Macromolecular iron-chelators via RAFT-polymerization for the inhibition of methicillin-resistant Staphylococcus aureus growth

Article abstract of DOI:10.1016/j.polymer.2016.01.073

A series of linear poly (glycidyl methacrylate) (PGMA) polymers were synthesized via RAFT polymerization and conjugated with amine-containing 3-hydroxypyridin-4-ones (HPOs) to generate a panel of HPO-containing materials with controlled structures and spe

Full text of DOI:10.1016/j.polymer.2016.01.073

Accepted Manuscript
Macromolecular iron-chelators via RAFT-polymerization for the inhibition of
methicillin-resistant Staphylococcus aureus growth
Junpei Li, Eniola D. Olaleye, Xiaole Kong, Tao Zhou, Yongmin Ma, Jagoda Jurach,
Osamah Al Rugaie, Robert C. Hider, Guoqing Zhang, Selwa Alsam, Vincenzo Abbate
PII:
S0032-3861(16)30073-8
10.1016/j.polymer.2016.01.073
JPOL 18425
DOI:
Reference:
To appear in: Polymer
Received Date: 29 September 2015
Revised Date: 23 January 2016
Accepted Date: 27 January 2016
Please cite this article as: Li J, Olaleye ED, Kong X, Zhou T, Ma Y, Jurach J, Al Rugaie O, Hider
RC, Zhang G, Alsam S, Abbate V, Macromolecular iron-chelators via RAFT-polymerization for
the inhibition of methicillin-resistant Staphylococcus aureus growth, Polymer (2016), doi: 10.1016/
j.polymer.2016.01.073.
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ACCEPTED MANUSCRIPT
Polymeric iron scavengers deprive MRSA organisms of essential iron

ACCEPTED MANUSCRIPT
Macromolecular Iron-Chelators via RAFT-Polymerization for the Inhibition of
Methicillin-Resistant Staphylococcus aureus Growth
Junpei Lia, Eniola D. Olaleyeb, Xiaole Kongc, Tao Zhoud, Yongmin Mae, Jagoda
c
Jurachc, Osamah Al Rugaieb,f, Robert C. Hider , Guoqing Zhanga* , Selwa Alsamb
and Vincenzo Abbatec*
a Hefei National Laboratory for Physical Sciences at the Microscale, University of Science and
Technology of China, Hefei, Anhui, 230026, P. R. China.
b School of Biological Sciences, University of Essex, Wivenhoe Park, Colchester, CO4 3SQ, UK
c Institute of Pharmaceutical Science, King’s College London, Franklin-Wilkins Building, 150
Stamford Street, London, SE1 9NH, UK.
d School of Food Science and Biotechnology, Zhejiang Gongshang University, 18 Xuezheng Street,
Hangzhou, Zhejiang, 310018, P. R. China.
e Zhejiang Chinese Medical University, 548 Binwen Road, Hangzhou, 310053, P R China.
f Unizah College of Medicine and Medical Sciences (UCM), Qassim University, P.O. Box 991,
AlQassim 51911, Saudi Arabia.
Abstract: A series of linear poly (glycidyl methacrylate) (PGMA) polymers were
synthesized via RAFT polymerization and conjugated with amine-containing
3-hydroxypyridin-4-ones (HPOs) to generate a panel of HPO-containing materials
with controlled structures and specific iron-binding functions. The structures of the
1
resulting polymers were characterized via H-NMR, GPC and FT-IR and their
chelating capacity for iron was investigated using UV-Vis spectrophotometric
titration of the iron(III) complexes. In vitro antimicrobial studies of selected
ligand-containing homopolymers demonstrate that the homopolymers are capable of
inhibiting the growth of methicillin-resistant Staphylococcus aureus (MRSA). It is
proposed that the inhibition activity of MRSA is derived from the iron-chelating
capability of the iron-binding polymers.
Keywords: MRSA; Hydroxypyridinones; Iron-binding polymers.
1

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1. Introduction
In recent years, macromolecular iron chelators have received increasing attention
as human therapeutic agents.[1, 2] Polymeric chelators have been used for water
treatment,[3] pollution control,[4] recovery of metals,[5, 6] active packaging[7, 8] and
analytical chemistry.[9, 10] Recently several iron binding polymer applications in the
biomedical field have been reported.[11] As iron is an essential element for all living
processes, in principle targeting iron is a useful approach for the treatment of
microbial infectious diseases.[12, 13] Macromolecular iron chelators also have
potential in the treatment of acute iron overload.[14] Although most of the
present-day iron-chelating therapeutic agents are designed as small molecules, there
are a number of advantages associated with polymeric therapeutics that cannot be
readily achieved with low molecular weight drugs. For example, when taken orally,
the high-molecular-weight characteristic of polymers render them largely
non-absorbed by the gastrointestinal tract, thus potentially extending the drug
half-life.[1]
The design of iron chelating polymers is presently based on several strategies, one
involving the immobilization of natural chelators (e.g., desferrioxamine B) onto
activated supports[15, 16], a second involving the conjugation of bidentate ligands
with activated polymers[17, 18] and
a
third, copolymerization of
1-(-acrylamidoethyl)-3-hydroxy-2-methyl-4(1H)-pyridinone (AHMP) with other
cross-linking agents.[19] The former structures are presently limited to carbohydrate
matrices and, as a consequence, are expensive to prepare, and high binding capacities
are difficult to achieve. Furthermore the synthetic chemistry for the second and third
approaches is difficult to control, generating polydisperse polymers and crosslinked
materials with poor solubility. It is therefore desirable to develop a simple
preparative method for iron-binding polymers of well-defined structure.
Poly(glycidyl methacrylate) (PGMA) is a well-known polymer for both industrial
and biomedical applications because it is reactive, inexpensive, hydrophilic,
biocompatible, and generally nontoxic. Furthermore it permits fast and efficient
post-polymerization modifications.[20, 21] RAFT polymerization was successfully
employed for the synthesis of well-defined PGMA polymers, exerting remarkable
control over their molecular weight and affording polymers with low
polydispersities.[22, 23] Recently, both linear- and star-shaped PGMAs were
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modified with different amines by a nucleophilic ring-opening reaction of the epoxy
group.[24, 25] 3-Hydroxypyridin-4-ones (HPOs) are one of the main classes of
candidates for the development of clinically useful iron chelators, and
1,2-dimethyl-3-hydroxypyridin-4-one (deferiprone, Figure 1) is currently available for
clinical use (Apotex Inc., Toronto, Canada as Ferriprox).[26] We therefore sought to
utilize amine-containing HPOs, such that the amine functionality can be subsequently
added onto a pre-formed PGMA, in a ring-opening process to obtain novel
macromolecular iron chelators.
1.1 Iron restriction and Staphylococcus activity
Staphylococcus aureus is a widespread and dangerous pathogen that is responsible
for many human infections worldwide.[27] Some characteristics that help account for
the prevalence of S. aureus as a human pathogen include its genetic diversity, ability
to acquire new exogenous genes, a remarkable propensity to acquire resistance to
multiple antimicrobial agents, high virulence and efficient quorum sensing
mechanisms.[28] S. aureus infections range from moderately severe infections of the
skin or the respiratory tract to life-threatening diseases such as necrotizing pneumonia,
necrotizing fasciitis, osteomyelitis, endocarditis and toxic shock syndrome.[29, 30]
These infections impose a high and increasing burden on healthcare resources[31] and
are associated with high morbidity and mortality[32] as well as a longer hospital
stay.[33, 34]
The global spread of methicillin-resistant S. aureus (MRSA) has become one of
the most serious contemporary challenges to the treatment of hospital-acquired
infections (HAIs).[35] MRSA bear the mecA gene that encodes a 78-kDa
penicillin-binding protein 2a (PBP2a) which has low-affinity for methicillin and all
β-lactam antibiotics.[29] MRSA is the principal cause of nosocomial infections
caused by a single bacterial pathogen in the United States and many other parts of the
world.[36-39] An estimated 44% of all HAIs can be attributed to MRSA.[40]
Furthermore, the emergence and dramatic increase of MRSA infections in otherwise
healthy individuals with no risk factors has become an issue of increasing
concern.[41]
MRSA has acquired resistance to multiple antibiotic classes and this has limited
the therapeutic options available for its treatment.[41, 42] Vancomycin is considered
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the workhorse of parenteral anti-infective agents for MRSA, yet, some strains of S.
aureus exhibit reduced susceptibility to vancomycin.[43-45] Many of the new
antimicrobial agents available for the treatment of MRSA infections (e.g. linezolid,
quinupristin-dalfopristin, daptomycin, tigecycline and telavancin) are associated with
dose-limiting adverse events, emerging resistance issues, and high cost.[46] This
emphasizes the need for further research to identify new antimicrobial drug targets in
MRSA as well as to develop new antimicrobial agents with novel mechanisms of
action against MRSA.
All microorganisms require iron for both growth and replication. Most
microorganisms have developed efficient methods of absorbing iron from the
environment and many microorganisms secrete siderophores in order to scavenge iron.
The role of these compounds is to scavenge iron from the environment and render it
available to the microbial cell. Hence, limiting the amount of available iron should, in
principle, inhibit microbial growth. It has been established that both bidentate and
hexadentate iron chelators can control the growth of a wide variety of
microorganisms.[47, 48] In this work we have investigated the possible application of
polymeric bound chelators on the growth of S. aureus. Thus, the synthesis of a panel
of HPO bidentate ligands with different amine-substituents and PGMA of two
molecular weights, as well as the post-polymerization modification by nucleophilic
ring-opening reaction of the epoxy group is reported. For the first time, RAFT
polymerization[49, 50] has been employed to generate well-defined polymeric
HPO-based macromolecular chelators. The physicochemical properties of the
monomer bidentate ligands and the iron-chelating properties of the resultant
PGMA-HPO chimera have been investigated. Preliminary in vitro antimicrobial
activity against MRSA is reported.
2. Materials and methods
2.1 Materials
All chemicals were purchased from Aldrich or Merck Chemical Company and
used without any further purification. Column chromatography purifications were
1
13
performed on Merck silica gel 60 (0.04−0.063 mm). H NMR and C NMR spectra
were recorded using a Bruker Avance 400 (400 MHz) NMR spectrometer. Chemical
shifts (δ) are reported in ppm downfield from the internal standard tetramethylsilane
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(TMS). J values are in hertz (Hz), and splitting patterns are designated as follows: s
(singlet), bs (broad singlet), d (doublet), t (triplet), and m (multiplet). The ESI-MS
analyses were performed using a Waters/Micromass ZQ mass spectrometer
(Manchester, UK). HRMS were carried out by the Mass Spectrometry Facility,
School of Biomedical and Health Science, King’s College London. Purity was
determined via HPLC analysis.
2.2 Polymerization of glycidyl methacrylate (GMA)
The following conditions were used for the synthesis of PGMA using
[GMA]/[CPBD] ratios of 65:1 and 100:1, in anhydrous toluene, at 62ꢀ°C. A solution
of GMA (4.9 g, 3.43ꢀ×ꢀ10−2 mol, 65 equiv.), CPBD (11.6ꢀ×ꢀ10−2 g, 5.28ꢀ×ꢀ10−4 mol,
1.0 equiv.), and AIBN (2.9ꢀ×ꢀ10−2 g, 1.76ꢀ×ꢀ10−4 mol, 0.33 equiv.) or a solution of
GMA (5.0 g, 3.52ꢀ×ꢀ10−2 mol, 100 equiv.), CPBD (7.8ꢀ×ꢀ10−2 g, 3.52ꢀ×ꢀ10−4 mol, 1.0
equiv.), and AIBN (1.9ꢀ×ꢀ10−2 g, 1.17ꢀ×ꢀ10−4 mol, 0.33 equiv.) were prepared to obtain
the [GMA]/[CPBD] ratios of 65:1 or 100:1, respectively. The solutions were degassed
using three freeze-pump-thaw cycles to remove traces of oxygen. The Schlenk tubes
were placed in an oil bath preheated at 62ꢀ°C and the polymerizations were carried out
for 5hrs and 2hrs, respectively. After completion, the polymerization mixture was
precipitated into ethyl ether to remove the unreacted monomer followed by filtration
1
and drying under vacuum. Monomer to polymer conversions was determined by H
NMR spectroscopy of the samples taken from the polymerization mixture at different
1
time intervals. The products were characterized by GPC and H NMR. DMF
(N,N-Dimethylformamide) GPC analysis coupled to a refractive-index detector (vs.
PS standards) indicated a Mn of 12,800 g mol−1 and an Mw/Mn of 1.07 for 7, and an Mn
of 9,300 g mol−1 and an Mw/Mn of 1.07 for 8.
2.3 Synthesis of iron-binding polymers
For the preparation of an iron-binding polymer using 1 or 3 as the
amine-containing HPO ligands, 0.2 g of PGMA (7 or 8) and 1 mL of triethylamine
were dissolved in 8 mL of DMF. The reaction mixture was stirred at 55 °C for 36 h to
produce 7.1, 8.1, 7.3 and 8.3. The final reaction mixture was precipitated with excess
diethyl ether and the resulting polymer-drug conjugate was purified via dialysis for
5

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three days against excess distilled water with frequent water changes. For the
preparation of iron-binding polymers using HPOs 2, 4, 5 or 5b, 0.2 g of PGMA (7 or
8) was dissolved in 8 mL of DMF. 2, 4, 5 or 5b and 1 mL of triethylamine were then
added. The reaction mixtures were stirred at 80 °C for 2 h to produce polymers 7.2,
8.2, 7.4, 8.4, 7.5, 8.5 and 7.5b. The final reaction mixtures were precipitated with
excess diethyl ether and the resulting polymer-drug conjugates were purified via
dialysis for three days as described above. 7.4 and 8.4 were found to be insoluble in
all solvents investigated. 7.5b, 7.5, 8.5, 7.3 and 8.3 were soluble in DMSO: H2O (1:1,
v/v). The Mn of the conjugated products was calculated as derived from the estimated
Mn (NMR) of the corresponding PGMA and assuming 100% conjugation of the
epoxide ring-opening reactions, as judged by 1H NMR and FT-IR data.
2.4 pKa determination
Equilibrium constants of protonated ligands were determined using an automated
computerized system, consisting of a UV/vis spectrophotometer (Perkin-Elmer
Lambda 5), an autoburette (Metrohm Dosimat 665), a pH meter (Corning Delta 225),
and a peristaltic pump (Watson-Marlow 101U/R M2) all interfaced to a computer. A
blank titration of 0.1 M KCl (20 mL) was carried out to determine the electrode zero
and slope using GLEE.[49] The solution (0.1 M KCl, 20 mL), contained in a jacketed
titration cell, was acidified by the addition of 0.15 mL of 0.2 M HCl. Titration was
carried out against 0.3 mL of 0.2 M KOH using 0.01 mL increments dispensed from
the dosimat. All solutions were maintained at 25 ± 0.5 °C under an argon atmosphere.
The above titration was repeated in the presence of bidentate ligands. The data
obtained from titration were analyzed using the pHab software.[52]
2.5 Determination of stability constant of the iron(III) complex with bidentate ligands
In a typical experiment, iron(III) (35.9 µM) and bidentate ligand (180 µM) in 0.1
o
M KCl (20 mL) were alkalimetrically titrated from pH 1.6 to 7.6 at 25 C. Each pH
observation was taken after standing for 30 minutes to achieve equilibrium. The
titration data were analyzed using pHab software.[52] The affinity constants were
used to provide pFe3+ values for the following conditions: [Fe3+]total = 1 µM, [ligand]
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= 10 µM, pH =7.4. A typical spectral series for compound 1 is presented in
total
Figures S1 and S2 (SI).
2.6 Chelating capacity of iron(III)-binding homopolymers
Dry chelating homopolymer (7.1, 4.25 mg, or 6.15 mg, 8.5) was added to 0.4 mL
DMSO to prepare a stock solution. 5 µL aliquots of the above stock solution were
added to 1.4 mL water to yield the final solution to be assayed (the concentrations of
homopolymers 7.1 and 8.5 were 0.75 and 6.15 µM, respectively). Iron(III) aqueous
solution (0.8953 mM, FeCl3) was placed in a stoppered vessel ready for titration.
Batch spectrophotometric determinations were performed on a UV spectrophotometer
scanning from 250 to 600 nm. Automatic titration and spectral scans adopted the
following strategy: 5 µL iron solution was added for each point, until absorbance
remained unchanged for 30 min.
2.7 Preparation of polymers for antimicrobial activity determination
Stock solutions of polymers used in this study were prepared by dissolving in
different solvents as appropriate e.g. 20% DMSO in water for 7.1 and 50% DMSO in
water for 7.5 and 7.5b.
2.8 Bacterial culture
One strain of MRSA (a nasal isolate obtained from the Colchester Hospital
University Foundation NHS Trust) was used in this study. MRSA was inoculated in
o
10 mL Luria-Bertani (LB) broth medium and cultured without shaking at 37 C in
aerobic conditions for 6 hours. Optical Density (OD) was measured at wavelength
595 nm to give a colony forming unit (CFU)/mL of 108 (OD595 0.22 = 108
CFU/mL). The reference strains MRSA NCTC 12493 and S. aureus NCTC 6571 were
used in this study.
Antibiotic susceptibility testing
The susceptibility of the MRSA isolate used in this study to a number of antibiotics
(as listed in Table 3 in result section) was determined using the disk diffusion
(Kirby-Bauer) method. The OD of a 6 hr broth culture of the isolate was adjusted to
within the range of >0.1 - 0.3 at wavelength 500 nm in a spectrophotometer. 40 µl of
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the standardized culture was then added to 5 ml sterile distilled water following which
sterile swab sticks were dipped into the bacterial suspension and swabbed on the
entire surface of nutrient agar plates. Plates were left to dry for about 5 mins
following which antibiotics were placed on the surface of the nutrient agar plates. All
o
plates were then incubated at 37 C for 18 hrs. The zones of inhibition around the
antibiotic discs were measured and the results interpreted as resistance or susceptible
based on the interpretative standard of the British Society for Antimicrobial
Chemotherapy - BSAC (BSAC, 2015).
Identification of the MRSA isolate
PCR was done in order to confirm the identity of the MRSA isolate used in this
study. DNA was extracted using the QIAamp DNA Mini kit (QIAGEN) according to
manufacturer’s instructions. To confirm that the isolate was S. aureus, primers nuc1
and nuc2 were used in PCR to amplify the S. aureus-specific thermonuclease (nuc)
o
gene. PCR amplification consisted of a 5 min initial denaturation step at 95 C
o
followed by 37 cycles of denaturation at 95 oC for 30 secs, annealing at 55 C for 30
o
o
secs and extension at 72 C for 60 secs, with a final extension step at 72 C for 10
mins. Furthermore, primers mec1 and mec2 and specific to a 162 bp region of DNA
specific to the mecA gene were used in PCR to confirm that the isolate was indeed
MRSA. PCR amplification consisted of a 4 min initial denaturation step at 94˚C
o
followed by 35 cycles of denaturation at 94 C for 30 secs, annealing at 53˚C for 30
secs and extension at 72 oC for 1 min, with a final extension step at 72˚C for 4 min.
Agarose gel electrophoresis
PCR products and extracted DNA were analysed in 1% agarose gels to which
SafeView nucleic acid stain (nbs biologicals) had been added for fluorescent
visualisation of DNA bands. Gels were run in 1X TAE electrophoresis buffer at 150
V for 15 mins in a Mini-Sub cell GT electrophoresis apparatus (Bio-Rad) were
visualised in a Syngene InGenius3 gel documentation system.
2.9 Antimicrobial assay
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The incubation medium was water and all assays were performed in 96-well plates
with 107 CFU/mL of bacteria per well in the absence or presence of different
o
concentrations of the polymers. Plates were incubated at 37 C under aerobic
conditions for 24 hours after which the number of viable bacteria in each well was
determined by their growth on Cysteine Lactose Electrolyte Deficient (CLED) agar.
The bactericidal rate was calculated as follows:
[ꢁꢂ – ꢁꢃ]
(ꢀ =
× 100)
ꢁꢂ
where R is the bactericidal rate, X0 the number of bacteria without polymer (control),
and Xt the number of bacteria after the treatment with polymer. Minimum inhibitory
concentration (MIC) endpoints were determined as the lowest concentration of the
antimicrobials that inhibited the visible growth of bacteria after 24 hours incubation
using the micro-dilution method recommended by the British Society for
Antimicrobial Chemotherapy (BSAC)[53]. Measurements were carried out in
triplicate.
3. Results and discussion
3.1 Chemistry
The 1-substituted-2-methyl-3-hydroxy-4(1H)-pyridinones in this study (1-4,
Figure 1) were synthesized utilizing the methodology described by Dobbin and
co-workers[54, 55] (Schemes S1-S3 in the SI). The commercially available
2-methyl-3-hydroxy-4(1H)-pyranone, maltol, was benzylated in 90% aqueous
methanol
to
give
3-(benzyloxy)-2-methyl-4H-pyran-4-one
(1a).[56]
3-Methoxy-2-methyl-5-((methylamino)methyl)pyridin-4(1H)-one (5a) was readily
prepared from commercially available maltol in a four-step reaction by following an
established procedure.[57] The resulting protected HPO was found to partition from
aqueous solution (pH 7) into chloroform and was finally purified by column
chromatography. 2a was synthesized according to a reported procedure[58] and
protected via the Mannich reaction. Removal of the protecting benzyl group was
achieved by catalytic hydrogenolysis invariably to yield the corresponding bidentate
chelators as the free bases. The bidentate ligand 5 was obtained after removal of
methyl groups by treatment of 5b with boron trichloride. The final bidentate ligands
9

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(1-5, Schemes S1-3 in the SI and Figure 1) were obtained as a pale-yellow solid or a
1
brown oil, and characterized using H, 13C NMR, and high-resolution mass
spectroscopy. The purity of the compounds was determined by analytical RP-HPLC
using a C18 column. The protected intermediate 5b was employed to prepare a
control polymer 7.5b, thus not possessing iron-chelating ability.
Figure 1. Structure of bidentate ligands.
Glycerol monomethacrylate (GMA) was polymerized in anhydrous toluene at
62 °C using an azo initiator (AIBN) and a dithiobenzoate-based RAFT agent (CPBD)
to produce well-defined RAFT PGMA with epoxy functionalized groups that enabled
further post-polymerization modifications. GPC was used to determine Mn and PDI
values for PGMA 7 and 8. As expected by the RAFT technology, nearly
monodisperse materials were obtained. As a consequence, it was assumed that the
subsequent conjugation with amine-HPOs would yield well-defined macromolecular
iron chelators. The bidentate ligands 1-5 were used as nucleophilic reagents to
perform ring-opening reactions of PGMA to yield the final RAFT-based iron-binding
polymers (Scheme 1). According to the HPO used, we were able to produce a panel of
polymers with various properties (Table 1). Conjugation of PGMA with primary and
secondary amine-containing HPOs (e.g. 3 and 4) resulted in materials with either poor
water solubility (7.3 and 8.3) or with general insolubility properties (7.4 and 8.4);
clearly, removal of hydrogen bonding capability when transforming a primary amine
(3) to a secondary amine (4) markedly decreased the solubility of the resultant
macromolecular chelators. Using 5, as slightly more hydrophilic HPO-ligand, yielded
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polymers 7.5 and 8.5 with improved solubility in aqueous DMSO solutions. This
prompted the design of compound 1 (Figure 1) in order to produce the more
hydrophilic polymers 7.1 and 8.1 which possessed good water solubility properties. In
order to prepare a control polymer with similar macromolecular structure to that of
7.5, we employed the protected intermediate 5b to produce polymer 7.5b, which
lacked iron-chelating ability due to methylation of the 3-hydroxyl group on the HPO
ring. For the polymer-7 series, the number of chelating units per polymer chain is 106;
the corresponding number for the polymer-8 series is 28.
Table 1. Physical properties of iron-binding polymers1
.
2
Polymer PGMA
Ligand Solubility
Mn
34,700
50,100
38,900
33,100
36,100
33,100
13,400
10,400
7.5b
7.1
7.2
7.3
7.4
7.5
8.1
8.2
7
7
7
7
7
7
8
8
5b
1
H2O/DMSO (1:1, v/v)
H2O
2
H2O/DMSO (1:1, v/v)
H2O/DMSO (1:1, v/v)
None
3
4
5
H2O/DMSO (1:1, v/v)
H2O
1
2
H2O/DMSO (1:1, v/v)
8,900
9,700
8,900
8.3
8.4
8.5
8
8
8
3
4
5
H2O/DMSO (1:1, v/v)
None
H2O/DMSO (1:1, v/v)
1
For the polymer 7 series, the number of chelating units per polymer chain has been
calculated to be 106; the corresponding figure for the 8 series is 28.
2 The Mn of iron-binding polymers was derived from the calculated PGMA
a quantitative conjugation of the HPO ligand to the PGMA epoxide units.
Mn and assuming
To demonstrate the versatility of this synthetic approach, we designed compound
2 as a nucleophilic ligand to prepare fluorescent polymer-chelator chimera (7.2 and
8.2) where drug and fluorophore were embedded in the same structural unit. This
11

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strategy was preferred to the conventional approach to the preparation of fluorescent
RAFT polymers.[59, 60] Such methods usually make use of post-polymerization
chemistries to transform the RAFT agent into a fluorophore. Our approach was
therefore faster, producing multifluorescent moieties per polymer chain. Fluorescent
spectra confirmed the fluorescent properties of both compound 2 and the resultant
polymers 7.2 and 8.2 (SI, Figures S9-S11). These polymers will be useful to establish
their cellular localization in future microbial studies.
Scheme 1. Synthesis of PGMA and iron-binding polymers1, 2
(a) AIBN and GMA; 62 oC, toluene; (b) 55 oC, DMF, 36h; (c) 80 oC, DMF, 2h.
1 Polymer nomenclature: X.Y, where X is the parent PGMA and Y is the conjugated HPO
2 For 7.3 and 8.3, R1 = 3; for 7.1 and 8.1, R1 = 1; for 7.5 and 8.5, R2 = 5; for 7.2 and 8.2, R2 =
2; for 7.4 and 8.4, R2 = 4; and for 7.5b, R2 = 5b.
GPC analysis under various conditions (water, DMF or their mixtures) was not
found to be practical for the HPO-PGMA conjugates; therefore the final Mn of
conjugates was estimated by assuming a 100% epoxide ring-opening reaction, as
confirmed by FT-IR and 1H NMR data. Figure S3 (SI) shows the 1H NMR spectra of
the PGMA, which reveals that the epoxy ring remained intact during the
polymerization reaction. The peaks at δ = 7.44 – 8.14 ppm correspond to the aromatic
protons of the CPBD units, indicating that the resulting RAFT polymers had
incorporated the chain-transfer agent. The peaks at δ = 3.23 ppm, 2.63 and 2.83 ppm
can be assigned to the protons of the oxirane ring. The two protons were in different
chemical environments, resulting in two different resonances. End-group analysis was
used to calculate the mean degree of polymerization (DP) for this reaction, polymer 7
with a DP of 106 and Mn = 15,200, and polymer 8 with a DP of 28 and Mn = 4,200.
The disappearance of peaks corresponding to the epoxy group (C–O–C asymmetric
12

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stretching 750–950 cm-1) in the IR spectrum (Figure S3) and the appearance of a
broad peak at 3400–3700 cm-1 pertaining to the –OH stretching vibration (Figure S4
in the SI) confirmed quantitative epoxide ring-opening reaction. Furthermore, The
NMR spectrum of 8.1 (Figure S6 in the SI) clearly shows the protons of pyridine ring
at 7.3, 6.3 ppm, the shift of the proton adjacent to the oxirane ring and the appearance
of the hydroxyl proton at 5.0 ppm. This suggests successful and quantitative
conjugation of the HPO-ligands to the PGMA. In summary, fine-tuning of the
structural features of the HPO ligands followed by RAFT-mediated PGMA
preparation allowed the development of a small library of polymer-chelator materials
with defined Mn and physico-chemical properties.
3.2 Determination of pKa and logKFe3+ values for hydroxypyridinones
The overall results for the five bidentate ligands are presented in Table 2. The
pFe3+ values for 1, 2, 4 and deferiprone are all similar, the slightly lower value for 3
results from the electron withdrawing influence of the β-amino function on the
1-substituent, which is para to one of the coordinating oxygens.
Table 2. Physico-chemical parameters of bidentate HPO-ligands.
Deferiprone
20.3
1
2
3
4
5
pFe3+
pKa3
pKa2
pKa1
19.47
10.12
9.72
3.45
19.77
9.32
6.62
1.41
18.7
7.56
9.68
3.09
19.2
9.95
9.03
3.14
15.9
5.74
9.17
3.99
9.77
3.68
The spectrophotometric titration of 1 is presented in SI (Figure S1 and S2). The
pFe3+ of 5 is much lower than the other five HPOs and this is probably associated
with the ability of 5 to exist in two tautomeric forms, one of which is catechol-like
(Figure 2). Such tautomerism is not possible with the other analogues. Significantly
the pFe3+ value for catechol is 15, which is close to that of 5.
13

ACCEPTED MANUSCRIPT
Figure 2. Tautomeric forms of compound 5.
3.3 Characterization of bidentate-containing homopolymers
In order to evaluate the iron(III) binding properties of polymers, compounds 7.1
and 8.5 were selected as model compounds due to their different Mn and chelating
units. UV–vis spectra of a range of solutions containing various polymer-iron(III)
ratios were determined scanning between 300 and 600 nm (Figure 3 and SI Figure
S13). Based on calculated Mn and number of chelating units per polymer chain (Table
1), the effective concentrations of the polymer-bound chelator on the homopolymers
7.1 and 8.5 were 81 and 172 µM. Bidentate iron chelators are known to form a 3:1
ligand:metal complexes with iron(III)[26]. As a consequence, it was expected that the
metal complex saturation point for 7.1 and 8.5 would be reached at 27 and 57 µM
Fe(III), respectively. In each polymer investigated the intensity of absorbance at 460
nm increased until a plateau. Indeed, the plateau for the polymer 7.1: iron(III) curve is
reached at around 27 µM iron(III) (Figure 3a), demonstrating that this polymer
possesses high affinity for iron, and that all the chelating units appended to the PGMA
backbone are available for metal binding.
0.12
0.10
0.08
0.06
0.04
0.02
0.12
0.10
0.08
0.06
0.04
0.02
0.00
8.5
7.1
a
b
0
5
10 15 20 25 30 35 40 45
0
5
10 15 20 25 30 35 40 45 50 55 60
Fe3+
]
[
Fe3+
]
(µM)
(µM)
[
Figure 3. Plots of absorbance versus equivalent ratios of iron(III) to homopolymers
7.1 and 8.5 at 460 nm. The concentrations of homopolymers 7.1 and 8.5 were 0.75 and
6.15 µM, respectively. The solvent system was MOPS buffer.
14

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In contrast, the saturation point for polymer 8.5 occurred at around 43 µM Fe(III),
somewhat lower than the theoretical metal concentration (Figure 3b). This was
attributed to the different metal chelator (namely compound 5) attached to the PGMA
in 8.5. Such a chelator is conjugated much closer to the PGMA backbone, thus
generating steric hindrance that may not permit efficient metal chelation[57].
Furthermore, the affinity for iron(III) is lower with this chelating unit when compared
to 1. The iron chelating capacities of 7.1 and 8.5 were calculated to be 843 and 350
ꢁmolg, respectively. Thus the iron chelating capacity of the homopolymers in this
study is greater than Chelex 100 (110 ꢁmolg).[57]
Antibiotic susceptibility profile of the MRSA isolate
The isolate was seen to be resistant to only 4 of the 13 antibiotics used in this
study (Table 3). Resistance of the isolate to Cefoxitin (10 µg), a β-lactam antibiotic,
tentatively confirmed that the isolate was MRSA.
Table 3. Antibiotic susceptibility profile of the MRSA isolate used in this study
Antibiotics
Susceptibility
profile
Cefoxitin (10 µg)
Linezolid (10 µg)
Fusidic acid (10 µg)
Tetracycline (10 µg)
Clindamycin (2 µg)
R
S
R
S
S
R
S
R
S
Ciprofloxacin (1 µg)
Neomycin (10 µg)
Erythromycin (5 µg)
Fosfomycin/Trometamol (200 µg)
Tigecycline (15 µg)
Quinupristin/Dalfopristin (15 µg)
R
S
Rifampicin (2 µg)
Gentamicin (10 µg)
S
S
R = Resistant, S = Susceptible
Identity of the isolate
15

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The MRSA isolate used in this study was confirmed to be S. aureus as PCR
amplification with primers nuc1 and nuc2 yielded an ~270 bp band of DNA (Figure 4)
which corresponds to the nuc gene of S. aureus.
M
MRSA-C SA-C blank-C
2
Nasal Abdo Shin
1000 bp
500 bp
250 bp
270 bp nuc gene
Figure 4. Gel showing products from PCR amplification of the ~270 bp S. aureus-specific
nuc gene in four clinical MRSA isolates: Lane M: 1 kb Generuler; Lane MRSA-C = MRSA
NCTC 12493 reference strain; Lane SA-C = S. aureus NCTC 6571 reference strain; Lane
blank C = Blank control (PCR with no DNA). Lanes 2, Nasal, Abdo and Shin: Products
obtained from the amplification of the nuc gene in six UK clinical MRSA isolates. *Nasal is
the MRSA isolate used in this study
PCR amplification of a 162 bp mecA specific DNA using primers mec 1 and mec 2
in the MRSA isolate used in this study (as seen in Figure 5) conclusively confirmed
that the isolate is indeed methicillin resistant.
M
+ve C
-ve C
blank C
Nasal
Abdo
1000 bp
500 bp
250 bp
162 bp mecA
specific DNA
Figure 5. Gel showing products from PCR amplification of a 162 bp region of the mecA gene
in two clinical S. aureus isolates: Lane M: 1 kb Generuler; +ve C = Positive control (MRSA
NCTC 12493); -ve C = Negative control (S. aureus NCTC 6571); blank C = Blank control
(PCR with no DNA); Lanes Nasal and Abdo: Products obtained from the amplification of
the mecA specific DNA in both clinical MRSA isolates. *Nasal is the MRSA isolate used in
this study
16

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3.4 Antimicrobial effect of selected polymers on MRSA
In this work, we selected a range of polymers for biological evaluation, based on
their physical properties such as Mn and acceptable solubility in water or aqueous
DMSO systems, namely 7.1, 7.5, 8.1 and 8.5.(Table 1). In addition, 7.5b was
evaluated as a non-chelating control to assess the importance of the iron-chelating
activity of polymers with respect to their biological properties. The concentrations of
the polymers used in this study are presented in Table 4. The inhibitory effects on
MRSA are given in Figures 6 and 7 and their bactericidal effects are listed in Table 5.
The number of viable cells was determined following incubation of bacteria for 24
hours in the presence and absence of various concentrations of the polymers. The
greatest MRSA inhibitory effect was observed with 7.1, which showed greater
potency than 7.5. Both polymers totally inhibited the growth of MRSA at 24 µM. 7.1
(12 µM) was found to exhibit a bactericidal rate of 100%. 7.1 is a more effective
inhibitor of MRSA than 7.5 (MIC_7.1 = <12µM; MIC_7.5 = <24 µM) (Figure 6).
This is in line with both their iron binding properties (Table 2) and their
hydrophilicities, 7.1 being more hydrophilic than 7.5 (Table 1). The lower Mn
counterparts of 7.1 and 7.5, namely 8.1 and 8.5, showed reduced bactericidal
influence (Figure 7 and Table 5). Indeed, inhibitory effects for such polymers were
only observed at concentrations above 48 µM. This suggests that the density of the
chelating units on the macromolecular scaffold determines the potency of the
compounds against MRSA. To further demonstrate that MSRA inhibition was derived
by an iron-chelating mechanism, the control polymer 7.5b was investigated and found
to be much less effective at inhibiting MSRA growth than 7.5 (Figure 7 and Table 5).
Polymer 7.5b lacks iron-chelating ability due to methylation of phenolic moieties
present on the HPO units. It is clear that the chelating properties of the active
polymers are of sufficient strength to be able to compete with the growing
microorganism for iron.
17

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MICs of 7.1, 7.5 and 7.5b on MRSA after 24 hour incubation under
aerobic conditions
10
9
8
7
6
5
4
3
2
1
0
7.1
7.5
7.5b
MRSA
without
polymer
6
8
12
16
24
32
48
64
96
126
Concentrations of polymers (µM)
Figure 6. Antimicrobial effect of various concentrations of 7.1, 7.5 and 7.5b on
MRSA after 24 hour incubation. Results are representatives of several independent
experiments performed in duplicate. Error bars represent standard error.
MICs of 8.5 and 8.1 on MRSA after 24 hour incubation under aerobic
10
9
8
7
6
5
4
3
2
1
0
conditions
8.5
8.1
MRSA
without
polymer
48
64
96
126
192
256
300
350
400
Concentrations of polymers (µM)
Figure 7. Antimicrobial effect of various concentrations of 8.5 and 8.1 on MRSA
after 24 hour incubation. Results are representatives of several independent
experiments, error bars represent standard error.
18

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Table 4. Concentrations of the polymers used in the course of the study1.
7.1
7.5
8.5
(Mn = 8900)
7.0
(Mn = 34,600)
8.1
(Mn = 50,100)
(Mn = 33,100)
(Mn = 13,400)
µM mg/mL µM mg/mL µM
mg/mL
0.427
0.570
0.854
1.121
1.709
2.278
2.670
3.115
3.560
µM
48
64
mg/mL
1.661
2.214
3.322
4.360
6.643
8.858
10.380
12.110
13.840
µM mg/mL
6
0.301
0.401
0.601
0.802
1.202
1.603
2.405
3.207
4.811
6
0.199
0.265
0.397
0.530
0.794
1.059
1.589
2.119
3.179
48
64
48
64
0.643
0.858
1.287
1.688
2.573
3.430
4.020
4.690
5.360
8
8
12
16
24
32
48
64
96
12
16
24
32
48
64
96
96
96
96
126
192
256
300
350
400
126
192
256
300
350
400
126
192
256
300
350
400
1 MICs are highlighted in yellow
Table 5. Bacterial inhibition rate of various concentrations of polymers on MRSA
after 24 hour incubation in an O2 incubator.
Bacterial
inhibition
rate after 24
hour
incubation
in an O2
incubator
(%)
Bacterial
inhibition
rate after 24
hour
incubation
in an O2
incubator
(%)
Bacterial
inhibition
rate after 24
hour
incubation in
an O2
Bacterial
inhibition
rate after
24 hour
incubation
in an O2
incubator
(%)
Bacterial
inhibition rate
after 24 hour
incubation in
an O2
7.1
(µM)
7.5
(µM)
8.5
(µM)
7.5b
(µM)
8.1
(µM)
incubator (%)
incubator
(%)
48
32
24
16
12
8
100
100
48
32
24
16
12
8
100
100
48
64
99.59942
99.85965
99.93199
99.98988
99.99648
99.99927
99.99967
99.99979
100
48
64
91.12392
99.89625
99.99356
100
48
64
99.85187
99.97349
99.99144
99.99954
100
100
100
96
96
96
100
99.99774
99.89395
99.31988
96.77233
-
126
192
256
300
350
400
126
192
256
300
350
400
126
192
256
300
350
400
100
100
99.99735
99.93545
-
100
100
6
6
100
100
-
-
100
100
-
-
-
-
100
100
4. Conclusions
RAFT polymerization of GMA in the presence of CPBD as the chain-transfer
agent was adopted for the synthesis of PGMA with well-defined properties and low
19

ACCEPTED MANUSCRIPT
polydispersities (PDI < 1.1). HPO-functionalized iron chelating homopolymers were
synthesized by ring-opening reaction of RAFT-based PGMA using HPOs with
various amine-substituents. The synthetic method is simple with high yield and low
cost, and thus the approach could be applied to the syntheses of other related types of
functional materials. The resulting materials possess different chelating capacity
depending upon the type and amount of incorporated HPO. Water soluble, insoluble
and/or fluorescent polymer-drug chimera are easily accessible using the described
technology. The homopolymers showed high affinity for iron(III) and some were
identified as new antimicrobial agents against MRSA. Polymer molecular weight and
HPO ligand-type are important features in the determination of the potency of t
macromoleculs for MRSA inhibition.
Supporting Information
Details of the synthesis and characterization of the HPO-ligands and iron-binding
polymers are provided in the Supporting Information.
Corresponding Authors
*
(V. A.) E-mail: vincenzo.abbate@kcl.ac.uk
* (G. Z) E-mail: gz6m@virginia.edu
Present Address
Institute of Pharmaceutical Science, King’s College London, 150 Stamford Street,
London SE1 9NH
Acknowledgement
The authors are grateful for financial support from National Natural Science
Foundation of China (NSFC, 21222405).
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Highlights
·HPO-functionalized iron chelating polymers were synthesized from RAFT-based PGMA.
·The synthetic method of the resulting materials is simple with high yield and low cost.
·Homopolymers were identified as new antimicrobial agents against MRSA.
·This versatile synthetic procedure can be adopted to prepare a wide range of polymeric chelators.