Amphiphilic Fluorescence Resonance Energy-Transfer Dyes: Synthesis, Fluorescence, and Aggregation Behavior in Water

Article abstract of DOI:10.1002/chem.202000107

Amphiphilic pyrene/perylene bis-chromophore dyes were synthesized from unsymmetrically substituted perylene bisimide dyes, which were obtained through three synthetic methods. The optical and aggregation behaviors of these functional dyes were studied by means of UV/Vis absorption and fluorescence spectroscopy, dynamic light scattering, and TEM. These dyes are highly fluorescent and cover the whole visible-light region. A donor/acceptor dye displays intramolecular fluorescence resonance energy transfer (FRET), with a high efficiency of up to 96.4 percent from pyrene to perylene bisimide chromophores, which leads to a high fluorescence color sensitivity to environmental polarity. Under a λ=365 nm UV lamp, the light-emitting colors of the donor/acceptor dye change from green to yellow with increasing solvent polarity, which demonstrates application potential as a new class of FERT probes. The donor/acceptor dye in water was assembled into hollow vesicles with a narrow size distribution. The bilayer structure of the vesicular wall was directly observed by means of TEM. These vesicular aggregates in water are fluorescent at λ=650–850 nm within the near-infrared region.

Full text of DOI:10.1002/chem.202000107

A Journal of
Accepted Article
Title: Amphiphilic Fluorescence Resonance Energy Transfer Dyes:
Synthesis, Fluorescence and Aggregation Behaviors in Water
Authors: Shilei Dou, Ying Wang, and Xin Zhang
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Amphiphilic Fluorescence Resonance Energy Transfer Dyes:
Synthesis, Fluorescence and Aggregation Behaviors in Water
Shilei Dou, Ying Wang and Xin Zhang*
Abstract: Amphiphilic pyrene/perylene bischromophore dyes were
synthesized from unsymmetrically substituted perylene bisimide
dyes, which were obtained by three synthetic methods. The optical
behaviors and aggregation behaviors of these functional dyes were
studied by UV/vis absorption and fluorescence spectroscopy,
dynamic light scattering and transmission electron microscopy.
These dyes are highly fluorescent covering whole visible light region.
(p-phenylenevinylene) donors and acceptors connected by
supramolecular weak H-bonds [1a, 1b, 6, 9] or covalent bonds.[10]
Perylene bisimides dyes (PBIs), one class of rylene dyes
based on naphthalene units, possess outstanding optical
properties such as high extinction coefficients and absorption at
visible light region, strong electron-accepting ability,
fluorescence quantum yields close to unity, and excellent
chemical, photo-, thermal- stability.[11] Owing to these
outstanding optical properties, perylene bisimide dyes recently
have been extensively studied and their applications have been
expanded tremendously in the last 25 years from traditional
industrial dyes[12] into various new fields including optoelectronic
and n-type semiconductor materials,[13] photovoltaic devices[11]
and solar cells,[14] light-emitting diodes,[15] field effect
transistors[16] and near-infrared dyes.[17] More recently, owing to
the strong - stacking interactions, perylene bisimide dyes have
been regarded as versatile building blocks for various
supramolecular assemblies and aggregates in organic
solution,[17a, 18] even in water.[19]
A
donor/acceptor dye displays intramolecular fluorescence
resonance energy transfer with a high efficiency up to 96.4% from
pyrene to peylene bisimide chromophores, which leads to a high
fluorescence color sensitivity to environment polarity. Under 365 nm
UV lamp, the light-emitting colors of donor/acceptor dye change from
green to yellow with increasing solvent polarity, which demonstrates
application potentials as a new class of fluorescence resonance
energy transfer probes. The donor/acceptor dye in water was
assembled into hollow vesicles with narrow size distribution. The
bilayer structure of vesicular wall was directly observed by
transmission electron microscopy. These vesicular aggregates in
water are fluorescent at 650-850 nm within near-infrared region.
In our previous work, we have reported on precise control
of dye aggregate morphologies including micelles, rods and
vesicles in water by tuning molecular curvatures.[21]
Subsequently, we further developed water-soluble, white-light
emitting intermolecular energy transfer system, where bispyrene
donors were encapsulated in acceptor vesicles, and energy
transfer efficiency is up to 74%.[7a] Towards the purpose of
creating an intramolecular energy transfer system, here, we
synthesized a new amphiphilic energy transfer perylene bisimide
dye where the acceptor and donor are covalently linked together
in a single molecule (Figure 1). The optical properties and
aggregation behaviors of the new intramolecular energy transfer
dye were investigated in water.
Introduction
Fluorescence (Förster) resonance energy transfer (FRET) is a
phenomenon of radiationless energy transfer occurring between
two chromophores, specifically, a donor chromophore transfer
energy to an acceptor chromophore through nonradiative
dipole–dipole coupling when the donor is photo-excited.[1]
Fluorescence resonance energy transfer is involved in
photosynthesis system in nature.[2] Recently, it has attracted
much attention due to diverse applications such as artificial
photosynthetic light harvesting systems, bioimaging, biosensors
and fluorescence probes,[3-5] and white light-emitting materials.[6.
7a]
Generally, the energy transfer efficiency mainly depends on
Results and Discussion
three factors:[4b,c] 1) donor-to-acceptor distance, 2) the spectral
overlap of the acceptor absorption and the donor emission
spectra,[7] and 3) the chromophore orientation of the donors and
the acceptors. The distance dependence of the energy transfer
efficiency has been frequently used in chemical, biological and
8]
biophysical fields as spectroscopic ruler[4b,
to measure the
biomacromolecular distance generally in the range from 1 to 10
nm. Encouraged by these unique emission characteristics,
chemists have designed and synthesized various functional dye
molecules, and prepared their supramolecular assemblies to
construct fluorescence resonance energy transfer systems.
Recently, Ajayaghosh and coworkers have systemically studied
highly efficient energy transfer systems constructed from oligo
S. Dou, Y. Wang, Prof. Dr. X. Zhang
School of Chemical Engineering and Technology, Collaborative Innovation
Center of Chemical Science and Chemical Engineering (Tianjin)
Tianjin University, Tianjin, 300072, (China)
Figure 1. Chemical structures of energy donor 1, acceptor 2 and donor/
acceptor 3.
E-mail: xzhangchem@tju.edu.cn
1
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Figure 2. Three synthetic routes towards unsymmetrical perylene bisimide 2.
Synthesis. In this work, we first synthesized unsymmetrically
substituted perylene bisimide dye 2 as energy acceptor (Figures
1 and 2). Compared with symmetrically substituted dyes,
unsymmetrically substituted perylene bisimide dyes not only
require more complex reaction steps and purification processes
but also have lower yield. We thus developed three synthetic
routes for unsymmetrically substituted dyes (Figure 2). First
synthetic route is based on one-pot reaction procedure. Two
different substituent groups, i. e., 3, 4, 5-tris(3-(2-(2-
ethoxyethoxy)ethoxy)-1-propyl)-aniline and 6-amino-hexanol,
are mixed with the perylene-3, 4, 9, 10-tetracarboxylic
dianhydride in a pot to perform the substitution reaction.[21]
Owing to the competition of symmetric substitution, the first
synthetic method eventually produces considerable by-products,
resulting in difficulty in purification and extremely low yield (2%).
The second and third synthetic routes are based on two-step
reaction procedure. 3, 4, 5-Tris(3-(2-(2-ethoxyethoxy)ethoxy)-1-
propyl)-aniline was reacted with perylene- 3, 4, 9, 10-
tetracarboxylic dianhydride to produce monoanhydride
monoimide and then 6-aminohexanol was reacted with
monoanhydride monoimide at another imide position. In the third
synthetic route, the reaction order of 3, 4, 5-tris(3-(2-(2-
ethoxyethoxy)ethoxy)-1-propyl)-aniline and 6-aminohexanol is
opposite with the second route. Higher reaction yields (29~42%)
were obtained by the two-step reaction of the second and third
synthetic routes.
was attached to one imide side, and the alkyl amino group was
attached to the other imide side. The aromatic and alkyl amino
groups have extremely different reaction activity, thus, our
unsymmetrical substitution is a synthetic challenge. By using the
third route, we obtained the highest reaction yield (42%, Figure 2)
for unsymmetrical dye 2. The dye 3 consists of pyrene donor 1
and perylene bisimide acceptor 2, which was synthesized by the
esterification of energy donor 1-pyrenylbutyric acid 1 with the
hydroxyl group of alkyl chain of acceptor 2 (Figure 3).
Figure 3. Synthetic route towards donor/acceptor dye 3.
Fluorescence resonance energy transfer. To investigate the
energy transfer of these dyes, we performed UV/vis absorption
and fluorescence spectroscopic measurements. The optical
data including maximum UV/vis absorption and fluorescence
emission wavelengths, extinction coefficients, fluorescence
lifetimes and fluorescence quantum yields of these dyes are
listed in Table 1. The dichloromethane solution of donor 1 is
transparent. 2 and 3 are slightly pink in dichloromethane (Figure
4a inset). Donor 1 and acceptor 2 show characteristic absorption
of pyrene and perylene bisimide chromophores with well-defined
For the most reported unsymmetrically substituted perylene
bisimide dyes,[11a] although the substituent groups at two imide
positions are different, but they are based on the same class of
amino substituent groups, i.e. both aromatic amino groups or
both alkyl amino groups at two imide positions. In our
unsymmetrically substituted synthesis, the aromatic amino group
2
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fine structures and maximum absorption wavelengths at 344 nm
and 525 nm, respectively (Figure 4a). In addition, the absorption
bands of 1 and 2 are well separated, therefore, 1 and 2 can be
blue-purple light in dichloromethane with a light-emiting color
coordinate (0.17, 0.04) in CIE 1931 chromaticity diagram (Figure
4b inset). Acceptor 2 displays green-yellow fluorescence from
perylene bisimide chromophore with well-defined fine structure
at 534 nm and 577 nm in dichloromethane. When
donor/acceptor 3 was excited at 330 nm where only pyrene
donor was excited, the fluorescence of pyrene donor was almost
not observed, and enhanced fluorescence of perylene bisimide
acceptor was observed (Figure 4b). Under 365 nm UV lamp, 3
emits only acceptor green-yellow light in dichloromethane with a
light-emiting color coordinate (0.36, 0.56) (Figure 4b inset).
These results suggest the fluorescence energy transfer
occurring from pyrene donor to perylene acceptor in 3.[22] To
confirm the energy transfer, we performed the excitation-
dependent 3D fluorescence spectral measurements (Figure 4 c
and d). When 3 was selectively excited at 450-530 nm for its
perylene acceptor, or at 310-360 nm for its pyrene donor, we
observed the same fluorescence spectra from perylene bisimide
acceptor. These results further reveal high efficient energy
transfer occurring from pyrene donor to perylene acceptor in dye
3.
selectively excited. Donor/acceptor dye
3
shows two
characteristic absorption bands at 345 nm and 525 nm, which
are obviously arose from pyrene and perylene bisimide
chromophores, respectively. At the same concentration of these
dyes in solution, the absoprtion spectrum of 3 is consistent with
the absorption spectra of donor 1 plus acceptor 2 (Figure 4a),
suggesting that although pyrene and perylene chromophores
are connected by covalent bonds with a close distance in 3, they
have no interaction in the ground state and still maintain their
independent optical properties.
Table 1. Optical data of energy donor 1, acceptor 2 and donor/acceptor 3 in
CH2Cl2 (c = 2 ×10-6 M): maximum absorption wavelength (A), maximum
fluorescence emission wavelength (F), extinction coefficient (), fluorescence
lifetime () and fluorescence quantum yield ().
Dyes
A /nm
344
 /104 M-1cm-1
5.44

F /nm
 /ns
31.45
3.59

1
2
3
376
0.17
0.54
0.18
525
7.19
534
345, 525
3.53, 7.32
533
0.99
To quantify the energy transfer of 3, the efficiency (E) of
energy transfer was calculated by the following equations:[4c]
(1)
(2)
(3)
Figure 4. UV/vis absorption (a) and fluorescence emisson spectra (b) of
energy donor 1, acceptor 2 and donor/acceptor 3 in dichloromethane. ex
=
330 nm. c = 2×10-6 M. Inset in (a): Photograph of dichloromethane solution of
1, 2 and 3 under normal light. Inset in (b): Photograph of dichloromethane
solution of 1 and 3 under 365 nm UV lamp and CIE 1931 chromaticity diagram.
These points indicated by circles are the fluorescence color coordinates for 1
where  is the wavelength, FD () is donor’s fluorescence
spectrum, A is acceptor’s extinction coefficient, J () is the
overlap integral of acceptor absorption and donor emission, k2 is
the orientation factor and usually takes the value of 2/3.[23], η is
the refractive index of the medium, D is the fluorescence
quantum yield of donors without acceptors, R0 is the Förster
distance, that is, the distance between donor and acceptor when
the efficiency of energy transfer is 50%. rDA is the donor-to-
acceptor distance. The energy-minimized molecular structure of
3 (semi-empirical AM1 calculations) indicates that donor-to-
acceptor distance (rDA) is 14.6 Å (Supporting information S11).
J() was calculated to be 1.087×1014 cm-1M-1nm4 according to
the equation (1) and R0 was then calculated to be 25.3 Å
according to the equation (2). We finally obtained the energy
transfer efficiency (E) as high as 96.4% according to the
(0.17, 0.04) and
3 (0.36, 0.56). c) and d) Excitation-dependent 3D
fluorescence spectra of 3 in dichloromethane. [3]= 2×10-6 M.
All these dyes 1-3 are highly fluorescent covering whole
visible light region. The fluorescence quantum yields for donor 1,
acceptor 2 and donor/acceptor 3 were measured to be 0.17,
0.54 and 0.18, respectively. Fluorescence lifetimes were
measured to be 31.45 ns, 3.59 ns and 0.99 ns for donor 1,
acceptor 2 and donor/acceptor 3, respectively (Table 1). Donor 1
shows blue-purple fluorescence of pyrene chromophore with
well-defined fine structure at 376 nm and 397 nm in
dichloromethane (Figure 4b). Under 365 nm UV lamp, 1 emits
3
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equation (3). This is a rare example for such high intramolecular
efficient energy transfer. Energy transfer efficiency (E) could
also be calculated on the basis of fluorescence lifetimes by
changes in different polar solvents (Figure 5b, Table 2). Under
365nm UV lamp, light-emitting colors of 3 change from green to
yellow with increasing solvent polarity (Figure 5d). The light-
equation:[4c] E = 1- DA/D, where DA is 0.99 ns and D is 31.45 ns, emitting color coordinates in CIE 1931 chromaticity diagram
which are fluorescence lifetimes of donor/acceptor 3 and donor
1, respectively. Energy transfer efficiency (E) was obtained to be
96.8% from fluorescence lifetimes, which well agrees with our
move from (0.39, 0.57) in green region for 3 in ethyl acetate, to
(0.42, 0.53) in yellow region for 3 in polar n-butanol (Figure 5e).
We further used the empirical Lippert equations (4) and (5)[4c] to
above result (96.4%) obtained from the equations (1), (2) and (3). quantify the environment solvent polarity sensitivity:
Fluorescence resonance energy transfer probe for
environmental polarity sensitivity. Donor /acceptor 3 is a new
dye molecule. We further explore its application potentials as a
new class of fluorescence resonance energy transfer probes.
Generally, the emission wavelength of a fluorophore is longer
than the absorption wavelength. This difference is known as
Stokes’ shift. The emission energy lever of a dye fluorophore is
lower than its absorption energy lever. This energy loss is
caused by several dynamic processes occurring during light
absorption, such as vibration relaxation, the changes of dipole
moments of excited state fluorophores, reorientation or
relaxation of the solvent dipoles after excitation.[4c, 24]
(4)
(5)
Where F and A are the wavenumbers (cm-1) of the emission
and absorption, respectively, G and E are the dipole moments
of dye 3 in the ground state and the excited state, respectively, c
is the light speed, h is Planck's constant, a is the molecular
radius of dye 3, n and ε are the refractive index and the dielectric
constant of the solvents, respectively, and f is the orientation
polarizability.
Table 2. Solvent environment polarity sensitivity. Stokes’ shifts (A-F or F-A)
of 3 in various polar solvents. [3] = 2×10-6 M. f is solvent parameter
(orientation polarizability),
Solvents
f a
A
/nm
F
/nm
A - F
=F-A
/nm
/cm-1
Cyclohexane
Chloroform
Ethyl acetate
THF
-0.0026
0.1523
0.1997
0.2103
0.2182
0.2644
0.3086
519
527
519
521
525
527
528
521
534
526
529
533
536
538
74
2
7
248
256
290
285
318
352
7
8
Dichloromethane
n-Butanol
8
9
Methanol
10
a Δf = [(ε-1)/(2ε + 1)] - [(n2-1)/(2n2 + 1)]
Stokes’ shifts of dye 3 show significantly increase with
increasing the solvent orientation polarizability (f), revealing
that 3 is very sensitive to polarity of the solvents. We plotted the
Stokes’ shifts (A-F) versus solvent polarity parameters (f) and
a linear relationship was obtained (Figure 5c). According to the
Lippert equations, the value of (2/hc)[(µE - µG)2/a3] was obtained
from the slope of the Lippert plot. The value of a is 22.7 Å. The
change (µ= µE - µG) in dipole moment between the excited
state and the ground state was thus calculated to be 32.1 D,
which is comparable to the dipole moment change from one unit
charge’s (4.8×10-10 esu) separation by 6.7 Å. These results
reveal that the excited state dipole moment of 3 changes
significantly with the polarity of the solvents, which is due to the
existence of fluorescence resonance energy transfer in 3 from
pyrene to perylene chromophores. From all these results, we
conclude that dye 3 is sensitivity to environment polarity, which
can be regarded as a new class of fluorescence resonance
energy transfer probes.
Figure 5. UV/vis absorption (a) and fluorescence emission spectra (b) of
donor/acceptor 3 in cyclohexane (CYH), chloroform (CHCl3), ethyl acetate
(EtOAc), dichloromethane (DCM), tetrahydrofuran (THF), n-butanol (n-BuOH)
and methanol (MeOH). [3] = 2×10-6 M, ex= 344nm. c) Lippert plot for 3 in
various solvents. d) Photograph of 3 in various solvents under 365 nm UV
lamp. e) CIE 1931 chromaticity diagram. These points indicated by circles are
the fluorescence color coordinates for
3 in ethyl acetate (0.39, 0.57),
tetrahydrofuran (0.41, 0.55), dichloromethane (0.42, 0.54), n-butanol (0.42,
0.53), methanol (0.35, 0.43).
To study the environment polarity sensitivity of 3, we
chosen the different polar solvents: cyclohexane (CYH),
chloroform (CHCl3), ethyl acetate (EtOAc), dichloromethane
(DCM), tetrahydrofuran (THF), n-butanol (n-BuOH) and
methanol (MeOH). UV/vis absorption and fluorescence emission
spectra of 3 were measured at a concentration of 2×10-6
(Figure 5a, b). Fluorescence behaviors of 3 display obviously
M
4
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Donor/acceptor 3 is an amphiphilic dye composed of three
hydrophilic oligo-ethylene glycol chains and hydrophobic
pyrene/perylene bischromophores. We further investigated its
aggregation behaviors in water by UV/vis absorption (Figure 7a)
and fluorescence emission spectroscopy (Figure 7b, c).
Tetrahydrofuran is a good solvent for 3, thus, 3 dissolves in
tetrahydrofuran (THF) in molecularly dissolved state at low
concentration. Water is a poor solvent. As the fraction of water in
tetrahydrofuran solution increases, the absorption maximum
wavelength was blue-shifted from 525 nm to 500 nm.
Simultaneously, a decrease in the extinction coefficient and a
loss of the spectral fine structures were observed (Figure 7a).
Moreover, a new broad absorption band appears at a longer
wavelength of around 550 nm. When water fraction is larger
than 50% (>50%, v/v), UV/vis absorption spectroscopic baseline
Supramolecular assembly and aggregation behaviors in
water towards nanosystem fluorescence sensors. We further
explore the application of our novel fluorescence probe in
biologically relevant environment, i.e. water. Before the study on
the aggregation in water, we first investigated the
supramolecular assembly behaviors of 3 in organic solvents.
Concentration-dependent UV/vis absorption spectra were
measured in tetrahydrofuran (Figure 6). At low concentration
(<10-6M), 3 displays the absorption spectrum of molecularly
dissolved state. With increasing the concentration, the
absorption spectra become broad, the extinction coefficient
decreases, and the maximum absorption wavelength is blue-
shifted about 33 nm. At the same time, a new weak absorption
band appears at about 553 nm, and an isosbestic point was
observed at 537 nm (Figure 6). These absorption spectral
changes reveal molecular aggregation occurring with increasing
concentration in tetrahydrofuran through - stacking between
the aromatic chromophores.
of
3 does not go to zero, indicating the suspension
characteristics of the aggregate formation.
Figure 6. UV/vis absorption spectra of
3 in tetrahydrofuran at different
concentration from 1×10-6 M to 1×10-2 M. The arrows indicate changes of the
spectra with increasing concentration. Inset: plot of mole fraction of
aggregated molecules (agg) versus concentration in tetrahydrofuran. The
curve was acquired by fitting the data according to the isodesmic model.[25, 27]
Figure 7. UV/Vis absorption (a) and fluorescence emission spectra (b) of 3 in
THF/H2O.

ex= 344nm. [3] = 2×10-6 M. The arrows indicate the spectral
changes upon increasing the fraction of water. Inset in (b): CIE 1931
chromaticity diagram. These points indicated by circles are the fluorescence
color coordinates for 3 in THF/H2O: 100/0 (0.36, 0.61), 80/20 (0.37, 0.59),
50/50 (0.38, 0.56), 40/60 (0.36, 0.52), 35/65 (0.27, 0.30), 10/90 (0.20, 0.11). c)
Fluorescence spectra of
Photograph of 3 in THF/H2O under 365 nm UV lamp. [3] = 2×10-6 M.
The mole fraction of aggregated molecules (αagg) is obtained
by the following equation (6): [26]
3 in THF/H2O. [3] = ex=344nm. d)
2×10-6 M.
(6)
As the solvents change from nonpolar THF to polar water,
the fluorescence intensity of dye 3 gradually decreases (Figure 7
b), a new fluorescence band appears, and the maximum
emission wavelength at 529 nm is red-shifted to 755 nm, which
falls within near infrared region (Figure 7c). The large red-shift
(226 nm) of emission wavelength with increasing solvent polarity
further confirms our above conclusion, that is, environment
polarity sensitivity of dye 3. The new broad near-infrared
where ε is the extinction coefficient and subscripts mon and agg
are monomers and aggregates in solution respectively. It is
assumed here that the molecules are fully aggregated at the
highest concentration and all dissolved in molecular state at the
lowest concentration. According to equation (6), the plot of the
mole fraction of aggregated molecules (αagg) versus the
concentration in solution, reveals the changes from molecularly
dissolved state to aggregated state in tetrahydrofuran. We
subsequently measured concentration-dependent fluorescence
spectroscopy (Figure S13). A new structureless and broad
fluorescence band at long wavelength appears and increases
gradually with increasing the concentration, which further
confirms the aggregate formation in tetrahydrofuran.
fluorescence band becomes more obvious at
a higher
concentration in water (Figure S14). Figure 7d shows a
photograph of 3 from molecularly dissolved state to aggregating
state in THF/water under 365nm UV lamp. Fluorescence color
coordinates of 3 in CIE 1931 chromaticity diagram in THF/water
are shown in Figure 7b inset, which indicates the color changes
5
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with increasing the water fraction. All these spectral results
reveal the large aggregate formation of 3 in aqueous solution.
We subsequently performed theoretical analysis by using
Israelachvili's models for aggregates of 3 in water.[21, 28] These
theoretical models are based on molecular curvatures to
produce a minimum in free energy for assembling morphology in
polar solvents by using Israelachvili's critical packing parameter
Pc = V/al, where V and l are the effective volume and optimal
length of molecule, respectively, a is the area occupied by
hydrophilic chains. This Israelachvili's model is particularly
suitable to predict the assembly of shape-preserving robust
molecules, where no change in molecular length and the
hydrophilic occupied area. According to Israelachvili's models,
with increasing critical packing parameter Pc, the spontaneous
curvature increases on the surface between aqueous and
organic phases, and assembling morphologies change from
spherical micelles (Pc < 0.33), cylindrical or disk-shaped micelles
(0.33  Pc 0.5), vesicular structures (0.5  Pc  1.0), to inverted
or reversed micelles (Pc  1.0). For dye 3, critical packing
parameter (Pc) was calculated to be 0.61, which theoretically
predicts that vesicular aggregates should be favorably formed in
polar solvents.
The aggregate morphologies of 3 in water were then
observed by transmission electron microscopy (TEM). The TEM
sample of 3 was prepared as follows: 3 (0.36 mg) was dissolved
in THF (6 mL), and then the deionized water (3 mL) was slowly
added into the above solution. The suspension solution was
exposed to air at room temperature for several days to remove
tetrahydrofuran by volatilization. The suspension solution of 3 in
water was finally obtained at a concentration of 0.12 mg/ml,
which was then used for TEM measurement. Our theoretical
prediction was confirmed by TEM observation, which reveals the
formation of hollow vesicles with an average size of about 62 nm
in water (Figure 8a-b). The wall thickness is uniform (9-10 nm),
which is corresponding to twice the length of one molecule (5.3
nm in the extended conformation of alkyl spacer, 4.5 nm in the
coiled conformation of alkyl spacer). Interestingly, the bilayer
structures were directly observed by TEM (Figure 8a inset). The
thickness of outer and inner layer is 5-6 nm and 4-5 nm,
respectively, implying the extended alkyl spacer for outer layer
molecules, and the coiled alkyl spacer for inner layer molecules.
To the best of our knowledge, this is the first time to clearly and
directly observe bilayer structures of vesicle by TEM. By dividing
the volume of the double layer by the volume of a single
molecule, the aggregate numbers of the outer and inner layers
were calculated to be approximate 4.1×105 and 3.5×105,
respectively. These vesicles are well separated and isolated,
close but not connected together, implying that these vesicles
are not adhesive, and the bilayer membrane is rigid and no
molecular exchanges occur intermembrane.
The above TEM results were further confirmed by dynamic
light scattering (DLS) measurements. We observed a narrow
scattering intensity distribution for aggregates of 3 in water
(Figure 8c), suggesting the size monodispersity and structural
homogeneity of these aggregates. DLS analysis indicates the
average diameter of these vesicular aggregates is 149 nm in
deionized water. DLS result is slightly larger than that from TEM,
which can be attributed to the measured states of samples.[29] In
the case of DLS measurements, the samples were detected in
situ in water. In the case of TEM measurements, the samples
were observed in the dried state in vacuum chamber. Then we
performed the excitation-dependent 3D fluorescence spectral
measurement (Figure 8e), which indicates that these vesicular
aggregates are light-emitting from 630 nm to 850 nm in near-
infrared region. Under 365 nm UV lamp, aggregates of 3 in
water emits slightly dark red light (Figure 8d), which is not easily
observed by naked eyes, because the light-emitting color
beyond the visible light region, which falls within near-infrared
region.
Conclusion
We designed and synthesized new pyrene/perylene
bischromophore dyes from unsymmetrically substituted perylene
bisimide dyes by three synthetic routes. The new dye
demonstrates a high efficient fluorescence resonance energy
transfer from pyrene donor to perylene bisimide acceptor. Owing
to energy transfer effect, the fluorescence behaviors of the
donor/acceptor dye are sensitive to polarity changes of
microenvironment, which demonstrates application potentials as
novel fluorescence probe. The donor/acceptor dye assembled
into bilayer vesicles in water with narrow size distribution
revealed by UV/vis absorption and fluorescence spectroscopy,
Figure 8. a) TEM images of aggregates of 3 prepared from deionized water.
Top and inset: magnified TEM images of one typical bilayer vesicle. The outer
and inner layers are composed of approximate 4.1×105 and 3.5×105 molecules,
respectively. b) Size distribution of the aggregates calculated from 99
aggregates according to TEM measurements. c) Size distribution of
aggregates of 3 obtained from dynamic light scattering in water. d) Photograph
of aggregates of 3 in water under 365 nm UV lamp. e) Excitation-dependent
3D fluorescence spectra of aggregates of 3 in deionied water. [3] = 0.12
mg/ml.
6
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10.1002/chem.202000107
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and transmission electron microscopy and dynamic light
scattering measurements. Our results may encourage the effort
to develop new class of fluorescence probes and functional dye
aggregates in water.
We acknowledge the National Natural Science Foundation of
China (Grant No. 21674079 and Grant No. 21975177) for
financial support.
Conflict of interest
Experimental Section
The authors declare no conflict of interest.
Dye 2: 3, 4, 5-tris(3-(2-(2-ethoxyethoxy)ethoxy)-1-propyl)-aniline (0.22 g,
0.45 mmol) , monoanhydride monoimide (0.13 g, 0.23 mmol) and zinc
acetate (0.04 g, 0.22 mmol) were mixed with imidazole (3.47 g, 0.05 mol).
The reaction mixture was heated under nitrogen at 140C. After 12 hr,
the reaction mixture was cooled to room temperature. 1M HCl (75 mL)
was added to the above reaction mixture. The resulting mixture was
extracted with dichloromethane (75 mL) for several times. Then the
solution was dried with anhydrous MgSO4 and concentrated under
reduced pressure, the crude product was purified by silica gel column
chromatography using dichloromethane/methanol (40/1, v/v) as eluent to
give a red solid (150mg, 60%). Rf = 0.31 (CH2Cl2/CH3OH=20/1, v/v). 1H
NMR (400MHz, CDCl3, ppm)(Figure S5): = 8.56-8.72 (m, 8H, ArH in
perylene ring), 7.04 (s, 2H, ArH in phenyl ring), 4.21 (t, J=7.6 Hz, 2H, -
NCH2-), 3.73-3.54 (m, 32H, 15-OCH2-, -CH2OH), 3.51 (q, J=2.8 Hz,
J=17.2 Hz, 6H, 3-OCH2CH3), 2.78 (t, J=8.00 Hz, 6H, 3-PhCH2-), 2.00-
1.93 (m, 4H, 2-PhCH2CH2-), 1.91- 1.77(m, 4H, -PhCH2CH2-, -NCH2CH2),
1.54-1.46(m, 4H, -CH2CH2CH2OH) 1.23 (t, J= 7.2 Hz, 6H, 2-CH3), 1.17 (t,
J= 6.8 Hz, 3H, -CH3). MALDI-TOF MS (matrix: α-cyano-4-
hydroxycinnamic acid) calculated for C83H94N2O15 1088.561 m/z, found
1111.737 [M+Na]+.
Keywords: Fluorescence • Fluorescence resonance energy
transfer (FRET) • Perylene bisimides • Supramolecular assembly
Aggregates • Dyes
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Entry for the Table of Contents
An amphiphilic bischromophore dye was synthesized by three optimized synthetic routes. The new dye demonstrates high efficient
intramolecular fluorescence resonance energy transfer from pyrene donor to perylene bisimide acceptor. Near-infrared fluorescent,
narrow size distribution bilayer vesicles were prepared in water towards nanosystem fluorescence sensors.
9
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