Melamine driven supramolecular self-assembly of nucleobase derivatives in water

Article abstract of DOI:10.1002/pola.28954

Water-soluble supramolecular polymers, especially made up of biomolecules, are ideally suited to build new biomaterials that can mimic or interact with dynamic, biological environments. Here, two derivatives from thymine (T), that is N-[2-(3,4-Dihydro-5-methyl-2,4-dioxo-1(2H)-pyrimidinyl)acetyl]-L-phenylalanine (T-phe) and N-(2-Aminoethyl)-3,4-dihydro-5-methyl-2,4-dioxo-1(2H)-pyrimidineacetamide (T-NH₂) were synthesized. Then the optimal condition for self-assembly of T-phe and T-NH₂ driven by melamine (M) was explored. It was observed that M/T kept at 1:3 with equivalent T-phe and T-NH₂ under neutral environment resulted in long fibers (>1 μm) with extremely high aspect ratios, which suggested that electrostatic and π-stacking interactions could be effectively orchestrated by hydrogen bonds to direct the hierarchical assembly. Furthermore, hydrogels were spontaneously generated with a concentrated solution of T-phe, T-NH₂, and M due to the fibril entanglement. Given its biomimetic nature and efficient self-assembly process, this newly developed supramolecular polymer stacked by tetrameric structures represented an innovative concept and pathway for novel bio-inspired materials.

Full text of DOI:10.1002/pola.28954

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Melamine Driven Supramolecular Self-Assembly of Nucleobase
Derivatives in Water
Yan Xiao , Jiaxiao Zhang, Meidong Lang
Shanghai Key Laboratory of Advanced Polymeric Materials, Key Laboratory for Ultrafine Materials of Ministry of Education,
School of Materials Science and Engineering, East China University of Science and Technology, Shanghai 200237, China
Correspondence to: Y. Xiao (E-mail: yxiao@ecust.edu.cn) or M. Lang (E-mail: mdlang@ecust.edu.cn)
Received 3 November 2017; accepted 2 January 2018; published online 00 Month 2018
DOI: 10.1002/pola.28954
ABSTRACT: Water-soluble supramolecular polymers, especially
made up of biomolecules, are ideally suited to build new bio-
materials that can mimic or interact with dynamic, biological
environments. Here, two derivatives from thymine (T), that is
N-[2-(3,4-Dihydro-5-methyl-2,4-dioxo-1(2H)-pyrimidinyl)acetyl]-
L-phenylalanine (T-phe) and N-(2-Aminoethyl)-3,4-dihydro-5-
methyl-2,4-dioxo-1(2H)-pyrimidineacetamide (T-NH2) were syn-
thesized. Then the optimal condition for self-assembly of T-phe
and T-NH2 driven by melamine (M) was explored. It was
observed that M/T kept at 1:3 with equivalent T-phe and T-NH2
under neutral environment resulted in long fibers (>1 lm) with
extremely high aspect ratios, which suggested that electrostatic
and p-stacking interactions could be effectively orchestrated by
hydrogen bonds to direct the hierarchical assembly. Further-
more, hydrogels were spontaneously generated with a concen-
trated solution of T-phe, T-NH2, and
M due to the fibril
entanglement. Given its biomimetic nature and efficient self-
assembly process, this newly developed supramolecular poly-
mer stacked by tetrameric structures represented an innovative
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concept and pathway for novel bio-inspired materials.
2018
Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem.
2018, 00, 000–000
KEYWORDS: fiber; melamine; nucleobase derivatives; supramo-
lecular polymer; water-soluble
hydrophobic alkyl pocket was spaced and shielded.8 Boue
ꢀ
INTRODUCTION Rapid progress has been made in supramo-
lecular polymers since Lehn and coworkers firstly reported
that bifunctional diamidopyridines and uracil derivatives
formed linear chains via intermolecular force.1 Different
from conventional polymers linked by covalent bonds of
repeating units, supramolecular polymers are held together
by highly directional and reversible non-covalent interac-
tions, including coordination, hydrogen bond, p–p interac-
tion, and so on.2–4 Among them, hydrogen bonding is
considered as one of the most versatile interactions in fabri-
cation of functional materials due to its specificity and direc-
tionality. For instance, poly(ethylene/butylene) functionalized
with self-complementary quadruple hydrogen-bonded
ureido-pyrimidone (UPy) units preserved the combinational
advantages of excellent mechanical properties and feasible
processibility.5 However, H-bonding usually weakens under
polar conditions like in water, where assemblies purely
based on H-bonds become unstable.6 Therefore, a hydropho-
bic microenvironment is usually built for H-bonding motifs
to promote their assembling in water.7 For example, hydro-
philic polyethylene glycol (PEG) end-capped with UPy units
and coworkers reported that alkylene spacers were placed in
between bis-urea motif and water-soluble ethylene oxide
oligomers to create a hydrophobic environment and thereby
formed long rigid filaments.9 Alternatively, electrostatic or p-
stacking interactions were more often combined with hydro-
gen bonding to obtain stable assemblies, owing to the strong
non-covalent force.10–12 Besenius and coworkers mixed the
oppositely charged dendritic peptide amphiphiles in a 1:1
ratio to supramolecular copolymers through a combination
of Coulomb attractive forces and hydrogen-bonding interac-
tions.13 Lloyd and coworkers reported that a family of aro-
matic carboxylic acid benzene 1,3,5-tricarboxamide (BTA)
modifications showed spontaneous nucleation and one-
dimensional growth resulting from the molecules stacking
into fibers through p-stacking interactions and amide–amide
hydrogen bonding.14
In nature, molecular recognition including duplex DNA via
Watson–Crick base-pairing is ubiquitous. Such specific inter-
action is originated from non-covalent bonding between two
or more biomolecules. Over the last few decades, chemists
have been to go beyond the natural realm and to use
could not self-assemble into the nanofibers unless
a
Additional Supporting Information may be found in the online version of this article.
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EXPERIMENTAL
complementary nucleobase-pairing to construct novel supra-
molecular assemblies.15 Fenniri and colleagues pioneered the
supramolecular polymers with the heteroaromatic bicyclic
base GÙC, which resulted in remarkable helical rosette nano-
tubes.16–18 Zhu et al. reported that adenine-terminated
poly(E-caprolactone) and uracil-terminated poly(ethylene gly-
col) assembled in water on association of their complemen-
tary termini to form well-defined micelles capable of
doxorubicin uptake.19 However, small molecules including
canonical nucleobases and their corresponding mononucleo-
sides do not form H-bonded base-pairing in water.20 A nota-
ble exception is the G-tetrad formed by guanosine
compounds with the aid of cation coordination, leading to
polymeric structures in water and ultimately hydrogels.21,22
Such stable assemblies were attributed to the hydrophobic
surface with an area greater than about 1 nm2, which was
beneficial for stacking to build supramolecular polymers.23
Therefore, the combination of nucleobases with other moie-
ties was necessary to enlarge the hydrophobic surface and
thereby access higher-order supramolecular structure in
water.24 Recently, Sleiman and coworkers have reported that
cyanuric acid (CA) with three thymine-like faces reprog-
rammed the assembly of unmodified poly(adenine) (poly(A))
into long and abundant fibers with cyclic hexamers as inter-
nal structure.25 Hud et al. have demonstrated that
adenosine-50-monophosphate (AMP) was able to form supra-
molecular assemblies of stacked hexads with CA in water.26
Up to date, most existing supramolecular polymers based on
nucleobases have focused on six-membered supermacro-
cycles as intermediate structure.
Materials
Thymine (99%), bromoacetic acid (99%), L-phenylalanine
(98%), and N-Boc-ethylenediamine (98%) were purchased
from Aladdin reagent and used without further purification.
N,N-Dimethylformamide (DMF) was refluxed with calcium
hydride (CaH2) and distilled in vacuum. 1-Ethyl-3-(3-
dimethyl-aminopropyl)-1-carbodiimide hydrochloride (EDCꢀHCl)
and N-hydroxysuccinimide (NHS) were purchased from Aldrich
and used as received. All other reagents and solvents were pur-
chased from Aladdin and used without further purification.
General Characterization
1H NMR (400 MHz) spectra in dimethyl sulfoxide (DMSO-d6)
and 13C NMR (100 MHz) spectra in deuteroxide (D2O) were
recorded at room temperature on a Bruker Avance series
instrument. UV/Vis absorbance spectra were recorded on a
Perkin Elmer Lambda Bio40 UV spectrometer with different
concentration. Dynamic light scattering (DLS) measurements
were carried out using a Brookhaven BI-200SM goniometer
equipped with a BI-9000AT digital correlator. The aggregates
in aqueous solution were filtered through a 0.45 mm cellu-
lose membrane filter before analysis. Potentiometric Titrant
(ZDJ-4A) was used to measure the pKa values of both T-phe
and T-NH2. 0.01 molꢀL21 NaOH and HCl were used as stan-
dard titrated solution of T-phe and T-NH2, respectively. The
end point of titration was determined by the jump of pH
value. AFM imaging was performed on freshly cleaved mica
using a MultiMode 8 microscope with a Nanoscope V con-
troller (Bruker) in ScanAsyst mode. A 5-ml mixture solution
was dropped and dried at 15 8C overnight. TEM (JEOL/JEM-
1400EXII) was used to observe the morphology of the aggre-
gates at an acceleration voltage of 60 keV. Typically, a drop
of mixture solution was added on the copper grid followed
by drying at room temperature before observation. The ESI
mass spectra were recorded in CH3OH/H2O (1/1) with a
Bruker Daltonics microTOFTM focus spectrometer. SEM (S-
4800N) was used to investigate the morphology of the
hydrogels. The hydrogel samples were put on the silicon
wafer and dried at room temperature overnight.
Melamine (M) is a versatile molecule as scaffolding components
in the field of supramolecular chemistry due to its nine H-
bonding sites and aromatic planar ring structure.27 Zhang et al.
reported the synthesis of 2D organic micro sheets using dialky-
lated M derivatives with halogen acid.28 Kitamura et al. devel-
oped
a well-defined columnar structure based on CA-
functionalized perylene bisimide and substituted M.29 Among
the natural nucleobases, thymine (T) contains complementary
NHꢀꢀꢀO and NHꢀꢀꢀN hydrogen bonds with M.30 Intrigued by this
recognition unit, Liu et al. synthesized amphiphilic polystyrene-
co-poly(N-acryloylthymine), which could be used for detecting
M.31 Besides, Bong et al. reported that M-displaying a-peptides
were able to mediate discrete assembly of T-rich DNA/
RNA.32–34 However, this molecular recognition has rarely been
applied to supramolecular polymer in aqueous environment
owing to poor water-solubility of T and M.
Synthesis of (Thymine-1-yl) Acetic Acid
According to the literature, a typical procedure for synthesis
of (thymine-1-yl) acetic acid was described as follows.35 Thy-
mine (3.78 g, 30 mmol) was dissolved in 20 ml KOH
(6.458 g, 115 mmol) aqueous solution, which was then kept
at 40 8C. Afterwards, a solution of bromoacetic acid (6.25 g,
45 mmol) in deionized water (10 ml) was slowly added over
30 min. The reaction mixture was stirred for another 30 min
at 40 8C. Then it was cooled to room temperature and pH
was adjusted to 5.5 with HCl. The solution was then put in a
refrigerator for 2 h. Any precipitate formed was removed by
filtration. The solution was then adjusted to pH 2 with HCl
and put in a freezer for 2 h. The resultant white precipitate
was isolated by filtration, washed with water and dried to
afford the white product. 1H NMR (400 MHz, DMSO-d6, d):
7.50 (s, AC(CH3)@CHA), 4.37 (s, ACH2COOH), 1.76 (s,
AC(CH3)@CHA). 13C NMR (100 MHz, D2O, d): 11.9
In this paper, two derivatives from T, bearing terminal amide
and carboxyl groups respectively, were proposed. M was
expected to drive self-assembly of nucleobase derivatives in
water to build supramolecular assembly by the combina-
tional interactions of H-bonds, p-stacking, and Coulomb
attractive forces. The optimal condition of self-assembly in
water was extensively investigated. The fibrillar structures
were observed and mechanism of self-assembly was further
suggested. We also expected to fabricate hydrogel based on
the self-assembled system in concentrated solution.
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SCHEME 1 Syntheses of T-NH2 (1), T-phe (2) and hydrogen-bonding recognition between melamine (M) and thymine (T). [Color
figure can be viewed at wileyonlinelibrary.com]
(AC(CH3)@CHA), 51.4 (ACH2COOH), 111.3 (AC(CH3)@CHA),
141.8 (AC(CH3)@CHA), 160.0 (ACH2COOH), 176.4, 177.6
(ACONHCOA).
1 h and vacuum dried. Then 30 ml of deionized water was
added and basified to pH 11, the resulted solution was
obtained by filtration. After evaporation, the product was
Synthesis of N-[2-(3,4-Dihydro-5-Methyl-2,4-Dioxo-1(2H)-
Pyrimidinyl)Acetyl]-L-Phenylalanine (T-phe)
According to the literature, (thymine-1-yl) acetic acid
(184 mg, 1 mmol) was added to a 25 ml Schlenk flask fol-
lowed by addition of 10 ml dry DMF.36 After the solid was
completely dissolved in DMF, EDCꢀHCl (210.9 mg, 1.1 mmol)
and NHS (126.5 mg, 1.1 mmol) were introduced and stirred
at room temperature for 1 h. L-Phenylalanine (166 mg, 1
mmol) and Na2CO3 (84.8 mg, 0.8 mmol) were dissolved in
20 ml of water with stirring and added to the above DMF
solution. The resulted reaction mixture was stirred at room
temperature for 24 h followed by vacuum drying. Then
30 ml of deionized water was added and acidified to pH 3,
the resulted product was obtained by filtration, washed with
deionized water, and then dried in vacuo. The white solid
was purified by HPLC using water–acetonitrile as eluent
(from 8:2 to 4:6). 1H NMR (400 MHz, DMSO-d6, d): 8.55 (s,
ACH2CONHA), 7.45 (m, AC(CH3)@CHA), 7.35 (m, PhAH),
4.43 (m, ANHCH(COOH)CH2A), 4.19 (m, ACH2CONHA),
3.04, 2.89 (dd, ACH2Ph), 1.73 (s, AC(CH3)@CHA). 13C NMR
(100 MHz, D2O, d): 11.8 (AC(CH3)@CHA), 37.6 (ACH2Ph),
55.9 (ACH2CONHA), 59.3 (ANHCH(COOH)CH2A), 111.3
(AC(CH3)@CHA), 126.8, 128.6, 129.0, 135.2 (Ar), 142.2
(AC(CH3)@CHA),
160.0
(NHCH(COOH)CH2A),
169.4
(ACH2CONHA), 177.2, 177.8 (ACONHCOA).
Synthesis of N-(2-Aminoethyl)-3,4-Dihydro-5-Methyl-2,4-
Dioxo-1(2H)-Pyrimidineacetamide (T-NH2)
(Thymine-1-yl) acetic acid (184 mg, 1 mmol) was added into
a 25 ml Schlenk flask followed by addition of 10 ml dry
DMF. After the solid was dissolved in DMF completely,
EDCꢀHCl (210.9 mg, 1.1 mmol) and NHS (126.5 mg, 1.1
mmol) were introduced and stirred at room temperature for
1 h. N-Boc-ethylenediamine (160 mg, 1 mmol) was added to
the above DMF solution. The resulted reaction mixture was
stirred at room temperature for 24 h and then vacuum-
dried. 2 ml DCM and 1 ml TFA were added into the reaction
mixture, which was kept stirring at room temperature for
FIGURE 1 (a) Diameters from DLS and (b) UV spectra of mix-
ture of M/T at different molar ratios: 0:3, 0.3:3, 0.6:3, 1:3, 3:3,
and 6:3. [Color figure can be viewed at wileyonlinelibrary.com]
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procedures reported by Katritzky and coworkers, (thymine-
1-yl) acetic acid was prepared by the substitution reaction of
T and bromoacetic acid.35 (Thymine-1-yl) acetic acid was
subsequently reacted with L-phenylalanine to yield T-phe.
Meanwhile, (thymine-1-yl) acetic acid could also react with
N-Boc-ethylenediamine followed by deprotection to prepare
T-NH2. The molecular structures of T-phe and T-NH2 were
1
1
characterized by H NMR and 13C NMR spectroscopy. The H
NMR spectrum of (thymine-1-yl) acetic acid and the corre-
sponding peak assignments were shown in Supporting Infor-
mation Figure S1a. After amidation with L-phenylalanine,
new signals at 2.89, 3.04, and 7.35 ppm related to T-phe
appeared (Supporting Information Fig. S1b). If amidation
was carried out with N-Boc-ethylenediamine followed by
deprotection, it was found in Supporting Information Figure
S1c that additional peaks at 2.86 and 3.3 ppm were attrib-
uted to T-NH2. The 13C NMR spectra of T-phe and T-NH2
shown in Supporting Information Figure S2 further con-
firmed the structure of the target products. Consequently, all
FIGURE 2 (a) Diameters from DLS, (b) UV spectra of mixture of
T-phe and T-NH2 with M while the molar ratio of T-phe and T-
NH2 was different in phosphate buffer. [Color figure can be
viewed at wileyonlinelibrary.com]
recrystallized from methanol as purified and colorless mate-
1
rial. H NMR (400 MHz, DMSO-d6, d): 8.53 (s, ACH2CONHA),
7.50 (d, AC(CH3)@CHA), 4.34 (s, ACH2CONHA), 3.35
(ACH2CH2NH2 1 H2O), 2.86 (t, ACH2CH2NH2), 1.75 (s,
AC(CH3)@CHA). 13C NMR (100 MHz, D2O, d): 11.2
(AC(CH3)@CHA), 36.9 (ACH2CH2NH2), 39.0 (ACH2CH2NH2),
50.6 (ACH2CONHA), 111.0 (AC(CH3)@CHA), 143.1
(AC(CH3)@CHA), 152.3, 166.9 (ACONHCOA), 170.2
(ACH2CONHA).
RESULTS AND DISCUSSION
Synthesis and Characterization
Two derivatives of thymine (T), i.e. N-[2-(3,4-Dihydro-5-
methyl-2,4-dioxo-1(2H)-pyrimidinyl)acetyl]-L-phenylalanine
(T-phe)
and
N-(2-Aminoethyl)-3,4-dihydro-5-methyl-2,4-
FIGURE 3 (a) Diameters from DLS, (b) UV spectra of mixture of
T-phe and T-NH2 with M at pH 0.9–13.5. [Color figure can be
viewed at wileyonlinelibrary.com]
dioxo-1(2H)-pyrimidineacetamide (T-NH2), were designed
and synthesized as described in Scheme 1. Following the
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in the process of denaturation and renaturation of polynu-
cleotides and nucleic acids, owing to H-bonds and stacking
interactions between the bases.37,38 Herein, UV absorbance
at 270 nm was employed to evaluate the efficiency of base
stacking [Fig. 1(b)]. It was found that the peak intensity
gradually decreased with increasing M concentration until
1M-3T reached. Therefore, 1M-3T was the appropriate ratio
for the mixture, where their self-assembly was pronounced.
Meanwhile, a solution of M (0.1 mM) was added into the
phosphate buffer solutions with different molar ratios of T-
phe and T-NH2. The size of the mixture by DLS as a function
of the molar ratio of T-phe and T-NH2 was also plotted and
presented in Figure 2(a). When T-phe concentration was
gradually increased with a fixed 1M-3T, the dimension was
firstly increased and then dropped quickly. When T-phe was
equivalent to T-NH2, a maximum size was reached due to the
strongest Coulomb attractive force between amide and car-
boxyl groups. Figure 2(b) showed the UV spectra of different
molar ratios of T-phe and T-NH2. It was observed that the
peak at 270 nm was the lowest when the ratio of T-phe to T-
NH2 was 1.5:1.5 (1.5T-phe–1.5T-NH2), where the hypochro-
mic effect was the most significant. Consequently, the opti-
mum ratio for the most efficient recognition and base
stacking was determined as 1M-1.5T-phe–1.5T-NH2. It was
ascribed that the strongest attraction was resulted from the
equivalence of T-phe and T-NH2, which were interacted with
three complementary faces of M.
FIGURE 4 Diameters from DLS for T-phe and T-NH2 with M at
various concentrations ranging from 0.0025 mM to 0.2 mM
([M]) in phosphate buffer.
NMR results demonstrated that two derivatives of T bearing
terminal amide and carboxyl groups were successfully
synthesized.
Self-Assembly Behavior of T-phe, T-NH2, and M
To optimize the self-assembly condition for three compo-
nents including M, T-phe, and T-NH2, DLS and UV/Vis spec-
trophotometry were used to estimate the assemblies when
varying their proportion. First, different M solutions ranging
from 0 mM to 0.6 mM were added into the phosphate buffer
solution of T-phe (0.15 mM) and T-NH2 (0.15 mM) based on
the molar ratios 0:3, 0.3:3, 0.6:3, 1:3, 3:3, and 6:3. As shown
in Figure 1(a), the hydrodynamic diameter of the mixture
measured by DLS as a function of the molar ratio of M and T
(T-phe and T-NH2) was plotted. With increasing M concentra-
tion, the mixture size dramatically increased from around
50 nm to 680 nm. An equilibrium was found at a molar ratio
of 1:3 (1M-3T), which indicated the optimal proportion. The
hypochromic effect is reflected by the UV absorption change
Self-Assembly at Different pH Values and Concentrations
In the following study, assemblies formed from 1M-1.5T-
phe–1.5T-NH2 were generally applied for other investigations
unless otherwise noted. Figure 3(a) revealed the change of
assembly size by DLS against pH variation. A maximum size
of assemblies was observed at neutral condition (pH 7.2),
while both T-phe (pKa 5 3.45, Supporting Information Fig.
S3a) and T-NH2 (pKa 5 9.65, Supporting Information Fig. S3b)
were in their charge state. However, the size decreased
quickly at either pH < 3.45 (T-phe was uncharged, T-NH2 was
FIGURE 5 Images of T-phe and T-NH2 with M supramolecular self-assemblies. (a) TEM image and (b) AFM micrograph of self-
assembled T-phe and T-NH2 with M fibers. [M] 5 0.1 mM, [T-phe] 5 [T-NH2] 5 0.15 mM in phosphate buffer. [Color figure can be
viewed at wileyonlinelibrary.com]
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SCHEME 2 Model of the proposed self-assembly process. [Color figure can be viewed at wileyonlinelibrary.com]
positively charged) or pH > 9.65 (T-phe was negatively
charged, T-NH2 was uncharged) environment owing to the
disrupted electrostatic equilibrium. UV spectra of mixture
[Fig. 3(b)] further confirmed the change of base stacking
under extremely acidic and basic environment. Compared
with other curves, it was found that negative band at 236 nm
changed sharply at both conditions, indicating the non-
efficient self-assembly of M. However, it was also observed a
peak arising at 236 nm only for pH at 0.9, which might be
caused by free M as shown in Supporting Information Figure
S4. Therefore, it could be concluded that the size and base
stacking of self-assembly were able to be adjusted by chang-
ing solution pH owing to the varying Coulomb attractive
forces, which agreed with results reported in literature.13
presented here was in the range of similar assembled mech-
anism. A rough calculation was also carried out for an M-3T
structure, which included 1.2 nm as reported non-substitu-
tion39 plus approximately 1.5 nm substitution. So a theoreti-
cal diameter of 2.7 nm for the assemblies was estimated, in
accordance with the AFM results.
Proposed Mechanism for Supramolecular Assembly
Herein, M-driven nucleobase derivatives assembly via non-
covalent interactions led to the fibrous structure, suggesting
that the process was close to a supramolecular polymeriza-
tion.40,41 It may thus occur via two distinct mechanisms: iso-
desmic (step-growth) or cooperative polymerization
(nucleation–growth).42 The self-assembly at different concen-
trations (Fig. 4) showed that the self-assembly only occurred
above a critical concentration, which was more inclined to
cooperative nucleation–growth polymerization.25,43 Consider-
ing the fiber’s diameter of the supramolecular structure, we
proposed that in the nucleation process tetrameric struc-
tures formed by multiple hydrogen bonds between M and T,
which was consistent with the literature.44 Subsequently, the
tetrameric structures were extended by p-stacking and elec-
trostatic interaction during the elongation process.
We further investigated the effect of concentration on assem-
blies by DLS. As described in Figure 4, the hydrodynamic
diameter of mixture as a function of the concentration of M
was presented. In general, the lower the concentration, the
smaller size of the assembly. In particular, a sharp increment
of diameter was observed at the concentration of 0.03 mM,
which was suggested as the critical concentration for self-
assembly of the mixture. Sleiman and coworkers have
reported that the process of CA-mediated poly(A) assembly
on dilution occurred with a sharp transition as a function of
concentration, which indicated a cooperative assembly onset
at a critical concentration of the small molecule.25 Therefore,
our result presented here hinted that the process of M-
driven T derivatives assembly exhibited a similar cooperative
nucleation–elongation pathway.
However, the tetrameric structures may differ in the nucle-
ation process. As illustrated in Scheme 2, there were two
Microscopic Images on Supramolecular Assembly
The morphology of the assemblies was firstly visualized by
transmission electron microscopy (TEM) as shown in Figure
5(a), which revealed fiber structures with extremely high
aspect ratios in phosphate buffer solution. Furthermore,
image from atomic force microscope (AFM) as shown in Fig-
ure 5(b) implied the accurate diameter of assemblies. For
example, a diameter between 2.6 and 3.2 nm was detected
for a single fiber perpendicular to the image plane (i.e., the
height). It has also been reported that the diameter of the
self-assembly fiber from 2,4,6-triaminopyrimidine modified
with succinate group (TAPAS) and cyanuric acid (CA) was
between 1.5 and 2.2 nm.39 Moreover, the diameter of the
nanotubes from the GÙC motif was between 3.1 and
3.7 nm.18 Thus, it could be speculated that the diameter
FIGURE 6 ESI-MS of a 0.1 mM solution of T-phe and T-NH2
with M. The peaks at 127.1, 227.1, 354.1, 376.1, 458.2, 602.2,
685.2, 950.6, and 1016.3 corresponded to the non-covalent
intermediate species of the self-assembled structures (1-mer to
4-mer). [Color figure can be viewed at wileyonlinelibrary.com]
6
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FIGURE 7 (a) Inverted bottle test demonstrating gelation as a function of concentration. Each solution kept the ratio of [M]:[T]
(1:3) unchanged while [T-phe] was equal to [T-NH2], all buffered at pH 7.2, (b) SEM image of self-assembled T-phe 1 T-NH2 with M
gel at 20 wt %. [Color figure can be viewed at wileyonlinelibrary.com]
CONCLUSIONS
possible combinations including 1M-3T-phe/1M-3T-NH2
(alternate self-assembly) and 1M-2T-phe–1T-NH2/1M-T-phe–
In summary, T-phe and T-NH2, bearing the terminal amide
2T-NH2 (synergistic self-assembly) in view of the repulsive
and carboxyl group respectively, have been successfully syn-
or attractive Coulomb interactions.13 To confirm the nucle-
thesized. The optimal condition for self-assembly of T-phe
ation process, electrospray ionization mass spectrometry
and T-NH2 driven by M was determined at 1M-1.5T-phe–
(ESI-MS) was conducted to investigate the composition of
1.5T-NH2 under neutral environment. These molecules self-
the non-covalent intermediate species (1-mer to 4-mer) of
assembled into fibrous supramolecular structure with a few
the parent tetrameric structure. As shown in Figure 6, two-
nanometers in diameter. It was preliminarily demonstrated
weak signals at m/z 5 950.6 [1M-T-phe–2T-NH2 1 H2O 1
that the self-assembly process was similar to cooperative
nucleation–growth polymerization and the nucleation pro-
cess was mainly promoted by synergistic self-assembly.
Na]1 and m/z 5 1016.3 [1M-2T-phe–1T-NH2 1 H]1 were
found. In contrast, the signals of 1M-3T-phe and 1M-3T-NH2
were not shown, possibly attributing to the concentrated
Moreover, hydrogels could be spontaneously generated with
charge of 3T-phe-M or 3T-NH2-M. Therefore, it was suggested
increased concentrations of building molecules of T and M at
that the process preferred to occur through a synergistic
their optimized ratio. This efficient self-assembly system
based on biomimetic compounds would have great potential
rather than an alternate self-assembly.
in supramolecular polymers and bio-inspired materials.
Gelation of the Supramolecular Assembly
It has been known that DAP-Cyco4 assemblies formed linear
fibers in dilute solutions and a metastable hydrogel in con-
centrated solutions.26 Therefore, we hypothesized that fibril-
This work was supported by the National Natural Science Foun-
ACKNOWLEDGMENTS
dation of China (21674037) and the Shanghai Scientific and
Technological Innovation Project (14520720600).
lar structure presented here could also generate hydrogels at
high concentrations. To verify the assumption, the concentra-
tions of M, T-phe, and T-NH2 were further increased at their
optimized ratio. As determined by the test tube inversion
method, assemblies displayed critical gel concentration
(CGC) at 20 wt %. Further increasing the concentrations to
30 wt % led to an opaque hydrogel [Fig. 7(a)], which was
attributed to the poor solubility of M. However, 30 wt %
solution of T-phe and T-NH2 in water without M was not
able to form gel (Supporting Information Fig. S5). Further-
more, SEM image of the transparent hydrogel was presented
in Figure 7(b), which revealed a highly branched network of
interlinked fibers owing to the entanglement. This observa-
tion was consistent with the formation of non-covalent cross-
linked supramolecular assemblies.26 Consequently, the self-
assembly system presented here demonstrated that the
nucleobase-based complex could be used as a building block
to generate hydrogels, which would be favorable for bio-
inspired material design and applications.
REFERENCES AND NOTES
1 C. Fouquey, J. M. Lehn, A. M. Levelut, Adv. Mater. 1990, 2,
254.
2 Z. Zhang, L. Shao, Y. Zhou, J. Yang, J. Polym. Sci. Part A:
Polym. Chem. 2017, 55, 3529.
3 A. J. P. Teunissen, J. A. Berrocal, C. Corbet, E. W. Meijer, J.
Polym. Sci. Part A: Polym. Chem. 2017, 55, 2971.
4 W. Seo, K. L. Carpenter, J. A. Gaugler, W. Shao, K. A.
Werling, P. M. Fournier, D. S. Lambrecht, A. Star, J. Polym.
Sci. Part A: Polym. Chem. 2017, 55, 1095.
5 B. J. B. Folmer, R. P. Sijbesma, R. M. Versteegen, J. A. J. van
der Rijt, E. W. Meijer, Adv. Mater. 2000, 12, 874.
6 J. M. Zayed, N. Nouvel, U. Rauwald, O. A. Scherman, Chem.
Soc. Rev. 2010, 39, 2806.
7 E. Krieg, M. M. Bastings, P. Besenius, B. Rybtchinski, Chem.
Rev. 2016, 116, 2414.
JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2018, 00, 000–000
7

JOURNAL OF
POLYMER SCIENCE
ARTICLE
WWW.POLYMERCHEMISTRY.ORG
8 P. Y. W. Dankers, T. M. Hermans, T. W. Baughman, Y.
Kamikawa, R. E. Kieltyka, M. M. C. Bastings, H. M. Janssen, N. A.
J. M. Sommerdijk, A. Larsen, M. J. A. van Luyn, A. W. Bosman,
E. R. Popa, G. Fytas, E. W. Meijer, Adv. Mater. 2012, 24, 2703.
26 C. Li, B. J. Cafferty, S. C. Karunakaran, G. B. Schuster, N. V.
Hud, Phys. Chem. Chem. Phys. 2016, 18, 20091.
27 B. Roy, P. Bairi, A. K. Nandi, RSC Adv. 2014, 4, 1708.
28 J. Xu, G. Wu, Z. Wang, X. Zhang, Chem. Sci. 2012, 3,
9 E. Obert, M. Bellot, L. Bouteiller, F. Andrioletti, C. Lehen-
3227.
ꢀ
Ferrenbach, F. Boue, J. Am. Chem. Soc. 2007, 129, 15601.
29 S. Yagai, M. Usui, T. Seki, H. Murayama, Y. Kikkawa, S.
Uemura, T. Karatsu, A. Kitamura, A. Asano, S. Shu, J. Am.
Chem. Soc. 2012, 134, 7983.
10 T. H. Rehm, C. Schmuck, Chem. Soc. Rev. 2010, 39, 3597.
11 N. Jiang, J. Shang, X. Di, M. K. Endoh, T. Koga, Macromole-
cules 2014, 47, 2682.
30 W. J. Qi, D. Wu, J. Ling, C. Z. Huang, Chem. Commun.
2010, 46, 4893.
12 Q. He, S. Narayanan, D. T. Wu, M. D. Foster, ACS Macro
Lett. 2016, 5, 999.
31 Y. Tao, Y. Yang, D. Shi, M. Chen, C. Yang, X. Liu, Polymer
2012, 53, 1551.
13 H. Frisch, J. P. Unsleber, D. Ludeker, M. Peterlechner, G.
Brunklaus, M. Waller, P. Besenius, Angew. Chem. Int. Ed. Engl.
2013, 52, 10097.
32 Y. Zeng, Y. Pratumyot, X. Piao, D. Bong, J. Am. Chem. Soc.
2012, 134, 832.
14 R. C. Howe, A. P. Smalley, A. P. Guttenplan, M. W. Doggett,
M. D. Eddleston, J. C. Tan, G. O. Lloyd, Chem. Commun. 2013,
49, 4268.
33 X. Piao, X. Xia, D. Bong, Biochemistry 2013, 52, 6313.
34 J. Mao, C. DeSantis, D. Bong, J. Am. Chem. Soc. 2017, 139,
9815.
15 J. L. Sessler, C. M. Lawrence, J. Jayawickramarajah, Chem.
Soc. Rev. 2007, 36, 314.
35 A. R. Katritzky, T. Narindoshvili, Org. Biomol. Chem. 2008, 6,
3171.
16 H. Fenniri, P. Mathivanan, K. L. Vidale, D. M. Sherman, K.
Hallenga, K. V. Wood, J. G. Stowell, J. Am. Chem. Soc. 2001,
123, 3854.
36 X. Li, Y. Kuang, J. Shi, Y. Gao, H. C. Lin, B. Xu, J. Am.
Chem. Soc. 2011, 133, 17513.
37 M. Gentner, M. G. Allan, F. Zaehringer, T. Schirmer, S.
Grzesiek, J. Am. Chem. Soc. 2012, 134, 1019.
17 J. G. Moralez, J. Raez, T. Yamazaki, R. K. Motkuri, A.
Kovalenko, H. Fenniri, J. Am. Chem. Soc. 2005, 127, 8307.
38 V. I. Danilov, FEBS Lett. 1974, 47, 155.
18 G. Borzsonyi, R. L. Beingessner, T. Yamazaki, J.-Y. Cho, A.
J. Myles, M. Malac, R. Egerton, M. Kawasaki, K. Ishizuka, A.
Kovalenko, H. Fenniri, J. Am. Chem. Soc. 2010, 132, 15136.
39 B. J. Cafferty, I. Gallego, M. C. Chen, K. I. Farley, R. Eritja, N.
V. Hud, J. Am. Chem. Soc. 2013, 135, 2447.
40 P. Besenius, J. Polym. Sci. Part A: Polym. Chem. 2017, 55,
34.
19 D. Wang, Y. Su, C. Jin, B. Zhu, Y. Pang, L. Zhu, J. Liu, C. Tu,
D. Yan, X. Zhu, Biomacromolecules 2011, 12, 1370.
41 L. Brunsveld, B. Folmer, E. Meijer, R. Sijbesma, Chem. Rev.
2001, 101, 4071.
20 P. O. P. Ts’O, I. S. Melvin, A. C. Olson, J. Am. Chem. Soc.
1962, 85, 1289.
42 S. Ogi, T. Fukui, M. L. Jue, M. Takeuchi, K. Sugiyasu,
Angew. Chem. Int. Ed. Engl. 2014, 53, 14363.
21 J. T. Davis, Angew. Chem. Int. Ed. Engl. 2004, 43, 668.
22 Y. Yu, D. Nakamura, K. Deboyace, A. W. Neisius, L. B.
Mcgown, J. Phys. Chem. B 2008, 112, 1130.
43 T. F. A. D. Greef, M. M. J. Smulders, M. Wolffs, A. P. H. J.
Schenning, R. P. Sijbesma, E. W. Meijer, Chem. Rev. 2009, 109,
5687.
23 D. Chandler, Nature 2005, 437, 640.
24 B. J. Cafferty, N. V. Hud, Isr. J. Chem. 2015, 55, 891.
44 R. F. Lange, F. H. Beijer, R. P. Sijbesma, R. W. Hooft, H.
Kooijman, A. L. Spek, J. Kroon, E. Meijer, Angew. Chem. Int.
Ed. Engl. 1997, 36, 969.
25 N. Avakyan, A. A. Greschner, F. Aldaye, C. J. Serpell, V.
Toader, A. Petitjean, H. F. Sleiman, Nat. Chem. 2016, 8, 368.
8
JOURNAL OF POLYMER SCIENCE, PART A: POLYMER CHEMISTRY 2018, 00, 000–000