Vinyl-diazine triphenylamines and their N-methylated derivatives: Synthesis, photophysical properties and application for staining DNA

Article abstract of DOI:10.1016/j.dyepig.2012.04.009

In this paper we describe the synthesis of vinyl-diazine triphenylamines. These compounds exhibit strong green-yellow fluorescence in dichloromethane solution, important emission solvatochromism and acidochromism. Regioselective N-alkylation of these dyes provides cationic compounds that exhibit affinity for double-stranded DNA. Binding to the biopolymer results in a strong bathochromic shift and increase of the emission intensity.

Full text of DOI:10.1016/j.dyepig.2012.04.009

Dyes and Pigments 95 (2012) 400e407
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Vinyl-diazine triphenylamines and their N-methylated derivatives: Synthesis,
photophysical properties and application for staining DNA
,
,
b
,
a
,
b
,
,b
Ana I. Aranda a b, Sylvain Achelle a , Fabien Hammerer c, Florence Mahuteau-Betzer a
**
,
*
Marie-Paule Teulade-Fichou a
,b,
a UMR 176 CNRS/Institut Curie, Institut Curie, Bât 110, Centre Universitaire, Univ. Paris-Sud, F-91405 Orsay, France
b Institut Curie, Section de Recherches, Centre Universitaire, Univ. Paris-Sud, F-91405 Orsay, France
c Université Paris Sud, Centre Universitaire, F-91405 Orsay, France
a r t i c l e i n f o
a b s t r a c t
Article history:
In this paper we describe the synthesis of vinyl-diazine triphenylamines. These compounds exhibit
strong greeneyellow fluorescence in dichloromethane solution, important emission solvatochromism
and acidochromism. Regioselective N-alkylation of these dyes provides cationic compounds that exhibit
affinity for double-stranded DNA. Binding to the biopolymer results in a strong bathochromic shift and
increase of the emission intensity.
Received 14 March 2012
Received in revised form
17 April 2012
Accepted 19 April 2012
Available online 4 May 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Diazines
Pyrimidine
Pyrazine
Triphenylamine
Fluorescence
DNA strainers
1. Introduction
vinylpyridinium triphenylamines DNA stainers, TP-2Py and TP-3Py
(Fig. 1) [3].
Over the past decade, there has been a great interest in the
conception of fluorescent probes for the detection of nucleic acids
[1]. Among the analytic applications of these probes, fluorescent
labeling of DNA is widely used in microscopy to stain the nuclei of
cells. The requested characteristics for a good fluorescent DNA
probe are a better affinity for DNA than for other cell components,
a large increase in quantum yield upon binding with DNA, a low
mutagenicity and strong emission inside what is currently known
as the physiological optical window between 700 and 1300 nm
where biological tissues absorb less [2]. In a previous paper some of
us described the synthesis and optical properties of two
Triphenylamine derivatives are well-known as fluorescent
materials. Even if the quantum yield of the triphenylamine itself is
not high, judiciously substituted derivatives with strong electron-
acceptor groups and
p-conjugation extension exhibit strong
emission properties with high quantum yields and Stokes shifts [4].
Such structures have found applications as chemosensors [5],
components for Organic Light Emitting Diodes (OLEDs) [5d,6],
Photovoltaic materials [7] and Two-Photon Absorption (TPA)
chromophores [4ab,8].
Recently, diazine rings and in particular pyrimidine and pyr-
azine heterocycles, have been subject to intensive research, due to
their interest as building blocks for the synthesis of functionalized
p-conjugated materials [9]. In particular, star-shaped and V-shaped
structures with a pyrimidine central core substituted with electron-
withdrawing groups exhibit intense fluorescence emission [10] and
TPA properties [11]. Linear [12] as well as V-shaped [13] structures
incorporating a pyrazine moiety exhibit also interesting emission
properties. Therefore, we speculated that the combination of the
two scaffolds will afford compounds of practical interest for fluo-
rescence applications.
* Corresponding author. Institut des Sciences Chimiques de Rennes, UMR CNRS
6226, IUT Lannion, B.P. 30219, Rue E. Branly, 22302 Lannion, France. Tel.: þ33 0 2 96
46 94 46; fax: þ33 0 2 96 46 93 54.
** Corresponding author. UMR 176 CNRS/Institut Curie, Institut Curie, Bât 110,
Centre Universitaire, Univ. Paris-Sud, F-91405 Orsay, France. Tel.: þ33 0 1 69 86 30
86; fax: þ33 0 1 69 07 53 81.
E-mail addresses: sylvain.achelle@univ-rennes1.fr (S. Achelle), mp.teulade-
fichou@curie.u-psud.fr (M.-P. Teulade-Fichou).
0143-7208/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved.
doi:10.1016/j.dyepig.2012.04.009

A.I. Aranda et al. / Dyes and Pigments 95 (2012) 400e407
401
The aim of this paper is to describe the synthesis and photo-
physical properties of new chromophores with a triphenylamine
core substituted with vinyldiazines and diazinium moieties at the
periphery. Application of the vinylpyrimidinium triphenylamine
derivatives to DNA labeling is also discussed.
(71%) of 3 as a yellow solid. Mp: 207e208 ꢀC. 1H NMR (CDCl3): 9.14
(s, 2H, H-2pyrim), 8.64 (d, 2H, J ¼ 5.1 Hz, H-5pyrim), 7.84 (d, 2H,
J ¼ 15.9 Hz, H-vinylpyrim), 7.50 (d, 4H, J ¼ 8.4 Hz, H-mNPh3), 7.33e7.27
(m, 3H, H-5pyrim þ H-mNPh3), 7.18e7.10 (m, 7H, H-oNPh3 þ H-pNPh3),
6.95 (d, 2H, J ¼ 15.9 Hz, H-vinylNPh3
)
13C (CDCl3): 162.4 (C-4pyrim),
158.9(C-2pyrim), 157.3(C-6pyrim), 148.4 (C-1NPh3), 146.5 (C-1NPh3),
136.8 (C-vinylpyrim), 130.0 (C-4NPh3), 129.7(C-mNPh3), 128.9 (C-
mNPh3), 125.8 (C-pNPh3), 124.6 (C-vinylpyrim), 123.8 (C-oNPh3), 123.5
(C-oNPh3), 118.5(C-5pyrim). MS (ESþ): 453.1 (Mþ, 100%). Anal. Cald for
C30H23N5 (453.54): C, 79.45; H, 5.11; N, 15.44; Found C, 79.23; H,
5.53; N, 15.31.
2. Experimental
2.1. General experimental
All solvents (reagent grade) and the starting materials were
acquired from SigmaeAldrich and were used without purification.
Column chromatography was performed with the indicated
solvents using Acros neutral aluminum oxide Brockmann l (particle
2.3.2. Synthesis of 4,40-di[(E)(2-thiomethylpyrimidin-4-yl)vinyl]
triphenylamine (4)
size 50e200
m
m). Yields refer to chromatographically and spec-
Condensation reaction according to general procedure gave
after purification by column chromatography (ethyl acetate: n-
hexane 2:3),109 mg (20%) of 4 as a yellow solid. Mp: 149e151 ꢀC. 1H
troscopically pure compounds. 1H and 13C NMR spectra were
recorded at 300 and 75.3 MHz respectively, on a Bruker AC-300
spectrometer at room temperature using an internal deuterium
lock. Chemical shift values are given in ppm relative to tetramethyl
silane (TMS). Acidic impurities in CDCl3 were removed by treat-
ment with anhydrous K2CO3. Assignments of the resonance to
individual protons were based on integration and selective homo-
nuclear correlation (COSY). Heteronuclear multiple coherence
(HSQC, HMBC) spectra were obtained for all compounds and the
carbon resonances were assigned. Melting points were determined
on Electrothermal 1100 element. Quantitative UVevisible spectra
were recorded with a UVIKON xm SECOMAM spectrometer. Fluo-
rescence spectra were recorded using Spex FluoroMax-3 Jobin-
Yvon Horiba apparatus. Measurements were performed at room
temperature with solutions of OD < 0.1 to avoid re-absorption of
the emitted light, and data were corrected with a blank and from
the variations of the detector with the emitted wavelength. Fluo-
rescence quantum yield were measured according to Crosby
comparative method [14] using quinine bisulfate in 1 M H2SO4
NMR (CDCl3)
d
8.43 (d, 2H, J ¼ 5.2 Hz, H-5pyrim), 7.82 (d, 2H,
J ¼ 15.7 Hz, H-vinylpyrim), 7.48 (d, 4H, J ¼ 8.7 Hz, H-mNPh3), 7.16e7.10
(m, 7H, J ¼ 8.6 Hz, H-oNPh3 þ H-pNPh3), 6.93 (d, 2H, J ¼ 5.2 Hz, H-
6pyrim), 6.88 (d, J ¼ 15.8 Hz, H-vinylNPh3), 2.62 (s, 6H, Me). 13C NMR
(CDCl3)
d 172.33 (C-3pyrim), 162.55 (C-1pyrim), 157.28 (C-5pyrim),
148.31 (C-1NPh3), 146.53 (C-1NPh3), 136.82 (C-vinylpyrim), 130.05 (C-
4NPh3), 129.64 (C-mNPh3), 128.84 (C-mNPh3), 125.76 (C-pNPh3), 124.50
(C-vinylpyrim), 123.79 (C-oNPh3), 123.50 (C-oNPh3), 113.64 (C-6pyrim),
14.16 (C-Me). MS (ESþ): 546 (Mþ). HRMS (ESI) Anal. Cald for
C32H27N5S2 [M þ H]þ 546.1781, found 546.1788.
2.3.3. Synthesis of 4-bromo-40,400-di[(E)(pyrimidin-4-yl)vinyl]
triphenylamine (5)
Condensation reaction according to general procedure gave
after purification by column chromatography (ethyl acetate: n-
heptane 2:3), 225 mg (42%) of 5 as a yellow solid. Mp: 237e238 ꢀC.
1H NMR (CDCl3): 9.15 (s, 2H, H-2pyrim), 8.65 (d, 2H, J ¼ 5.1 Hz, H-
6pyrim), 7.84 (d, 2H, J ¼ 15.9 Hz, H-vinylpyrim), 7.51 (d, 4H, J ¼ 8.4 Hz,
H-mNPh3), 7.42 (d, 2H, J ¼ 8.4 Hz, H-mNPh3), 7.29 (d, 2H, J ¼ 5.1 Hz, H-
5pyrim), 7.09 (d, 4H, J ¼ 8.4 Hz, H-oNPh3), 7.04 (d, 2H, J ¼ 8.4 Hz, H-
(
FF ¼ 0.54) or rhodamine in ethanol (FF ¼ 1.00) as reference. The
CD spectra were recorded on a Jasco J-710 spectrometer in a 1 cm
quartz cuvette thermostated at 20 ꢀC. Microanalyses were obtained
from ICSN-CNRS Elemental Analysis Center at Gif-sur-Yvette,
France. Mass spectrometry (Electrospray Ionization, ESI) was per-
formed using ZQ 2000 Waters apparatus.
oNPh3), 6.95 (d, 2H, J ¼ 15.9 Hz, H-vinylNPh3
)
13C (CDCl3): 162.3 (C-
4pyrim), 158.9(C-2pyrim), 157.3(C-6pyrim), 147.9 (C-1NPh3), 145.7 (C-
1NPh3), 136.6 (C-vinylpyrim), 132.7(C-mNPh3), 130.5 (C-4NPh3), 128.9
(C-mNPh3), 126.8 (C-vinylpyrim), 124.1 (C-oNPh3), 123.8 (C-oNPh3),
118.6(C-5pyrim), 117.0 (CeBr). MS (ESþ): 532.3 (MHþ, 100%), 534.3
2.2. Studies of interactions with DNA
The fluorimetric titrations were performed at a constant dye
concentration (1
m
M) with increasing concentrations of DNA and
I
I
the binding curves were obtained by plotting the fluorescence
enhancement F/F0 (F ¼ integrated fluorescence area of the complex
and F0 ¼ integrated fluorescence area of the free dye) versus the
concentration in DNA. The DNA used is drewAT, which is a 14 base
pairs oligonucleotide CGCGAAATTTCGCG.
N
N
N
2.3. General procedure for the dicondensation of aldehydes 1 and 2
and methyldiazine
A stirred mixture of methyldiazine (2.2 mmol) and the corre-
sponding 4,40-diformyltriphenylamine (1.0 mmol) in aqueous
sodium hydroxide (5 M, 15 mL) containing aliquat 336 (43 mg,
0.1 mmol) was heated under reflux for 1 h (4 h for compound 7) The
mixture was allowed to cool and the precipitate was filtered off,
washed with water, and purified either by crystallization from the
indicated solvent or chromatography on alumina gel column.
N
I
2.3.1. Synthesis of 4,40-di[(E)(pyrimidin-4-yl)vinyl]triphenylamine (3)
Condensation reaction according to general procedure gave
after purification by crystallization from MeOH/water, 321 mg
Fig. 1. Structure of model compounds TP-2Py (solid) and TP-3Py (dashed) previously
developed as DNA stainers.

402
A.I. Aranda et al. / Dyes and Pigments 95 (2012) 400e407
(MHþ, 100%). Anal. Cald for C30H22BrN5 (532.43): C, 67.67; H, 4.16;
N, 13.15; Found C, 67.29; H, 4.43; N, 13.31.
2.3.5. Synthesis of 4,40-di[(E)(1methylpyrimidin-4-ium)vinyl]
triphenylamine (7)
A mixture of compound 3 (20 mg, 0.04 mmol) and methyliodide
(1 mL) was stirred for 3 h. Compound 7 was filtrated and obtained
2.3.4. Synthesis of 4,40-di[(E)(pyrazin-2-yl)vinyl]triphenylamine (6)
Condensation reaction according to general procedure gave
after purification by column chromatography (ethyl acetate: n-
heptane 2:3), 228 mg (51%) of 6 as a yellow solid. Mp: 152e153 ꢀC.
1H NMR (CDCl3): 8.61 (s, 2H, H-3pyra), 8.52 (s, 2H, H-6pyra), 8.38 (s,
2H, H-5pyra), 7.70 (d, 2H, J ¼ 15.9 Hz, H-vinylpyrim), 7.49 (d, 4H,
J ¼ 8.4 Hz, H-mNPh3), 7.34e7.29 (m, 2H, H-mNPh3), 7.18e7.09 (m, 7H,
as a red solid. 1H NMR (DMSO):
d
9.48 (s, 2H), 9.04 (d, J ¼ 5.8 Hz,
2H), 8.28 (d, J ¼ 15.9 Hz, 2H), 8.12 (d, J ¼ 6.5 Hz, 2H), 7.83 (d,
J ¼ 8.0 Hz, 4H), 7.51e7.41 (m, 4H), 7.31 (t, J ¼ 7.7 Hz, 2H), 7.21 (d,
J ¼ 7.2 Hz, 2H), 7.12 (d, J ¼ 8.0 Hz, 4H), 4.14 (s, 6H). HRMS (ESI) Anal.
Cald for C32H29N5 [Mþ] 483.2417, found 483.2423.
H-oNPh3 þ H-pNPh3), 7.05 (d, 2H, J ¼ 15.9 Hz, H-vinylNPh3
)
13C (CDCl3):
2.3.6. Synthesis of 4,40-di[(E)(1methylpyrazin-4-ium)vinyl]
triphenylamine (8)
151.6 (C-2pyra), 148.0(C-1NPh3), 146.8 (C-1NPh3), 144.3 (C-6pyra), 143.6
(C-3pyra), 142.4 (C-2pyra), 134.5 (C-vinylNPH3), 130.5 (C-4NPh3), 129.6
(C-mNPh3), 128.4 (C-mNPh3), 125.6 (C-pNPh3), 124.3 (C-oNPh3), 123.7
(C-pNPh3), 122.4 (C-vinylpyra). MS (ESþ): 453.1 (100%, Mþ), 454.2
(55%, MHþ) Anal. Cald for C30H23N5 (453.54): C, 79.45; H, 5.11; N,
15.44; Found C, 79.40; H, 5.15; N, 15.44.
A mixture of compound 6 (20 mg, 0.04 mmol) and methyliodide
(1 mL) was stirred for 3 h. Compound 8 was filtered and obtained as
a red solid. 1H NMR (300 MHz, DMSO)
d 9.35 (s, 1H), 9.27 (s, 1H),
8.86 (s, 1H), 7.98 (d, J ¼ 15.7 Hz, 2H), 7.73 (d, J ¼ 7.5 Hz, 4H), 7.24 (t,
J ¼ 7.0 Hz, 1H), 7.19 (d, J ¼ 7.5 Hz, 2H), 7.11 (d, J ¼ 7.5 Hz, 4H), 4.36 (s,
Scheme 1. Synthesis of the triphenylamine based chromophores. a) aqueous NaOH, aliquat 336, reflux 1 h; b) CH3I 3 h.

A.I. Aranda et al. / Dyes and Pigments 95 (2012) 400e407
403
6H). HRMS (ESI) Anal. Cald for C32H29N5 [Mþ] 483.2417, found
483.2423.
3. Results and discussion
3.1. Synthesis
The main method described in the literature for the synthesis of
(E)-vinyldiazines consists in the condensation of aldehydes with
the corresponding methyldiazines [15,13c,10d]. This approach has
the advantages of a large range of commercially available or easily
accessible methyldiazines and the use of environmentally friendly
conditions. Triphenylamine building blocks 2 and 1 have been
obtained in good yield from triphenylamine according to proce-
dures described in the literature [16]. Condensation reactions have
been carried out with various pyrimidine derivatives and 2-
methylpyrazine on compounds 1 and 2 in boiling aqueous 5 M
NaOH using aliquat 336 as catalyst, according to a previously
reported procedure [15a]. Compounds 3e6 have been obtained in
good yields (Scheme 1). The N-methylation has been carried out
with iodomethane at reflux without solvent or under microwave
activation. As described in the literature [17]. N-methylation occurs
regioselectively on the diazine rings and dimethylated products
7e8 are obtained in good yield.
Fig. 2. Normalized absorption (solid line) and emission (broken line) of compound 3 in
CH2Cl2 with (red) or without (blue) addition of TFA. c ¼ 5.0 ꢂ 10ꢁ6 M. (For interpre-
tation of the references to color in this figure legend, the reader is referred to the web
version of this article.)
orientation polarizability [18.] In contrast, the absorption band is
not significantly shifted. This solvatochromic behavior is typical of
compounds that undergo an internal charge transfer upon excita-
tion leading to a highly polar charge separated emitting state
stabilized by polar solvents. This phenomenon has been fully
documented with donoreacceptor fluorophores [19,4b,10d].
As well, the nitrogen atoms of the prepared diazines are basic
centers that can be protonated (pKa ¼ 1.2 in water) [10d,13c,15d].
Thus, the effect of protonation on the optical properties of
compounds 3 and 6 in CH2Cl2 has been studied. The changes in the
UVevis spectra of 3 upon addition of trifluoroacetic acid are illus-
trated in Fig. 2. The spectra show the disappearance of the
absorption band at 400 nm upon addition of acid, whereas a new
band corresponding to the protonated species appears at 570 nm.
Similar changes have been observed for compound 6. Concerning
the photoluminescence, it is interesting to note that the protonated
form of pyrimidine derivative 3 remains emissive with a red-shifted
fluorescence, as it is illustrated in broken line in Fig. 2, while pyr-
azine derivative 6 becomes nonemissive.
3.2. UV/vis and PL spectroscopy
The optical properties of the synthetized vinyl-diazine triphe-
nylamine derivatives 3e6 have been investigated by UV/visible and
photoluminescence spectroscopy on CH2Cl2 solutions at room
temperature. The data obtained are summarized in Table 1. All
compounds were photostable and did not undergo cisetrans
isomerization under the analysis conditions. All the compounds
show similar absorption spectra with two bands: one in the visible
region of the spectrum (417e422 nm) and a second one of higher
energy (286e292 nm). Neutral derivatives show high fluorescence
emission in CH2Cl2, with quantum yields ranging from 0.60 to 0.94.
In particular, the emission band of the pyrazine derivative 6
exhibits a large bathochromic shift and high fluorescence quantum
yield compared with pyrimidine derivatives 3e5. As an example,
the spectra of compound 3 are shown in Fig. 2 (blue line).
Compounds 3, 4 and 5 show similar emission properties with
quantum yields between 0.6 and 0.7. Large Stokes shifts are
observed for the compounds under investigation, indicating
important differences (vibrational, electronic, geometric) between
the FranckeCondon state and the excited state from which the
emission starts.
The optical properties of pyrimidinium and pyrazinium salts 7
and 8 have been investigated in different solvents: water, glycerol
and DMSO (Table 2). As observed for model compound TP-2Py, the
new compounds are not fluorescent in aqueous media. This is due
to the existence of nonradioactive decay processes presumably
A
fluorosolvatochromic study has been carried out on
compound 3 (Fig. 3). A bathochromic shift of the emission band can
be observed while increasing solvent polarity, estimated by solvent
Table 1
UV/Vis and photoluminescence data of neutral compounds 3e6 and TP-2Py.
Compounda labs, nm (
3
, L molꢁ1 cmꢁ1
)
lem, nm VF
VD
SSd, cmꢁ1
b
c
3
4
5
6
288 (52,300) 422 (29,500) 555
286 (26,625) 430 (42,420) 540
292 (46,700) 417 (27,900) 555
292 (57,900) 420 (35,800) 569
0.73 0.07 5679
0.60 0.07 4737
0.69
e
5963
0.94 0.08 6235
0.64 0.03 4955
TP-2Py
e
402 (50,350) 502
a
All spectra were recorded in CH2Cl2 solutions at room temperature from
c ¼ 5.0 ꢂ 10ꢁ6 M to 1.0 ꢂ 10ꢁ5 M.
b
Fluorescence quantum yield (ꢃ10%); excitation at 420 nm.
c
Singlet oxygen quantum yields were measured relative to rose bengale in
CH2Cl2 as standard (VD ¼ 0.76); excitation at 420 nm.
d
Stokes shift.
Fig. 3. Normalized emission of compound 3 in various solvents. c ¼ 5.0 ꢂ 10ꢁ6 M.

404
A.I. Aranda et al. / Dyes and Pigments 95 (2012) 400e407
Table 2
coefficients ( ) of the diazine derivatives are higher than that of the
3
UV/Vis and photoluminescence data of salts 7, 8 and TP-2Py.
TP-2Py, being that more than twice in the case of compound 7
(76,640 Mꢁ1 cmꢁ1 for 7 versus 37,400 Mꢁ1 cmꢁ1 for TP-2Py).
Concerning the emission properties in glycerol, both 7 and 8 emit at
longer wavelengths than the TP-2Py, with respective shifts of 36
and 41 nm. However, the values of the quantum yields for the
diazine derivatives are very low compared to that of the TP-2Py.
The optical properties of these compounds have also been studied
in DMSO. In this case, we can observe that the maximum absorp-
tion wavelength is lower than in glycerol or aqueous media for both
7 and 8, with very low quantum yields. In the case of pyrimidine
salt 7, the maximum emission wavelength is strongly shifted to the
red compared with that in glycerol. Moreover, the Stokes shift of 7
in DMSO is especially high (6519 cmꢁ1).
The production of singlet oxygen by pyrimidine derivatives 3
and 4 as well as by pyrazine derivative 6 have been studied and
quantum yields between 0.07 and 0.08 were found (Table 1), which
is of the same order as for the previously reported molecule TP-2Py
(0.03). The singlet oxygen quantum yields obtained for compounds
3, 4 and 6 are too weak to consider these molecules as potential
photosensitizers. Nevertheless, it is worth noting that no significant
heavy atom effect was observed for 4 despite the presence of the
sulfur atoms on the pyrimidine rings.
b
Compound
Solventa
labs, nm
, L molꢁ1 cmꢁ1
)
lem, nm
VF
SSc, cmꢁ1
(
3
7
Buffer
Glycerol
DMSO
Buffer
Glycerol
DMSO
525 (59,810)
560 (76,640)
500 (45,601)
485 (33,980)
500 (38,400)
440 (57,340)
509 (31,400)
496 (37,400)
e
e
e
685
720
e
0.026
<0.01
e
<0.01d
<0.01
0.08
3259
6519
e
8
690
530
656
649
5507
3859
4402
4753
TP-2Py
Buffer
Glycerol
0.11
a
All spectra were recorded at room temperature and the concentration range was
c ¼ 3 ꢂ 10ꢁ6 Me15 ꢂ 10ꢁ5 M in lithium cacodylate buffer pH 7.2, 100 mM NaCl,
c ¼ 0.5 ꢂ 10ꢁ6 Me1.5 ꢂ 10ꢁ5 M in glycerol and c ¼ 0.5 ꢂ 10ꢁ6 Me3 ꢂ 10ꢁ5 M in
DMSO.
b
Fluorescence quantum yield (ꢃ10%).
c
Stokes shift.
d
Quantum yield obtained 0.001.
involving molecular motions around the vinyl bond and also to
interactions with water. The optical properties of these salts have
also been studied in glycerol, which is a viscous solvent that hinders
the vibration of the molecule. A weak fluorescence is observed in
these conditions, with quantum yields of 2.6% and 0.1% for
compounds 7 and 8 respectively. If we compare the maximum
absorption wavelength in glycerol of both pyrimidine and pyrazine
derivatives with that of the TP-2Py we observe that there is
a bathochromic shift, specially important in the case of the pyr-
imidime derivative 7 (60 nm). As well, the molar absorption
3.3. Interaction with DNA
As it was explained before [3], TP-2Py is virtually non fluores-
cent when free in water, whilst a strong fluorescence enhancement
is observed upon binding to DNA making this molecule
Fig. 4. Absorbance of compound 7 (left) and 8 (right) in sodium cacodylate buffer pH 7.2, 100 mM NaCl upon addition of 0e5 molar equivalents of drewAT c ¼ 1.0 ꢂ 10ꢁ5 M.
Fig. 5. Absorption (black) and emission (blue) spectra of compound 7 (left) and 8 (right) in sodium cacodylate buffer pH 7.2, 100 mM NaCl in presence of 1 molar equivalent of
drewAT. cabs ¼ 10 M and cem ¼ 1 M. Excitation at 560 nm for compound 7 and at 510 for compound 8. (For interpretation of the references to color in this figure legend, the reader
is referred to the web version of this article.)
m
m

A.I. Aranda et al. / Dyes and Pigments 95 (2012) 400e407
405
Table 3
Optical properties of the cationic dyes in the presence of 5 molar equivalents of
drewAT.
Compounda
labs, nm (
3
, L molꢁ1 cmꢁ1
)
lem, nm
VF
D
Sc, cmꢁ1
b
7
8
560 (38,330)
550 (28,870)
509 (31,400)
725
e
<0.01d
e
4064
e
TP-2Py
656
0.08
4403
a
All spectra were recorded in lithium cacodylate buffer pH 7.2, 100 mM NaCl at
room temperature at c ¼ 0.5 ꢂ 10ꢁ6 Me2.5 ꢂ 10ꢁ5 M.
b
Fluorescence quantum yield (ꢃ10%).
c
Stokes shift.
d
Exact value of the quantum yield of 7 in these conditions was 0.004.
a remarkable DNA light-up probe. The fluorescence restoration of
TP-2Py bound to DNA, is attributed to the restriction of molecular
motions around the vinyl bond and to hydrophobic effects namely
screening from water once the dye is inserted inside DNA. This
emission specifically turned-on in presence of the biopolymeric
matrix is a clear advantage for cellular imaging of nuclear DNA or
chromosomes, as it should result in a high imaging contrast.
It was therefore interesting to evaluate the affinity of the
analogs 7 and 8 for DNA. In this aim, the UVevis titration of both
salts 7 and 8 was carried out. Fig. 4 depicts the absorbance of
compounds 7 and 8 upon addition of DNA, pointing out a bath-
ochromism in absorbance typical of DNA interaction. A clear
isobestic point is obtained at 522 nm for compound 8 and at
552 nm for compound 7 reflecting the existence of two forms in
equilibrium.
For compound 7, a fluorescence signal is observed upon addition
of one equivalent of DNA (Fig. 5). Likely, the DNA matrix plays the
same role as glycerol by hindering the vibration mode around
double bonds thereby increasing the fluorescence. In these condi-
tions, the Stokes shift is important (3300 cmꢁ1) and the emission
wavelength is red-shifted as compared to the TP-2Py analog
(725 nm versus 650 nm). The quantum yield obtained for the pyr-
imidinium salt 7 in presence of an excess of DNA (Table 3) albeit
moderate is significant (0.4%). In contrast, no fluorescence resto-
ration is observed for the pyrazinium salt 8 upon addition of DNA
(Fig. 5).
This result is not unexpected as compound 8 was found to be
poorly fluorescent in glycerol (VF ¼ 0.001 versus VF ¼ 0.026 for
compound 7, Table 2). Nonetheless, an interaction between 8 and
DNA occurs as indicated by the red-shift and the pronounced
hypochromism of absorbance in presence of DNA (Fig. 4).
The fluorimetric titration of salt 7 with drewAT (Fig. 6) shows
a quite important fluorescence enhancement (F/F0), around 35. This
Fig. 7. Circular dichroism spectra of drewAT [at 3 mM] upon addition of compound 7
(up) 8 (down); concentration in dye (0e16 mM) in lithium cacodylate buffer, 10 mM,
pH 7.2, 100 mM NaCl.
fluorimetric titration has been fitted with a one-site model in order
to determine the affinity constant (Ka ¼ 8.9 ꢂ 105
M
ꢁ1).
As well, Circular Dichroism (CD) measurements were performed
with salts 7 and 8 in presence of drewAT at increasing dye/DNA
ratios. In these conditions, a strong induced CD (ICD) signal is
observed for both compounds. This signature that reflects the
association to the chiral DNA matrix, is more frequently observed
with dyes binding in the minor groove of the double helix [3,20]
(Fig. 7).
Fig. 6. (left) Fluorimetric titration of drewAT with compound 7 c ¼ 2
enhancement fitting of compound 7 upon addition of drewAT.
m
M in 10 mM lithium cacodylate buffer pH 7.2, 100 mM NaCl, [drewAT] ¼ 0e15
mM and (right) fluorescence

406
A.I. Aranda et al. / Dyes and Pigments 95 (2012) 400e407
2004;16:456e65;
4. Conclusion
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Four divinyldiazine-triphenylamine 3e6 derivatives have been
obtained in good yield from 4,40-diformyltriphenylamine deriva-
tives. Both pyrimidine and pyrazine derivatives exhibit strong
fluorescence in dichloromethane with quantum yields up to 0.94. A
strong emission solvatochromism and a dramatic color change in
acidic media have been observed. This behavior indicates that this
type of material could be developed as luminescent polarity and pH
sensors. The corresponding N-methyl pyrimidinium and pyr-
azinium salts (7 and 8) have also been synthesized. The two
compounds display affinity for double-stranded DNA as demon-
strated by UVevis and CD measurements. The two compounds are
not fluorescent in aqueous media but the fluorescence of the pyr-
imidinium salt 7 is restored when bound to DNA whilst the pyr-
azinium 8 remains quenched. Remarkably, the emission of 7
associated to DNA is strongly red-shifted peaking in the (very) near
infrared region lem ¼ 725 nm. Altogether, this makes this pyr-
imidinium derivative a new NIR DNA stainer of potential interest
for imaging.
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