Cyclodextrin-modified polyesters from lactones and from bacteria: An approach to new drug carrier systems

Article abstract of DOI:10.1021/ma1027627

Copper(I)-catalyzed cycloaddition of synthetic and bacterial copolyesters bearing pendant alkyne group with mono-(6-azido-6-desoxy)-β-cyclodextrin was carried out to synthesize β-CD-functionalized copolyester. The synthetic clickable copolyesters were obtained by the ring-opening copolymerization of the propargyl-modified lactones and ε-caprolactone. The bacterial copolyesters containing an alkyne group were biosynthesized from a mixture of 10-undecynoic acid and hexanoic acid by the Gram-negative bacteria Pseudomonas oleovorans. The modified products of the click reaction were characterized by FT-IR, ¹H NMR spectroscopy, and DSC. Furthermore, the host guest capability of covalently attached β-cyclodextrin moieties was proved by dynamic light scattering measurements.

Full text of DOI:10.1021/ma1027627

ARTICLE
pubs.acs.org/Macromolecules
Cyclodextrin-Modified Polyesters from Lactones and from Bacteria:
An Approach to New Drug Carrier Systems
Olga Jazkewitsch, Adam Mondrzyk, Rebecca Staffel, and Helmut Ritter*
Institute of Organic Chemistry and Macromolecular Chemistry II, Heinrich-Heine-University Duesseldorf, Universitaetsstr. 1, 40225
Duesseldorf, Germany
S Supporting Information
b
ABSTRACT: Copper(I)-catalyzed cycloaddition of synthetic
and bacterial copolyesters bearing pendant alkyne group with
mono-(6-azido-6-desoxy)-β-cyclodextrin was carried out to
synthesize β-CD-functionalized copolyester. The synthetic
“clickable” copolyesters were obtained by the ring-opening
copolymerization of the propargyl-modified lactones and
ε-caprolactone. The bacterial copolyesters containing an alkyne group were biosynthesized from a mixture of 10-undecynoic acid
and hexanoic acid by the Gram-negative bacteria Pseudomonas oleovorans. The modified products of the “click” reaction were
1
characterized by FT-IR, H NMR spectroscopy, and DSC. Furthermore, the host guest capability of covalently attached
β-cyclodextrin moieties was proved by dynamic light scattering measurements.
’ INTRODUCTION
reported about the synthesis and “click” reactions of several
alkynes onto azide-functionalized PCL. PHAs were modified by
e.g. chlorination,22 epoxidation,23 grafting,24 and cross-
linking.22,25 Langlois and co-workers modified oligomers from
a PHA containing “clickable” end groups with azide-terminated
PEG.26
Polymer analogous reactions of suitable polyesters with β-
cyclodextrin (β-CD) derivatives lead to potential carrier systems
for various substances. Cyclodextrins are cyclic oligomers of D-
glucose units that are joined by 1,4-R-glucosidic linkages. CDs
have the shape of a hollow cone and are able to include small
hydrophobic, molecular guests into their cavity.27 In this manner
many hydrophobic substances become water-soluble, e.g., ada-
mantane and its monosubstituted derivatives.28 The pharmaceu-
tical industry uses cyclodextrins to expand the bioavailability of
various hydrophobic drugs.29
Aliphatic polyesters have deeply impacted biomedical and
engineering fields, such as tissue scaffolding and therapeutic
delivery.1,2 Poly(lactide) (PLA), poly(glucolide) (PGA), poly-
(ε-caprolactone), and their respective copolymers combine
biocompatibility and biodegradability which make them leading
candidates for resorbable implant materials, prosthetics, surgical
sutures, vascular grafts, bone screws, and erodible polymers for
drug delivery systems.3-6 Pseudomonas oleovorans bacteria are
able to produce poly(3-hydroxy alkanoate)s (PHAs) from
suitable carbon sources with more than five carbon atoms in
low nutrient media.7 For example, PHAs from n-alkanes, n-
alkanoic acids, n-alkynoic acids, and ω-phenoxy-substituted
alkanoic acids have been produced and characterized.8,9 The
obtained PHAs are medium-chain-length (mcl) elastomers with
a low degree of crystallinity and a low melting temperature.9 In
comparison to their chemically produced counterparts, PHAs
from bacteria are chiral.10-12
An apparent drawback of standard aliphatic polyesters is the
lack of functionalities on the polymer backbone which allow the
tailoring of polyester properties for the specific applications or
introduction of bioactive moieties. In recent years, “click chem-
istry” became a very important tool for the preparation of novel
polymeric structures. Since the first report of Sharpless,13 Cu(I)-
catalyzed Huisgen-type14,15 cycloaddition of azides and alkynes
has been applied for synthesis of polymers with different
architectures.16 The “click” reaction is an attractive method for
preparation of functionalized aliphatic polyesters due to the
relatively mild reaction conditions and tolerance of the presence
other functional groups. Emrick and co-workers17-19 grafted
polyesters with PEG, phosphorylcholine, and azide-functiona-
lized camptothecin derivates via “click” reaction. Riva et al.20,21
Up to now, linear biodegradable copolyesters bearing cova-
lently attached cyclodextrin units have not been described in the
literature. Thus, the aim of the present work is to describe linear
polyesters bearing alkyne functionalities and their “click”-type
modification with mono-(6-azido-6-desoxy)-β-cyclodextrin.
’ EXPERIMENTAL SECTION
Materials. β-Cyclodextrin was obtained from Wacker-Chemie
GmbH (Burghausen, Germany) and used after drying overnight in a
vacuum oil pump over P4O10. ε-Caprolactone (ε-CL) was purchased
from Acros, dried over calcium hydride, distilled under reduced pressure,
Received: December 3, 2010
Revised:
January 18, 2011
Published: February 21, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ma1027627 Macromolecules 2011, 44, 1365–1371
|

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ARTICLE
and stored over 0.4 nm molecular sieves under an argon atmosphere.
Cyclohexanone was purchased from Aldrich, dried over magnesium
sulfate, and distilled before use. Propargyl bromide (80 wt % solution in
toluene) and lithium N,N-diisopropylamide solution (2.0 M in THF/
heptanes/ethylbenzene) were purchased from Acros Organics and from
Sigma-Aldrich, respectively. m-Chloroperbenzoic acid (70-75%) was
purchased from Sigma-Aldrich and used as received. Adipic acid was
obtained from Aldrich and dried overnight in a vacuum oil pump over
P4O10. Sodium ascorbate (AppliChem), copper(II) sulfate (Carl Roth
GmbH), N,N0-dicyclohexylcarbodiimide (AppliChem), and 2,2-diphe-
nylethanamine (96%, Acros Organics) were used as received. 10-Unde-
cynoic acid was purchased from Alfa Aesar in 96% purity. Hexanoic acid
was obtained from Aldrich in 99.5% purity. Commercially available
reagents and solvents were used without further purification.
ethylbenzene, 60 mL, 120 mmol) was added to the round-bottom flask
and stirred for 30 min. An argon-purged solution of cyclohexanone
(120 mmol) in 3 mL of THF was added dropwise to the LDA (lithium
N,N-diisopropylamide) solution so that the temperature of solution did
not exceed -70 °C. Propargyl bromide (120 mmol) in 10 mL of THF
was also added dropwise by syringe and then stirred for an additional
60 min. The reaction mixture was then warmed up to room temperature
and stirred overnight. The reaction was quenched with excess of aqueous
ammonium chloride, washed twice with diethyl ether, dried over
MgSO4, and concentrated via a rotary evaporator. The following dis-
tillation under vacuum (T = 45 °C, p = 0.34 mbar) afforded 1 as a
colorless liquid (56% yield). 1H NMR (500 MHz, CDCl3, ppm): δ 2.56
(ddd, J = 17.1 Hz, 4.6 Hz, 2.7 Hz, 1H), 2.48-2.42 (m, 1H), 2.39-2.34
(m, 2H), 2.30-2.23 (m 1H), 2.13 (ddd, J = 17.0 Hz, 8.4 Hz, 2.6 Hz, 1H),
2.07-2.03 (m, 1H), 1,92 (t, J = 2.6 Hz, 1H), 1.89-1.86 (m, 1H), 1.70-
1.55 (m, 2H), 1.37 (ddd, J = 25.4 Hz, 12.7 Hz, 3.6 Hz, 1H). 13C NMR
(125 Hz, CDCl3, ppm): δ 210.9, 82.7, 69.5, 49.6, 42.05, 33.3, 27.9, 25.2,
18.9. GC/MS: m/z (%) = 137 [M þ H]þ, 136 [M]þ. FT-IR (diamond):
ν = 3290 (w, C-H, -CtCH), 2930 (m, CH), 2859 (m, C-H), 1703
In this study, stock cultures of P. oleovorans (ATCC 29347) were
used. The strain was purchased from LGC Standards GmbH and main-
tained at -70 °C as glycerine culture (500 μL of glycerine, 500 μL of
bacterial culture) after incubation. Cultivation of the microorganisms
took place in following mineral medium: (NH4)2HPO4 (1.1 g/L),
K2HPO4 (5.8 g/L), KH2PO4 (3.7 g/L), MgSO4 solution (0.1 mol/L,
10 mL), microelement solution (1 mL). The microelement solution was
a 1 M HCl solution containing FeSO4 7H2O (2.78 g/L), MnCl2 4H2O
(s, CdO), 1450, 1424, 1129 cm-1
.
Synthesis of 3-/7-(Prop-2-ynyl)oxepan-2-one (2a/b). 2-Prop-2-
ynyl-cyclohexanone (60 mmol) was added to a solution of m-chlor-
operoxybenzoic acid (90 mmol) in 160 mL of methylene chloride. The
reaction mixture was refluxed for 48 h. After cooling to room tempera-
ture and filtration, the solution was washed twice with aqueous sodium
sulfite and with aqueous sodium hydrogen solution. Subsequent removal
of the solvent under reduced pressure and distillation under oil pump
vacuum yielded 80% the product as isomeric mixture (3- (70%) and
3
3
(1.98 g/L), CoSO4 7H2O (2.81 g/L), CaCl2 2H2O (1.67 g/L),
3
3
CuCl2 2H2O (0.17 g/L), ZnSO4 7H2O (0.29 g/L). The carbon source
was added in a concentration 10 mmol/L. The cultivation was carried
out in vigorously stirred flasks with baffles.
3
3
Measurements. 1H NMR and 13C NMR spectroscopies were
performed using a Bruker Avance DRX 500 spectrometer at 500.13
MHz and at 125.77 MHz in DMSO-d6 and CDCl3 as solvent. Chemical
shifts were referenced to the solvent value δ = 2.5 ppm for DMSO-d6
and δ = 7.26 ppm for CDCl3. IR measurements were performed using a
FT-IR spectrometer (Nicolet 6700 FT-IR) equipped with an ATR unit.
Molecular weight distributions were measured by size exclusion chro-
matography (SEC) using a HEMA-5 μm column set consisting of a
precolumn of 4 nm and main column of 103, 102, and 10 nm. Tetra-
1
7-(prop-2-ynyl)oxepan-2-one (30%)). H NMR (500 MHz, CDCl3,
ppm): δ 4.35-4.16 (m, 3H), 2.77-2.72 (m, 1H), 2.66-2.52 (m, 5H),
2.43 (ddd, J = 16.7 Hz, 8.1 Hz, 2.7 Hz, 1H), 2.31 (ddd, J = 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, ppm): δ 176.03 (1A), 174.62
(1B), 82.40 (2A), 80.02 (2B), 77.68 (3B), 71.36 (4B), 70.21 (4A), 68.55
(5A), 42.05 (3A), 34.71 (5B), 33.58 (6B), 28.97 (6A), 28.86 (9A), 28.02
(7A), 27.84 (8B), 26.11 (7B), 22.93 (9B), 21.96 (8A). FT-IR
(diamond): ν = 3277 (w, -CtCH, C-H), 2933, 2859 (m, C-H),
hydrofuran was used as eluent at a flow rate of 1 mL min-1. For online
3
detection, a Waters 486 tunable absorbance detector (λ = 256 nm) and a
Waters 410 differential refractometer were used. The system was
calibrated with polystyrene standards with a molecular range from
580 Da to 1186 kDa. Differential scanning calorimetry (DSC) measure-
ments were performed using a Mettler Toledo DSC 822 controler
apparatus in a temperature range between -50 and 350 °C with a
1723 (s, CdO), 1443, 1289, 1174, 1051 cm-1
.
Copolymerization of 3-/7-(Prop-2-ynyl)oxepan-2-one (2a/b), ε-CL,
and Adipic Acid (3). 3-/7-(Prop-2-ynyl)oxepan-2-one (3.2 mmol),
ε-CL (22.9 mmol), and previously dried adipic acid (0.12 mmol) were
weighted in a round-bottom flask. After homogenization, 0.5 mol % of
Sn(Oct)2 was added under an atmosphere of dry argon. The flask was
immersed in oil bath at 130 °C for 24 h. The cold reaction product was
dissolved in chloroform, precipitated twice in a cold mixture of diethyl
heating rate of 10 °C min-1. The melting point (Tm) values reported
3
are taken from the third heating cycle. Dynamic light scattering
measurements were carried out at 25 °C using Malvern Zetasizer Nano
ZS90 instrumentation. The polymer concentration was ∼0.25 mg mL-1
.
1
3
ether/hexane (50:50), and dried in vacuum. The yield was 98%. H
The solutions were filtered through 0.45 μm Filtropur syringe filters
prior to measurements. The particle size distribution was derived from a
deconvolution of the measured intensity autocorrelation function of the
sample by the general purpose mode algorithm included in the DTS
software. Optical density (OD) measurements were carried out at λ =
660 nm using a Varian Cary 50 scan UV/vis spectrometer. Specific
rotations were measured by Perkin-Elmer Polarimeter 341 at a wave-
length of λ = 589 nm and a temperature of 20 °C. The specific rotation
RTλ can be calculated by general equation
NMR (500 MHz, CDCl3, ppm): δ 4.91 (m, 1H) 4.04 (t, J = 6.7 Hz, 2H),
2.44 (m, 2H), 2.28 (t, J = 7.5 Hz, 2H), 1.98 (s, 1H), 1.63 (m, 4H), 1.36
(m, 2H). Content of 3-/7-(prop-2-ynyl)oxepan-2-one (2a and 2b) in
copolymer 10 mol %. FT-IR (diamond): ν = 3309 cm-1, 3283 cm-1
(m, -CtCH, C-H), 3020, 2945, 2860 (m, C-H), 1726 (s, CdO),
1463 (m), 1157 (s, C-O), 753, 669 cm-1. SEC (THF): M = 22 880,
hn
PD = 1.3. DSC: Tm = 44 °C, Tdecomposition = 313 °C.
Reaction of Copolymer 3 with 2,2-Diphenylethanamine (4). A
solution of 1.034 g of copolymer and 88 mg of 2,2-diphenylethanamine
(10-fold excess relating to end groups of polyester) in 4 mL of methylene
chloride was cooled in a ice bath. After 46 mg of N,N-dicyclohexylcar-
bodiimide (5-fold excess relating to end groups of polyester) was added,
the reaction mixture was warmed up to room temperature and then
stirred for 24 h at 40 °C. The cold reaction product was filtered,
precipitated twice in a cold methanol, and dried in a vacuum. The yield of
copolyester 4 was 56%. 1H NMR (500 MHz, DMSO-d6, ppm): δ 7.27
(m, 8H), 7.16 (m, 2H), 4.81 (m, 1H), 4.71 (m, 1H), 3.98 (t, J = 6.4 Hz,
2H), 2.54-2.43 (m, 3H), 2.27 (t, J = 7.2 Hz, 2H), 1.54 (m, 4H), 1.29
R
βd
T
½Rꢀλ
¼
where T is temperature [K], λ the wavelength of irradiated light [nm], R
the optical rotation [deg], β the concentration [mg/mL], and d the
length of cell [dm].
Synthesis of 2-Prop-2-ynylcyclohexanone (1). A 250 mL round-
bottom flask was purged with argon and cooled in a dry ice/acetone
bath. A solution of lithium diisopropylamide (2.0 M in THF/heptane/
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(m, 2H). FT-IR (diamond): ν = 3271 (m, -CtCH, C-H), 2940, 2863
(m, C-H), 1723 (s, CdO), 1366, 1292, 1241, 1164, 1087, 958 cm-1
.
SEC (THF): M = 23 930. DSC: Tm = 42 °C, Tdecomposition = 320 °C.
hn
Synthesis of Cyclodextrin Containing Copolymer 6 via “Click”
Reaction. Mono-(6-azido-6-desoxy)-β-cyclodextrin (5-fold excess re-
lating to the alkine group of polyester) was added to a solution of 233 mg
of polyester 4 in 3.5 mL of DMF. To the clear solution were added
10 mol % sodium ascobate and 5 mol % copper(II) sulfate pentahydrate.
The flask was immersed in oil bath at 95 °C for 24 h. The cold reaction
product was precipitated in a mixture of acetone and distilled water (1:3)
and dried in a vacuum. 1H NMR (500 MHz, DMSO-d6, ppm): δ 7.76-
7.73 (m, 1H), 5.87-5.56 (m, 14H), 5.03-4.76 (m, 7H), 4.59-4.31 (m,
6H), 3.98 (t, J = 6.4 Hz, 2H), 3.64-3.56 (m, 28H), 2.27 (t, J = 7.1, 2H,),
1.53 (m, 4H), 1.27 (m, 2H). FT-IR (diamond): ν = 3364 (m, OH),
2933, 2863 cm-1 (m, C-H), 1726 cm-1 (s, CdO), 1649 (w, CdC),
1549, 1150, 1029, 849 cm-1. DSC: Tm = 303 °C.
Biosynthesis of PHA. First a small-scale culture in 500 mL Erlen-
meyer flasks was prepared with sodium octanoate as carbon source and
used as inoculum. The main cultures were cultivated in 5 L flasks with
2 L medium. As carbon source, 0.83 g of 10-undecynoic acid and 1.74 g
of hexanoic acid (totaling 10 mmol/L) were used, and 20 mL of
inoculum was added. The flasks were stirred at 150 rpm. A temperature
of 28 °C was maintained. The moment of maximum growth was verified
by OD660 measurement (OD660 at maximum growth = 1.45). After 48 h
the cells reached stationary phase and were harvested by centrifugation
and lyophilized to yield 7.45 g dry cell mass. Extraction of the polymer
was carried out in a Soxhlet extractor with chloroform as solvent for
6 h. The organic phase was concentrated to ca. 20 mL and precipitated in
250 mL of methanol. The PHA was gathered and dried in vacuo for 12 h.
The extraction yielded 2.5 wt % polymer of the dry cell mass. 1H NMR
(500 MHz, CDCl3, ppm): δ 0.89 (t, J = 6 Hz, 3H), 1.23-1.73 (m, 14H),
1.93 (m, 1H), 2.17 (m, 2H), 2.51 (m, 4H), 5.16 (m, 2H). 13C NMR
(125 MHz, CDCl3, ppm): δ = 14.03, 18.42, 18.54, 22.70, 24.28, 25.14,
28.20, 28.52, 28.75, 29.02, 31.70, 33.42, 33.97, 36.18, 39.23, 39.11, 68.50,
68.87, 70.80, 76.81, 77.23, 77.43, 77.65, 84.30, 84.76, 169.57, 169.63.
FT-IR (diamond): ν = 3298 (m, -CtCH, C-H), 2934 (m, C-H),
2863 (m, C-H), 2331 (w, -CtCH, CtC), 1731 (s, CdO). SEC
Figure 1. Synthesis of 3- and 7-(prop-2-ynyl)oxepan-2-one (2a/b) by
Baeyer-Villiger oxidation of 2-prop-2-ynyl-cyclohexanone (1).
benzoic acid yielded the lactone mixture 2a/b (Figure 1).
Analysis by 13C NMR has revealed that the reaction rendered
an isomeric mixture of 70% 3- (2b) and 30% 7-(prop-2-ynyl)-
oxepan-2-one (2a). For the ring-opening polymerization, the
mixture of isomers was used without separation.
In our previous work the copolymerization of lactones 2a/b
with ε-caprolactone and the coupling of cyclodextrin azide
afforded a supramolecular cross-linked polymer.30 It was found
that the hydrophobic cavity of the polyester attached cyclodex-
trin included the polyester chain as shown in Figure 2.
In respect of these former findings, the new approach of this
work was to suppress the formation of pseudo-polyrotaxane by
capping the copolyester chain with bulky end groups before
the polyester will be functionalized by “click” reaction with
β-CD-N3.
The copolymerization of ε-caprolactone and lactone mixture
2a/b was promoted by Sn(Oct)2 in the presence of adipic acid as
co-initiator and afforded a copolyester with terminal acid groups
(Figure 3).
1
The obtained copolyester 3 was characterized by H NMR,
FT-IR, DSC, and SEC. The molar content of 2a/b incorporated
1
into the copolymers 3 was calculated by integration of the H
NMR spectral signals at δ = 1.97 ppm (CtCH from monomers
2a/b) and at δ = 4.9 ppm (OCH(CH2CtCH)) of the polymer
backbone from 2b) compared to the signal at δ = 4.03 ppm
(OCH2 of the polymer backbone from 2a and ε-CL). The spec-
troscopically calculated incorporation ratio is about 90 mol %
ε-CL and 10 mol % of 2a/b and differs just a little from the feed
ratio (88 mol % ε-CL:12 mol % of 2a/b), indicating a nearly
complete consumption of lactone 2a/b during the polymeriza-
tion. The thermal properties of 3 indicate that the obtained
copolyester is a random copolymer. The signals from adipic acid
protons CdOCH2 and CdOCH2(CH2)2 overlapped with the
peaks at 2.28 ppm (CdOCH2 of the polymer backbone from 2b
and ε-CL) and at 1.63 ppm (CdOCH2(CH2)CH2(CH2) of the
polymer backbone from 2a/b and ε-CL), respectively. Because
of molar ratio of adipic acid to lactones (2a/b and ε-CL) of
1:217, the IR spectrum of 3 revealed only few weak stretches at
3309 and 3020 cm-1, indicating the presence of acid end groups.
Synthesis of End-Capped Polyester 4 by Amidation with
2,2-Diphenylethanamine. As mentioned above, the amidation
of acid end groups of the polyester 3 with 2,2-diphenylethana-
mine in the presence of DCC was carried out (Figure 3). We
calculated the amount of acid groups in consideration of the
(THF): M = 166 000, PD = 3.4. DSC: T = -24.7 °C.
hn
g
Modification of PHA 7 with Mono-(6-azido-6-desoxy)-β-cyclodex-
trin. In a 10 mL pear-shaped flask 103.4 mg of PHA 7 (ca. 0.37 mmol
related to triple bond) were dissolved in 4 mL of DMF under heating to
50 °C. Sodium ascorbate (6.6 mg, 0.03 mmol), copper(II) sulfate
pentahydrate (4.3 mg, 0.02 mmol), and mono-(6-azido-6-desoxy)-β-
cyclodextrin (567.0 mg, 0.49 mmol) were added to the solution. The
solution was stirred at 90 °C (oil bath temperature) for 48 h. It was then
cooled to RT and precipitated in aqueous NaCl solution (20%). The
solid was separated by centrifugation. Afterward, it was diluted in DMSO
and precipitated in distilled water. The reaction yielded 181.1 mg of
clicked polymer (87.6% of theoretical yield). 1H NMR (500 MHz,
DMSO-d6, ppm): δ 0.81 (t, J = 6 Hz, 3H), 1.10-1.78 (m, 24H), 2.09
(m, 2H), 2.27-2.73 (m, 9H) 3.09-3.41 (m, 14H), 3.41-3.79 (m,
28H), 4.35-4.55 (m, 6H), 4.65-4.86 (m, 7H), 4.94-5.14 (s, 3H),
5.54-5.85 (m, 14H), 7.68 (s, 1H). FT-IR (diamond): ν = 3329 (m,
OH), 2932, 2864 (m, C-H), 2117 (w, CtC), 1726 (s, CdO), 1650 (w,
CdC), 1026 (vs, C-N) cm-1, DSC: Tg1 = -30.4 °C; Tg2 = 153.5 °C.
’ RESULTS AND DISCUSSION
Preparation of Copolyester with Pendent Alkyne Group.
The alkyne-functionalized lactones 3- and 7-(prop-2-ynyl)-
oxepan-2-one (2a/b) were prepared in two steps. The first step
was the nucleophilic substitution reaction of cyclohexanone with
propargyl bromide to yield 2-prop-2-ynyl-cyclohexanone (1).
Baeyer-Villiger oxidation of 1 with excess of m-chloroperoxy-
number-average molecular weight M = 22 900 (determined by
hn
SEC) of the copolyester 3. The 10-fold excess of 2,2-dipheny-
lethanamine and 5-fold excess of DCC relating to end groups of
polyester were used to ensure that all acid moieties would be
reacted. The obtained reaction product was filtered and pre-
cipitated in cold methanol to remove the byproduct and the rest
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Figure 2. Reaction product of Huisgen-type 1,3-dipolar cycloaddition of propargyl-functionalized copolyester and mono(6-azido-6-desoxy)-
β-cyclodextrin.30
Figure 4. Schematic representation of synthesis of cyclodextrin-func-
tionalized copolyester 6 by Huisgen-type 1,3-dipolar cycloaddition.
aromatic protons and signals at δ = 4.9 ppm (OC(CH2Ct
CH)H of the polymer backbone from 2b) and at δ = 4.03 ppm
(OCH2 of the polymer backbone from 2a and ε-CL). The
calculated ratio of end groups to 2a/b and ε-CL is about 0.5
mol %:99.5 mol %. In consideration that the theoretical ratio
calculated from the batch of 3 is 0.45 mol %:99.55 mol %, it could
be assumed that the adipic acid was completely incorporated
during the ring-opening polymerization.
Polyester-graft-β-CD Prepared by “Click” Chemistry.
Mono-(6-azido-6-desoxy)-β-cyclodextrin (β-CD-N3) 5 was syn-
thesized according to a method described in the literature.31 The
“click” reaction of alkyne-functionalized copolyester 4 with
mono-(6-azido-6-desoxy)-β-cyclodextrin (5) was performed in
Figure 3. Synthesis of end-capped copolyester 4 by Sn(Oct)2-initiated
ROP of lactones 2a/b and ε-caprolactone and following amidation.
of the educts. It was found that the average molar mass (M ) of
hn
copolyester was slightly increased after the amidation from
22 900 up to 23 900 according to SEC. The 1H NMR spectrum
of the end-capped polyester (measured in DMSO-d6) showed
the proton signals at 7.27 and 7.16 ppm ascribed to the both
aromatic rings as well as the characteristic proton signals from the
copolyester backbone at δ = 3.98, 2.27, 1.54, and 1.29 ppm. The
amount of end groups was calculated by comparing the signals of
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2113 cm-1 a weak CtC peak is noticeable. The H NMR
spectrum of 7 shows a triplet signal at δ = 0.85 ppm which
originates from the methyl protons of the 3-hydroxyhexanoate
part of the polymer. The signals at δ = 1.89 ppm and δ = 2.12
ppm can be assigned to the protons at the triple bond. The molar
ratio of unsaturated to aliphatic building blocks has been
determined by comparison of these peaks, and it was calculated
to 1:0.6 (molar ratio). This means that about 65 mol % of the
repeating units contain triple bonds. The difference between the
initial molar ratio of 10-undecynoic acid and hexanoic acid (1:3)
and the found molar ratio of the repeating units in the polymer
can be explained by the bacterium preferring 10-undecynoic acid
over hexanoic acid. As previously reported bacterial polyesters
are copolymers even if only one carbon source has been fed.7,9
Typically, in the β-oxidation cycle of bacterial metabolism
degradation or elongation of side chains by two C units takes
place.11 Thus, PHA 7 is a complex copolymer, too. The 13C
NMR spectrum of the bacterial polyester shows signals of
3-hydroxy-10-undecynoate, 3-hydroxy-10-nonynoate, and 3-hy-
droxyhexanoate. The thermal properties of copolyester 7 indi-
cate that it is a random copolymer. To simplify schemes and
figures, all building blocks except 3-hydroxy-10-undecynoate and
3-hydroxyhexanoate have been omitted.
“Click” Reaction of Bacterial Poly(3-hydroxy-10-unde-
cynoate-co-3-hydroxyhexanoate) 7 with Mono-(6-azido-6-
desoxy)-β-cyclodextrin. The “click” reaction of copolyester 7
with mono-(6-azido-6-desoxy)-β-cyclodextrin (5) has been car-
ried out according to Scheme 1. The obtained product 8 is
soluble in DMSO, indicating linear structures without supramo-
lecular cross-linking as observed in case of the smaller propargyl-
modified poly(ε-caprolactone) (Figure 2). Thus, the longer side
chains act as barrier groups comparable with the diphenyl end
groups in polyester 4. It can be admitted that, comparing the IR
spectra of 5 and product 8, the azide peak at 2100 cm-1
disappeared after reaction. Further, the signal of the carbon-
nitrogen vibration appears at 1027 cm-1. The IR spectrum indi-
cates that the reaction was successful and proves that no remains
of 5 are found in the product.
1
Figure 5. DSC thermogram of the precursor copolyester 3 (—), end-
capped copolyester 4 (—), bacterial copolyester 7 ( ), and CD-
3 3 3
functionalized copolyester 6 (—) and 8 ( ). The heating rate was
3 3 3
10 °C min-1
.
DMF as a solvent by the addition of copper(II) sulfate and
sodium ascorbate as a catalyst system (Figure 4).
The isolated compound 6 was characterized by 1H NMR and
FT-IR spectroscopy. In contrast to the previously described
product without bulky end groups (Figure 2), the product 6
1
was soluble in DMSO. The H NMR spectrum confirmed the
proposed structure of product 6. The amount of CD in 6
1
calculated from H NMR data by comparing the signal of the
triazole proton (at 7.76 ppm) and the signals of polyester
backbone protons in the region of 1.5-2.5 ppm was about 9
mol %. Considering the fact that the amount of acetylene groups
of 10 mol % in copolyester 4 indicates that the “click” reaction
takes place in a yield of at least 90%. Furthermore, the success of
the functionalization of 4 with β-CD was also proven by the
disappearance of the characteristic azide vibration at 2100 cm-1
for mono-(6-azido-6-desoxy)-β-cyclodextrin 5 in the FT-IR
spectrum of the product 6. Instead, a new peak was observed
at 1647 cm-1 assigned to carbon-carbon double bond vibration,
which proved the existence of the triazole ring in association with
The 1H NMR spectrum of the product 8 (Figure 6) shows the
signal of the methyl protons of the saturated side chains. The
conversion of the reaction has been determined by comparison
of the peak at δ = 2.09 ppm and the peak at δ = 7.68 ppm which
are assigned to the methylene group in proximity to the triple
bond and the 1,2,3-triazole proton, respectively. The result is that
9.5 mol % cyclodextrin moieties are attached to the polymer.
The [R]2D0 values of mono-(6-azido-6-desoxy)-β-cyclodextrin
5 and polymers 7 and 8 were measured (Table 1). As expected,
the calculated specific rotation of a pure mixture of the educts
5 and 7 differs significantly from the measured specific rotation
of 8.
The DSC curve of 8 in Figure 5 shows two glass transition
temperatures at Tg1 = -30.4 °C and at Tg2 = 153.5 °C. Jasse and
Akc-atel showed that two or more glass transitions can occur in
polyesteramides if H-bridge bonds are formed.32 The reason for
this behavior is an increase in stiffness of the polymer chain
through H-bridge bonding. The modified polymer 8 has 9.5% of
cyclodextrin attached to it. The cyclodextrin groups form inter-
chain H-bridge bonds which are destroyed at Tg2.
the vibration of carbon-nitrogen bond at 1027 cm-1
.
The thermal behavior of polymers 3, 4, and 6 was determined
by DSC in the temperature range from -50 °C up to 350 °C
(Figure 5). The DSC graph shows that the melting point of
copolyester decreased from 44 °C down to 42 °C after the amida-
tion with 2,2-diphenylethanamine. This suggests that already a
small amount of the bulky end groups disrupt the formation of
crystalline domains. Thus, the incorporation of β-CD by “click”
reaction of polyester 4 changed significantly its thermal proper-
ties as expected. The DSC curve of 6 exhibits a melting point at
303 °C, which is closely followed by decomposition of the
polymer. The reason for the high melting point could be the
interchain formation of hydrogen bonds between OH groups of
cyclodextrins.
Poly[(3-hydroxy-10-undecynoate)-co-(3-hydroxyhexano-
ate)] from P. oleovorans. Pseudomonas oleovorans was grown
on a low nutrient medium and a 1:3 molar mixture of 10-
undecynoic acid and hexanoic acid. After harvest of cells poly(3-
hydroxy-10-undecynoate)-co-(3-hydroxyhexanoate) 7 has been
obtained with an number-average weight of 166 ꢁ 103 g/mol via
extraction with chloroform. The FT-IR spectrum of the copo-
lyester 7 shows the characteristic CdO peak at 1731 cm-1. At
Further, the host guest capability of β-CD-modified copolye-
sters 6 and 8 should be demonstrated by complexation with an
appropriate guest. In this respect a preliminary experiment
carried out showed that in DMSO sparingly soluble salts such
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ARTICLE
Figure 6. 1H NMR (500 MHz) spectrum of β-CD-modified bacterial copolyester 8 in DMSO-d6.
Scheme 1. Huisgen-Type “Click” Reaction of Poly[(3-hydroxy-10-undecynoate-)-co-(3-hydroxyhexanoate)] 7 with β-CD-N3 (5)
Table 1. Measurements of Optical Rotations of Substances
Mono-(6-azido-6-desoxy)-β-cyclodextrin (5), Copolyester 7,
and Copolyester 8
than the synthetic copolyester 6. In comparison to the copolye-
ster 6, the polyester 8 possesses long hydrophobic side chains
that lead to a greater entanglement of the polyester molecules in a
polar solvent such as DMSO.
By repeating the DLS measurement of polymers 6 and 8 in
the presence of adamantyl carboxylate as guest, the hydro-
dynamic diameter increased substantially in both cases. This
increase can be attributed to the electrostatic repulsion of
negatively charged carboxylate groups leading to chain exten-
sion. As expected, the hydrodynamic diameter of bac-
terial copolyester 8 increased stronger (from 8.7 nm up
compound
specific rotation [R]2D0 [deg L/dm g]a
3 3
5
132.4
2.8
7
8
47.3
a The error of the measurement is (0.2.
as potassium adamantyl carboxylate were dissolved by the
addition of solid β-CD. Thus, the size distributions of the
polymers 6 and 8 were investigated by DLS in presence of
adamantyl carboxylate (Figure 7). It was observed that the
bacterial copolyester 8 possess a smaller hydrodynamic diameter
to 227.8 nm) due to the greater molecular weight (M =
hn
170 000) compared to copolyester 6 (M = 23 900), which
hn
hydrodynamic diameter increased from 12.9 nm up to
83.4 nm.
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Macromolecules
ARTICLE
’ AUTHOR INFORMATION
Corresponding Author
*Fax þ49-211-8115840; e-mail h.ritter@uni-duesseldorf.de.
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3
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’ CONCLUSION
In summary, we described a new synthesis of β-CD-functio-
nalized synthetic and bacterial polyester with biodegradable
backbone. The synthesis of synthetic polyester relies on the
ring-opening polymerization of unknown to literature alkyne-
functionalized lactone followed by the Huisgen-type cycloaddi-
tion with mono-(6-azido-6-desoxy)-β-cyclodextrin. The end-
capping of the copolyester chain with bulky groups inhibits the
formation of pseudo-polyrotaxane and the consequent cross-
linking. Furthermore, the bacterial copolyester bearing alkyne
groups in the side chain was produced and also modified with β-
cyclodextrin azide by the “click” reaction. NMR and FT-IR
measurements confirmed the success of these reactions. The
ability of covalently attached cyclodextrins to include guest
molecules was proven by DLS measurements in the presence
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’ ACKNOWLEDGMENT
The authors gratefully acknowledge Prof. Dr. Lutz Schmitt
and his group of the Institute of Biochemistry I, Heinrich-Heine-
University Duesseldorf for the support in the microbiological
work.
’ ASSOCIATED CONTENT
S
Supporting Information. Figures 1-5. This material is
b
available free of charge via the Internet at http://pubs.acs.org.
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dx.doi.org/10.1021/ma1027627 |Macromolecules 2011, 44, 1365–1371