Rapid Self-Healing and Thixotropic Organogelation of Amphiphilic Oleanolic Acid-Spermine Conjugates

Article abstract of DOI:10.1021/acs.langmuir.0c03335

Natural and abundant plant triterpenoids are attractive starting materials for the synthesis of conformationally rigid and chiral building blocks for functional soft materials. Here, we report the rational design of three oleanolic acid-triazole-spermine

Full text of DOI:10.1021/acs.langmuir.0c03335

pubs.acs.org/Langmuir
Article
Rapid Self-Healing and Thixotropic Organogelation of Amphiphilic
Oleanolic Acid−Spermine Conjugates
¶
¶
¶
¶
¶
̌
̈
́
́
́
Zulal Ozdemir, David Saman, Kia Bertula, Manu Lahtinen, Lucie Bednarova, Markéta Pazderkova,
́
́
̌
Lucie Rarova, Nonappa,* and Zdenek Wimmer*
Cite This: Langmuir 2021, 37, 2693−2706
Read Online
sı
*
Metrics & More
Article Recommendations
Supporting Information
ACCESS
ABSTRACT: Natural and abundant plant triterpenoids are attractive starting
materials for the synthesis of conformationally rigid and chiral building blocks for
functional soft materials. Here, we report the rational design of three oleanolic acid−
triazole−spermine conjugates, containing either one or two spermine units in the
target molecules, using the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition
reaction. The resulting amphiphile-like molecules 2 and 3, bearing just one spermine
unit in the respective molecules, self-assemble into highly entangled fibrous networks
leading to gelation at a concentration as low as 0.5% in alcoholic solvents. Using step-
strain rheological measurements, we show rapid self-recovery (up to 96% of the initial
storage modulus) and sol ⇔ gel transition under several cycles. Interestingly,
rheological flow curves reveal the thixotropic behavior of the gels. To the best of our
knowledge, this kind of behavior was not shown in the literature before, neither for a
triterpenoid nor for its derivatives. Conjugate 4, having a bolaamphiphile-like
structure, was found to be a nongelator. Our results indicate that the position and number of spermine units alter the gelation
properties, gel strength, and their self-assembly behavior. Preliminary cytotoxicity studies of the target compounds 2−4 in four
human cancer cell lines suggest that the position and number of spermine units affect the biological activity. Our results also
encourage exploring other triterpenoids and their derivatives as sustainable, renewable, and biologically active building blocks for
multifunctional soft organic nanomaterials.
new gelators. Extensive research, pursued over the last three
decades on diverse building blocks, has offered the possibilities
toward the rational design of molecular gelators with unique
material properties. The building blocks ranging from long
chain hydrocarbons,15 peptides,25 polyaromatics,18 ste-
roids,26−32 carbohydrates,33 and metal complexes19−21 have
been studied extensively. Molecular gels are generally
considered as kinetically trapped metastable states, and the
gelation process can be controlled by tuning the supra-
molecular interactions and self-assembly pathways to obtain
different material properties.34,35 It has been shown that there
is a delicate balance between gelation and crystallization.
Therefore, gelation is often considered as failed crystalliza-
tion.36,37 It has been shown that this balance between gel state
and the crystallization depends on the activation barrier.38
Molecular gels with a wide range of morphologies including
tapes, ribbons, fibers, and microcrystalline networks have been
INTRODUCTION
■
Supramolecular gels resulting from the self-assembly of low
molecular mass organic compounds represent an important
class of soft materials having their potential in applications in
functional materials, catalysis, optoelectronics, and sensors.1−5
Low molecular weight gelators (LMWGs) undergo hierarchical
assembly into highly entangled fibrous networks capable of
immobilizing a large number of solvent molecules.6 Despite
being predominantly comprised of liquids, the resulting gels
display solidlike mechanical properties in their rheological
behavior.7 Importantly, in general, molecular organo- and
hydrogels offer reversible control of their mechanical proper-
ties when perturbed by external stimuli, such as temperature,7
light,8 electric field,9 pH,10 oxidation−reduction,11 or mechan-
ical stress.12 The reversible mechanical properties are
consequences of the structural reorganization of gelators.4
The microscopic changes occurring in molecular gels are
attributed to noncovalent intermolecular interactions respon-
sible for the molecular self-assembly. The noncovalent
interactions such as hydrogen bonding,13 electrostatic effect,14
hydrophobic effect,15 London dispersion forces,16 van der
Waals interactions,17 charge-transfer complexation,18 metal
coordination,19−21 halogen bonding,22 and fluorine−fluorine
interactions23,24 have been explored effectively for designing
Received: November 19, 2020
Revised: January 22, 2021
Published: February 17, 2021
https://dx.doi.org/10.1021/acs.langmuir.0c03335
© 2021 American Chemical Society
Langmuir 2021, 37, 2693−2706
2693

Langmuir
pubs.acs.org/Langmuir
Article
reported in the literature.1−5 More importantly, self-assembly
of chiral gelator molecules has also been shown to exhibit
amplification of chirality upon gelation,14,39−42 and in some
cases, formation of helical fibers, twisted tapes, or ribbons has
been visualized using electron microscopy (EM).43,44 Molec-
ular gels also display self-healing and thixotropic behavior
making them attractive for industrial, pharmaceutical, and
cosmetic applications.45,46 Self-healing gels recover their initial
properties, e.g., modulus or shape, after being subjected to
physical, chemical, or mechanical perturbations.47−49 Self-
healing properties of molecular gels can be determined using
visual demonstration, oscillatory rheology, and cyclic com-
pression/tensile testing.50 While visual demonstration is a
qualitative method, step-strain rheological experiments allow
quantitative determination of structure recovery. Importantly,
step-strain experiments are suitable for low molecular gels even
with very low solid content.
On the other hand, thixotropic gels undergo a transition
from gel to solution upon applying stress. However, they
recover to the original gel state when there is no applied
shear.51,52 According to the IUPAC definition, thixotropy is
defined as “the continuous decrease of viscosity with time when
flow is applied to a sample that has been previously at rest, and the
subsequent recovery of viscosity when flow is discontinued”.53
Unlike shear thinning behavior, i.e., decrease in viscosity upon
increasing shear stress, thixotropy is a time dependent
property. Thixotropic behavior can be studied by time
dependent step-strain and rate dependent shear stress versus
shear rate oscillatory rheological experiments.47 Recovery of
the gel is observed after shearing in rate dependent shear stress
versus the shear rate experiment. However, when the gel
recovers to its original modulus, it does not follow the exact
path, instead it shows a hysteresis loop, indicating the
thixotropic behavior of the gels. The thixotropic properties
are generally exhibited by molecular gels composed of the
noncrystalline network.54−56 However, there is no specific
design strategy available to prepare such gels.57
Irrespective of the nature of the gels and the diversity of
building blocks, amphiphilicity has been a common property in
a large number of LMWGs.5 Furthermore, a certain class of
gelators such as cholesterol,27 bile acids,26 plant sterols,58−62
and sorbitol displays varying degrees of conformational rigidity,
which facilitates a reduction in the entropic losses upon self-
assembly.63,64 Contrary to other gelators, strong hydrogen
bonding interactions are absent in several conformationally
rigid gelators, and even if they are present, they contribute to a
lesser extent toward self-assembly.5 However, in the field of
soft materials, plant triterpenoids are a much less explored class
of conformationally rigid, naturally abundant, renewable, and
sustainable resources.65 Triterpenoids, being structurally differ-
ent from steroids, are a class of triterpenes, compounds with 30
carbon atoms, either in linear structure or fused rings, but
containing additional heteroatoms or functional groups.66
They are precursors for steroids, which compounds, contrary
to triterpenoids, are characterized by at least 17 carbon atoms
and bear tetracyclic systems with three six-membered rings (A,
B, and C) and a five-membered ring (D).67 This class of
compounds, including cholesterol (a sterol), and bile acids, has
already been extensively studied for their self-assembly and
gelation properties.1−5,10,26−32
Figure 1. Chemical structures of the target compounds studied in this
work. Oleanolic acid (1) and its space filling model based on the X-
ray single crystal structure (CSD entry: VUZVUI; top left), space
filling model of the triazole−spermine conjugate based on its energy
minimized structure (top right), and oleanolic acid−triazole−
spermine conjugates 2−4 along with their energy minimized
structures.
especially in Panax ginseng root and Sambucus chinensis
(Chinese elder) in high concentrations.68,69 It displays a
wide range of modest biological activities, including antitumor
effects.70−73 Studies on the self-assembly of triterpenoids have
been limited to betulinic acid, arjunolic acid, ursolic acid,
glycyrrhetinic acid, and oleanolic acid in a few selected organic
solvents.74−76 At relatively high concentrations, gelation of
oleanolic acid has also been reported in organic solvents.74,75
Oleanolic acid is a lipophilic compound displaying low
bioavailability,68 and the functional group chemistry of
oleanolic acid has not yet been fully exploited for the synthesis
Oleanolic acid [(3β)-3-hydroxyolean-12-en-28-oic acid, 1] is
a pentacyclic triterpenoid (Figure 1) found throughout the
plant kingdom in more than 120 plant species, of which
2694
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
a
Scheme 1. Synthesis of Oleanolic Acid−Triazole−Spermine Conjugates 2−4
a
a) Benzyl bromide, K2CO3, in DMF, rt; b) propargyl bromide, NaH (60% dispersion in mineral oil), in THF, rt; c) 15, CuSO4·5H2O/TBTA,
sodium ascorbate, in DCM/H2O, rt; d) Pd/C (10%), H2, in THF/EtOH rt; e) 1.0 M HCl, in ethyl acetate, rt; f) propargyl bromide, K2CO3, in
DMF, rt.
of its derivatives. Therefore, it is desirable to address whether
naturally abundant oleanolic acid can be functionalized to
obtain amphiphilic building blocks. Such a design requires a
careful and selective chemical transformation. Toward this end,
naturally occurring sterol−spermine conjugates, such as
squalamine, has inspired the synthesis of spermine conjugates
of fatty acids and steroids as polycationic amphiphiles.77,78 The
spermine conjugates have been shown to efficiently bind to
DNA and act as nonviral gene carriers and ionophores.79−84
The synthetic spermine conjugates have been shown to exhibit
a broad spectrum of antimicrobial activities85−87 and are
reported to self-assemble into molecular gels.88,89 Previously,
the synthesis of different triterpenoid−polyamine conjugates
via the amide bond formation has been studied, and the
compounds were subjected to the selected pharmacological
screening tests.90−92
Herein, we aimed to explore the possibilities to design
oleanolic acid−triazole−spermine conjugates as polycationic
amphiphiles, to study their self-assembly behavior, potential
gelation, morphological features, and mechanical properties.
To achieve this objective, we have undertaken the following
tasks: (a) The Cu(I)-catalyzed Huisgen 1,3-dipolar cyclo-
addition reaction has been used as one of the key steps to
prepare oleanolic acid−triazole−spermine conjugates (Figure
1). Accordingly, three derivatives were prepared by appending
spermine conjugates at C(3)-OH (2), C(17)-COOH (3), and
both C(3)-OH and C(17)-COOH (4) by selective chemical
transformations (Scheme 1). (b) We show that the synthesized
conjugates 2 and 3 act as LMWGs when tested in alcoholic
solvents, resulting in highly entangled fibrous networks. The
VC and VT NMR spectroscopy, IR spectroscopy, X-ray
powder diffraction techniques, thermal analysis, and electron
microscopy were used to investigate supramolecular self-
assembly of the target compounds. (c) Using extensive
oscillatory rheological measurements and using a visual
demonstration, we have shown that the gels exhibit rapid
self-healing and thixotropic behavior. (d) Finally, the target
compounds 2−4 were subjected to cytotoxicity screening tests
in four cancer cell lines, exhibiting differences in the
cytotoxicity values that depended on the structure of the
conjugates.
EXPERIMENTAL SECTION
■
General Methods and Materials. The 1H and 13C nuclear
magnetic resonance (NMR) spectra were recorded on a Bruker
AVANCE 600 MHz spectrometer at 600.13 and 150.90 MHz in
CDCl3 or CD3OD, using tetramethylsilane (δ = 0.0) as internal
reference. 1H NMR spectral data are presented in the following order:
chemical shift (δ) expressed in ppm, multiplicity (s, singlet; d,
doublet; t, triplet; q, quartet; m, multiplet), coupling constants in
Hertz, number of protons. For an unambiguous assignment of both
1
1H and 13C signals, 2D NMR H,13C gHSQC and gHMBC spectra
were measured using standard parameters sets and pulse programs
delivered by the producer of the spectrometer. Infrared spectra (IR)
were measured with a Nicolet 6700 FT-IR spectrometer (USA)
2695
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
equipped with a standard mid-IR source, KBr beam splitter, and
DTGS detector and with the cell compartment purged by dry
nitrogen during all the measurements. Mass spectra (MS) were
measured with a Waters ZMD mass spectrometer in a positive or
negative electrospray ionization mode. Optical rotations were
measured on an Autopol IV instrument (Rudolph Research
Analytical, USA) at 589 nm wavelength, and the values were
corrected to 20 °C. All other instruments used in a detailed
investigation of supramolecular systems (scanning electron micros-
copy, transmission electron microscopy, rheometer, X-ray diffrac-
tometer, thermogravimetric analyzer) are described in the Supporting
Information. Thin-layer chromatography (TLC) was carried out on
silica gel plates (Merck 60F254), and the visualization was performed
using UV detection and by spraying with the methanolic solution of
phosphomolybdic acid (5%) followed by heating. For column
chromatography, silica gel 60 (0.063−0.200 mm) from Merck was
used. All chemicals and solvents were purchased from regular
commercial sources in analytical grade, and the solvents were purified
by general methods before use. Oleanolic acid was purchased from
Synthesis of Compound 2. A solution of HCl (gas; 1.0 M in ethyl
acetate; 48 mL, 47.41 mmol) was added to a solution of 8 (0.63
mmol, 1 equiv), and the reaction mixture was stirred at r.t. for 24 h.
The resulting white residue was filtered and washed with ethyl acetate
to furnish 2 as a white solid in a 95% yield.
Procedure for Preparation of Compound 3. Synthesis of
Compound 9. Propargyl bromide (115 μL, 1.31 mmol) and K2CO3
(182 mg, 1.31 mmol) were added to a solution of 1 (400 mg, 0.88
mmol) in DMF (5 mL). The reaction mixture was stirred at r.t. for 48
h. The reaction was monitored using TLC and quenched by the
addition of water to the reaction medium. The mixture was extracted
using benzene and washed with a brine solution (5 × 50 mL). The
organic layer was dried by Na2SO4, filtered, and evaporated under
vacuum. The crude product obtained after aqueous workup was
column purified on silica gel using chloroform to afford 9 in a 98%
yield as a white solid.
Synthesis of Compound 10. Compound 15 (340 mg, 0.64 mmol)
was added to a stirred solution of 9 (266 mg, 0.54 mmol) in DCM
(5.4 mL), followed by a CuSO4·5H2O/TBTA (1:1) complex solution
(0.27 mmol, 5.4 mL) and stirred at r.t. After stirring for 5 min, sodium
ascorbate (106 mg, 0.54 mmol) was added to the mixture, and the
content was mixed at r.t. for 27 h. It was extracted into CHCl3 and
washed with H2O. After drying the organic layer over anhydrous
Na2SO4, the volatiles were removed under reduced pressure. The
crude product obtained after aqueous workup was column purified on
silica gel using ethanol in chloroform [from 100/0 v/v (%) to 100/4
v/v (%)] affording 10 as a white foamy solid in a 99% yield.
Synthesis of Compound 3. A solution of HCl (gas; 1.0 M in ethyl
acetate; 31 mL, 31.30 mmol) was added to a solution of 8 (433 mg,
0.42 mmol), and the reaction mixture was stirred at r.t. for 18 h. The
resulting white residue was filtered using a sintered glass funnel and
washed with diethyl ether, and white crystals were obtained with a
97% yield.
̌
Dr. Jan Sarek−Betulinines (www.betulinines.com).
Procedure for the Preparation of Compound 2. Synthesis of
Compound 5. Benzyl bromide (235 μL, 1.97 mmol) and K2CO3 (272
mg, 1.97 mmol) were added to a solution of 1 (600 mg, 1.31 mmol)
in DMF (10 mL) in a round-bottom flask. The mixture was stirred at
r.t. for 17 h. The reaction was monitored using TLC, and upon
complete conversion of the starting materials to product, the reaction
was quenched by adding water to the reaction medium. The reaction
mixture was extracted into benzene and washed with brine solution (5
× 50 mL) using a separatory funnel. The organic layer was dried over
anhydrous Na2SO4, filtered, and removed under reduced vacuum to
obtain the crude product. The crude product was column purified on
silica gel using 50/50 v/v (%) petroleum ether/chloroform to 100%
chloroform to afford 5 in a 94% yield as a white solid.
Synthesis of Compound 6. Sodium hydride (60% dispersion in
mineral oil; 1049 mg, 26.24 mmol) was added to a solution of 5 (896
mg, 1.64 mmol) in dry THF (15 mL) as two portions within 2-h
intervals in a round-bottom flask. The mixture was stirred at r.t. for 4
h. When the color of the solution turned creamy gray, propargyl
bromide (425 μL, 4.91 mmol) was added to reaction mixture drop by
drop under the argon atmosphere. After stirring at r.t. for 5 days,
excess sodium hydride was neutralized by the slow addition of water.
The reaction mixture was extracted to chloroform using a separatory
funnel and dried over anhydrous Na2SO4. The volatiles were removed
under reduced pressure. The crude product obtained after aqueous
workup was column purified on silica gel using 50/50 v/v (%)
petroleum ether/chloroform to 100% chloroform to afford 6 in a 76%
yield as a white-yellowish solid.
Synthesis of Compound 7. Compound 15 (529 mg, 1.0 mmol)
was added to a stirred solution of 6 (450 mg, 0.77 mmol) in DCM (7
mL) in a round-bottom flask, followed by a CuSO4·5H2O/TBTA
(1:1) complex solution (7.7 mL, 0.39 mmol) and stirred at r.t. for 5
min. To the above mixture, sodium ascorbate (152 mg, 0.77 mmol)
was added, and the contents were mixed at r.t. for 20 h. The reaction
mixture was then extracted by CHCl3 and washed with water. After
drying the organic layer over anhydrous Na2SO4, the volatiles were
removed under reduced pressure. The crude product obtained after
aqueous workup was column purified on silica gel using ethanol in
chloroform [from 100/0 v/v (%) to 100/5 v/v (%)] to afford 7 in a
99% yield as a white foam.
Synthesis of Compound 8. Catalyst (Pd/C (10%); 550 mg, 0.52
mmol) was added to a solution of 7 (860 mg, 0.77 mmol) in a THF/
ethanol mixture (10/10 mL). The reaction vessel was repeatedly
degassed under vacuum and flushed with argon four times. The
catalytic hydrogenation was carried out under a hydrogen atmosphere
at r.t. for 27 h. The reaction mixture was filtered using a sintered glass
funnel to remove the solid. The filtrate was collected and evaporated
under reduced pressure to furnish the white solid that was purified by
column chromatography using a gradient mobile phase ethanol/
chloroform [from 100/0 v/v (%) to 100/2 v/v (%)] to afford 8 in a
90% yield as a white foam.
Procedure for the Preparation of Compound 4. Synthesis of
Compound 11. Sodium hydride (60% dispersion in mineral oil; 129
mg, 3.23 mmol) was added to a solution of 9 (100 mg, 0.20 mmol) in
dry THF in two portions within 2-h intervals. The reaction mixture
was stirred at r.t. for 4 h. When the color of the solution turned
creamy gray, propargyl bromide (70 μL, 0.80 mmol) was added to the
reaction mixture drop by drop under the argon atmosphere. After
stirring at r.t. for 4 days, an excess of sodium hydride was neutralized
by a slow addition of water. The mixture was extracted into an organic
layer using chloroform. After drying the organic layer over anhydrous
Na2SO4, the volatiles were removed under reduced pressure. The
crude product obtained after aqueous workup was column purified on
silica gel using petroleum ether in chloroform [from 50/50 v/v (%) to
0/100 v/v (%)] to afford 11 in a 91% yield as a pale yellowish solid.
Synthesis of Compound 12. Compound 15 (338 mg, 0.64 mmol)
was added to a stirred solution of 11 (142 mg, 0.27 mmol) in DCM,
followed by a CuSO4·5H2O/TBTA (1:1) complex solution (5.33 mL,
0.27 mmol). After stirring the reaction mixture for 5 min, sodium
ascorbate (105 mg, 0.53 mmol) was added to the mixture and stirred
at r.t. for 24 h. It was then extracted into CHCl3 and washed with
brine. After drying the organic layer over anhydrous Na2SO4, the
volatiles were removed under reduced pressure. The crude product
obtained after aqueous workup was column purified on silica gel using
ethanol in chloroform [from 100/1.5 v/v (%) to 100/4.5 v/v (%)] to
afford 12 in a 96% yield as a white foam.
Synthesis of Compound 4. A solution of HCl (gas; 1.0 M in ethyl
acetate, 37 mL, 36.69 mmol) was added to a solution of 12 (389 mg,
0.25 mmol), and the reaction mixture was stirred at r.t. for 25 h. The
resulting residue was filtrated using a sintered glass funnel and washed
with diethyl ether to furnish 4 as a white solid in a 95% yield.
Procedure for the Preparation of Compound 15. Synthesis
of Compounds 13 and 14. Sulfuryl chloride (13.5 g, 100 mmol) was
added dropwise to an ice-cooled suspension of NaN3 (6.5 g, 100
mmol) in acetonitrile (100 mL) resulting in a white salt formation
(13). After stirring the mixture at r.t. overnight, it was cooled over an
ice bath, imidazole (12.9 g, 100 mmol) was added in portion-wise,
and the resulting slurry was stirred at r.t. for 3 h. The initial addition
2696
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
of imidazole resulted in a pinkish color which turned white upon
complete addition. The mixture was diluted with ethyl acetate (200
mL) and H2O (200 mL), and the aqueous layer was separated and
discarded. The organic layer was washed with H2O (200 mL) and
then with saturated aqueous NaHCO3 (2 × 200 mL), dried over
MgSO4, and filtered. The yellow oily liquid 14 obtained after
evaporating the solvent was used for the next step without further
purification.
Synthesis of 14·H2SO4. The reaction was carried out using a
slightly modified literature procedure.93,94 Sulfuric acid [93% (w/w),
5.8 mL in diethyl ether (50 mL)] was added slowly to a solution of
imidazole-1-sulfonyl azide 14 [100 mmol in diethyl ether (50 mL)] at
0 °C. The reaction mixture was stirred at r.t. for 30 min. The
precipitate was filtered, washed with diethyl ether, and dried in
vacuum to give a white crystalline powder 14·H2SO4 with an 80%
yield.
Synthesis of Compound 15. Imidazole-1-sulfonyl azide hydrogen
sulfate (14·H2SO4, 647 mg, 2.39 mmol) was added to a mixture of the
commercially available N1,N4,N9-tris(Boc)spermine (1.0 g, 1.99
mmol), K2CO3 (0.5 mmol), and CuSO4·5H2O (5 mg, 0.02 mmol)
in methanol (22 mL), and the resulting reaction mixture was stirred at
r.t. for 30 h. It was diluted with H2O (50 mL) and extracted with ethyl
acetate (3 × 50 mL). After drying the organic layer over anhydrous
Na2SO4, the volatiles were removed under reduced pressure. The
crude product obtained after aqueous workup was column purified on
silica gel using ethanol in chloroform [from 100/0 v/v (%) to 100/2
v/v (%)] to afford 15 in a 93% yield as an oily liquid.
carried out by 1.0 M HCl (gas) in ethyl acetate, furnishing 4.
Detailed synthetic and purification procedures are described in
the Experimental Section. The spectral data, 1D (1H and 13C),
2D NMR (1H−13C correlation spectroscopy), and electrospray
ionization MS, IR spectra, and other related characterizations
of intermediate and target products are presented in the
Supporting Information (see also Figures S3−S15).
Gelation Studies. The presence of the lipophilic core and
the cationic spermine side chains imparts amphiphilic nature to
the conjugates 2 and 3. The oleanolic acid core has an end-to-
end length of 1.45 nm based on its solid-state structure, and
the triazole linked spermine in its fully stretched conformation
has a length of 1.97 nm (Figure 1 and Figure S1). Though 2
and 3 have a similar overall structure, they differ in the position
of spermine substituents, i.e., they are positional isomers.
Conjugate 2 has a free carboxylic acid group, whereas 3 has a
free hydroxyl group. On the other hand, 4 has both of these
groups functionalized, resulting in a bolaamphiphile-like
molecule. More importantly, oleanolic acid−triazole−spermine
conjugates 2−4 differ from conventional surfactant molecules
due to the conformationally rigid and chiral triterpenoid core.
Natural bile salts (steroid compounds) and their synthetic
derivatives are classical examples for this type of unconven-
tional surfactants and are known for their unique aggregation
behavior.26−32 Depending on the number and position of
hydroxyl groups and functional transformation, bile salts and
their derivatives are known to form globules, tubules, and
fibrous gel networks.10,26−32 Therefore, it is desirable to
investigate the self-assembly behavior of the novel amphiphilic
building blocks based on the plant triterpenoid 1. The
solubility of 2−4 was tested in 22 different solvents (Table
1). Interestingly, 2 and 3 underwent gelation−mostly in polar
protic solvents−when tested at 1.0% (note: from hereafter all
percent values (%) are in w/v of gelator/solvent), as suggested
by a vial inversion test (we have used 1.0% as an upper limit for
gelation tests). Conjugate 2 was gelled at a concentration as
low as 0.5% in 1-butanol and 1.0% in 1-propanol, 2-propanol,
1-pentanol, 1-hexanol, 1-heptanol, and in polar aprotic
solvents, pyridine and N,N-dimethylformamide (DMF).
Similarly, gels were obtained for 3 in 2-propanol, 1-butanol,
1-pentanol, 1-heptanol, in polar aprotic solvents, pyridine,
DMF, and acetonitrile, and, surprisingly, in a nonpolar solvent
(1,4-dioxane) at a concentration of 1.0%. The resulting gels
were translucent or opaque, depending on the concentration
and the type of organic solvent used for gelation (Figure 2). In
all other tested organic solvents, both 2 and 3 remained in the
solutions or precipitated after having been allowed to stand at
r.t., or they remained insoluble (Table 1). However, 4 is either
insoluble or remains in a solution; no gelation was observed.
This finding already suggests that the gelation is strongly
affected by the number and position of the spermine units, as
well as by the presence of the unconjugated hydroxyl or
carboxylic acid groups. The driving force of the gel formation is
connected with the presence of electrostatic interactions
between a quaternary amino group present in the spermine
chain and a chloride anion,61 which is present in all three
conjugates. However, intermolecular hydrogen bonds between
the carbonyl and amino groups represent an important factor
when a nongelator property of 4 is taken into consideration
contrary to those of 2 and 3.
RESULTS AND DISCUSSION
■
Synthesis and Characterization. First, we discuss the
synthesis of oleanolic acid−triazole−spermine conjugates 2−4
(Scheme 1). To design novel structures, we used the Cu(I)-
catalyzed Huisgen 1,3-dipolar cycloaddition reaction providing
a facile route for 1,2,3-triazoles by selective functionalization of
either the hydroxyl group [C(3)-OH] or the carboxylic acid
group [C(17)-COOH].95 Apart from the increased yield, the
1,4-disubstituted 1,2,3-triazoles also share certain similarities in
their structural as well as physicochemical properties with the
amide bond.96,97 The functionalization at C(3)-OH of 1 was
achieved by carrying out the benzyl protection of carboxyl acid
group and yielded 5. Compound 5 was then reacted with
propargyl bromide in the presence of sodium hydride in THF,
and 6 was obtained.98 Simultaneously, the tert-butoxy (Boc)-
protected azide derivative of spermine derivative 15 was
prepared according to the slightly modified literature
procedures93,94 (see the Experimental Section and Scheme
S1 in the Supporting Information). Compound 6 upon the in
situ generated Cu(I)-catalyzed Huisgen 1,3-dipolar cyclo-
addition reaction98 with 15 in DCM/H2O at r.t. furnished 7.
Hydrogenation of 7 using 10% Pd/C as a catalyst under H2
atmosphere in a THF/EtOH mixture resulted in a
deprotection of carboxylic acid, furnishing 8. Acid hydrolysis
of 8 using 1.0 M HCl (gas) in ethyl acetate99 resulted in a
preparation of 2. Compound 3 was synthesized by reacting 1
with propargyl bromide in the presence of K2CO3 to furnish 9
in the first step. Ester 9 was then subjected to a series of
transformation steps that include the Cu(I)-catalyzed Huisgen
1,3-dipolar cycloaddition reaction to obtain 10, followed by a
removal of the Boc-protecting groups using 1.0 M HCl (gas) in
ethyl acetate to get conjugate 3. To synthesize 4, intermediate
9 was reacted with propargyl bromide in the presence of
sodium hydride to obtain 11. Subsequently, the Cu(I)-
catalyzed Huisgen 1,3-dipolar cycloaddition reaction was
applied under the above-described conditions, yielding 12. A
removal of the Boc-protecting groups, present in 12, was
Morphological Characterization. Scanning Electron
Microscopy (SEM). To investigate the structure of the
supramolecular assemblies, we carried out electron microscopy
2697
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
concentration on morphology, SEM micrographs of the 1-
butanol gels of 2 at four different concentrations (0.5, 1.0, 1.5,
and 2.0%) were obtained (see Supporting Information, Figure
S17). However, the fiber diameter changed slightly upon
increasing the concentration, and the fibers tend to be densely
packed (see Supporting Information, Figure S18 and Table
S2), a property that is consistent with the literature reports.100
Moreover, for gels at higher concentrations, due to dense
packing the determination of the lateral dimensions of
individual fibers was challenging. In all the solvents tested
for gelation, 2 showed similar morphological features with a
highly entangled network of fibers (Figure S16). A
representative SEM micrograph from the 1,4-dioxane gel
(1.0%) of 3 with highly entangled fibrous networks is shown in
Figure 2f. The fiber morphologies in aerogels of 3 are similar to
those of 2. However, the lateral dimensions of the fibers were
different from those for 2. For example, aerogels derived from
Table 1. Solvents (Nonpolar, Polar Protic, Polar Aprotic,
Acids, and Bases) Tested for Gelation Studies of 1−4
a
solvent/compound
1
2
3
4
benzene
I
I
I
I
chlorobenzene
toluene
S
SS
SS
SS
P
SS
I
P
TG
S
SS
I
SS
OG
S
I
I
I
I
I
I
I
PI
S
S
S
I
P
I
PI
PI
PI
I
PI
PI
S
S
PI
P
S
S
S
OG
OG
S
S
S
S
S
S
carbon tetrachloride
1,4-dioxane
chloroform
dichloromethane
acetonitrile
DMF
DMSO
pyridine
methanol
1-propanol
2-propanol
1-butanol
1-pentanol
1-hexanol
I
OG
TG
S
CG
S
VS
OG
OG
OG
VS
OG
P
pCG
S
CG
TG
TG
TG
pTG
TG
P
DMF gels of 2 and 3 showed the lateral dimensions of 43
5
nm and 29 5 nm, respectively (see Supporting Information,
Figure S19 and Table S3).
Transmission Electron Microscopy (TEM). To obtain more
information about fiber morphology, TEM imaging was
performed for all gels (see the Supporting Information for
sample preparation). TEM micrographs of all the gels studied
in this work showed the presence of fibrous networks. Figure 3
shows the representative TEM micrographs of the 1-propanol
gel of 2 (1.0%) and the 1-heptanol gel of 3 (1.0%). More
importantly, in some cases, the helical twist was observed
(Figures 3b and 3d) with a pitch length of about 90 nm and
lateral dimension varying from 15 to 25 nm (Figure 3e).
Oleanolic acid being the core structure of studied derivatives is
inherently chiral (many stereogenic center). This can be one of
the reasons for the helical twist observed for 2 and 3 (although
there exist achiral gelators that produce helical fibers with both
handiness).101 Chiral self-assembly can be expressed consid-
ering the packing pattern of chiral molecules as well. In general,
chiral molecules do not pack parallel to their neighbors but
rather pack at a slight angle with respect to their neighbors
which can arise from repulsive and/or attractive interactions
leading to a twist in the orientation of the molecules.102 In the
literature, fibers with helical twists are reported for steroid
based systems102 and esters of arjunolic acid (another type of
1-heptanol
1-octanol
formic acid
triethylamine
N-methylmorpholine
PI
S
S
S
I
I
S
P
I
S
I
I
a
Gelation concentration c = 1.0%. Abbreviations: TG = translucent
gel, OG = opaque gel, pTG = partial translucent gel, pCG = partial
clear gel, P = precipitated upon cooling, S = soluble at r.t., I =
insoluble at boiling point, PI = partially soluble at boiling point, SS =
suspension, VS = viscous solution.
(EM) imaging of all the studied gels of 2 and 3 (Note: only
representative micrographs are presented in the main text.).
Ambient drying of gels (xerogels) resulted in filmlike structures
due to drying artifacts (Figure S16). Therefore, aerogels were
prepared using a liquid nitrogen freeze-drying technique (see
the Supporting Information for details). The SEM image of the
methanol gel of 1 (Figure 2a) showed microcrystalline
structures (Figure 2). A representative SEM image of the
0.5% 1-butanol gel of 2 is shown in Figure 2e, displaying highly
entangled fibrous networks with an average lateral dimension
of 35 5 nm and with indefinite length. To study the effect of
Figure 2. Gelation and scanning electron microscopy. Electron micrographs of representative gels: a) 1 in methanol, 1.0%, b) 2 and 3 in different
solvents, 1.0%, c) 2 in 1-butanol at different concentrations; d) SEM micrograph of the methanol gel of 1; e) SEM micrograph of the freeze-dried
1-butanol gel of 2, 0.5%, and f) SEM micrograph of the freeze-dried 1,4-dioxane gel of 3, 1.0%.
2698
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
chirality is expressed in the morphology of the fiber when the
molecules possess some chemical functions capable of directed
noncovalent interactions and/or rigid segments which govern
the pseudocrystalline organization of the molecules in the
fibrous structures.105 The lateral dimensions observed under
TEM imaging are comparable with the results obtained from
SEM imaging. The TEM micrographs from all other gels are
presented in the Supporting Information (Figure S20).
X-ray Powder Diffraction and Thermal Analysis. To gain
further insight about solid state and thermal properties of the
conjugates, thermogravimetric analysis (TGA), differential
scanning calorimetry (DSC), and X-ray powder diffraction
(XRPD) studies of oleanolic acid (1), synthetic solid
compounds 2−4, but aerogels from the 1-butanol gel of 2
(1.0%) and the 1-pentanol gel of 3 (1.0%) were performed
(see the Supporting Information for experimental details and
results). TGA and DSC results revealed a decreased
decomposition temperature for the conjugates and the aerogels
compared to parent 1 (see Supporting Information, Table S4
and Figures S21 and S22). XRPD studies revealed the
semicrystalline nature of 1. However, the target conjugates
2−4 showed broad featureless patterns in their powder X-ray
patterns (see Supporting Information, Figure S23). These
results suggest that the synthetic solids as well as the self-
assembled nanostructures in gels are amorphous.
Figure 3. Transmission electron microscopy: a,b) TEM micrographs
of the 1.0% 1-propanol gel of 2, c,d) TEM micrographs of the 1.0% 1-
heptanol gel of 3, and e) a single fiber with a helical twist is shown
(left) together with its graphical representation (right).
Mechanical Properties of Gels. For rheological measure-
ments, only those gels that are suitable for repeated rheological
measurements were used that can be transferred onto the
rheometer without losing their structures. Accordingly, the
premade and stabilized 1-propanol, 2-propanol, 1-butanol, and
1-heptanol gels of 2 were studied (Figure 4, see also the
pentacyclic triterpenoid).103 When we look at the structures of
2 and 3, besides having many chiral centers, they are
amphiphilic in nature. Amphiphilicity has also been pointed
out as another factor causing the formation of chiral self-
assemblies.104 Additionally, it is known in the literature that
Figure 4. Rheological properties of gels: a) time sweep experiments of gels derived from 2, b) frequency sweep experiments of gels derived from 2,
c) stress sweep experiments derived from 2, d) strain sweep experiments derived from 2 [(full circles indicate G′, empty circles indicate G′′, 1-
butanol gel (black), 1-propanol gel (red), 2-propanol gel (blue), 1-heptanol gel (green)], and e, f) the effect of the concentration on mechanical
properties of the 1-butanol gel of 2.
2699
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
Figure 5. Self-recovery and thixotropic properties: a) step-strain experiments showing self-healing properties of the 1-butanol gel of 2, flow tests
showing viscosity vs the shear rate and shear stress vs the shear rate for b) the 2-propanol gel of 2 and c) for the 1-pentanol gel of 3, and d, e)
snapshots from video S1 demonstrating thixotropy by manual shaking and vortexing of the 1-butanol gel of 2.
Supporting Information for experimental details). The gels
have already shown that the storage modulus (G′) is several
folds higher than that of the loss modulus (G′′) suggesting that
the systems under investigation are already in the gel state
(viscoelastic) and remained constant under time sweep (30
min) experiments (Figure 4a). The frequency sweep experi-
ment provides insights into the nature and lifetime of the
bonds that are involved in network junctions.106 It is well-
known that for gels, the G′ is independent of frequency,
whereas for viscous fluids, the G′ is frequency dependent. The
storage modulus (G′) of gels derived from 2 and 3 only slightly
varies as a function of the frequency under the frequency
sweep experiment (Figure 4b) confirming soft-solid-like
behavior. The slight frequency dependence of the storage
and loss moduli observed for gels derived from 2 and 3
indicates the presence of junction points and temporary bonds
that form the networks. Such temporary bonds are sensitive to
the frequency of the mechanical stress and the lifetime of the
bonds. Our results suggest that there are junction networks as
supported by SEM and TEM micrographs (Figures 2 and 3).
In the literature, various hypotheses have been presented and
supported using experimental as well as computational
simulation studies on how the fibers are constrained at
junction points and how fibrous networks are formed.107−112
According to Raghavan and Douglas,113 there are three
possible scenarios, i.e., attraction at junctions, junction zones
or bundling, and jamming at junctions. A closer look at the EM
images (Figures 2 and 3) indicates that in our system both
attractions at junctions and junction zone or bundling exists.
Further, the EM images also suggest that fibers become
entangled and penetrated through each other all over the entire
network. The fiber entanglements and the branching sites are
junctions of networks (the former being the transient junctions
and the latter being the physically permanent junctions).
At a given concentration, the stiffness of the gels varied in
the following order: 1-butanol > 1-propanol > 2-propanol > 1-
heptanol with the G′ values of 1.4, 0.7, 0.5, and 0.025 kPa,
respectively (see Supporting Information, Figure S24c1). For a
given solvent, the modulus was increased as a function of
concentration, a property that is typical for low molecular
weight gelators. Figures 4e and 4f show the concentration
dependent, stress and strain sweep experiments for the 1-
butanol gels of 2 (for time and frequency sweep experiments
see Supporting Information, Figures S24a and S24b). For the
1-butanol gels of 2, the storage modulus was increased from
0.22 kPa for 0.5% to 6.5 kPa for 2.0%, which is a 30-fold
increase in the stiffness (see Supporting Information, Figure
S24c2). The yield stress values were determined by the tangent
analysis method. The yield stress values of the 1-butanol, 1-
propanol, 2-propanol, and 1-heptanol gels of 2 were found to
be 30, 24, 13, and 8 Pa, respectively (Figure 4c). Similarly, the
yield stress values for the 1-butanol gels of 2 at different
concentrations were determined as 3, 16, 40, and 95 Pa for 0.5,
1.0, 1.5, and 2.0%, respectively (Figure 4f). For compound 3,
gels that are suitable for rheological measurements were
obtained from 1-pentanol and 1-heptanol. Time, frequency,
strain, and stress sweep experiments were performed for the 1-
heptanol and 1-pentanol gels of 3, and the details are presented
in Supporting Information, Figure S24. For 1.0% gels, the G′
values were found to be 1.4 and 0.3 kPa for 1-pentanol and 1-
heptanol, respectively. The only common solvent for
rheological measurements of 2 and 3 was 1-heptanol, in
which both compounds formed a gel that could be transferred
onto the rheometer without losing its structure. The storage
modulus of the 1.0% 1-heptanol gel was found to be 0.025 kPa
(for 2) and 0.95 kPa (for 3), respectively (see Supporting
Information, Figure S24c1 and S24d). The yield stress values
of the 1-pentanol and 1-heptanol gels of 3 were found to be 51
and 35 Pa, respectively (see Supporting Information, Figure
2700
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
Figure 6. VC 1H NMR graph of a) 2 in CD3OD at 40 °C and b) expanded version of the VC 1H NMR graph of 2 in four different regions (right).
S24f). These results show us that 2 forms stronger gel than 3 in
alcoholic solvents, in general. Furthermore, the stiffness of gels
decreases with the increasing number of carbon atoms in the
alcoholic solvents. The structure of these carbon chains present
in alcoholic solvents, i.e., whether they are branched or linear,
has also an impact on the stiffness of gels. Linear chains
bearing alcoholic solvents seem to form stiffer gels than the
alcoholic solvents with branched carbon chains. Therefore, to
induce gelation, a delicate balance exists between the carbon
chain length and its structural property in the used alcohols.
Self-Healing Properties. Step-strain rheological measure-
ments were performed to investigate the reversible gel ⇔ sol
transition and self-recovery, under several cycles. For the step-
strain experiments, controlled strains of 0.1% and 150% were
applied for 60 s, respectively. The gels showed an immediate
response to the increased strain by turning into sol indicated
by the rapid decrease in G′ by several folds and well below that
of G′′ (Figure 5a). The application of the increased strain also
appears to break the structure further during the 60 s
experiment as shown by the gradually decreasing elastic
moduli values in the subsequent cycles. The gels recovered
their original mechanical strength almost instantaneously upon
switching to a lower strain (0.1%), i.e., rapid self-recovery.
Importantly, the process can be repeated for several cycles.
However, slightly lower G′ was observed after the first high−
low strain cycle, which remained constant in subsequent cycles,
a property typically observed for several low molecular weight
gelators. Importantly, the gels were able to recover up to 96%
of their original storage moduli even after four cycles. The
percentage recovery of the storage moduli of the 1-butanol, 1-
propanol, and 2-propanol gels of 2 was found to be 96, 89, and
78%, respectively (Figure 5a, see also the Supporting
Information, Figure S25 for the other gels of 2 and 3).
Similarly, for the 1-pentanol and 1-heptanol gels of 3, the
percentage recovery of the original storage moduli was found
to be 76 and 75%, respectively.
respectively. A clear hysteresis loop showing a clockwise turn
was observed suggesting the gels under investigation display
thixotropic properties. At a macroscopic level, by manual
shaking, the gel resulted in a gel ⇔ sol transition recovering
within 60 s, showing a rapid recovery. Figures 5d and 5e show
selected snapshots of the 1-butanol gel (1.0%) of 2,
demonstrating a rapid recovery and thixotropic properties by
either shaking or vortexing (see video S1). The thixotropic
nature of gels can be explained on the basis of the amorphous
fibrous network. X-ray diffraction studies of the aerogels
suggest the amorphous nature of the gel network (Figure S23).
This observation is in agreement with other thixotropic gels
reported in the literature.54−56
The observed mechanical behavior of the gels derived from
compounds 2 and 3 can be based on several factors. First,
compounds 2 and 3 differ in the position of the spermine
groups. Compound 2 has a free carboxylic acid group (favors
hydrogen bonding dimerization), whereas 3 has a free hydroxyl
group (relatively weak H-bonding group). These differences in
the position and functional groups affected their solubility
behavior, gelation, and gel preparation method (see the
Supporting Information for details). To achieve gelation,
compound 2 was subjected for sonication in a solvent of
interest to achieve partial dissolution followed by heating close
to the boiling point of the solvent to obtain a clear solution.
Whereas for 3, a clear solution was obtained after sonication,
and heating destabilized the gels. This suggests that gels
derived from 2 are thermodynamically more stable, whereas
those from 3 are in a kinetically trapped metastable form.
Furthermore, at a given concentration, larger fibers were
observed for gels derived from 2 compared to that of 3 (see
Supporting Information, Figure S19). Further, it is well
documented in the literature that the difference in the
chemical structure affects greatly the solute−solvent inter-
actions such as dispersion, dipole−dipole, and hydrogen
bonding interactions.114,115 This affects both the fiber
morphology, junction networks which is reflected in the
rheological properties. Therefore, the above factors together
contribute to the observed differences in gelation behavior and
mechanical properties of 2 and 3. The amorphous, semiflexible
nature of the fibers and the presence of transient and
permanent junction points imply the self-recovery and
thixotropic behavior of the gels.
Thixotropic Properties. To evaluate the thixotropic
property of the gels of 2 and 3, their flow curves were also
measured (Figures 5b and 5c). The measurement was carried
out as follows: the shear stress was measured while increasing
the shear rate from 0 to 100 s−1 and then decreasing it back to
0 s−1. Figures 5b and 5c show a change in the viscosity and
shear stress as a function of the shear rate for the 2-propanol
gel (1.0%) of 2 and the 1-pentanol gel (1.0%) of 3,
2701
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
Table 2. Cytotoxicity (IC50 [μM]) of 2−4 in Four Cancer Cell Lines and Normal Human Fibroblasts after 72 h
cytotoxicity (IC50 [μM]; 72 h)
compound
MW
CEM
MCF7
HeLa
G-361
BJ
2
3
4
723.1
723.1
989.5
300.1
29.8 3.3
2.1 0.8
7.1 1.1
0.8 0.1
28.0 1.0
3.7 1.0
2.7 0.7
7.7 1.7
12.6 2.8
3.7 0.5
2.2 0.1
11.4 3.8
27.8 0.9
6.6 0.4
0.8 0.0
4.5 0.6
14.2 0.4
2.3 0.1
1.9 0.0
6.9 0.9
a
CDDP
a
cis-Diamminedichloridoplatinum(II) (cisplatin), a pharmacologically used agent for treating different cancer types, a positive reference compound.
Variable Concentration and Variable Temperature NMR
FT-IR Spectroscopy. Inter- and intramolecular interactions
can be investigated using IR spectroscopy, which is a
frequently used technique in gel studies. With the aim to
better understand the driving forces leading to the gel
formation, IR spectra of the aerogels prepared from 2 and 3
in different solvents were measured. The spectra of aerogels
were compared with those of the corresponding synthetic
solids in their powder form (see Supporting Information,
Figure S29). The IR spectra of the aerogels, as well as those of
the synthetic solids, exhibited similar characteristic peaks. This
finding suggests that the molecular structure in the gel state
resembles that of the synthetic powder form. These findings
are in agreement with the XRD results. In addition, no spectral
differences were observed in the IR spectra when aerogels,
prepared in different solvents, were compared. This result is in
agreement with the SEM and TEM micrographs, in which
similar entangled fibrous networks were obtained in each
solvent used for the gels preparation. This type of observation
has already been described for steroidal gelators in the
literature;58 however, it is novel knowledge found with the
triterpenoid-based gelators described here.
Cytotoxic Studies. The target compounds 2−4 showed
cytotoxicity in the four tested cancer cell lines (Table 2; for
experimental details see the Supporting Information). CDDP
(cisplatin) was used as a positive reference compound. An
important difference was observed in the cytotoxicity values of
2 in comparison with those of 3 and 4. It seems that the
location and number of the spermine units on the triterpenoid
skeleton play a key role for cytotoxicity. Substitution of the
C(17)-COOH group with the spermine substituent in 3
(C(3)-OH group remains free) and in 4 led to the one order
increase of cytotoxicity in comparison with the data obtained
for 2. Surprisingly, high cytotoxicity was found in tests of 4 in
malignant melanoma cells (G-361). Melanoma cancer belongs
among the most aggressive cancer types. Even though 3 and 4
were toxic toward human fibroblasts, they still exhibited a
better cytotoxicity profile than CDDP (a positive control)
against several cancer cell lines (3 against MCF7 and Hela and
4 against MCF7, Hela, and G-361).
Spectroscopy. The VC 1H NMR spectra of 2−4 in CD3OD at
40 °C (to mimic the conditions of gel preparation) were
measured to gain insights into the interactions involved in self-
assembly. The reason why CD3OD was chosen for the VC 1H
NMR spectra measurements was the observation of a gel even
at a concentration as low as 0.5% in the NMR tube when used
with solvents other than CD3OD, e.g., DMF. Because 2 and 3
form gels mostly in alcoholic solvents, this finding prompted us
1
to check their behavior in CD3OD. Relatively sharp H NMR
resonance peaks, which arise presumably from the non-
aggregated molecules, were observed at a low concentration
for 2−4. As the concentration increased, the peaks turned to
become broader or even remained NMR silent with a slight
downfield shift. The broadening, along with the invisible
nature of certain signals, is possibly due to a self-assembly of
2−4. Figure 6 shows the VC 1H NMR spectrum in a
concentration range from 0.15% to 2.15% of 2 (see Supporting
Information, Figure S26 for 3 and 4). The signal broadening is
more pronounced for 2 compared to that of 3 and 4. The fact
that certain NMR signals turned to become NMR silent above
a certain concentration is attributed to the large size of the
aggregates. The VT 1H NMR spectra of 2−4 were also
measured to see the effect of temperature on aggregates.
However, the maximal temperature limit of 40 °C can be
reached when CD3OD is used as an NMR solvent, due to its
boiling point. As a consequence of this maximum temperature
limitation, the VT 1H NMR experiments carried out
accordingly revealed no important information about aggre-
gates. In other words, no changes in the chemical shift values
1
of the VT H NMR resonance peaks were observed (see
Supporting Information, Figure S27). To overcome the
1
limitations, we further studied the VT H NMR of 2 and 3
in DMF-d7 below gelation concentration (0.12%) between the
20 and 90 °C temperature range with a 10 °C increment (see
Supporting Information, Figure S28). The observable chemical
shift changes for 2 (Δδ90−20) were as follows: deshielding of H-
26 (−CH3 of oleanolic acid backbone) by 0.053 ppm and
shielding of H-3′ (from the triazole ring) and H-6′7′10′11′13′
(spermine chain) by −0.124 ppm and −0.452 ppm,
respectively (see Supporting Information, Tables S4 and S5).
The deshielding of 26-CH3 protons suggests that the
molecules initially undergo aggregation via hydrophobic
interaction where the methyl groups are buried in the
hydrophobic pocket at room temperature. At high temper-
ature, the aggregates are disrupted, and consequently, the
methyl groups are deshielded. In contrast, at higher temper-
ature, the triazole proton and the methylene protons from
spermine chains were shielded. This is attributed to the
possible involvement of the triazole proton and spermine arm
in H-bonding. Similar observations have been made previously
for gels derived from bile acid−amide conjugates.116
CONCLUSION
■
Our results showed that modifications of triterpenoids allow
the rational design of novel building blocks with unique self-
assembly and material properties. The synthetic approach
presented here supports that the Cu(I)-catalyzed Huisgen 1,3-
dipolar cycloaddition represents a convenient synthetic
method for the facile chemical transformation of sterically
hindered functional groups of triterpenoids. In this study,
compounds 2−4 were synthesized starting from 1, and their
structures were characterized. Conjugates 2 and 3 exhibited
solvent dependent self-assembly resulting in thixotropic and
self-healing organogels with helical fibrous networks. Gelation
2702
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
properties of the studied compounds were affected by the
position and the number of the spermine units. Bolaamphi-
phile-like compound 4 was found to be a nongelator and
displayed different behavior than 2 and 3 that were found to be
gelators. Gels obtained from 2 and 3 exhibited different
stiffness values. Our results suggest that the structural
modification of gelator and the solvents significantly affect
gelation and mechanical properties of gels. The gels, xerogels
and aerogels, were characterized using multiple and comple-
mentary microscopic and spectroscopic methods. Further, the
mechanical property of gels was extensively studied using
oscillatory rheological measurements.
The cytotoxicity of 2−4 was tested in four different cancer
cell lines within this investigation. The cytotoxicity depends on
the way of substitution of the oleanolic acid with the spermine
side chains. Importantly, compounds bearing spermine as a
substituent in the C(17)-COOH group, i.e., 3 and 4, showed
higher cytotoxicity. Furthermore, 3 (against MCF7 and HeLa)
and 4 (against MCF7, HeLa, and G-361) showed a somewhat
improved cytotoxicity profile compared to that of positive
control CDDP.
Prague, 16028 Prague 6, Czech Republic; Isotope
Laboratory, Institute of Experimental Botany of the Czech
Academy of Sciences, 14220 Prague 4, Czech Republic;
orcid.org/0000-0002-9083-4919
̌
David Saman − Institute of Organic Chemistry and
Biochemistry of the Czech Academy of Sciences, 16610
Prague 6, Czech Republic
Kia Bertula − Department of Applied Physics, Aalto
University, FI-02150 Espoo, Finland; orcid.org/0000-
0002-7134-3591
Manu Lahtinen − Department of Chemistry, University of
Jyväskylä, FI-40014 Jyväskylä, Finland; orcid.org/0000-
0001-5561-3259
́
́
Lucie Bednarova − Institute of Organic Chemistry and
Biochemistry of the Czech Academy of Sciences, 16610
Prague 6, Czech Republic
́
Markéta Pazderkova − Institute of Organic Chemistry and
Biochemistry of the Czech Academy of Sciences, 16610
Prague 6, Czech Republic; Institute of Physics, Faculty of
Mathematics and Physics, Charles University, 12116 Prague
2, Czech Republic
́
́
Overall, this work shows that there is a potential to utilize
naturally abundant sustainable oleanolic acid for new func-
tional materials, and this approach can be extended to other
triterpenoids. More importantly, this work also opens
possibilities to theoretical and computational chemists to
investigate the self-assembly of this new class of amphiphiles.
Each partial investigation in this work contributed to a
formation of a complex view on characteristics of the studied
supramolecular systems with the ability to form supramolecular
gels exhibiting a potential to become promising next-
generation materials for a broad range of technical and
biomedical applications.
Lucie Rarova − Laboratory of Growth Regulators, Institute of
Experimental Botany of the Czech Academy of Sciences, and
Faculty of Science, Palacký University, CZ-78371 Olomouc,
Czech Republic
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.langmuir.0c03335
Author Contributions
¶The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
̌
the manuscript. D.S., K.B., M.L., L.B., and M.P. contributed to
different analytical methods.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.langmuir.0c03335.
■
Notes
sı
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Additional details on analytical data of prepared
compounds, on gelation studies, SEM, TEM, rheology
studies, VC and VT NMR spectra, IR spectra, powder
diffraction analysis, TG/DSG analysis, and in silico
energy calculations (PDF)
The authors are thankful for the funding of this research from
̈
the MPO through grants FV10599 (Z.O.) and FV30300
(Z.W.). We acknowledge the provision of facilities and
technical support by the Aalto University Nanomicroscopy
Center (Aalto-NMC). We thank Dr. S. Hietala for useful
discussions on rheology. Part of this work was also carried out
under the framework of the Centre of Excellence in Molecular
Engineering of Biosynthetic Hybrid Materials (HYBER 2014-
2019) and the Photonics Research and Innovation (PRIEN)
Video S1 (MP4)
AUTHOR INFORMATION
Corresponding Authors
Nonappa − Department of Applied Physics, Aalto University,
FI-02150 Espoo, Finland; Faculty of Engineering and
Natural Sciences, Tampere University, FI-33101 Tampere,
Finland; Email: nonappa@aalto.fi, nonappa@tuni.fi
■
̈
flagship program. Z.O. thanks the Erasmus+ program of the
EU for funding her study visit to Aalto University, Finland.
L.R. thanks the Czech Science Foundation for grant no. 19-
01383S.
̌
Zdenek Wimmer − Department of Chemistry of Natural
Compounds, University of Chemistry and Technology in
Prague, 16028 Prague 6, Czech Republic; Isotope
Laboratory, Institute of Experimental Botany of the Czech
Academy of Sciences, 14220 Prague 4, Czech Republic;
orcid.org/0000-0002-4512-0116;
ABBREVIATIONS
■
DCM, dichloromethane; DMF, N,N-dimethylformamide;
THF, tetrahydrofuran; TBTA, tris[(1-benzyl-1H-1,2,3-triazol-
4-yl)methyl]amine; TLC, thin-layer chromatography; CEM,
T-lymphoblastic leukemia; MCF7, breast carcinoma; HeLa,
cervical carcinoma; G-361, malignant melanoma; BJ, human
foreskin fibroblasts
Email: zdenek.wimmer@vscht.cz, wimmer@biomed.cas.cz
Authors
̈
Zulal Ozdemir − Department of Chemistry of Natural
Compounds, University of Chemistry and Technology in
2703
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
REFERENCES
pubs.acs.org/Langmuir
Article
̌
́
(21) Cametti, M.; Dzolic, Z. New frontiers in hybrid materials: noble
metal nanoparticles - supramolecular gel systems. Chem. Commun.
2014, 50, 8273−8286.
■
(1) Weiss, R. G. Molecular Gels: Structure and Dynamics; Royal
Society of Chemistry: Cambridge, 2018; pp 1−376, DOI: 10.1039/
9781788013147.
(22) Meazza, L.; Foster, J. A.; Fucke, K.; Metrangolo, P.; Resnati, G.;
Steed, J. W. Halogen-bonding-triggered supramolecular gel formation.
Nat. Chem. 2013, 5, 42−47.
(2) Chivers, P. R. A.; Smith, D. K. Shaping and structuring
supramolecular gels. Nat. Rev. Mater. 2019, 4, 463−478.
(3) Weiss, R. G. The Past, Present, and Future of Molecular Gels.
What Is the Status of the Field, and Where Is It Going? J. Am. Chem.
Soc. 2014, 136, 7519−7530.
(4) Weiss, R. G.; Terech, P. Molecular Gels: Materials with Self-
Assembled Fibrillar Networks; Springer: Netherlands, 2006; pp 1−938,
DOI: 10.1007/1-4020-3689-2.
(5) Escuder, B.; Miravet, J. F. In Functional Molecular Gels, The
Design of Molecular Gelators; Zweep, N., van Esh, J. H., Eds.; Royal
Society of Chemistry: Cambridge, 2013; pp 1−29, DOI: 10.1039/
9781849737371-00001.
(6) Kuroiwa, K.; Shibata, T.; Takada, A.; Nemoto, N.; Kimizuka, N.
Heat-Set Gel-like Networks of Lipophilic Co (II) Triazole Complexes
in Organic Media and Their Thermochromic Structural Transitions. J.
Am. Chem. Soc. 2004, 126, 2016.
(7) Xie, F.; Qin, L.; Liu, M. A dual thermal and photo-switchable
shrinking-swelling supramolecular peptide dendron gel. Chem.
Commun. 2016, 52, 930−933.
(8) Ma, X.; Cui, Y.; Liu, S.; Wu, J. A thermo-responsive
supramolecular gel and its luminescence enhancement induced by
rare earth Y3+. Soft Matter 2017, 13, 8027−8030.
(9) Zheng, Y.; Wang, D.; Cui, J.; Mezger, M.; Auernhammer, G. K.;
Koynov, K.H.-J.; Butt, H.-J.; Ikeda, T. Redox-Responsive and
Thermoresponsive Supramolecular Nanosheet Gels with High
Young’s Moduli. Macromol. Rapid Commun. 2018, 39, 1800282.
(10) Banerjee, S.; Das, R. K.; Maitra, U. Supramolecular gels ‘in
action’. J. Mater. Chem. 2009, 19, 6649−6687.
(11) Ke, H.; Yang, L.-P.; Xie, M.; Chen, Z.; Yao, H.; Jiang, W. Shear-
induced assembly of a transient yet highly stretchable hydrogel based
on pseudopolyrotaxanes. Nat. Chem. 2019, 11, 470−477.
(12) Zhang, M.; Xu, D.; Yan, X.; Chen, J.; Dong, S.; Zheng, B.;
Huang, F. Self-Healing Supramolecular Gels Formed by Crown Ether
Based Host-Guest Interactions. Angew. Chem., Int. Ed. 2012, 51,
7011−7015.
(13) Dastidar, P. Designing Supramolecular Gelators: Challenges,
Frustrations, and Hopes. Gels 2019, 5, 15.
(14) Berthier, D.; Buffeteau, T.; Leger, J.-M.; Oda, R.; Huc, I. From
Chiral Counterions to Twisted Membranes. J. Am. Chem. Soc. 2002,
124, 13486−13494.
(15) Mihajlovic, M.; Staropoli, M.; Appavou, M.-S.; Wyss, H. M.;
Pyckhout-Hintzen, W.; Sijbesma, R. P. Tough Supramolecular
Hydrogel Based on Strong Hydrophobic Interactions in a Multiblock
Segmented Copolymer. Macromolecules 2017, 50, 3333−3346.
(16) Abdallah, D. J.; Weiss, R. G. n-Alkanes Gel n-Alkanes (and
Many Other Organic Liquids). Langmuir 2000, 16, 352−355.
(17) Suzuki, M.; Nakajima, Y.; Yumoto, M.; Kimura, M.; Shirai, H.;
Hanabusa, K. Effects of Hydrogen Bonding and van der Waals
Interactions on Organogelation Using Designed Low-Molecular-
Weight Gelators and Gel Formation at Room Temperature. Langmuir
2003, 19, 8622−8624.
́
(23) Arnedo-Sanchez, L.; Nonappa Bhowmik, S.; Hietala, S.;
Puttreddy, R.; Lahtinen, M.; De Cola, L.; Rissanen, K. Rapid self-
healing and anion selectivity in metallosupramolecular gels assisted by
fluorine-fluorine interactions. Dalton Trans. 2017, 46, 7309−7316.
(24) Kolari, K.; Bulatov, E.; Tatikonda, R.; Bertula, K.; Kalenius, E.;
Nonappa Haukka, M. Self-healing, luminescent metallogelation driven
by synergistic metallophilic and fluorine-fluorine interactions. Soft
Matter 2020, 16, 2795−2802.
(25) Muraoka, T.; Cui, H.; Stupp, S. I. Quadruple Helix Formation
of a Photoresponsive Peptide Amphiphile and Its Light-Triggered
Dissociation into Single Fibers. J. Am. Chem. Soc. 2008, 130, 2946−
2947.
(26) Tamminen, J.; Kolehmainen, E. Bile Acids as Building Blocks of
Supramolecular Hosts. Molecules 2001, 6, 21−46.
(27) Virtanen, E.; Kolehmainen, E. Use of Bile Acids in
Pharmacological and Supramolecular Applications. Eur. J. Org.
Chem. 2004, 2004, 3385−3399.
(28) Mukhopadhyay, S.; Maitra, U.; Krishnamoorthy, I. G.; Schmidt,
J.; Talmon, Y. Structure and Dynamics of a Molecular Hydrogel
Derived from a Tripodal Cholamide. J. Am. Chem. Soc. 2004, 126,
15905−15914.
(29) Nonappa; Maitra, U. Unlocking the potential of bile acids in
synthesis, supramolecular/materials chemistry and nanoscience. Org.
Biomol. Chem. 2008, 6, 657−669.
(30) Svobodova, H.; Noponen, V.; Kolehmainen, E.; Sievanen, E.
Recent Advances in Steroidal Supramolecular Gels. RSC Adv. 2012, 2,
4985−5007.
(31) Zhang, M.; Strandman, S.; Waldron, K. C.; Zhu, X. X.
Supramolecular hydrogelation with bile acid derivatives: structures,
properties and applications. J. Mater. Chem. B 2016, 4, 7506−7520.
(32) Zhang, M.; Fives, C.; Waldron, K. C.; Zhu, X. X. Self-Assembly
of a Bile Acid Dimer in Aqueous Solutions: From Nanofibers to
Nematic Hydrogels. Langmuir 2017, 33, 1084−1089.
(33) Grassi, S.; Carretti, E.; Dei, L.; Branham, C. W.; Kahr, B.;
Weiss, R. G. D-Sorbitol, a structurally simple, low molecular-mass
gelator. New J. Chem. 2011, 35, 445−452.
(34) Ramos Sasselli, I.; Halling, P. J.; Ulijn, R. V. T.; Tuttle, T.
Supramolecular Fibers in Gels Can Be at Thermodynamic
Equilibrium: A Simple Packing Model Reveals Preferential Fibril
Formation versus Crystallization. ACS Nano 2016, 10, 2661−2668.
(35) de Jong, J. J. D.; Lucas, L. N.; Kellogg, R. M.; van Esch, J. H.;
Feringa, B. L. Reversible Optical Transcription of Supramolecular
Chirality into Molecular Chirality. Science 2004, 304, 278−281.
(36) Terech, P. Metastability and Sol Phases: Two Keys for the
Future of Molecular Gels? Langmuir 2009, 25, 8370−8372.
(37) Wang, Y.; Tang, L.; Yu, J. Investigation of Spontaneous
Transition from Low-Molecular-Weight Hydrogel into Macroscopic
Crystals. Cryst. Growth Des. 2008, 8, 884−889.
(38) De Rudder, J.; Bergé, B.; Berghmans, H. Competition between
Gelation and Crystallization in Solutions of Syndiotactic Polystyrene
in cis-Decalin. Macromol. Chem. Phys. 2002, 203, 2083−2088.
(39) Shimizu, T.; Masuda, M.; Minamikawa, H. Supramolecular
Nanotube Architectures Based on Amphiphilic Molecules. Chem. Rev.
2005, 105, 1401−1443.
(40) Ziserman, L.; Lee, H.-Y.; Raghavan, S. R.; Mor, A.; Danino, D.
Unraveling the Mechanism of Nanotube Formation by Chiral Self-
Assembly of Amphiphiles. J. Am. Chem. Soc. 2011, 133, 2511−2517.
(41) Oliveira, I. S.; Lo, M.; Araujo, M. J.; Marques, E. F.
Temperature-responsive self-assembled nanostructures from lysine-
based surfactants with high chain length asymmetry: from tubules and
helical ribbons to micelles and vesicles. Soft Matter 2019, 15, 3700−
3711.
́
̈
(18) Das, R. K.; Banerjee, S.; Raffy, G.; Guerzo, A. D.; Desvergne, J.-
P.; Maitra, U. Spectroscopic, microscopic and first rheological
investigations in chargetransfer interaction induced organogels. J.
Mater. Chem. 2010, 20, 7227−7235.
(19) Tatikonda, R.; Bertula, K.; Nonappa Hietala, S.; Rissanen, K.;
Haukka, M. Bipyridine based metallogels: an unprecedented differ-
ence in photochemical and chemical reduction in the in-situ
nanoparticle formation. Dalton Trans. 2017, 46, 2793−2802.
̈
(20) Tatikonda, R.; Bulatov, E.; Ozdemir, Z.; Nonappa Haukka, M.
Infinite Coordination Polymer Networks: Metallogelation of Amino-
pyridine Conjugates and in situ Silver Nanoparticle Formation. Soft
Matter 2019, 15, 442−451.
2704
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
(42) Palmans, A. R. A.; Meijer, E. W. Amplification of Chirality in
Dynamic Supramolecular Aggregates. Angew. Chem., Int. Ed. 2007, 46,
8948−8968.
(64) Watase, M.; Nakatani, Y.; Itagaki, H. On the Origin of the
Formation and Stability of Physical Gels of Di-O-benzylidene-D-
sorbitol. J. Phys. Chem. B 1999, 103, 2366−2373.
(43) Li, Z.-T.; Zhao, X. Scanning Electron Microscopy. In
Supramolecular Chemistry: From Molecules to Nanomaterials; Gale, P.
A., Steed, J. W., Eds.; John Wiley & Sons: Chichester, 2012; Vol. 2 -
Techniques, pp 619−631, DOI: 10.1002/9780470661345.smc044.
(44) Kikuchi, J.-I.; Yasuhara, K. In Supramolecular Chemistry: From
Molecules to Nanomaterials; Gale, P. A., Steed, J. W., Eds.; John Wiley
& Sons: Chichester, 2012; Vol. 2 - Techniques, pp 633−645,
DOI: 10.1002/9780470661345.smc022.
́
́
̌
́
(65) Dzubak, P.; Hajduch, M.; Vydra, D.; Hustova, A.; Kvasnica, M.;
̌
́
Biedermann, D.; Markova, L.; Urban, M.; Sarek, J. Pharmacological
activities of natural triterpenoids and their therapeutic implications.
Nat. Prod. Rep. 2006, 23, 394−411.
(66) James, J. T.; Dubery, I. A. Pentacyclic Triterpenoids from the
Medicinal Herb, Centella asiatica (L.) Urban. Molecules 2009, 14,
3922−3941.
(67) Kasal, A. Structure and Nomenclature of Steroids. Steroid Analysis;
Makin, H. L. J., Gower, D. B., Eds.; Springer Netherlands: Dordrecht,
2010; pp 1−25, DOI: 10.1023/b135931_1.
(45) Mallia, V. A.; Weiss, R. G. Correlations between thixotropic and
structural properties of molecular gels with crystalline networks. Soft
Matter 2016, 12, 3665−3676.
(46) Dawn, A.; Kumari, H. Low Molecular WeightSupramolecu-
larGels Under Shear: Rheologyasthe Tool for Elucidating Structure-
Function Correlation. Chem. - Eur. J. 2018, 24, 762−776.
(47) Wool, R. P. Self-healing materials: a review. Soft Matter 2008, 4,
400−408.
(48) Hager, M. D.; Greil, P.; Leyens, C.; Zwaag, S. V. D.; Schubert,
U. S. Self-Healing Materials. Adv. Mater. 2010, 22, 5424−5430.
(49) Yu, X.; Cao, X.; Chen, L.; Lan, H.; Liu, B.; Yi, T. Thixotropic
and self-healing triggered reversible rheology switching in a peptide-
based organogel with a cross-linked nano-ring pattern. Soft Matter
2012, 8, 3329−3334.
(50) Strandman, S.; Zhu, X. X. Self-Healing Supramolecular
Hydrogels Based on Reversible Physical Interactions. Gels 2016, 2, 16.
(51) Bauer, W. H.; Collins, E. A. In Rheology: Theory and
Application; Eirich, F. R., Ed.; Academic Press: New York, 1967;
Ch. 8, DOI: 10.1016/B978-1-4832-2941-6.50014-3.
(52) Larson, R. G.; Wei, Y. A review of thixotropy and its rheological
modeling. J. Rheol. 2019, 63, 477−501.
(53) IUPAC Compendium of Chemical Terminology; McNaught, A.
D., Wilkinson, A., Eds.; Blackwell Scientific: Hoboken, NJ, 1997.
(54) Weng, W.; Jamieson, A. M.; Rowan, S. J. Structural origin of the
thixotropic behavior of a class of metallosupramolecular gels.
Tetrahedron 2007, 63, 7419−7431.
(55) Mukhopadhyay, P.; Fujita, N.; Takada, A.; Kishida, T.;
Shirakawa, M.; Shinkai, S. Regulation of a Real-Time Self-Healing
Process in Organogel Tissues by Molecular Adhesives. Angew. Chem.,
Int. Ed. 2010, 49, 6338−6342.
(68) Pollier, J.; Goossens, A. Oleanolic Acid. Phytochemistry 2012,
77, 10−15.
(69) Sporn, M. B.; Liby, K. T.; Yore, M. M.; Fu, L.; Lopchuk, J. M.;
Gribble, G. W. New Synthetic Triterpenoids: Potent Agents for
Prevention and Treatment of Tissue Injury Caused by Inflammatory
and Oxidative Stress. J. Nat. Prod. 2011, 74, 537−545.
(70) Shanmugam, M. K.; Dai, X.; Kumar, A. P.; Tan, B. K. H.; Sethi,
G.; Bishayee, A. Oleanolic acid and its synthetic derivatives for the
prevention and therapy of cancer: Preclinical and clinical evidence.
Cancer Lett. 2014, 346, 206−216.
(71) Kamble, S. M.; Goyal, S. N.; Patil, C. R. Multifunctional
pentacyclic triterpenoids as adjuvants in cancer chemotherapy: a
review. RSC Adv. 2014, 4, 33370−33382.
(72) Liu, J. Pharmacology of oleanolic acid and ursolic acid. J.
Ethnopharmacol. 1995, 49, 57−68.
(73) Wang, X.; Ye, X.-I.; Liu, R.; Chen, H.-L.; Bai, H.; Liang, X.;
Zhang, X.-D.; Wang, Z.; Li, W.-I.; Hai, C.-X. Antioxidant activities of
oleanolic acid in vitro: Possible role of Nrf2 and MAP kinases. Chem.-
Biol. Interact. 2010, 184, 328−337.
(74) Bag, B. G.; Paul, K. Vesicular and Fibrillar Gels by Self-
Assembly of Nanosized Oleanolic Acid. Asian J. Org. Chem. 2012, 1,
150−154.
(75) Bag, B. G.; Majumdar, R. Self-assembly of Renewable Nano-
sized Triterpenoids. Chem. Rec. 2017, 17, 841−873.
(76) Dash, S. K.; Chattopadhyay, S.; Dash, S. S.; Tripathy, S.; Das,
B.; Mahapatra, S. K.; Bag, B. G.; Karmakar, P.; Roy, S. Self-assembled
nano fibers of betulinic acid: A selective inducer for ROS/TNF-alpha
pathway mediated leukemic cell death. Bioorg. Chem. 2015, 63, 85−
100.
(77) Viola, J. R.; Leijonmarck, H.; Simonson, O. E.; Oprea, I. I.;
Frithiof, R.; Purhonen, P.; Moreno, P. M. D.; Lundin, K. E.;
Stromberg, R.; Smith, C. I. E. Fatty acid-spermine conjugates as DNA
carriers for nonviral in vivo gene delivery. Gene Ther. 2009, 16, 1429−
1440.
(78) Behr, J. P. Gene Transfer with Synthetic Cationic Amphiphiles:
Prospects for Gene Therapy. Bioconjugate Chem. 1994, 5, 382−389.
(79) Geall, A. J.; Taylor, R. J.; Earll, M. E.; Eaton, M. A. W.;
Blagbrough, I. S. Synthesis of Cholesteryl Polyamine Carbamates: pKa
Studies and Condensation of Calf Thymus DNA. Bioconjugate Chem.
2000, 11, 314−326.
(80) Blagbrough, I. S.; Hadithi, D. A.; Geall, A. J. Cheno-, Urso- and
Deoxycholic Acid Spermine Conjugates: Relative Binding Af®nities
for Calf Thymus DNA. Tetrahedron 2000, 56, 3439−3447.
(81) Smith, D. K. From fundamental supramolecular chemistry to
self-assembled nanomaterials and medicines and back again - how
Sam inspired SAMul. Chem. Commun. 2018, 54, 4743−4760.
(82) Merritt, M.; Lanier, M.; Deng, G.; Regen, S. L. Sterol-
Polyamine Conjugates as Synthetic Ionophores. J. Am. Chem. Soc.
1998, 120, 8494−8501.
(83) Janout, V.; Regen, S. L. Bioconjugate-Based Molecular
Umbrellas. Bioconjugate Chem. 2009, 20, 183−192.
(84) Lu, Y. M.; Deng, L. Q.; Huang, X.; Chen, J. X.; Wang, B.; Zhou,
Z. Z.; Hua, G. S.; Chen, W. H. Synthesis and anionophoric activities
of dimeric polyamine-sterol conjugates: the impact of rigid vs. flexible
linkers. Org. Biomol. Chem. 2013, 11, 8221−8227.
(56) Lescanne, M.; Grondin, P.; D’Aléo, A.; Fages, F.; Pozzo, J.-L.;
Monval, O. M.; Reinheimer, P.; Colin, A. Thixotropic Organogels
Based on a Simple N-Hydroxyalkyl Amide: Rheological and Aging
Properties. Langmuir 2004, 20, 3032−3041.
(57) Suzuki, M.; Hayakawa, Y.; Hanabusa, K. Thixotropic
Supramolecular Gel Based on L-Lysine Derivatives. Gels 2015, 1,
81−93.
(58) Nonappa; Maitra, U. Simple esters of cholic acid as potent
organogelators: direct imaging of the collapse of SAFINs. Soft Matter
2007, 3, 1428−1433.
(59) Ikonen, S.; Nonappa Valkonen, A.; Juvonen, R.; Salo, H.;
Kolehmainen, E. Bile acid-derived mono- and diketalssynthesis,
structural characterization and self-assembling properties. Org. Biomol.
Chem. 2010, 8, 2784−2794.
(60) Noponen, V.; Nonappa Lahtinen, M.; Valkonen, A.; Salo, H.;
̈
Kolehmainen, E.; Sievanen, E. Bile acid-amino acid ester conjugates:
gelation, structural properties, and thermoreversible solid to solid
phase transition. Soft Matter 2010, 6, 3789−3796.
́
(61) Svobodova, H.; Nonappa Wimmer, Z.; Kolehmainen, E.
Design, Synthesis and Stimuli Responsive Gelation of Novel
Stigmasterol-Amino Acid Conjugates. J. Colloid Interface Sci. 2011,
361, 587−593.
́
(62) Svobodova, H.; Nonappa Lahtinen, M.; Wimmer, Z.;
Kolehmainen, E. A Steroid-Based Gelator of A(LS)2 Type: Tuning
Gel Properties by Metal Coordination. Soft Matter 2012, 8, 7840−
7847.
(63) Lin, Y. C.; Weiss, R. G. A Novel Gelator of Organic Liquids and
the Properties of Its Gels1. Macromolecules 1987, 20, 414−417.
2705
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706

Langmuir
pubs.acs.org/Langmuir
Article
(85) Sadownik, A.; Deng, G.; Janout, V.; Regen, S. L. Rapid
Construction of a Squalamine Mimic. J. Am. Chem. Soc. 1995, 117,
6138−6139.
(86) Kim, H. S.; Choi, B. S.; Kwon, K. C.; Lee, S. O.; Kwak, H. J.;
Lee, C. H. Synthesis and Antimicrobial Activity of Squalamine
Analogue. Bioorg. Med. Chem. 2000, 8, 2059−2065.
(87) Chen, W. H.; Shao, X. B.; Moellering, R.; Wennersten, C.;
Regen, S. L. A Bioconjugate Approach toward Squalamine Mimics:
Insight into the Mechanism of Biological Action. Bioconjugate Chem.
2006, 17, 1582−1591.
(88) Dewangan, R. P.; Jain, A.; Tanwar, S.; Yar, M. S.; Pasha, S. Self-
assembly and hydrogelation of spermine functionalized aromatic
peptidomimetics against planktonic and sessile methicillin resistant S.
aureus. RSC Adv. 2016, 6, 112656−112666.
(89) Gabrielson, N. P.; Cheng, J. Multiplexed Supramolecular Self-
Assembly for Non-Viral Gene Delivery. Biomaterials 2010, 31, 9117−
9127.
(105) Yashima, E.; Ousaka, N.; Taura, D.; Shimomura, K.; Ikai, T.;
Maeda, K. Supramolecular Helical Systems: Helical Assemblies of
Small Molecules, Foldamers, and Polymers with Chiral Amplification
and Their Functions. Chem. Rev. 2016, 116, 13752−13990.
(106) Sollich, P. In Molecular Gels, Materials with Self-Assembled
Fibrillar Networks; Weiss, R. G., Terech, P., Eds.; Springer: Dordrecht,
Netherlands, 2006; pp 161−192, DOI: 10.1007/1-4020-3689-2_6.
(107) Li, J.-L.; Zhang, Z.-S.; Liu, X.-Y. Effects of kinetics on
structures of aggregates leading to fibrillar networks. In Molecular gels:
structure and dynamics; Weiss, R. G., Ed.; RSC: Cambridge, UK, 2018;
pp 88−128, DOI: 10.1039/9781788013147-00088.
(108) Raghavan, S. R. Distinct Character of Surfactant Gels: A
Smooth Progression from Micelles to Fibrillar Networks. Langmuir
2009, 25, 8382−8385.
(109) Wang, R. Y.; Liu, X. Y.; Narayanan, J.; Xiong, J. Y.; Li, J. L.
Architecture of Fiber Network: From Understanding to Engineering
of Molecular Gels. J. Phys. Chem. B 2006, 110, 25797−25802.
(110) Douglas, J. F. Theoretical Issues Relating to Thermally
Reversible Gelation by Supermolecular Fiber Formation. Langmuir
2009, 25, 8386−8391.
(111) Hashemnejad, S. M.; Huda, M.; Rai, N.; Kundu, S. Molecular
Insights into Gelation of Di-Fmoc-l-Lysine in Organic Solvent-Water
Mixtures. ACS Omega 2017, 2, 1864−1874.
(112) Hashemnejad, S. M.; Kundu, S. Probing Gelation and
Rheological Behavior of a Self-Assembled Molecular Gel. Langmuir
2017, 33, 7769−7779.
(113) Raghavan, S. R.; Douglas, J. F. The conundrum of gel
formation by molecular nanofibers, wormlike micelles, and
filamentous proteins: gelation without cross-links? Soft Matter 2012,
8, 8539−8546.
(114) Wu, S.; Gao, J.; Emge, T. J.; Rogers, M. A. Influence of solvent
on the supramolecular architectures in molecular gels. Soft Matter
2013, 9, 5942−5950.
(115) Diehn, K. K.; Oh, H.; Hashemipour, R.; Weiss, R. G.;
Raghavan, S. R. Insights into organogelation and its kinetics from
Hansen solubility parameters. Toward a priori predictions of
molecular gelation. Soft Matter 2014, 10, 2632−2640.
(116) Bunzen, H.; Kolehmainen, E. In situ formation of steroidal
supramolecular gels designed for drug release. Molecules 2013, 18,
3745−3759.
̌
́
́
́
̌
(90) Bildziukevich, U.; Vida, N.; Rarova, L.; Kolar, M.; Saman, D.;
̌
̌
Havlícek, L.; Drasar, P.; Wimmer, Z. Polyamine Derivatives of
Betulinic Acid and β-Sitosterol: A Comparative Investigation. Steroids
2015, 100, 27−35.
̌
́
̈
(91) Bildziukevich, U.; Kaletova, E.; Saman, D.; Sievanen, E.;
̌
Kolehmainen, E. T.; Slouf, M.; Wimmer, Z. Spectral and Microscopic
Study of Self-assembly of Novel Cationic Spermine Amides of
Betulinic Acid. Steroids 2017, 117, 90−96.
̈
́
́
́
(92) Bildziukevich, U.; Malík, M.; Ozdemir, Z.; Rarova, L.; Janovska,
̌
̌
̌
L.; Slouf, M.; Saman, D.; Sarek, J.; Nonappa Wimmer, Z. Spermine
Amides of Selected Triterpenoid Acids: Dynamic Supramolecular
Systems Formation Influences Cytotoxicity of the Drugs. J. Mater.
Chem. B 2020, 8, 484−491.
(93) Goddard-Borger, E. D. R. V.; Stick, R. V. An Efficient,
Inexpensive, and Shelf-Stable Diazotransfer Reagent: Imidazole-1-
sulfonyl Azide Hydrochloride. Org. Lett. 2007, 9, 3797−3800.
̈
(94) Fischer, N.; Goddard-Borger, E. D.; Greiner, R.; Klapotke, T.
M.; Skelton, B. W.; Stierstorfer, J. Sensitivities of Some Imidazole-1-
sulfonyl Azide Salts. J. Org. Chem. 2012, 77, 1760−1764.
(95) Huisgen, R. 1,3-Dipolar Cycloadditions Past and Future.
Angew. Chem., Int. Ed. Engl. 1963, 2, 633−645.
(96) Li, H.; Aneja, R.; Chaiken, I. Click Chemistry in Peptide-Based
Drug Design. Molecules 2013, 18, 9797−9817.
(97) Tron, G. C.; Pirali, T.; Billington, R. A.; Canonico, P. L.; Sorba,
G.; Genazzani, A. A. Click Chemistry Reactions in MedicinalChem-
istry: Applications of the 1,3-dipolar Cycloaddition Between Azides
and Alkynes. Med. Res. Rev. 2008, 28, 278−308.
(98) Cheng, K.; Liu, J.; Liu, X.; Li, H.; Sun, H.; Xie, J. Synthesis of
glucoconjugates of oleanolic acid as inhibitors of glycogen
phosphorylase. Carbohydr. Res. 2009, 344, 841−850.
̈
̌
̌
(99) Ozdemir, Z.; Bildziukevich, U.; Saman, D.; Havlícek, L.;
́
́
́
́
Rarova, L.; Navratilova, L.; Wimmer, Z. Amphiphilic Derivatives of
(3β,17β)-3-Hydroxyandrost-5-ene-17-carboxylic Acid. Steroids 2017,
128, 58−67.
̈
(100) Bertula, K.; Martikainen, L.; Munne, P.; Hietala, S.; Klefstrom,
J.; Ikkala, O. Nonappa Strain-Stiffening of Agarose Gels. ACS Macro
Lett. 2019, 8, 670−675.
(101) Brizard, A.; Oda, R.; Huc, I. Chirality Effects in Self-assembled
Fibrillar Networks. In Low Molecular Mass Gelator. Topics in Current
Chemistry; Springer: Berlin, Heidelberg, 2005; pp 167−213,
DOI: 10.1007/b107174.
(102) Spector, M. S.; Selinger, J. V.; Schnur, J. M. Chiral Molecular
Self-Assembly. In Materials-Chirality; Denmark, S. E., Siegel, J., Green,
M. M., Nolte, R. J. M., Meijer, E. W., Eds.; John Wiley & Sons:
Washington, DC, 2003; pp 281−372, DOI: 10.1002/
0471471895.ch5.
(103) Bag, B. G.; Dinda, S. K.; Dey, P. P.; Mallia, V. A.; Weiss, R. G.
Self-Assembly of Esters of Arjunolic Acid into Fibrous Networks and
the Properties of their Organogels. Langmuir 2009, 25, 8663−8671.
(104) Liu, M.; Zhang, L.; Wang, T. Supramolecular Chirality in Self-
Assembled Systems. Chem. Rev. 2015, 115, 7304−7397.
2706
https://dx.doi.org/10.1021/acs.langmuir.0c03335
Langmuir 2021, 37, 2693−2706