Flapping viscosity probe that shows polarity-independent ratiometric fluorescence

Article abstract of DOI:10.1039/c7tc01533j

A variety of fluorescent molecular viscosity probes have been widely used for mapping the local viscosity in cells and for monitoring the microenvironments in materials. However, their viscosity-sensing structural design still relies strongly on molecular

Full text of DOI:10.1039/c7tc01533j

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Materials Chemistry C
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PAPER
Flapping viscosity probe that shows
polarity-independent ratiometric fluorescence†
Cite this:DOI: 10.1039/c7tc01533j
a
b
c
a
Ryota Kotani,
Yumi Nakaike,
Hikaru Sotome,
Hajime Okajima,
Soichi Yokoyama,
a
d
d
d
Akihiro Kashiwagi, Chigusa Mori, Yuki Nakada,
d
a
c
Shigehiro Yamaguchi,
Hiroshi Miyasaka * and Shohei Saito
Atsuhiro Osuka,
Akira Sakamoto,
*
b
ae
*
A variety of fluorescent molecular viscosity probes have been widely used for mapping the local viscosity
in cells and for monitoring the microenvironments in materials. However, their viscosity-sensing structural
design still relies strongly on molecular rotors featuring intramolecular rotational dynamics. Here we report
flapping molecules (FLAP) as a ratiometric viscosity-sensing fluorophore that shows polarity-independent
dual fluorescence. Viscosity-sensing mechanism is based on a unique V-shaped-to-planar conformational
Received 10th April 2017,
Accepted 10th May 2017
1
change in the singlet excited state (S ), in which the flexible motion of an eight-membered ring plays an
important role. Fast conformational dynamics have been studied by time-resolved spectroscopies, and the
viscochromic properties have been quantitatively analyzed. Application of FLAP to monitoring the curing
process of epoxy resins has also been demonstrated, in which other typical environment-sensitive dyes
did not work as a local viscosity probe.
DOI: 10.1039/c7tc01533j
rsc.li/materials-c
intensity of fluorogenic molecular rotors increases. These fluoro-
phores have been applied to the study of microenvironments in
Introduction
2,4
5
Viscosity imaging with designed fluorescent probes is a useful polymeric materials and cellular organelles. Later, quantitative
technique because it enables mapping of the local viscosity of measurement of the local viscosity was developed with the ratio-
1
,2
1,6–8
heterogeneous media. Although the local viscosity estimated metric FL technique,
in which the local viscosity can be
by using the molecular probes does not simply correspond to estimated based on the intensity ratio of two FL bands at different
the bulk viscosity measured by viscometers and rheometers, wavelengths. Recently, the FL lifetime imaging microscopy (FLIM)
these properties are significantly related in homogeneous technique has also been an important alternative to quantitative
1
,3
solvents (particularly in Newtonian fluids). Mapping spatial viscosity analysis, in which the viscosity-dependent FL lifetime of
heterogeneity is an important advantage for the molecular probe molecules is measured for mapping the heterogeneity in
8a,b,9
viscosity probe compared with those instruments.
cells and materials.
With these techniques, a variety of
Molecular rotors have long been recognized as representa- sophisticated molecular rotors that show viscosity-sensing FL with
1
–3
10
tive viscosity probes. Since the dynamic internal rotation in small polarity dependence are now applied to quantitative
6
S
1
is suppressed in highly viscous media, the fluorescence (FL) viscosity imaging, for example, CVJ (cyanovinyljulolidine)-based,
1g,8b,9
BODIPY (boron-dipyrromethene)-based,
and Cy (cyanine)-
probes (Fig. 1a). However, structural design of these
viscosity probes still relies on molecular rotors featuring the
intramolecular rotational dynamics in S (including a bond twist-
ing which leads to trans–cis photoisomerization).
8a,c,9d,e
a
based
Department of Chemistry, Graduate School of Science, Kyoto University,
Kitashirakawa Oiwake, Sakyo, Kyoto 606-8502, Japan.
E-mail: s_saito@kuchem.kyoto-u.ac.jp
1
b
c
11–13
Division of Chemistry, Graduate School of Engineering Science, Osaka University,
Toyonaka, Osaka 560-8531, Japan
Here we
report unique flapping fluorophores as a new series of viscosity
probes that show polarity-independent ratiometric FL properties.
Our molecular design is different from molecular rotors but
based on the hybridization of a flexible eight-membered ring
cyclooctatetraene, COT) and rigid fluorescent aromatic wings
Fig. 1b), which we have given the trivial name FLAP (flexible
Department of Chemistry and Biological Science, College of Science and Engineering,
Aoyama Gakuin University, 5-10-1, Fuchinobe, Chuo, Sagamihara,
Kanagawa 252-5258, Japan
d
e
Department of Chemistry, Graduate School of Science, Nagoya University, Furo,
Chikusa, Nagoya 464-8602, Japan
(
(
Japan Science and Technology Agency (JST), PRESTO, Kitashirakawa Oiwake,
Sakyo, Kyoto 606-8502, Japan
14
and aromatic photofunctional systems).
†
Electronic supplementary information (ESI) available: Experimental procedures,
FLAP takes a V-shaped conformation in the ground state,
while it is expected to undergo conformational relaxation in S
1
theoretical calculations, time-resolved spectroscopies are included. See DOI:
0.1039/c7tc01533j
1
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Subsequent deprotection of the TMS groups proceeded nicely
2
3
0
upon treatment with AgNO
to afford 5 in 98% yield. Precursor
6
with a fused COT structure was obtained in gram scale in 51%
1
8
yield from 5 using Ni catalyzed [2+2+2+2] cycloaddition and
subsequent DDQ oxidation. Tetraester 6 was converted to tetra-
4
aldehyde 9 by reduction with LiAlH and subsequent Swern
oxidation.
FLAP1, a standard model compound of the FLAP systems,
2
1
was obtained from 9 with an acene-elongation reaction
2
2
reported by Lin et al. To suppress strong aggregation, bulky
substituents were introduced at the terminal maleimide groups
in FLAP1. FLAP2, having a methyl group on the COT core, was
synthesized in order to confirm an impact of conformational
flexibility of the COT moiety on the FL properties. On the basis
1
8b
of the reported method,
subsequent methylation reaction were performed efficiently in
Fig. 1 Viscosity-sensing structural dynamics of (a) molecular rotors and 93% and 86% yields, respectively. Finally, hydrophilic FLAP3
mono-bromination of 6 and the
(
b) FLAP system. (c) Schematic energy diagram of FLAP.
bearing six triethylene glycol groups was synthesized by the
reaction of 9 with maleimide 12. FLAP3 was soluble in a mixed
solvent system of DMSO/glycerol with a wide range of composi-
tions, which have been commonly used media for quantitative
to form a planar conformation (Fig. 1c). A unique feature of
FLAP is possible dual FL from V-shaped and planar conformations.
In this study, the importance of the COT flexibility in the dual FL
mechanism has been demonstrated experimentally, and the photo-
excited state dynamics of FLAP have been studied by time-resolved
FL/IR spectroscopies as well as time-dependent density functional
theory (TD-DFT) calculations. Polarity-independent ratiometric FL
viscochromism of FLAP shows its efficacy in quantitative micro-
viscosity analysis, and application to monitoring epoxy resin curing
has also been demonstrated in comparison with other photo-
responsive probes such as TICT (twisted intramolecular charge
23
evaluation of viscosity-independent properties.
Preliminary X-ray crystal structure determined that FLAP2
had V-shaped geometry (Scheme 1, inset). The two anthrace-
neimide moieties were connected with two cis-olefins to form
the tub-shaped COT ring. The torsion angle y, defined by the
neighboring carbon atoms C1, C2, C3, and C4 of the COT ring
(Fig. 2, inset), was ca. 61 and 651, in which the presence of
the methyl group on the C3 or C8 carbon atom of the COT ring
was crystallographically disordered. These torsion angles are
comparable to those of the previously reported FLAP series
1
5
transfer)-type and ESIPT (excited-state intramolecular proton
transfer)-type dyes.
14,22
(y = 51–641).
1
6
Steady-state fluorescence
Reflecting the structural similarity, the absorption spectra of
FLAP1 and FLAP2 are almost the same, but the FL properties
are remarkably different (Fig. 3). FLAP1 showed intense green
Results and discussion
Synthesis
Scheme 1 outlines the synthetic route of a series of FLAP. This FL in tetrahydrofuran (THF) with peaks at 520, 561, and 607 nm
1
4
scheme was modified from the previously reported synthesis.
A large-scale synthesis was prevented in the previous protocol line). The large Stokes shift (4580 cm ) of FLAP1 suggested a
because it included a photochemical rearrangement of 9,10- large structural change in S . Notably, a weak broad emission
f
and fluorescence quantum yield (F ) of 0.34 (Fig. 3b, green
ꢀ
1
1
17
dihydro-9,10-ethenoanthracene to form a central eight-membered band was also observed in the blue region (450–500 nm) of the
COT ring as a key step. In this photoreaction, the concentration of FL spectrum at room temperature. In the frozen glass of
substrate solution had to be kept considerably low for efficient UV 2-methyl tetrahydrofuran (MTHF) medium at 77 K, the green
excitation, which hampered large-scale preparation. Instead, here we emission band disappeared and the blue emission bands with
18
use Ni catalyzed [2+2+2+2] cycloaddition, reported by Wender & vibronic peaks at 437, 467, and 494 nm increased in intensity
Christie, to form a COT core.
Precursor 4 (Scheme 1) was obtained in decagram scale by
Diels–Alder reaction of 1,3-butadiene and dimethyl acetylene- 440 and 464 nm and F
(Fig. 3c).
In contrast, FLAP2 exhibited blue FL in THF with peaks at
= 0.28 (Fig. 3b, red line). The Stokes
f
ꢀ
1
dicarboxylate, DDQ oxidation, and Wohl–Ziegler bromination. shift was 1200 cm , much smaller than that of FLAP1, which
Introduction of TMS-ethynyl groups into 4 could not be achieved indicated that the structural change of FLAP in S was largely
1
1
8
with TMS-ethynyl Grignard reagent and CuI. We presumed suppressed by introduction of only a methyl group onto the
that the failure was due to undesirable side reactions at the ester COT ring, and that the conformational flexibility of the COT
groups, and thus attempted reaction of 4 with TMS-acetylene ring was key in determining the dynamics of FLAP systems. At
1
9
4 2 3
under milder reaction conditions (CuI, n-Bu NI and Cs CO ),
room temperature in MTHF, FLAP2 showed similar absorption
which led to successful TMS-ethynylation in 71% yield. and FL spectra to those taken in THF. In MTHF matrix at 77 K,
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Scheme 1 Modified synthesis of a FLAP series. Reagents and conditions: (a) dimethyl acetylenedicarboxylate (1.0 equiv.), 2,3-dimethyl-1,3-butadiene
1.0 equiv.), toluene, 70 1C, 18 h; then DDQ (1.1 equiv.), toluene, 70 1C, 6 h, 98%; (b) NBS (2.2 equiv.), BPO (5 mol%), a,a,a-trifluorotoluene, 100 1C,
0 h, 78%; (c) CuI (2.0 equiv.), n-Bu NI (2.0 equiv.), Cs CO (2.1 equiv.), TMS acetylene (5.0 equiv.), MeCN, 50 1C, 20 h, 71%; (d) AgNO (10 equiv.), CH Cl
acetone/H O, RT, 1 h; then conc. HCl aq. (excess), RT, 1 h, 98%; (e) Zn (50 mol%), NiBr (dme) (25 mol%), THF/H O, 60 1C, 2 h; then DDQ (4.2 equiv.),
toluene, RT, 30 min, 51%; (f) Br (1.1 equiv.), CH Cl , 40 1C, 2 h; then DBU (10 equiv.), benzene, 80 1C, 1 h, 93%; (g) MeB(OH) (4.0 equiv.), K PO
(40 mol%), Pd(OAc) (10 mol%), THF, reflux, 24 h, 86%; (h) LiAlH (4.5 equiv.), THF, 60 1C, 4 h, 80%; (i) (COCl) (4.4 equiv.), DMSO
, ꢀ78 1C, 6 h; then NEt (35 equiv.), 0 1C, 2 h, 63%; (j) maleimide 11 (2.4 equiv.), PBu (2.6 equiv.), DBU (0.20 equiv.), 1,2-dichloroethane,
0 1C, 40 h, 29% for FLAP1 and 39% for FLAP2; (k) maleimide 12 (2.4 equiv.), PBu (2.4 equiv.), DBU (0.10 equiv.), 1,2-dichloroethane, 80 1C, 3 h, 13%.
(
1
4
2
3
3
2
2
/
2
2
2
2
2
2
2
3
4
(
4.0 equiv.), PPh
8.8 equiv.), CH Cl
3
2
4
2
(
2
2
3
3
8
3
DDQ = 2,3-dichloro-5,6-dicyano-1,4-benzoquinone, NBS = N-bromosuccinimide, BPO = benzoyl peroxide, TMS = trimethylsilyl, THF = tetrahydro-
furan, dme = 1,2-dimethoxyethane, DBU = 1,8-diazabicyclo[5.4.0]undec-7-ene, DMSO = dimethyl sulfoxide.
0
however, the blue FL bands of FLAP2 became sharper, and the (Fig. S5 and S6, ESI†). The S energy profile was delineated
maximum wavelengths were further blue-shifted to 435, 465, using constrained DFT structural optimization with fixed COT
and 492 nm (Fig. 3d). This vibronic structure agreed well with dihedral angle y (Fig. S7, ESI†). The S energy became higher as
0
that of FLAP1 in the frozen medium, indicating that the structural the molecular shape came close to the flat form. Inversion
change in S
1
0
of FLAP2 was completely suppressed in the frozen behavior in S between V- and L-shaped conformers was
medium, and the vibronic levels of the most stable S
0
geometry suggested for both FLAP1 and FLAP2.
were reflected in the FL spectra at 77 K (see Fig. S1–S4, ESI† for
1
The S energy profile was also described using TD-DFT
further information of the steady-state fluorescence).
constrained optimization with fixed y from 01 to 801 at 51
0
intervals (Fig. 4). The S
1
profile of FLAP1 suggested that a
Calculated energy profile
shallow V-shaped structure and a planar one corresponded to
Calculated energy profiles explained well the distinct FL proper- different energy minima, and there was a small energy barrier
ties of FLAP1 and FLAP2. The corresponding FLAP structures between them. These energy-minimal structures were fully
0
0
FLAP1 and FLAP2 were used for the calculations, in which the optimized as a shallow V-shaped conformation with y = 54.51
terminal bulky substituents were replaced by hydrogen atoms and the planar conformation with y = 01, and it is suggested
(
Fig. 2). The structural optimizations were performed using that the planar form was slightly more stable (Fig. 4a).
DFT for S and TD-DFT for S at the CAM-B3LYP/6-31+G(d) On the basis of the calculated S profile, the observed intense
level. The most stable geometries in S
0
1
1
2
4
0
were obtained with green FL at 520 nm of FLAP1 was assigned to emission from the
0
the V-shaped conformation (y = 60.01 for FLAP1 and y = 66.41 planar form, while the weak FL band observed in the blue region
for FLAP2 ), and the structural features were largely consistent (450–500 nm) was assigned to emission from the shallow
0
with the X-ray crystal structures. In the optimized structures, V-shaped form, respectively (Fig. 2). In the shallow V-shaped
owing to the bent conformation of the COT ring, the bond form, the electronic configuration arising from HOMO - LUMO
1
alternation around the cis-olefins was explicit and therefore the transition was predominant in the S electronic structure. These
two anthraceneimide moieties were not efficiently p conjugated related orbitals were located on the anthraceneimide moieties
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Fig. 2 Excited-state dynamics of FLAP1. The structural optimization of
0
FLAP1 at the energy minimum points (a V-shaped form in S
0
, a shallow
) was performed using DFT for
at the (CAM-)B3LYP/6-31+G(d) level.
V-shaped form in S
1 1
, and a planar form in S
S
0
and TD-DFT for S
1
without effective p conjugation through the COT joint (Fig. S8,
ESI†). On approaching the flat conformation, p-orbital inter-
action on the COT ring became more significant, inducing a
configurational change of S
FL. According to previous theoretical studies, the emissive planar
form (y = 01) in S has a trapezoid structure with C2v symmetry
Fig. S9, ESI†) rather than a rectangle structure with D2h symmetry
1
that was responsible for the green
1
Fig. 3 (a) Fluorescence of FLAP1 and FLAP2 in THF under 365 nm UV
lamp irradiation. Ar = 2,6-diisopropylphenyl. (b) Absorption and FL spectra
(
1
4b,25
(Fig. S10, ESI†).
The S
1
electronic structure of the C2v planar of FLAP1 and FLAP2 in THF at room temperature. (c) Continuous change
of the FL spectrum of FLAP1 in MTHF during temperature decrease from
form had contributions from the HOMO - LUMO, HOMOꢀ1 -
LUMO, HOMO - LUMO+1 configurations, in which molecular
orbitals delocalized over the entire p-conjugated frame were
1
53 K to 77 K, and (d) that of FLAP2. Freezing point of MTHF is 137 K.
involved. The broad FL band observed in the blue region whose large Stokes shift has been explained by conforma-
(
1
450–500 nm) suggested the presence of multiple emissive tional planarization in S originating from the excited-state 8p
2
8
points around the shallow V-shaped form. The observed clear aromaticity.
vibronic bands in the green FL indicated that the vertical S
point of the green-emissive planar form was located near a local
0
Time-resolved spectroscopy
energy minimum in the S energy surface (Fig. 2).
To directly elucidate the conformational dynamics of FLAP in
the excited state, we employed time-resolved FL and IR spectro-
0
0
In sharp contrast, the calculated S energy profile of FLAP2
1
afforded a single minimum with y = 66.61 (Fig. 4b and Fig. S11, scopies. Fig. 5 shows time-resolved FL spectra of FLAP1 in
S12, ESI†). The energy became increasingly higher upon DMSO solution excited with a 400 nm laser pulse. A broad
decreasing y value in S
the COT ring gave rise to steric hindrance for the conforma- after the excitation. Since this broad emission appeared within
tional planarization in S (as well as in S ). The observed blue the response time of the apparatus (ca. 40 ps), this emission
FL of FLAP2 with smaller Stokes shift thus can be assigned to band was attributed to the shallow V-shaped form. With an
emission from the V-shaped structure. increase of the delay time after the excitation, this broad band
It should be noted that the photochemical reactions gradually decreased with t = 550 ps, followed by the appearance
1
, suggesting that the methyl group on emission band was observed around 450–500 nm immediately
1
0
2
6
reported for several COT derivatives were not expected here, of prominent bands around 530 and 570 nm. Then, the FL
because the most probable photoinduced events of FLAP would bands at 530 and 570 nm decayed with t = 12.5 ns.
be conformational changes rather than chemical reactions. The FL
behavior of FLAP is rather similar to that of dibenzo[b, f ]oxepin,
The weak FL intensity around 450–500 nm still remained
during the decay of the strong band at ca. 530 and 570 nm.
27
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0 0
1
S energy profile of (a) FLAP1 and (b) FLAP2 . The constrained
Fig. 4
structural optimizations in S
B3LYP/6-31+G(d) level.
1
were performed using TD-DFT at the CAM-
Fig. 6 (a) Time-resolved IR spectra of FLAP1 in DMSO-d
6
, excited at
330 nm. (b) Calculated IR absorption bands of the shallow V-shaped form
at the local minimum in S
1
(red bars), the planar form at the global
minimum in S
bars) with a scaling factor of 0.98.
1
(blue bars), and the most stable V-shaped form in S (black
0
ꢀ
1
process. The IR band at 1358 cm remained even after 1500 ps,
at which time the conformational planarization in S must be
1
complete according to the time-resolved FL spectroscopy, and
therefore this band was assigned to the planar form at the global
minimum in S . In comparison with the calculated IR absorption
1
spectra of the respective optimized structures of FLAP1 including
bulky terminal substituents at the B3LYP/6-31G(d) level (Fig. 6b),
these assignments were well supported; the observed bands at
Fig. 5 Time-resolved FL spectra of FLAP1 in DMSO, excited at 400 nm.
a) 50 ps–2 ns and (b) 2–50 ns.
ꢀ1
1353, 1358, and 1380 cm correspond to the n227 of the shallow
(
V-shaped form in S , the n of the planer C2v trapezoid form in
1
227
1 0
S , and the n229 of the V-shaped form in S , respectively (Fig. 6b).
All of them are C–N stretching vibrations between the terminal
bulky substituents and the terminal imide moieties (Fig. S15,
Coexistence of these bands in the decay process indicated that
the planar form was in equilibrium with the shallow V-shaped
form. The presence of the multi-emissive points around the
1
ESI†). Moreover, the calculated spectrum of S (Fig. 6b) showed
shallow V-shaped form in S
the analysis of the FL time profiles in the wavelength range of
1
has been confirmed on the basis of
good agreements with the transient IR bands observed at
ꢀ
1
2.5–100 ps in the 1260–1330 cm
region attributed to the
430–680 nm (Fig. S13, ESI†).
shallow V-shaped form in S . As a consequence of these time-
1
Time-resolved IR spectroscopy of FLAP1 provided results
resolved spectroscopies, the excited-state dynamics of FLAP1 in
consistent with the other analyses. In the wavenumber range
Fig. 2 were directly elucidated.
ꢀ
1
of 1300–1400 cm , well-resolved IR bands for each species
were observed (Fig. 6a). Immediately after the excitation
ꢀ
1
Polarity-independent viscochromism
(
2.5 ps), the ground-state bleaching at 1380 cm was confirmed
ꢀ1
and, at the same time, the IR absorption band at 1353 cm
Viscochromism of the FLAP system has been studied using the
assignable to the shallow V-shaped form at the local minimum hydrophilic molecule FLAP3 because FLAP1 is not soluble in
in S was observed. commonly used viscous media such as a mixed solvent of
1
2
3
The IR band gradually shifted to the higher wavenumber DMSO/glycerol. This mixed solvent system is a well-studied
side with a time constant of B500 ps (Fig. S14, ESI†), which Newtonian fluid, whose viscosity is constant regardless of the
corresponds to the time constant measured by the time-resolved shear rate (namely, the rate of a rotating spindle on a visco-
FL spectroscopy and is much longer than the vibrational cooling meter). Polarity-independent FL properties were confirmed for
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FLAP3 as well as FLAP1. As shown in Fig. 7a, the peak viscosity were prepared. As the viscosity became higher, the
wavelength of the green emissive band showed a negligible relative intensity of the blue FL band (450–500 nm) gradually
shift within the 520–526 nm region in nonpolar to polar organic increased compared with the green FL band (525 nm). According
1
,3d
solvents such as toluene, tetrahydrofuran (THF), dichloro- to the F ¨o rster–Hoffmann equation,
methane (DCM), dimethyl formamide (DMF), acetonitrile sities at 461 nm and 525 nm (I461/I525) was plotted versus viscosity
MeCN), and dimethyl sulfoxide (DMSO). These solvents cover measured at 25 1C in a double logarithmic graph (Fig. 7c). A
a wide range of relative dielectric constants e from 2.4 to 46.7 at linear relationship with a slope of 0.91 was observed in the
room temperature, while the viscosity values are within a small viscosity range from 2.2 to 100 cP. In comparison with other
the ratio of the FL inten-
(
r
6
–8
range of 0.4–2.2 cP. In spite of the wide range of relative reported viscosity probes,
the viscosity-sensitive range is
dielectric constants, the spectral features of FLAP3 were smaller, but the sensitivity in this range is remarkably high
well preserved. The polarity independence suggested that the (Fig. S17, ESI†), which may originate from the different struc-
emissive structures in S are not charge-polarized. The calculated tural motion and the bulkier hydrophilic substituents of FLAP3
1
ES
m
(excited-state dipole moments) and Dm (the difference compared with other molecular rotors. The polarity-independent
between excited state (ES) and ground state (GS) dipoles in the ratiometric viscochromism of the FLAP fluorophore enables
0
vertical geometry) of FLAP1 are 1.20 and 1.30 D for the planar real-time visualization of local viscosity change without sensing
C
2v trapezoid structure at the S
values are 6.46 and 1.10 D, respectively, for the shallow V-shaped
structure at the S local minimum (Fig. S16, ESI†). These Dm
values (1.30 and 1.10 D) are significantly small, compared with
the typical TICT systems having electron-donor and acceptor The efficacy of FLAP as a polarity-independent viscosity probe
1
global minimum, while these the local polarity change.
1
Monitoring epoxy resin curing
2
9
moieties.
was confirmed in the application to the real-time monitoring of
4
e,g
On the other hand, FLAP3 showed a remarkable viscosity epoxy resin curing.
Epoxy resin is a suitable platform for this
dependence in FL (Fig. 7b). Simply by changing the volume purpose because both the local viscosity and polarity increase
ratio of DMSO/glycerol, mixed solvents having a wide range of in the curing process. Reaction of epoxy groups with curing
agents leads to generation of a number of polar hydroxy groups,
while the microviscosity is expected to increase with the
decrease of free volume. First, we used a prepolymer of bisphenol
A diglycidyl ether E1, pentaerythritol tetrakis(2-mercaptoacetate)
H1, and n-Bu N as epoxy agent, hardener, and catalytic accelerator,
3
respectively (Fig. 8). This epoxy resin was cured in two steps: first,
the epoxy prepolymer E1 was mixed with the hardener H1, and
second, a catalytic amount of n-Bu N was added to accelerate the
3
cross-linking polymerization. In advance of the curing, a 0.1 wt%
ratio of FLAP1 was doped into E1. A fluid of E1 doped with FLAP1
showed green FL at 60 1C. After the addition of H1 to E1, the green
FL was still preserved at the same temperature, but the FL color
turned into blue after the addition of n-Bu N, indicating the
3
conformational planarization in S1 of FLAP1 was eventually
suppressed.
Fig. 7 Polarity-independent viscochromism of FLAP3. FL spectra (a) in
common organic solvents and (b) in mixed solvents of DMSO/glycerol.
Relative dielectric constants e
described as solvent indices. (c) F o¨ rster–Hoffmann equation plot. I461 and
525 describe the FL intensities at 461 and 525 nm, respectively.
r
and viscosity Z at room temperature are
Fig. 8 (a) Chemical structures of epoxy prepolymer E1 and thiol hardener
H1. (b) Monitoring epoxy resin curing by using FLAP1 as a molecular
viscosity probe.
I
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0
-(2 -hydroxyphenyl)benzoxazole ESIPT1 (Fig. 9a). These dyes are
2
12
13
known for the typical behaviors of TICT and ESIPT, respectively.
Since TICT1 has a more charge-polarized structure after the con-
formational change in S , the FL band with a large Stokes shift
1
increased after the addition of the thiol hardener H1 (Fig. 9c),
which demonstrated TICT1 did not work as a viscosity probe but
rather as a polarity probe in the first curing step. In the case of
ESIPT1, the FL band with a large Stokes shift was still observed
even after the epoxy curing was completed (Fig. 9d). This result
suggested that the structural change of the intramolecular proton
transfer was too small to be suppressed by the external environ-
mental change during the epoxy resin curing. These spectral
tendencies of TICT1 and ESIPT1 were distinct from FLAP1, and
the competence of the FLAP systems has been demonstrated as a
polarity-independent ratiometric fluorescent probe.
Conclusions
In conclusion, flapping fluorophores (FLAP) have been explored as
new viscosity probes with characteristic viscosity-sensing structural
design, which is different from molecular rotors. FLAP displays
polarity-independent ratiometric viscochromism as a single fluoro-
phore, since the relative FL ratio of the V-shaped conformer versus
the planar conformer depends on microviscosity and both the
species are not charge-polarized. Fast conformational relaxation
from the V-shaped to the planar conformation in the excited state
of FLAP1 has been revealed by time-resolved FL and IR analyses as
well as theoretical calculations. The observed small Stokes shifts of
FLAP2 bearing a methyl group at the COT core indicated that even
a small structural modification at the COT core has a large impact
on its fluorescence. Polarity-independent viscochromism of FLAP3
has been demonstrated in DMSO/glycerol mixed solvents, indicat-
ing that the flapping motion in the excited state does not involve an
intramolecular charge transfer process. Quantitative viscosity eval-
uation has been realized with high sensitivity in the viscosity range
of 2.2–100 cP. Finally, the competence of FLAP as a molecular
viscosity probe has been indicated by monitoring epoxy resin
curing with FLAP1. Improvement of the water solubility is currently
underway for biochemical applications of the FLAP series.
Fig. 9 (a) Chemical structures of the epoxy monomer E2 and photo-
responsive fluorophores TICT1 and ESIPT1. Spectral change in the FL of
(
b) FLAP1, (c) TICT1, and (d) ESIPT1 during the epoxy resin curing.
For quantitative evaluation, we selected ethylene glycol diglycidyl
ether E2 as epoxy monomer, whose viscosity is so low (20 cP) at 25 1C
that the spectroscopic monitoring can be performed at room
temperature (Fig. 9a). After the addition of H1, the I461/I525 ratio of
FLAP1 changed from 0.22 to 0.50 (Fig. 9b, left), which corresponds to
a local viscosity change from 20 to 49 cP according to the F ¨o rster–
Hoffmann plot obtained above (Fig. 7c). Further addition of the
n-Bu
process, the I461/I525 ratio increased steadily and saturated at 1.30
Fig. 9b, right). Although the estimated local viscosity was over the
sensing limit (2.2–100 cP), the spectral analysis revealed that the
curing process took 60 min after the addition of n-Bu N.
3
N accelerator induced a gradual spectral shift. In the curing
(
3
It should be noted here that, particularly in the case of non-
Newtonian fluids such as concentrated polymer solutions, the
local viscosity estimated by the chemical analysis must be
regarded as a different index from the macroscopic viscosity Author contributions
measured by a physical method using a viscometer with a
R. K., S. Yokoyama, Y. N., A. K., C. M. and Y. N. performed the
rotating spindle. The local viscosity, the so-called microviscosity,
depends on the degree to which the excited-state structural change
of photoresponsive fluorophores is suppressed. This should be
closely related to the remaining free volume in condensed medium
synthesis and the measurements of the steady-state absorption and
FL. H. S. and H. M. conducted the time-resolved fluorescence
spectroscopy. H. O. and A. S. performed the time-resolved IR spectro-
scopy. R. K., H. O., and S. S. performed the DFT and TD-DFT
calculations. R. K., H. S., H. O. A. O., A. S. and S. S. co-wrote the
manuscript, and S. Yamaguchi and H. M. proofread it. S. S. designed
the FLAP molecules and directed the research project.
2,4g
as well as the glass transition temperature.
On the other hand,
the macroscopic viscosity is defined from the physical aspect,
namely the resistance to deformation of medium when a shear
force is applied. The resistance mainly originates from the entan-
30
glement of polymer chains, which is a different measure from the
free volume remaining in the polymer matrix.
Acknowledgements
To compare the performance of FLAP1 with those of other
photoresponsive fluorophores, the same experiments were This work was supported by JST/PRESTO Grant Numbers
conducted using 4-dimethylaminobenzonitrile TICT1 and JPMJPR12K5 and JPMJPR16P6 to S. S., by JSPS KAKENHI Grant
This journal is ©The Royal Society of Chemistry 2017
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Numbers JP15H05482 and JP15H01083 to S. S., by JSPS
KAKENHI Grant Numbers JP26107010 and JP16H00849 to
A. S., and by JSPS KAKENHI Grant Number JP26107002 to
H. M. The authors acknowledge Prof. Hideki Yorimitsu (Kyoto
University) for the high resolution mass spectroscopy measure-
ments, and thank Prof. Yuki Kurashige (Kyoto University) and
Prof. Yoshikatsu Sato (ITbM, Nagoya University and ABiS) for
fruitful discussion on the theoretical analysis and viscosity
imaging, respectively.
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