Hyaluronan derivatives bearing variable densities of ferulic acid residues

Article abstract of DOI:10.1039/c3tb21824d

A synthetic procedure has been developed to conjugate ferulic acid (FA) to an important natural polysaccharide derivative such as hyaluronic acid (HA). The activation of FA with 1,1′-carbonyldiimidazole (CDI) has been investigated. Two reactive intermedia

Full text of DOI:10.1039/c3tb21824d

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DOI: 10.1039/C3TB21824D
ARTICLEꢀ
ꢀ
Hyaluronan Derivatives Bearing Variable Densities of
Ferulic Acid Residues
Cite this: DOI: 10.1039/x0xx00000x
A. Cappelli,*,a G. Grisci,a M. Paolino,a G. Giuliani,a A. Donati,a R. Mendichi,b R. Artusi,c
M. Demiranda,c A. Zanardi,c G. Giorgi,a and S. Vomero.a
Received 00th January 2012,
Accepted 00th January 2012
A synthetic procedure has been developed to conjugate ferulic acid (FA) to an important
natural polysaccharide derivative such as hyaluronic acid (HA). The activation of FA with
1,1’-carbonyldiimidazole (CDI) has been investigated. Two reactive intermediates, namely
monoimidazolide 2 [i. e. (E)-3-(4-hydroxy-3-methoxyphenyl)-1-(1H-imidazol-1-yl)prop-2-en-
DOI: 10.1039/x0xx00000x
www.rsc.org/
1-one] and bisimidazolide
3
[i. e. (E)-4-(3-(1H-imidazol-1-yl)-3-oxoprop-1-enyl)-2-
methoxyphenyl 1H-imidazole-1-carboxylate] were characterized from the point of view of
their structure and reactivity. The ready isolation of bisimidazolide 3 and its reactivity support
its potential usefulness in the feruloylation of molecular or macromolecular materials bearing
hydroxyl moieties. Bisimidazolide derivative 3 has been found to be an effective reagent in the
feruloylation of HA to give HAFA graft copolymers showing different grafting degrees (GD),
which could be modulated by varying the reaction conditions. A series of HAFA derivatives
showing different GD values has been prepared and submitted to an extensive macromolecular
and rheological characterization in order to ascertain that the grafting of HA with FA does not
degrade the polysaccharide backbone and to evaluate the role of GD in affecting solubility and
rheological properties. The results suggested that relatively low GD values were sufficient to
confer physical cross-linking capabilities resulting in the features of strong gel of HAFA
dispersions.
Ferulic acid (FA, 4-hydroxy-3-methoxycinnamic acid, 1) is a
hydroxycinnamic acid derivative that is naturally abundant in
Introduction
cell wall of several plants (i. e. in the seeds of coffee, apple,
peanut, and orange, as well as in both seeds and cell walls of
rice, wheat, oats, and pineapple).6 FA residues are inserted in
the cell wall polysaccharides through ester linkages involving
its carboxylic group and the primary alcohol group of arabinose
side chains of arabinoxylans playing the important role of
cross-linking polysaccharides and proteins during lignin cell
wall synthesis.6-9 In particular, feruloylation is an esterification
reaction mediated by specific enzymes showing an high degree
of structural specificity. Furthermore, FA residues have been
proposed to undergo peroxidase-mediated oxidative coupling
(or dimerization and photoisomerism by UV light) to form a
large variety of dehydrodiferulate dimers capable of bridging
different polysaccharide chains.6-9
FA has been described to exhibit a wide range of biological and
pharmacological properties including anti-inflammatory, anti-
diabetic, anti-carcinogenic, anti-bacterial, anti-aging, and
neuroprotective activities.6 It is used in traditional Chinese
medicine in the prevention of thrombosis and in the treatment
of cardiovascular diseases. Owing to the presence of a phenol
moiety linked to the conjugated side chain, FA is able to form
resonance-stabilized phenoxy radical species, which accounts
Hyaluronic acid (HA, also called hyaluronan or hyaluronate) is
a glycosaminoglycan performing several functions in human
body, from the water regulation in tissues to the cellular
proliferation, adhesion, differentiation, and migration. HA is
one of the main components of the extracellular matrix and,
because of its peculiar physicochemical features such as
hydrophilicity and stiffness, plays a key role in the organization
of cartilage proteoglycans into aggregates, providing
a
viscoelastic medium in the vitreous humor of the eye and
synovial fluid. Moreover, the interaction of HA with other
components of the extracellular matrix is of fundamental
importance in giving the necessary compactness to derma.1-4
HA is synthesized by integral membrane proteins called
hyaluronan synthases by repeated addition of glucuronic acid
(GlcA) and N-acetylglucosamine (GlcNAc) and undergoes to a
rapid turnover because enzymes called hyaluronidases actively
operate its enzymatic degradation. Interestingly, the structure
manipulation of the repeating unit has been reported to affect
hyaluronidase activity, so that the chemical modification (e. g.
crosslinking) of the biopolymer represents an interesting
strategy for the modulation of in vivo stability.5
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for the potent antioxidant activity. Indeed, FA protects
membranes from lipid peroxidation and is capable of
neutralizing (scavenging) a broad range of radical species.
Topical administration of FA was shown to inhibit UVB-
induced erythema suggesting photo-protective features.10
Finally, FA shows a strong absorption in the UV spectrum
(maximum at 307 nm)11 that can result into sunscreen features.
The work described in the present paper is finalized to the
1,1’-carbonyldiimidazole (CDI, 13.4 g, 82.6 mmol) was added.
The reaction mixture was heated under reflux for 3.5 h and then
cooled to room temperature. The wDhOitIe: 10p.1r0e3Vc9iei/pwCiA3tarTttiBce2le1O8wn2l4ianDse
collected by filtration, washed in sequence with dry THF and
ether, and dried under reduced pressure to obtain compound 3
as a yellowish white solid (10 g, yield 72%, mp 180-182 °C).
1H-NMR (400 MHz, CDCl3): 3.91 (s, 3H), 7.03 (d,
1H), 7.15 (m, 2H), 7.23 (s, 1H), 7.30 (m, 2H), 7.55 (s, 1H),
7.61 (s, 1H), 8.02 (d, = 15.4, 1H), 8.28 (s, 1H), 8.30 (s, 1H).
1H-NMR (400 MHz, DMSO-d6): 3.91 (s, 3H), 7.15 (s, 1H),
7.18 (s, 1H), 7.53 (d, = 8.3, 1H), 7.62 (d, = 8.3, 1H), 7.69 (d,
= 15.5, 1H), 7.80 (m, 2H), 7.93 (s, 1H), 8.02 (d, = 15.4, 1H),
J = 15.4,
development of
a synthetic procedure for the chemical
modification of HA with FA in order to obtain new advanced
materials (i. e. HAFA graft copolymers) combining the
outstanding properties of the two natural compounds. For
instance, HAFA derivatives have demonstrated interesting
wound healing properties both in vitro and in vivo preclinical
models.
J
J
J
J
J
8.48 (s, 1H), 8.76 (s, 1H). 13C-NMR (100 MHz, CDCl3): 56.2,
112.5, 116.0, 116.4, 117.6, 121.6, 123.2, 131.2, 131.4, 133.7,
136.3, 137.6, 141.0, 146.3, 148.5, 151.5, 161.5. 13C-NMR (150
MHz, DMSO-d6): 56.4, 113.2, 116.8, 117.2, 118.0, 122.6,
123.2, 130.7, 130.9, 134.0, 137.4, 137.9, 140.3, 146.3, 147.5,
151.0, 161.9. HRMS (ESI) calculated for [C17H14N4O4 + H+]
requires 339.1088, found 339.1082.
Experimental Section
Materials and methods
Hyaluronan starting materials were purchased from Shandong
Freda Biopharm Co., Ltd, Biophil Italia SpA and used without
further purifications. Melting points were determined in open
capillaries and are uncorrected. Merck silica gel 60 (230-400
mesh) was used for column chromatography. Merck TLC
plates, silica gel 60 F254 were used for TLC. NMR spectra were
recorded in the indicated solvents with TMS as internal
standard: the values of the chemical shifts are expressed in ppm
Reaction of 3 with benzyl alcohol - Method A. To mixture of
3 (0.10 g, 0.296 mmol) in dry THF (10 mL), benzyl alcohol
(0.124 mL, 1.2 mmol) and triethylamine (TEA, 0.17 mL, 1.22
mmol) were added. The reaction mixture was stirred at room
temperature under argon atmosphere for 5 days and then
concentrated under reduced pressure. The resulting residue was
purified by flash chromatography with dichloromethane-ethyl
acetate (2:1) as the eluent to give benzyl 1-
imidazolecarboxylate (4) as a colorless oil (0.031 g, yield 52%).
The spectroscopic (1H and 13C NMR spectra) features of this
compound were in agreement with those described in the
literature.12,13 The same flash chromatography purification of
the reaction mixture with dichloromethane-ethyl acetate (1:2) as
the eluent afforded compound 2 as a pale yellow oil, which
crystallized spontaneously to form a whitish yellow solid
(0.027 g, yield 37%).
and the coupling constants (
J) in Hz.
(E
)-3-(4-Hydroxy-3-methoxyphenyl)-1-(1H-imidazol-1-
yl)prop-2-en-1-one (2). To a solution of ferulic acid 1 (Sigma
Aldrich, 0.39 g, 2.0 mmol) in dry THF (8.0 mL), 1,1’-
carbonyldiimidazole (CDI, 0.32 g, 2.0 mmol) was added. The
reaction mixture was heated under reflux for 3.5 h and then
concentrated under reduced pressure (or used in the synthesis of
HAFA derivatives without any isolation or purification).
Purification of the residue by flash chromatography with
dichloromethane-ethyl acetate (1:2) as the eluent gave
compound
2 as a pale yellow oil, which crystallized
Reaction of 3 with benzyl alcohol - Method B. To mixture of 3
(0.10 g, 0.296 mmol) in dry THF (10 mL), benzyl alcohol (0.124
mL, 1.2 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU, 0.18
mL, 1.2 mmol) were added. The reaction mixture was refluxed under
argon atmosphere for 3 h and then concentrated under reduced
pressure. The resulting residue was partitioned between chloroform
and water and the organic layer was washed with water, dried over
sodium sulfate and concentrated under reduced pressure. Purification
of the final residue by flash chromatography with dichloromethane
as the eluent gave benzyl 3-(4-hydroxy-3-methoxyphenyl)-2-
propenoate (5)14 as a colorless oil (0.037 g, yield 43%). 1H-NMR
(400 MHz, CDCl3): 3.90 (s, 3H), 5.24 (s, 2H), 5.92 (br s, 1H), 6.33
(d, J = 15.9, 1H), 6.89 (d, J = 8.2, 1H), 7.02 (d, J = 1.5, 1H), 7.07
(dd, J = 1.5, 8.2, 1H), 7.39 (m, 5H), 7.65 (d, J = 15.9, 1H). 13C-NMR
(100 MHz, CDCl3): 55.9, 66.3, 109.4, 114.8, 115.2, 123.2, 127.0,
128.3, 128.6, 136.2, 145.3, 146.8, 148.1, 167.1. MS(ESI): m/z 307
(M+Na+).
spontaneously to form a whitish yellow solid (0.18 g, yield
37%). An analytical sample was obtained by recrystallization
from chloroform by slow evaporation (mp 209 °C dec). 1H-
NMR (400 MHz, CDCl3): 3.98 (s, 3H), 6.22 (br s, 1H), 6.88 (d,
J
= 15.3, 1H), 6.98 (d,
(s, 1H), 7.23 (dd, = 1.6, 8.3, 1H), 7.62 (s, 1H), 8.00 (d,
15.3, 1H), 8.30 (s, 1H). H-NMR (400 MHz, DMSO-d6): 3.86
J = 8.3, 1H), 7.10 (d, J = 1.4, 1H), 7.15
J
J =
1
(s, 3H), 6.85 (d,
8.2, 1H), 7.43 (d,
J
J
= 8.2, 1H), 7.12 (s, 1H), 7.34 (dd,
= 15.5, 1H), 7.54 (s, 1H), 7.91 (m, 2H), 8.72
J = 1.2,
(s, 1H). 13C-NMR (100 MHz, CDCl3): 56.1, 110.4, 111.8,
115.2, 116.4, 124.1, 126.2, 131.0, 136.2, 147.1, 149.6, 150.2,
162.0. 13C-NMR (150 MHz, DMSO-d6): 55.9 112.1, 115.6,
116.7, 124.9, 125.5, 130.4, 137.2, 148.0, 149.5, 150.6, 162.2.
MS(ESI, negative ions): m/z 243 (M-H+).
(E
)-4-(3-(1
H
-Imidazol-1-yl)-3-oxoprop-1-enyl)-2-
-imidazole-1-carboxylate (3). To
methoxyphenyl
1
H
a
solution of ferulic acid 1 (8.0 g, 41 mmol) in dry THF (50 mL),
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General procedure for the synthesis of HAFA derivatives. A Chromatographic system. The characterization of the
mixture of HA sodium salt in formamide (20 mL/1 g HA) was molecular weight distribution (MWD) of two native HA
View Article Online
heated at about 58-60 °C until the complete dissolution and samples and six HAFA derivatives was performed by a multi-
DOI: 10.1039/C3TB21824D
cooled to room temperature. To the resulting viscous solution, angle laser light scattering (MALS) absolute detector on-line to
TEA (1 equivalent) and the appropriate amount of the suitable a size exclusion chromatographic system (SEC). The MWD
activated derivative of FA were added in sequence. In was obtained by a modular multi-detector SEC system. The
particular, compound 2 was used as activation reaction mixture SEC system consisted of an Alliance 2695 separation module
without any isolation or purification (Table 1), whereas equipped with two on-line detectors: a MALS Dawn DSP-F
compound 3 was added as a finely powdered solid (entry 1 of photometer and a 2414 differential refractometer (DRI) used as
Table 2) or as a dispersion in formamide (entries 2-9 of Table concentration detector. The setup of this multi-detector SEC
2). The reaction mixture was stirred overnight at room system was serial in the following order: Alliance-MALS-DRI.
temperature and then diluted with a 5% w/v NaCl solution (5 In a multi-detector SEC system the different on-line detectors
mL/1 g HA). The resulting mixture was stirred at room have an intrinsic temporal delay because they are located in
temperature for 10-15 min and MeOH was added to decrease different positions of the fluidic path. Consequently an
mixture viscosity when necessary. Finally, the polymer was alignment of the different signals is needed. The experimental
obtained by treatment of the mixture with acetone and purified methodology for a reliable use of the SEC-MALS system has
by washing with MeOH for three times. The final solid was been described in detail in the literature.17-21
dried under reduced pressure to afford the expected HAFA The SEC-MALS experimental conditions were the following: 3
derivative.
TSKgel PW (G6000-G5000-G4000) from Tosoh Bioscience as
column set; 80% 0.20M NaCl + 20% dimethylsulfoxide
X-ray crystallography. A single crystal of 2 was submitted to (DMSO) as mobile phase; 0.8 mL/min of flow rate; 150 µL of
X-ray data collection by using a four-circle diffractometer at injection volume; ca. 1 mg/mL of sample concentration.
293K, equipped with a graphite monochromated Mo-Kα The MALS detector uses a vertically polarized He-Ne laser, λ =
radiation (λ = 0.71069 Å). The structure was solved by direct 632.8 nm, and simultaneously measures the intensity of the
methods implemented in the SHELXS-97 program.15 Data scattered light at 18 fixed angular locations ranging in mixed
refinement was carried out by full-matrix anisotropic least- solvent from 12.4° to 155.4°. It is well known that an on-line
squares on F2 for all reflections for non-H atoms by means of MALS detector coupled to a concentration detector allows to
the SHELXL-97 program.16
obtain the true molecular weight M and the size, i. e. the root
Crystallographic data (excluding structure factors) for the mean square radius <s2> in short to hereafter denoted as radius
structure in this paper have been deposited with the Cambridge of gyration Rg, of each fraction of the eluting polymer. The
Crystallographic Data Centre as supplementary publication no. MALS calibration constant was calculated using Toluene as
CCDC 950542. Copies of the data can be obtained, free of standard assuming a Rayleigh Factor R(θ)=1.406•10-5 cm-1. The
charge, on application to CCDC, 12 Union Road, Cambridge normalization of the photodiodes was performed by measuring
CB2 1EZ, UK; (fax: + 44 (0) 1223 336 033; or e-mail: the scattering intensity in the mobile phase of a bovine serum
½
deposit@ccdc.cam.ac.uk).
albumin (BSA: M = 66.4 kg/mol, Rg = 2.9 nm) globular protein
assumed to act as an isotropic scatterer. The MALS photometer
Mass Spectrometry. Electrospray measurements have been has been described in detail in the literature.19,20
carried out on a LCQ-DECA ion trap (ThermoFinnigan, The specific refractive index increment, dn/dc, with respect to
Bremen, D) and on
a LTQ Orbitrap XP instrument the mobile phase was measured by a Chromatix KMX-16
(ThermoFinnigan, Bremen, D). Operating conditions for the differential refractometer. The dn/dc value in 0.20
M
ESI source were as follows: spray voltage +/- 4.5 kV; capillary NaCl+DMSO (80:20) solvent at 35 °C of temperature was:
temperature 200°C; sheath gas (nitrogen) flow rate, ca. 0.75 0.115 mL/g for native HA; 0.110 mL/g for HAFA derivatives.
L/min.
MSn product ion experiments have been carried out inside the Rotational Rheometer. The rheological properties in
ion trap by isolating the precursor ion and then by applying a oscillation mode (frequency sweep) were measured by means
supplementary potential for collision induced dissociations; of an AR 2000 rotational rheometer from TA Instrument using
collision gas: He; collision energy: 20-40% arbitrary units. The
a cone/plate geometry. The rheological properties were
mass window was 1 or 2 u. The Orbitrap analyzer was measured on concentrated solution/dispersion of native HA or
operating in the resolution range 30,000-100,000 FWHM and HAFA derivative samples. The sample temperature was
calibrated using the manufacturer’s calibration mixture maintained at 20 °C by a Peltier Plate. The rheological
obtaining mass accuracies < 2 ppm. 30-50 Scans were recorded experimental conditions were the following: i) rotor: cone/plate,
and averaged for accurate mass measurements.
stainless steel material, 4 cm diameter, 1° angle; ii) temperature
Methanolic solutions of the different compounds (ca. 1x10-4 M) 20 °C; iii) method: frequency (ω) sweep from 0.01 rad/s to
have been introduced via direct infusion at a flow rate of 5 620.3 rad/s; iv) sample concentration 2%.
µL/min.
Results and Discussion
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Synthesis and characterization of activated derivatives of
The crystal structure of 2 (Figure 1) confirmed the trans
FA. The reaction of FA (1) with one equivalent of 1,1’- configuration of the double bond. Bond lengths and bond
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carbonyldiimidazole (CDI) in refluxing dry THF led to the angles were found to be normal.
DOI: 10.1039/C3TB21824D
formation of the corresponding
N
-acylimidazole 2 (Scheme 1),
As an example, the bond distances C(6)-N(1) and C(6)-C(7)
which was characterized by NMR spectroscopy, mass were 1.421(4) and 1.460(4) Å, respectively. The 2-methoxy-4-
spectrometry (MS), and by crystallography (see below). vinylphenol moiety was planar but the imidazole ring was out
However, purification of this important intermediate required of the plane. The dihedral angle between the least squared
the application of chromatographic techniques (i. e. flash planes defined by the phenyl and the imidazole was 23.2(2)°.
chromatography) and the yields were found to be relatively The crystal structure consisted of chains of molecules linked by
low.
hydrogen bonding interactions O(3)-H(x,y,z)…N(3) (3/2+x,1/2-
On the other hand, when two equivalents of CDI were y,1/2+z) with the H…N distance equal to 2.678(4) Å. Unusual
employed, we observed the formation of a conspicuous amount hydrogen bond interactions C(5)-H(5)….O(1) (1/2-x,1/2+y,1/2-
of a yellowish-white precipitate, which was easily collected by z) linked the chains together with the H…O distance equal to
filtration, washed with dry THF and diethyl ether, dried under 2.875(4) Å.
reduced pressure, and stored into a tightly closed vial.
CDI (1 eq)
THF
reflux, 3.5 h
CH3
O
O
CH3
O
O
OH
N
N
HO
1
HO
2
CDI (2 eq)
THF
reflux, 3.5 h
Method A
C6H5CH2OH (4 eq)
TEA, THF
25 °C, 5 days
2
O
CH3
O
N
Figure 1. X-ray crystal structure of 2. Ellipsoids enclose 50%
probability.
O
O
N
+
N
N
O
3
4
Mass spectrometry was used to evaluate the reactivity of
protonated 2 and 3 in the gas phase. Owing to electrospray in
positive mode, only the protonated molecules of 2 and 3 were
observed. Low-energy collision-induced dissociation MSn
experiments, carried out inside an ion trap, were used for
elucidating the reactivity of different ionic species (Figure 2).
N
O
Method B
C6H5CH2OH (4 eq)
DBU, THF
N
reflux, 3 h
CH3
O
O
O
HO
5
Scheme 1. Activation of FA and reactivity of compound 3.
The solid was characterized by both NMR spectroscopy (1H,
13C, COSY, NOESY, HSQC, and HMBC experiments) and
MS. Accurate mass measurements suggested the formula
C17H14N4O4 that was also supported by NMR experiments. The
results were easily explained in terms of capability of the
phenol group of 2 to react with the second equivalent of CDI to
give 1-imidazolecarboxylate 3. The easy isolation of 3 as a
solid from the reaction mixture led us to consider this
compound a promising intermediate for the insertion of ferulic
acid residues in different molecular or macromolecular
materials. The reaction of 3 with low-molecular weight
alcohols such as benzyl alcohol revealed the higher reactivity of
the activated carbamate moiety with respect to the
N-
acryloylimidazole one. In fact, the reaction of 3 with benzyl
alcohol in THF in the presence of triethylamine (TEA) at room
temperature (Method A) gave a mixture of 2 and benzyl 1-
imidazolecarboxylate (4).12,13 In other words, benzyl alcohol
behaved as a scavenger in restoring the phenol hydroxyl
moiety. The same reaction with 1,8-diazabicyclo[5.4.0]undec-
7-ene (DBU, in place of TEA) in refluxing THF (Method B)
afforded benzyl ester 514 supporting the potential usefulness of
Figure 2. Low-energy collision-induced dissociation MSn
spectra obtained by selecting different precursor ions produced
3
for introducing ferulic acid residues in molecular or
by 3.
ꢀ
macromolecular materials bearing hydroxyl moieties.
The MS2 spectrum, obtained by selecting [3+H]+ (m/z 339)
as precursor ions, showed product ions at m/z 271 attributable
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to elimination of imidazole. This behavior was identical of that (as reaction mixture, without any purification) in the grafting
shown by [2+H]+ in which only an imidazole residue is present. reaction.
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DOI: 10.1039/C3TB21824D
In the case of 3, it was of interest to determine which of the two
OH
OH
imidazolyl moieties was involved in this fragmentation. The
MS3 spectrum, obtained by selecting the species at m/z 271 as
precursor ions, showed the most abundant product ions at m/z
227 and 177, due to elimination of 44 and 94 u, respectively.
The former are due to loss of CO2, thus evidencing that the
imidazole bound to the carboxyl group is the first to be
eliminated by [3+H]+.
Ions at m/z 177 are due to the elimination of an
imidazolecarbaldehyde molecule. These latter can further
decompose (MS4) through an unusual loss of a methyl radical
from an even-electron species, or losing methanol, yielding the
ionic species at m/z 162 and 145, respectively. The radical
cations at m/z 162 can further decompose (MS5) by elimination
of 28 u yielding ions at m/z 134.
O
O
O
O
HO
HO
O
NH
OH
O
n
HA
CH3
O
O
N
N
R
OH
O
O
2 or 3
TEA, HCONH2
25 °C, overnight
O
O
OH
OH
OH
O
O
Reaction of FA activated derivatives 2 and 3 with HA
O
O
O
O
NH
O
HO
HO
O
HO
HO
O
In the development of an easily scalable, industrially-viable
functionalization procedure, commercially available sodium
hyaluronate was dissolved into formamide at 58-60 °C, and the
resulting viscous solution was cooled to room temperature and
treated in sequence with triethylamine (TEA) and the reaction
mixture containing activated FA derivative 2.
O
OH
NH
OH
O
O
y
x
HAFA
Scheme 2. Esterification of HA into HAFA derivatives.
The stoichiometric ratio between 2 and HA (2/HA ratio) was
varied from 1:1 to 1:4 (i. e. 100, 50, and 25%, see Table 1) in
order to obtain graft copolymers showing a different grafting
degree (GD). Moreover, different amounts of the starting
materials were used in the aim of evaluating the productive
conversion of the 1-acylimidazole derivative 2 into ferulate.
The data shown in Table 1 suggest that GD was substantially
regulated by 2/HA stoichiometric ratio, which affected also the
productive conversion of the 1-acylimidazole derivative 2 into
ferulate. In fact, low 2/HA ratios (i. e. 25%) produced low GD
(i. e. 7%) and relatively high conversion values (i. e. 28%),
whereas high 2/HA ratios (i. e. 100%) led to higher GD (i. e.
15%) and lower conversion values (i. e. 15%). However, when
HAFA samples thus obtained were submitted to hydrolytic
conditions, the presence of impurity traces was observed in the
1H NMR spectra. This result was related to the use of crude 2
The easy isolation of 3 in its pure form and the reactivity
shown above led to evaluate the use of this compound in HA
grafting reaction. Thus, the viscous solution of sodium
hyaluronate in formamide was treated in sequence with TEA
and activated FA derivative 3. Owing to the limited solubility
shown by 3 in formamide, the reactant was added to the
reaction mixture as a finely powdered solid or as a dispersion in
formamide. The stoichiometric ratio between 3 and HA (3/HA
ratio) was varied from 75 to 10% (see Table 2) with a number
of five replicates (with different HA amounts) in 3/HA ratio of
20% in the aim of optimizing the conditions to obtain a desired
grafting degree (GD) and to maximize the productive
conversion of 3 into ferulate.
Table 1. Reaction parameters in the functionalization of HA polymer to HAFA graft copolymers with
N-acylimidazole 2.
graft copolymer
HA
(mmol)
convers.(c)
(%)
entry
HA(a) (g)
2/HA ratio (%) grafting degree(b) (%)
1
2
3
4
HAFA-01
HAFA-02
HAFA-03
HAFA-04
3.0
3.0
3.0
10
7.5
7.5
7.5
25
100
50
15
11
7
15
22
28
24
25
50
12
(a) All HAFA graft copolymers were obtained by using HA-02 as the starting material. (b) A rough estimate of the grafting degree
was made by 1H-NMR spectroscopy after hydrolysis with NaOD in D2O as described below in the main text. (c) The conversion
into ferulate was calculated from the substitution degree and stoichiometric ratio 2/HA.
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Table 2. Reaction parameters in the functionalization of HA polymer to HAFA graft copolymers with activated FA derivative 3.
graft copolymer
HA
(mmol)
convers.(c)
HA(a) (g)
3/HA ratio (%) grafting degree(b) (%D)OI: 10.1039/C3TB21824D
View Article Online
entry
(%)
1
2
3
5
4
6
7
8
9
HAFA-07
HAFA-08
HAFA-09
HAFA-10
HAFA-14
HAFA-11
HAFA-15
HAFA-12
HAFA-13
3.0
10
7.5
25
75
60
40
20
20
20
20
20
10
17
15
12
8
23
25
30
40
40
40
25
20
30
1.6
3.0
9.0
10
4.0
7.5
22.4
25
8
8
10
25
5
15
37.4
75
4
30
3
(a) All HAFA graft copolymers were obtained by using HA-01 polymer except the HAFA-15 derivative, obtained by using HA-02
polymer. (b) A rough estimate of the grafting degree was made by 1H-NMR spectroscopy after hydrolysis with NaOD in D2O as
described below in the main text. (c) The conversion into ferulate was calculated from the substitution degree and stoichiometric
ratio 3/HA.
The comparative analysis of the data reported in Tables 1 the signals attributed to the trans-double bond protons (in
and 2 suggested that the use of 3 produced GD values higher particular to H in position beta of the acryloyl moiety) in
than those obtained with 2. Moreover, as observed in HA agreement with the assumption that the ferulate residues are
grafting with 2, the 3/HA stoichiometric ratio affected both GD linked through ester bonds to HA.
and the productive conversion of 3 into ferulate in such a way
that high 3/HA ratios (i. e. 75%) led to high GD (i. e. 17%) and
to relatively low conversion values (i. e. 23%), while gradual spectroscopy
Determination of HAFA grafting degree by 1H NMR
decreases in GD values and increases in the productive
In order to estimate GD of HAFA derivatives, the hydrolysis
conversion were observed with the decrease of 3/HA ratios. of their ester linkages with FA moieties were performed in D2O
1
The data obtained with the five replicates with 3/HA ratio of by using NaOD and it was followed by H NMR spectroscopy.
20% appeared to suggest that the scalability of the process was Thus, HAFA samples (ca. 6 mg) were swollen in D2O (ca. 0.7
not optimized. Owing to the relatively low solubility of 3 in mL) and a drop of a 40% NaOD in D2O was added (Figure 4).
formamide, the heterogeneity of the reaction mixture requires 1H NMR spectra of the dispersions were recorded at regular
additional work to be performed in order to optimize further the time intervals until the end of the hydrolysis. The down-field
conversion of the imidazolide into ferulate.
signals attributed to ferulate were integrated and the integral
values were compared with that of the methyl group of -acetyl
residue of HA. During the performing of this experiment we
N
Structure of HAFA derivatives
1
The structure of HAFA derivatives was characterized by H observed that the addition of 40% NaOD solution to the HAFA
NMR spectroscopy using D2O as the solvent. The NMR dispersion in water produced marked modifications in the
analysis of all the graft copolymers mentioned in the present samples. In particular, the starting colorless transparent gel
paper confirmed the successful coupling between HA and FA sample became a viscous yellow solution immediately after the
in our reaction conditions. In fact, beside the distinctive profile adding up of the base and the color disappeared in the
of HA in the up-field region, signals attributable to the FA following time with the completion of the hydrolysis reaction.
residues were well manifest in the down-field region of the These results were rationalized by assuming that the basic
recorded spectra. Moreover, the addition of free FA to HAFA- environment was capable of deprotonating the phenol hydroxyl
14 dispersion in D2O (Figure 3) showed that the signals of free of ferulate residues and breaking the H-bond network, which
FA displayed chemical shift values different from those of FA stabilizes the gel dispersion.
bound to the polysaccharide. The highest difference involved
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View Article Online
DOI: 10.1039/C3TB21824D
1
Figure 3. Comparison of H NMR spectra obtained with HAFA-14 derivative (D2O, 400 MHz) in the absence (bottom trace),
or in the presence (middle trace) of free FA (added) with that obtained with a physical mixture of HA and FA (top trace). The
peak intensity of the aromatic regions was magnified (x 2) for the sake of clarity.
1
Figure 4. Effects produced by the NaOD addition on the H NMR spectra (D2O, 400 MHz) of Figure 3 samples. The dispersions
of HAFA-14 derivative (bottom trace), HAFA-14 containing free FA (middle trace), and the physical mixture of HA and FA (top
1
trace) were treated with a drop of a 40% NaOD in D2O and H NMR spectra were recorded at regular time intervals until the end
of the hydrolysis. The peak intensity of the aromatic regions was magnified (x 2) for the sake of clarity.
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PolymerꢀChemistryꢀ
ꢀ
Macromolecular characterization of HAFA derivatives
weight (Mw), and the dispersity Mw/Mn where Mn denotes the
numeric-average of the molecular weight.
View Article Online
Solubility of HAFA derivatives
DOI: 10.1039/C3TB21824D
Specifically for the macromolecular characterization, but Table 3. Solubility data of HA native and HAFA derivative
also in general for the derivatives processing and the samples calculated from the Recovered Mass.
knowledge of complex solution properties, the solubility of
grafting
degree (%)
0.2M NaCl 0.2M NaCl-DMSO
HAFA graft copolymers in aqueous solvents is very important.
The solubility of HAFA derivatives was tested in two different
solvents: 1) 0.2 M NaCl (usual aqueous solvent for HA native
biopolymers); 2) 80% 0.2 M NaCl + 20% DMSO mixed
solvent (an expected better solvent for HAFA derivatives). The
solubility data of native HA and HAFA derivatives are
reported in Table 3. The solubility data were obtained by
quantification of the Recovered Mass,22 that is the mass of
polymeric sample eluting from the SEC columns expressed in
% with respect to the injected mass (calculated from the
product of sample concentration and injection volume). The
Recovered Mass is calculated from the area of the
chromatogram of the differential refractometer (DRI)
concentration detector after accurate calibration. In general it is
well known that the Recovered Mass is lower than 100% in
consequence of the presence in the sample of solvents,
additives and impurity. In particular HA samples always
contain a meaningful fraction of water (ca. 5-15%).
polymer
(%)
(80:20) (%)
HA-01
88
89
92
HA-02
94
HAFA-07
HAFA-08
HAFA-09
HAFA-10
HAFA-11
HAFA-14
HAFA-15
HAFA-12
HAFA-13
17
15
12
8
Insoluble
Insoluble
Insoluble
4.2
Insoluble
Insoluble
Insoluble
8.2
8
3.0
8.9
8
9.4
32
The two native HA samples were completely soluble both in
0.2M NaCl aqueous solvent and in the mixed solvent
containing 20% of organic DMSO. On the contrary, HAFA
derivatives showed very different behaviors. The solubility
degree of HAFA derivatives is substantially related to GD
value.
5
37
68
4
87
88
3
81
81
Specifically, HAFA derivatives showing the lowest GD
value (e. g. HAFA-12 and HAFA-13: GD ≤ 4%) were
completely soluble, whereas HAFA-15 showing a GD value
around 5% was partially soluble, and those showing higher GD
Table 4. Macromolecular features of the HAFA derivatives
compared to the starting HA.
values (e. g. HAFA-10, HAFA-11, and HAFA-14: GD = 8%)
grafting
degree
(%)
Sol.
(%)
Mp
Mw
Mw/
could be considered practically insoluble. Furthermore, a little
better solubility of HAFA derivatives was obtained by using
the mixed solvent 0.20 M NaCl-DMSO (80:20), whereas a
polymer
(kg/mol) (kg/mol) Mn
more significant increase in solubility was obseved when
N,N-
HA-01
89
92
81
88
68
32
8.9
164.2
211.9
181.7
213.1
225.4
143.6
100.2
249.8
271.2
299.9
283.3
365.1
232.1
119.2
3.2
3.1
2.8
3.0
3.9
3.8
2.4
dimethylacetamide (DMA) was used as the co-solvent in the
(50:50) ratio with 0.05M NaCl water solution (data not shown).
In consideration of the hydrogen bond acceptor features, which
are generally recognized to DMA, this result suggests that the
feruloylation of HA into HAFA graft copolymers induced the
formation of physical cross-linking based on the establishment
of interchain hydrogen bonds, which are broken by the addition
of substantial amounts of a strong acceptor such as DMA. In
conclusion, the density of ferulate residues appears to affect
significantly the intermolecular interactions of HAFA
derivatives and their behavior in water environment.
HA-02
HAFA-13
HAFA-12
HAFA-15
HAFA-14
HAFA-11
3
4
5
8
8
Macromolecular and conformational characterization
The macromolecular and conformational characterization of
HAFA derivatives as well as of the HA starting biopolymer
were performed by means of a multi-angle laser light scattering
(MALS) absolute detector on-line to
chromatography (SEC) system.
a
The
size exclusion
SEC-MALS
characterization was performed by using as mobile phase the
mixed solvent 0.2 M NaCl-DMSO (80:20), considering the
little better solubility in this solvent of the HAFA derivatives.
Table 4 summarizes the most important SEC-MALS results
obtained with native HA and HAFA samples. Specifically,
Table 4 reports the molecular weight of the peak of the
chromatogram (Mp), the weight-average of the molecular
In the analysis of macromolecular data reported in Table 4
some important concerns have to be considered. Obviously the
SEC-MALS results (MWD, relative averages and dispersity)
only refer to the soluble fraction of the HAFA derivative
samples. The direct consequence of the partial solubility is that
the molecular weight and dispersity of some derivatives could
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be underestimated. Furthermore, no information on the MWD obtained using concentrated solution or dispersion of native HA
of the completely insoluble HAFA derivatives could be and HAFA derivatives in the mixed solvent 0.2M NaCl-DMSO
View Article Online
obtained. Despite the above mentioned problems in the (80:20). The specimens subjected to the rheological test were
DOI: 10.1039/C3TB21824D
solubility of HAFA-derivatives, the macromolecular SEC- substantially gels with 2% of sample concentration.
MALS results (Table 4) for native HA and HAFA derivatives Specifically, the rheological characterization allows to
convey important information. The molecular weight of the two overcome the solubility problems of HAFA derivatives
native HA samples is high (Mw average ca. 260 kg/mol) and showing high GD values.
their MWD is broad (Mw/Mn ca. 3). Furthermore, their MWD
1.0E+04
were found to be substantially similar. In general, the
comparison of the differential MWD of completely soluble
samples (HA-01, HA-02, HAFA-12, and HAFA-13, see Table
4) clearly shows that the derivatization of HA does not change
HA-01
HA-02
HAFA-10
1.0E+03
HAFA-11
HAFA-12
HAFA-13
the molecular weight of HA macromolecules. In other words,
1.0E+02
1.0E+01
1.0E+00
1.0E-01
1.0E-02
HAFA-14
HAFA-15
the derivatization does not degrade the HA polysaccharide
backbone. Some little differences in MWD could be explained
by the partial solubility (HAFA-14) and by the presence in the
solution of some fractions of HAFA macromolecules showing
low physical cross-linking density (HAFA-15).
Table 5 reports the parameters (e. g. the average size Rg of
macromolecules, the intercept K and the slope α of the
conformation plot) of the experimental function Rg = ƒ(M)
generally known as conformation plot. The conformation plot,
Rg = K⋅Mα, is the scaling law between the size Rg and chain
length (molecular weight) of macromolecules. In general the
conformation plot is very important because is related with the
conformation, branching, and eventual derivatization or
chemical modification of the polymer.
1.0E-02
1.0E-01
1.0E+00
Frequency
1.0E+01
[rad/s]
1.0E+02
1.0E+03
!
Figure 5. Complex dynamic viscosity |η*| as a function of the
frequency ω of native HA samples and HAFA derivatives.
The comparison of the experimental |η*|=ƒ(ω) rheological
curve of the two native HA samples with six HAFA derivatives
with different GD values (see Figure 5) shows a very different
rheological behavior apparently related to GD of HAFA
derivatives. The rheological curve of the two native HA
samples are very similar in consequence of the quite similar
MWD (see data in Table 3). The rheological curve of two
HAFA derivatives with low GD (HAFA-12, GD = 4; HAFA-
13, GD = 3) is a little shifted toward lower complex dynamic
viscosity values without meaning modifications of the pseudo
plastic behavior of the polymer. Evidently, in these HAFA
derivatives showing low GD values, the grafting with FA
residues is particularly effective in affecting their rheology. The
rheological curve of HAFA derivatives with intermediate GD
Table 5. Size and conformation results of starting HA and of
some HAFA derivatives.
grafting
degree
(%)
Sol.
Mw
Rg
polymer
K
α
(%) (kg/mol) (nm)
HA-01
89.2
81.2
87.6
68.2
32.1
249.8
299.9
283.3
365.1
232.1
49.6 3.46E-02 0.58
51.4 4.33E-02 0.56
HAFA-13
HAFA-12
HAFA-15
HAFA-14
3
4
5
8
48.9 3.75E-02 0.57 values is only shifted toward higher complex viscosity values.
Evidently, when GD > 4% also a low degree of physical cross-
linking is present without compromising the complete solubility
of samples. Finally, when GD ≥ 8% the physical cross-linking
degree increases, the complex viscosity strongly increase, and
the solubility also in the mixed solvent is only partial.
54.1 6.59E-02 0.52
41.1 4.49E-02 0.55
Figures 6 and 7 show the comparison between the elastic or
in-phase (G’) moduli and the viscous or out-phase moduli (G”)
for the native HA-02 sample and for the HAFA-10 derivative
The derivatization of HA with FA produces only little
changes in the conformation of the native HA macromolecules.
Indeed, the slope α of the conformation plot in the mixed
solvent decreases a little from 0.58 to 0.55. Thus, HAFA
macromolecules are a little more compact with respect to the
native HA ones. Furthermore, the conformation plot confirms
that in the HAFA-15 derivative there is a little extent of
physical cross-linking (more compact conformation, slope α ca.
0.52). Obviously, the conformation plot concerns only the
soluble macromolecule fractions of HAFA samples.
respectively. The native HA-02 sample shows a typical
mechanical spectrum of a high molecular weight biopolymer
with a crossover point (G’=G”). On the contrary the mechanical
spectrum of the HAFA-10 derivative (GD = 8%) is completely
different with the elastic moduli G’ higher than the viscous
moduli G” for the whole range of frequency explored. This
behavior is typical of a “strong” gel. This rheological result
confirms the presence of physical cross-linking in HAFA
derivatives showing GD values higher than around 8%.
Therefore, the insolubility of HAFA derivatives showing the
highest GD values (GD > 10%) can be rationalized in terms of
presence of a high degree of physical cross-linking, which
could be originated by the non-covalent interaction of ferulate
residues building a stable H-bond network.
Rheological properties
Figure
5 shows the comparison of the experimental
rheological curve of complex dynamic viscosity |η*| as a
function of the frequency ω for two HA native samples and six
HAFA derivatives. The |η*|=ƒ(ω) rheological curves were
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1.0E+04
1.0E+03
1.0E+02
1.0E+01
1.0E+00
1.0E-01
1.0E-02
1.0E-03
1.0E-04
GD = 3%) and a high esterification degree (HAFA-08, GD =
G'
HA-02
15%).
G''
View Article Online
DOI: 10.1039/C3TB21824D
The experiments were carried out according to conventional
procedures. Shortly, the polysaccharide solution maintained at
37 °C (pH 7.4) and containing the enzyme in an amount equal
to 0.1 mg/mL (bovine testicular hyaluronidase, type I-S, Sigma,
1000 U/mg) was incubated at 37 °C. Two different substrate
(S)-enzyme (E) ratios (i. e. S/E = 100 and S/E = 300) were
tested. At regular time intervals, in the range 0-120 min,
samples in the amount of 0.5 mL were taken, heated at 100 °C
for 5 min, filtered to remove the enzyme, and thereafter
analyzed by means of SEC-MALS analysis in order to evaluate
the molecular weight distribution.
Already after 10 min incubation with the enzyme, the native
HA samples, at both enzyme concentrations, exhibited a rather
complete degradation, changing from an average molecular
weight of about 300 KDa to a value about 10 times lower. Also
HAFA-13 sample showed a strong degradation (i. e. 60% and
90% after 10 and 30 min of incubation) already at the lower
enzyme concentration. On the contrary, the degradation of
HAFA-08 sample was much lower [i. e. 18%, 44%, and 67% at
10, 30, and 60 min, respectively for the S/E = 300 (Figure 8)
and 29%, 61%, and 77% at the same times for the ratio S/E =
100].
1.0E-05
1.0E-02
1.0E-01
1.0E+00
Frequency
1.0E+01
[rad/s]
1.0E+02
1.0E+03
!
Figure 6. Comparison between elastic (G’) and viscous (G”)
moduli for native HA-02 sample.
ꢀ
1.0E+04
HAFA-10
G'
G''
1.0E+03
1.0E+02
1.0E+01
1.0E+00
1.0E-01
1.0E-02
1.0E-01
1.0E+00
Frequency
1.0E+01
[rad/s]
1.0E+02
1.0E+03
!
Figure 7. Comparison between elastic (G’) and viscous (G”)
moduli for HAFA-10 derivative.
This assumption was supported by the effects observed in
the rheological behavior when
N,N-dimethylacetamide was
used as the co-solvent in the (50:50) ratio with 0.05M NaCl
solution. In short, DMA was capable of decreasing the apparent
cross-linking degree of HAFA derivatives up to the
corresponding values shown by native HA samples (data not
shown). Thus, in the aqueous dispersions of HAFA derivatives,
ferulic residues are prone to establish interchain hydrogen
bonds, which are broken by DMA.
Figure 8. Comparison of differential MWD of HAFA-8
derivative after the enzymatic action of hyaluronidase.
Conclusions
Based on the natural feruloylation of arabinose side chains of
arabinoxylans in the cell wall of plants, a synthetic procedure
was developed in order to conjugate the natural
hydroxycinnamic acid derivative FA together with HA
polysaccharide macromolecule. The activation of FA with CDI
was extensively studied and the two reactive intermediates 2
(monoimidazolide) and 3 (bisimidazolide) were thoroughly
characterized from the point of view of their structure and
reactivity. The easy isolation of bisimidazolide 3 as a solid
from the activation reaction mixture makes this compound an
interesting intermediate for the insertion of ferulic acid moieties
in different materials. Moreover, the reactivity characterization
by using benzyl alcohol as a model compound supports the
potential usefulness of 3 for the feruloylation of molecular or
macromolecular materials bearing hydroxyl moieties. As
expected, 3 demonstrated to be an effective reagent in the
feruloylation of HA producing HAFA graft copolymers, which
Resistance against enzymatic degradation
The presence of microorganisms on the epidermis causes a
relevant biological activity on the skin, activity which takes the
form of various enzymatic functions, including the one carried
out by the enzyme hyaluronidase. HA is naturally degraded due
to the presence of the mentioned enzyme, which catalyzes its
degradation by randomly cleaving the 1,4 beta glycosidic bonds
between the monomeric units of the polysaccharide chain.
HAFA derivatives are resistant to the enzymatic action of
hyaluronidase, and their resistance grows with the increase of
the esterification degree.
This enzymatic degradation study was performed using the
commercial native HA sodium salt (molecular weight about
300 KDa) and two HAFA samples showing a low (HAFA-13,
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showed different grafting degrees in the range 3-17%
depending on the amount of the reactants used. In particular,
the stoichiometric ratio 3/HA affected both GD and the
conversion of 3 into ferulate in such a manner that high 3/HA
ratios led to relatively high GD and low conversion values,
whereas a gradual decrease in GD values and an increase in the
conversion percentage was obtained with the decrease of 3/HA
ratios. NMR studies confirmed the successful coupling between
HA and FA in our reaction conditions, and the release of intact
FA in hydrolytic conditions. The results of the macromolecular
and rheological characterization of HAFA derivatives
suggested that the grafting of HA with FA does not degrade the
polysaccharide backbone and affects solubility and rheological
properties. In fact, low GD values are related to high solubility
and to complex viscosity values lower than those shown by
native HA samples, whereas high GD values are linked to low
solubility and higher complex viscosity values. Relatively low
GD values (e. g. ≥ 8%) are sufficient to confer physical cross-
linking properties resulting in the features of strong gel of
HAFA-10 dispersions. Moreover, the presence of acid ferulic
residues in HAFA derivatives produces an increased resistance
to the enzymatic action of hyaluronidase, and this resistance
grows with the increase of the grafting degree.
In conclusion, new materials were obtained by the covalent
coupling of two natural products: a macromolecular species
such as HA and a small molecule such as FA. A wide range of
applications can be envisioned for these materials on the basis
of the outstanding (physico-chemical, biological, and
pharmacological) properties of the natural products. For
instance, HAFA derivatives have demonstrated interesting
wound healing properties both in vitro and in vivo preclinical
models. Moreover, the covalent linking of FA to HA in HAFA
graft copolymers provides additional features such as a physical
cross-linking, which constitutes an added value in biological
applications (e. g. as dermal fillers or in tissue engineering).
4
T. Coviello, P. Matricardi, C. Marianecci and F. J. Alhaique, Control.
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5
6
A. Tezel and G. H. J. Fredrickson, Cosmet. Laser TherV.ie2w00A8rt,ic1le0O, n3l5in.e
DOI: 10.1039/C3TB21824D
M. M. de O. Buanafina, Mol. Plant 2009, 5, 861 and references cited
therein.
7
J. H. Grabber, J. H. Ralph and R. D. Hatfield, J. Agric. Food Chem.
2000, 48, 6106.
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G. Wallace and S. Fry, Int. Rev. Cytol. 1994, 151, 229.
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Acknowledgements
Thanks are due to Italian MIUR (Ministero dell’Istruzione,
dell’Università e della Ricerca) for financial support.
21 R. Mendichi, L. Soltes, A. Giacometti Schieroni, Biomacromolecules
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22 Recovered Mass (Recov. Mass) is the sample mass (g) eluting (i. e.
recovered) from the SEC columns, expressed as % of the sample
injected mass (sample concentration times injection volume). The
Recovered Mass is calculated from the area of the DRI concentration
detector after accurate calibration.
Notes and references
Dipartimento di Biotecnologie, Chimica e Farmacia and European
a
Research Centre for Drug Discovery and Development, Università degli
Studi di Siena, Via A. Moro 2, 53100 Siena, Italy.
b
Istituto per lo Studio delle Macromolecole (CNR), Via E. Bassini 15,
20133 Milano, Italy
c
Rottapharm Madaus, Via Valosa di Sopra 7, 20900 Monza, Italy
*Corresponding
author.
Tel:
+39
0577
234320.
E-mail:
andrea.cappelli@unisi.it.
Electronic Supplementary Information (ESI) available: NMR spectra of
compounds 2, 3 and 5, crystallographic data for compound 2, and MWD
and conformational features of the native HA and HAFA derivatives. See
DOI: 10.1039/b000000x/
1
A. Donati, A. Magnani, C. Bonechi, R. Barbucci and C. Rossi,
Biopolymers 2001, 59, 434.
2
3
J. A. Burdick and G. D. Prestwich, Adv. Mater. 2011, 23, H41.
K. L. Goa and P. Benfield, Drugs 1994, 47, 536.
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ2012ꢀ
J.ꢀMater.ꢀChem.ꢀB,ꢀXXXX,ꢀXX,ꢀX‐Xꢀ|ꢀ11ꢀ

Journal of Materials Chemistry B
Page 12 of 12
JournalꢀofꢀMaterialsꢀChemistryꢀBꢀ
ARTICLEꢀ
View Article Online
DOI: 10.1039/C3TB21824D
TOC
A synthetic procedure has been developed to conjugate ferulic acid (FA) to an important natural polysaccharide derivative such as
hyaluronic acid (HA).
12ꢀ|J.ꢀMater.ꢀChem.ꢀB,ꢀXXXX,ꢀXX,ꢀX‐Xꢀ
Thisꢀjournalꢀisꢀ©ꢀTheꢀRoyalꢀSocietyꢀofꢀChemistryꢀ2012ꢀ