A systematic analysis of aromatic heterocyclic rings in solvatochromic fluorophores

Article abstract of DOI:10.1246/cl.2011.378

The SuzukiMiyaura cross-coupling was found to be effective for the modification of aromatic heterocyclic rings in solvatochromic fluorophores, thereby providing quantitative evaluation of the ring effects on photophysical properties. The effect of heteroatom, a β-substituent, and the number of rings in the aromatic moiety were investigated systematically.

Full text of DOI:10.1246/cl.2011.378

doi:10.1246/cl.2011.378
378
Published on the web March 12, 2011
A Systematic Analysis of Aromatic Heterocyclic Rings in Solvatochromic Fluorophores
Sang-Hyun Son,1 Yuko Abe,2 Maiko Yuasa,1 Yutaka Yamagishi,1 Naoki Sakai,2 Tokiyoshi Ayabe,2 and Koji Yamada*1
1Section of Materials Science, Faculty of Environmental Earth Science, Hokkaido University,
Kita-ku, Sapporo, Hokkaido 060-0810
2Department of Cellular Life Science, Faculty of Advanced Life Science, Hokkaido University,
Kita-ku, Sapporo, Hokkaido 001-0021
(Received January 12, 2011; CL-110031; E-mail: yamada@ees.hokudai.ac.jp)
The Suzuki-Miyaura cross-coupling was found to be
effective for the modification of aromatic heterocyclic rings in
solvatochromic fluorophores, thereby providing quantitative
evaluation of the ring effects on photophysical properties. The
effect of heteroatom, a ¢-substituent, and the number of rings in
the aromatic moiety were investigated systematically.
[
]
A fluorescent indicator is a powerful tool for the imaging of
various living biological systems due to its high sensitivity,
good spatial and time resolution, and low invasiveness.1 Among
these indicators, solvatochromic fluorophores, which exhibit
modest changes in their absorption spectra but large changes in
their emission spectra through their electronic interaction with
solvents or solute molecules, provide a range of information on
the surrounding microenvironment.2 For this reason, fluoro-
phores have been used in the monitoring of various biological
processes that demonstrate polarity changes, such as antigen-
antibody reactions, denaturation and renaturation of DNA,3 and
cell membrane dynamics,4 through the measurement of two
different emission wavelengths. To date, several solvatochromic
fluorophores, such as N,N-dimethyl-6-propionyl-2-naphthyl-
amine (PRODAN),5 5-(dimethylamino)naphthalene-1-sulfonyl
ethylenediamine (dansyl EDA),6 4-fluoro-7-nitrobenzofurazan
(NBD),7 and N-(2-aminoethyl)-4-[5-[4-(dimethylamino)phenyl]-
2-oxazolyl]benzenesulfonamide (Dapoxyl SEDA),8 have been
reported. All of these compounds are composed of three
moieties: electron-donating, aromatic, and electron-withdraw-
ing. However, the chemical structures of these compounds are
not consistent and, therefore it is difficult to evaluate the effects
of each moiety. Furthermore, these compounds are excited by
near UV light, which limits their applications in biological
fields.9 Thus, to broaden their biological applications, it remains
necessary to develop new fluorophores that can be excited by
visible light at relatively longer wavelengths.
[
]
Scheme 1. Synthesis of fluorophores 1-4.
advantageous to replace the aromatic moiety, regardless of the
electron-donating and -withdrawing moieties. In this paper, we
have investigated new synthetic approaches to the synthesis of
solvatochromic fluorophores: the three starting moieties were
synthesized separately, before being connected directly by the
hetero-coupling reactions.
In order to further improve their photophysical properties,
various combinations of aromatic heterocyclic rings with both
electron-donating and -withdrawing aromatic groups need
systematic investigation. However, solvatochromic fluorophores
are usually synthesized in a stepwise manner and electron-
donating and -withdrawing moieties cannot be freely substituted,
making it difficult for systematic studies on the effects of
molecular structures.
The Suzuki-Miyaura cross-coupling reaction provides a
feasible approach to the condensation of aromatic ring systems
from stable and easily manipulated boronic acid derivatives.10
This reaction is widely used, not only in electronics and
pharmacology, but also for the synthesis of photofunctional
molecules.11 To develop solvatochromic fluorophores, it is
Herein, we describe the synthesis of a series of fluorophores
1-4 with different aromatic rings via the Suzuki-Miyaura cross-
coupling reaction (Scheme 1). Four different five-membered
heterocycles, thiophene,12 furan,13 bithiophene,12 and 3,4-ethyl-
enedioxythiophene (EDOT),14 were selected as the aromatic
heterocyclic rings. A 4-dimethylaminophenyl group was used as
the electron-donating moiety in the same manner as dansyl
amide and Dapoxyl SEDA. Furthermore, a 4-(neopentyloxysul-
fonyl)phenyl group was chosen as the electron-withdrawing
group for the following three reasons: (1) the sulfonyl group is
strongly electron-withdrawing; (2) the neopentyl group im-
proves solubility in organic solvents thereby facilitating purifi-
cation; and (3) a 4-(neopentylsulfonyloxy)phenyl group can be
easily cleaved to form a sulfonic acid group15 thereby improving
Chem. Lett. 2011, 40, 378-380
© 2011 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

379
Table 1. Spectral properties fluorophores 1-4 in different solventsa
Toluene Dichloromethane
DMF
Fluorophore
-
¾
-
/nm
-
/nm
¾
-
/nm
-
¾
-
em
/nm
abs
em
abs
em
abs
Φfl
Φfl
Φfl
¹1
¹1
¹1
/nm /M¹1 cm
/M¹1 cm
/nm /M¹1 cm
1
2
3
4
394
388
404
419
27000
30000
34000
32000
482
468
482
515
0.60
0.67
0.58
0.37
394
391
405
421
27000
30000
35000
33000
517
512
518
563
0.44
0.48
0.41
0.22
397
392
407
424
27000
30000
34000
33000
551
541
546
590
0.24
0.28
0.26
0.06
aQuantum yields were measured using quinine sulfate (Φfl = 0.546 in 0.5 M H2SO4) as the standard.
10500
1.2
1
1.2
1
8500
6500
4500
2500
L
J
0.8
0.6
0.4
0.2
0
0.8
0.6
0.4
0.2
0
(MeOH)
I
H
G
E
F
D
C
K
B
(EtOH)
A
30
40
50
60
ET(30)/kcalmol−1
340 365 390 415 440 465 490 515 540 565 590 615
Wavelength/nm
Figure 2. The plot of the Stokes shift of fluorophores 1-4
versus solvent polarity parameter ET (30). A: cyclohexane;
B: toluene; C: 1,4-dioxane; D: tetrahydrofuran; E: ethyl acetate;
F: dichloromethane; G: acetone; H: N,N-dimethylformamide;
Figure 1. Normalized absorption (solid lines) and emission
spectra (dashed lines) for fluorophores 1-4. : fluorophore 1,
: fluorophore 2, : fluorophore 3, and : fluorophore 4.
I: dimethyl sulfonate; J: acetonitrile;
: fluorophore 1;
: fluorophore 2; : fluorophore 3; : fluorophore 4. The
water solubility and the ability of the resultant probes to attach to
amino group in proteins.
slopes for the fits to aprotic solvents are 253 (R2 = 0.906), 244
¹1
(R2 = 0.939), 230 (R2 = 0.947), and 191 cm (0.805), respec-
With the above in mind, we investigated the synthesis of
novel fluorophores 1-4.16 We found that they were readily
synthesized in four steps from commercially available substan-
ces by Suzuki-Miyaura cross-coupling reactions of dibromo
derivatives, with the boronic ester of the electron-withdrawing
and the boronic acid of the electron-donating components
(Supporting Information; SI18). Their structures were fully
tively.
polar protic solvents, such as methanol and ethanol, however,
the plots for 1 deviate from linearity. A similar phenomenon
was observed for Dapoxyl dye. The absorption maximum of
fluorophore 1 was shifted further to the red end of the spectrum
than was that of Dapoxyl dye. Fluorophore 2, in which the
hetero atom in the aromatic ring was replaced, has almost the
same plot pattern. The absorption maximum of 2 is slightly more
blue-shifted than that of 1. Moreover, the synthetic yield of
fluorophore 2 was low, probably due to the relative instability of
the furan intermediate. The furan ring appears to be useless as
the aromatic ring. Fluorophore 3, in which substituent groups at
the ¢-positions of thiophene were introduced, showed the same
slope as, but has a lower intercept than 1. The absorption
maximum of 3 is red-shifted slightly. Fluorophore 4, having
bithiophene as the aromatic heterocyclic ring, exhibits a similar
extinction coefficient and both the absorption and emission
maxima of 4 were more strongly red-shifted. However, the slope
of the plot was the lowest value (Figure 2) and the fluorescent
quantum yield (Φfl) was lower than those of fluorophore 1-3.
These results suggest that bithiophene was an unfavorable
aromatic ring for the solvatochromic fluorophores. As men-
tioned above, the strategy for the improvement of solvatochro-
1
characterized by H, 13C NMR spectra, and FAB-HRMS data.
The synthetic pathways to fluorophores 1-4 are given in the SI.18
The photophysical properties of fluorophores 1-4 measured
in solvents with different polarities are summarized in Table 1
and SI,18 while Figure 1 shows the normalized absorption and
emission spectra in toluene. The excitation wavelengths of the
absorption spectra were found to be more favorable for
biological samples compared with Dapoxyl dye, because these
fluorophores would be excited efficiently using a common
405 nm blue diode laser. The absorption maxima -abs of 1-4
changed little, allowing improved ratiometric determination
using only a single beam. On the other hand, the emission
maxima -em of 1-4 exhibited large bathochromic shifts,
increasing solvent polarity. All fluorophores showed fluorescent
solvatochromism.
We also evaluated the effect of the aromatic heterocyclic
ring moiety on fluorescent solvatochromic properties.17 The plot
for 1 shows a good linear correlation with aprotic solvents. In
Chem. Lett. 2011, 40, 378-380
© 2011 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

380
mic fluorescent probes could be identified through a comparison
of fluorophores 1-4, containing different heterocyclic aromatic
rings.
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In conclusion, we have efficiently synthesized a series of
solvatochromic fluorophores 1-4 with different aromatic hetero-
cyclic rings via Suzuki-Miyaura cross-coupling reactions. The
effect of heteroatom, a ¢-substituent, and the number of rings in
the aromatic moiety were investigated systematically. Our future
research will focus on the modification of the structure of these
fluorophore, employing alternative electron-donating and -with-
drawing moieties, to obtain various photophysical properties.
5
6
7
8
9
This work was supported by a Grant-in-Aid for Knowledge
Cluster Phase II, Sapporo Bio-S and a Grant-in-Aid for
Scientific Research on Priority Areas from The Ministry of
Education, Culture, Sports, Science and Technology of Japan
and a Grant-in-Aid for Frontier Technology Research from
Northern Advancement Center for Science and Technology
(NOASTEC) of Japan.
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Chem. Lett. 2011, 40, 378-380
© 2011 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/