Concentration-dependent dye aggregation and disassembly triggered by the same artificial helical foldamer

Article abstract of DOI:10.1016/j.polymer.2019.02.063

Constructing and regulating the cyanine dye aggregates have been constantly pursued because of their wide applications in photosensitizers, biological fluorescence probes, nonlinear optics, etc. However, to realize such a process using a single additive still remains a formidable challenge. Here, the aggregation and disassembly of positively charged 3,3-diethyloxadicarbocyanine iodide (DiOC₂(5)) have been demonstrated just by changing the concentration of an artificial poly(phenylenediethynylene)-based helical foldamer (Poly-1) bearing L-alanine sodium pendants with an amide linker. Poly-1 possessed the negatively charged pendants and hydrophobic helical grooves. Upon simple mixing of equal DiOC₂(5) and Poly-1, DiOC₂(5) molecules were mainly enriched on Poly-1 surface driven by electrostatic interaction and arranged along the main chain of Poly-1, resulting in the formation of chiral H-aggregates. But, the excessive Poly-1 would attract some DiOC₂(5) molecules in H-aggregates via electrostatic interaction. The isolated monomers were easy to orderly disperse into the helical grooves due to hydrophobic interaction. Meanwhile, during the aggregation and disassembly, the solution color changed from peachblow to purple red and then to purple and finally to navy blue.

Full text of DOI:10.1016/j.polymer.2019.02.063

Polymer170(2019)7–15
Contents lists available at ScienceDirect
Polymer
journal homepage: www.elsevier.com/locate/polymer
Concentration-dependent dye aggregation and disassembly triggered by the
same artificial helical foldamer
Yuan Qiua, Haisi Hua, Dongxu Zhaoa, Jing Wanga, Hong Wanga, Qin Wanga, Haiyan Penga,b
,
Yonggui Liaoa,b,∗, Xiaolin Xiea,b
a Key Laboratory of Material Chemistry for Energy Conversion and Storage, Ministry of Education, Hubei Key Laboratory of Materials Chemistry and Service Failure, School
of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan, 430074, China
b National Anti-counterfeit Engineering Research Center, Huazhong University of Science and Technology, Wuhan, 430074, China
H I G H L I G H T S
A novel strategy to achieve chiral H-aggregation and disassembly of cyanine dyes via changing [foldamer]/[dye] ratio.
The new foldamer contains multiple binding sites, i.e., negatively charged pendants, helical grooves and cavities, for dyes.
During the aggregation and disassembly, the solution color changes from peachblow to navy blue.
•
•
•
A R T I C L E I N F O
A B S T R C T
Keywords:
Artificial helical foldamer
Cyanine dye
Constructing and regulating the cyanine dye aggregates have been constantly pursued because of their wide
applications in photosensitizers, biological fluorescence probes, nonlinear optics, etc. However, to realize such a
process using a single additive still remains a formidable challenge. Here, the aggregation and disassembly of
positively charged 3,3-diethyloxadicarbocyanine iodide (DiOC2(5)) have been demonstrated just by changing
the concentration of an artificial poly(phenylenediethynylene)-based helical foldamer (Poly-1) bearing L-ala-
nine sodium pendants with an amide linker. Poly-1 possessed the negatively charged pendants and hydrophobic
helical grooves. Upon simple mixing of equal DiOC2(5) and Poly-1, DiOC2(5) molecules were mainly enriched
on Poly-1 surface driven by electrostatic interaction and arranged along the main chain of Poly-1, resulting in
the formation of chiral H-aggregates. But, the excessive Poly-1 would attract some DiOC2(5) molecules in H-
aggregates via electrostatic interaction. The isolated monomers were easy to orderly disperse into the helical
grooves due to hydrophobic interaction. Meanwhile, during the aggregation and disassembly, the solution color
changed from peachblow to purple red and then to purple and finally to navy blue.
H-aggregate
1. Introduction
biopolymers [7,8] and some artificial polymers [9,10] have been used
as the additives to construct the assembled cyanine dye aggregates. In
Cyanine dyes are a class of organic ionic compounds containing two
heterocyclic components connected by a polymethine bridge with an
odd number of conjugated carbon atoms [1]. Their aggregates have
attracted considerable attention for their wide applications including
photosensitizers [2], biological fluorescence probes [3] and nonlinear
optics [4]. To realize these applications, the construction and regulation
of the cyanine dye aggregates are of great importance. Due to the ionic
nature as well as π-π stacking and van der Waals forces, cyanine dyes
can self-aggregate in aqueous solution by changing the concentration,
ionic strength, pH or temperature [5]. Besides, liposomes [6],
many cases, J- or H-aggregates can be obtained when the dyes stack in a
head-to-tail or face-to-face way, respectively [11]. In spite of many
methods to construct J- or H-aggregates, rare attempts to further reg-
ulate the aggregates have been reported. For example, enhanced J-ag-
gregation of a cyanine dye has been achieved in the presence of a large
excess carboxymethyl amylose, leading to a super-helical complex of
cyanine dye/carboxymethyl amylose [12]. J-aggregates for pseudoiso-
cyanine or H-aggregates for pinacyanol are decreased by the moderate
binding of cucurbit[7]uril with pseudoisocyanine or pinacyanol [13]. A
cycling switch among four kinds of aggregates for a cyanine dye have
∗ Corresponding author. Key Laboratory of Material Chemistry for Energy Conversion and Storage, Ministry of Education, Hubei Key Laboratory of Materials
Chemistry and Service Failure, School of Chemistry and Chemical Engineering, Huazhong University of Science and Technology, Wuhan, 430074, China.
E-mail address: ygliao@mail.hust.edu.cn (Y. Liao).
https://doi.org/10.1016/j.polymer.2019.02.063
Received 29 January 2019; Received in revised form 26 February 2019; Accepted 28 February 2019
Availableonline05March2019
0032-3861/©2019ElsevierLtd.Allrightsreserved.

Y. Qiu, et al.
Polymer170(2019)7–15
2.2. Characterization
1H and 13C NMR spectra were recorded using a Bruker AVANCE III-
400 instrument at room temperature. FTIR spectra were obtained with
a Bruker Equinox 55 spectrometer. The number-average molecular
weight (Mn) and the polydispersity (Mw/Mn) were determined on a
size exclusion chromatography (SEC) apparatus equipped with a JASCO
PU-980 Intelligent pump and a JASCO RI-930 Intelligent RI detector.
DMF containing lithium chloride (0.01 M) was used as the eluent at a
flow rate of 0.4 mL min−1 at 40 °C. The molecular weight calibration
curve was obtained with polystyrene standard. The solution pH was
measured with an AS-600 pH meter. The electron spray ionization mass
spectra (ESI-MS) were measured on a Bruker micrOTOF-Q II spectro-
meter. The specific optical rotation values ([α]20 D) were obtained
from a WZZ-2B polarimeter (Shanghai ShenGuang Instrument Co.,
Ltd.). The circular dichroism (CD) spectra were recorded on a JASCO J-
810 spectropolarimeter. The ultraviolet–visible (UV–vis) absorption
spectra were recorded on a PerkinElmer Lambda 35 spectrophotometer.
The fluorescence spectra were measured on a JASCO FP-6500 spec-
trofluorescence. Isothermal titration calorimetry (ITC) measurements
Scheme 1. Illustrative process of concentration-dependent DiOC2(5) aggrega-
tion and disassembly.
were carried out at 25.00
0.01 °C on a MicroCal iTC200 apparatus.
been achieved by adding Tb3+ and then EDTA [5]. Obviously, the
addition of extra additives can further regulate the aggregates.
In recent years, artificial foldamers have garnered a lot of interest
because they can mimic the folding behavior that occurs in the sec-
ondary structure of biopolymer [14]. Thus, these three-dimensional
foldamers possess a characteristic asymmetric groove and cavity [15].
When guest molecules are bound to the foldamers, they can insert into
the groove or cavity [16,17]. In this regard, the foldamers can provide
multiple binding sites and thus emerge as new additives to regulate
cyanine dye aggregates. On the other hand, the foldamers can also be
used to construct the aggregates at the same time when the charged
pendants are conjugated to the main chain. The electrostatic interac-
tions between the charged pendants and dyes may promote the dye
aggregation. For this purpose, we have designed a poly(phenylene-
diethynylene)-based foldamer (Poly-1) bearing L-alanine sodium pen-
dants with an amide linker as a novel additive for constructing and
regulating the cyanine dye aggregates (Scheme 1a). The helical folding
behavior of Poly-1 in water was driven by the hydrophobic interaction.
This helical structure was further stabilized via π-π stacking between
phenylenediethynylene units and electrostatic repulsion between the
adjacent negatively charged pendants. In addition, Poly-1 helix fea-
tures π-π stacked aromatic residues similar to the π-π stacked bases in
DNA and the negatively charged pendants like the phosphate groups on
DNA. Herein, we use Poly-1 to complex with the cyanine dye, 3,3-
diethyloxadicarbocyanine iodide (DiOC2(5), Scheme 1a), in aqueous
solution. The system demonstrates that the concentration-dependent
aggregation and disassembly of the dye have been achieved. Mean-
while, the color changes during the process can be directly observed by
naked eye.
Atomic force microscope (AFM) images were acquired in tapping mode
with a Shimadzu SPM-9700 instrument, performed at ambient tem-
perature in air using standard silicon cantilevers with a spring constant
of about 40 N/m, a tip radius of 5–10 nm, and a resonance frequency of
∼300 kHz.
2.3. Synthesis of compound 2
Thionyl chloride (12.2 mL, 168.5 mmol) was added to methanol
(66 mL) at 0 °C, and the resulting solution was stirred for 10 min. L-
Alanine (3.0 g, 33.7 mmol) was then added to the solution, and the
resulting mixture was stirred at room temperature overnight. The sol-
vent was evaporated and dried in vacuum to obtain 2 as colorless oil
(4.56 g, 97%). [α]20 D = +6.8° (c = 2 g/dL, in methanol). 1H NMR
(400 MHz, DMSO-d6, TMS, ppm): δ = 8.71 (s, -NH3Cl, 3H), 4.05 (q,
J = 7.2 Hz, -CH-CH3, 1H), 3.74 (s, -O-CH3, 3H), 1.43 (d, J = 7.2 Hz,
-CH-CH3, 3H).
2.4. Synthesis of compound 1a
To a mixture of 3,5-diethynyl-benozoic acid (1.0 g, 5.80 mmol) and
2 (1.05 g, 7.54 mmol) in anhydrous THF (29 mL) were added 4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (DMT-
MM) (3.21 g, 11.6 mmol) and dry TEA (4.2 mL, 30.2 mmol). The mix-
ture was stirred at room temperature for 24 h and the solvent was
concentrated under reduced pressure. The residue was dissolved in
ethyl acetate, and washed with 0.5 M HCl, saturated solution of sodium
bicarbonate, and brine and dried over anhydrous sodium sulfate. After
filtration, the solvent was removed by evaporation, and the residue was
then purified by column chromatography (SiO2, petroleum ether/ethyl
acetate = 5/1, v/v) to afford 1a as a white solid (1.08 g, 72%). [α]20
D = +5.9° (c = 2 g/dL, in DMF). 1H NMR (400 MHz, DMSO-d6, TMS,
ppm): δ = 9.01 (d, J = 6.8 Hz, -NH-, 1H), 8.01 (s, Ar-H, 2H), 7.75 (s,
Ar-H, 1H), 4.52–4.45 (m, -NH-CH-, 1H), 4.40 (s, -C≡CH, 2H), 3.65 (s,
-O-CH3, 3H), 1.41 (d, J = 7.6 Hz, -CH-CH3, 3H). 13C NMR (100 MHz,
DMSO-d6, TMS, ppm): δ = 173.4 (-CO-(ester)), 164.8 (-CO-(amide)),
137.4 (aromatic), 135.0 (aromatic), 131.4 (aromatic), 123.1 (aro-
matic), 82.9 (-C≡CH), 82.2 (-C≡CH), 52.4 (-O-CH3), 48.9 (-CH-NH-),
17.1 (-CH-CH3). IR (KBr, cm−1): 3311 (≡C-H), 3285 (N-H), 2106
(C≡C), 1729 (C=O), 1634 (C=O). HRMS (ESI-MS): m/z calcd for [M
(C15H13NO3) + Na]+, 278.0895; found 278.0882.
2. Experimental section
2.1. Materials
L-Alanine and thionyl chloride were purchased from Sahn Chemical
Technology Co., Ltd. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-
morpholinium chloride (DMT-MM) was purchased from J&K Chemical
Co., Ltd. Copper(I) chloride, N,N,N′,N′-tetramethylethylenediamine
(TMEDA) and ultrapure water were purchased from Alfa Aesar.
Triethylamine (TEA) was dried over potassium hydroxide and distilled
under nitrogen. Tetrahydrofuran (THF) was dried over sodium benzo-
phenone ketyl and distilled onto LiAlH4. N,N-dimethylformamide
(DMF) was distilled from calcium hydride. 3,5-Diethynyl-benozoic acid
was prepared according to a previous literature [15].
2.5. Synthesis of compound 1
To a solution of 1a (0.3 g, 1.18 mmol) in THF (5 mL) was added a
8

Y. Qiu, et al.
Polymer170(2019)7–15
Scheme 2. Synthetic route for foldamer Poly-1.
solution of sodium hydroxide (0.05 g, 1.18 mmol) in water (5 mL). The
mixture was stirred at room temperature overnight and the solvent was
concentrated under reduced pressure. The residue was dissolved in
water, and washed with chloroform three times. The water was re-
moved by evaporation, and the residue was purified by recrystallization
from water/methanol (8/2, v/v) to afford 1 as a white crystalline solid
(0.19 g, 60%). [α]20 D = +27.5° (c = 2 g/dL, in water). 1H NMR
(400 MHz, DMSO-d6, TMS, ppm): δ = 8.23 (d, J = 6.4 Hz, -NH-, 1H),
7.90 (s, Ar-H, 2H), 7.69 (s, Ar-H, 1H), 4.38 (s, -C≡CH, 2H), 4.03–3.96
(m, -NH-CH-, 1H), 1.28 (d, J = 6.8 Hz, -CH-CH3, 3H). 13C NMR
(100 MHz, DMSO-d6, TMS, ppm): δ = 175.8 (-CO-(carboxylate)), 163.5
(-CO-(amide)), 137.0 (aromatic), 136.6 (aromatic), 131.0 (aromatic),
122.9 (aromatic), 82.7 (-C≡CH), 82.3 (-C≡CH), 51.0 (-CH-NH-), 19.4
(-CH-CH3). IR (KBr, cm−1): 3321 (≡C-H), 3289 (N-H), 2105 (C≡C),
1627 (C=O), 1583 (C=O). HRMS (ESI-MS): m/z calcd for [M
(C14H10NO3Na) + Na]+, 286.0456; found 286.0446.
(C=O), 1579 (C=O).
2.8. Sample preparation for CD, UV–vis absorption and fluorescence
measurements
Ultrapure water was degassed with nitrogen and a borate buffer (pH
7.8) was made for these measurements. The concentration of monomer
unit was chosen as the concentration of Poly-1.
2.9. Isothermal titration calorimetry (ITC) measurements
In a typical run, 200 μL of 0.1 mM aqueous solution of DiOC2(5)
was placed in the sample cell and then titrated dropwise with 2 μL of
1 mM Poly-1 in water. The titrations consist of the first 0.4 μL injection
and followed 19 injection of 2 μL with 20 s duration at 150 s equili-
bration time interval. The titration of Poly-1 in syringe into water in the
sample cell without DiOC2(5) was used as a control to obtain the di-
lution heat. All solutions were degassed prior to titration. A binding
isotherm curve was obtained by plotting the total heat per injection (kJ
mol−1 of injectant) as a function of the molar ratio ([Poly-1]/
[DiOC2(5)]). All the data were analyzed with Microcal Origin 7.0
software.
2.6. Polymerization of 1a
1a (0.8 g, 3.11 mmol), N,N,N′,N′-tetramethylethylenediamine
(TMEDA) (1.08 g, 9.32 mmol), and CuCl (0.09 g, 0.932 mmol) were
dissolved in DMF (31 mL). The mixture was stirred at 40 °C with oxygen
bubbling. After 24 h, the solution was precipitated into a large amount
of acetone acidified with 37 wt % HCl solution. The precipitate was
collected by centrifugation, washed with acetone, and then dried in
vacuum to afford Poly-1a as a pale yellow solid (0.64 g, 80%).
Mn = 2.07 × 104, Mw/Mn = 2.89. 1H NMR (400 MHz, DMSO-d6, TMS,
ppm): δ = 9.03–7.84 (m, Ar-H, 3H), 4.46 (brs, -NH-CH-, 1H), 3.66 (brs,
-O-CH3, 3H), 1.42 (brs, -CH-CH3, 3H). IR (KBr, cm−1): 3301 (N-H),
2212 (C≡C), 1732 (C=O), 1640 (C=O).
3. Results and discussion
3.1. Synthesis of foldamer Poly-1
The foldamer was synthesized via the route illustrated in Scheme 2.
At first, the condensation of 3,5-diethynyl-benozoic acid with com-
pound 2 proceeded using 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-me-
thylmorpholinium chloride (DMT-MM) as a catalyst to afford the novel
monomer 1a. Then, 1a was polymerized through Glaser-Hay coupling
reaction with CuCl and N,N,N′,N′-tetramethylethylenediamine
(TMEDA) in DMF under an oxygen atmosphere. The disappearance of
the acetylene protons of 1a at 4.38 ppm demonstrated the successful
polymerization (Figs. S1 and S5). The resultant Poly-1a was soluble in
DMF and DMSO, while insoluble in dichloromethane, chloroform, THF,
methanol and water. The number-average molecular weight (Mn) and
polydispersity (Mw/Mn) were estimated to be 2.07 × 104 and 2.89 (Fig.
S7), respectively. Finally, the hydrolysis of Poly-1a afforded the am-
phiphilic Poly-1 containing the hydrophobic phenylenediethynylene
backbone and a hydrophilic side chain. 1H NMR analysis failed to
confirm the completion of the reaction because the signal of the methyl
2.7. Hydrolysis of Poly-1a
To a solution of Poly-1a (0.64 g) in DMF (24.9 mL) was added a
solution of sodium hydroxide (0.60 g, 14.9 mmol) in water (24.9 mL).
The reaction mixture was stirred at room temperature overnight and
dialyzed against ultrapure water with 3.5 kDa. After 72 h, the solvent
was concentrated by evaporation, and the residue was precipitated into
a large amount of acetone. The resultant precipitate was collected by
centrifugation, washed with acetone, and dried under vacuum to afford
Poly-1 as a brown solid (0.57 g, 89%). 1H NMR (400 MHz, DMSO-d6,
TMS, ppm): δ = 9.07–7.69 (m, Ar-H, 3H), 4.40 (brs, -NH-CH-, 1H), 1.42
(brs, -CH-CH3, 3H). IR (KBr, cm−1): 3301 (N-H), 2213 (C≡C), 1633
9

Y. Qiu, et al.
Polymer170(2019)7–15
Fig. 1. CD (up) and UV–vis absorption (bottom) spectra of 1 and Poly-1 in
water. All spectra were measured in a 1 mm quartz cell at ambient temperature.
[1] = [Poly-1] = 0.62 mM.
Fig. 2. FTIR spectra of
[1] = [Poly-1] = 20 mM.
1
and Poly-1 in D2O at ambient temperature.
spectrum of Poly-1 showed amide I band at 1639 cm−1 and amide II'
band at 1448 cm−1 instead of amide II band in D2O, also implying that
NH groups were accessible to water. Namely, hydrogen bonding was
not a stabilizing force to the helical structure in Poly-1.
ester moiety at 3.66 ppm would be shielded by water peak (Fig. S6).
Fortunately, FTIR spectra demonstrated the hydrolysis by the absence
of absorption band at 1732 cm−1 for C=O stretching due to methyl
ester and presence of absorption band at 1579 cm−1 for C=O
stretching due to carboxylate sodium (Fig. S8) [18]. Poly-1 was in-
soluble in dichloromethane, chloroform, THF and DMF, but partially
soluble in DMSO and methanol, while quite in water. In particular, the
number-average degree of polymerization (N) of Poly-1 was considered
to be the same as that of Poly-1a because the conditions for hydrolysis
reaction were mild to the backbone structure.
To confirm whether π-π stacking interaction between the phenyle-
nediethynylene units was the stabilizing force to the helical structure in
Poly-1, we investigated the fluorescence spectra of Poly-1 in mixed
DMSO/water. Compared to water, DMSO was a good solvent for the
phenylenediethynylene backbone. As shown in Fig. 3a, the fluorescence
intensity of Poly-1 at around 570 nm significantly decreased with in-
creasing water content. The low fluorescence intensity in water was a
typical result of helical folding attributed to the strong π-π stacking
interaction between phenylenediethynylene units of Poly-1 [24,25].
Also, this result was further confirmed by the UV–vis absorption
spectra. When the content of water increased, an apparent hypochromic
effect was observed with a slight red shift from 338 to 340 nm in the
main chain (Fig. 3b), which was described as the promotion of π-π
stacking interaction [26]. Obviously, π-π stacking interaction was a
stabilizing force to the helical structure in Poly-1.
Interestingly, the electrostatic repulsion existed between the ad-
jacent negatively charged pendants in Poly-1. To determine its role in
stabilizing the helical structure, we investigated the effect of different
NaCl concentration on the degree of the preferred-handedness of Poly-
1 at [θ]max wavelengths. In particular, the degree of the preferred-
handedness was evaluated by the Kuhn dissymmetry factor g (= △ε/ε,
in which △ε = [θ]/3298) [27]. Fig. S9 showed that the g-value de-
creased by 12% with increasing NaCl concentration, indicating that the
helix-forming ability of Poly-1 was decreased [22]. The presence of
NaCl was anticipated to shield the charged pendants so that the elec-
trostatic repulsion was weakened. Meanwhile, the dehydration of NaCl
attenuated the hydration of the pendants [28]. As a result, the polymer
chain started to shrink, which was unfavorable for the helix formation.
Therefore, these results demonstrated that the helical folding behavior
of Poly-1 in water was mainly driven by the hydrophobic effect and this
helical structure was further stabilized via π-π stacking interaction and
electrostatic repulsion.
3.2. Helical folding of Poly-1
The helical folding behavior of Poly-1 was studied by CD and
UV–vis absorption spectra. The concentration of monomer unit was
chosen as the concentration of Poly-1. As shown in Fig. 1, monomer 1
exhibited weak absorption peak at 300 nm, which was ascribed to
phenyl rings [19], and no absorption was observed above 318 nm.
However, three absorption peaks were found at 300, 318 and 340 nm
for Poly-1. The former absorption peak was again assigned to phenyl
rings, while the latter two to the polymer main chain. In addition,
monomer 1 showed no Cotton effects in water at wavelengths greater
than 300 nm. But strong negative Cotton effects were observed at 323
and 344 nm. These results indicated that the observed Cotton effects for
Poly-1 were not attributed to the chiral center of 1 or intermolecular
chiral assembly, but attributed to the helical chirality of the main chain
[20]. In other words, Poly-1 main chain definitely possessed a pre-
ferred-handed folded helix in water.
Next, we investigated helical folding mechanism of Poly-1. Due to
the amphiphilic nature of Poly-1, the helical folding in water could be
driven by hydrophobic effect [21]. Moreover, this behavior would
occur in a preferred-handed screw sense directed by L-alaninate sodium
pendants. Usually, the folded helical structures could be stabilized by
intramolecular hydrogen bonding and/or the π-π stacking interaction
[22]. The H/D exchange experiments based on solution-state FTIR
spectra of monomer 1 and Poly-1 were conducted in the dilute solution
(20 mM) to determine the presence/absence of hydrogen bonding
(Fig. 2). For monomer 1, the absorption band at 1637 cm−1 corre-
sponded to the C=O stretching amide I band. Interestingly, no amide II
band of N-H bending was observed in D2O at the range from 1570 to
1515 cm−1, but the amide II' absorption band of N-D bending at
1446 cm−1 appeared due to the deuterated NH groups [23]. These re-
sults suggested that the amide groups in monomer 1 formed hydrogen
bonds with water. Similar results could be found for Poly-1 in D2O. The
Fig. 3c showed that the Cotton effects for Poly-1 were varied at
different DMSO/water ratios. The relatively weak Cotton effects were
observed at DMSO/water of 9/1, indicating that Poly-1 adopted a he-
lical structure to some extent. However, the Cotton effect intensities
increased by about 4-fold in the presence of 80 vol% of water. Further
addition of water led to ∼14.2% decrease. These results implied that
the degree of the preferred-handedness for Poly-1 could be modulated
in the mixtures of DMSO and water. In contrast, the g-values of Poly-1
10

Y. Qiu, et al.
Polymer170(2019)7–15
Fig. 3. Fluorescence (a; λex = 360 nm), UV–vis absorption (b) and CD spectra (c) of Poly-1 in DMSO/water (v/v). (d) Effect of water content on g-values of Poly-1 at
the [θ]max wavelengths. All spectra were measured in a 1 mm quartz cell at ambient temperature. [Poly-1] = 0.62 mM.
at [θ]max wavelengths were also calculated. As shown in Fig. 3d, the g-
value reached a maximum at a DMSO/water ratio of 2/8, which in-
dicated that the helix-forming ability of Poly-1 was enhanced at this
ratio [22]. However, Poly-1 still showed a good helix-forming ability in
water with a g value as high as 2.38 × 10−3, only 14.7% less than the
maximum value. In order to simplify the system, we chose water as
solvent for the subsequent investigations.
implied a higher aggregate beyond the dimeric stage for DiOC2(5) was
obtained [31]. The solution color changed from peachblow to purple
red. Plotting the absorption band at 527 nm versus the concentration of
Poly-1 revealed that the formation process of the aggregates was ac-
complished in the presence of 1.0 equiv of Poly-1, corresponding to a
ratio of one monomer unit per DiOC2(5) (Fig. S10b). However, the
aggregates started to disassemble into dimer (540 nm) and monomer
(598 nm) in the presence of 2.0 equiv of Poly-1, resulting in a light
purple solution. Upon further addition of Poly-1 to 4.0 equiv, the ab-
sorption band in the dimer was red-shifted to 552 nm, accompanied
with a slight hyperchromic effect. At the same time, the absorption
band at 598 nm also showed the hyperchromic effect. As a result, the
light purple solution transformed to deep purple. Interestingly, the
spectral shape seemingly returned to the original one by loading 16
equiv of Poly-1, in spite of about 22 nm red-shift from the original one.
So, a navy blue solution was obtained. Figs. S10b and S10c showed,
upon further increasing Poly-1, there were almost no consistent red-
shift and hyperchromic effect of the resultant absorption band, clearly
indicating that the disassembly process completed.
3.3. Concentration-dependent DiOC2(5) aggregation and disassembly
Fig. 4a showed that the aqueous solutions of the pure Poly-1 and
DiOC2(5) were colorless and peachblow, respectively. Upon mixing
Poly-1 into DiOC2(5) aqueous solution, various colors from peachblow
to purple red and then to purple and finally to navy blue could be
observed immediately when the concentration of Poly-1 increased from
0 to 16 equiv (Fig. 4a). This result suggested that there existed some
specific interactions between Poly-1 and DiOC2(5) and these interac-
tions would be changed at different ratios.
UV–vis absorption spectra of the pure Poly-1 and DiOC2(5) with
different content of Poly-1 in water were measured (Fig. 4b and Fig.
S10a). For Poly-1, no absorption peak was found in the wavelength
range of 450–700 nm. The pure DiOC2(5) featured the monomer ab-
sorption band at 576 nm with a vibrational shoulder at about 548 nm
[29]. When the concentration of Poly-1 increased from 0 to 0.6 equiv,
the absorption at 576 nm gradually decreased and finally blue-shifted to
533 nm, indicative of the transformation of DiOC2(5) monomers to
face-to-face π-stacked dimers (H-dimers) [30]. In addition, an isosbestic
point at 530 nm clearly suggested an equilibrium between monomer
and dimer. As the concentration of Poly-1 was increased to 1.0 equiv,
the absorption curves did not pass the isosbestic point and the dimeric
absorption band at 533 nm took a blue-shift to 527 nm. These results
To determine the type of the aggregates, CD measurement was
conducted. As shown in Fig. 5, the pure DiOC2(5) displayed no CD
signal. However, upon addition of Poly-1 from 0 to 1.0 equiv, the po-
sitive CD signals at 520 nm for H-dimers and the negative at 476 nm
were observed, which was centered at 505 nm. Such biphasic CD signals
were attributed to the exciton coupling interaction between the ad-
jacent H-dimers. This suggested that the dimers were arranged on the
top of each other, resulting in formation of the higher aggregates, i.e. H-
aggregates [32]. When an excess of Poly-1 was added, the biphasic CD
signals disappeared due to the absence of exciton coupling interaction
among dimers [33]. Meanwhile, an increasing intensity of CD signal at
606 nm for the monomeric DiOC2(5) and a decreasing at 520 nm could
11

Y. Qiu, et al.
Polymer170(2019)7–15
signal from 611 to 644 nm along with a modest intensity enhancement.
This was in good agreement with the disassembly of H-aggregates,
because the isolated DiOC2(5) molecules did not show aggregation-
caused quenching (ACQ) behavior any more [35].
3.4. Binding mode of DiOC2(5) to Poly-1
The above UV–vis absorption, CD and fluorescence spectral changes
revealed that DiOC2(5) could be bound to Poly-1. For insight under-
standing of the binding behavior, the binding modes of DiOC2(5) to
Poly-1 were explored. We initially performed the AFM measurements
for Poly-1 in the absence and presence of DiOC2(5). Samples for AFM
measurements were prepared by spin coating dilute solutions onto
freshly-cleaved mica substrates and then dried in vacuum overnight. As
shown in Fig. 6a and b, Fig. S12a and S12b, the average heights of Poly-
1
and
DiOC2(5)
were
approximately
1.66
0.21
and
0.43
0.04 nm, respectively. The swollen height for Poly-1 with fully
conjugated backbone could be estimated using the radius of gyration of
a worm-like chain [36,37], Rg = (Nb2/12)1/2, where the degree of
polymerization N = 82 for Poly-1 and average statistical segment
length b = 0.68 nm [38–41] were taken.
Moreover, the height of the dried polymer in vacuo was usually
about 40% of the diameter (2Rg) of swollen one [42,43]. As a result, the
estimated dried height was about 1.42 nm, which was in agreement
with that obtained from AFM observation at ambient atmosphere. This
result also implied that single chain of Poly-1 was well dispersed in
such a dilute aqueous solution. The average height increased to
Fig. 4. (a) Sample colors corresponding to the pure Poly-1 and DiOC2(5) with
different content of Poly-1. (b) UV–vis absorption spectra of the pure Poly-1
and DiOC2(5) in the presence of Poly-1 in water. All spectra were measured in
a 2 mm quartz cell at ambient temperature. The concentrations of the pure
Poly-1 and DiOC2(5) were 832 and 52 μM, respectively. (For interpretation of
the references to color in this figure legend, the reader is referred to the Web
version of this article.)
2.04
0.25 nm after complexation with 1.0 equiv of DiOC2(5), in-
dicative of the mode of surface binding (Fig. 6c and Fig. S12c) [44]. In
other words, DiOC2(5) H-aggregates were arranged on the periphery of
Poly-1 helix. In contrast, after the complete disassembly of H-ag-
gregates, the complex featured an average height of 1.67
0.23 nm
which was similar to that of pure Poly-1 (Fig. 6d and Fig. S12d). Such a
case excluded the possibility of surface binding.
Then, the 1H NMR spectra measurements of DiOC2(5) in absence
and presence of Poly-1 (Fig. 7) were also conducted. The obvious re-
sonance absorptions of aromatic protons assigned to DiOC2(5) ap-
peared ranging from 8 to 7 ppm in D2O, but disappeared completely
after the addition of 16 equiv of Poly-1. These results indicated that
DiOC2(5) was entrapped within Poly-1 helix, and thus the aromatic
signals were shielded [30]. Interestingly, these signals appeared again
when the system was treated with DMSO-d6 (D2O/DMSO-d6 = 6/4),
which still gave rise to a good helix-forming ability of Poly-1 (Fig. 3d).
In other words, the shielding effect at this moment was weakened be-
cause DiOC2(5) molecules were released from the well-defined Poly-1
helix. These results implied a fact that DiOC2(5) was inserted into the
helical groove or the cavity of Poly-1 helix in water.
However, we still needed to distinguish whether DiOC2(5) was
bound to Poly-1 via intercalation or groove binding. Fluorescence
quenching experiments using negatively charged iodide ion as a
quencher would clarify the binding mode. Fluorescence quenching
could be analyzed using the Stern-Volmer equation, F0/F = 1 + Ksv[Q],
where F0 and F were fluorescence intensities in the absence and pre-
sence of the quencher Q. The dynamic quenching constant (Ksv) was
calculated if plot of F0/F against [Q] displays a linear trend [45]. In
general, intercalative binding of a fluorophore led to a reduction in
fluorescence quenching because intercalation could protect the fluor-
ophore, to which the approach of the quencher was restricted [46]. In
contrast, for groove binding, the quenching efficiency should be im-
proved in the addition of a quencher like KI. The phenomenon was
associated with the change in ionic strength. The increase in the ionic
strength resulted in the release of the fluorophore from the groove and
thus a decrease in fluorescence yield [47]. As shown in Fig. 8, in aqu-
eous solution KI quenched the fluorescence of DiOC2(5) effectively and
Ksv value of DiOC2(5) was estimated to be 24.9 M-1. More importantly,
the Stern-Volmer plot obtained by addition of KI to the complex of
Fig. 5. CD spectra of DiOC2(5) in the presence of Poly-1 in water. The inset
showed the range from 440 to 560 nm. All spectra were measured in a 1 mm
quartz cell at ambient temperature. [DiOC2(5)] = 52 μM.
be seen. At last, only a single CD signal at 606 nm remained, indicative
of the complete disassembly of H-aggregates to monomers.
Fluorescence spectra were also performed to further confirm the
formation and disassembly of H-aggregates. As shown in Fig. S11a, the
pure DiOC2(5) showed a fluorescence at 611 nm in aqueous solution
upon excited at 470 nm. However, the addition of Poly-1 gradually
decreased the fluorescence intensity, which reached a minimum in the
presence of 1.0 equiv of Poly-1 (Fig. S11b). This result also verified the
formation of H-aggregates which usually acted as the poor emitters
[34]. Further addition of Poly-1 resulted in a red-shift of fluorescence
12

Y. Qiu, et al.
Polymer170(2019)7–15
Fig. 6. Typical tapping-mode AFM height images of pure Poly-1 (a) and DiOC2(5) (b), and the mixing with DiOC2(5) at a [Poly-1]/[DiOC2(5)] ratio of 1/1 (c) and
16/1 (d), respectively. Height profiles measured along the dashed lines were also shown underneath the images. The concentration of pure Poly-1 and DiOC2(5) was
30.4 μM in water. The substrate was mica.
Poly-1 and DiOC2(5) displayed an upward curve, indicative of both
static and dynamic quenching occurring [48]. These results showed
that KI quenching effect increased when DiOC2(5) was bound to Poly-
1, which was in consistent with the groove binding mode.
electrostatic interactions between the positively charged DiOC2(5)
molecules and the negatively charged pendants of Poly-1 [49].
Usually, the cyanine dye aggregation was primarily triggered by
hydrophobic and van der Waals interactions [50]. In this system, Poly-
1 could act as an electrostatic three-dimensional scaffold and partly
neutralized the electrostatic repulsive forces among DiOC2(5) mole-
cules. These positively charged molecules were located on the nega-
tively charged Poly-1 chains, allowing the removal of water molecules
from the vicinity of DiOC2(5) aromatic groups [9]. Afterwards, the
hydrophobic and van der Waals interactions played crucial roles, and
thus promoted the aggregation. As a result, we found the formation of
face-to-face H-dimers for DiOC2(5). The [DiOC2(5)]/[Poly-1] ratio of
1:1 was saturated for H-dimers to polymer chains. Moreover, H-dimers
would be arranged on the top of each other based on the screw sense of
templated Poly-1, resulting in the formation of chiral H-aggregates.
Notably, this type of foldamer featured ∼6 monomer units per turn
[20], which could complex with three dimers. This specific
3.5. Mechanism of concentration-dependent DiOC2(5) aggregation and
disassembly
Obviously, the above concentration-dependent aggregation de-
pended on the binding of DiOC2(5) to Poly-1. To determine the driving
force for the binding, we conducted isothermal titration calorimetry
(ITC) measurements. Fig. 9 showed that the saturation of binding
stoichiometry was at around 1:1, i.e., one monomer unit of Poly-1 per
DiOC2(5) molecule, or two monomer units per dimer. In addition, the
binding constant was calculated as 1.21 × 104 M−1. Besides, the ne-
gative ΔG value of −5.57 × 103 cal mol−1 revealed the binding of
DiOC2(5) to Poly-1 to be a spontaneous process, which was based on
13

Y. Qiu, et al.
Polymer170(2019)7–15
Fig. 7. 1H NMR spectra of DiOC2(5) in D2O (a) and DMSO-d6 (b) Poly-1 in D2O
(c), and DiOC2(5) with Poly-1 in D2O (d) and D2O/DMSO-d6 (6/4, v/v) (e) at
ambient temperature. [Poly-1] = 3.8 mM; [DiOC2(5)] = 0. 24 mM.
Fig. 9. Plots of heat rate versus time (up) and heat versus molar ratio of Poly-1
to DiOC2(5) (bottom) obtained from ITC experiments at 25 °C.
small, chiral DiOC2(5) H-aggregates were enriched on Poly-1 surface.
In contrast, when the ratio was large, DiOC2(5) molecules were in-
serted into the helical groove of Poly-1 in a single-molecule form.
Meanwhile, the solution color underwent an obvious change. The pre-
sent investigation is crucial not only in further insighting the aggrega-
tion and diassembly of cyanine dye but in widening applications ran-
ging from sensors to high anti-countfeits.
Fig. 8. Quenching of DiOC2(5) fluorescence by KI in the presence and absence
of Poly-1 in water at ambient temperature. [DiOC2(5)] = 52 μM; [Poly-1]/
[DiOC2(5)] = 16.
Acknowledgments
We acknowledge the financial support of the National Natural
Science Foundation of China (51433002 and 51773070) and Research
on geological technology and application of Hubei province (KJ2018-
21). We also thank the HUST Analytical and Testing Center for CD and
AFM Measurements.
complexation was illustrated in Scheme 1b. Interestingly, despite the
formation of chiral H-aggregates, the aggregates were disassembled by
further addition of Poly-1. The increase of negative charges from newly
added Poly-1 led to more anion binding sites, which attracted
DiOC2(5) molecules in H-aggregates. As a result, it gave rise to the
disassembly of H-aggregates. In particular, these disassembled dyes
were inserted into the helical grooves of Poly-1, probably due to the
hydrophobic effect, which decreased the local polarity around
DiOC2(5). Consequently, the energy gap between HOMO and LUMO of
the dye reduced, leading to a 22 nm red-shifted absorption band similar
to the original one [51]. The H-aggregates could be completely dis-
assembled to the monomeric molecules. More importantly, these
monomers were still dispersed within the helical grooves of Poly-1 and
exhibited a specific chirality, as illustrated in Scheme 1b.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.polymer.2019.02.063.
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