Design and syntheses of highly emissive aminobenzopyrano-xanthene dyes in the visible and far-red regions

Article abstract of DOI:10.1021/ol403262x

New derivatives of aminobenzopyrano-xanthene (ABPX) dyes have been designed and synthesized with high fluorescence quantum yields in the visible and far-red regions. It was kinetically demonstrated that the structurally rigid conjugation of the xanthene moiety, which is closely related to the reduction of the nonradiative deactivation process, is an effective molecular design for the drastic enhancement of fluorescence emission efficiency.

Full text of DOI:10.1021/ol403262x

Letter
pubs.acs.org/OrgLett
Design and Syntheses of Highly Emissive Aminobenzopyrano-
xanthene Dyes in the Visible and Far-Red Regions
Shinichiro Kamino,*,†,‡ Miho Murakami,‡ Masaru Tanioka,‡ Yoshinao Shirasaki,‡ Keiko Watanabe,†,‡
Jun Horigome,§ Yousuke Ooyama,∥ and Shuichi Enomoto*,†,‡
†Next-generation Imaging Team, RIKEN-CLST, Kobe-shi, Hyogo 650-0047, Japan
‡Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, Okayama-shi, Okayama 700-8530, Japan
§Hitachi High-Tech Science Co., Ltd., Hitachinaka-shi, Ibaraki 312-8504, Japan
∥Department of Applied Chemistry, Graduate School of Engineering, Hiroshima University, Higashi-Hiroshima 739-8527, Japan
S
* Supporting Information
ABSTRACT: New derivatives of aminobenzopyrano-xan-
thene (ABPX) dyes have been designed and synthesized
with high fluorescence quantum yields in the visible and far-
red regions. It was kinetically demonstrated that the
structurally rigid conjugation of the xanthene moiety, which
is closely related to the reduction of the nonradiative
deactivation process, is an effective molecular design for the
drastic enhancement of fluorescence emission efficiency.
arge planar π-conjugated fluorescent organic dyes, such as
1
2
3
1c,4
Lxanthenes, acenes, boron-dipyromethenes, perylenes,
and others,5 have received a great deal of attention because of
their potential applications as fluorescent probes for life
sciences, photoactive medicines, photonics, and optoelec-
tronics. The advantages of large planar π-conjugated organic
molecules over linearly bonded fluorescent molecules such as
cyanine dyes tend to include a high fluorescence quantum yield
(ϕfl) and a high molar extinction coefficient (ε).6 On the other
hand, the main drawbacks of large planar π-conjugated organic
molecules are generally their low solubility in conventional
polar or nonpolar solvents and concentration-dependent
quenching (CQ) because of attractive dipole−dipole inter-
actions and/or effective intermolecular aromatic interactions,
which rule out their use in solution.7 Therefore, large π-
conjugated organic molecules possessing high solubility in
various solvents and desirable optical characteristics are
continually required and their development has led to the
rapid proliferation of advanced fluorescence-based techniques,
the biggest growth areas being biology, medicine, chemistry,
and material science.
Figure 1. Chemical structure of aminobenzopyrano-xanthene (ABPX)
dye. ABPX01H22+ 1. The cis- and trans-stereoisomers of the dicationic
form are described for ABPX01 (1a and 1b), respectively.
low in various solvents. To achieve good performance in
modern bioimaging techniques and solution-processable device
applications, a high fluorescence quantum yield and high
solubility in various media are desired. Therefore, the
development of new ABPX derivatives with high fluorescence
emission efficiencies is the task at hand. Herein, we report the
design, syntheses, and fluorescence properties of new ABPX
dyes having a rigid structural conformation to increase ϕfl.
We speculated that a possible rationale for the reduction of
ϕfl of 1 is that energy is lost from the excited state via thermal
pathways involving the rotation of the aromatic substituents.
The intramolecular mobilities of fluorophores, such as
molecular vibration and rotation, are intimately related to the
kinetic balance between fluorescence emission and nonradiative
deactivation processes. It is a well-established design concept
that the rigid conjugation of the π-conjugated skeleton by the
incorporation of ring-fused structure enhances ϕfl of mole-
cules.7,11 Therefore, the restriction of the intramolecular
mobility (RIM) of the xanthene moiety of 1 is a design
Aminobenzopyrano-xanthene (ABPX) dyes were newly
developed as one of the alternative π-conjugated fluorescent
organic dyes8 because ABPX01 1, the first ABPX dye to be
developed as shown in Figure 1, is very soluble in a wide array
of solvents and emits far-red fluorescence in a wide
concentration range (<ca. 10−3 M), whereas conventional
rhodamine dyes cannot practically emit fluorescence emission
due to the CQ effect.9 The exceptional properties of ABPX
dyes should make them good candidates for optical
microscopy, biological imaging, and sensing.10 However, the
fluorescence quantum yield of alkyl-substituted 1 is significantly
Received: November 12, 2013
Published: December 10, 2013
© 2013 American Chemical Society
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Letter
Scheme 1. Syntheses of ABPX Dyes
strategy. As RIM creates efficient nonradiative deactivation
pathways for the decay of the excited states, modification of the
xanthene moiety such that these intramolecular mobilities are
restricted could increase the fluorescence emission intensity.
Then, we focused on the structurally rigid conjugations around
the diethylamino chains of 1. Single bonds connected to the
diethylamino group of ABPX01 freely rotate in solution. Hence,
restriction of the free rotation of the diethylamino chains would
lead to further enhancement of the fluorescence emission
intensity. Based on these hypotheses, we designed and
synthesized new ABPX derivatives in which the amino groups
are incorporated into rigid six-membered rings. Then, the
structure−fluorescence emission relationships are discussed.
The synthetic routes of ABPX101−103 are outlined in
Scheme 1. Alkylation of 3-methoxy-N-methylaniline 2 using 1-
chloro-3-methyl-2-butene in the presence of K2CO3 gave 3-
methoxy-(3-methyl-2-butenyl)aniline 4 in 54% yield. Cycliza-
tion of compound 4 using neat MeSO3H at 95 °C gave
regioselective quinoline derivatives 5 in 38% yield and 11 in
15% yield. Demethylation of 5 and 11 using aqueous HBr in
CH3COOH gave 6 and 12, respectively. New benzophenone
derivative 8-(2-carboxymethylbenzoyl)-7-hydroxy-1,1-dimethyl-
N-methylquinoline 7 or 8-(2-carboxybenzoyl)-9-hydroxy-1,1-
dimethyl-N-methylquinoline 13 was then synthesized by the
acylation of 6 or 12 with phthalic anhydride. 9-(2-Carboxy-
benzoyl)-8-hydroxyjulolidine 18 was similarly synthesized by
the acylation of 8-hydroxjulolidine 17 with phthalic anhydride
according to the literature.12 ABPX101, ABPX102, and
ABPX103 were prepared by reacting the corresponding
benzophenone derivatives with resorcinol in CH3SO3H,
followed by simple purification by silica gel normal phase
chromatography via conversion into the spirolactone form. The
cis- and trans-stereoisomers of the spirolactone form were
obtained for ABPX101 (8a and 8b), 102 (14a and 14b), and
103 (19a and 19b), respectively.
To identify the relationships among chemical species, color,
and fluorescence emission in solution, the absorption spectra
and the fluorescence emission spectra of 5 μM ABPX dyes were
measured in THF and CHCl3 with various volume fractions of
trifluoroacetic acid (TFA), as shown in Figures S1 and S2. The
absorption spectra of the nonfluorescent spirolactone forms of
8, 14, and 19 showed peaks in the ultraviolet region. Chemical
species transformation and spectral red shifts derived from the
stepwise protonation of the xanthene moiety were observed in
the visible region as the volume fraction of TFA was increased.
The absorption bands of the nonfluorescent monocationic
forms of ABPX101H+ 9, ABPX102H+ 15, and ABPX103H+ 20
appeared in the visible wavelength region. The absorption
2+
bands of the dicationic forms of ABPX101H2
10,
2+
2+
ABPX102H2 16, and ABPX103H2 21 as shown in Figure
2 appeared in the long wavelength region. In all solvents, the
absorption spectra of 10, 16, and 21 commonly exhibited
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Letter
by excitation at 366 nm using reference RB dye in ethanol (ϕfl
= 0.73). The relative fluorescence quantum yields of 10 (10a,
ϕfl = 0.70; 10b, ϕfl = 0.69), 16 (16a, ϕfl = 0.50; 16b, ϕfl = 0.49),
and 21 (21a, ϕfl = 0.55; 21b, ϕfl = 0.54) were drastically
increased relative to that of 1 (1a, ϕfl = 0.17; 1b, ϕfl = 0.16).
The excellent ϕfl of 10 was observed in the solvents examined
and was comparable to that of highly fluorescence emitting RB
(ϕfl = 0.71). All ABPX derivatives commonly exhibited well-
developed bimodal fluorescence emission spectra over a wide
wavelength region that included the visible and far-red regions
and a significant spectral red shift relative to the spectrum of
RB. The fluorescence emission spectra of 16a (λfl0−0 = 640 nm,
λfl0−1 = 693 nm) and 21a (λfl0−0 = 635 nm, λfl0−1 = 683 nm)
Figure 2. Chemical structures of dicationic forms of ABPX dyes. The
cis- and trans-stereoisomers of the dicationic form are described for
ABPX101 (10a and 10b), 102 (16a and 16b), and 103 (21a and 21b),
respectively.
were red-shifted relative to that of 1 (λfl0−0 = 622 nm, λfl0−1
=
671 nm). In contrast, the fluorescence emission spectrum of
10a (λfl0−0 = 612 nm, λfl0−1 = 658 nm) was blue-shifted. The
results are summarized in Table 1 (see Table S1 for trans-ABPX
vibronic bands. The longest-wavelength band was attributed to
the 0−0 transition, and the second-longest band, to the 0−1
transition. The profiles of the absorption spectra and
fluorescence emission spectra were almost symmetric to each
other. Based on the spectral studies, we concluded that the
fluorescent dicationic form is appropriate for investigations of
the optical properties in solution. The protolytic equilibria of
these ABPX dyes are summarized in Scheme S1. The profiles of
the absorption spectra and fluorescence emission spectra of 5
μM cis-ABPX dyes of 10a, 16a, 21a, and rhodamineB (RB) in
CHCl3 containing 2.5% TFA are shown in Figure 3, and a
solution of them exhibits a vivid absorption color and bright
fluorescence emission.
Table 1. Optical Properties of cis-ABPX Dyes and RB Dye in
Chloroform Containing 2.5% TFA
λabs0−0
[nm]
λfl0−0
[nm]
λabs0−1
[nm]
λfl0−1
ε0−0
a
dye
[nm] [M−1 cm−1
]
Φfl*
ABPX101
590
617
611
612
640
635
545
567
562
658
693
683
130 300
108 200
117 000
0.70
0.50
0.55
10a
ABPX102
16a
ABPX103
21a
ABPX01 1a
598
552
622
576
553
671
128 650
129 000
0.17
0.71
RB
−
−
a
The fluorescence quantum yields of the ABPX dyes were determined
10a, 16a, and 21a were well dissolved and exhibited strong
fluorescence in CHCl3 containing 2.5% TFA. Subsequently, the
optical properties of 1 μM ABPX derivatives were closely
investigated in CHCl3 containing 2.5% TFA. The relative
fluorescence quantum yields of these derivatives were measured
by using the reference standard dye RB (ϕfl = 0.73 in ethanol).
of 10b, 16b, and 21b). 10, 16, and 21, which have almost the
same absorption/fluorescence emission maxima in CHCl3,
exhibited high ϕfl values in various solvents (see Figure S3 and
Table S2). Both absorption and fluorescence emission spectra
were almost unaffected by the change of polarity of the solvents
(see Figure S4). Such high quantum yields in various solvents
underscore the potential of ABPX dyes for highly sensitive and
high-resolution analyses.
A drastic improvement in ϕfl was achieved by simple
modification of ABPX01. However, no marked differences in
the molar extinction coefficient (ε) were noted among the
ABPX derivatives in Table 1. The rate constant of radiative
transition (kf) is proportional to ε of the ABPX derivatives. The
results suggested that the kf values of the respective ABPX
derivatives showed little differences. To further verify the
hypotheses that the structurally rigid conjugations around the
nitrogen atoms of the xanthene moiety, which enhanced the
fluorescence emission efficiency, are related to the reduction of
the nonradiative deactivation process, the fluorescence lifetimes
(τobs) of 10a, 16a, and 21a were measured in CHCl3
(containing 2.5% TFA), and kf and the rate constant of
nonradiative transition (knr) were calculated as shown in Table
2 (see Table S3 for 10b, 16b, and 21b). The fluorescence
lifetimes of all the ABPX dyes showed a monoexponential
decay (τobs = 0.72−2.48 ns) in Figures S5−S6. The modified
ABPX dyes exhibited longer fluorescence lifetimes than 1. The
knr values of 10a, 16a, and 21a were much lower than that of 1a
although little difference in kf was seen among the ABPX
derivatives. As we had expected, the enhancement of ϕfl of 10a,
16a, and 21a was dependent on the marked decrease in knr.
Figure 3. (a) Absorption and (b) fluorescence emission spectra of cis-
ABPX dyes and RB in chloroform containing 2.5% TFA (black, RB;
red, ABPX101 10a; orange, ABPX01 1a; blue, ABPX103 21a; green,
ABPX102 16a). Iex = 365 nm. (c) Absorption (upper row) and
fluorescence emission (bottom row) colors of RB and cis-ABPX dyes
in chloroform containing 2.5% TFA.
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Letter
thank Ms. Ayako Tohzaka of Okayama University for fruitful
discussions.
Table 2. Fluorescence Quantum Yields, Lifetimes, and
Kinetic Constants of cis-ABPX Dyes
dye
ϕfl
τobs [ns]
kf [ns−1
]
knr [ns−1
]
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0.72
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2.41
0.24
0.30
0.20
0.23
1.06
0.30
0.32
0.32
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ASSOCIATED CONTENT
* Supporting Information
Experimental procedures, spectrofluorometric and -photo-
metric spectra, and additional photophysical data for ABPX
dyes. This material is available free of charge via the Internet at
http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Authors
■
*E-mail: skamino@riken.jp.
*E-mail: senomoto@pharm.okayama-u.ac.jp.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
(12) Sauers, R. R.; Husain, S. N.; Piechowski, A. P.; Bird, G. R. Dyes
Pigment. 1987, 7, 35.
This work was supported by the Adaptable and Seamless
Technology Transfer Program through Target-driven R&D
(Grant AS242Z01483M) of Japan Science and Technology
(JST). This research was also supported in part by a Grant-in-
Aid for Challenging Exploratory Research (Grant 24659019)
from the Ministry of Education, Culture, Sports, Science and
Technology (MEXT) of the Japan Society for the Promotion of
Science (JSPS). We thank Dr. Hiroyuki Koshino of RIKEN for
NMR measurement and Dr. Yayoi Hongo and Dr. Takemichi
Nakamura of RIKEN for MS measurement. We would also like
to thank Dr. Toshihiro Shirasaki of Hitachi High-Tech Science
Co., Ltd. for support on fluorescence emission analysis. We also
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