Novel poly(ε-caprolactone)s bearing amino groups: Synthesis, characterization and biotinylation

Article abstract of DOI:10.1016/j.reactfunctpolym.2010.03.008

A novel functional ε-caprolactone monomer containing protected amino groups, γ-(carbamic acid benzyl ester)-ε-caprolactone (γCABεCL), was successfully synthesized. A series of copolymers [poly(CL-co-CABCL)] were prepared by ring-opening polymerization of

Full text of DOI:10.1016/j.reactfunctpolym.2010.03.008

Reactive & Functional Polymers 70 (2010) 400–407
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Reactive & Functional Polymers
journal homepage: www.elsevier.com/locate/react
Novel poly(
e
-caprolactone)s bearing amino groups: Synthesis, characterization
and biotinylation
*
Jinliang Yan, Yi Zhang, Yan Xiao, Yan Zhang, Meidong Lang
Shanghai Key Laboratory of Advanced Polymeric Materials, Key Laboratory for Ultrafine Materials of Ministry of Education,
School of Materials Science and Engineering, East China University of Science and Technology, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel functional
ester)- -caprolactone (
CL)] were prepared by ring-opening polymerization of
tin (II)-2-ethylhexanoate [Sn(Oct)2] as catalyst. The morphology of the copolymers changed from semi-
crystalline to amorphous with increasing CAB CL monomer content. They were further converted into
e
-caprolactone monomer containing protected amino groups,
CAB CL), was successfully synthesized. A series of copolymers [poly(CL-co-CAB
-caprolactone (CL) and CAB CL in bulk using
c-(carbamic acid benzyl
Received 6 October 2009
Received in revised form 21 March 2010
Accepted 22 March 2010
Available online 27 March 2010
e
c
e
e
c
e
c
e
deprotected copolymers [poly(CL-co-ACL)] with free amino groups by hydrogenolysis in the presence of
Pd/C. After deprotection, the free amino groups on the copolymer were further modified with biotin. The
monomer and the corresponding copolymers were characterized by 1H NMR, 13C NMR, FT-IR, mass, GPC
and DSC analysis. The obtained data have confirmed the desired monomer and copolymer structures.
Ó 2010 Elsevier Ltd. All rights reserved.
Keywords:
c
-(Carbamic acid benzyl ester)-e-
caprolactone
Ring-opening polymerization
Poly(e-caprolactone)
Baeyer–Villiger reaction
Amino groups
Biotinylation
1. Introduction
In general, there are two main strategies available for modifying
PCL with pendant functional groups. The first approach is postpo-
In the past few decades, there has been a paradigm shift in the
biomedical and pharmaceutical fields from biostable biomaterials
to biodegradable biomaterials [1,2]. Among the various families of
biodegradable polymers, aliphatic polyesters [3–5], such as poly
lymerization modification. For example, Philippe Lecomte and
coworkers have synthesized PCL bearing pendant chloride substit-
uents, which could be easily converted into azides. These pendant
azides made it possible to graft a variety of substituents onto PCL
using the ‘‘click reaction” [14]. The second route is polymerization
of functionalized monomers. In order to prevent inter- and intra-
molecular side reactions, the functional monomers have to be
protected prior to polymerization and deprotected afterwards. Lav-
(e-caprolactone) (PCL), poly(L-lactide) (PLA) and polyglycolide
(PGA), have taken a leading position over the others because of their
excellent biodegradability, biocompatibility and mechanical prop-
erties. PCL especially has been widely explored due to its distin-
guished properties of nontoxicity and permeability to a wide
range of drugs [6,7]. However, PCL degrades extremely slowly
in vitro and in vivo due to its high hydrophobicity and crystallinity.
Moreover, the lack of pendant reactive functional groups to which
bioactive compounds can be covalently attached has severely lim-
ited its biomedical application. In recent years, different chemical
methods have been applied to introduce hydroxyl [8,9], carboxylic
acid [10], carbonyl [11], vinyl [12] and halogen [9,13] substituents
onto the PCL chain. The introduction of these pendant functional
groups regulates and adjusts the physico-chemical properties of
the material, such as crystallinity, hydrophilicity, biodegradation
rate, and biological activity. Therefore, modification of PCL is of
great necessity.
asanifar et al. synthesized a novel monomer,
a-benzyl carboxylate-
e
-caprolactone, from -caprolactone by deprotonation using LDA
e
followed by an electrophilic substitution with benzyl chlorofor-
mate. The copolymerization of the monomer with CL was then
carried out using methoxy PEO as an initiator and tin (II)-2-ethyl-
hexanoate [Sn(Oct)2] as a catalyst to get block copolymers. The
block copolymers bearing carboxyl groups were finally obtained
by catalytic debenzylation [10]. Moreover, these pendant func-
tional groups open the door for further modification such as graft-
ing, crosslinking and attaching bioactive substances or drugs for
biomedical applications [15,16].
Biomolecule–polymer conjugates are widely used in tissue
engineering and drug delivery [17–19]. Moreover, it would be
desirable for biomaterials to have the ability to recognize and
immobilize specific proteins or drugs. The biotin–avidin (or strep-
tavidin) system is extensively used currently because of their high
* Corresponding author. Fax: +86 21 64253432.
E-mail address: mdlang@ecust.edu.cn (M. Lang).
1381-5148/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.reactfunctpolym.2010.03.008

J. Yan et al. / Reactive & Functional Polymers 70 (2010) 400–407
401
binding affinity (1013–15 Mꢀ1) [20]. Once biotin is attached to a
polymer, it can combine with avidin (or streptavidin) due to the
specific recognition, which facilitates the combination of various
proteins or drugs without complicated synthesis. For example,
Narain has directly prepared well-defined protein–glycopolymer
bioconjugates via avidin–streptavidin binding without many pro-
tection and deprotection steps [21]. However, the biotin is usually
connected to the polymer terminus, which is quite limited in a
polymer [21–23]. Therefore, conjugating biotin with the pendant
groups on a polymer chain would be favorable.
After extraction, the combined organic layers were washed with
0.5 M HCl and brine, dried over Na2SO4 and filtered. The filtrate
was evaporated until 1 began to crystallize. The product was
recrystallized overnight in the refrigerator and collected as white
needles by suction filtration (17.8 g, 71.4 mmol, 80%).
1H NMR (CDCl3): d = 1.12–2.15 [m, 8H, –(CH2)2–CH(OH)–
(CH2)2–], 3.43 (m, 1H, –CH–NH–), 3.58 (m, 1H, –CH–OH–), 4.62
(s, 1H, –NH–), 5.09 (s, 2H, –CH2–Ph), 7.30–7.37 (m, 5H, –C6H5).
IR (KBr): 3389 cmꢀ1 (OH), 3341 cmꢀ1 (NH), 1688 cmꢀ1
(–CONH–), 780 cmꢀ1, 758 cmꢀ1, 724 cmꢀ1, 693 cmꢀ1 (CH).
MS (70 eV): m/z = 249.2 (M+), 158.1 (M+ꢀC6H5CH2), 114.1
(M+ꢀCOOCH2C6H5), 91.1 (C6H5CHþ2 ).
In this paper, we report the following achievements: (1) synthe-
sis of a novel functional monomer [
-caprolactone, CAB CL] with protected amino groups; (2)
ring-opening copolymerization of the preprotected functional
monomer with -caprolactone (CL) to prepare PCL-based poly-
c-(carbamic acid benzyl ester)-
e
c
e
2.4. Benzyl 4-oxocyclohexane carbamate (2)
e
mers; (3) removal of the protective carbobenzoxy (Cbz) groups
by hydrogenolysis in the presence of Pd/C to obtain the corre-
sponding copolymers with free amino groups; (4) grafting of biotin
onto the copolymer through amide coupling reactions. To the best
of our knowledge, this functional monomer with protected amino
groups is a novel compound that has not been reported, and this
work is the first report introducing amino functional groups onto
the PCL backbone by protection and deprotection methods.
A suspension of 1 (17.4 g, 70 mmol) in acetone (250 mL) was
placed in a 500-mL flask. The mixture was cooled in an ice bath,
and Jones’ reagent (20 mL) was added dropwise over a few min-
utes. When the addition of the Jones’ reagent was complete, the
reaction mixture was allowed to warm slowly to room tempera-
ture, stirred overnight and quenched by addition of isopropyl alco-
hol. After stirring for 5 min, the mixture was filtered. The filtrate
was concentrated, and a proper amount of sodium bicarbonate
solution was added until the pH of the reaction mixture was tested
neutral. The orange aqueous mixture was extracted three times
with ethyl acetate. Then the combined organic layers were washed
with brine, dried over MgSO4, filtered, concentrated, and purified
by recrystallization (EtOAc/hexane = 1:1) to afford the white crys-
talline solid 2 (12.4 g, 50 mmol, 71%).
2. Experimental
2.1. Materials
Trans-4-aminocyclohexanol (Longshan Chemical Co., Ltd., Zhe-
jiang, China, 99.9%), benzyl chloroformate (CbzCl, 98%), chromium
trioxide (CrO3) and N-hydroxysuccinimide (NHS) were used as re-
ceived. Palladium-coated charcoal (Pd/C, 10%) was obtained from
Sinopharm Chemical Reagent Co., Ltd. m-Chloroperoxybenzoic acid
(m-CPBA, Acros, 70–75%) was purified according to a method de-
1H NMR (CDCl3): d = 1.65–2.45 [m, 8H, –(CH2)2–(O)–(CH2)2–],
3.99 (m, 1H, –CH–NH–), 4.80 (s, 1H, –NH–), 5.14 (s, 2H, –CH2–
Ph), 7.30–7.38 (m, 5H, –C6H5).
IR (KBr): 3343 cmꢀ1 (NH), 1718 cmꢀ1 (C@O), 1688 cmꢀ1 (–CONH–),
777 cmꢀ1, 756 cmꢀ1, 723 cmꢀ1, 695 cmꢀ1 (CH).
MS (70 eV): m/z = 247.2 (M+), 140.1 (M+ꢀC6H5CH2O), 112.1
scribed in the literature and stored under vacuum [24].
e-Caprolac-
(M+ꢀCOOCH2C6H5), 91.1 (C6H5CHþ2 ).
tone ( -CL, Acros, 99%) was dried over calcium hydride for 48 h
e
followed by vacuum distillation prior to use. Tin (II)-2-ethylhex-
anoate [Sn(Oct)2, Sigma, 95%] and d-biotin (Sigma) were used
without further purification. All the other solvents and reagents
were of AR grade and were used as received.
2.5. c-(Carbamic acid benzyl ester)-e-caprolactone (cCABeCL) (3)
A 500-mL, round-bottomed flask was charged with 2 (24.7 g,
0.1 mol) and 100 mL of CH2Cl2. The mixture was stirred vigorously
in an ice bath while a suspension of m-chloroperoxybenzoic acid
(25.3 g, 0.11 mol) in 250 mL of CH2Cl2 was added dropwise. After
the addition of the peracid, the reaction mixture was stirred for
an additional 14 h at room temperature. The solution was then
washed successively with sodium thiosulfate solution (three
times), bicarbonate solution (three times) and sodium chloride
solution (three times). After the organic layer was dried over
Na2SO4, it was filtered, concentrated and purified by recrystalliza-
tion (EtOAc/hexane = 3:2) to obtain white needle crystals
(18.41 g, 70 mmol, 70%), m.p. 116–117 °C, 99.9% purity as deter-
mined by HPLC analysis (recrystallized from dry toluene three
times).
2.2. Measurements
NMR spectra were recorded at room temperature on a Bruker
Avance series instrument (400 MHz). The molecular weights and
distributions were determined by gel permeation chromatography
(GPC) measurements on a Waters GPC system equipped with a
Waters 2414 HPLC solvent pump, three Ultrastyragel columns
0
(2 ꢁ 105, 105, and 5 ꢁ 104 ÅA) in series and a refractive detector. Tet-
rahydrofuran (THF) was used as the eluent and delivered at a flow
rate of 1.0 mL/min at 35 °C. The molecular weight was calibrated
with polystyrene standards. Differential scanning calorimetry
(DSC) was used to study the thermal behavior of the copolymers
on a DSC2910 Modulated DSC system. FT-IR spectra were recorded
on a Nicolet 5700 instrument.
1H NMR (CDCl3): d = 1.52–2.29 [m, 4H, –CH2–CH(NH)–CH2–],
2.53–2.62 (m, 2H, –C(O)–CH2–CH2–), 3.82 (s, 1H, –CH–NH–),
4.14–4.31 (m, 2H, –O–CH2–CH2–) 4.76 (s, 1H, –NH–), 5.09 (s, 2H,
–CH2–Ph), 7.30–7.37 (m, 5H, –C6H5).
2.3. Benzyl 4-hydroxycyclohexanecarbamate (1)
13C NMR (CDCl3): d = 29.5 [–CH2–CH2–C(O)O–], 30.9 [–CH2–
CH2–O–C(O)–], 36.1 [–CH2–CH2–C(O)O–], 51.5 [–CH2–CH(NH)–
CH2–], 66.1 [–CH2–CH2–O–C(O)–], 67.4 [C6H5–CH2–O–C(O)–],
128.7, 128.8, 129.1, 136.8 (–C6H5), 156.0 (–O–CO–NH–), 175.6
(–CH2–CO–O–).
A solution of sodium bicarbonate (18.8 g, 224 mmol) and trans-
4-aminocyclohexanol (10.3 g, 89.4 mmol) in 600 mL of water was
added into a 1000-mL flask. The solution was cooled in an ice/
water bath, and benzyl chloroformate (16.6 mL, 116 mmol) was
added dropwise with continuous stirring over 10 min. The reaction
mixture was then heated to 45 °C, and the stirring was continued
for 4 h. The mixture was extracted with ethyl acetate and butanol.
IR (KBr): 3337 cmꢀ1 (NH), 1719 cmꢀ1 (–CO(O)–), 1688 cmꢀ1
(–CONH–), 780 cmꢀ1, 758 cmꢀ1, 724 cmꢀ1, 693 cmꢀ1 (CH).
MS (70 eV): m/z = 263.1 (M+), 156.1 (M+ꢀC6H5CH2O), 128.1
(M+ꢀC6H5CH2O–CO), 91.1 (C6H5CHþ).
2

402
J. Yan et al. / Reactive & Functional Polymers 70 (2010) 400–407
2.6. Copolymerization of CL and
c
CABe
CL
mixture was stirred at room temperature for 12 h, then precipi-
tated in cold diethyl ether, isolated by filtration and dried in
vacuum (yield: 80%).
The copolymer poly(CL-co-CABCL) was synthesized by ROP of
CL and CAB CL using trace water as initiator and Sn(Oct)2 as cat-
c
e
alyst. A typical example of the synthetic procedure is described as
2.9. Biotinylation
follows. Under the protection of argon, 1.71 g of CL (15 mmol),
0.44 g of
cCABeCL (1.67 mmol) and a stirring bar were quickly
A
solution of biotin (50 mg, 0.21 mmol) and N-hydroxy-
put into a flame-dried polymerization tube. The tube was con-
nected to a Schlenk line, where an exhausting–refilling process
was repeated several times. The tube was then placed into an oil
bath at 50 °C with vigorous stirring until the mixture became
homogeneous and clear. To obtain the desired monomer/catalyst
molar ratio, Sn(Oct)2 in dry toluene was added to the polymeriza-
tion tube by using a microliter syringe, and the exhausting–refill-
ing process was conducted again to remove the toluene. The tube
was then sealed under vacuum and placed in an oil bath at
130 °C for 24 h. The polymerization was stopped by cooling the
tube to room temperature. The resulting product was dissolved
in chloroform and precipitated by addition to cold methanol under
vigorous stirring. The copolymer was collected and dried in vacuo
before characterization.
succinimide (NHS, 23.6 mg, 0.21 mmol) in 6 mL anhydrous DMF
was cooled in an ice bath, and then N,N0-dicyclohexylcarbodiimide
(DCC, 55 mg, 0.27 mmol) was added portion-wise. The solution
was stirred overnight at room temperature, during which time a
white precipitate (DCU) was formed. The precipitate was then re-
moved by filtration. The copolymer (0.5 g) with free amino groups
was dissolved in 5 mL DMF and added drop-wise to the filtrate. To
the mixture was added triethylamine (0.41 mL, 2.9 mmol), and the
reaction was stirred for another 24 h under N2. The resulting reac-
tion solution was filtered, dialyzed in DMF with a dialysis mem-
brane (cutoff Mn 3000) and condensed in vacuum. The
condensation product was precipitated in a large amount of diethyl
ether, filtered and washed with diethyl ether several times. The
white precipitates were dried under vacuum (yield: 70%).
1H NMR (CDCl3): d = 1.33–1.40 (2H, –CH2–CH2–CH2–CH2–O–),
1H NMR (DMSO-d6): d = 1.33–1.40 (2H, –CH2–CH2–CH2–CH2–
O–), 1.25–1.67 (6H, biotin, –CH2–CH2–CH2–CH–), 1.64–1.86 [8H,
–CH2–CH2–CH2–CH2–CH2–O–, –CH2–CH2–CH(NH)–CH2–CH2–O–],
2.21 [2H, biotin, –C(O)–CH2–], 2.29–2.37 [4H, –C(O)–CH2–CH2–
CH2–, –C(O)–CH2–CH2–CH(NH)–], 2.54–2.86 (2H, biotin, –S–CH2–),
3.1 (1H, biotin, –S–CH–), 3.77 [1H, –CH2–CH(NH)–CH2–], 4.03–4.15
[4H, –CH2–CH2–CH2–O–, –CH(NH)–CH2–CH2–O–], 4.15–4.35 (2H,
biotin, –NH–CH–CH–NH–), 6.25–6.45 [2H, biotin, –NH–C(O)–
NH–], 7.6 (1H, –CONH–).
1.62–1.85
[8H,
–CH2–CH2–CH2–CH2–CH2–O–,
–CH2–CH2–
CH(NH)–CH2–CH2–O–], 2.27–2.40 [4H, –C(O)–CH2–CH2–CH2–,
–C(O)–CH2–CH2–CH(NH)–], 3.76 [1H, –CH2–CH(NH)–CH2–], 4.03–
4.17 [4H, –CH2–CH2–CH2–O–, –CH(NH)–CH2–CH2–O–], 4.82 (1H,
–CONH–), 5.06–5.14 (2H, –CH2–Ph), 7.30–7.40 (5H, –C6H5).
13C NMR (CDCl3): d = 24.5 (–CH2–CH2–CH2–CH2–O–), 25.5
(–CO–CH2–CH2–CH2–), 28.3 (–CH2–CH2–CH2–O–), 30.2 [–CO–
CH2–CH2–CH(NH)–], 30.8 [–CH(NH)–CH2–CH2–O–], 34.0 (–CO–
CH2–CH2–CH2–), 34.2 [–CO–CH2–CH2–CH(NH)–], 48.6 (–NH–CH–),
61.2 [–CH(NH)–CH2–CH2–O–], 64.1 [–CH(NH)–CH2–CH2–O–], 66.6
(–CH2–C6H5), 128.1, 128.3, 128.5, 136.5 (–C6H5), 156.0 (–CO–
NH–), 173.3 [–CO–CH2–CH2–CH(NH)–], 173.5 (–CO–CH2–CH2–
CH2–).
3. Results and discussion
In the past few years, there have been several reviews of func-
tional PCLs based on functionalized
e-caprolactone monomers,
IR (KBr): 3364 cmꢀ1 (NH), 3064 cmꢀ1
,
3034 cmꢀ1 (C–H),
mainly focusing on the introduction of hydroxyl, carboxylic acid,
carbonyl and halogen substituents [25,26]. To date, there have been
few reports on PCL modified with pendant amino groups. Our work
fills this gap and could potentially broaden the applications of PCL
in biomedical fields due to the presence of the amine active site.
1732 cmꢀ1 [–C(O)O–], 740 cmꢀ1, 699 cmꢀ1 (C–H).
2.7. Deprotection of poly(CL-co-CABCL) by hydrogenolysis
A solution of the protected copolymer poly(CL-co-CABCL) (0.5 g
in 20 mL of THF) was placed into a 50-mL round-bottomed flask.
Then, 0.2 g of Pd/C (10%) suspension in CH3OH (10 mL) was added
to the solution. The flask was degassed and saturated with hydro-
gen. The reaction mixture was stirred vigorously under a hydrogen
atmosphere (balloon) for 48 h at 50 °C. Then the mixture was cen-
trifuged at 8 ꢁ 103 rpm to remove most of the catalyst. The resid-
ual catalyst was removed by filtration through Celite using THF as
the eluent. The filtrate was collected, concentrated and precipi-
tated in cold diethyl ether to obtain the deprotected copolymer,
3.1. Synthesis of
CAB CL)
c-(carbamic acid benzyl ester)-e-caprolactone
(c
e
The novel functional monomer,
c-(carbamic acid benzyl ester)-
e-caprolactone ( CAB CL), was prepared by a three-step reaction
c
e
from trans-4-aminocyclohexanol. The reaction pathway is shown
in Scheme 1. Benzyl chloroformate was used to protect the amino
group. Then the resultant alcohol 1 was oxidized into the corre-
sponding ketone 2 by the use of Jones’ reagent. By a Baeyer–Villiger
oxidation, ketone 2 was converted into the corresponding lactone 3
by using m-chloroperoxybenzoic acid at room temperature. The
products in each step were easily purified by recrystallization,
and the yields were between 70% and 80%. The structure of the
[poly(CL-co-ACL)] bearing
c-amino groups. The obtained product
was washed with diethyl ether several times to remove impurities
and dried in vacuo at room temperature (yield: 70%).
1H NMR (CDCl3): d = 1.33–1.40 (2H, –CH2–CH2–CH2–CH2–O–),
1.64–1.86 [8H, –CH2–CH2– CH2–CH2–CH2–O–, –CH2–CH2–CH-
(NH2)–CH2–CH2–O–], 2.29–2.37 [4H, –C(O)–CH2–CH2–CH2–,
–C(O)–CH2–CH2–CH(NH)–], 3.77 [1H, –CH2–CH(NH2)–CH2–],
4.03–4.15 [4H, –CH2–CH2–CH2–O–, –CH(NH2)–CH2–CH2–O–].
IR (KBr): 3368 cmꢀ1, 737 cmꢀ1 (NH2), 1 733 cmꢀ1 (–C(O)O–).
2.8. (Cbz)reprotection of poly(CL-co-ACL)
A typical procedure for the reprotection of poly(CL-co-ACL) with
benzyl chloroformate is described as follows: poly(CL-co-ACL)
(0.5 g) was dissolved in 15 mL of CH2Cl2, then 0.5 mL of triethyl-
amine and 0.25 mL of CbzCl were added to the solution. The
Scheme 1. The general synthetic route for the monomer (
NaHCO3, H2O; (b) acetone, Jones’ reagent, 0 °C; (c) CH2Cl2, m-CPBA, 0 °C.
cCABeCL): (a) CbzCl,

J. Yan et al. / Reactive & Functional Polymers 70 (2010) 400–407
403
monomer was confirmed by the combined analysis of 1H NMR, 13
NMR, FT-IR, and mass spectrometry. The 1H NMR spectrum of
CA-
CL, shown in Fig. 1A, was consistent with the desired structure:
peaks g and h correspond to the benzyl protons in the Cbz group,
peak f is the proton of amide bond, and the remaining peaks are as-
C
3.2. Copolymerization of cCABeCL with CL
c
Be
Novel copolymers poly(CL-co-CABCL) with different composi-
tions were synthesized by ring-opening polymerization of CA-
CL and CL at 130 °C in bulk. Sn(Oct)2 was chosen as the
c
Be
signed to the corresponding protons on the
e
-caprolactone ring.
catalyst because of its high efficiency for lactones and low toxicity.
Scheme 2 shows the synthesis and the subsequent deprotection of
poly(CL-co-CABCL). Table 1 summarizes the results of the copoly-
The chemical structure was also confirmed by the 13C NMR spec-
trum, as shown in Fig. 1B. The peak at 175.57 ppm is the signal
of the ester carbonyl carbon, and the peak at 156.04 ppm is the
amide carbonyl carbon. The remaining peaks are assigned to the
merization of
cCABeCL and CL with various molar ratios of the
two monomers. It has been reported that the percentage contents
of the functional monomer for the further modification of polyes-
ters by attaching bioactive substances or drugs, such as RGD, biotin
and paclitaxel, was normally between 5 mol% and 10 mol% [27–
30]. In this article, with the goal of modifying PCL by incorporating
a certain amount of amino groups, we focused on the investigation
carbons of the benzyl group and the e-caprolactone ring. The infra-
red spectrum (Fig. 1C) showed two sharp peaks at 1719 and
1688 cmꢀ1 corresponding to the carbonyl groups of the ester and
amide bonds, respectively. In mass spectrometry, the presence of
the molecular ion (M+) peak at m/z = 263.1 and other fragment
ion peaks at m/z 156.1, m/z 128.1, m/z 91.1 (data not shown)
provided additional evidence for the chemical structure
of the monomer. Based on the HPLC analysis, the purity
of the molar feed ratios of cCABeCL/(cCABeCL + CL) below 25 mol%,
which was sufficient for the further biomodification [29]. Using 1H
NMR (Fig. 2A), the copolymer composition could be calculated
from the relative peak areas of the phenyl ring (–C6H5) protons
(99.9%) of cCABeCL was proved to be high enough for the success-
ful polymerization.
at 7.30–7.40 ppm of the
(–CH2–O–) protons at 1.3–1.43 ppm of the CL repeat units. It can
be seen that the molar fraction of the CAB CL unit in the copoly-
mer was slightly lower than the monomer feed ratio, indicating
that the reaction activity of CL was a little higher than that of CA-
CL under the reaction conditions we used. The polymerization
yields were relatively high, ranging from 80% to 95%. The products
we obtained were white solids [ CAB CL/( CAB CL + CL) < 19 -
mol%] or transparent viscous liquids [ CAB CL/( CAB CL + CL) >
cCABeCL repeat units and the methylene
c
e
c
Be
c
e
c
e
c
e
c
e
19 mol%]. The GPC curves of all the copolymers with different com-
positions exhibited a unimodal peak, indicating that the copoly-
mers had been exclusively obtained without homopolymers of
PCL or PCABCL. The molecular weight of the copolymers was in
the range of 1.9–2.4 ꢁ 104, and the polydispersity was 1.6–2.4.
The polydispersities of these polymers were relatively high, which
may be attributed to the difference in the reactivities of the two
monomers. The Cbz substituent at the
c position of the monomer
c
CAB CL may cause steric hindrance and lead to a decrease in reac-
e
tivity accordingly. Fig. 3 [C(I)] displays a typical GPC trace (Table 1,
entry 1).
The structure of the copolymers was characterized by measur-
ing 1H and 13C NMR spectra in CDCl3. Fig. 2A shows a typical 1H
NMR spectra of copolymer P(CL-co-CABCL) (Table 1, entry 3). The
Fig. 1. (A) 1H NMR spectrum of
c
CABe
CL monomer in CDCl3. (B) 13C NMR spectrum
CAB CL monomer. The arrows
of
c
CABe
CL monomer in CDCl3. (C) IR spectrum of
c
e
indicate the characteristic peaks of the monomer.
Scheme 2. Synthesis and biotinylation of poly(CL-co-ACL).

404
J. Yan et al. / Reactive & Functional Polymers 70 (2010) 400–407
based on their 13C NMR spectra analysis. The full 13C NMR spec-
Table 1
Bulk copolymerization of CL with
c
CAB
e
CL catalyzed by Sn(Oct)2.a
Copolymer
trum of the copolymer (Table 1, entry 3) is shown in Fig. 2B, and
all the corresponding peaks have been marked. As reported in pre-
vious literature studies [31,32], the sequences formed by the copo-
lymerization were thought to be the following four possible diads:
CL–CL, CABCL–CL, CL–CABCL, CABCL–CABCL. Fig. 2C shows the
comparison of the expanded 13C NMR spectrum of the ester car-
bonyl carbons of both homopolymers and copolymer (Table 1,
entry 6). Four resonance signals are observed in the carbonyl
region (173.48, 173.33, 173.29 and 173.07 ppm). The peak at
173.48 ppm is assigned to the diad CL-CL, which is the major se-
quence distribution in the copolymer. The peaks at 173.33 and
173.29 ppm are due to the CABCL–CL and CL–CABCL diads. The
equal intensity of these two diads illustrates the identical probabil-
ity of CL and CABCL linkage. The diad CABCL–CABCL corresponds to
the peak at 173.07 ppm, which shows a low intensity because of
Entry
c
CAB
e
CL Content (mol%)
Yield (%)
In polymerb
Mn
Mw
Mw/Mn
c
c
c
In feed
1
2
3
4
5
6
5
4.8
6.1
11.8
13.4
18.6
22.2
23,100
19,100
21,900
21,200
23,500
21,200
43,400
32,200
41,800
35,900
58,300
41,900
1.88
1.68
1.91
1.69
2.40
1.97
95
88
90
89
85
80
7.5
12.5
14
19.5
24.5
a
Copolymerization was carried out in bulk at 130 °C for 24 h. Molar ratio of
Sn(Oct)2 to monomer (CL + cCABeCL) was 1/1000.
b
Calculated using 1H NMR (CD Cl3).
Calculated using GPC (THF as eluent).
c
signals at 7.30–7.40 ppm and 5.10 ppm are assigned to the protons
of the Cbz group present in the CAB CL repeat units. The signal at
the low cCABeCL content in the feed composition in the present
c
e
study. In addition, the sequence distribution of the copolymer
can also be assessed by 13C NMR analysis in the range of 60–
65 ppm for the oxymethylene carbon atom, displayed in Fig. 2D.
Four resonance peaks were found (64.32, 64.07, 61.36 and
61.15 ppm). Compared with the homopolymers of PCL (diad CL–
CL: 64.06 ppm) and PCABCL (diad CABCL–CABCL: 61.39 ppm) [the
4.8 ppm is assigned to the protons of the amide bond. The remain-
ing peaks are the signals for the corresponding protons of the
copolymer backbone. In order to determine the sequence distribu-
tion of the copolymer, we also synthesized the homopolymers of
PCL and PCABCL. The distribution of the major resonances was
Fig. 2. (A) 1H NMR spectrum of copolymer P(CL-co-CABCL) in CDCl3 (Table 1, entry 3). (B) 13C NMR spectrum of P(CL-co-CABCL) in CDCl3 (Table 1, entry 3). (C) Expanded 13
C
NMR spectrum in the carbonyl carbon region (Table 1, entry 6) compared with the homopolymers of PCL and PCABCL. (D) Expanded 13C NMR spectrum in the oxymethylene
carbon region (Table 1, entry 6) compared with the homopolymers of PCL and PCABCL. (E) Expanded 13C NMR spectrum in the carbonyl carbon region (Table 1, entry 3). C
represents CL, B represents CABCL.

J. Yan et al. / Reactive & Functional Polymers 70 (2010) 400–407
405
CL diad was observed at this level. From these results, the
copolymers poly(CL-co-CABCL) are considered to be statistical
copolymers [28].
3.3. Deprotection by hydrogenolysis
The Cbz group is one of the most commonly used amino pro-
tecting groups in a variety of synthetic strategies. Several methods
to remove Cbz groups have been reported in the literature [33,34].
In this paper, deprotection of the NH2 groups was carried out by
hydrogenolysis with Pd/C (10%) as the catalyst and H2 as the
hydrogen donor in THF/CH3OH. 1H NMR and FT-IR spectra con-
firmed the successful removal of the Cbz groups in the copolymer.
As shown in Fig. 3A, no proton signals are observed at 7.30–7.40
and 5.10 ppm, which are the regions corresponding to the aromatic
and the methylene protons of the Cbz group, respectively, yet the
other signals are basically unchanged. In the FT-IR spectra
(Fig. 3B), the mCH vibration at 3064.9, 3034.5 cmꢀ1 and the cCH
vibration at 740.0, 699.4 cmꢀ1 of the benzene rings disappeared
after hydrogenation, also indicating the absence of the Cbz group
in the P(CL-co-ACL) copolymer [27]. The vibration at 737.5 cmꢀ1
is the wagging vibration of the primary amine. Reprotection of
poly(CL-co-ACL) with benzyl chloroformate was carried out to
investigate the possibility of degradation during the reaction. Typ-
ical GPC curves of these copolymers are shown in Fig. 3C. The slight
change in the molecular weight between the protected and depro-
tected polymer (from 23,100 to 18,000) could be attributed to
either the loss of Cbz groups or the interaction between the amino
groups of polymer and the filler (PS) of GPC [35]. After reprotec-
tion, the reprotected copolymer had a similar molecular weight
to the protected copolymer. Additionally, the GPC trace of the
deprotected copolymer still exhibits a unimodal peak, which hints
that the degradation of the copolymer could be negligible [27]. In
summary, there is no obvious chain scission occurring during
hydrogenation. All these results demonstrated that the Cbz groups
were completely removed, and the deprotection was successful.
3.4. Thermal properties of the copolymers
The thermal behavior of the copolymers before and after depro-
tection was studied by DSC analysis. Table 2 summarizes the com-
position dependence of Tg, Tm and
DHm of the polymers measured
by DSC. Fig. 4A shows the DSC curves of some typical samples be-
fore protection. As expected, the homopolymer PCL was a semi-
crystalline material with a melting temperature (Tm) at 58.1 °C
and a glass transition temperature (Tg) at ꢀ62.9 °C. The homopoly-
mer PCABCL was completely amorphous, with a Tg at ꢀ2.4 °C. The
melting temperature was found to decrease with the increase of
the
c
CABe
CL content in the copolymer. Especially when the con-
tent of
c
CAB CL increased to about 20 mol%, no melting peaks
e
were observed, and the morphology of the copolymers changed
from semi-crystalline to amorphous. This change might be attrib-
Fig. 3. (A) 1H NMR spectrum of copolymer P(CL-co-CABCL) (Table 1, entry 1) after
deprotection in CDCl3. (B) FT-IR spectra of (I) P(CL-co-CABCL) and (II) P(CL-co-ACL)
(Table 1, entry 5). (C) GPC curves of (I) P(CL-co-CABCL), (II) P(CL-co-ACL) and (III)
reprotection of P(CL-co-ACL) (Table 1, entry 1).
utable to the random
cCABeCL component in the copolymers
intensively disrupting the crystallinity of the PCL chain [29].
In addition, we also studied the thermal properties of the depro-
tected copolymers, as shown in Fig. 4B. The Tg of the deprotected
polymers was higher than that of PCL (Tg = ꢀ62.8 °C). This result
was due to the formation of intermolecular hydrogen bonds be-
tween the amino groups, which hindered the movement of the
PCL chain [37]. The fact that Tm of the deprotected copolymer
was sharper and higher than that of the protected one was attrib-
uted to the increase of the weight percentage of CL units after re-
moval of Cbz group [38]. As shown in Fig. 4, a unique Tg was
detected for both the protected and deprotected copolymers,
which further indicates that the copolymers have a random
structure [27,36].
illustrations in Fig. 2D], the additional peaks observed in the ex-
panded 13C NMR spectrum of the copolymer are assigned to the
diads of CL–CABCL (64.32 ppm) and CABCL–CL (61.15 ppm) due
to the sensitivity of the resonance of the oxymethylene carbon of
each unit. The expanded carbonyl group region of the 13C NMR
spectrum of the copolymer (Table 1, entry 3) is shown in Fig. 2E.
Compared with Fig. 2C, we found that the signal intensity of the
CABCL–CL and CL–CABCL diads weakened with decreasing the
cCABeCL molar content to 12% in copolymers, and no CABCL–CAB-

406
J. Yan et al. / Reactive & Functional Polymers 70 (2010) 400–407
Table 2
Melting temperature, glass transition temperature, and enthalpy melting for copolymers P(CL-co-CABCL) and P(CL-co-ACL).
Entry
F
c
CABe
CLa
P(CL-co-CABCL)
Tm (°C)b
P(CL-co-ACL)
Tm (°C)b
Tg (°C)b
D
Hm (J/g)b
Tg (°C)b
D
Hm (J/g)b
1
2
3
4
5
6
7
0
4.5
9.5
14
18.5
22.5
100
58.1
55.3
43.6
38.2
–
ꢀ62.9
ꢀ55.5
ꢀ44.9
ꢀ43.0
ꢀ40.7
ꢀ35.6
ꢀ2.4
83.8
77.4
47.5
20.2
–
60.9
49.9
42.7
16.0
–
ꢀ52.4
ꢀ50.4
ꢀ41.4
ꢀ39.5
ꢀ39.0
ꢀ11.8
83.5
59.4
23.2
3.6
–
–
–
–
–
–
–
a
The molar content of
DSC carried out under N2 at 10 °C/min from ꢀ80 to 100 °C (second heating run).
c
CAB
e
CL in the copolymer determined by 1H NMR.
b
Fig. 5. 1H NMR spectra of biotin (A) and the P(CL-co-ACL)/biotin conjugate (B) in
DMSO-d6 (Table 1, entry 4).
carboxyl groups of biotin disappeared, and the proton peaks of
the amide bond appeared at 7.6 ppm. Because the unreacted biotin
or NHS-biotin could be removed by dialysis, the 1H NMR spectrum
indicates that biotin was grafted onto the copolymer P(CL-co-ACL)
successfully [28,39]. Analogously, other bioactive molecules could
also be introduced onto PCL.
4. Conclusion
In conclusion, we have designed and synthesized a novel func-
tional lactone,
CL), in three steps. The copolymers poly(CL-co-CABCL) were
prepared by ring-opening polymerization of CAB CL and CL using
c-(carbamic acid benzyl ester)-e-caprolactone (cCA-
Be
Fig. 4. (A) DSC trace of P(CL-co-CABCL) polymers. (I) Entry 1, (II) entry 2, (III) entry
3, (IV) entry 5, (V) entry 7 from Table 2. (B) DSC trace of P(CL-co-ACL) polymers. (I)
Entry 2, (II) entry 3, (III) entry 5, (IV) entry 7 from Table 2.
c
e
Sn(Oct)2 as catalyst in bulk at 130 °C. In addition, the structures of
the monomer and copolymers were extensively investigated. The
incorporation of the cCABeCL unit into PCL resulted in an alteration
of the morphology from semi-crystalline to amorphous. The Cbz
protecting groups were successfully removed by hydrogenolysis
in the presence of Pd/C to obtain PCL with free primary amino
groups along the backbone. The pendant amino groups were used
for the attachment of biotin to the polymer. Further investigation
of the biological properties of the novel polymer is still in progress.
3.5. Biotinylation
The free amino groups on the copolymer chains are capable of
functionalization, which provides opportunities for covalent
attachment of biological and drug molecules. To explore the activ-
ity of the free amino groups, biotin was chosen to conjugate with
the amino groups of the copolymer. Fig. 5 shows the 1H NMR spec-
tra of biotin (A) and the P(CL-co-ACL)/biotin conjugate (B). Com-
paring Figs. 3A and 5A, the characteristic peaks of biotin and
P(CL-co-ACL) could all be observed in the 1H NMR spectrum of bio-
tinylated P(CL-co-ACL). In Fig. 5B, the signals at 11.8 ppm for the
Acknowledgements
This research was supported by the Fundamental Research
Funds for the Central Universities, the National Natural Science

J. Yan et al. / Reactive & Functional Polymers 70 (2010) 400–407
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