Antimicrobial Hydantoin-grafted Poly(ε-caprolactone) by Ring-opening Polymerization and Click Chemistry

Article abstract of DOI:10.1002/mabi.201200238

Novel degradable and antibacterial polycaprolactone-based polymers are reported in this work. The polyesters with pendent propargyl groups are successfully prepared by ring-opening polymerization and subsequently used to graft antibacterial hydantoin moieties via click chemistry by a copper(I)-catalyzed azide-alkyne cycloaddition reaction. The well-controlled chemical structures of the grafted copolymers and its precursors are verified by FT-IR spectroscopy, NMR spectroscopy, and GPC characterizations. According to the DSC and XRD results, the polymorphisms of these grafted copolymers are mostly changed from semicrystalline to amorphous depending on the amount of grafted hydantoin. Antibacterial assays are carried out with Bacillus subtilis and two strains of Escherichia coli and show fast antibacterial action.

Full text of DOI:10.1002/mabi.201200238

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Full Paper
Antimicrobial Hydantoin-grafted
Poly(e-caprolactone) by Ring-opening
Polymerization and Click Chemistrya
Licheng Tan, Samarendra Maji, Claudia Mattheis, Yiwang Chen,
Seema Agarwal*
Novel degradable and antibacterial polycaprolactone-based polymers are reported in this
work. The polyesters with pendent propargyl groups are successfully prepared by ring-
opening polymerization and subsequently used to graft antibacterial hydantoin moieties
via click chemistry by a copper(I)-catalyzed azide-
alkyne cycloaddition reaction. The well-controlled
chemical structures of the grafted copolymers and
its precursors are verified by FT-IR spectroscopy,
NMR spectroscopy, and GPC characterizations. Accord-
ing to the DSC and XRD results, the polymorphisms of
these grafted copolymers are mostly changed from
semicrystalline to amorphous depending on the
amount of grafted hydantoin. Antibacterial assays
are carried out with Bacillus subtilis and two strains
of Escherichia coli and show fast antibacterial action.
1. Introduction
including absorbable implant materials, biodegradable
surgical sutures, vascular grafts, bone screws, and carriers
in drug delivery.[1–6] Preparation of polyesters containing
pendent functional groups has attracted much attention in
recent years. Pendent functionalization of aliphatic poly-
esters can be achieved by polymerization of functionalized
lactones via ring-opening polymerization (ROP), post-
polymerization modification, or a combination of these
two approaches.[7] A series of polymers based on PCL
with pendent allyl,[8] cyclopentene,[9] propargyl,[10–12] and
hydroxyl,[13,14] have been prepared, which possess func-
tional groups for covalent substitution and grafting of
functional units. This is highly desirable for properties
modification and/or for additional functionalities in
polyesters.
Due to biodegradability, biocompatibility, as well as
excellent shaping and molding properties, aliphatic poly-
esters such as polylactide (PLA), poly(e-caprolactone) (PCL),
and their respective copolymers have been increasingly
used in biomedical and engineering fields in recent years,
Prof. S. Agarwal, L. Tan, S. Maji, C. Mattheis
Department of Chemistry and Center of Materials Science,
Philipps-Universit¨at Marburg, Hans-Meerwein-Straße, D-35042
Marburg, Germany
E-mail: agarwal@staff.uni-marburg.de
L. Tan, Prof. Y. Chen
Department of Chemistry, Nanchang University, 999 Xuefu
Avenue, Nanchang 330031, China
Recently, the ‘‘click’’ reaction of copper(I)-catalyzed
Huisgen 1,3-dipolar cycloaddition[15,16] between azide
and alkyne groups has been developed and represented
a Supporting Information for this article is available from the Wiley
Online Library or from the author.
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an attractive method for the preparation of clickable and
biodegradable polyesters for potential biomedical applica-
tions due to the relatively mild reaction conditions and
tolerance of other functional groups.[17–22] Such polyesters
were generally synthesized by ROP and subsequently
modified via a click reaction. For example, Suksiriworapong
et al. prepared poly(e-caprolactone)–poly(ethylene glycol)–
poly(e-caprolactone) with pendent azide groups on the
PCL backbone via ROP, and further click modified the
polyesters with nicotinic acid and p-aminobenzoic acid
(PABA) moieties containing alkyne groups.[23]
term stabilities, non-toxicity to humans, biocidal activity
against a broad range of microorganisms, and regenerable
properties upon exposure to household bleach.[27,28]
Both low and high molecular weight N-halamine-based
antibacterial materials are known in the literature. In
one of the approaches, either N-halamine containing
polymers or low molecular weight N-halamine derivatives
were added as additives to impregnate the respective
polymers in order to obtain antibacterial materials.[29–33]
The other recent approaches involve functionalization
by chemical reactions to introduce N-halamine structures
into the desired matrix polymers.[34–38] Worley and
coworkers have done extensive work on antibacterial
materials based on N-halamine. They have provided
N-halamine based new materials and many novel
concepts of attaching N-halamine moieties to various
polymers either by copolymerization or by polymer
analogous reactions and layer-by-layer process.[39]
For many application areas of biodegradable polymers
like sutures, implants, wound coatings, etc. on one side
and packaging, food storage, etc. on the other side, it is
reasonable to have antibacterial functionality besides
degradability. For such applications antibacterial func-
tionality could provide self-sterilizing articles and
packaging, etc. and complete elimination of bacterial
growth just by contact during the use period. For such
applications the antimicrobial moiety should be
active while attached to the polymer. The concept of
having a biodegradable antimicrobial polymer with
hydrolysable moiety together with antibacterial units
in its inactive form is reported in the literature for
the smart release of active ingredients on demand.
Such systems are important for targeted antibacterial
action. They make use of hydrolysable ester units either
as linker between the biocide and polymer backbone or
in the backbone attaching biocide units together.[24]
Poly(vinyl alcohol-peptide linker-gentamicin) is one
such polymer,[25] which has degradable peptide units
attaching biocide (gentamicin) to the polymer backbone.
The enzyme proteinases from wounds infected with
bacteria like Pseudomonas aeruginosa cleaved the linker
releasing the antibiotic. In these cases the biocides are
present in their inactive form in the polymers and
were released after degradation of either linker or
polymer backbone and brought biocides in the active
form. There is hardly any literature on dual functional
polyesters (hydrolysable and antibacterial) with inherent
antimicrobial functionality.
In this work we show the possibility of introducing
antibacterial functionality to polycaprolactone, a highly
studied biomaterial by a combination of ROP and click
chemistry. A series of novel antibacterial poly(e-capro-
lactone)-graft-hydantoin copolymers have been success-
fully prepared by a copper(I)-catalyzed click reaction
of azide-functionalized hydantoin with PCL bearing
pendent alkyne functionalities and characterized in terms
of structure, properties like thermal, mechanical, anti-
bacterial, and degradability.
2. Experimental Section
2.1. Materials
Cyclohexanone was purchased from Aldrich, dried over magne-
sium sulfate and distilled before use. e-Caprolactone (e-CL, Aldrich,
97%) was dried over calcium hydride, distilled under reduced
pressure and stored over molecular sieves (0.4 nm) under an
argon atmosphere. Stannous(II)octoate (tin bis-2-ethylhexanoate,
Aldrich, 95%) was degassed at high vacuum and used as received.
Propargyl bromide (80 wt.-% solution in toluene) and 5,5-
dimethylhydantoin (DMH) were purchased from Alfa Aesar Co.,
Ltd. 1-Bromo-2-chloroethane, lithium N,N-diisopropylamide (LDA)
solution (2.0 M in THF/heptanes/ethylbenzene), m-chloroperben-
zoic acid (70–75%), (þ)-sodium-L-ascorbate and copper(II)sulfate
were purchased from Sigma–Aldrich and used as received. 1,8-
Diazabicyclo[5.4.0]undec-7-ene (DBU) (98%), sodium azide (99%)
were purchased from Acros and the lipase of Pseudomonas cepacia
was acquired from Sigma–Aldrich. Tetrahydrofuran (THF) was
refluxed over a benzophenone-sodium mixture to remove water
and distilled before use. N,N-Dimethylformamide (DMF) was
distilled from calcium hydride under reduced pressure. Dichloro-
methane (DCM) was dried over calcium chloride and stored in
presence of molecular sieves (0.4 nm). All other chemicals and
solvents of analytical grade were used as received without
further purification.
Recently, we have made a dual functional antimicrobial
biodegradable polyester using N-halamine as the biocidal
unit. For the first time, polyesters of dimethyl succinate
(DMS), 1,4-butanediol (BDO), and 3-(N,N-di-b-hydroxyethyl-
aminoethyl)-5,5-dimethylhydantoin (H-diol) in varied
molar ratios were made via a two-step melt polycondensa-
tion reaction. 3-(N,N-di-b-hydroxyethylaminoethyl)-5,5-
dimethylhydantoin (H-diol) was used as antibacterial
comonomer. The resulting polyesters were biocompatible
as determined by cell viability studies and showed an
antibacterial activity against Escherichia coli.[26]
N-Halamines are compounds containing one or more
covalent nitrogen-halogen bonds, which exhibit long-
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2.2. Methods
2.3.2. Synthesis of 3-/7-(Prop-2-ynyl)oxepan-2-one
FT-IR spectra were recorded on a Digilab Excalibur Series system
using a Pike Miracle attenuated total reflection (ATR) unit.
1H (300 MHz) and 13C (75 MHz) NMR spectra were recorded on a
Bruker DRX-300 spectrometer using deuterated chloroform (CDCl3)
as solvent. 1H–13C heteronuclear multiple quantum correlation
(HMQC) experiments were performed on a Bruker DRX-500
spectrometer, with a 5 mm multinuclear gradient probe and using
gs-HMQCpulsesequences.Molecularweightsofthepolymerswere
determined by GPC using a Knauer system equipped with one PSS-
SDV (linear XL, 5 mm, 8.0 mm ꢀ 300 mm) column, a differential
refractive index detector, CHCl3 as eluent at a flow rate of
0.5 mL ꢁ minꢂ1. Thermogravimetric analysis (TGA) was performed
on a TGA/SDTA 851e (Mettler Toledo) at a heating rate of
10 8C ꢁ minꢂ1 under nitrogen from 25–800 8C with a sample size
of8–10 mg. Differentialscanningcalorimetry(DSC)measurements
of polymers were carried out on a DSC 821e unit (Mettler Toledo)
under a nitrogen flow at a rate 80 mL ꢁ minꢂ1. About 7 mg sample
were encapsulated in the DSC aluminum pan, heated quickly to
150 8C and held for 5 min to erase the thermal history. Then, the
samples were cooled to ꢂ100 8C and subsequently heated to 150 8C
at a rate of 5 8C ꢁ minꢂ1. The corresponding glass transition
temperature (Tg), melting temperature (Tm), crystallization tem-
perature (Tc), melting enthalpy (DHm) and crystallization enthalpy
(DHc) were recorded, respectively. X-ray diffraction patterns of the
samples were recorded with a Siemens goniometer D5000 using
2-Prop-2-ynyl-cyclohexanone (60 mmol) was added to a solution of
m-chloroperbenzoic acid (90 mmol, 20.18 g) in 160 mL dichloro-
methane and then refluxed for 48 h. After cooling to room
temperature and filtration, the solution was washed twice with
aqueous sodium sulfite and aqueous sodium hydrogen solutions,
the solvent was evaporated. Subsequent distillation under oil
pump vacuum yielded 64% of the product as an isomeric mixture
(3-(70%) and 7-(prop-2-ynyl)oxepan-2-one (30%)), named as alkyne
CL. 1HNMR(300 MHz,CDCl3):d(ppm) ¼ 4.35–4.16(m, 3H), 2.77–2.72
(m, 1H), 2.66–2.52 (m, 5H), 2.43 (ddd, J ¼ 16.7 Hz, 8.1 Hz, 2.7 Hz, 1H),
2.31 (ddd, J ¼ 17.1 Hz, 9.2 Hz, 2.6 Hz, 1H), 2.18–2.06 (m, 2H), 2.00–
1.88 (m, 5H), 1.72–1.37(m, 6H). 13C NMR (125 Hz, CDCl3):
d(ppm) ¼ 176.0, 174.6, 82.0, 79.5, 78.2, 71.4, 70.8, 68.6, 42.5, 34.8,
33.4, 28.9, 28.8, 28.3, 28.1, 26.1, 22.9, 21.9.
2.3.3. Copolymerization of Alkyne CL and e-CL via
Ring-opening Polymerization
The mixture of 3-/7-(prop-2-ynyl)oxepan-2-one (alkyne CL), e-CL
(ratio 1:9, 1.5:8.5, 2:8) and 0.5 mol-% of Sn(Oct)2 as catalyst were
placed in a Schlenk flask purged with argon for 10 min. Then, the
polymerization was carried out in an oil bath at 100 8C for 24 h
under continuous stirring. After cooling to room temperature,
the solution was dissolved in dichloromethane, precipitated in
cold methanol and dried in vacuum.
˚
Cu Ka-radiation of l ¼ 1.54 A. The crystal morphology of the
2.3.4. Synthesis of 3-(20-Chloroethyl)-5,5-
dimethylhydantoin (DMH-Cl)
polymers was analyzed by a Leica DMRX polarized optical
microscope (POM) fitted with
a Leica DC200 camera at a
magnification of 1:100. Mechanical testing was done using an
Instron testing machine (model 4411) using dumbbell-shaped
samples (35 mm length, 2 mm width). Films with a thickness of
d ¼ 0.08–0.1 mm were prepared from chloroform solution.
5,5-Dimethylhydantoin (DMH) (96.1 g, 0.75 mol) was dissolved in a
solution of 42.1 g (0.75 mol) KOH in 500 mL ethanol. 1-Bromo-2-
chloroethane (215.1 g, 1.5 mol) was added in one portion to the
solution and then refluxed at 80 8C for 8 h. Evaporation of the
reaction mixture resulted in a white solid which was shaken with
ethyl acetate and water in a separatory funnel. The organic layer
wasseparated, washed(10%aqueousNaHCO3), dried (Na2SO4), and
evaporatedtoisolatetheproduct.Recrystallizationfromtoluene/2-
propanol (v/v ¼ 1:9) resulted in white crystals with a yield of 92%.
This compound was identified as 3-(20-chloroethyl)-5,5-dimethyl-
hydantoin. 1H NMR (300 MHz, CDCl3): d(ppm) ¼ 1.46 (6H, CH3), 3.75
(2H, CH2), 3.84 (2H, CH2), 6.61 (1H, NꢂH).
2.3. Synthetic Procedures
2.3.1. Synthesis of 2-Prop-2-ynyl-cyclohexanone
A solution of lithium diisopropylamide (LDA, 120 mmol) was
added to a 250 mL round-bottom flask purged with argon, cooled in
a dry ice/acetone bath, and stirred for 30 min. An argon-purged
solution of cyclohexanone (120 mmol, 11.78 g) in 3 mL THF was
added dropwise to the LDA solution at ꢂ70 8C. Propargyl bromide
(120 mmol, 14.28 g) in 10 mL THF was also added dropwise by a
syringe and the solution was stirred for additional 60 min. The
reaction mixture was then warmed up to room temperature and
stirred overnight. Excess aqueous ammonium chloride was added,
and the reaction mixture was washed twice with diethyl ether,
dried over MgSO4 and concentrated. The following distillation
under reduced pressure resulted in a colorless, viscous liquid
(61% yield). 1H NMR (300 MHz, CDCl3): d(ppm) ¼ 2.56 (ddd,
J ¼ 17.5 Hz, 4.8 Hz, 2.7 Hz, 1H), 2.48–2.42 (m, 1H), 2.39–2.34 (m,
2H), 2.30–2.21 (m, 1H), 2.14 (ddd, J ¼ 16.8 Hz, 8.7 Hz, 2.7 Hz, 1H),
2.07–2.03 (m, 1H), 1.92 (t, J ¼ 2.7 Hz, 1H), 1.89–1.86 (m, 1H), 1.70–
1.55 (m, 2H), 1.38 (ddd, J ¼ 25.8 Hz, 12.3 Hz, 3.6 Hz, 1H). 13C NMR
(75 Hz, CDCl3): d(ppm) ¼ 210.5, 82.6, 69.3, 49.4, 42.1, 33.4, 27.9,
25.2, 18.7.
2.3.5. Synthesis of 3-(20-Azido-ethyl)-5,5-
dimethylhydantoin (DMH-N3)
3-(20-Chloroethyl)-5,5-dimethylhydantoin (1.90 g, 10mmol) was
initiallydissolvedin6 mLDMFbeforesodiumazide(0.91 g,14mmol)
in 3 mL distilled water was added dropwise with a syringe. The
reaction mixture was stirred under argon atmosphere at 808C for
48h. After removal of DMF under reduced pressure, the residue was
dissolved in diethyl ether and washed with distilled water three
times before drying over anhydrous MgSO4. The product was
concentrated by rotary evaporation and obtained in a yield of 93%.
2.3.6. Synthesis of Poly(e-caprolactone)-graft-Hydantoin
Polymers by Click Chemistry
DMH-N3 (1.3 equiv. compared to the alkyne group of alkyne PCL)
was added to a solution of 400 mg alkyne PCL in DMF (4 mL) in a
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flask with a magnetic stirring bar, and the solution was bubbled
with argon for 30 min. 5 mol-% CuSO4 ꢁ 5H2O and 10 mol-% sodium
ascorbate (NaAsc) was individually dissolved in 3 mL of water,
followed by bubbling with argon for 10 min. After dropping
aqueousCuSO4 andNaAscsolutionsseparatelyinsequenceintothe
Schlenk flask with a syringe under vigorous stirring at room
temperature, 10 mol-% DBU was added. The reactive mixture was
degassed by three freeze–pump–thaw cycles, stirred at 35 8C
for 24 h, and then exposed to air. After that, the solution was
passed through a neutral aluminum oxide column to remove the
copper catalyst and evaporated. The final product was dried in a
vacuum oven at room temperature.
extract (5 g ꢁ Lꢂ1 peptone and 3 g ꢁ Lꢂ1 meat extract in distilled
water) for B. subtilis. To evaluate the antibacterial properties of the
grafted polymers, films exhibiting
a size of approximately
5 mm ꢀ 5 mm were inoculated with 10 mL inoculum and covered
with a second film each. After given time intervals (5, 15, and
30 min), these ‘‘sandwiches’’ were vigorously washed by immer-
sionin10 mLofa0.03%sodiumthiosulfatesolutioninsterilewater.
A 100 mL aliquot of each solution was then serially diluted with
sterile potassium buffer solution (pH 7.4), 100 mL of each dilution
were spread on nutrient agar plates. By counting of the formed
coloniesafterincubationat37 8Cfor24 h, thenumberofviablecells
in the solutions was determined and the reduction was calculated
respective to the inoculum in the appropriate dilution stage.
2.4. Enzymatic Degradation
3. Results and Discussion
To investigate the enzymatic degradation, polymer films
(10 mm ꢀ 10 mm ꢀ 0.08 mm), cast from chloroform solutions, were
placed in vials containing 10 mL of phosphate buffer solution
(Na2HPO4/KH2PO4, pH 7.0 at 25 8C) with 0.20 mg ꢁ mLꢂ1 Pseudo-
monas cepacia lipase at a constant temperature of 37 8C. At
predetermined degradation time intervals, the specimens were
removedfromthemedium, rinsedwithdistilledwater, dried under
vacuum at room temperature for 48 h and weighed. Weight loss
percentages of the copolyesters were obtained according to the
relationship:
3.1. Synthesis of Poly(e-caprolactone)-graft-
Hydantoin Polymers by Ring-opening Polymerization
and Click Chemistry
By following a procedure of the known approach by
Ritter and coworkers,[6] the functionalized lactone with
propargyl groups was prepared in two steps. Firstly, 2-prop-
Weight loss ð%Þ
¼ ðW0 ꢂ WrÞ=W0 ꢀ 100%
(1)
where W0 is the initial weight, and Wr is the
dry weight of the specimens after degrada-
tion.
2.5. Antibacterial Test
To obtain inocula for the antibacterial tests, a
single colony of the respective test organism,
preserved on tryptic soy agar at 4 8C, was
transferred to the corresponding liquid nutri-
ent with an inoculation loop. The inoculated
broth was incubated with shaking at 37 8C
until the optical density at 578 nm had
increased by 0.125, indicating a density of
viablecellsof107–108 cfu ꢁ mLꢂ1,anddilutedto
an approximate concentration of 106–107
cfu ꢁ mLꢂ1 to result in the final inoculum; the
exact number of colony forming units per mL
was determined subsequently by the spread
plate method. E. coli (DSM No. 1077, K12 strain
343/113,DSZMBraunschweig),E.coli(DSMNo.
1058, ML3 strain, ATCC 15223, DSMZ), and
Bacillus subtilis (B. subtilis, DSM No. 1088, IFO
13169, DSZM) were employed as test organ-
isms; tryptic soy broth (TSB, Sigma–Aldrich;
c ¼ 30 g ꢁ Lꢂ1 in distilled water) was used as
nutrient for E. coli strains and peptone/meat
Scheme 1. Preparation of side-chain hydantoin-containing PCL by ROP and click
chemistry.
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