluorescence
and the light sources are less expensive. In our laboratory the first
red fluorophore (sulforhodamine B)-nitroxide adduct was synthe-
sized for the purposes of studying the interaction of singlet
molecular oxygen and a double sensor [21].
The continuation of this research was inspired by the fact that
application of long wavelength emitting fluorophores has become
widespread in the past decade [22,23], however to find an ideal
fluorophore is not easy and always determined by the application.
Water-solubility, chemical stability, sensitivity toward polarity of
* Corresponding author. Tel.: þ36 72 536 221.
E-mail address: kalman.hideg@aok.pte.hu (K. Hideg).
0143-7208/$ e see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2010.03.030

B. Bognár et al. / Dyes and Pigments 87 (2010) 218e224
219
calculated on the equation
F
¼ (I/I0)(A0/A)(n/n0)
F
0, where I0, A0, and
Flu
Flu
N
Flu
F0 are the integrated emission, absorbance (at the excitation
wavelength), and quantum yield of the reference sample, respec-
tively. n0 is the refractive index of the solvent used for reference
ox
ROS
N
H
N
O
red
sample. I, A, n,
F are related to the sample with the same definitions
OH
"b-form"
applied to reference sample.
"a-form"
"c-form"
high
no
Flu intensity:
EPR signal:
high
low
yes
2.3. Dyes
no
2.3.1. Synthesis of BODIPY core 3a and 3c
Fig. 1. The fluorescence intensity and EPR signal change depending on nitrogen
oxidative status in nitroxide-fluorophore adducts.
To a deoxygenated solution of compound 2a or 2c (5.0 mmol)
and compound 1 (10.0 mmol 1.23 g) in CH2Cl2 (30 mL) trifluoro-
acetic acid (57 mg, 0.5 mmol for compound 3c and 627 mg,
5.5 mmol for compound 3a) was added and the mixture was stirred
at rt. overnight (e10 h) in dark under nitrogen. Then DDQ (1.13 g,
5.0 mmol) was added and after 30 min i-Pr2EtN (8.0 mL) and
microenvironment, intrinsic fluorescence of the environment,
Stokes shift, quantum yield, fluorescence lifetime are the possible
parameters for consideration. The objective of this work was to
synthesize new double sensor compounds with different, long
wavelength emitting fluorophores (Nile Red (C.I. Basic Blue 12),
BODIPY and metal-ligand complex) attached by C]C bond to
a nitroxide unit thereby achieving redox probes utilizable in bio-
logical systems.
.
BF3 Et2O (8.0 mL) was added at 0 ꢂC and the solution was stirred for
40 min at this temperature. The deep red solution was washed with
sat. NaHCO3 solution (20 mL), with brine (20 mL), the organic phase
was separated, dried (MgSO4). In the case of compound 3c PbO2
(478 mg, 2.0 mmol) was added and O2 was bubbled through. The
solutions were filtered, evaporated and the residue was purified by
flash column chromatography (hexane/EtOAc or CHCl3/Et2O) to
afford the BODIPY dyes in 10e35% yield as redepurple crystals.
2. Experimental
2.3.1.1. 1-Oxyl-2,2,5,5-tetramethyl-3-[4,4-difluoro-1,3,5,7-tetramethyl-
2,6-diethyl-4-bora-3a,4a-diaza-s-indacen-8-yl]-2,5-dihydro-1H-pyrr-
ole radical (3c). Yield: 220 mg (10%), mp 150e152 ꢂC, Rf 0.47
(hexane/EtOAc, 2:1). MS: m/z (%): 442 (Mþ, 25), 427 (62), 412 (50)
370 (100), 355 (86). Anal. Calcd. for C25H35BF2N3O: C 67.88; H 7.97; N
9.50. Found: C 67.87; H 7.86; N 9.53.
Melting points were determined with a Boetius micro melting
point apparatus and are uncorrected. Elemental analyses (C, H, N, S)
were performed on Carlo Erba EA 1110 CHNS elemental analyzer.
Mass spectra were recorded on an Automass Multi or VG TRIO-2
instruments in the EI mode (70 eV, direct inlet). ESI-TOF MS
measurements were performed with a BioTOF II instrument
(Bruker Daltonics, Billerica, MA). 1H NMR spectra were recorded
with Varian UNITYINOVA 400 WB spectrometer. Chemical shifts are
referenced to Me4Si, the exchangeable NH signal was not observed.
Measurements were run at 298 K probe temperature in CDCl3
solution. ESR spectra were obtained from 10ꢀ5 molar solutions
(CHCl3), using a Magnettech MS200 spectrometer, and all mono-
radicals gave triplet signal aN ¼ 14.5e14.7 G.). Preparative flash
column chromatography was performed on Merck Kieselgel 60
(0.040e0.063 mm). Qualitative TLC was carried out on commer-
cially available plates (20 ꢁ 20 ꢁ 0.02 cm) coated with Merck
2.3.1.2. 2,2,5,5-Tetramethyl-3-[4,4-difluoro-1,3,5,7-tetramethyl-2,6-
diethyl-4-bora-3a,4a-diaza-s-indacen-8-yl]-2,5-dihydro-1H-pyrro-
le (3a). Yield: 750 mg (35%), mp 135e137 ꢂC, Rf: 0.30 (CHCl3:Et2O:
MeOH, 8:3:1). MS: m/z (%): 427 (Mþ, 51), 412 (9), 370 (100), 355
(66). 1H NMR (CDCl3):
d: 5.75 (s, 1H), 2.50 (s, 6H), 2.26 (s, 6H), 2.33
(m, 4H), 1.81 (s, 6H), 1.69 (s, 6H), 1.02e0.98 (m, 6H). 13C NMR
(CDCl3): 154.82, 137.19, 136.79, 134.40, 133.08, 131.22, 130.89,
74.47, 69.28, 26.99, 26.74, 17.19, 17.07, 15.21, 14.31, 14.05, 12.63.
Anal. Calcd. for C25H36BF2N3: C 70.26; H 8.49; N 9.83. Found: C
70.20; H 8.46; N 9.75.
Kieselgel GF254
.
2.3.2. General procedure for dyes (4a, 4c, 5a, 5c)
2.1. Materials
A solution of compound 3a or 3c (1.0 mmol) and 4-(N,N-dime-
thylamino)benzaldehyde (596 mg, 4.0 mmol), piperidine (0.6 mL)
and AcOH (0.5 mL) in toluene (50 mL) was heated under reflux in
a Dean and Stark apparatus for 24 h. Crude product was then
concentrated under vacuum and purified by flash column chro-
matography (hexane/EtOAc or CHCl3/Et2O) to give the green or blue
colored fractions in 10e45% yield.
Calf thymus B-DNA sodium salt was purchased from Sigma and
3260
concentration was estimated spectrophotometrically
(
¼
6600 Mꢀ1 cmꢀ1). Compounds 2a [24], 2c [25], 6 [26], 7 [27], 9 [28],
10 [29], 12 [30] were prepared as published earlier and all other
reagents and compounds were purchased from Aldrich or Fluka.
2.2. Spectroscopic measurements
2.3.2.1. 1-Oxyl-2,2,5,5-tetramethyl-3-[3-(4-dimethylaminostyryl)-4,4
-difluoro-1,5,7-tetramethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacen
-8-yl]-2,5-dihydro-1H-pyrrole radical (4c). Yield: 57 mg (10%), mp
200e202 ꢂC, Rf 0.29 (hexane/EtOAc, 2:1). MS ESI: 573 [M þ H]þ.
Anal. Calcd. for C34H44BF2N4O: C 71.20; H 7.73; N 9.77. Found: C
71.13; H 7.73; N 9.75.
The UV spectra were taken with a Specord 40 (Jena Analytic), the
molar extinction coefficients (3) at absorption maxima were
obtained from slope of absorbance vs concentration using five
solutions of different concentrations. Fluorescence spectra of
compounds dissolved in dioxane or MeOH or NaCl/Tris buffer were
measured with Perkin Elmer LS50B spectrofluorimeter, with 10 nm
slits, with correction of instrumental factors by means of a rhoda-
mine B quantum counter and correction files supplied by the
manufacturer. Quantum yields were referred to Cresyl Violet dis-
solved in MeOH (lex ¼ 640 nm, F0 ¼ 0.54) or fluorescein dissolved
2.3.2.2. 1-Oxyl-2,2,5,5-tetramethyl-3-[3,5-bis(4-dimethylaminostyryl)
-4,4-difluoro-1,7-dimethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacen-
8-yl]-2,5-dihydro-1H-pyrrole radical (5c). Yield: 320 mg (45%), mp
222e223 ꢂC, Rf 0.22 (hexane/EtOAc, 2:1). MS ESI: 704 [M]þ. Anal.
Calcd. for C43H53BF2N5O: C 73.29; H 7.58; N 9.94. Found: C 73.18; H
7.53; N 9.90.
in 0.1 M NaOH
(
lex ¼ 496 nm, F0 ¼ 0.95). The values were

220
B. Bognár et al. / Dyes and Pigments 87 (2010) 218e224
Q
N
2, 3, 4, 5
Q
CHO
a
b
c
H
1) cat. TFA, DCM, rt.
2
+
OH
N
F
N
F
N
Q
N
H
1
Na-ascorbate
B
2) DDQ
O
.
3)i-PrEt N, BF Et O
2
3
2
3
2
N
CHO
cat. AcOH, piperidine
toluene, reflux
Q
Q
N
N
+
N
F
N
F
N
B
N
F
B
F
5
N
N
4
N
Scheme 1. Synthesis of BODIPY-based redox sensitive dyes 4 and 5.
2.3.2.3. 2,2,5,5-Tetramethyl-3-[3-(4-dimethylaminostyryl)-4,4-diflu-
oro-1,5,7-trimethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacen-8-yl]-
2,5-dihydro-1H-pyrrole (4a). Yield: 84 mg (15%), mp 137e139 ꢂC, Rf
0.29 (CHCl3/Et2O/MeOH, 8:3:1). MS ESI: 559 [M þ H]þ. 1H NMR
warmed up to 70 ꢂC until the reaction started and the mixture was
stirred at rt. for 60 min. After diluting with water (20 mL) the solu-
tion was decanted from iron residue and in a 250 mL baker the
solution was made alkaline (pH ¼ 9) by solid K2CO3 (intense foam-
ing!). The mixture was extracted with CHCl3 (2 ꢁ 15 mL), the organic
phase was dried (MgSO4), filtered and evaporated. The residue was
purified by flash column chromatography (CHCl3:MeOH) to yield the
title compound 112 mg (51%), reddishepurple crystals, mp
233e235 ꢂC, Rf: 0.31 (CHCl3:MeOH, 9:1). MS (EI): m/z (%): 441 (Mþ,
(CDCl3):
d
: 7.72e7.65 (m, 4H), 7.64 (d, J ¼ 5.5 Hz, 2H), 5.80 (s, 1H),
3.18 (s, 6H), 2.67e2.59 (q, J ¼ 7.8 Hz, 2H), 2.56 (s, 3H), 2.52e2.48 (m,
2H), 2.33 (s, 3H), 2.30 (s, 3H),1.92 (s, 6H),1.71 (d, J ¼ 6.3 Hz, 6H),1.17
(t, J ¼ 7.5 Hz, 3H), 1.03 (t, J ¼ 7.7 Hz, 3H). Anal. Calcd. for
C34H45BF2N4: C 73.11; H 8.12; N 10.03. Found: C 73.15; H 8.25; N
10.01.
12), 426 (100), 396 (13), 309 (18), 206 (44). 1H NMR (CDCl3):
d: 8.49
(s, 1H), 8.12 (d, J ¼ 7.2 Hz, 1H), 7.73 (d, J ¼ 9.7 Hz, 1H), 7.64 (d,
J ¼ 9.1 Hz, 1H), 6.85 (d, J ¼ 11.2 Hz, 1H), 6.67 (s, 1H), 6.30 (s, 1H), 6.25
(s, 1H), 3.52e3.47 (q, J ¼ 6.9 Hz, 4H), 1.70, 1.61 (2s, 12H), 1.15
(t, J ¼ 7 Hz, 6H). 13C NMR (CDCl3): 182.69, 152.14, 150.78, 146.86,
144.57, 139.11, 135.51, 132.19, 131.89, 131.47, 131.01, 129.06, 126.18,
125.33, 123.07, 110.36, 105.74, 105.43, 72.04, 67.90, 45.27, 28.40,
27.64, 12.32. Anal. Calcd. for C28H31N3O2: C 76.16; H 7.08; N 9.52.
Found: C 76.10; H 7.01; N 9.43.
2.3.2.4. 2,2,5,5-Tetrametil-3-[3,5-bis(4-dimethylaminostyryl)-4,4-difl-
uoro-1,7-dimethyl-2,6-diethyl-4-bora-3a,4a-diaza-s-indacen-8-yl]-2,5
-dihydro-1H-pyrrole (5a). Yield: 280 mg (40%), mp >360 ꢂC, Rf 0.28
(CHCl3/Et2O/MeOH, 8:3:1). MS ESI: 690 [M þ H]þ. 1H NMR (CDCl3):
d
: 7.85e7.60 (m, 8H), 7.55e7.41 (m, 4H), 5.86 (s, 1H), 3.18 (s, 12H),
2.70e2.63 (m, 4H), 2.35 (s, 6H), 1.92, 1.70 (2s, 12H), 1.20 (t, J ¼ 6.9 Hz,
6H). 13C NMR (CDCl3): 154.73, 149.12, 138.26, 135.63, 135.12, 134.43,
133.12,131.22,130.11,127.72,123.28,117.32, 74.42, 69.33, 40.32, 27.12,
26.82, 17.22, 17.10, 15.21, 14.10, 12.65. Anal. Calcd. for C43H54BF2N5: C
74.88; H 7.89; N 10.15. Found: C 74.83; H 7.76; N 10.10.
2.3.4. Synthesis of paramagnetic ligand (11)
2.3.4.1. 2-(1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol-3-yl)
dipyrido[3,2-a:20,30-c]quinoxaline radical. A solution of 5,6-dia-
mino-1,10-phenantroline 10 (210 mg, 1.0 mmol) and compound 9
2.3.3. Synthesis of Nile Red derivatives (8c and 8a)
2.3.3.1. 9-Diethylamino-2-(1-oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H
-pyrrol-3-yl)-5H-benzo[a]phenoxazin-5-one radical (8c). To a deoxy-
genated solution of compound 6 (466 mg, 1.0 mmol) in dioxane
(10 mL) Pd(PPh3)4 (50 mg, 0.05 mmol) was added and the mixture
was stirred at rt. for 10 min. Then compound 7 (184 mg, 1.0 mmol)
and aq. 10% Na2CO3 (5 mL) was added and the mixture was stirred
and refluxed for 10 h under N2. After cooling the dioxane was
evaporated off and the red residue was partitioned between brine
(10 mL) and EtOAc (20 mL). The organic phase was separated, the
aqueous phase was extracted with EtOAc (20 mL). The combined
organic phase was dried (MgSO4), filtered, evaporated and the
residue was purified by flash column chromatography (hexane/
EtOAc) to yield compound 8c 173 mg (38%), red crystals,þ mp
240e242 ꢂC, Rf 0.50 (CHCl3/Et2O, 2:1). MS (EI): m/z (%): 441 (M -15,
14), 426 (100), 396 (12), 277 (12), 206 (36). Anal. Calcd. for
C28H30N3O3: C 73.66; H 6.62; N 9.20. Found: C 73.48; H 6.56; N 9.15.
Table 1
Optical properties of compound 3ae13c synthesized.
Compound
Solvent
labs nm
3
Mꢀ1 cmꢀ1
lex nm
lem nm
Fa
3a
MeOH
Dioxane
MeOH
Dioxane
MeOH
Dioxane
MeOH
Dioxane
MeOH
Dioxane
MeOH
Dioxane
MeOH
Dioxane
MeOH
537
541
536
536
600
600
629
638
648
648
730
731
564
525
564
530
448
3.59 ꢁ 104
4.79 ꢁ 104
2.82 ꢁ 104
2.81 ꢁ 104
1.49 ꢁ 104
1.42 ꢁ 104
3.32 ꢁ 104
1.55 ꢁ 104
1.36 ꢁ 104
541
540
535
541
600
615
629
626
642
648
730
733
573
529
567
530
453
560
559
558
561
697
676
695
678
788
772
785
766
640
578
638
584
600
0.315
0.417
0.194
0.308
0.002b
0.052b
0.005b
0.025b
0.001b
0.039b
0.001b
0.011b
0.220
0.795
0.038
0.185
0.053
3c
4a
4c
5a
5c
8a
8c
2.3.3.2. 9-Diethylamino-2-(2,2,5,5-tetramethyl-2,5-dihydro-1H-pyrrol
Dioxane
MeOH
13c
-3-yl)-5H-benzo[a]phenoxazin-5-one (8a). To
a
solution of
a
compound 8c (228 mg, 0.5 mmol) in glacial acetic acid (5 mL) Fe
powder (140 mg, 2.5 mmol) was added and the mixture was
Referred to fluorescein in 0.1 M NaOH at 496 nm.
Referred to Cresyl Violet in MeOH at 640 nm, n ¼ 3, accuracy ꢃ10%.
b

B. Bognár et al. / Dyes and Pigments 87 (2010) 218e224
221
(196 mg, 1.0 mmol) in anhydr. EtOH (10 mL) was heated under
condenser for 4 h. After evaporation of the solvent the residue was
purified by flash column chromatography (CHCl3/MeOH) to yield
compound 11 259 mg (70%), yellow solid, mp 230e232 ꢂC, Rf 0.29
(CHCl3/MeOH, 9:1). MS (EI) m/z (%): 370 (Mþ, 12), 356 (25), 340
(100), 325 (23). Anal. Calcd. for C22H20N5O: C 71.33; H 5.44; N 18.91.
Found: C 71.16, H 5.49, N 18.94.
B(OH)2
N
O
N
O
N
O
N
O
Pd(PPh )
3 4
+
N
O
7
dioxane, aq. Na2CO3
OTf
8
6
8
N
Q
a
H
OH
O
b
Na-ascorbate
c
2.3.5. Synthesis of Ru-complex (13c)
2.3.5.1. [Ru(phen)2 2-(1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydro-1H-
pyrrol-3-yl)dipyrido[3,2-a:20,30-c]quinoxaline](PF6)2 salt radical. A
solution of compound 11 (185 mg, 0.5 mmol) and Ru-complex (12)
(380 mg, 0.5 mmol) in anhydr. EtOH (75 mL) was heated under
reflux condenser for 2 h. After cooling the solution was filtered and
the filtrate was treated with NH4PF6 upon which the complex
precipitated and the solution was allowed to stay in a refrigerator
(ꢀ18 ꢂC) overnight. The precipitate was filtered, washed with Et2O
(20 mL) to give orangeebrown solid 202 mg (36%), mp >360 ꢂC, MS
ESI: 977 [M2þ þ PF6ꢀ]þ. Anal. Calcd. for C46H36F12N9OP2Ru: C 49.25;
H 3.23; N 11.24 Found: C 49.02, H 3.20, N 11.08.
Scheme 2. Synthesis of Nile Red redox sensitive dye 8.
and 146 nm for 4a and 5a dyes, respectively and the quantum yields
decreased because of strong charge transfer caused by amines [32].
3.2. Synthesis and characterization of Nile Red based redox probes
Nile Red is a phenoxazine dye, which fluoresces intensely and
has been used for histochemical detection of lipids [33]. Very
recently Nile Red linked with chemiluminescent donor has been
used in the construction of energy-transfer cassettes [26] and in
fluorescent probe molecules in a silicate matrix [34]. In our
approach the 2-triflate of Nile Red 6 was coupled with para-
magnetic boronic acid 7 [27] using a Suzuki coupling in aq. dioxane
in the presence of Na2CO3 and Pd(PPh3)4 to yield compound 8c.
Subsequent reduction with Fe powder in AcOH [24] afforded
compound 8a (Scheme 2). These dyes emit at 640 nm in MeOH and
at a shorter wavelength 584 nm in dioxane. The fluorescence
quantum yield is increased fourfold for the diamagnetic derivative
8a over the paramagnetic derivative 8c. These dyes seem to be an
ideal redox sensor emitting at longer wavelengths.
3. Results and discussion
3.1. Synthesis and characterization of BODIPY-based sensors
For the synthesis of new BODIPY derivatives the mixture of
paramagnetic aldehyde (2c) [25] and 2,4-dimethyl-3-ethylpyrrole
(1) was treated with a catalytic amount of trifluoroacetic acid (TFA)
in CH2Cl2 followed by treatment with 2,3-dichloro-5,6-dicyano-
1,4-benzoquinone (DDQ), i-Pr2EtN and boron trifluoride diethyl
etherate at ambient temperature to give compound 3c. To
synthesize the diamagnetic derivative (3a) aldehyde 2a [24] and 1.1
equivalent TFA was used [20]. The emission wavelength of these
derivatives (3a and 3c) are in the shorter wavelength region
(w560 nm), and Stokes shift are also small, 20 nm. Only a slight
difference was noted in the excitation and emission spectra when
recorded in polar (MeOH) and apolar (dioxane) solvents. We hoped
that extension of conjugation would shift both excitation and
emission toward longer wavelength. These BODIPY cores (3a and
3c) were condensed with 4-(N,N0-dimethylamino)benzaldehyde in
benzene in the presence of a catalytic amount of acetic acid and
piperidine under azeotropic removal of water [31] to give mixture
of the 3-monostyryl derivative 4c and 3,5-distyryl 5c and
diamagnetic 4a and 5a compounds, respectively (Scheme 1). The
3,5-distyryl derivatives 4a and 5a exhibited long (788 nm) emis-
sions (Table 1). The Stokes shift of these compounds in MeOH are 97
3.3. Synthesis and characterization of paramagnetically
modified Ru(II) complex
The metal-ligand complexes have been recognized as long-
lifetime luminescent probes and as new diagnostic and therapeutic
agents [31,35]. Polypyridyl complexes of Ru(II) are intensely colored
owing to well-characterized, localized metal-to-ligand charge
transfer transitions. The nitroxide moiety can be attached only to
a polypyridyl ligand via a C]C bond to a modified 1,10-phenan-
throline, e.g. dipyrido[3,2-a:20,30-c]quinoxaline ligand (11) which
was obtained by condensing 1,2-dicarbonyl compound (9) [28]
with 5,6-diamino-1,10-phenanthroline (10) [29]. Treatment of [Ru
(phen)2-(O3SCF3)2] complex (12) [30] with compound 11 in
O
EtOH, reflux
CHO
H2N
N
N
N
N
N
N
+
H2N
N
O
N
O
9
10
11
Q
N
+
2
13
N
1) EtOH, reflux
-
N
Ru
(PF6)22
N
[Ru(phen)2-(O3SCF3)2]+ 11
12
N
N
N
2) NH PF
4
6
N
b
c
OH
O
N
13
Scheme 3. Synthesis of Ru-complex-based redox sensitive dye 13.

222
B. Bognár et al. / Dyes and Pigments 87 (2010) 218e224
18
16
14
12
10
8
14
12
10
8
100 µM
100 µM
6
0
6
0
4
4
2
2
0
500
0
740
760
780
800
nm
820
840
860
550
600
650
700
750
nm
Fig. 4. Fluorescence emission spectra of compound 13c (50
m
M) in MeOH with sodium
Fig. 2. Fluorescence emission spectra of compound 5c (50 mM) in MeOH with sodium
ascorbate (dissolved in MilliQ water) 0, 5, 10, 15.5, 25, 40, 50 and 100 mM
(final concentration). lex: 730 nm.
ascorbate (dissolved in MilliQ water) 0, 10, 25, 50 and 100
453 nm.
mM (final concentration), lex:
increase (Fig. 2 and Table 2), from the paramagnetic Nile Red
derivative (8c) compound 8b was formed with 29% fluorescence
quantum yield raise (Fig. 3) and from 13c compound 13b formed
with a 31% fluorescence quantum yield enhancement (Fig. 4). In all
cases the nitroxide was reduced to N-hydroxylamine with
approximately one equivalent amount of ascorbate.
Table 2
Quantum yield increase upon reduction of 5c, 8c, 13c nitroxides dissolved in 10%
H2O (V) containing MeOH.
Compound
Fa
Compound
Fa
5c
5b
8c
8b
0.0018b
0.0041b
0.024
13c
13b
13c þ B-DNA
0.050
0.065
0.134
3.5. Characterization of fluorescent dye 13c
in the presence of B-DNA
0.031
a
Referred to fluorescein in 0.1 M NaOH at 496 nm.
Referred to Cresyl Violet in MeOH at 640 nm, n ¼ 3, accuracy ꢃ10%.
b
Ru-complexes were reported to bind B-DNA resulting in a fluo-
rescence increase, and for paramagnetically modified Ru-
complexes EPR line broadening also was described. This broad-
ening is attributed to surface bound complexes and intercalatively
bound complexes [36]. Adding a solution of Calf thymus B-DNA
refluxing ethanol followed by precipitation with NH4PF6 yielded
compound 13c, which exhibits emission at 600 nm with low
(F
¼ 0.05) quantum yield (Scheme 3).
(w270 mM) in 50 mM NaCl and 5 mM Tris buffer to a solution of 13c
3.4. Characterization of fluorescent dyes
in the presence of ascorbate
caused a 169% quantum yield increase in fluorescence (Table 2,
Fig. 5). This can not be attributed exclusively to reduction of 13c to
hydroxylamine (13b) because the treatment the solution of 13c
with sodium ascorbate produced minimal fluorescence increase in
Tris buffer (Fig. 5). Although the intensity of the EPR lines
decreased, some broadening of the EPR bands and the appearance
of a high field peak (arrow in Figs. 6 and 7) also confirms the
binding of compound 13c with B-DNA.
Solutions of compounds 5c, 8c and 13c (50 mM) in MeOH were
titrated with sodium ascorbate dissolved in MilliQ water. The
fluorescence intensity increased in all cases, from compound 5c the
N-hydroxylamine (5b) was formed with a 132% quantum yield
80
200
150
100
50
60
40
20
0
70 µM
0
0
500
550
600
650
700
750
800
550
600
650
700
750
800
nm
nm
Fig. 3. Fluorescence emission spectra of compound 8c (50
m
M) in MeOH with sodium
Fig. 5. Fluorescence emission spectra of 50
5 mM Tris), 50 M 13c and 1.0 mM sodium ascorbate ($ $ $ $) in a buffer, 50
270 M Calf thymus B-DNA (- - - -) in a buffer, lex: 453 nm.
m
M 13c (d) in a buffer (50 mM NaCl and
ascorbate (dissolved in MilliQ water) 0, 15.5, 50 and 70
567 nm.
mM (final concentration). lex
:
m
mM 13c and
m

B. Bognár et al. / Dyes and Pigments 87 (2010) 218e224
223
30000
25000
20000
15000
10000
5000
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Acknowledgements
This work was supported by a grant from the Hungarian
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