Development of a new azido-oxazoline monomer for the preparation of amphiphilic graft copolymers by combination of cationic ring-opening polymerization and click chemistry

Article abstract of DOI:10.1016/j.reactfunctpolym.2013.04.013

A new monomer (2-(5-azidopentyl)-2-oxazoline) bearing an azido group was synthesized. The cationic ring-opening copolymerization of this monomer with 2-methyl-2-oxazoline resulted in a well-defined linear polymer backbone with pendant azido groups. Alkynyl-poly(D,L-lactide) was grafted onto the azido groups of poly(oxazoline) via a Huisgen 1,3-dipolar cycloaddition reaction to give a novel amphiphilic graft copolymer [poly(2-methyl-2-oxazoline-co-2-pentyl- 2-oxazoline)-g-poly(D,L-lactide)] (P[(MeOx-co-PentOx)-g-LA]). Different graft copolymers were prepared with PLA of different lengths. Preliminary results of the self-association of this copolymer in water indicated the formation of nanoparticles, which suggests this copolymer may have applications as vehicles for drug delivery.

Full text of DOI:10.1016/j.reactfunctpolym.2013.04.013

Reactive & Functional Polymers 73 (2013) 1001–1008
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Reactive & Functional Polymers
journal homepage: www.elsevier.com/locate/react
Development of a new azido-oxazoline monomer for the preparation
of amphiphilic graft copolymers by combination of cationic ring-opening
polymerization and click chemistry
⇑
Thanh-Xuan Lav, Pierre Lemechko, Estelle Renard, Catherine Amiel, Valérie Langlois, Gisèle Volet
Systèmes Polymères Complexes, ICMPE, UMR 7182, CNRS, 2, rue Henri Dunant, 94320 Thiais, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A new monomer (2-(5-azidopentyl)-2-oxazoline) bearing an azido group was synthesized. The cationic
ring-opening copolymerization of this monomer with 2-methyl-2-oxazoline resulted in a well-defined
Received 22 February 2013
Received in revised form 25 April 2013
Accepted 28 April 2013
linear polymer backbone with pendant azido groups. Alkynyl-poly(
groups of poly(oxazoline) via a Huisgen 1,3-dipolar cycloaddition reaction to give a novel amphiphilic
graft copolymer [poly(2-methyl-2-oxazoline-co-2-pentyl-2-oxazoline)-g-poly( -lactide)] (P[(MeOx-co-
D,L-lactide) was grafted onto the azido
Available online 9 May 2013
D,L
PentOx)-g-LA]). Different graft copolymers were prepared with PLA of different lengths. Preliminary
results of the self-association of this copolymer in water indicated the formation of nanoparticles, which
suggests this copolymer may have applications as vehicles for drug delivery.
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Poly(2-methyl-2-oxazoline)
Cationic ring-opening polymerization
Huisgen 1,3-dipolar cycloaddition
Click chemistry
Graft copolymers
Poly(D,L-lactide)
1. Introduction
grafting-through macromonomer method [24]. The grafting-from
approach has been used to prepare graft copolymers of poly(2-
Amphiphilic block or graft copolymers that consist of hydro-
philic and hydrophobic segments are attracting increasing interest
because original nanostructures are generated by the self-associa-
tion of these compounds in aqueous solution or at interfaces
[1–11]. Poly(2-methyl-2-oxazoline) (PMeOx) is an important
hydrophilic and nonionic polymer that exhibits biocompatibility
and a lack of toxicity, as evidenced by previous studies related to
oxazoline)s (2-methyl-, 2-ethyl- and 2-isopropenyl-2-oxazoline)
from the dual-functional monomer (2-isopropenyl-2-oxazoline)
[25]. In all of these works, the graft copolymers contained a hydro-
phobic polymer backbone bearing poly(2-oxazoline) grafts.
The objective of this work was to prepare a new form of graft
copolymer architecture composed of a POx backbone and hydro-
phobic PLA grafts. Such an architecture should exhibit interesting
properties as potential nonpolar drug carriers, and no such
architecture has yet been reported in the literature. The novel
amphiphilic graft copolymer [poly(2-methyl-2-oxazoline-co-2-
its biomedical applications [12–16]. Also, poly(D,L-lactide) (PLA) is
an important aliphatic polyester that has been extensively studied
for surgical and medical applications [17,18] because of its biode-
gradability and hydrophobicity. A large number of synthetic
copolymers for biomedical application are composed of either
poly(2-oxazoline) (POx) or PLA blocks. Diblock [19] and triblock
[20–22] copolymers based on poly(2-ethyl-2-oxazoline) and
poly(lactide) have been prepared. To our knowledge, few reports
that describe the synthesis of graft copolymers based on poly(2-
oxazoline)s have appeared in the literature. Guillerm et al. [23]
have developed the grafting-onto technique for the synthesis of
amphiphilic copolymers with pendant poly(2-methyl-2-oxazoline)
pentyl-2-oxazoline)-g-poly(D,L-lactide)] (P[(MeOx-co-PentOx)-g-
LA]) was prepared using the grafting-onto approach through
copper-catalyzed azide–alkyne cycloaddition (CuAAC) [26,27]. This
approach first involved the synthesis of a new monomer: 2-(5-
azidopentyl)-2-oxazoline. This monomer was copolymerized by
cationic ring-opening polymerization with 2-methyl-2-oxazoline
to give azido-functionalized copolymers poly(2-methyl-2-oxazo-
line-co-2-(5-azidopentyl)-2-oxazoline)
(P(MeOx-co-N3PentOx).
Second, alkyne end-functionalized poly(D,L-lactide) (alkynyl-PLA)
arms on a polyester backbone (poly-
ethyl-2-oxazoline)methacrylate]s were synthesized using the
e
-caprolactone). Poly[oligo(2-
was prepared in a one-step reaction via the transesterification
from high-molecular-weight PLA with propargyl alcohol using
dibutyltin dilaurate as a catalyst. Finally, we prepared different
graft copolymers by coupling the alkynyl-PLA onto the
copolymer-bound azides to form 1,2,3-triazoles via the Huisgen
⇑
Corresponding author. Tel.: +33 1 49 78 12 35; fax: +33 1 49 78 12 08.
E-mail address: volet@icmpe.cnrs.fr (G. Volet).
1381-5148/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.reactfunctpolym.2013.04.013

1002
T.-X. Lav et al. / Reactive & Functional Polymers 73 (2013) 1001–1008
1,3-dipolar cycloaddition reaction [26,27] and conducted a preli-
minary study of the self-association of this copolymer by dynamic
light scattering experiments.
(m, 2H, N3CH2CH2), 1.61 (m, 2H, CH2CH2COO), 1.47 (m, 2H, CH2-
CH2CH2CH2CH2) ppm.
13C NMR (100 MHz, CDCl3): d = 169.15 (NC@O), 168.39 (OC@O),
51.10 (N3CH2), 30.79 (N3CH2CH2CH2CH2CH2), 28.39 (N3CH2CH2),
25.88 (N3CH2CH2CH2), 25.59 (O@CCH2CH2C@O), 24.14 (N3CH2CH2-
CH2CH2) ppm.
2. Experimental section
FTIR (ATR mode): N@N@N 2091 cmꢁ1
.
2.1. Materials
2.2.3. Third step: Synthesis of N-(2-chloroethyl)-6-azidohexanamide
(C)
6-Bromohexanoic acid (purity 97%), sodium azide (BioUltra,
purity P 99.5%), N-hydroxysuccinimide (NHS, purity 98%),
N-(3-dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride
(EDC, commercial grade), 2-chloroethylamine hydrochloride (pur-
ity 99%), iodobutane (99%), anhydrous methanol, dichloromethane,
acetonitrile (99.8%), 1-iodobutane (purity 99%), copper(I) iodide
To a flask containing 13.0 g (51.1 mmol) of N-succinimidyl-6-
azidohexanoate (B) and 5.93 g (51.1 mmol) of 2-chloroethylamine
hydrochloride, 150 mL of dry dichloromethane was added. The
mixture was ice-cooled, and 18 mL (129 mmol) of triethylamine
was added dropwise. After the mixture was stirred for 30 min at
0 °C, it was allowed to equilibrate to room temperature and was
stirred an additional 72 h. Fifty milliliters of water was added,
and the organic layer was removed and extracted four times with
50 mL of water. The combined organic layers were dried over
MgSO4, and the solvent was evaporated. The product was dried un-
der vacuum (9.1 g, yield: 81%).
(purity
99.5%),
N,N,N0,N00,N00-pentamethyldiethylenetriamine
(PMDETA, purity 99%), propargyl alcohol (purity 99%) and dibutyl-
tin dilaurate (DBTDL, 95%) were purchased from Aldrich (Milwau-
kee WI, USA) and were used as received. Dimethylsulfoxide
(DMSO), diethyl ether and dichloromethane were purchased from
Carlo Erba (Val de Reuil, France) and were used without further
purification. Triethylamine was purified by distillation. Potassium
hydroxide from VWR (Fontenay-sous-bois, France) was dried un-
der vacuum at 40 °C before use. The monomer 2-methyl-2-oxazo-
line (Aldrich, Milwaukee WI, USA, purity 99%) was dried overnight
over calcium hydride and purified by distillation under a nitrogen
1H NMR (400 MHz, CDCl3): d = 5.86 (s, broad, 1H, NH), 3.60 (m,
4H, CH2CH2N), 3.25 (t, J = 8 Hz, 2H, N3CH2), 2.20 (t, J = 8 Hz, 2H,
CH2CON), 1.78–1.60 (m, 4H, N3CH2CH2CH2CH2), 1.41 (m, 2H, N3-
CH2CH2CH2) ppm.
13C NMR (100 MHz, CDCl3): d = 172.82 (C@O), 51.23 (N3CH2),
44.24 (CH2Cl), 41.13 (NHCH2), 36.34 (N3CH2CH2CH2CH2CH2),
28.61 (N3CH2CH2), 26.32 (N3CH2CH2CH2), 25.05 (N3CH2CH2CH2-
CH2) ppm.
atmosphere. The high molecular weight poly(D,L-lactide)
(Mn = 8.72 ꢀ 104 g molꢁ1, Mw/Mn = 2) was obtained from Cargill
Laboratories (Cerdar Grove, NJ).
FTIR (ATR mode): N@N@N 2091 cmꢁ1
.
2.2. Synthesis of 2-(5-azidopentyl)-2-oxazoline
2.2.4. Fourth step: Synthesis of 2-(5-azidopentyl)-2-oxazoline (D)
We prepared a solution of KOH in MeOH under nitrogen by dis-
solving 2.33 g of KOH into 20 mL of dry MeOH. N-(2-Chloroethyl)-
6-azidohexanamide (C) (9.1 g, 41.6 mmol) was dissolved in dry
MeOH, and the KOH/MeOH solution was added dropwise to the
N-(2-chloroethyl)-6-azidohexanamide solution. The reaction mix-
ture was stirred at 50 °C under a nitrogen atmosphere for 72 h.
The salt was removed by filtration, and the solvent was evaporated.
Diethyl ether was added to dissolve the final product and to allow
filtration of the salt. After the solvent was removed, the colorless
liquid was dried under vacuum (6.61 g, yield: 87%).
The synthesis of 2-(5-azidopentyl)-2-oxazoline involved four
steps starting from 6-bromohexanoic acid.
2.2.1. First step: Synthesis of 6-azidohexanoic acid (A)
6-Bromohexanoic acid (25.0 g, 128.2 mmol) and sodium azide
(41.7 g, 641.4 mmol) were dissolved in 250 mL of DMSO and stir-
red at 60 °C for 18 h. Then, 100 mL of distilled water was added
to the reaction mixture. After the solution was transferred into a
decantation funnel, 100 mL of distilled water and 75 mL of dichlo-
romethane were added. The organic layer was removed, extracted
four times with 75 mL of water, and then dried over magnesium
sulfate. The solvent was removed by rotary evaporation. The
slightly yellow product was dried under vacuum (17.5 g, yield:
87%).
1H NMR (400 MHz, CDCl3): d = 4.20 (t, J = 10 Hz, 2H, OCH2CH2-
N), 3.80 (t, J = 10 Hz, 2H, OCH2CH2N), 3.25 (t, J = 8 Hz, 2H, N3CH2),
2.27 (t, J = 8 Hz, 2H, CH2CON), 1.65-1.60 (m, 4H, N3CH2CH2CH2-
CH2), 1.41 (m, 2H, N3CH2CH2CH2) ppm.
1H NMR (400 MHz, CDCl3): d = 11.11 (s, broad, 1H, COOH), 3.25
(t, J = 8 Hz, 2H, N3CH2), 2.33 (t, J = 8 Hz, 2H, CH2COOH), 1.61 (m, 4H,
CH2CH2CH2CH2CH2), 1.40 (m, 2H, CH2CH2CH2CH2CH2) ppm.
13C NMR (100 MHz, CDCl3): d = 179.06 (C@O), 51.17 (N3CH2),
33.82 (CH2COOH), 28.51 (N3CH2CH2), 26.13 (N3CH2CH2CH2),
24.16 (CH2CH2COOH) ppm.
13C NMR (100 MHz, CDCl3): d = 168.24 (OC@N), 67.21 (OCH2-
CH2N), 54.37 (OCH2CH2N), 51.25 (N3CH2), 28.55 (N3CH2CH2),
27.77 (N3CH2CH2CH2CH2CH2), 26.30 (N3CH2CH2CH2), 25.47 (N3-
CH2CH2CH2CH2) ppm.
FTIR (ATR mode): CAH 2938, 2879 cmꢁ1; N@N@N 2091 cmꢁ1
;
C@N 1666 cmꢁ1; CAN 1456 cmꢁ1; CAO 1240 cmꢁ1
.
FTIR (ATR mode): N@N@N 2091 cmꢁ1
.
Mass spectrometry, m/z (relative intensity): 183 ([MAH]+,
3.3%); 140 (N@+CA(CH2)5AN3, 53.7%); 112 (+CH2A(CH2)4AN3,
9.5%); 98 (+CH2A(CH2)3AN3, 100%); 85 (+CH2ACO(CH2)2N, 91%).
Elemental analysis: C (51.9 wt%), H (7.7 wt%), N (29.9 wt%), O
(9.9 wt%).
2.2.2. Second step: Synthesis of N-succinimidyl-6-azidohexanoate (B)
6-Azidohexanoic acid (A) (12.0 g, 76.4 mmol), NHS (14.06 g,
122.2 mmol), EDC (17.56 g, 91.6 mmol) and 200 mL of dichloro-
methane were combined in a flask. The mixture was stirred for
18 h at room temperature. After the solvent was evaporated, the
residue was dissolved in a mixture of diethyl ether and water (3/
1 v/v) and extracted six times with 50 mL of water. The combined
organic layers were dried over MgSO4, and the solvent was re-
moved. After the product was dried under vacuum, a yellowish
oil was obtained (16.6 g, yield: 86%).
2.3. Polymer syntheses
2.3.1. Synthesis of poly(2-(5-azidopentyl)-2-oxazoline)
Before being polymerized, the monomer 2-(5-azidopentyl)-2-
oxazoline was dried overnight over calcium hydride and was puri-
fied by distillation under a nitrogen atmosphere. The procedure for
the homopolymerization is given as follows. A solution of 5-azid-
opentyl-2-oxazoline (1 mL, 5.87 mmol) was prepared in dry
1H NMR (400 MHz, CDCl3): d = 3.27 (t, J = 8 Hz, 2H, N3CH2), 2.81
(s, 4H, O@CCH2CH2C@O), 2.60 (t, J = 8 Hz, 2H, CH2COO), 1.76

T.-X. Lav et al. / Reactive & Functional Polymers 73 (2013) 1001–1008
1003
acetonitrile (ACN) (3 mL) under dry nitrogen, and 24
l
l
the graft copolymer (G1) was 1.8 ꢀ 104 g molꢁ1, and its dispersity
was 1.4. The other copolymers were prepared similarly using alky-
nyl-PLA samples with different molecular weights.
(0.21 mmol) of iodobutane (BuI) was added to this solution. The
reaction mixture was stirred for 24 h at 80 °C under a nitrogen
atmosphere. The polymerization was stopped by the addition of
an excess of MeOH into the reaction mixture. Finally, the polymer
was purified by precipitation into diethyl ether and was dried un-
der vacuum. The final product was a tacky yellow viscous oil
(0.41 g, yield: 91%). The number-average molecular weight of the
FTIR (ATR mode): CAH 3016, 2968 cmꢁ1
;
C@O (ester)
1757 cmꢁ1
1186 cmꢁ1
;
.
C@O (amide) 1653 cmꢁ1
;
CAN 1458 cmꢁ1
;
CAO
2.5. Preparation of graft copolymer nanoparticles
poly(2-(5-azidopentyl)-2-oxazoline) was 5.5 ꢀ 103 g molꢁ1
.
FTIR (ATR mode): CAH 2935, 2862 cmꢁ1; N@N@N 2089 cmꢁ1
;
The P[(MeOx-co-PentOx)-g-LA] nanoparticles were prepared via
nanoprecipitation. The graft copolymer (20 mg) was first dissolved
in 10 mL of tetrahydrofuran one day prior to its use to ensure that
the sample was equilibrated. This copolymer solution was added
dropwise to 2 mL of deionized water under stirring. After all the
copolymer solution was added to the water, a homogeneous nan-
odispersion was observed. Tetrahydrofuran was removed from
the dispersion via evaporation under reduced pressure. The final
C@O 1733, 1635 cmꢁ1; CAN 1453 cmꢁ1
.
2.3.2. Synthesis of poly(2-methyl-2-oxazoline-co-2-(5-azidopentyl)-2-
oxazoline)
For the synthesis of the copolymer, a procedure similar to that
used for the synthesis of the homopolymer was used. A mixture
that contained 0.4 mL (2.35 mmol, 7 mol%) of 5-azidopentyl-2-
oxazoline, 2.6 mL (30.7 mmol, 93 mol%) of 2-methyl-2-oxazoline
copolymer concentration was 10 g Lꢁ1
.
and 9 mL of dry ACN was prepared under dry nitrogen, and 35 lL
of BuI (0.3 mmol) was added to this solution. The reaction mixture
was maintained at 80 °C for 24 h. The polymerization was
quenched and the copolymer was purified as previously described
for the homopolymer. A white powder was finally obtained (2.89 g,
yield: 95%).
2.6. Analytical methods
The monomer and the polymers were characterized by 1H NMR
on a Bruker Advance 400 spectrometer; the samples were dis-
solved in deuterated water or deuterated chloroform. FTIR spectra
were recorded on a Bruker Tensor 27 instrument equipped with a
Digi Tect DLaTGS detector; 32 scans were collected at a resolution
of 1 cmꢁ1 using an ATR accessory (single-reflection horizontal ATR,
Miracle, Pike Technologies). Gas chromatography–mass spectrom-
etry (GC–MS) analyses were performed with a Thermo Scientific
ITQ 700 GC with a MS detector operated in electronic impact
(EI+) ionization mode. The GC was fitted with a fused-silica capil-
lary nonpolar column (5% phenyl, DB-5MS) from Agilent Technol-
ogies (Les Ulis, France). The oven temperature was increased
from 60 to 240 °C at a rate of 8 °C minꢁ1. Size exclusion chromatog-
raphy (SEC) was performed on a chromatograph equipped with a P
100 pump (Spectra-Physics, Fremont, CA, USA) and a Rheodyne
injector. Two sets of columns were used: One was a set of two col-
umns (PL-aquagel OH-30 and OH-40, Polymer Laboratories, Shrop-
shire, UK) for the analysis of polymers with aqueous 0.1 mol Lꢁ1
LiNO3 used as the eluent; the other was a set of two columns
(PL-gel Mixed C) for the analysis of polymers in tetrahydrofuran
or chloroform. Two detectors were connected in series at the end
of the columns: a RI 71 differential refractometer (Shodex, Japan)
and a MiniDawn light scattering detector (Wyatt Technology, Santa
Barbara, CA, USA). The MiniDawn instrument (LS) uses a polarized
semiconductor diode laser (k = 690 nm) and measures the scat-
tered light at three angles (45°, 90° and 135°). The chromato-
graphic analyses of polymers were performed with polymer
solutions at a concentration of 10 mg mLꢁ1. Differential scanning
calorimetry (DSC) measurements were performed on a Perkin El-
mer Diamond apparatus using the following procedure: heating
The number-average molecular weight of the poly(2-methyl-2-
oxazoline-co-2-(5-azidopentyl)-2-oxazoline) was 1.0 ꢀ 104 g molꢁ1
,
and its dipersity was 1.2.
FTIR (ATR mode): CAH 2938, 2938 cmꢁ1; N@N@N 2097 cmꢁ1
;
C@O 1735, 1618 cmꢁ1; CAN 1417 cmꢁ1
.
2.3.3. Synthesis of alkynyl-terminated poly(
D,L-lactide)
Alkynyl-terminated poly( -lactide) oligomers were prepared
D,L
via the transesterification of high-molecular-weight PLA. The gen-
eral procedure was as follows: PLA (2 g mol, 27.8 mmol mono-
meric units), propargyl alcohol (400
lL, 6.94 mmol) and DBTDL
(147 L, 0.24 mmol) were dissolved in 20 mL of chloroform in a
l
seal-capped tube equipped with a magnetic stirring bar. The reac-
tion was performed at 100 °C for 3.5 h. At the end of the reaction,
the mixture was quickly cooled at room temperature, and the poly-
mer was precipitated into petroleum ether. The solid obtained was
dried under vacuum at 80 °C overnight.
The number-average molecular weight of the alkynyl-termi-
nated poly(
was 1.9.
D
,
L
-lactide) was 4.8 ꢀ 103 g molꢁ1, and its dispersity
1H NMR (400 MHz, CDCl3): d = 5.14 (q, 1H, CH(CH3), 4.7 (m, 2H,
CH2CCH), 4.31 (m, 1H, HOCHCH3), 2.66 (s, 1H, OH), 2.48 (t, 1H,
HCC), 1.56 (d, 3H, CH3) ppm.
13C NMR (100 MHz, CDCl3): d = 175.3 (HOCH(CH3)C@O),
169.6(C@O), 77.4 (CCH), 75.8 (CCH), 69.3 (CH(CH3)), 66.9
(HOCH(CH3)), 53.1 (CH2CCH), 20.7 (HOCH(CH3)), 16.8 (CH3) ppm.
FTIR (ATR mode): CAH 2996, 2946 cmꢁ1; C@O 1754 cmꢁ1; CAO
1180 cmꢁ1
.
from ꢁ50 to 150 °C at 20 °C minꢁ1
,
cooling at ꢁ50 °C at
2.4. Preparation of the graft copolymer by click-chemistry
The coupling of the alkynyl-terminated poly( -lactide) onto
200 °C minꢁ1 and heating again to 150 °C at 20 °C minꢁ1. The re-
ported glass-transition temperatures (Tgs) correspond to the sec-
ond run. Thermogravimetric analysis (TGAs) measurements were
recorded on a Setaram Setsys Evolution 16 apparatus. The samples
were heated from room temperature to 600 °C under an argon
atmosphere at a rate of 10 °C minꢁ1. Dynamic light scattering
(DLS) measurements were performed using a Zetasizer Nano ZS
(Malvern Instruments) equipped with a He–Ne laser with a wave-
length k = 633 nm. The angle of detection was set at h = 173°. The
measurements were performed at 25 °C. CONTIN algorithms were
used in the Laplace inversion of the autocorrelation function to ob-
D,L
poly(2-methyl-2-oxazoline-co-2-(5-azidopentyl)-2-oxazoline)
was catalyzed by CuI/PMDETA. The general procedure is as follows:
P(MeOx-co-N3PentOx) (100 mg, 76
(16 L, 76 mole), CuI (20 mg, 0.10 mmol), CH2Cl2 (6 mL) and alky-
nyl-PLA (350 mg, 76 mole alkynyl groups) were added to a
lmole azido groups), PMDETA
l
l
l
Schlenk tube. The click reaction was performed at 50 °C for 48 h.
The solvent was removed under reduced pressure. The crude prod-
uct was dissolved in DMSO (50 mL) and dialyzed against water.
After the product was freeze-dried, a white powder was obtained
(322 mg, yield: 72%). The number-average molecular weight of
tain the distribution of the relaxation times (
tion that the solutions were dilute, the distributions were
s). Under the assump-

1004
T.-X. Lav et al. / Reactive & Functional Polymers 73 (2013) 1001–1008
represented as a function of the hydrodynamic radius using the fol-
initiator. The structure of the polymers was confirmed by 1H NMR
(Fig. S1, Supporting Information). The 1H NMR molecular weight
(MNnMR) was determined via the analysis of 1H NMR spectra. For
the poly(2-(5-azidopentyl)-2-oxazoline), P(N3PentOx), the molar
ratio of the repeating unit was determined from the peak integra-
tion ratio of the methylene protons of the pendant group of the
polymer backbone (2.2–2.5 ppm) and the methyl protons of the
butyl group (0.94 ppm) at one end of the macromolecular chain.
A good agreement was observed between this molecular weight
and the expected molecular weight calculated from the feed ratio
(Menxp ¼ 5:1 ꢀ 103 g moLꢁ1). The molecular weight was not deter-
mined by SEC because the P(N3PentOx), which is soluble in CHCl3,
interacts with the columns when dissolved in this solvent. In the
FTIR spectra (Fig. 2), the peak of the azido group of the repeating
unit is clearly visible at 2090 cmꢁ1, which indicates that the azido
group is preserved during the polymerization reaction.
lowing relationship between
s and rh:
q2kBT
6pg0
rh
¼
s
ð1Þ
where kB is the Boltzmann constant, T is the absolute temperature,
g0 is the solvent viscosity, and q is the scattering vector.
3. Results and discussion
3.1. Monomer synthesis
The new monomer 2-(5-azidopentyl)-2-oxazoline was synthe-
sized in four steps starting from commercially available 6-bromo-
hexanoic acid (Scheme 1), as described in Section 2. The first step is
a nucleophilic substitution of 6-bromohexanoic acid with sodium
azide. The product 6-azidohexanoic acid (A) was obtained with a
yield of 87%, and its structure was confirmed by 1H NMR (t, 2H,
N3CH2 at 3.25 ppm) and infrared (azide signal at 2091 cmꢁ1) spec-
troscopies. Steps 2–4 are modified versions of a previously re-
ported procedure [28]. In the second step, the 6-azidohexanoic
acid is activated by N-hydroxysuccinimide (NHS) and N-(3-
dimethylaminopropyl)-N0-ethylcarbodiimide hydrochloride (EDC).
The third step is the formation of an amide function from which
the cyclization occurs, giving 2-(5-azidopentyl)-2-oxazoline (D)
(Scheme 1). In step 4, the reaction was monitored and the purity
of the monomer (D) was controlled using gas chromatography.
All the products were obtained with good yields (>81%), and the
overall yield for the monomer (D) synthesis was 53%. The GC purity
of 2-(5-azidopentyl)-2-oxazoline was as high as 99%, and its chem-
ical structure was confirmed by 1H NMR spectroscopy (Fig. 1) and
by infrared spectroscopy (azide signal at 2091 cmꢁ1). The molecu-
lar weight determined by mass spectrometry was the expected
molecular weight. The elemental analysis indicated a molecular
formula of C8H14N4O, as expected. The monomer 2-(5-azidopen-
tyl)-2-oxazoline contains two reactive sites that exhibit orthogonal
reactivity. The oxazoline group allows the cationic ring-opening
polymerization of this monomer, and the azide group allows the
Huisgen 1,3-dipolar cycloaddition reaction between the azide
and a terminal alkyne on another polymer.
The copolymer poly(2-methyl-2-oxazoline-co-2-(5-azidopen-
tyl)-2-oxazoline) (P(MeOx-co-N3PentOx), which is composed
mostly of 2-methyl-2-oxazoline, is soluble in water and could
be characterized by SEC using aqueous 0.1 mol Lꢁ1 LiNO3 as
the eluent. This method allows the determination of the absolute
number-average molecular weight (MSnEC). The MnSEC was equal to
the expected molecular weight (Menxp ¼ 1:01 ꢀ 104 g moLꢁ1) with
a dispersity of 1.2, which indicates that the polymer exhibits a
narrow molecular-weight distribution. We calculated the compo-
sition of the copolymer (values n and m, Scheme 2) via 1H NMR
analysis by considering the peak integration ratio of the methyl
protons of the 2-methyl-2-oxazoline unit (2.07 ppm) or the
methylene protons of the 2-(5-azidopentyl)-2-oxazoline unit
(2.35 ppm) and the methyl protons of the butyl group for the
values n or m, respectively. The fraction of azido group (fN3) cal-
culated from the ratio m/(n + m) is 6.7 mol%. This fraction could
also be calculated from the FTIR spectrum (Fig. 2) on the basis of
the ratio of both absorption bands that correspond to the azido
group (2097 cmꢁ1
) and the carbonyl of the amide function
(1618 cmꢁ1). A value of 8.5% was obtained, which is close to
the values obtained from feed ratio and from the NMR results.
The glass-transition temperature (Tg) was measured for the
poly(2-methyl-2-oxazoline), the poly(2-(5-azidopentyl)-2-oxazo-
line) and the copolymer P(MeOx-co-N3PentOx). The value of Tg
measured for the poly(2-methyl-2-oxazoline) was 51 °C. This re-
sult is intermediate between the values reported in the literature
(46 °C [23] and 79 °C [29–31]). The copolymer exhibited only
one glass-transition temperature, which was between the Tg
measured for the poly(2-methyl-2-oxazoline) and that measured
for the poly(2-(5-azidopentyl)-2-oxazoline) (Table 1). The
appearance of only one Tg may indicate that the two monomers
3.2. Synthesis and characterization of azido-polymers
The monomer 2-(5-azidopentyl)-2-oxazoline (N3PentOx) was
both polymerized alone and copolymerized with 2-methyl-2-oxaz-
oline (MeOx) (Scheme 2). In each case, iodobutane was used as an
(B)
(A)
(D)
(C)
Scheme 1. Synthesis route of 2-(5-azidopentyl)-2-oxazoline (D).

T.-X. Lav et al. / Reactive & Functional Polymers 73 (2013) 1001–1008
1005
Fig. 1. 1H NMR spectrum of 2-(5-azidopentyl)-2-oxazoline.
Scheme 2. Schemes of synthesis of poly(2-methyl-2-oxazoline-co-2-(5-azidopentyl)-2-oxazoline) by CROP, alkynyl-PLA by transesterification with propargyl alcohol and
[poly(2-methyl-2-oxazoline-co-2-pentyl-2-oxazoline)-g-poly( -lactide)] by click-chemistry.
D,L
MeOx and N3PentOx are well miscible. Based on this preliminary
study, we do not know if the copolymer is a random or gradient
copolymer. Copolymers of MeOx and 2-ethyl-2-oxazoline (EtOx)
or 2-nonyl-2-oxazoline (NonOx) have been investigated previ-
ously [32]. The kinetic plots have revealed a slightly faster incor-
poration of MeOx as compared to the incorporation of EtOX and
NonOx, which resulted in a gradient copolymer. In the present
case, given that the N3PentOx contains a pendant pentyl group
with a length intermediate between the length of a pendant
ethyl and a nonyl group, a gradient copolymer could also be ex-
pected. However, further investigations would be required to
determine the distribution of the copolymer P(MeOx-co-
N3PentOx).
3.3. Preparation of alkynyl-terminated PLA
Alkynyl-functionalized PLAs were prepared by a direct transe-
sterification reaction with propargyl alcohol in chloroform at
100 °C (Scheme 2). This method offers the major advantage of pro-
ducing oligomers with an alkynyl end group in a one-step reaction.
We performed a series of reactions in dry chloroform to study the
influence of the amount of catalyst and the reaction time on the
rate of the transesterification reaction. The amount of propargyl
alcohol was maintained at 0.25 equivalents relative to the quantity
of the PLA monomeric units. The amount of catalyst was increased
linearly with the reaction time to give the best compromise
between a sufficient reaction rate and control of the molecular

1006
T.-X. Lav et al. / Reactive & Functional Polymers 73 (2013) 1001–1008
weight of the oligomers. The amount of catalyst was 1 mol% for
each hour of reaction. The polymers were characterized by 1H
NMR (in CDCl3) and SEC (in tetrahydrofuran). The results are re-
ported in Table S1 (see Supporting Information). The chemical
structures of the alkynyl-PLA samples were confirmed by the
appearance of new signals in the 1H NMR spectra at 4.70 ppm
and 2.48 ppm, which are characteristic of the CH2 and AC„CAH
protons added at the end of the macromolecular chain. We calcu-
lated number-average molecular weights (MNnMR) based on the peak
integration ratio of the hydroxyl protons borne by the other end of
the macromolecular chain (4.31 ppm) and the CH proton of the
backbone (5.25 ppm). Different reaction times, which ranged from
0.5 to 8 h, led to alkynyl-PLA samples with molecular weights, as
determined by SEC (MSnEC), that ranged from 4.8 ꢀ 104 to 1.6 ꢀ 103 -
g moLꢁ1 (Table S1, Supporting Information). As illustrated in Fig. S2
(see Supporting Information), the molecular weight of the oligo-
mers decreased as the reaction time corresponding amount of cat-
alyst were increased. A decrease of the dispersity (Mw/Mn) was also
observed when smaller molecular weights were obtained because
of the precipitation purification step. The transesterification reac-
tion was thus successfully realized, and the molecular weight of
the alkynyl-PLA could be controlled through adjustments to the
reaction conditions.
Ν3
4000
3500
3000
2500
2000
1500
1000
500
Fig. 2. FTIR spectra of poly2-(5-azidopentyl)-2-oxazoline) (P(N3PentOx)), poly(2-
methyl-2-oxazoline-co-2-(5-azidopentyl)-2-oxazoline) (P(MeOx-co-N3PentOx))
and [poly(2-methyl-2-oxazoline-co-2-pentyl-2-oxazoline)-g-poly( -lactide)]
D,L
(P[(MeOx-co-PentOx)-g-LA]).
Table 1
Synthesis conditions for and characteristics of poly(2-(5-azidopentyl)-2-oxazoline) (P(N3PentOx)) and poly(2-methyl-2-oxazoline-co-2-(5-azidopentyl)-2-oxazoline)
(P(MeOx-co-N3PentOx)).
MNnMRg moLꢁ1
MSnEC g moLꢁ1
Polymer
[M]0/[I]0 feed ratio
[N3PentOx]0/[MeOx]0 feed ratio
Mw/Mn
fN3 (% moL)
Tg (°C)
P(N3PentOx)
P(MeOx-co-N3PentOx)
28
110
–
0.07
5.5 ꢀ 103
–
–
1.2
100
7
ꢁ30
1.0 ꢀ 104
1.0 ꢀ 104
28
(a) : Estimated by 1H NMR.
(b) : Estimated by SEC and light scattering detector.
Table 2
Characteristics of [poly(2-methyl-2-oxazoline-co-2-pentyl-2-oxazoline)-g-poly(D,L-lactide)] (P[(MeOx-co-PentOx)-g-LA]).
PLA wt%expected
PLA wt%TGA
Mw/Mn
NMR
PLA
MNnPMLRAg moLꢁ1
MSnEC g moLꢁ
1
P[(MeOx-co-PentOx)-g-LA]
s
(% moL)
G1
G2
G3
4.8 ꢀ 103
8.9 ꢀ 103
1.9 ꢀ 104
8
7
9
78
88
94
75
84
92
1.8 ꢀ 104
1.4
1.4
1.6
3.2 ꢀ 104
4.4 ꢀ 104
Fig. 3. 1H NMR spectrum of [poly(2-methyl-2-oxazoline-co-2-pentyl-2-oxazoline)-g-poly(
D,L-lactide)].

T.-X. Lav et al. / Reactive & Functional Polymers 73 (2013) 1001–1008
1007
Fig. 4. Size distribution of [poly(2-methyl-2-oxazoline-co-2-pentyl-2-oxazoline)-g-poly(D,L-lactide)] (G2).
3.4. Synthesis and characterization of graft copolymers
with a core mainly composed of the PLA grafts and a shell made
of the hydrophilic PMeOx chains can be anticipated; such struc-
tures have already been demonstrated for PLA-PEG copolymers
[33]. The original architecture of the PMeOx shell, as schematized
in Fig. 4, should be outlined with the presence of PMeOx loops in
the interfacial area. These first results appear promising because
such nanoparticles could be used as carriers for nonpolar drugs
that can be loaded into the biocompatible PLA core with the hydro-
philic PMeOx shell potentially ensuring stealth properties. This
preliminary study and the biological evaluation of these nanopar-
ticles will be reported in a forthcoming publication.
The graft copolymers were prepared via the Huisgen 1.3-dipolar
cycloaddition reaction of the pendant azido groups on the block
copolymers P(MeOx-co-N3PentOx) and the alkynyl group at the
end of the PLA. The click reaction was catalyzed by CuI/PMDETA
in dichloromethane at 50 °C. The [N3]/[C„C] feed ratio was main-
tained at 1/1, and the reaction was conducted for 48 h. The prod-
ucts of the azide–alkyne coupling reaction were checked by SEC,
FTIR, 1H NMR and TGA. The graft copolymers that contain a large
proportion of PLA (Table 2) are soluble in tetrahydrofuran, which
was the solvent in which the SEC analyses were performed. Typical
chromatograms, such as those given in Fig. S3 (see Supporting
Information), from both light-scattering and refractometer detec-
tors showed monomodal distributions and contained no molecu-
lar-weight peaks due to unreacted reactants. The dispersity
values of P[(MeOx-co-PentOx)-g-LA] determined by SEC are close
to that of P(MeOx-co-N3PentOx) (Table 2). The number-average
molecular weight (MnSEC) measurements showed that the MSnEC in-
creased as the length of the PLA side chain increased. In the IR
spectra of each sample, the absorption at 2097 cmꢁ1, which is char-
acteristic of the azido group, completely disappeared after the
cycloaddition (Fig. 2), thereby confirming that the azido groups
were consumed quantitatively during the coupling reaction. For
example, the 1H NMR spectra of [poly(2-methyl-2-oxazoline-co-
4. Conclusion
Well-defined amphiphilic graft copolymers made of a poly(2-
oxazoline) backbone and PLA grafts were prepared using a combi-
nation of ring-opening polymerization, transesterification and click
chemistry. The synthesis strategy was to couple an alkynyl-termi-
nated poly(D,L-lactide) onto a POx backbone bearing some pendant
azido groups. This approach first involved the synthesis of a new
oxazoline monomer bearing an azido group and its copolymeriza-
tion with the 2-methyl-2-oxazoline monomer. Proper character-
izations by NMR, IR, SEC and DSC showed that copolymers
P[(MeOx-co-PentOx)-g-LA] with varying PLA chain lengths were
successfully synthesized and exhibited well-defined architectures.
This new synthesis strategy opens the field of polyoxazoline-based
polymers for the design of well-defined graft polymers with a POx
backbone and hydrophobic grafts. The self-association of these
amphiphilic copolymers may result in new nanostructures. The
physico-chemical analysis of the self-associating properties as well
as the evaluation of their suitability for drug delivery will be the
subject of a future publication.
2-pentyl-2-oxazoline)-g-poly(D,L-lactide)] are shown in Fig. 3.
According to the 1H NMR analysis results for PLA, the peak of the
HC„C-proton at 2.48 ppm disappeared entirely. The degree of
PLA grafting calculated from 1H NMR (sPNLMAR) was determined from
the peak integration ratios of the CH proton in the PLA repeating
unit at 5.14 ppm and the methyl protons in the PMeOx repeating
unit at 2.10 ppm. The results, which are reported in Table 2, are
in agreement with the fraction of azido group (fN3) incorporated
in the copolymer precursor. The weight percent of PLA incorpo-
rated into graft copolymers, as determined by TGA, was in good
agreement with the expected weight percent (Table 2), which con-
firms the quantitative grafting of alkynyl-PLA onto the linear POx
backbone bearing azido anchoring groups in its repeating unit.
All of these results prove the successful synthesis of [poly(2-
Acknowledgments
Christine Jakubowicz, Patrice Renevret and Isabelle Lachaise of
the analytical and preparative chromatography platform of ICMPE
are thanked for the GC-MS analyses. The authors thank the Institut
de Chimie des Substances Naturelles (Gif-sur-Yvette, France) for
elemental analyses.
methyl-2-oxazoline-co-2-pentyl-2-oxazoline)-g-poly(D,L-lactide)].
To test the self-association properties of the copolymers that
are not soluble in water, we adapted a nanoprecipitation method
that has already been used in the analysis of pure PLA and PLA-
PEG block copolymers [33] for P[(MeOx-co-PentOx)-g-LA] copoly-
mers. After the copolymers were dissolved in THF and added to
pure water, nanodispersions spontaneously formed. Dynamic light
scattering was used to evaluate the properties in water of the nan-
odispersions obtained after the THF was removed. Irrespective of
the composition of the copolymers (G1–G3), well-defined mono-
modal size distributions were obtained, with an average hydrody-
Appendix A. Supplementary material
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.reactfunctpolym.
2013.04.013.
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