Efficient RAFT polymerization of N-(3-aminopropyl)methacrylamide hydrochloride using unprotected "clickable" chain transfer agents

Article abstract of DOI:10.1016/j.reactfunctpolym.2014.04.001

The reversible addition fragmentation chain transfer (RAFT) of N-(3-aminopropyl)methacrylamide hydrochloride (APMA) using unprotected "clickable" chain transfer agents in water/dioxane mixtures is reported. The controlled character of the polymerization was confirmed by the linear increase of the polymer molecular weight with monomer conversion, the narrow molecular weight distribution (≤ 1.1) and by chain extension experiments. The alkyne-terminated PAPMA was further functionalized by "click" chemistry with an azido-functionalized coumarin derivative. The method reported here will be useful for the preparation of novel PAPMA based materials for biomedical applications using a strategy that does not require challenging protection/deprotection steps.

Full text of DOI:10.1016/j.reactfunctpolym.2014.04.001

Reactive & Functional Polymers 81 (2014) 1–7
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Reactive & Functional Polymers
journal homepage: www.elsevier.com/locate/react
Efficient RAFT polymerization of N-(3-aminopropyl)methacrylamide
hydrochloride using unprotected ‘‘clickable’’ chain transfer agents
Patrícia V. Mendonça a, Arménio C. Serra a, Anatoliy V. Popov b, Tamaz Guliashvili a, Jorge F.J. Coelho a,
⇑
a CEMUC, Department of Chemical Engineering, University of Coimbra, Polo II, Pinhal de Marrocos, 3030-790 Coimbra, Portugal
b Department of Radiology, University of Pennsylvania, Philadelphia, PA 19104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
The reversible addition fragmentation chain transfer (RAFT) of N-(3-aminopropyl)methacrylamide
hydrochloride (APMA) using unprotected ‘‘clickable’’ chain transfer agents in water/dioxane mixtures
is reported. The controlled character of the polymerization was confirmed by the linear increase of the
polymer molecular weight with monomer conversion, the narrow molecular weight distribution
(Ð 6 1.1) and by chain extension experiments. The alkyne-terminated PAPMA was further functionalized
by ‘‘click’’ chemistry with an azido-functionalized coumarin derivative. The method reported here will be
useful for the preparation of novel PAPMA based materials for biomedical applications using a strategy
that does not require challenging protection/deprotection steps.
Received 24 January 2014
Received in revised form 31 March 2014
Accepted 2 April 2014
Available online 13 April 2014
Keywords:
N-(3-aminopropyl)methacrylamide
hydrochloride
Reversible addition fragmentation chain
transfer
Ó 2014 Elsevier Ltd. All rights reserved.
‘‘Click’’ chemistry
Primary amine polymer
Coumarin
1. Introduction
which is typically catalyzed by copper(I) complexes [15]. This type
of ‘‘click’’ chemistry reaction became very attractive due to its high
Reversible deactivation radical chain polymerization (RDRP) [1]
has been studied for the past two decades as a powerful tool for the
preparation of complex polymer structures with well-defined
molecular weights, molecular weight distributions, compositions,
architectures, topologies and chain-end functionalities. Reversible
addition fragmentation chain transfer (RAFT) [2] is one of the most
versatile RDRP techniques due to its tolerance to different func-
tionalities, the range of monomers that can be polymerized and
the diversity of affordable polymer architectures [3]. Several poly-
mers, such as poly(meth)acrylates [4,5], poly(meth)acrylamides
[6–9] and polystyrene [10,11], have been successfully synthesized
by RAFT polymerization and used in numerous applications. Spe-
cial attention has been given to polymers with functionalized
chain-ends that can be used in further post-modification reactions
[12–14] to produce unique polymer structures.
yield, mild reaction conditions and absence of by-products [16].
Click strategies are particularly useful for the synthesis of well-
defined block copolymers [17]. Using this technique, it is possible
to prepare block copolymers that combine homopolymers with
very distinct natures, derived from monomers with different reac-
tivity. Additionally, it is possible to maintain the low dispersity (Ð)
of block copolymers because the building blocks can be synthe-
sized separately using suitable RDRP techniques and further cou-
pled by click reactions [18]. The combination of RDRP and click
chemistry has been extensively reported in the literature [19–
23], as it allows the design of high performance materials with very
unique structures and properties that can be applied in relevant
areas (e.g., the biomedical field) [24–26].
Polymers with pendant primary amino groups, which can be
used for post-polymerization modification reactions [27–29], such
as Michael addition reactions, are of prime importance for the
development of new materials for biomedical applications. For
One of the most popular post-polymerization reactions involv-
ing polymers prepared by RDRP is the Huisgen 1,3-dipolar cycload-
dition between azide and acetylene chain-end functionalities,
instance,
N-(3-aminopropyl)methacrylamide
hydrochloride
(APMA) has been used in the preparation of copolymers and
cross-linked micelles for gene delivery [30,31] and drug delivery
[32–34]. The controlled synthesis of this polymer is usually per-
formed by RAFT polymerization mediated by 4-cyano-4-(phen-
ylcarbonothioylthio)pentanoic acid (CTP) [35] or by RAFT
⇑
Corresponding author. Address: Department of Chemical Engineering, Univer-
sity of Coimbra, Polo II, Pinhal de Marrocos, 3030-790 Coimbra, Portugal. Tel.: +351
239 798 744; fax: +351 239 798 703.
E-mail address: jcoelho@eq.uc.pt (J.F.J. Coelho).
http://dx.doi.org/10.1016/j.reactfunctpolym.2014.04.001
1381-5148/Ó 2014 Elsevier Ltd. All rights reserved.

2
P.V. Mendonça et al. / Reactive & Functional Polymers 81 (2014) 1–7
copolymerization with other (meth)acrylamide monomers [30–
32,36,37] or with 2-(diisopropylamino)ethyl methacrylate
[33,34]. The post-polymerization modification of controlled/‘‘liv-
ing’’ poly(3-aminopropyl methacrylamide hydrochloride) (PAPMA)
typically involves reaction of the amino groups to either prepare
shell cross-linked micelles [34] or to attach molecules of interest
(e.g., D-glucuronic acid sodium salt) [38] for the studied applica-
tion. To the best of our knowledge, the chain-ends of controlled/
‘‘living’’ PAPMA have never been used in post-polymerization mod-
ification reactions.
5-mm TIX triple resonance detection probe in D2O. The conversion
of the monomers was determined by integration of the monomer
and polymer NMR signals using MestRenova software version
6.0.2-5475.
Fourier transform infrared attenuated total reflection (FTIR-
ATR) spectroscopy was performed using a Jasco model 4000 UK
spectrometer. The samples were analyzed with 64 scans and
4 cmꢀ1 resolution between 500 and 3500 cmꢀ1
.
2.3. Procedures
Here, we report the successful synthesis of PAPMA by RAFT
polymerization mediated by non-protected acetylene or azide
functionalized chain transfer agents (CTAs) to allow further modi-
fication of the PAPMA chain-ends. An alkyne-terminated PAPMA
was coupled with a biocompatible coumarin derivative via cop-
per(I) catalyzed azide alkyne cycloaddition to demonstrate the
usefulness of the strategy. The primary goals of this work were
to develop suitable polymerization conditions and a facile proce-
dure for the preparation of controlled chain-end functionalized
PAPMA in order to allow further chemical modifications, expand-
ing the range of applications of this polymer [39].
2.3.1. The synthesis of the alkyne-CTP
The synthesis of the alkyne-terminated chain transfer agent
was adapted from a procedure described in the literature [41].
Briefly, a mixture of CTP (700 mg, 2.51 mmol), PgOH (170 mg,
3.01 mmol) and DCM (40 mL) was added to a round bottom flask
equipped with a magnetic bar and a rubber stopper. The solution
was cooled to 0 °C and purged with argon. A solution of EDC
(720 mg, 3.76 mmol) and DMAP (50 mg, 0.38 mmol) in DCM
(10 mL) was added to the flask under argon atmosphere. The mix-
ture was allowed to react at 0 °C for 2 h and then at room temper-
ature overnight. The reaction mixture was washed with water
(100 mL, 3 times) and dried over anhydrous Na2SO4. The DCM
was removed under reduced pressure and the crude product was
purified by column chromatography (SiO2, CH2Cl2 and hexane/
ethyl acetate = 4/1 (v/v)). The pure product (0.60 g, 75%) was ana-
lyzed by 1H NMR (400 MHz, CDCl3), d (TMS, ppm): 1.94 (s, 3H,
(CN)CACH3); 2.49 (t, 1H, HCBC); 2.5–2.8 (t(x2), 4H, ACH2ACH2);
4.72 (d, 2H, CH2AOAC); 7.4–7.9 (t (x2), d, 5H, ArH).
2. Experimental section
2.1. Materials
Glacial acetic acid (Fisher Scientific, 99.79%), acetone (Fisher
Scientific, HPLC grade), APMA (Polysciences, >98%), deuterated
chloroform (CDCl3, Euriso-top, 99.50% D), 3-chloro-1-propanol
(Aldrich, 98%), copper (II) sulfate pentahydrate (P98%, Aldrich),
CTP (Sigma–Aldrich, >97%), dichloromethane (DCM) (Fisher Scien-
tific, 99.99%), diethyl ether (Fisher Scientific, 99.85%), 4-dimethyl-
aminopyridine (DMAP, Acros Organics, 99%), deuterium oxide
(D2O, Euroiso-top, 99.90% D), 1-ethyl-3-(3-dimethylaminopro-
pyl)carbodiimide (EDC, Sigma–Aldrich, P98.0%), ethanol (EtOH,
absolute, Fisher Chemical), ethyl acetate (99.98%, Fisher Scientific),
hexane (Fisher Scientific, 99.05%), propargyl alcohol (PgOH)
2.3.2. The synthesis of the azido-CTP
The synthesis of the azide-terminated chain transfer agent was
adapted from a procedure described in the literature [41]. First, 3-
chloro-1-propanol (3 g, 31.7 mmol) and NaN3 (3.5 g, 54.0 mmol)
were dissolved in a mixture of acetone (50 mL) and water (5 mL)
and refluxed overnight. The acetone was removed under reduced
pressure, and 35 mL of water were added to the remaining solu-
tion. The product was extracted with diethyl ether (3 ꢁ 70 mL),
the organic layer was dried over anhydrous Na2SO4, and the 3-
azido-1-propanol was obtained as a colorless oil (1.6 g, 50%) after
solvent removal under reduced pressure. The product was ana-
(Aldrich, 99%), silica gel (Panreac, 63–200 lm), sodium ascorbate
(crystalline, P98%, Aldrich), sodium azide (Panreac, 99%), and
sodium sulfate (anhydrous) were used as obtained.
1,4-Dioxane (Sigma–Aldrich, 99+%) was passed through an acti-
vated alumina column before use to remove any peroxides.
4,40-Azobis(4-cyanovaleric acid) (ACVA) (Aldrich, 75%) was
recrystallized from methanol before use.
lyzed by FTIR:
m
max/cmꢀ1 3340, 2949, 2884, 2098, 1449, 1262,
1051, 955, 900. In a second step, a mixture of CTP (700 mg,
2.51 mmol), 3-zido-1-propanol (380 mg, 3.76 mmol) and DCM
(40 mL) was added to a round flask equipped with a magnetic
bar and a rubber stopper. The solution was cooled to 0 °C and
purged with argon. A solution of EDC (720 mg, 3.76 mmol) and
DMAP (50 mg, 0.38 mmol) in DCM (10 mL) was added to the flask
under argon atmosphere. The mixture was allowed to react at 0 °C
for 2 h and then at room temperature overnight. The reaction mix-
ture was washed with water (100 mL, 3 times) and dried over
anhydrous Na2SO4. The DCM was removed under reduced pres-
sure, and the crude product was purified by column chromatogra-
phy (SiO2, CH2Cl2 and hexane/ethyl acetate = 4/1 (v/v)). The pure
product (0.61 g, 67%) was analyzed by 1H NMR (400 MHz, CDCl3),
d (TMS, ppm): 1.94 (s, 3H, AC(CN)CH3); 2.04–2.18 (m, 2H, ACH2-
ACH2AN3); 2.30–2.80 (m, 4H, (CN)CACH2ACH2AC(@O)); 3.60 (t,
2H, CH2AN3); 4.28 (t, 2H, ACH2ACH2A CH2AN3); 7.40–7.90 (t
(x2), d, 5H, ArH).
Deionized water was obtained from a Milli-QÒ Millipore reverse
osmosis unit (resistivity = 18.0 MO).
3-azido-7-diethylaminocoumarin was synthesized as reported
in the literature [40].
2.2. Techniques
The polymers were analyzed by a size exclusion chromatogra-
phy (SEC) system equipped with an online degasser, a refractive
index (RI) detector and a set of columns comprising a Shodex
OHpak SB-G guard column and OHpak SB-804HQ and OHpak SB-
804HQ columns. The polymers were eluted at a flow rate of
0.5 mL/min with 0.1 M Na2SO4 (aq)/1 wt% acetic acid/0.02% NaN3
at 40 °C. Before the injection (50
through polytetrafluoroethylene (PTFE) membrane with
0.45
lL), the samples were filtered
a
l
m pores. The system was calibrated with five narrow PEG
2.3.3. Typical procedure for the RAFT polymerization of APMA with
[APMA]0/[CTP]0/[AVCA]0 = 1/1/0.5 ([APMA]0 = 1.87 M; H2O:1,4-
dioxane = 2:1 (v:v))
APMA (0.5 g, 2.80 mmol) was dissolved in deionized water
(1 mL), CTP (7.82 mg, 0.028 mmol) was dissolved in 1,4-dioxane
(0.5 mL), and both solutions were inserted into a Schlenk tube
standards, and the polymer molecular weights (MSnEC) and Ð (Mw/
Mn) were determined by conventional calibration using Clarity
software version 2.8.2.648.
400 MHz 1H NMR spectra of the reaction mixture samples were
recorded on a Bruker Avance III 400 MHz spectrometer with a

P.V. Mendonça et al. / Reactive & Functional Polymers 81 (2014) 1–7
3
reactor. AVCA (1.57 mg, 0.0056 mmol) was weighed and added to
the reactor, which was subsequently sealed and frozen in liquid
nitrogen. The Schlenk tube reactor containing the reaction mixture
was deoxygenated with four freeze–vacuum–thaw cycles and
purged with nitrogen. The Schlenk tube reactor was placed in an
oil bath at 70 °C under stirring (700 rpm). Different reaction mix-
ture samples were collected during the polymerization using an
airtight syringe and purging the side arm of the Schlenk reactor
with nitrogen. The samples were analyzed by 1H NMR spectros-
copy to determine the monomer conversion and by aqueous SEC
to determine the molecular weights and dispersity of the polymers.
The final reaction mixture was dialyzed against deionized water,
and the polymer was obtained after freeze drying.
segments, mainly with the purpose to be used on the biomedical
field [30–34,36,37]. In this work, the RAFT system described in
the literature for the successful polymerization of PAPMA [35,36]
was used as a starting point for the evaluation of the synthesized
‘‘clickable’’ CTAs chain transfer activity. Alkyne and azido CTP
derivatives (Fig. 1) were prepared by esterification with PgOH
(with no protection of the alkyne termini) and 3-azido-1-propanol,
respectively, following procedures reported elsewhere [41] (see
Fig. S1 in the ESI ). The chemical structure and purity of the CTAs
were confirmed by 1H NMR spectroscopy (see Fig. S2 in the ESI ) to
ensure that all RAFT polymerizations were performed using the
same conditions and were free of significant impurities, allowing
data comparison. The polymerizations were conducted at 70 °C
using a solvent mixture of water:1,4-dioxane = 2:1 (v:v) and ACVA
as the initiator under nitrogen atmosphere. The pH of the reaction
mixture was measured before the polymerization to ensure a value
between 4 and 5 in order to avoid CTP hydrolysis and/or aminolysis
[43]. Notably, no pH adjustment was necessary during the poly-
merization. The monomer conversions were determined using 1H
NMR spectroscopy by comparing the integrals of the vinyl protons
of the monomer at 5.80–5.35 ppm and those of the methylene pro-
tons of the PAPMA backbone at 1.25–0.70 ppm.
The other RAFT polymerizations were conducted employing the
same procedure described but using alkyne-CTP or azido-CTP as
the chain transfer agent.
2.3.4. Typical procedure for the chain extension of PAMA
A sample of alkyne-terminated PAPMA (MSnEC ¼ 10; 400; Mw/
Mn = 1.19), obtained after dialysis and freeze drying, was used as
the macro-CTA in a new RAFT polymerization. Briefly, the macro-
CTA (43.7 mg, 0.0042 mmol) and APMA (300 mg, 1.70 mmol) were
Initially, both alkyne-CTP and azido-CTP were tested in the
RAFT polymerization of APMA for different targeted degrees of
polymerization (DP) using the same experimental conditions. The
kinetic parameters presented in Table 1 shows that both CTAs were
able to mediate successful RAFT polymerization of APMA for differ-
ent chain lengths, as judged by the very low Ð values. Polymeriza-
tion control decreased for shorter polymer chains, as expected, but
still afforded PAPMA with a narrow molecular weight distribution
(Ð ꢂ 1.2).
dissolved in
a mixture of water (1.93 mL) and 1,4-dioxane
(0.97 mL) inside a Schlenk tube reactor. Subsequently, AVCA
(0.59 mg, 0.0021 mmol) was weighed and added to the reactor,
which was immediately frozen in liquid nitrogen. The Schlenk tube
reactor containing the reaction mixture was deoxygenated with
four freeze–vacuum–thaw cycles, purged with nitrogen and placed
in an oil bath at 70 °C under stirring (700 rpm). After 24 h of reac-
tion, a sample was collected and analyzed by SEC in order to
observe the movement of the SEC trace toward a higher molecular
weight relative to that of the macro-CTA.
Kinetic studies were conducted to evaluate the usefulness of the
synthesized CTAs for the control of the PAPMA molecular weight
during polymerization. In our hands and under the experimental
conditions used in this work, the maximum monomer conversion
achieved (red symbols in Fig. 2) in the RAFT of APMA mediated
by CTP was lower than that reported in the literature [35]. Taking
this into consideration, the reaction was repeated three times, and
the same results were obtained in all cases. Nevertheless, the con-
trol over the polymer molecular weight was very good, as the Ð
was always lower than 1.1, with the exception of the first kinetic
point (Fig. 2(b)). In addition, the SEC traces (see Fig. S3 in the
ESI ) of the different samples were unimodal and symmetric with
no tailing, suggesting that no termination reactions occurred dur-
ing polymerization. In terms of the monomer conversion evolution,
the results were consistent with the literature, showing first-order
kinetics with respect to monomer conversion (Fig. 2(a)).
It has been reported that the use of unprotected alkyne-termi-
nated CTAs can interfere with the RAFT polymerization process
[41]. The synthesized unprotected alkyne-CTP was used to mediate
the RAFT polymerization of APMA under the same conditions pre-
viously described for the CTP. The kinetic results presented in Fig. 2
(black symbols) show that the alkyne-CTP had higher chain trans-
fer activity than the CTP, with high monomer conversion (ꢂ95%)
achieved after 4 h of reaction. This behavior can be attributed to
the strong dependence of the efficiency on the CTA R group, con-
sidering that the two CTAs have the same Z group [3]. The polymer
molecular weight was well controlled throughout polymerization
2.3.5. Click reaction between alkyne-terminated PAPMA and (3-azido-
7-diethylaminocoumarin)
An alkyne-terminated PAPMA sample (MSnEC ¼ 8:7 ꢁ 103;
Ð = 1.08) was purified by dialysis against deionized water, and
the polymer was recovered after freeze drying. A solution of 3-
azido-7-diethylaminocoumarin (0.6 mg; 2.2
nated PAPMA (15.6 mg; 1.79 mol) in deionized water (100
EtOH (300 L), and a stock solution of CuSO4.5H2O (28 mM;
100 L) in deionized water were placed in a round-bottom flask
lmol), alkyne-termi-
l
l
L)/
l
l
equipped with a magnetic stir bar and sealed with a rubber sep-
tum. The mixture was bubbled with nitrogen for 20 min to remove
oxygen. Lastly, a degassed stock solution of sodium ascorbate in
water (11 mM; 100 lL) was injected into the flask under nitrogen
atmosphere. The reaction was allowed to proceed with stirring
(700 rpm) at room temperature for 96 h. The product was purified
by dialysis against water, followed by dialysis against ethanol and
a second dialysis against water, then recovered by freeze drying.
The functionalized polymer was analyzed by 1H NMR
spectroscopy.
3. Results and discussion
3.1. RAFT homopolymerization of APMA
CTP, which contains a phenyl Z group that strongly stabilizes
the intermediate radical, has previously been used for the success-
ful RAFT polymerization of a wide range of activated monomers,
including both acrylamide and methacrylamide monomers
[8,35,42]. To the best of our knowledge, there are only two reports
that show kinetic data regarding the RAFT of APMA using CTP as a
chain transfer agent [35,36]. Other publications have reported the
synthesis of responsive block copolymers including PAPMA
Fig. 1. Chemical structures of both alkyne-CTP and azido-CTP.

4
P.V. Mendonça et al. / Reactive & Functional Polymers 81 (2014) 1–7
Table 1
Kinetic parameters for the RAFT polymerization of APMA using azido-CTP or alkyne-CTP in water:1,4-dioxane = 2:1 (v:v) at 70 °C. Conditions: [APMA]0 = 1.87 M; [ACVA]0 = 0.5
(molar ratio in comparison to the CTA number of moles).
MSnEC ꢁ 10ꢀ3
Entry
CTA
Targeted DP
Time (h)
Conv. (%)
Ð
a
BPAPMA25
BPAPMA50
BPAPMA100
N3PAPMA25
N3PAPMA100
Alkyne-CTP
Alkyne-CTP
Alkyne-CTP
Azido-CTP
Azido-CTP
25
50
100
25
5
18
4
4
4
82
98
95
94
50
10.4
29.4
25.1
16.2
19.1
1.19
1.08
1.07
1.21
1.11
100
a
An ACVA molar ratio of 0.2 was used in this polymerization.
Fig. 2. (a) Kinetic plots of ln[M]0/[M] vs. time and (b) plot of number–average molecular weight (MSEC) and Ð (Mw/Mn) vs. monomer conversion (the dashed line represents
n
the theoretical molecular weight at a given conversion) for the RAFT of APMA at 70 °C in a water:1,4-dioxane = 2:1 (v:v) mixture using CTP (red symbols) and alkyne-CTP
(black symbols) as chain transfer agents (CTA). Reaction conditions: [APMA]0/[CTA]0/[ACVA]0 = 100/1/0.5; [APMA]0 = 1.87 M. (For interpretation of the references to color in
this figure legend, the reader is referred to the web version of this article.)
(Ð < 1.1), and the molecular weight distribution was characterized
by unimodal SEC traces (see Fig. S4 in the ESI ). Despite the low Ð
values, the experimental molecular weights of PAPMA determined
by SEC were not in close agreement with the corresponding theo-
retical ones (Fig. 2(b)). The observed differences can be explained
considering that the molecular weight values were determined
by conventional calibration using PEG standards, which have a dif-
ferent hydrodynamic volume than PAPMA. These results are extre-
mely promising, demonstrating that alkyne-terminated PAPMA
can be prepared and directly used in further click reactions without
the need for deprotection steps.
showed that azide groups can undergo 1,3-cycloaddition to the
double bonds of some monomers in the absence of a catalyst, espe-
cially for long reaction times and high temperatures [44]. In the
case of APMA RAFT polymerization, the results suggested that
the occurrence of this type of side reaction can be neglected, as
the
Ð of the polymer decreased with polymerization time
(Fig. 3(b)). The low reactivity of acrylamides as well as the pres-
ence of the methylene group at the double bond of APMA, which
causes steric hindrance, may prevent this type of side reaction. In
addition, it was possible to confirm the polymer functionality by
identifying the ACH2 protons near the N3 group by 1H NMR spec-
troscopy (Fig. 5(b)).
Alternatively, to use the alkyne functionality, one can take
advantage of azide terminated polymers for click chemistry pur-
poses. An experimental study reported by Ladmiral and co-workers
Nevertheless, the kinetic results showed that the azido-CTP did
not allow the same level of control over the PAPMA molecular
Fig. 3. (a) Kinetic plots of ln[M]0/[M] vs. time and (b) plot of the number–average molecular weight (MSEC) and Ð (Mw/Mn) vs. monomer conversion (the dashed line
n
represents the theoretical molecular weight at a given conversion) for the RAFT of APMA at 70 °C in water:1,4-dioxane = 2:1 (v:v) (black symbols) and water:1,4-
dioxane = 1:1 (v:v) (red symbols) mixtures. Reaction conditions: [APMA]0/[azido-CTP]0/[ACVA]0 = 100/1/0.5; [APMA]0 = 1.87 M. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)

P.V. Mendonça et al. / Reactive & Functional Polymers 81 (2014) 1–7
5
weight as did both CTP and alkyne-CTP for the same experimental
conditions (black symbols in Fig. 3). The Ð of the polymers was
slightly higher, primarily due to the presence of tailing in the
molecular weight distribution curves (Fig. 4(a)). This observation
could be due to radical termination reactions that occurred in the
beginning of the polymerization. By comparing the area of the
low molecular weight shoulder with the total area of the SEC chro-
matogram, it was possible to estimate that after 30 min of poly-
merization, there were ꢂ27% dead polymer chains. We
anticipated that the poor control exhibited by the azido-CTP could
be associated with the low solubility of this compound in the reac-
tion solvent mixture, even at 70 °C. In comparison with both CTP
and alkyne-CTP, the azido-CTP R group has a longer aliphatic chain,
which decreases its solubility in water. To confirm this hypothesis,
the effect of the solvent mixture composition on the control of the
RAFT polymerization of PAPMA mediated by the azido-CTP was
also examined. The percentage of dioxane in the solvent mixture
was increased up to 50%, and the other experimental conditions
were conserved. The results showed that significant improvements
were achieved in terms of polymerization control, with a 64%
decrease in the PAPMA dead chains (Fig. 4(b)) as well as a 39%
increase in the maximum monomer conversion (red symbols in
Fig. 3). In addition, better control was achieved, as evidenced by
the lower Ð of the PAPMA and the agreement between the exper-
imental molecular weights and the theoretical ones (dashed line in
Fig. 3(b)). These results corroborate the idea that the poorer control
exhibited by the azido-CTP in this particular RAFT system was
related to its solubility in the reaction solvent mixture. Some
authors have suggested that the marked chain transfer activity dif-
ferences between clickable CTAs derived from the same molecule
Fig. 4. SEC chromatograms of the PAPMA samples during the RAFT polymerization at 70 °C in (a) a water:1,4-dioxane = 2:1 (v:v) mixture and (b) a water:1,4-dioxane = 1:1
(v:v) mixture. Conditions: [APMA]0/[azido-CTP]0/[ACVA]0 = 100/1/0.5; [APMA]0 = 1.87 M.
Fig. 5. 400 MHz 1H NMR spectra in D2O of (a) the alkyne-terminated PAPMA obtained by RAFT polymerization using [APMA]0/[alkyne-CTP]0/[ACVA]0 = 25/1/0.2 (molar
ratios) in water:1,4-dioxane = 2:1 (v:v) at 70 °C for 5 h. Conv. = 82%; Mnth ¼ 3:7 ꢁ 103; MSnEC10:4 ꢁ 103; Ð = 1.19; (b) azide-terminated PAPMA obtained by RAFT polymerization
using [APMA]0/[azido-CTP]0/[ACVA]0 = 25/1/0.5 (molar ratios) in water:1,4-dioxane = 2:1 (v:v) at 70 °C for 4 h. Conv. = 94%; Mth ¼ 4:5 ꢁ 103; MnSEC ¼ 16:3 ꢁ 103; Ð = 1.21.
n

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P.V. Mendonça et al. / Reactive & Functional Polymers 81 (2014) 1–7
polymerization is re-initiated from a macro-CTA. A sample of
alkyne-terminated PAPMA (MSnEC ¼ 10:4 ꢁ 103; Ð = 1.19) prepared
by RAFT polymerization was isolated by extensive dialysis against
deionized water (pH ꢂ 5). This polymer was then used as the
macro-CTA in a self-blocking experiment with a targeted DP of
400. Fig. 6 shows the clear shift of the molecular weight distribu-
tion curve of the macro-PAPMA towards higher molecular weight
values, confirming the living character of the polymer. It is also
important to note that the resultant extended PAPMA presented
some low molecular weight tailing as well as a higher Ð than the
macro-CTA. This result can be due to the occurrence of some rad-
ical termination reactions or due to partial macro-CTA hydrolysis
during the purification process by dialysis, which may have
decreased the macro-CTA efficiency, as reported by other authors
for similar polymers [42]. The same experiment was performed
using an azide-terminated PAPMA sample, and the results obtained
were similar to those of the alkyne-terminated PAPMA chain
extension (see Fig. S5 in the ESI ).
Fig. 6. SEC traces of the macro-PAPMA (alkyne-terminated) and extended PAPMA
obtained by RAFT polymerization at 70 °C in water:1,4-dioxane = 2:1 (v:v).
could be related to both the leaving ability and the re-initiating
ability of each R group [9]. However, this work shows that it is also
important to consider other reaction parameters, namely, the type
of solvent, and make small adjustments in order to improve the
CTA efficacy.
3.3. Functionalization of alkyne-PAPMA by click chemistry
To prove the reactivity of the click moieties introduced in the
PAPMA structure, an alkyne-terminated PAPMA sample was func-
tionalized with a biocompatible azide-terminated coumarin deriv-
ative, 3-azido-7-diethylaminocoumarin, via a copper(I) catalyzed
azide alkyne cycloaddition. The success of the click reaction was
confirmed by the appearance of the triazole ring proton signal (g)
at 8.47 ppm [46] in the 1H NMR spectrum of the functionalized
polymer (Fig. 7). In addition to the functionalization through click
chemistry, the PAPMA structure allows further modification
through other strategies involving the reaction of the amino
groups, such as Michael addition reactions [37], to expand the
range of structures/applications of this polymer. Due to the biorel-
evance of coumarin [47] as well as the possibility of the double
functionalization of alkyne-functionalized PAPMA, we anticipate
that the strategy reported here will allow the preparation of com-
plex polymeric structures useful for biomedical applications.
3.2. 1H NMR analysis and chain extension reaction
The chemical structure of low molecular weight clickable poly-
mers prepared by RAFT polymerization was confirmed by 1H NMR
spectroscopy. Fig. 5(a) presents the 1H NMR spectrum of a purified
alkyne-terminated PAPMA sample. The characteristic signals of the
PAPMA structure at 3.23 ppm (f) (2H, ACH2ACH2ANH2), 3.06 ppm
(d) (2H, ANHACH2ACH2A), 1.91 ppm (e) (2H, ACH2ACH2ANH2),
1.76 ppm (b) (2H, CACH3ACNACH2ACA) and 1.25–0.70 ppm (c)
(3H, ACCH3SA) were in agreement with the data reported in the
literature [35,36]. It was also possible to identify the alkyne
chain-end (a) (1H, CHBC, 2.63 ppm) [45] from the alkyne-CTP,
which confirms the retention of the functionality. The same analy-
sis was conducted for the N3-terminated PAPMA (Fig. 5(b)), which
proved to have the methylene protons (i and g) associated with the
azido-CTP R group (4H, N3ACH2ACH2ACH2A), while the signal of
the methylene protons (h) (2H, N3ACH2ACH2ACH2A) was over-
lapped with signals (m) and (j). It was not possible to determine
neither the percentage of functionality nor the average-number
molecular weight of the PAPMA (MNnMR) by 1H NMR spectroscopy
because the protons of the aromatic ring (Z group) were partially
overlapped with the –NH protons of the polymers.
4. Conclusions
We reported the synthesis of clickable poly(N-(3-aminopro-
pyl)methacrylamide) (PAPMA) by RAFT polymerization using
azido-CTP or alkyne-CTP as chain transfer agents without any pro-
tection/deprotection steps. Both clickable CTAs exhibited similar or
higher chain transfer activity compared to the commercial ana-
logue CTP under the experimental conditions used. The control
over the PAPMA molecular weight was very good, as the polymer
One of the most effective ways to demonstrate the ‘‘livingness’’
of polymers is to perform a chain extension reaction, in which the
Fig. 7. 400 MHz 1H NMR spectrum in D2O of the purified coumarin-functionalized PAPMA obtained by a click reaction.

P.V. Mendonça et al. / Reactive & Functional Polymers 81 (2014) 1–7
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