Novel 3-hydroxypyridin-4-one hexadentate ligand-based polymeric iron chelator: Synthesis, characterization and antimicrobial evaluation

Article abstract of DOI:10.1039/c5md00264h

A novel 3-hydroxypyridin-4-one (HPO) hexadentate monomeric chelator 14 has been synthesized and incorporated into polymers by copolymerization with 2-hydroxyethyl acrylate (HEA), using azobisisobutyronitrile (AIBN) as an initiator. The monomeric chelator was found to possess very high affinity for iron(III), with the log stability constant of the iron complex (log K₁) = 33.61 and pFe³⁺ = 30.37. The iron binding capacity and monomer recovery of the copolymers were determined using spectrophotometry. The in vitro antimicrobial activity of monomeric chelator 14 and polymeric chelator 16-4 against both Gram-positive bacteria (Staphylococcus aureus and Bacillus subtilis) and Gram-negative bacteria (Escherichia coli, Salmonella spp., and Pseudomonas aeruginosa) was evaluated by inhibition zone and minimum inhibitory concentration (MIC) assays, which demonstrated that both monomeric and polymeric chelators possess inhibitory activity. The polymeric chelators possess potential application for the treatment of wound infection.

Full text of DOI:10.1039/c5md00264h

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Novel 3-hydroxypyridin-4-one hexadentate ligand-based polymeric
iron chelator: synthesis, characterization and antimicrobial
evaluation
Received 00th January 20xx,
Accepted 00th January 20xx
Ying-Jun Zhoua, Xiao-Le Kongb, Jun-Pei Lib, Yong-Min Mac, Robert C Hiderb and Tao Zhoua*
DOI: 10.1039/x0xx00000x
www.rsc.org/
Abstract:
A novel 3-hydroxypyridin-4-one (HPO) hexadentate monomeric chelator 14 has been synthesized and
incorporated into polymers by copolymerization with 2-hydroxyethyl acrylate (HEA), using azobisisobutyronitrile (AIBN) as
an initiator. The monomeric chelator was found to possess very high affinity for iron(III), with a log stability constant of
iron complex (logK1)=33.61 and pFe3+=30.37. The iron binding capacity and monomer recovery of the copolymers was
determined using spectrophotometry. In vitro antimicrobial activity of the monomeric chelator 14 and polymeric chelator
16-4 against both Gram-positive bacteria (Staphyloccocus aureus, Bacillus subtilis) and Gram-negative bacteria (Escherichia
coli, Salmonella spp., Pseudomonas aeruginosa) was evaluated by inhibition zone and minimum inhibitory concentration
(MIC) assays, which demonstrated that both monomeric and polymeric chelators possess inhibitory activity. The polymeric
chelators possess potential application for the treatment of wound infection.
Introduction
agents must be extraordinarily high, so that they can efficiently
out-compete the native siderophores for iron. The structure of
Bacterial and fungal resistance to antimicrobial agents is a
growing problem worldwide.1-3 Thus there is an urgent need
for the development of novel types of antimicrobial agents
targeting unique mechanisms and pathways. Iron is an
essential cofactor of many biochemical pathways in both
prokaryotic and eukaryotic species.4 Hence, limiting the
amount of available iron should, in principle, inhibit microbial
growth.5 Many microorganisms have evolved strategies to
scavenge and absorb iron from the environment by the
production and secretion of siderophores, which are generally
hexadentate low-molecular-weight compounds (500–1500 Da)
possessing a high affinity and selectivity for iron(III).6 For
instance, Staphylococcus aureus has been demonstrated to
use two hydroxycarboxylate-type siderophores, staphyloferrin
A and staphyloferrin B, via the transporters Hts and Sir,
respectively, to access the transferrin iron pool.7,8
Pseudomonas aeruginosa produces two extensively
characterized siderophores, pyochelin, and pyoverdin.9 Such
uptake can be interrupted by the introduction of high-affinity
iron-selective chelating agents.10 The iron affinity of these
antimicrobial chelators should differ appreciably from those of
siderophores, otherwise the resulting iron-chelator complex
will be recognized by iron-siderophore receptors and thereby
utilized by the microorganism.11 In our previous work, 3-
hydroxypyridin-4-ones (HPOs), which have a different iron-
binding moiety to those of siderophores, have been
demonstrated to inhibit the growth of both Gram-positive and
Gram-negative bacteria,12-15 and clinical isolates of methicillin
resistant Staphylococcus aureus and Pseudomonas strains.16
Wound infections are probably the next most commonly
treated condition after urinary tract infections. Two clinically
used iron chelators, desferrioxamine and deferiprone, have
been reported to enhance wound healing.17,18 Although all
currently adopted iron chelating therapeutics are based on
small molecules, macromolecular iron chelators also have
potential application in the treatment of both chronic and
acute iron overload.19,20 A re-evaluation of the attributes of
polymers has revealed a number of advantages associated
with polymeric therapeutics that cannot be readily achieved
with traditional small-molecule drugs. Generally, in
comparison with low molecular weight chelators, high-
molecular-weight polymeric chelators possess low toxicity due
to the fact that they are not absorbed by the gastrointestinal
tract.21 Most of reported iron chelating polymers are based on
bidentate ligands,22,23 and as it is difficult for each bidentate
ligand to form part of an ideal octahedral iron(III) coordination
site, the complexation of three bidentate ligands with iron will
not be consistently strong, partial chelation of iron being likely,
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O
O
N
O
for instance, where only two bidentate ligands bind an iron
atom.24
O
O
OBn
OBn
OH
OBn
a
Previously,
we
reported
a
hexadentate
c
b
hydroxypyridinone-based iron binding resin which possesses a
high iron(III) affinity.24 The 3-hydroxypyridin-4-one class of
molecule typically possesses a logKeqFe(III) (β3) value greater
than 35, the corresponding values for Cu(II) and Zn(II) are
much lower, namely 22 and 14.25 Consequently, the 3-
hydroxypyridin-4-one class of molecule is highly selective for
iron(III), this being particularly true for hexadentate chelators.
N
O
1
OBn
OH
2
4
3
O
N
O
O
OBn
CHO
d
e
OBn
OH
f
OBn
NH2
N
In this study, using maltol (1) as a starting material, a novel
N
HPO hexadentate monomeric chelator has been synthesized
and its incorporation into copolymer structures is reported. An
investigation of the physicochemical characterization of the
monomeric hexadentate ligand and the characterization of
polymers has been undertaken. In vitro antimicrobial activity
of the monomeric chelator and polymeric chelator against the
five tested strains was evaluated by inhibition zone and MIC
assays.
OBn
OBn
OBn
5
6
7
Scheme 1. Reagents and conditions
：(a) BnCl, NaOH, MeOH,
reflux, 8h; (b) Ethanolamine, NaOH, MeOH/H2O, reflux, 2h; (c)
BnCl, NaH, THF, reflux, 5h; (d) SeO2, CH3COOH/(CH3CO)2O, 90-
100oC, 3-4h; (e) NaBH4, EtOH, rt, 3h; (f) i) SOCl2, rt, 2h; ii)
NH3·H2O, MeOH, 4h.
presence
of
O-(6-chlorobenzotriazol-1-yl)-N,N,N′,N′-
Results and discussion
Chemistry
tetramethyluronium hexafluorophosphate (HCTU) and
triethylamine provided the desired triester 11 in 64% yield,
which was hydrolyzed by treatment with formic acid to give
Synthesis of benzyl-protected bidentate ligand (7).
the triacid 12. Amine
7 was conjugated to the triacid 12 via
Hexadentate ligands can be formed by conjugating three
bidentate ligands onto a suitable tripodal backbone. However,
in order for the ligand to adopt the correct geometry for
iron(III) binding, it is essential that the backbone be connected
to the ring at the ortho position relative to one of the chelating
oxygen anions,26 namely position-2 or -5 on the pyridinone
ring. In this work, we firstly synthesized a benzyl protected
bidentate HPO containing a free amino group at position-2
amide bonds in the presence of HCTU and
diisopropylethylamine (DIPEA) in DMF at room temperature,
providing the protected hexadentate ligand 13 in 75% yield.
O
OBu-t
O
10
H2N
OBu-t
O
H
N
NH2(CH2)2COOEt
a
OBu-t
Cl
COOH
(Scheme 1). The benzylation of maltol (
treatment with benzyl chloride to obtain
Treatment of with ethanolamine under basic conditions
provided the HPO derivative in 85% yield. The benzylation of
hydroxyl group in provided in 90% yield, which underwent
1) was achieved by the
O
O
b
2
in 82% yield.
9
2
8
3
O
O
OH
OBu-t
O
3
4
H
O
H
H
H
N
N
N
N
selective oxidation of the methyl group at position-2 with
selenium dioxide in acetyl anhydride to generate the aldehyde
d
OH
c
OBu-t
O
O
O
O
O
O
OH
OBu-t
N
5
in 63% yield. Reduction of
hydroborate as a reducing agent, producing alcohol
yield. Compound was treated with thionyl chloride to obtain
5
was carried out using sodium
11
12
6
in 85%
O
O
6
N
BnO
O
HO
the corresponding chlorinated product, which was then
treated with ammonia in methanol, generating benzyl
protected bidentate ligands with a free amino group at
OBn
BnO
OH
NH
O
NH
O
O
HO
N
O
O
H
H
H
e
H
N
N
N
N
N
N
N
H
H
position-2 (7) in 59% yield.
O
O
O
O
O
O
BnO
HO
HN
HN
BnO
HO
Synthesis of hexadentate HPO (14).
O
O
N
N
OBn
OH
14
The synthetic route for the hexadentate HPO (14) is outlined in
13
Scheme 2. Firstly we synthesized tripodal compounds (triacids
Scheme 2. Reagents and conditions
2h, then rt, overnight; ii) NaOH, MeOH, 0°C, 2h, then nutrilized
with Amberlite IR-120 resin (H form), 67% (two steps); (b) 10
：(a) i) CH2Cl2, Et3N, 0°C,
12). Treatment of acryloyl chloride (8) with β-alanine ethyl
ester hydrochloride gave ethyl 3-acrylamidopropanoate which
was hydrolyzed by dilute alkali solution, followed by
,
HCTU, Et3N, DMF, rt, overnight, 64%; (c) HCOOH, rt, overnight,
100%; (d) 7, HCTU, DIPEA, DMF, rt, overnight, 75%; (e) BCl3,
CH2Cl2, 0°C to rt, 6h, 92%.
neutralization with Amberlite IR-120 resin (H form) to obtain
in 67% yield. Coupling of compound 9
to amine 1027 in the
9
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ARTICLE
H2
C
H
C
H
C
Table 1. Preparation of Copolymers
CH2
CH
C
H2C
m
O
n
CH
C
H2C
C
O
C
O
O
O
O
+
HN
HN
O
Mole ratio
Mole
AIBN
O
Iron
OH
N
O
N
of 14 to
HEA in
Weight
recovery
(%)
M
onomer
ratio of
14 to
HN
O
N
HN
HO
OH
Polymer
code
binding
capacity
(μmol/g)
HO
H
H
recovery
(%)a
H
HO
N
H
HO
N
N
N
O
O
HN
O
O
O
reaction
mixture
HEA in
polymer
O
N
15 (HEA)
OH
HN
OH
OH
OH
OH
OH
N
O
HO
O
N
HO
O
O
16-1
16-2
16-3
16-4
16-5
16-6
0:100
1:99
3:97
5:95
7:93
9:91
85.3
80.2
74.0
67.0
64.2
50.0
0
14
16
Scheme 3. Synthesis of copolymeric chelators
65.0
65.0
56.2
55.9
48.1
39.9
0.7:99.3
2.1:97.9
3.8:96.2
4.4:95.6
6.0:94.0
160.5
261.7
297.1
370.1
Deprotection of benzyl groups on 13 was achieved in the
presence of boron trichloride, generating the hexadentate
chelators 14 as hydrochloride salts in excellent yield (92%). All
of the compounds were fully characterized by 1H NMR and MS.
For compound 14, the 13C NMR and HRMS analysis were also
performed.
aMonomer recovery means the percentage of monomer
incorporated in the copolymer in relation to the amount of
monomer used in the polymerization reaction. It was
calculated from the capacity of the copolymer based on the
assumption that all the hexadentate ligand units in copolymer
bind one iron atom.
Copolymerization of monomeric chelator 14 with 2-hydroxyethyl
acrylate.
The synthesis and a schematic structure of copolymeric
chelators are presented in Scheme 3. Chelating copolymers
were prepared by varying the initial mole percentage of
monomer 14 from 0 to 9% of the total composition (Table 1) in
the presence of initiator AIBN. Copolymer 16-1 was obtained
in almost quantitative yield, whereas the weight recovery of
ligand, the stability constant of the 14-iron(III) complex (K1)
can be expressed as follows:
the copolymers 16-2–16-5 decreased with increasing the
[FeL][H+]3
percentage of the hexadentate ligand monomer 14, indicating
that the bulky and polar 14 group renders the polymerization
more difficult.
Fe3+ + LH3
K1 =
(1)
FeL + 3H+
[Fe3+][LH3]
where L represents hexadentate ligand. Thus, the addition of
base favours the formation of complex FeL by consuming
protons. However, the addition of a large amount of base
could result in dissociation of FeL as a result of competition
with hydroxide anions:
Physico-chemical properties of chelating monomer 14.
To demonstrate the iron(III) affinity of hexadentate chelator
14, we determined its pKa values and stability constant for the
corresponding iron(III) complex using an automated titration
system.28 The titration data were analyzed with pHab
program29 and HYSS program.30
FeL + 4OH-
Fe(OH)4- + L3-
(2)
pKa values. The pH-dependent UV spectra of 14 (Figure 1) were
recorded between 250 and 350 nm over the pH range 5.327-
11.631 for the free ligand. The speciation spectra demonstrate
a clear shift in λmax from 280 to 310nm, which reflects the pH
dependence of the ligand ionization equilibrium. The ligand 14
can be considered as a trimer of the corresponding bidentate
ligand and it therefore possesses two sets of intrinsic pKa
values. Using the spectrophotometric titration method, the
pKa values of 14 obtained from nonlinear least squares
regression analysis were found to be 2.75, 3.21, 3.76, 8.21,
8.79 and 9.31. Of the six pKa values, the three lower values
correspond to the 4-oxo functions and the higher three
correspond to the 3-hydroxyl functions.
0.80
0.70
0.60
0.50
pH 11.631
0.40
0.30
0.20
0.10
pH 5.327
0.00
250
290
310
270
330
Wavelength (nm)
Iron(III) affinity. The stability constant of an iron-ligand complex
is one of the key parameters related to the chelation efficacy
of a ligand. As iron(III) forms a 1:1 complex with a hexadentate
Figure 1. UV spectra of 14. [14] = 49μM (in 20.060 ml of 0.1 M
KCl), pH was changed from 5.327 to 11.631 by the addition of
KOH at 25°C.
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of the copolymers, the mole ratios of monomer 14 to HEA in
the copolymers were calculated (Table 1).
0
0.45
Iron binding affinity. To compare the iron(III) affinity of the
polymeric chelator and monomeric chelator 14, a study
associated with competition between a fluorescent chelator
CP69132 and hexadentate 14 or polymeric chelator 16-4 was
performed.33 When CP691 is mixed with iron, fluorescence is
quenched to a low level, but is gradually increased on the
addition of a competing ligand (either hexadentate 14 or
polymeric chelator 16-4). When the competition reaches
equilibrium, the relative fluorescence intensity of the
corresponding solutions were found to be similar. The pFe
values of monomer 14 and polymer 16-4 were calculated to be
29.7 and 29.8 respectively, which indicates that the
hexadentate moieties on the soluble polymer possess a similar
affinity for iron(III) as hexadentate 14. A similar result was
observed between a hexadentate HPO and hexadentate HPO-
based dendrimers.20 It should be noted that the pFe value
obtained by this method is slightly lower than that obtained by
calculation using pKa and logK1 values (30.4).
pH 10.043
0
0.35
0
0.25
0
0.15
pH 12.784
450
0
0.05
400
600
550
500
Wavelength (nm)
Figure 2. UV spectra of 14 in the presence of iron(III). [14] =
28.1μM, [Fe] = 26.8μM (in 20.143ml of 0.1 M KCl), pH was
changed from 10.043 to pH 12.784 by the addition of KOH (1.0
M) at 25°C
Indeed, the stability constant of the 14-iron(III) complex can be
determined by the competition of iron binding between
chelator 14 and hydroxyl anion. The UV spectra of 14 in the
presence of iron at different pH values are shown in Figure 2.
With the increase of pH, the absorbance at 458nm decreased,
suggesting the decrease of iron complex FeL concentration.
The logK1 value was determined to be 33.61±0.02.
Molecular weight. To estimate the molecular weight
distribution of each fraction, its weight-average molecular
weight (Mw) and number-average molecular weight (Mn)
were measured by gel permeation chromatography (GPC). The
polydispersity (PD) was determined from the ratio of Mw to
Mn to be 1.14 for polymer 16-4. The data suggests that the
samples possess narrow dispersity, and that the weight-
molecular weight of polymer is about 180 KDa. Taking into
consideration the molar ratio of the two monomers in polymer
16-4, the approximate composition of this polymer is HEA
(n=1200) and 14 (n=50).
The pFe3+ value, defined as the negative logarithm of
concentration of the free iron(III) in solution (when [Fe3+]Total
=
10-6 M; [Ligand]Total = 10-5 M; pH = 7.4), is a more suitable
parameter for comparison of ligands than the stability
constant, since it takes into account the effect of ligand
basicity, denticity and the degree of protonation. The pFe3+
value of 14, calculated by using the measured stability
constant logK1 and pKa values, is extremely high, being 30.4
(the figure for desferrioxamine is 26.631).
Antimicrobial assay.
In vitro antimicrobial activity of the monomeric chelator and
polymeric chelator against the five tested strains (two Gram-
Characterization of hexadentate-containing copolymers.
Table 2. Inhibition zone diameters of chelators (concentration
Iron chelating ability. Iron chelating capacity and utilization rate
of monomeric chelator of polymers are shown in Table 1. The
chelating capacity of 14-HEA copolymers depended on the
initial monomer 14 concentration in the polymerization
6.53mM) against five strains of bacteria
Inhibitory zone diameter (mm)
Bacterial
strains
Control Monomer 14 Polymer 16-4
EDTA
reaction, the larger the initial concentration of monomer 14
,
the greater the chelating capacity of copolymer. However the
utilization rate of monomeric chelator and the weight recovery
decreased with increasing the percentage of the hexadentate
ligand monomer. For instance, when the initial mole ratio of
monomer 14 was 9% of the total composition, the iron
chelating capacity of the obtained copolymer reached
370.1µmol/1g of copolymer, which is the highest, whereas the
monomer recovery and weight recovery were 39.9% and
50.0%, respectively, which were the lowest. On consideration
of these factors (including iron chelating capacity, utilization
rate of monomeric chelator and yield), the polymer obtained
with the molar ratio of 5% (polymer 16-4) was selected for
more detailed investigation. Based on iron binding capacities
E. coli
0
0
0
18.0 b ± 0.4
24.1 e ± 0.6
24.4 e ± 0.9
17.1 b ± 0.4
23.6 e ± 1.1
24.3 e ± 0.5
10.1 a ± 0.2
10.6 a ± 0.3
10.3 a ± 0.4
S. aureus
B. subtilis
Salmonella
spp.
0
0
21.8 d ± 1.2
19.6 c ± 1.2
21.9 d ± 1.1
19.8 c ± 0.5
9.9 a ± 0.3
9.8 a ± 0.6
P. aeruginosa
Note: For polymer 16-4
, the concentration refers to
hexadentate binding sites. The different letters within each
group indicate significant differences (p < 0.05).
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Table 3. The MICs of chelators against five strains of bacteria for iron(III) and antimicrobial potencies, indicates that all the
(μM)
polymer-bound chelator moieties are active. The potential
advantage of the polymeric chelator over the monomeric form
is two fold: one, polymeric species may be stably coupled to
wound dressing materials; two, polymeric chelators are less
likely to gain access to the systemic circulation, when
administered topically.
Bacterial strains
Monomer 14
Polymer 16-4a EDTA
E. coli
82
41
41
41
82
82
41
41
82
82
340
170
170
340
340
S. aureus
B. subtilis
Conclusions
Salmonella spp.
P. aeruginos
In conclusion, HPO hexadentate functionalized iron
chelating copolymers
have been
synthesized
by
copolymerization of monomeric chelator 14 and HEA in the
presence of AIBN. Polymer 16-4 was found to possess
appreciable inhibitory activity against the growth of both
Gram-positive and Gram-negative bacteria and by virtue of its
macromolecular nature could find application in the treatment
of wound healing. Work along such lines is continuing.
a. The concentration refers to hexadentate binding sites.
positive bacteria Staphyloccocus aureus and Bacillus subtilis
and three Gram- negative bacteria Escherichia coli, Salmonella
spp. and Pseudomonas aeruginosa) was evaluated in
comparison with EDTA, a well-known hexadentate chelator, by
inhibition zone and MIC assays. The results are presented in
Table 2 and Table 3. All the experimental data concerning the
various antimicrobial treatments were statistically analyzed
using analysis of variance test (SPSS version 16.0). Statistical
analyses were performed using one-way ANOVA. Significant
differences between the treatments were examined by
Duncan’s multiple range test and p < 0.05 was considered
statistically significant.
Acknowledgments
This research work was financially supported by Science
Technology Department of Zhejiang Province of China (No.
2013C24006), Zhejiang Provincial National Natural Science
Foundation of China (No. LY12B02014), the National Natural
Science Foundation of China (No. 20972138), and
Postgraduate Science and Technology Inovation Project from
Zhejiang Gongshang University (1110XJ1513121). The authors
acknowledge Dr. Vincenzo Abbate at King’s College London for
the help with molecular weight determination of polymeric
chelator.
Inhibitory zone. As shown in Table 2, all of the three chelators
exhibited inhibitory effect against the five strains. However,
the monomer 14 and polymer 16-4 exerted superior inhibitory
activity on all of the five tested strains when compared to
EDTA at the same molar concentration (6.53 mM) (p < 0.05).
However, no significant difference between monomer 14 and
polymer 16-4 were observed (p > 0.05). Both monomer 14 and
polymer 16-4 were found to show stronger inhibitory effects
on the growth of the two Gram-positive bacterial strains (S.
aureus and B. subtilis) than the three Gram-negative bacteria
(E. coli, Salmonella spp. and P. aeruginosa) (p < 0.05).
Notes and references
1.
2.
3.
4.
H. Nikaido and H. I. Zgurskaya, Curr. Opin. Infect. Dis.,
1999, 12, 529-536.
L. A. Mitscher, S. P. Pillai, E. J. Gentry and D. M. Shankel,
Med. Res. Rev., 1999, 19, 477-496.
D. Hogan and R. Kolter, Curr. Opin. Microbiol., 2002,
472-477.
5,
M.G. Thompson, B. W. Corey, Y. Z. Si, D. W. Craft and D. V.
Zurawski, Antimicrob. Agents Chemother., 2012, 56, 5419-
5421.
MIC assay. As can be seen in Table 3, EDTA possesses the
weakest inhibition compared with monomer 14 and polymer
16-4 against all of the five tested bacterial strains, which can
be attributed to its relatively lower affinity for iron(III) (pFe
=23.4).6 It was found that monomer 14 and polymer 16-4
possessed the similar MIC value against the four bacterial
strains except Salmonella spp., which is more sensitive to
monomer 14 (MIC 41 μM) than polymer 16-4 (MIC 82 μM).
Overall, the MIC determination indicated that the monomeric
chelator exhibited a similar inhibitory activity to polymeric
chelator with same amount of iron binding centres, suggesting
that they both inhibit bacterial growth by removing iron from
the bacterial environment. The different sensitivities of the
five tested bacteria towards the iron chelator may result from
the different iron affinities of siderophores secreted by the
respective bacteria. That the polymer 16-4 and corresponding
monomer 14 investigated in this study have similar affinities
5.
Y. Shibata, K. Hiratsuka, M. Hayakawa, T. Shiroza, H.
Takiguchi, Y. Nagatsuka and Y. Abiko, Biochem. Biophy.
Res. Commun., 2003, 300, 351-356.
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