A new hyaluronan modified with β-cyclodextrin on hydroxymethyl groups forms a dynamic supramolecular network

Article abstract of DOI:10.3390/molecules24213849

A new hyaluronan derivative modified with β-cyclodextrin units (CD-HA) was prepared via the click reaction between propargylated hyaluronan and monoazido-cyclodextrin (CD) to achieve a degree of substitution of 4%. The modified hyaluronan was characterize

Full text of DOI:10.3390/molecules24213849

molecules
Article
A New Hyaluronan Modified with β-Cyclodextrin on
Hydroxymethyl Groups Forms a Dynamic
Supramolecular Network
1
Jelica Kovacˇevic´
, Zdenˇka Prucková 1 , Tomáš Pospíšil 2 , Veˇra Kašpárková 3,
Michal Rouchal 1 and Robert Vícha 1,
*
1
Department of Chemistry, Faculty of Technology, Tomas Bata University in Zl
Czech Republic; jelica.kovacevic1@gmail.com (J.K.); pruckova@utb.cz (Z.P.); rouchal@utb.cz (M.R.)
Centre of the Region Han for Biotechnological and Agricultural Research, Department of Chemical Biology
ín, Vavrecˇkova 275, 760 01 Zlin,
2
á
and Genetics, Faculty of Science, Palacký University Olomouc, Šlechtitelu˚ 241/27, CZ-783 71 Olomouc,
Czech Republic; tomas.pospisil@upol.cz
Department of Fat, Surfactant and Cosmetics Technology, Faculty of Technology, Tomas Bata University in
Zlín, Vavrecˇkova 275, 760 01 Zlin, Czech Republic; vkasparkova@utb.cz
Correspondence: rvicha@utb.cz; Tel.: +420-5-7603-1103
3
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 11 September 2019; Accepted: 23 October 2019; Published: 25 October 2019
Abstract: A new hyaluronan derivative modified with β-cyclodextrin units (CD-HA) was prepared
via the click reaction between propargylated hyaluronan and monoazido-cyclodextrin (CD) to achieve
a degree of substitution of 4%. The modified hyaluronan was characterized by 1H-nuclear magnetic
1
resonance spectroscopy (NMR) and size exclusion chromatography. Subsequent H-NMR and
isothermal calorimetric titration experiments revealed that the CD units on CD-HA can form virtual
1:1, 1:2, and 1:3 complexes with one-, two-, and three-site adamantane-based guests, respectively.
These results imply that the CD-HA chains used the multitopic guests to form a supramolecular
cross-linked network. The free CD-HA polymer was readily restored by the addition of a competing
macrocycle, which entrapped the cross-linking guests. Thus, we demonstrated that the new
CD-HA polymer is a promising component for the construction of chemical stimuli-responsive
supramolecular architectures.
Keywords: cyclodextrin; sodium hyaluronan; click reaction; host-guest systems; supramolecular
network
1. Introduction
Biopolymers are defined as polymers that are produced under natural conditions, from natural
sources, or by chemical synthesis from biological materials. Biopolymers are synthesized within cells by
enzymatic processes [1]. Biopolymers from the glycosaminoglycan series, namely, hyaluronic acid (HA),
are of particular interest since they are water soluble, biodegradable, biocompatible, and abundant
in nature. Biopharmaceutical properties, such as biocompatibility, biodegradability, and absence of
immunotoxicity and cytotoxicity, make biopolymers ideal candidates for use in drug delivery [
2,
3],
as drug carriers to prolong the effects of a drug [4,5], or for use as bioresorbable scaffolds in tissue
engineering [6,7].
Natural polymers can be chemically modified to introduce different functional groups in the
polymer chain, improve the properties of the polymer, and enable new functions. Studies on the chemical
modifications of HA have been mainly concerned with cross-linking and grafting. The chemical
structure of HA provides the three most commonly used sites for covalent modification, i.e., carboxylate
groups, hydroxyl groups, and -NHCOCH3 groups [8]. Hydroxyl groups are usually cross-linked via an
Molecules 2019, 24, 3849; doi:10.3390/molecules24213849
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ether linkage, and carboxylic acid groups are cross-linked via an ester linkage. The chemical modification
of the -NHCOCH3 group includes deacetylation, amidation, and hemiacetylation. Amidation methods
are used for the deacetylation of the N-acetyl groups, and these reactions are usually performed
using hydrazine sulphate [
leads to the preparation of HA-based hydrogels [12
9
–
11]. The most common modification of HA is cross-linking, which
13]. Hyaluronic acid can be cross-linked by
,
bisepoxide [14], divinylsulphone derivatives under alkaline conditions [15], or glutaraldehyde [16],
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride [16], and biscarbodiimide [17] under
acidic conditions. In these cases, covalent bonds are used for the cross-linking of the polymer chains,
and, therefore, any reversible modification of the polymer properties is disabled. In contrast, when the
supramolecular host-guest approach is employed, the degree of cross-linkage can be controlled due to
the reversible nature of supramolecular interactions [18].
Cyclodextrins (CDs), i.e., natural macrocyclic oligosaccharides with six to eight d-glucose units,
have been studied in host-guest chemistry for the construction of supramolecular aggregates due to
their hydrophobic cavities. Supramolecular polymers employing β-CD (a macrocycle consisting of
seven glucose units) as a host have been constructed to improve stimuli-responsive properties [19].
Cyclodextrins can be easily modified on their narrow rim, which is decorated by primary hydroxyl
groups. There are three strategies for constructing
-CD-based host-guest monomers to assemble a polymer chain [20], incorporating
in the side chain of the polymer [21] or assembling with a guest polymer and utilizing
blocks with extensible sites to construct supramolecular polymers [22]. The grafting of
has been achieved by covalent approaches, including the conjugation of amine-modified
β
-CD-based supramolecular polymers, such as using
-CD macrocycles
-CD as building
-CD to HA
-CD to the
β
β
β
β
β
carboxylic acid group of HA [23
groups of β-CD [25], or by supramolecular assembly [26,27].
To the best of our knowledge, only one study has thus far been reported that describes the
preparation and properties of HA modified with -CD via a triazole linkage [28]. However, in this
case, the cyclodextrin units were grafted onto carboxylate functional groups, which are believed to
play an important role in the recognition of HA by cells [29 30]. The aim of this work was to examine
whether the natural hyaluronan, which was conveniently modified with -cyclodextrin units via
,24], the interaction of boric acid grafted to HA with the hydroxyl
β
,
β
the primary hydroxyl groups of HA, can form supramolecular networks with two- and three-site
adamantane-based guest motifs (for structures, see Figure 1) in reversible manner.
Figure 1. The adamantane-based supramolecular guests used in this study.
2. Results and Discussion
2.1. Chemistry
Initially, mono-6-(p-toluenesulphonyl)-β-cyclodextrin (2) was prepared from natural β-CD (1),
where selective monotosylation was performed in the C6-OH position (Scheme 1). The reaction
was performed in an alkaline aqueous solution according to a published procedure [31]. Covalent
linkage between the tosyl group and β-CD was indicated by DOSY spectrum as shown in Figure S1.

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Afterwards, tosylated
β-cyclodextrin (
2
) was readily converted to monoazido-CD (
3
) by an azidation
reaction [32]. The azido group located on the primary rim of the CD scaffold is a suitable anchoring
point for the introduction of an alkyne into the structure. Thus, a new CD-HA polymer was synthesized
that combined the chemical modification of the HA chain with terminal alkyne groups via the
sequence of partial selective oxidation, reductive amination, and final 1,3-dipolar cycloaddition
between monoazido-β-CD (3) and propargylated HA (6) (Scheme 2).
Scheme 1. Monosubstitution of β-cyclodextrin (CD) intermediates at the primary hydroxyl group.
Natural hyaluronan (
(4-acetamide-TEMPO) [33] using sodium hypochlorite as a primary oxidant to produce aldehyde
moieties (hydrated form of product ( ) is shown in Scheme 2). Oxidation occurred at the C6-OH
position, and the oxidized HA was grafted with primary amines carrying alkyne groups via Schiff
imine intermediate mediation to produce modified hyaluronan ( ). A new CD-HA polymer was
4) was oxidized with 2,2,6,6-tetramethyl-4-acetamidopiperidine-1-oxyl
5
6
prepared via the 1,3-dipolar cycloaddition between monoazido-CD and the propargyl derivative of HA.
The coupling between monoazido-CD and the alkyne-terminated derivative of HA was successfully
performed in 1 mM PBS at ambient temperature (Scheme 2).
Scheme 2. Synthesis of the cyclodextrin-modified hyaluronic acid (CD-HA) via a click reaction.
2.2. Characterization of Products
The chemical structures of the monosubstituted CD intermediates were assessed using 1H-NMR,
ESI-MS, and DOSY analyses (see the Supporting information and Materials and Methods section).
The propargylated HA was characterized by means of 1H-NMR. The 1H-NMR spectrum shows signals
for the N-acetyl group at 2.02 ppm. Signals of the HA skeletal H-atoms can be seen at 3.40–4.00 ppm.
The remaining detected signals at 3.10 and 2.85 ppm can be assigned to the methylene protons at position
C6. These signals confirmed the successful formation of the covalent linkage between propargylamine
and HA. The 1H-NMR spectrum of the new CD-HA polymer showed a peak at 5.06 ppm, which is
attributed to the H1 (
peak of the N-acetyl group at 2.00 ppm and the peak at 5.06 ppm, which is related to the anomeric
proton of
-cyclodextrin (for the 1H-NMR spectrum, see Figure S3). It can be clearly seen in the DOSY
β-CD). The substitution degree of 4% was calculated from the integration of the
β

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spectrum (Figure S2) that the signals of the anomeric H-atoms from the CD macrocycle, anomeric
H-atoms from the HA chain, skeletal H-atoms, and CH3 groups from the acetamido substituent lie
on one line, i.e., display the same diffusion coefficient. This observation implies that the CD units
are covalently linked to the HA polymer. Additionally, size exclusion chromatography was used to
determine whether the HA polymer chain was markedly cleaved during the chemical transformations.
As is shown in Figure S4, the peaks related to the original HA polymer, oxidized HA, propargylated
HA, and CD-HA appeared at essentially the same retention time. Therefore, it can be inferred that no
significant scission of the polymer backbone occurred.
2.3. Supramolecular Study of CD-HA
We started our examination of the supramolecular properties of CD-HA by isothermal titration
calorimetry (ITC) with the single-site guest G1 (for structure, see Figure 1). Since the chemical nature of
guest G1 is well-defined and non-hygroscopic, it can be used as a standard to allow us to determine the
actual concentration of available CD units. It was previously demonstrated that the guest G1 forms a 1:1
complex with natural β-CD with a sufficiently high binding constant [34] to allow for the unambiguous
analysis of titration data, namely, the stoichiometric parameter n, even at millimolar concentrations of
the supramolecular components. Therefore, we performed an ITC titration of the CD-HA polymer
with G1 and calculated the actual concentration of CD units using the abovementioned parameter n
from ITC and the degree of substitution, which was previously obtained by integrating the 1H-NMR
spectrum. This concentration value was used as the input concentration of CD for the data processing
of further titrations of CD-HA with G2 and G3. Figure 2 clearly shows that relative to guest G1
an equivalency point was reached after the addition of one-half and one-third of the molar quantities
of G2 and G3, respectively (see Table 1 for ITC data). This means that, for instance, ditopic guest G2
employs both its binding sites to saturate the CD units of CD-HA to form a supramolecular network,
as depicted in Figure 2.
,
Figure 2. ITC results and interpretation of titrations of CD-HA with guests G1, G2, and G3.

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Table 1. Thermodynamic data for the guests G1–G3.
−∆H [kJ·mol−1
]
∆S [J·mol−1·K−1
]
Guest
G1
Host
n 1
K 2 [dm3·mol−1
]
β-CD
CB7 3
CD-HA
0.96
ng 4
1
7.05 × 103
1.7 × 1014
1.12 × 104
24.5
80.6
13.9
−7
0
32
β-CD 5
0.49
0.56
0.43
9.20 × 104
1.35 × 1012
1.14 × 106
59.0
ng
11.4
−99
G2
CB7 5
ng
79
CD-HA
β-CD 6
0.37
0.39
0.31
1.02 × 105
4.84 × 1010
1.00 × 105
85.7
173.8
87.6
−187
−369
−193
G3
CB7 6
CD-HA
1
2
n values for CD-HA are given relative to the n value for G1
;
values of apparent K are given for CD-HA;
reference [35]; 4 not given, 1:1 stoichiometry was assumed; 5 reference [36]; 6 reference [37].
3
Complementary information regarding the nature of the interactions between the supramolecular
components was obtained using 1H-NMR titration. The stacked spectra that were recorded during
the titration of CD-HA with the guest G1 are displayed in Figure 3. Note that the signals of all
hydrogen atoms, which can be assigned to the adamantane cage, are significantly shifted downfield.
This phenomenon is well recognized and attributed to the deshielding effect of the cyclodextrin
interior cavity [38]. In addition, complexation-induced shifts of the guest signals are very similar to
those observed within the titration of G1 with natural β-CD. Similar results were obtained within the
1H-NMR titration of G2 and G3, respectively (see Figures S5 and S6). Therefore, we can conclude
that the adamantane cage is included inside the CD cavity in all examined cases, and the nature of
the interaction detected by ITC can be attributed to the formation of the supramolecular complex in a
host-guest manner.
Figure 3. Stacking plot of portions of the 1H-NMR spectra recorded within the titration of G1 with
CD-HA (D2O, 30 ◦C).
Afterwards, we performed a competitive experiment to determine whether the cross-linking
agents G2 or G3 can be withdrawn from the CD-HA complex. The 1H-NMR spectra, which were
recorded during this experiment, are shown in Figure 4. Initially, we added an equimolar quantity
of CD-HA to the solution of guest G2 to form a supramolecular network, as described above (lines i
and ii in Figure 4). Subsequently, two molar equivalents of cucurbit[7]uril (CB7), with respect to G2
,
were added in two portions (lines iii and iv in Figure 4). Since cucurbit[7]uril is a well-known strong
binding agent for the cationic derivatives of cage hydrocarbons [39,40], we expected that a complex of
G2 with CB7 would be preferred in this mixture. After the first addition of CB7, a new set of G2 signals

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appeared. Since the new signals related to the adamantane cage were significantly shifted upfield
(marked with in Figure 4), we infer that the complex of G2@CB7 formed. Note that the remaining
‡
signals of the adamantane cage are markedly shifted downfield (asterisked in Figure 4, line iii). This can
be explained by a change in the environment, as the supramolecular network is partially cleaved. After
the second addition of CB7, the signals related to the adamantane cage complexed inside the
β-CD
units completely disappeared. It should be noted that complex G2@CB7 was sparingly soluble in this
system, and a colorless precipitate was formed. Since we obtained similar results with the guest G3
we infer that our new hyaluronan derivative is capable of forming supramolecular networks that can
be modulated by a competitive approach.
,
Figure 4. Stacking plot of portions of the 1H-NMR spectra recorded within the competitive experiment
in D2O at 30 ◦C. Free guest G2
(i); G2 and 1 eq. of CD-HA (ii); G2, 1 eq. of CD-HA and 1 eq. of CB7
(
iii); G2, 1 eq. of CD-HA and 2 eq. of CB7 (iv). The signal of the residual propan-2-ol is marked with
†
and signals of the adamantane cage complexed with
β-CD and CB7 macrocycle are marked with * and
‡, respectively.
3. Materials and Methods
3.1. General
Sodium hyaluronate (HA, Mn = 67.26
by Contipro Pharma (Doln Dobroucˇ, Czech Republic) and characterized prior to use by size exclusion
chromatography (Breeze 1525, Waters, Milford, MA US; column Shodex OH pack SB-806 HQ, 30 cm;
detector RI; calibrated to pullulan 0.18–642 kg
mol−1). All other chemicals were purchased from Merck
(Darmstadt, Germany). The purification of the modified HA was performed using a dialysis tubing
cellulose membrane (14 kg
mol−1 MWCO, avg. flat width 33 mm, Merck, Darmstadt, Germany) and
SnakeSkin dialysis tubing (3.5 kg
mol−1 MWCO, avg. flat width 34 mm, Thermo Scientific, Waltham,
MA, USA). Milli-Q (Merck, Darmstadt, Germany) water was used for preparation of modified HA.
HA was purified by a Direct-Q3 UV purification system (Merck, Darmstadt, Germany). Guests G2 36
and G3 37] were prepared via a conventional quaternization reaction and were reported previously,
×
103 g
·
mol−1, Mw = 174.27
×
103 g
·
mol−1) was provided
í
·
·
·
[
]
[
while the guest G1 was purchased from Merck.
1H-NMR and 13C-NMR experiments were performed on an Avance TM-III 500 instrument
(Bruker BioSpin, Billerica, MA, US) operating at frequencies of 500.11 MHz (1H) and 125.77 MHz
1
(13C). H-NMR and 13C-NMR chemical shifts were referenced to the signal of the solvent [1H:
δ
(residual HDO) = 4.70 ppm,
δ (residual DMSO-d5) = 2.50 ppm]. The degree of substitution (DS)
was determined from 1H-NMR spectra as the average number of substituted disaccharides units per
100 units. Diffusion-ordered spectra (DOSY) were collected using an ECA-500 spectrometer (Jeol,
Tokyo, Japan) operating at a frequency of 500.16 MHz (1H) and a bpp-led-dosy-pfg pulse sequence.

Molecules 2019, 24, 3849
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The DOSY acquisition parameters were as follows: diffusion time 0.1 s, linear array of gradient strength
(from 20 to 300 mT
−1, 16 points), acquisition time 2.61 s, relaxation delay 8 s, 4 dummy scans and
·
m
1024–2048 total scans. Data were processed using the DELTA software package (ver. 5.0.5.1, Jeol, Tokyo,
Japan).
Fourier transform infrared spectra were recorded on an ALPHA FTIR spectrometer (Bruker Optik
GmbH, Ettlingen, Germany). The samples were measured in KBr pellets using 16 scans, and absorption
spectra were scanned within the range of 400–4000 cm−1
.
Electrospray mass spectra were recorded using an amaZon X ion-trap mass spectrometer (Bruker
Daltonik GmbH, Bremen, Germany) equipped with an electrospray ionization source. All experiments
were conducted in both positive and negative ion polarity mode. Each sample (5
into the ESI source in a CH3OH:H2O (1:1, v:v) solutions using a syringe pump with a constant flow rate
of 3
min−1. The instrumental conditions used for the measurements are as follows: electrospray
voltage of 4.2 kV, capillary exit voltage of
140 V, drying gas temperature of 220 ◦C, drying gas flow
µg
·
mL−1) was infused
µL
·
±
±
rate of 6.0 dm3·min−1, and nebulizer pressure of 55.16 kPa.
Size exclusion chromatography (Shimadzu Prominence UFLC, Kyoto, Japan) with a refractive
index detector was used to characterize the modified polymers. Samples of HA (5 mg
prepared in 0.2 m NaNO3 + 0.01 m NaH2PO4, pH = 7 buffer. Measurements were performed at 40 ◦C
·
mL−1) were
using PL aquagel-OH 60 and 40 (2
×
30 cm) columns (Agilent, Santa Clara, CA, USA) with a flow rate
of 0.8 mL·min−1
.
The association constants and thermodynamic parameters for the complexation of the guests
(
G1
titration calorimetry measurements were carried out in a 0.1 m NaNO3, 0.005 m NaH2PO4 buffer at
pH = 5.03. The CD-HA solution (0.62 mg
cm−3) was placed in the sample cell where the guest solution
was added in a series of 29 injections (10 L per injection). The concentrations of the G1 G2, and G3
, G2, G3) with CD-HA were determined using a VP-ITC instrument (MicroCal, US). Isothermal
·
µ
,
were 0.50 mM, 0.25 mM, and 0.18 mM, respectively. The raw experimental data were analyzed with
MicroCal ORIGIN software (ver. 7.0, OriginLab, Northampton, MA, USA).
3.2. Preparation of 6-O-Monotosyl-6-Deoxy-β-Cyclodextrin (2)
Compound
in alkaline aqueous medium according to the published procedure [31], with minor modifications.
-Cyclodextrin (1.00 g, 0.88 mmol) was dispersed in an aqueous 0.4 M NaOH solution. Afterwards,
2 was prepared by the reaction of β-CD with p-toluenesulphonyl chloride (TsCl)
β
p-TsCl (0.71 g, 3.72 mmol) was slowly added in portions, and the solution was vigorously stirred at
0 ◦C. After 2 h, the remaining solid TsCl was removed by suction. Hydrochloric acid (0.8 M) was added
dropwise to the filtrate to adjust pH to 8, and a white precipitate appeared. The mixture was stored in
a refrigerator at 4 ◦C overnight, and the precipitate was collected by filtration through a sintered glass
funnel. The colorless solid was washed with ice-cold water and dried under vacuum (6 torr) at 60 ◦C
to yield 0.81 g (72%) of monotosylated cyclodextrin. M.p. = 168–170 ◦C.
1H-NMR (DMSO-d6,
δ ppm): 7.75 (d, J = 8.7 Hz, 2H, Ph), 7.43 (d, J = 8.2 Hz, 2H, Ph), 5.62–5.86 (m, 14H,
OH2, OH3), 4.85 (s, 3H, H1), 4.77 (s, 2H, H1), 4.42–4.48 (m, 5H, OH6), 4.21–4.24 (m, 1H, OH6), 3.46–3.66
(m, 30H, H1, H3, H5, H6), 3.17–3.38 (m, 14H, H2, H4 overlapped with H2O), 2.44 (s, 3H, -CH3). ESI-MS
(pos.) m/z (%): 656.1 [M + H + Na]2+ (29), 664.1 [M + H + K]2+ (100), 1311.3 [M + Na]+ (15). ESI-MS
(neg.) m/z (%): 643.1 [M
1643 (C=C), 1030 (C-O).
−
2H]2− (100), 1287.3 [M
−
H]− (57). FTIR (KBr, cm−1) 3385 (O-H), 2928 (C-H),
3.3. Preparation of 6-O-Monoazido-6-Deoxy-β-Cyclodextrin (3)
Compound
2
(0.50 g, 0.38 mmol) was suspended in dimethylformamide (DMF), and the mixture
was heated at 80 ◦C for 30 min [32]. Subsequently, NaN3 (0.12 g, 1.84 mmol) was slowly added to the
solution, and the reaction mixture was stirred for 24 h at 80 ◦C. Afterwards, the crude product was

Molecules 2019, 24, 3849
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precipitated by the addition of acetone. The colorless precipitate was collected by suction and dried in
vacuum at 60 ◦C to yield 0.40 g (90%) of monoazidocyclodextrin. M.p. = 208–210 ◦C.
1H-NMR (DMSO-d6,
δ ppm): 5.58–5.85 (m, 14H, OH2, OH3), 4.87 (m, 1H, H1), 4.83 (m, 6H, H1),
4.40–4.57 (m, 6H, OH6), 3.49–3.73 (m, 28H, H3, H5, H6), 3.30 (s, 14H, H2, H4 overlapped with H2O).
ESI-MS (pos.) m/z (%): 591.7 [M + H + Na]2+ (23), 599.6 [M + H + K]2+ (100), 1182.4 [M + Na]+ (24).
ESI-MS (neg.) m/z (%): 578.6 [M
2922 (C-H), 2110 (N3), 1157 (C-N).
−
2H]2− (79), 1158.3 [M
−
H]− (100). FTIR (KBr, cm−1) 3383 (O-H),
3.4. Preparation of Oxidized Hyaluronan (5)
The selective oxidation of hyaluronic acid (4) was performed according to a slightly improved
procedure, which has been published previously [33]. Hyaluronic acid sodium salt (1.00 g, 2.50 mmol)
was dissolved in deionized water (50 mL). Subsequently, NaBr (0.13 g, 1.25 mmol) and Na2HPO4 12H2O
(1.943 g, 5.43 mmol) were added. The reaction mixture was cooled in an ice water bath to 0 ◦C and
stirred for 1 h. Finally, 4-acetamido-TEMPO (5.30 mg, 0.02 mmol) and 340 L of 11% NaOCl were
·
µ
added to the reaction mixture under nitrogen atmosphere. The reaction was carried out for 15 min
and quenched by the slow addition of 5% aqueous Na2S2O3. Afterwards, the solution was dialyzed
against a 0.5% solution of salts (NaCl, NaHCO3). Finally, the product was precipitated with ethanol.
Yield = 0.93 g (95%).
1H-NMR (HDO,
δ ppm): 5.25 (s, 1H, CHO), 4.5 (m, 2H), 3.4–4.0 (m, 10H, skeletal H-atoms of HA), 2.0
(s, 3H, CH3). FTIR (KBr, cm−1) 3419 (C-OH), 2923, 1656, 1614 (-HC=O), 1413 (-O-C=O), 1153, 1080
(C-OH), 1039, 607.
3.5. Preparation of Propargylated Hyaluronan (6)
Reductive amination of hyaluronic acid was performed according to a previously published
procedure [33] with minor modifications. The oxidized HA (0.20 g, 0.50 mmol) was dissolved in
deionized water. Afterwards, propargylamine hydrochloride (6.90 mg, 0.07 mmol) was slowly added,
and the reaction mixture was stirred for 5 h. Subsequently, picoline borane complex (8.00 mg, 0.07 mmol)
was added, and the reaction was carried out overnight at room temperature. The prepared solution
was dialyzed in a 0.5% solution of NaCl and NaHCO3. The product was isolated by precipitation with
ethanol. Yield = 0.18 g (92%).
1H-NMR (D2O,
δ ppm): 4.5 (m, 2H), 3.4–4.0 (m, 10H), 3.1 (m, CH2), 2.85 (m, CH2), 2.0 (s, CH3). FTIR
(KBr, cm−1) 3379 (C-OH), 2894, 2131 (C≡C), 1614, 1407, 1078, 613.
3.6. Conjugation of β-CD to HA
Hyaluronan polymer modified with
between monoazido-CD and propargylated hyaluronan
dissolved in 2 mL of 1 mmol phosphate buffer solution (PBS) at pH = 7.4 followed by the subsequent
addition of propargylated HA (7.72 mg, 0.01 mmol). Furthermore, CuSO4 5H2O (2.15 mg, 0.008 mmol)
and sodium ascorbate (1.70 mg, 0.008 mmol) were dissolved in 120 L of PBS and slowly added into
β-cyclodextrin (CD-HA) was obtained via a click reaction
3
6
. Compound (0.10 g, 0.08 mmol) was
3
·
µ
the reaction mixture. The reaction mixture was stirred for a few minutes and was diluted with 1 mL
of water. Afterwards, the solution was dialyzed against the 0.5 mmol EDTA in order to remove the
copper catalyst. The product was recovered by precipitation with ethanol and dried under vacuum to
yield 0.14 g (80%) of colorless powder.
1H-NMR (D2O,
δ ppm): 5.0 (s), 4.6 (d), 4.5 (d), 3.3–4.0 (m, skeletal H-atoms of HA), 2.0 (s, NHCOCH3).
FTIR (KBr, cm−1) 3394 (N-H), 3028 (C-H), 2785 (C-H), 1673 (C=O), 1627 (C=O), 1396 (C-O), 1032,
710, 550.

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4. Conclusions
In conclusion, a new hyaluronan polymer modified with
β-cyclodextrin units at the C6 position of
the N-acetylglucosamine ring was prepared via 1,3-dipolar cycloaddition between propargylated HA
and monoazido- -CD to reach a degree of substitution of 4%. Subsequent NMR and ITC experiments
revealed that -CD units on the CD-HA polymer form 1:1, 1:2, and 1:3 supramolecular aggregates
β
β
in a host-guest manner with adamantane-based mono-, di-, and tritopic guests, respectively. Finally,
we demonstrated that the supramolecular network can be readily cleaved to restore the original CD-HA
by the addition of a suitable competitor for the multitopic guests. These results confirm our initial
expectation that the new CD-HA polymer is a promising component for the construction of chemical
stimuli-responsive supramolecular architectures.
Supplementary Materials: The following are available online; Figure S1: DOSY spectrum of the crude product
of monotosylation of
Figure S4: SEC results of original hyaluronan (HA,
(Prop-HA, ), and final hyaluronane modified by β-CD units (CD-HA), Figure S5: Stacking plot of portions of
β
-CD, Figure S2: DOSY spectrum of CD-HA, Figure S3: 1H-NMR spectrum of CD-HA,
4
), oxidized hyaluronan (OX-HA, 5), propargylated hyaluronan
6
the 1H-NMR spectra recorded within the titration of G2 with CD-HA (D2O, 30 ◦C), Figure S6: Stacking plot of
portions of the 1H-NMR spectra recorded within the titration of G3 with CD-HA (D2O, 30 ◦C).
Author Contributions: Conceptualization, R.V.; methodology, J.K., Z.P., M.R., V.K., and R.V.; investigation, J.K.,
Z.P., T.P., V.K., M.R., and R.V.; writing—original draft preparation, J.K., Z.P., M.R., and R.V.; writing—review and
editing, J.K., Z.P., T.P., V.K., M.R., and R.V.; visualization, J.K., Z.P., T.P., V.K., M.R., and R.V.; supervision, R.V.;
project administration, R.V.; funding acquisition, J.K.
Funding: This research was funded by the Internal Funding Agency of the Tomas Bata University in Zl
grant number IGA/FT/2019/007.
ín,
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to
publish the results.
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Sample Availability: Samples of the compounds are not available from the authors.
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