RAFT polymer end-group modification and chain coupling/conjugation Via disulfide bonds

Article abstract of DOI:10.1071/CH09062

End-group modification of polymers prepared by reversible additionfragmentation chain transfer (RAFT) polymerization was accomplished by the conversion of trithiocarbonate or dithioester end-groups into a pyridyl disulfide (PDS) functionality. Several different polymers, such as poly(methyl methacrylate), polystyrene, poly(oligoethylene glycol-acrylate), poly(hydroxypropylacrylamide), and poly(N-isopropylacrylamide) were prepared by RAFT polymerization, and subjected to aminolysis in the presence of 2,2′-dithiodipyridine to yield thiol-terminated polymers with yields in the range 6590% dependent on the polymer structure. Furthermore, this PDS end-group was utilized to generate higher-order architectures, such as diblock copolymers with high yields and selectively. In addition, the PDS end-groups were used for the bioconjugation of different biomolecules, such as oligonucleotides, carbohydrates, and peptides. The successful modification of well-defined polymers was confirmed by a combination of UV-vis, NMR spectroscopy, and gel permeation chromatography.

Full text of DOI:10.1071/CH09062

RESEARCH FRONT
Full Paper
CSIRO PUBLISHING
Aust. J. Chem. 2009, 62, 830–847
www.publish.csiro.au/journals/ajc
RAFT Polymer End-Group Modification and Chain
Coupling/Conjugation Via Disulfide Bonds
A
A
B,C
and Thomas P. DavisA,C
Cyrille Boyer, Jingquan Liu, Volga Bulmus,
ACentre for Advanced Macromolecular Design (CAMD), School of Chemical Sciences and Engineering,
The University of New South Wales, Sydney, NSW 2052, Australia.
B
School of Biotechnology and Biomolecular Sciences (BABS), The University of New South Wales,
Sydney, NSW 2052, Australia.
Corresponding authors. Email: t.davis@unsw.edu.au; vbulmus@unsw.edu.au
C
End-group modification of polymers prepared by reversible addition–fragmentation chain transfer (RAFT) polymerization
was accomplished by the conversion of trithiocarbonate or dithioester end-groups into a pyridyl disulfide (PDS) func-
tionality. Several different polymers, such as poly(methyl methacrylate), polystyrene, poly(oligoethylene glycol-acrylate),
poly(hydroxypropylacrylamide), and poly(N-isopropylacrylamide) were prepared by RAFT polymerization, and subjected
ꢀ
to aminolysis in the presence of 2,2 -dithiodipyridine to yield thiol-terminated polymers with yields in the range 65–90%
dependent on the polymer structure. Furthermore, this PDS end-group was utilized to generate higher-order architec-
tures, such as diblock copolymers with high yields and selectively. In addition, the PDS end-groups were used for the
bioconjugation of different biomolecules, such as oligonucleotides, carbohydrates, and peptides. The successful modi-
fication of well-defined polymers was confirmed by a combination of UV-vis, NMR spectroscopy, and gel permeation
chromatography.
Manuscript received: 30 January 2009.
Final version: 25 March 2009.
Introduction
star polymers. In a previous communication we outlined two syn-
thetic strategies for exploiting the aminolysis reaction of RAFT
Living radical polymerization is an efficient and ver-
satile synthetic approach to well-defined polymers, such
polymers,[ which included aminolysis in the presence of 2,2 -
dithiodipyridine (DTP) to generate protected-thiol end-groups.
In this current paper, we expand our earlier work to a wider range
ofpolymerstodemonstratethattheexperimentalconditionsused
need to be tailored to the specific polymer structure. In this work
we demonstrate that aminolysis in the presence of DTP resolves
synthetic problems observed previously.[
66]
ꢀ
[
1–5]
as α-monofunctional polymers,
α,ω-functional telechelic
[
6,7]
[1,8–20]
polymers,
and block copolymers.
Like other living
radicalpolymerizations,[21–24]
reversibleaddition–fragmentation
chain transfer polymerization (RAFT) is a synthetic approach
that has been increasingly used to create functional poly-
mers for biological and nanotechnology applications in recent
25,37,39,67–70]
[
9,25–33]
Capping the thiol end-group, in situ, by DTP obviates disul-
fide formation and other known side-reactions to generate
pyridyl disulfide (PDS) functional polymers. This approach pre-
serves the thiol reactivity for subsequent reaction with other
thiol compounds, at room temperature, under mild condi-
tions to yield bioconjugates, such as a bovine serum albumin
years.
imbue instability and colour to the polymeric products.
overcome this problem, different methods have been proposed
However, the presence of RAFT end-groups can
[34]
To
[
31,35]
for RAFT end-group removal using thermolysis,
by radical
[
36]
[37]
and by radiation.[38]
transfer,
by oxidation to give sulfine,
The presence of thiocarbonylthio can be also exploited to
introduce functionality by a targeted modification approach. A
common synthetic route is the removal of RAFT end-groups
26,28]
[71–74]
(
BSA)-RAFT agent[
or bioconjugate polymers.
To
fully demonstrate the wide applicability of this approach, we
have chosen to synthesize different polymer structures, such as
(meth)acrylamide, styrene, and (meth)acrylate using two differ-
ent RAFT agents, such as a dithioester and a trithiocarbonate.
Initially, we report the aminolysis of the polymers synthesized
with these two RAFT agents in the presence of DTP. Second, we
reporttheuseofPDSend-groupstobuildhigher-orderpolymeric
structures with a range of functionalities.
[36,39–48]
by reduction or by aminolysis
to yield a thiol end-
group that can be further modified,[
41,43,49,50]
for example,
[
51]
thiol-ene chemistry to obtain more complex architectures,
[52]
such as those described by Hawker et al.
and by several
However, sidereactions, suchasinterchainattach-
ment canleadtobimodalchain populationsanda mixture ofthiol
[
53–63]
authors.
[21,42]
and disulfide functional polymers.
Recently, McCormick
[
64]
et al. suggested the use of tris(2-carboxyethyl)phosphine·HCl
(
TCEP) to reduce the disulfide bond formed in situ during the
[65]
Results and Discussion
thiol-ene reaction. Lowe and coworkers
proposed the use of
dimethylphenyl phosphine to prevent the oxidation of thiol to
disulfide and to catalyze the thiol-ene reaction to yield tri-arm
Two different RAFT group structures (i.e., a trithiocarbonate
and a dithioester) and five different monomer types (i.e.,
©
CSIRO 2009
10.1071/CH09062
0004-9425/09/080830

RESEARCH FRONT
RAFT Polymer End-Group Modification
831
H
S
S
CO H
2
R2
n
1
or 2
R1
S
AIBN
R1
R2
R1
HO C
S
2
CN
S
S
S
CO H
2
S
O
O
O
O
1
HN
HN
O
R ꢀ O
1
O
S
CN
8
HO
S
CO H
2
R ꢀ H or CH3
2
2
Scheme 1. Synthesis by RAFT polymerization of different monomers.
Table 1. Recapitulation of polymers used in this study
B
C
Mn,GPCD
−1
PDID
E
f F
[%]
Monomers
CTAA
T
C]
[M]0/[RAFT]0/
initiator0
αM
Mn,theor.
−1
Mn,1H NMR
−1
◦
[
[%]
[g mol
980
]
[g mol
]
[g mol
]
MMA
MMA
HPMA
HPMA
CDTB
CDTB
CDTB
CDTB
CDTB
CDTB
BSPA
BSPA
BSPA
BSPA
BSPA
BSPA
BSPA
BSPA
70
70
70
70
70
65
65
65
65
65
60
60
80
80
10.0/1.0/0.2
71.0/1.0/0.2
21.0/1.0/0.2
32.0/1.0/0.2
76.8/1.0/0.2
60.0/1.0/0.2
20.0/1.0/0.1
54.8/1.0/0.1
74.2/1.0/0.1
102.7/1.0/0.1
18.0/1.0/0.1
30.0/1.0/0.1
17.0/1.0/0.3
33.0/1.0/0.3
70
75
60
70
70
75
55
75
75
70
70
80
65
80
1080
1.13
1.14
1.09
1.04
1.06
1.06
1.06
1.12
1.08
1.09
1.21
1.21
1.12
1.08
1300
95
95
95
95
90
90
85
90
95
90
90
95
85
85
5470
1800
3400
6800
5400
1300
4900
6500
8300
6000
10800
1250
3000
5100
4200
8200
14500
5900
1200
5300
7500
13200
7200
12300
1200
2800
4740
1540
3200
7600
6300
1400
4700
6700
12000
6500
12500
1200
2900
HPMA
NIPAAm
NIPAAm
NIPAAm
NIPAAm
NIPAAm
OEG-A
OEG-A
Styrene
Styrene
A
Chain transfer agents. BSPA: 3-(benzylsulfanylthiocarbonylsulfanyl)propionic acid, and CDTB: 4-cyanopentanoic acid dithiobenzoate.
Monomer conversion determined by H NMR spectroscopy.
B
C
1
Theoretical MW calculated by Mn = ([M]0/[RAFT]0) × αM × MWmonomer + MWRAFTagent, where [M]0, [RAFT]0, MWmonomer, and MWRAFTagent represent
monomer and RAFT agent concentrations, molar mass of monomer and RAFT agent, respectively.
D
MW determined by GPC (DMAc GPC andTHF GPC were used for HPMA, OEG-A, and NIPAAm, and for styrene and MMA, respectively) (PS calibration).
E
F
1
MW calculated by H NMR.
RAFT end-group functionality [%] calculated by H NMR according to the following equations, for dithioester, = (I7.2–7.4 ppm/5)/ (I2.4–2.5 ppm/4); for trithio-
1
3
.5 ppm
7.2–7.4 ppm
carbonate, = (I
/2)/(I
/5) and by UV-vis spectroscopy according the following equation: functionality = [RAFT]0/[polymer]0 = (absorbance at
1
−
−1
3
05 nm/ε)/[polymer]0, where ε was the extinction coefficient of the RAFT agent, i.e., εdithiocarbonate and εtrithiocarbonate are 15000 and 22000 L mol cm ,
respectively.
(
meth)acrylic, (meth)acrylamide, and styrene), were used in this
properties of the resultant polymers are given inTable 1. Gel per-
meation chromatography (GPC) analyses confirmed low poly-
study. This is summarized in Scheme 1.
To ensure both the presence of trithiocarbonate or dithioester
RAFT end-groups on the polymers and to minimize ter-
mination reactions during the polymerizations, the molar
ratio of the initiator to the RAFT agent in the feed solu-
tions ([initiator]0/[RAFT]0) was fixed between 1/10 and 1/5.
Moreover, the polymerizations were stopped at low monomer
conversions (60–75%). The polymerization conditions and the
dispersity indexes (PDIs) (PDI <1.2) consistent with the known
traits of living radical polymerizations.[
75] 1
H NMR analyses
confirmed the presence of the trithio- and dithioester function-
1
ality (Z group) (higher than 90% assessed by H NMR and by
UV-vis spectroscopy, in all cases, functionality was defined by
the ratio Z group to R group). UV-Vis spectrophotometer anal-
ysis of the purified polymers also confirmed the presence of the

RESEARCH FRONT
8
32
C. Boyer et al.
R2
R1
S
S
CO H
R
2
n
S
or
R2
R1
S
R
n
S
NH2
DCM or anhydrous ethanol
Room temperature
Intermediate product
R2
R1
R2
R1
SH
O2
S
R
R
S
R
R2
n
n
m
R1
I
N
S
S
N
H
N
N
R2
S
S
ꢁ
R
S
n
R1
II
Scheme 2. Chemical modification of RAFT end-group by aminolysis in the presence of DTP to give a PDS-
functionalized polymer.
RAFT end-groups and high RAFT group functionality (>90%),
as calculated by the following equation:
The aminolysis reaction has already been adopted by others to
convert RAFT-end groups into thiol functionalities.[
39–48]
How-
ever, it is also well known that thiols can rapidly be oxidized by
trace amounts of oxygen, which leads to the undesired forma-
tion of interchain disulfides. Thus, the addition of DTP during
the aminolysis procedure prevents the formation of interchain
disulfide bonds, whilst converting thiol end-groups into stable
PDS groups (compound I, Scheme 2).
During the reaction of DTP with thiol end-groups (formed
upon aminolysis), a co-product, pyridine-2-thione, can be
observed using UV-vis spectrophotometer analysis.The analyses
of reaction mixtures confirmed the formation of pyridine-2-
thione (λmax = 370 nm in acetonitrile and 350 nm in ethanol).
The disappearance of RAFT end-groups can also be observed
at 305 nm for dithioester and at 307 nm for trithiocarbon-
ate (Fig. 1 gives an example for poly(N-isopropylacrylamide)
(PNIPAAm)). In the case of a dithioester, the release of pyridine-
2-thione was in accord with the theoretical value as calculated
by the equation:
functionality = [RAFT]0/[polymer]0
=
Absorbance at 305 nm/ε,
(1)
where ε is the extinction coefficient of the RAFT agent, i.e.,
εdithiocarbonate and εtrithiocarbonate are 15000 and 22000 L mol
−
1
−
1
cm , respectively, and [polymer]0 is the polymer concentration.
Both UV and NMR analyses were in accord, which confirms
the presence of the RAFT end-groups (Z group). In addition,
electrospray-ionization mass spectrometry (ESI-MS) confirmed
the presence of RAFT end-groups.
The RAFT end-groups, i.e., trithio- and dithioester groups,
were cleaved by aminolysis and simultaneously reacted
with DTP for 4–5 h (Scheme 2). The RAFT end group/
hexylamine/DTP molar ratio was 1/3/10 and 1/4/10. The con-
ditions were designed to limit any degradation of PDS in the
presence of amine, as it is known that a large ratio of amine
and a long reaction time can give by-products from the degra-
Abspyridine-2-thione = ε × [RAFT end-group]0,
(2)
[
76]
dation of PDS.
The end-group functionalization occurs in
two steps: first, the amine reacts with dithioester or trithiocar-
bonate to yield a thiol group that instantly reacts with DTP.
with [RAFT end-group]0 assessed by UV spectroscopy, and
where ε is the extinction coefficient of pyridine-2-thione. In the

RESEARCH FRONT
RAFT Polymer End-Group Modification
833
S
(A)
Z
2
0
S
H
S
N
R
1
2
0
0
0
0
.6
.4
.2
.0
400
600
1
2
20
30
40
Time [min]
300
400
Wavelength [nm]
500
600
Initial PNIPAAm
Aminolysis in the presence of DTP
Aminolysis without DTP (after 3 h)
Aminolysis without DTP (after 6 h)
Fig. 1. Evolution of the UV absorbance of PNIPAAm obtained by RAFT
polymerization in the presence of 3-(benzylsulfanylthiocarbonylsulfanyl)
1
propionic acid as RAFT agent and AIBN as initiator (Mn ( H NMR/GPC)
−
1
6
700/7500 g mol , PDI 1.08), (top) in the presence and (bottom) in the
absence of DTP. Data were collected every 5 min.
(
B)
case of the trithiocarbonate, the release of pyridine-2-thione was
twice that of the initial RAFT end-group concentration. This
observation can be explained by the formation of two thiols dur-
ing the cleavage of the trithiocarbonate. When aminolysis occurs
in the absence of DTP, the reaction solution loses its characteris-
ticyelloworredcolour, whileinthepresenceofDTP, thesolution
remains yellow for the trithiocarbonate and changes from red to
yellow for the dithioester as pyridine-2-thione is released.
Post-cleavage of the RAFT end-groups, analyses by GPC
were performed, as shown in Fig. 2 for PNIPAAm (number- aver-
1
−1
30
35
40
45
age molecular weight (Mn) ( H NMR/GPC) 6700/7500 g mol
,
Time [min]
PDI 1.08) obtained by RAFT polymerization in the pres-
ence of 3-(benzylsulfanylthiocarbonylsulfanyl)propionic acid
1
Aminolysis without DTP
Aminolysis in the presence of DTP
Initial PMMA
(
1
BSPA)andpoly(methylmethacrylate)(PMMA)(Mn ( HNMR)
300 g mol , PDI 1.14). For PNIPAAm, the polymer cleaved
−1
by aminolysis in the absence of DTP exhibited a shoulder at low
retention times consistent with the coupling of polymer chains
by a disulfide bond. The proportion of the disulfide coupled
polymer increased with time. After 24 h, there was 40% of a
Fig. 2. (A) DMAc GPC of PNIPAAm obtained by RAFT polymer-
ization in the presence of AIBN as initiator and 3-(benzylsulfanyl-
thiocarbonylsulfanyl)propionic acid as RAFT agent (before aminolysis,
1
disulfide-coupled polymer for PNIPAAm with a Mn by H NMR
1
−1
Mn ( H NMR/GPC) 6700/7500 g mol , PDI 1.08; after aminolysis in the
−1
of6700 g mol .Therelativeamountsofdisulfide-coupledpoly-
mer are dependent on the molecular weights and experimental
conditions used.[77] In contrast, when the aminolysis was per-
formed in the presence of DTP, no shoulder caused by coupling
was observed (Fig. 2A). This indicates that the thiol end-group
formed upon aminolysis was protected by DTP, at a faster rate
than the competing disulfide formation reaction. In all cases,
the PDI value is maintained before and after aminolysis in the
presence of DTP, while the PDI increases in the absence of DTP
because of interchain coupling (Table 2).
1
−1
presence of DTP, Mn ( H NMR/GPC) 7100/7400 g mol , PDI 1.08; after
1
−1
aminolysis without DTP (after 4 h), Mn ( H NMR/GPC) 7100/8400 g mol
,
PDI 1.14). (B)THF GPC of PMMA obtained by RAFT polymerization in the
presence of CDTB as RAFT agent andAIBN as initiator (before aminolysis,
1
−1
Mn ( H NMR/GPC) 1300/1200 g mol , PDI 1.14; after aminolysis in the
1
−1
presence of DTP, Mn ( H NMR/GPC) 1200/1100 g mol , PDI 1.14). The
polymer concentrations were kept constant.
1H NMRs of PHPMA in D2O are given in Fig. 4 for pre- and
post-aminolysis in the presence of DTP. Before aminolysis, char-
acteristic signals of RAFT agent were observed at 2.4 ppm and
at 7.4, 7.5, and 7.8 ppm attributed to the R and Z groups, respec-
tively. To assist quantification it was arbitrary decided to use
the ratio of Z/R groups to estimate the RAFT functionality. The
characteristic NMR signals at 7.4, 7.5, and 7.8 ppm attributed
to the phenyl ring of RAFT agent (Z group) were totally absent
after aminolysis. Contemporaneously, new peaks at 7.1, 7.6, and
The purified polymers after aminolysis in the presence and
1
absence of DTP were also analyzed by ESI-MS, H NMR,
and UV-vis spectrophotometry. First, UV-vis spectrophotometry
confirmed the presence of pyridyl rings by a characteristic
absorption wavelength of 272 nm and the disappearance of
RAFT signals of ∼305 nm. Fig. 3 gives a representative result
for PHPMA before and after cleavage of the RAFT agent using
DTP.

RESEARCH FRONT
8
34
C. Boyer et al.
Table 2. Recapitulation of modified polymers used in this study. Experimental conditions: [RAFT]0/[NH2-R]0/[DTP]0 = 1.0/3.0/10.0, at room
temperature
Monomers
CTAA
Exp. cond.B
Before modification
After modification
PDS functionality [%]
Mn,GPCC
PDI
C
Mn,1H NMRD
Mn,GPC
−1
C
PDIC
[g mol
Mn,1H NMRE
1
H NMR
F
UV-VisG
−
[
g mol 1]
−1
]
−1
[
g mol
]
[g mol
]
MMA
MMA
MMA
CDTB
CDTB
CDTB
CDTB
CDTB
CDTB
BSPA
BSPA
BSPA
BSPA
BSPA
BSPA
BSPA
DTP
DTP
No
DTP
DTP
No
DTP
DTP
No
DTP
No
1080
5100
1080
4200
8200
8200
5300
11200
5300
12300
12300
1200
2700
1.13
1.14
1.13
1.09
1.04
1.04
1.12
1.09
1.12
1.21
1.26
1.13
1.08
1300
4740
750
1050
1.12
1.13
1.16
1.09
1.09
1.09
1.11
1.09
1.16
1.21
1.21
1.18
1.08
1200
5100
–
1500
3100
–
5200
13500
–
12500
–
92 (± 5)
90 (± 5)
–
nd
90
–
85
nd
–
90
nd
–
85
–
5000
1200
4100
8100
8000
5100
11000
6800
12200
14000
1050
2900
HPMA
HPMA
HPMA
NIPAAm
NIPAAm
NIPAAm
OEG-A
OEG-A
Styrene
Styrene
1540
3200
3200
4700
12000
4700
12500
14500
1200
2900
90 (± 5)
95 (± 10)
–
95 (± 5)
90 (± 8)
–
90 (± 5)
–
DTP
DTP
1350
2800
85 (± 8)
92 (± 5)
nd
90
A
Chain transfer agents. BSPA: 3-(benzylsulfanylthiocarbonylsulfanyl)propionic acid, and CDTB: 4-cyanopentanoic acid dithiobenzoate.
In the presence or absence of DTP.
MW determined by DMAc GPC (PS calibration), except for styrene and MMA determined by THF GPC.
Calculated by the RAFT end-group.
B
C
D
E
F
Calculated by the pyridyl end-group.
Average values determined by 1H NMR using the ratio of pyridyl group signals to the R group signals (error was calculated by the difference of values
obtained according the used signals).
G
Assessed by UV-vis spectroscopy using the signal of the pyridyl group at 270 nm. nd: values not determined.
(
Fig. 5). Before chemical modification, the signals attributed to
the Z group of the RAFT agent, i.e., of S–CH2 and of CH2CO2H
at 3.5 and at 2.6 ppm, respectively, confirmed the presence of a
RAFT agent with high functionality.As before, the functionality
was defined by the ratio of Z group to R group. The function-
ality was found to be in excess of 90% for all the polymers.
After chemical modification (cleavage of the RAFT agent in the
0
0
0
.8
.4
.0
1
presence of DTP), H NMR confirmed the absence of Z group
signals (at 2.5 and 3.6 ppm), and, simultaneously, the presence
of PDS end-groups for PNIPAAm, PS, and poly(oligoethylene
glycol-acrylate) (POEG-A) (see Table 2). The functionality was
calculated using the signal at 8.4 ppm normalized by the signal
of the R group at 2.5 ppm.
Finally, we also tried this approach for PMMA. This polymer
is known to undergo different side-reactions that compete with
thiol formation under aminolysis conditions.The thiol generated
by aminolysis is easily degraded by side reactions that lead to
disulfide interchain coupling and a thiolactone with elimination
4
00
Wavelength [nm]
Fig. 3. UV spectra of (- - -) purified PHPMA before modification,
of methanol.[
42,78]
Fig. 6 shows the H NMR spectra of PMMA
1
(
) purified PHPMA after modification with PDS, (−) purified PHPMA
1
after aminolysis without DTP (before aminolysis, Mn ( H NMR/GPC)
before and after modification. After aminolysis in the presence
−
1
8
(
200/3200 g mol , PDI 1.04; after aminolysis in the presence of DTP, Mn
H NMR/GPC) 3200/8100 g mol , PDI 1.04).
of DTP, the presence of a pyridyl group was observed with high
1
−1
8.4 ppm 2.5 ppm
efficiency(close to1, calculatedbythe equation I
/I
).
GPC analysis confirmed the formation of a monomodal distri-
bution when the reaction was carried out in the presence of DTP
(see Fig. 2B).
8
.4 ppm confirmed the attachment of a pyridyl ring. As DTP is
water insoluble, the observation of pyridyl groups in deuterium
oxide is strong evidence for conjugation to the water soluble
poly(hydroxypropylacrylamide) (PHPMA).The functionality of
the PDS groups was defined as the ratio of signal attributed to
the pyridyl group divided by the signal of the R group, i.e.,
at 2.4 ppm. The functionality was above 80% regardless of the
molecular weight (MW) of the polymers (Table 2).
ESI-MS was also exploited to study the efficiency of the func-
tionalization reaction with PMMA. The ESI-MS spectra of the
1
−1
PMMAs, (Mn ( H NMR/GPC) 1300/1080 g mol , PDI 1.13),
is shown in Fig. 7A. In the absence of DTP, the formation of
thiolactone was observed, as expected.[ In the presence of
DTP, the aminolysis reaction occurred without this side reaction
and a principal population attributed to PDS-modified PMMA
was observed (Table 3). It is important to note that a different
population was also observed by ESI-MS when the aminolysis
42]
1
A similar result was observed by H NMR spectroscopy for
PS and PNIPAAm terminated by a trithiocarbonate end-group

RESEARCH FRONT
RAFT Polymer End-Group Modification
835
h
NC
N
b
f
g
b
c
HO C
S
2
i
S
f
n
a
h
hꢂ
O
NH
d
g
e
e
d
OH
i
f
b
a
c
1.0
2.0 1.0
21.3
4
43.8
3
3.7
196..7
1
f
0
9
8
c
7
6
5
2
1
a
i
h
b
NC
g
h
HO C
S
2
f
c
a
n
S
hꢂ
g
O
NH
d
e
e
d
OH
i
a b c
f
2
.0
20.4
4
40.8 3.7 37.5
124.3
1
10
9
8
7
6
5
3
2
1
Fig. 4.
H NMR spectra of PHPMA obtained by RAFT polymerization in the presence of CDTB as RAFT agent and ACVA as initiator; (bottom) before
1
−1
and (top) after aminolysis in the presence of DTP (before aminolysis, Mn ( H NMR/GPC) 3200/8200 g mol , PDI 1.04; after aminolysis in the presence of
1
−1
8.4 ppm 2.5 ppm
DTP, Mn ( H NMR/GPC) 3200/8100 g mol , PDI 1.04) (recorded in D2O, 300 MHz, 128 scans). PDS functionality was calculated by I
/(I
/4),
8
.4 ppm
2.5 ppm
where I
and I
are the integration of signals at 8.4 and at 2.5 ppm, respectively.
reaction was performed at higher hexylamine or 2-aminoethanol
concentrations and longer reaction times (data not shown). This
population (not attributed) can be explained by the degradation
of PDS in the presence of amine, as reported elsewhere,[ or
by the partial transamidation of PMMA. As a control exper-
Initially, we tested the PDS reactivity using a powerful reducing
agent dl-dithiothreitol (DTT). It is well known that DTT induces
a quick reduction of disulfide groups into thiols.The reduction of
PDS gives a by-product, pyridine-2-thione, easily detectable by
UV-vis spectrophotometry (Fig. 8). Analysis of different poly-
mer solutions (PMMA, PHPMA, PNIPAAm, and POEG-A at
76]
1
−1
iment, PNIPAAm (Mn ( H NMR/GPC) 1300/1200 g mol
,
−5 −4
PDI 1.06) was also analyzed by ESI-MS (Fig. 7B). After the
aminolysis in the absence of DTP, the formation of both thiol-
terminated and disulfide-coupled interchain populations were
observed clearly (in perfect agreement with GPC results, Mn
varying concentrations of 10 to 10 M) after incubation with
−
3
a large amount of DTT (3 × 10 ) for different reaction times
revealed that 90 and 80% of the PDS end-groups of PNIPAAm
and POEG-A, respectively, were reduced by DTT in pyridine-
2-thione after 10–15 min (Table 4). No significant effect on the
MW was observed. The reduction was found to be slower and
lower for PHPMA and PMMA (Table 4). These two polymers
present a very similar reactivity. Under the same conditions,
only 60–75% of PDS groups could be reduced to pyridine-2-
thione after 14 h. We do not have a complete explanation for this
observation. However, it is reasonable to conjecture that steric
hindrance may have a significant influence on PDS reactivity.
−
1
(
GPC) 1400 g mol , PDI 1.12). The disulfide-linked dimers
appeared as a population centred at twice the MW of the origi-
nal population. After the addition of DTP, a new population was
observed attributed to PDS-functionalized PNIPAAm.
In conclusion, this synthetic approach can be applied to the
modification of both dithioesters and trithiocarbonates, and also
to a wide range of polymers, typified by the range of monomers
reported herein.This approach yields high functionalities of PDS
(
close to 85–90%, see Table 2).
We have demonstrated clearly the presence of PDS end-
Biomodification to the PDS-Terminated Polymers
groups after aminolysis in the presence of DTP for all the poly-
mers with dithioester or trithiocarbonate functionalities. In what
follows, we demonstrate the facile reactivity of PDS end-groups
and their utility in building complex structures and conjugates.
The versatility of the simultaneous end-group modification
strategy in the biofunctionalization of polymers was shown
by conjugation of a variety of model biomolecules, e.g., a

RESEARCH FRONT
8
36
C. Boyer et al.
a
(
A)
(C)
f
f
f
c
f
b
d
S
S
e
NH
e
CO H
2
e
O
c
n
S
a
S
N
h
a
b
d
S
g
h
n
×
H O
2
a
i
CDCl3 ×
a
d
c
Acetone
dꢂ
×
d
5
.00 4.90 4.80 4.70 4.60 4.50
a, i
b
× 5
e
f
g
h
b
dꢂ
1
33.6
1.0
2.0
2.0
86.5
1.0
2.0 5.9
60.0 0.9
2.0
561.0
1
0
9
8
7
6
5
4
3
2
1
9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
g
f
f
a
f
h
e
(D)
(
B)
e
NH
c
d
S
O
c
O
S
S
N
a
b
b
g
f
n
d
S
S
OH
h
a
n
a, h
×
H O
2
CDCl3
×
e
c
c
8
.55
8.50
8.45
8.40
d
4
.00 3.90 3.80 3.70 3.60 3.50 3.40
dꢂ
d
×
5
f, g
1.9
a
g
b
h
b
e
dꢂ
4.8
0.8 60.0 2.0
2.0
540.0
0
.9
141.5
7
0.9
86.5
9
.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0
1
0
9
8
6
5
4
3
2
1
1
Fig. 5.
H NMR spectra of PS obtained by RAFT polymerization in the presence of AIBN as initiator and 3-(benzylsulfanylthiocarbonylsulfanyl)propionic
1
−1
1
acid as RAFT agent. (A) Before aminolysis (Mn ( H NMR/GPC) 2700/2900 g mol , PDI 1.08). (B) After aminolysis in the presence of DTP (Mn ( H
NMR/GPC) 2900/2800 g mol , PDI 1.08) (recorded in CDCl3, 300 MHz, 128 scans). H NMR spectra of PNIPAAm obtained by RAFT polymerization
in the presence of AIBN as initiator and 3-(benzylsulfanylthiocarbonylsulfanyl)propionic acid as RAFT agent. (C) After aminolysis in the presence of DTP
Mn ( H NMR/GPC) 7500/7800 g mol , PDI 1.08). PDS functionality was calculated as described in the caption for Fig. 4. (D) Before aminolysis (Mn ( H
NMR/GPC) 7300/7900 g mol , PDI 1.08).
−
1
1
1
−1
1
(
−
1
hexapeptide, a carbohydrate, and an oligodeoxyribonucleotide
to water-soluble polymers, POEG-A, PNIPAAm, and PHPMA
the yield of the reaction and is commonly used for the con-
jugation of DNA with polymers.[ Indeed, steric hindrance
between the polymer and DNA generally results in lower con-
jugation yields when compared with reactions between either
polymer or DNA with small compounds. Initially, the thiol was
deprotected by DTT reduction, and purified by several extrac-
tions (water/ethyl acetate).The thiol formation was confirmed by
Elman’s assay. Thiol-functionalized DNA was then reacted with
PDS terminal polymers. The resulting conjugates were analyzed
by agarose gel electrophoresis (Fig. 10).The conjugates (lanes 2,
79]
(
Scheme 3).
Peptide Conjugation
To demonstrate the versatility of this approach, we utilized a
1
hexapeptide (GNGRGC). After careful purification, H NMR
1
−1
analysis of PNIPAAm (Mn ( H NMR) 6700 g mol , PDI 1.08)
proves the presence of the peptide by the characteristics peptide
signals at 2.7 and 3.0 ppm (Fig. 9). The yield of the reaction is
above 85% as determined by UV-vis spectroscopy.This approach
was also successfully used for the bioconjugation of BSA (data
not shown).
4, 5, and 7) appeared as bands that have a higher MW compared
with DNA (lane 1). The yield of conjugation was calculated to
be 70–85% from the band intensities of non-reacted DNA and
conjugates. After incubation under reducing conditions (DTT)
for 3 h, the intensity of the oligonucleotide (ON)-PNIPAAm
conjugate band decreased, in parallel, the intensity of the ON
band increased, which confirms DNA release consistent with
conjugation through a disulfide bond (lanes 3 and 6). It is inter-
esting to note that the total cleavage of disulfide bonds between
ON and PNIPAAm required longer reaction times (attributed to
sterichindrance). GPCanalysesoftheDNA–polymerconjugates
(mobile phase: water) were performed in conjunction with a UV
detector.A shift to lower retention times was clearly observed for
all conjugates relative to DNA. In the case of PHPMA, the yield
Oligodeoxyribonucleotide Conjugation
ꢀ
A 5 -thiol-modified double-stranded oligodeoxyribonucleotide
ꢀ
(
duplex sequences, 5 A-AG CTG ACC CTG AAG TTC ATC
TGC ACC-3 and 5 -/5thioMC6-D/GGT GCA GAT GAA CTT
CAG GGT CAG-3 , MW of duplex 16887.6 Da) was used for
ꢀ
ꢀ
ꢀ
conjugation to PDS end-group terminated POEG-A, PNIPAAm,
and PHPMA. The conjugations were performed in a phosphate
buffer at pH 8.0 at a polymer-to-DNA feed molar ratio of 25/1
or 40/1 for 14 h at room temperature. This high ratio increased

RESEARCH FRONT
RAFT Polymer End-Group Modification
837
d
O
O
e
S
CN
a
b
g
b
c
OH
S
g
h
f
n
a
O
d
dꢂ
f
h
b
e
a
g
c
1
.9 3.0
30.0
3.9 19.8
34.5
10
9
8
7
6
5
g
4
3
2
1
d
O
O
CN
h
N
S
e
OH
a
b
g
S
c
f
n
O
b
d
Acetone
× CDCl
3
f
×e
h
b
g
a
c
1.0
1.8 0.9
30.0
3.9 18.2 39.8
10
9
8
7
6
5
4
3
2
1
Fig. 6. 1H NMR spectra of PMMA obtained by RAFT polymerization in the presence of CDTB as RAFT agent
andAIBN as initiator; (bottom) before and (top) after aminolysis in the presence of DTP (before aminolysis, Mn
1
−1
1
(
H NMR/GPC) 1300/1200 g mol , PDI 1.14; after aminolysis in the presence of DTP, Mn ( H NMR/GPC)
−
1
1200/1100 g mol , PDI 1.14) (recorded in CDCl3, 300 MHz, 128 scans). PDS functionality was calculated as
described in the caption for Fig. 4.
of DNA–polymer conjugate was lower (∼40%). Similar results
and 4.4 ppm confirmed the presence of carbohydrate end-groups
(Fig. 11).
were found for small-interfering RNA (si-RNA) conjugation.
Architecture Control
Carbohydrate Conjugation
The PDS groups can also be used for the synthesis of diblock
polymers or more complex architectures. As an example to
demonstrate this potential, we decided to couple a thiol-modified
poly(ethylene glycol) (PEG) using the previous modified poly-
mers. The thiol-modified PEG was obtained by chemical mod-
ification of PEG bearing one hydroxy group in the presence
of 3-mercaptopropionic acid disulfide and a traditional cou-
pling agent (DCC and DMAP). In a second step, the disulfide
was cleaved in the presence of 2-mercaptoethanol at room
temperature, and then, purified by several precipitations. The
coupling reaction with the free thiol of PEG (Mn (DMAc GPC)
A thiol-modified glucose was synthesized using the method
reported by Davis et al.[ Thiol-modified glucose was reacted
80]
−
1
withPDS-terminatedPNIPAAm(Mn 5900 g mol , PDI1.08)in
methanol at room temperature for 14 h, using a [thiol-glucose]0
to [pyridyl disulfide]0 ratio of 2/1. UV-Vis spectroscopy showed
the formation of pyridine-2-thione at 350 nm. The yield calcu-
lated (based on the spectroscopic measurement) was 85–90%.
1
After dialysis against water, the sample was analyzed by H
NMR spectroscopy.The absence of a signal attributed to the PDS
ring at 6.8, 7.2–7.4, and 7.8 ppm confirmed that the PDS end-
group had reacted. Moreover, the presence of signals at 3.3, 3.6,
−
1
1400 g mol , PDI 1.04) and the PDS of polymers obtained

RESEARCH FRONT
8
38
C. Boyer et al.
(
A)
(
A1)
M1
00 m/z
1
M1ꢂ
M1ꢃ
800
1200
1600
(
(
A2)
A3)
M2
1
00 m/z M2ꢂ
M2ꢃ
1000
1200
1400
1600
1800
2000
M3
100 m/z
M3
800
1000
1200
1400
m/z
1600
1800
2000
(
B)
(
B1)
N1
113 m/z
N2
500
1000
1500
2000
2500
3000
3500
(
B2)
N3
113 m/z
400
600
800
1000
1200
1400
1600
(
B3)
N4
113 m/z
500
1000
1500
m/z
Fig. 7. (A) ESI-MS spectra of PMMA: (A3) Before aminolysis (two majors populations were observed): living
and dead chain PMMA obtained by termination (dismutation and coupling) and (A2) after aminolysis in the
presence of DTP, and (A1) after aminolysis without DTP. (B) ESI-MS of PNIPAAm: (B3) Before aminolysis,
(
B2) after aminolysis in the presence of DTP, and (B1) after aminolysis in the absence of DTP.
by RAFT polymerization was carried out at room tempera-
ture in water or in acetonitrile as solvent depending on the
polymer solubility. Two polymers were utilized: PMMA (Mn
of new distributions at low retention times that corresponded
to diblock copolymers (Fig. 12). The presence of a shoulder
shows that the yield is not quantitative (yield close to 85%).
However, by selective precipitation, i.e., in the case of PMMA-
b-PEG diblock in methanol/pentane (80–20% v/v, this shoulder
−1
(
GPC) 5100 g mol , PDI 1.14) and PNIPAAm (Mn (GPC)
−1
5
300 g mol , PDI1.12). GPCanalysesconfirmedtheformation

RESEARCH FRONT
RAFT Polymer End-Group Modification
839
was partially removed. In the case of PNIPAAm, a long dialy-
sis using a membrane with a cut off of 6000 Da against water
yielded a pure diblock copolymer. H NMR confirmed the pres-
acid). GPC analysis confirmed the formation of a major product:
PNIPAAm-b-PEG-b-PNIPAAm (data not shown).
1
ence of a PEG block by the signal at 3.6 ppm. This methodology
can be extended to the synthesis of tri-block polymers by using
a telechelic PEG, i.e., α,ω-thiol-terminated PEG (by reacting
a α,ω-hydroxy-functionalized PEG with 3-mercaptopropionic
Conclusion
In this study, we follow up an initial communication[66] with a
much broader experimental protocol to demonstrate a general
Table 3. Attribution of signal observed in ESI-MS
Units Cation Structures
Polymer
PMMA
Peak
M1
m/z (theor.)
m/z (exp.)
O
S
CN
O
OH
9
8
Na
Na
1050.76
1050.51
n
O
O
N
O
O
O
O
O
CN
M2
M3
M4
1091.82
1092.06
S
OH
OH
S
n
O
O
CN
8
9
8
K
1107.5
1107.82
1049.30
1047.30
N
S
S
n
O
O
H
O
CN
Na
Na
1050.20
1047.54
OH
n
O
O
O
CN
ꢀ
M4
O
OH
nꢄ1
O
O
O
S
O
S
CN
M5
N1
8
8
1102.65
938.48
1102.1
938.02
OH
n
O
O
NH
PNIPAAm
Na
Na
SH
n
NH
O
N2
16
S
2076.84
2077.04
S
n
m
O
NH
O
NH
N3
N4
8
8
Na
Na
937.68
973.36
936.90
973.67
S
N
S
n
NH
O
S
O
S
n
S
OH

RESEARCH FRONT
8
40
C. Boyer et al.
and straightforward way to exploit the presence of dithioester
and trithiocarbonate functionalities in polymers generated by
RAFT polymerizations. We show a facile approach to generate
terminal-protected thiol groups by aminolysis reactions con-
ducted in the presence of DTP. The synthetic approach was
successfully used to functionalize a variety of polymers, i.e.,
PMMA, PHPMA, POEG-A, and PNIPAAm. Second, we demon-
strate that these polymers can be conjugated with biomolecules,
with yields that range from 40 to 90%, depending on the struc-
ture of the polymer and the nature of the biocompound. Finally
we show that the PDS terminal functionality can be exploited to
build higher-order structures by disulfide coupling.
(
A)
Experimental
Materials
N-Isopropylacrylamide (NIPAAm, 97%, Aldrich) was crys-
◦
tallized twice from hexane before use (mp 64 C). Methyl
0
.08
methacrylate (Aldrich, 99%) was distilled before use. Oligoethy-
−
1
lene glycol acrylate (OEG-A) (Mn 450 g mol , PDI 1.04) was
passed through an alumina column to remove inhibitor before
use. N-(2-Hydroxypropyl) methacrylamide was obtained from
Polysciences Inc. 2,2 -Azoisobutyronitrile (AIBN) and 4,4 -
azobis(4-cyanovaleric acid) (ACVA) were purchased fromWako
Chemicals. AIBN was crystallized twice from methanol before
use. All other chemicals were used as received. Membranes for
dialysis (MWCO 2000 and 12000–14000 Da) were purchased
from Fisher Biotec (Cellu SepT4, regenerated cellulose-Tubular
membrane).
A model peptide (sequence: GNGRGC, purity 95%) was
purchased from AUSPEP (Australia).
Oligonucleotide (duplex sequences, 5 -AAG CTGACC CTG
AAG TTC ATC TGC ACC-3 and 5 -/5thioMC6-D/GGT GCA
GAT GAA CTT CAG GGT CAG-3 , MW of duplex 16 887.6 Da,
ε 256750 L mol cm ) was purchased from Integrated DNA
ꢀ
ꢀ
0.00
3
00
400
Wavelength [nm]
ꢀ
(
B)
ꢀ
ꢀ
0
0
0
0
0
0
0
.030
.025
.020
.015
.010
.005
.000
y ꢀ 2217x
R2 ꢀ 0.9881
ꢀ
−1
−1
Technologies (IDT) (USA).
Analytical Techniques
Gel Permeation Chromatography (GPC)
Aqueous GPC was implemented using a Shimadzu modular
system comprising a DGU-12A solvent degasser, a LC-10AT
pump, a CTO-10 A column oven, and a RID-10A refractive
index detector and SPD-10A Shimadzu UV-vis detector (flow
−
1
rate: 1 mL min ). The column was equipped with a PL 5.0 mm
2
bead-size guard column (50 × 7.8 mm ) followed by three PL
aquagel-OH columns (50, 40, 30; 8 µm). Calibration was per-
0.0
.0 ꢅ 10ꢄ6
1.0 ꢅ 10ꢄ5
5
formed with poly(ethylene oxide) (PEO) standards that ranged
from 500 to 500000 g mol . GPC analyses of the polymers were
[
polymer]0
−1
also performed in N,N-dimethylacetamide (DMAc: 0.03% w/v
Fig. 8. Titration of the PDS end group of modified PMMA (Mn
◦
1
−1
LiBr, 0.05% 2,6-di-butyl-4-methylphenol (BHT)) at 50 C (flow
(
H NMR/GPC) 1200/1100 g mol , PDI 1.14) by reaction of DTT
−
1
rate: 1 mL min ) using a Shimadzu modular system compris-
ing an SIL-10AD auto-injector, a PL 5.0 mm bead-size guard
to yield pyridine-2-thione (reaction was carried out in acetonitrile,
εpyridine−2-thione = 2450 L mol− cm ). (A) UV spectra of different PDS-
1
−1
2
functionalized PMMA polymers in the presence of DTT. (B) Plot of the
absorbance (at 370 nm) versus polymer concentration (M).
column (50 × 7.8 mm ) followed by four linear PL (Styragel)
5
4
3
columns (10 , 10 , 10 , and 500 Å) and an RID-10A differential
Table 4. Yield of bioconjugations obtained with PDS-modified polymers
Polymers
Mn,1H NMR
PDI
PDS functionalityA
[%]
Yield with DTTB
[%]
Ratio [polymer]0/
[DNA-SH]0
Yield with ONC
[%]
−
1
[
g mol
]
PNIPAAm
PNIPAAm
PHPMA
PHPMA
POEG-A
5200
13500
1500
3200
12500
1.11
1.09
1.09
1.04
1.21
95
90
90
75
85
90
90
60
55
90
20/1
20/1
–
40/1
20/1
85
75
–
40
80
A
1
Calculated by H NMR (Table 2).
B
C
−1
−1
Calculated by the release of pyridine-2-thione measured at 350 nm (yield = [Abs at 350 nm/8080 L mol cm ]/[pyridine disulfide termini polymer]0).
Calculated by agarose gel.

RESEARCH FRONT
RAFT Polymer End-Group Modification
841
R2
R2
R1
S
S
PEG
S
R
S
R
n
n
R1
DNA
SH
PEG-SH
N
R2
R1
S
S
R
n
Peptide
HS
CO H
HO
HO
2
H
HS
S
SH
H
H
H
O
HO
HO
R2
R2
R1
Peptide
S
H
S
CO H
H
2
S
R
R
OH
OH
n
O
R1
H
n
R2
H
S
S
H
R
H
HO
OH
n
R1
Scheme 3. Chemical and bioconjugation of polymers functionalized with PDS.
◦
refractive-index detector and in tetrahydrofuran (THF) at 40 C
(
60/40 v/v mixture of THF/methanol with an acetic acid concen-
−
1
−3
flow rate: 1 mL min ) using a Shimadzu modular system
comprising an SIL-10AD auto-injector, a PL 5.0 mm bead-
tration of 0.4 × 10 M. 112 Spectra were recorded in positive
ion mode with an instrumental resolution of 0.1 Da. All reported
molecular weights were calculated using the program package
CS ChemDraw 6.0 and monoisotopic. The theoretical MW over
charge ratios (m/z, assuming z of +1) was calculated using the
exact molecular mass of the predominant isotope within the
structure.
2
size guard column (50 × 7.8 mm ) followed by four linear PL
5
4
3
(
Styragel) columns (10 , 10 , 10 , and 100 Å). Calibration was
achieved with commercial PS standards that ranged from 500 to
6
−1
.
1
0 g mol
UV-Vis Spectroscopy
Syntheses
UV-Vis spectra were recorded using a CARY 300 spectro-
Synthesis of Thiol-Terminated PEG
Poly(ethylene glycol) methyl ether (provided by Fluka, 2.2 g,
photometer (Bruker) equipped with a temperature controller.
−
1
MW 1100 g mol , PDI 1.02, 0.002 mol) was dissolved in dried
NMR Spectroscopy
1H and 13C NMR spectra were recorded on a BrukerACF300
ꢀ
acetone (25 mL), and 3,3 -mercaptopropionic acid disulfide
(
0.3 g, 0.014 mol) (purchased from Aldrich (98%)), dimethyl-
(
300 MHz) or ACF500 (500 MHz) spectrometer, with D2O or
−
4
aminopyridine (23 mg, 2 × 10 mol) (purchased from Aldrich
CDCl3 used as solvents. NIPAAm monomer conversion was
ꢀ
(
(
98%)), and N,N -dicyclohexylcarbodiimide (0.44 g, 0.02 mol)
determined by comparing the vinyl proton signal (δ ∼5.4–6.3,
DCC, purchased from Aldrich (99%)) were added. The solu-
3
H per mol NIPAAm monomer) to the total isopropyl methylene
tion was stirred at room temperature for 14 h. The solution was
filtered to remove DCC. The product was then precipitated in
signal (δ ∼3.6–3.85 1H per mol NIPAAm).
◦
diethyl ether (three times) at 0 C. The trace of solvent was
Mass Analysis
evaporated under vacuum (yield 70%).
1
ESI-MS experiments were carried out using a Thermo Finni-
gan LCQ Deca ion trap mass spectrometer (Thermo Finnigan,
San Jose, CA, USA). The instrument was calibrated with caf-
feine, Met–Arg–Phe–Ala acetate (MRFA), and Ultramark 1621
H NMR spectroscopy confirmed the expected structure.
δH (300 MHz, CDCl3): 2.5 (2H, t, CH2–CO2), 2.6 (2H, t, –S–
CH2–CH2), 3.3 (2H, s, CH3–O), 3.6 (4H, s, CH2O), 4.1 (2H,
−
4
m, CH2O(CO)). Modified PEG (0.55 g, 5 × 10 mol) was dis-
solved in 10 mL of acetone at concentration of 0.05 M of DTT,
and the solution was mixed for 3 h. The product was precipitated
in diethyl ether (three times), dried, and stored in a fridge (yield
75%).
(
all from Aldrich) in the mass range of 195–1822 Da. All spec-
tra were acquired in positive ion mode over the mass to charge
range, m/z, of 100–2000 with a spray voltage of 5 kV, a capillary
◦
voltage of 44V, and a capillary temperature of 275 C. Nitrogen
was used as the sheath gas while helium was used as the auxiliary
1H-NMR confirmed the expected structure. δH (300 MHz,
CDCl3, TMS) SH 1.5 ppm (0.9H, t, SH), 2.5 (2H, t, CH2–CO2),
−
1
gas. The sample (1 mg mL ) was prepared by dissolving in a

RESEARCH FRONT
8
42
C. Boyer et al.
NH2
p
O
H
N
oNH
q
O
n
HN
O
OH
O
×D2O
O
k
l
NH
j
SH
m
HN
O
i g
NH2 HN
NH2
q, h, j
h
O
NH
i, n
g
p
m
o
l
k
8
.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
NH2
p
o
O
H
N
NH
q
O
f
O
OH
O
n
e
NH
HN
O
O
S
j
b
a
e
k
l
NH
d
c n
S
f
m
HN
O
i
HN
g
h
NH2
NH2
c
NH
O
d, k
g
q, j
m, p
l
a
dꢂ, i, n
o
b
5.0
0.8 63.8
4.3 5.0 1.9
436.1
8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5
Fig. 9. (Top) 1H NMR spectrum of the model peptide (sequence: GNGRGC) (recorded in deuterium oxide, 298 K, 128
1
1
scans). (Bottom) H NMR spectrum of purified PNIPAAm conjugated with the peptide ( H NMR was carried out by saturation
1
−1
of deuterium oxide, 600 MHz, 298 K, 128 scans). Pre-conjugation with peptide, Mn ( H NMR/GPC) 7100/7400 g mol , PDI
.08. Post-conjugation, Mn ( H NMR/GPC) 7500/8200 g mol , PDI 1.07.
1
−1
1
2
.6 (2H, m, HS–CH2–CH2), 3.3 (2H, s, CH3–O), 3.6 (4H, s,
under ambient conditions. The solvent was partially evaporated
under vacuum and the product was precipitated in diethyl ether
to yield an orange oil. The process was repeated three times.
The orange oil was separated from diethyl ether and traces of
CH2O), 4.1 (2H, m, CH2O(CO)).
Synthesis of BSPA[13,81]
1
diethyl ether were removed under vacuum (yield 80%). H NMR
3
,3-Mercaptopropionic acid (4 mL, 46 mmol) was placed in
confirmed the structure expected. δH (300 MHz, CDCl3, TMS)
1.2 (18H, t, CH3 of TEA), 2.5 (2H, t, –CH2–S–), 2.7 (2H, t,
–CH2–COO ), 2.9 (12H, q, N–CH2– of TEA).
Benzyl bromide (10.5 g, 0.046 mol) was dissolved in DCM
(50 mL) and stirred with a 1.2-fold excess of dithiocarboxysul-
fanyl propionic acid salt (20.50 g, 55.45 mmol) for 16 h at room
a round-bottomed flask containing dichloromethane (DCM,
5
1
−
0 mL). The solution was placed in an ice bath and stirred for
0 min, followed first by the dropwise addition of triethylamine
(
(
Aldrich, 99%) (12 mL, 96 mmol), and then by carbon disulfide
6 mL, 62 mmol). The resultant orange oil was stirred for 5 h

RESEARCH FRONT
RAFT Polymer End-Group Modification
843
(
A)
(B)
1
2
3
4
5
6
7
20
22
Retention time [min]
Fig. 10. DNA–polymer conjugates. (A)Agarose gel electrophoresis of conjugates. Lane 1: thiol functionalized oligonucleotide
−
1
(
ON), lanes 2 and 5: ON–PNIPAAm conjugate (Mn of PNIPAAm 5200 and 13500 g mol , respectively), lanes 3 and 6: ON–
−
1
PNIPAAm (Mn 5200 and 13500 g mol , respectively) after incubation with DTT (0.1 M for 3 h), lane 4: ON–PHPMA conjugate
Mn 3200 g mol ), and lane 7: ON–POEG-A conjugate (Mn 12500 g mol ). (B) GPC traces of (—) ON before conjugation,
) ON–PNIPAAm conjugate (Mn 5200 g mol ), ON–POEG-A conjugate
Mn 12500 g mol ). Note: the experimental method for agarose gel: ON solution (10 µL, 10 M) was mixed with TBE/urea
−
1
−1
(
(
(
−
1
−1
ꢀ) ON–PHPMA conjugate (Mn 3200 g mol ), (
•
−
1
−5
solution (10 µL).
H
d
OH
OH
H O
×
2
a
O
H
NH
H
O
f
b
g
H
c
g
S
H
e
OH
OH
S
n
8
.40 8.20 8.00 7.80 7.60 7.40 7.20
g
d
8
.40 8.20 8.00 7.80 7.60 7.40 7.20
a
e
f
c
b
2
.4
51.8
6.0
3.5
2.3
443.6
1.5
6.0
5.5
5.0
4.5
4.0
3.0
2.5
2.0
1.0
0.5
1
1
−1
Fig. 11.
H NMR of glucose-conjugated PNIPAAm (Mn ( H NMR) 5900 g mol , PDI 1.08) in D2O. Inset: (top) before reaction and (bottom) after reaction
and purification by dialysis.

RESEARCH FRONT
8
44
C. Boyer et al.
6
4
2
dropwise to the sodium dithiobenzoate using an addition funnel
over a period of 1 h under vigorous stirring conditions. The red
precipitate was filtered off and washed with deionized water
until the washings became colourless. The solid was dried under
vacuum at room temperature overnight.
Synthesis of CDTB
Distilled ethyl acetate (80.0 mL) was placed in a round-
bottomed flask. To the flask dry di(thiobenzoyl) disulfide
ꢀ
(
5.84 g, 21.0 mmol), 4,4 -azobis(4-cyanopentanoic acid) (4.25 g,
1
4.0 mmol), and ethyl acetate (50 mL) were added. The reac-
◦
tion solution was stirred at 75 C for 14 h. The ethyl acetate was
then removed under vacuum. The crude product was isolated by
column chromatography (silicagel 60 Å, 70–230 mesh) using
ethyl acetate/hexane (2/3) as eluent. Fractions that were red in
colour were combined and dried over anhydrous sodium sulfate
overnight. The solvent mixture was removed under vacuum, and
3
0
40
Retention time [min]
Fig. 12. GPC traces of (—) thiol-terminated PEG, (- - -) PNIPAAm,
- · - · -) crude product (PEG-b-PNIPAAm), (· · · ) after dialysis against water.
(
◦
the red oily residue placed in a freezer at −20 C, whereupon
it crystallized. The target compound was recrystallized from
temperature. The triethylammonium bromide was filtered off
and washed with DCM (5 mL). The product was purified by
extraction two times with deionized water (20 mL) and three
times with acidic water (pH 2). The organic phase was dried
over sodium sulfate (Na2SO4) and the solvent evaporated. The
product, a yellow oil, was purified by a silica gel column
chromatography (chloroform) (yield 65%).
benzene.
1H NMR spectroscopy confirmed the expected structure.
δH (300 MHz, CDCl3, TMS) 2.1 (3H, s, CH3), 2.5 (2H, dt,
–
CH2–CH2CO2H), 2.7 (2H, t, –CH2–COOH), 7.2 (2H, aromatic
group), 7.6 (1H, aromatic group), 7.8 (2H, aromatic group). δC
75 MHz, CDCl3, TMS) 224, 175, 145, 135, 130, 128, 126, 120,
5, 35, 32, 25.
(
4
δH (300 MHz, CDCl3, TMS) 2.85 (2H, t,
J
6.8,
CH2C(O)OH), 3.62 (2H, t, CH2CH2), 4.61 (2H, s, CH2-Ph),
RAFT Polymerizations
7
1
.31 (5H, m, CH(C6H5)). δC (75 MHz, CDCl3, TMS) 224.1,
79.2, 136.2, 130.7, 130.1, 129.2, 42.9, 34.4, 32.3.
RAFT Polymerization of N-(2-Hydroxypropyl)
Methacrylamide (HPMA) in the Presence of CDTB[
38]
Synthesis of 4-Cyanopentanoic Acid Dithiobenzoate
◦
Polymerizations were conducted at 70 C for 6 h employ-
ing ACVA as the primary radical source and CDTB as the
RAFT chain-transfer agent. All polymerizations with HPMA
were performed directly in an acetate buffer at pH 5.2,
[
82]
(CDTB)
Synthesis of Dithiobenzoic Acid (DTBA)
Sodium methoxide (30% solution in methanol, 180.0 g,
.0 mol) was added to a thoroughly dried, three-necked round-
− −1
1
(
0.27 mol L acetic acid and 0.73 mol L sodium acetate)
1
with an initial monomer concentration ([M]0) of 1 M in a
round-bottom flask that was purged with nitrogen for 30 min
before the polymerization. Typical HPMA polymerization con-
ditions were: [HPMA]0/[CDTB]0/[ACVA]0 = 40/1/0.2. HPMA
bottomed flask equipped with a magnetic stir bar, addition funnel
250.0 mL), thermometer, and a rubber septum for liquid trans-
(
fers. Anhydrous methanol (250.0 g) was then added to the flask
via a cannula, followed by rapid addition of elemental sulfur
−
4
(
(
0.716 g, 0.005 mol), CDTB (34.5 mg, 1.23 × 10 mol), ACVA
5
(
32.0 g, 1.0 mol). Benzyl chloride (63.0 g, 0.5 mol) was added
−
6.9 mg, 2.46 × 10 mol), and acetic buffer (10 mL) were
drop-wise by the addition funnel, over a period of 1 h, at room
temperature under a dry nitrogen atmosphere. The reaction mix-
mixed. The solutions were dialyzed against water for 3 h to
remove salt (water was changed every hour), and then freeze-
dried. The solid was dissolved in ethanol and the polymer
precipitatedindiethylether.Theprecipitationwasrepeatedtwice
to remove any unreacted monomer and RAFT agent. The pink
powder was dried under vacuum and characterized by DMAc
◦
ture was heated in an oil bath at 67 C for 10 h. Subsequently, the
◦
reaction mixture was cooled to 0 C. The precipitated salt was
removed by filtration and the solvent removed under vacuum.
Deionized water (500 mL) (resistivity 18.0 Mꢀ) was added to
the residue. The solution was filtered a second time and trans-
ferred to an extraction funnel. The crude sodium dithiobenzoate
solution was washed with diethyl ether (3 × 200.0 mL). Diethyl
ether (200.0 mL) and 1.0 N HCl (500.0 mL) were added, and
dithiobenzoic acid was extracted into the ethereal layer. Deion-
ized water (300.0 mL) and 1.0 N NaOH (600.0 mL) were added,
and sodium dithiobenzoate was extracted into the aqueous layer.
This washing process was repeated two more times to finally
yield a solution of sodium dithiobenzoate.
1
and aqueous GPC and by H NMR spectroscopy in D2O.
RAFT Polymerization of NIPAAm in the Presence
of BSPA
Typical NIPAAM polymerization conditions were:
[
0
3
NIPAAm]0/[CTA]0/[AIBN]0 = 55/1/0.2. NIPAAm (1.13 g,
−
4
.01 mol), BSPA (50 mg, 1.82 × 10 mol), AIBN (5.9 mg,
−
5
.6 × 10 mol), and acetonitrile (10 mL) were mixed.The solu-
◦
tions were maintained at 0 C and purged with nitrogen for
Synthesis of Di(thiobenzoyl) Disulfide
◦
30 min. Subsequently, the solutions were heated to 65 C for 5–
Potassium ferricyanide(iii) (32.93 g, 0.1 mol) was dissolved
in deionized water (500.0 mL). Sodium dithiobenzoate solution
6 h.The solution was partially evaporated under vacuum, and the
polymer was precipitated in cold diethyl ether (at 0 C).The oper-
◦
(
350.0 mL) was transferred to a conical flask equipped with a
ation was repeated three times to remove any unreacted RAFT
agent and traces of monomers. The polymer was dried under
magnetic stir bar. Potassium ferricyanide solution was added

RESEARCH FRONT
RAFT Polymer End-Group Modification
845
vacuum to yield a yellow powder. Samples were characterized
by DMAc GPC and by H NMR spectroscopy (D2O).
solution was diluted 20 times and analyzed by UV-vis spec-
troscopy (yield: 85% by UV). The purified polymer samples
were further characterized by H NMR spectrometry.
1
1
Chemical Modification of End-Groups by Aminolysis
in the Presence of DTP
A thiol-modified glucose was prepared following the proce-
dure described by Davis et al.[ The conjugation was carried
out using conditions similar to that of peptide conjugation and
was purified by dialysis.
80]
Typical RAFT end-group modification conditions for PNIPAAm
−
5
were as follows: PNIPAAm (0.1 g, 2.12 × 10 mol, Mn
−1
−3
4
700 g mol ) and DTP (46 mg, 2.10 × 10 mol) were dis-
Bioconjugation with ON or si-RNA
solved in acetone (2 mL). The solution was purged for 10 min
under nitrogen to remove oxygen. Hexylamine (0.15 mL, 64 mg
Thiol-functional DNA or si-RNA was purchased from
IDTDNA. These nucleoside products contain protected thiols,
i.e., the thiol is protected as a disulfide bond. To regenerate
the thiol functionality, the disulfides were reduced by DTT. ON
−
5
dilute in 1 mL of acetone, 9.5 × 10 mol) was then added under
nitrogen. The solution was shaken for 4–5 h, until a permanent
yellow was observed. The polymers were precipitated in cold
diethyl ether (four times) and dialyzed against an acetone/water
mixture (50/50% v/v ) for 2 × 3 h to remove any excess DTP, and
dialyzed against water for 2 × 3 h. The polymer was freeze-dried
and analyzed by NMR in deuterium oxide.
−
9
(
20 µL, 10 × 10 mol), triethylamine (2 µL), water (45 µL),
and DTT solution (50 µL, 7.7 mg dilute in 0.5 mL in RNAse
−
6
free water, 5 × 10 mol of DTT) were gently shaken. DTT
was removed by a series of extractions using ethyl acetate
(
>
4 × 400 µL) to yield thiol DNA (yield 75%, thiol functionality
95%). DNA and si-RNA were used directly for bioconjuga-
tions.
A large excess of polymer ([DNA/si-RNA]0/[PNIPAAm]0 =
/20) was used. Typically, PDS-terminated PNIPAAm (2.2 mg,
Typical RAFT end-group modification conditions for
−
5
PHPMA were as follows: PHPMA (0.1 g, 3.13 × 10 mol,
−1
−4
Mn 3200 g mol ) and DTP (69 mg, 3.2 × 10 mol) were dis-
solved in dimethylformamide (DMF, 2 mL). The solutions were
purged for 10 min under nitrogen to remove oxygen. Hexyl-
amine (0.170 mL, 64 mg dilute in 1 mL in dimethylformamide
1
−
1
−9
Mn 4700 g mol , 470 × 10 mol) was diluted in phosphate
buffer (200 µL, pH 8.0, 1 M). PNIPAAm solution (80 µL) was
added to the ON solution. The solution was gently shaken
−
3
(
DMF), 0.9 × 10 mol) was then added under nitrogen. The
solution was shaken for 3–4 h until a permanent yellow colour
was observed. The polymers were precipitated in cold diethyl
ether (four times), dialyzed against a methanol/water mixture
◦
overnight at 37 C. The product was dialyzed against water and
freeze-dried. Finally, the product was re-dispersed in water and
its concentration was determined by absorbance at 260 nm. The
conjugate was analyzed by aqueous GPC and agarose gel elec-
(
50/50%, v/v ) for 2 × 3 h to remove any excess DTP, and dia-
lyzed against water for 2 × 3 h. The polymer was freeze-dried
−
5
trophoresis. The ON solution (10 µL, 10 M) was mixed with
a Tris/Borate/EDTA (TBE)/urea solution (10 µL). The gel was
run at 100V for 45–50 min. (Note: the oxidation of thiol under
basic conditions takes longer than at neutral pH.)
1
and analyzed by H NMR spectroscopy in D2O.
Bioconjugations
Conjugation of Peptides and Thiol-Modified
Carbohydrate
A large excess of polymer ([DNA/si-RNA]0/[PHPMA]0 =
1
/40) was used. Typically, PDS-terminated PHPMA (2.2 mg,
−
1
−9
A typical example of conjugation is given as follows for
Mn 3200 g mol , 688 × 10 mol) was diluted in phosphate
the NGR peptide: the peptide was conjugated to ω-DTP-
buffer (200 µL, pH 8.0, 1 M). This solution (100 µL) was added
−
1
−9
functionalized PNIPAAm (Mn 4700 g mol ), DTP function-
to a solution of ON (10.0 × 10 mol), and was gently shaken
◦
ality 0.90 (assessed by NMR and by UV-vis). In a typical
overnight at 37 C. The product was dialyzed against water
−
6
conjugation experiment, PNIPAAm (10 mg, 2.12 × 10 mol)
was diluted in phosphate buffer (1 mL) at pH 8.The solution was
degassed with nitrogen for 5 min and was incubated with NGR
and freeze-dried. Finally, the product was re-dispersed in water
and its concentration was determined by absorbance at 260 nm.
The conjugate was analyzed by aqueous GPC and agarose gel
electrophoresis.
−
6
peptide (1.5 mg, 2.65 × 10 mol) for 6 h at room temperature.
Purification of the NGR peptide conjugated polymers was car-
ried out by dialysis against water using a membrane with a MW
cutoffof1000 Dafor1day.TheNGRconjugationcouldbequan-
tified by monitoring the release of pyridine-2-thione by UV-vis
Synthesis of Diblock Polymers
−
4
PDS-functionalized PNIPAAm (1.0 g, 2.12 × 10 mol, Mn by
1
−1
−1
H NMR = 4700 g mol , Mn by GPC = 5300 g mol , PDI
−
1
−1
−4
spectrophotometry monitoring at 343 nm (ε 8080 M cm in
water).
1.12) and thiol-terminated PEG (0.23 g, 2.12 × 10 mol, Mn
[
68,83]
1
−1
The solution was diluted 20 times and analyzed by
( H NMR/GPC) 1100/1400 g mol , PDI 1.04, reaction con-
ditions: [PEG]0/[PNIPAAm]0 = 1.0) were dissolved in water
(5 mL). The solution was purged with nitrogen for 15 min, and
then stirred at room temperature for 14 h. The crude product
was analyzed by UV-vis spectroscopy, DMAc GPC, and by
UV-vis spectroscopy (yield: 85% by UV). The purified polymer
1
samples were further characterized by H NMR spectrometry.
Atypicalexampleofconjugationisgivenasfollows:theNGR
peptide was conjugated to ω-DTP-functionalized PHPMA (Mn
−1
1
3
200 g mol ), DTP functionality 0.90 (assessed by NMR and
H NMR spectrometry. The product was purified by dialysis
UV-vis). In a typical conjugation experiment, PHPMA (10 mg,
against water (membrane cut off 3500 Da) for 2 days. The prod-
uct was freeze-dried and analyzed by DMAc GPC and NMR
spectrometry.
Theyieldwascalculatedbymeasuringthereleaseofpyridine-
2-thione(assessedbyUV-visspectroscopy):yield = [pyridine-2-
thione]/[PDS] = (Abs/ε)/[PDS], where [pyridine-2-thione] and
[PDS] are the concentration of pyridine-2-thione assessed
by UV-vis spectroscopy and PDS-functionalized PNIPAAm,
respectively. The yield was 85%.
−6
3
.12 × 10 mol)wasdilutedinphosphatebufferatpH8(1 mL).
The solution was degassed and incubated with NGR peptide
−
6
(
2.15 mg, 2.65 × 10 mol) for 6 h at room temperature. Purifi-
cation of the NGR peptide-conjugated polymers was carried out
by dialysis against water using a membrane with a MW cut off
of 1000 Da for 1 day. NGR conjugation could be quantified by
monitoring the release of pyridine-2-thione via a UV-vis spec-
−
1
−1
[68,83]
trophotometer at 343 nm (ε 8080 M cm in water).
The

RESEARCH FRONT
8
46
C. Boyer et al.
−
4
1
PDS-functionalized PMMA (1.0 g, 1.75 × 10 mol, Mn ( H
[23] G. David, C. Boyer, J. Tonnar, B. Ameduri, P. Lacroix-Desmazes,
B. Boutevin, Chem. Rev. 2006, 106, 3936. doi:10.1021/
CR0509612
[24] C. J. Hawker, A. W. Bosman, E. Harth, Chem. Rev. 2001, 101, 3661.
doi:10.1021/CR990119U
−
1
NMR/GPC) 4740/5100 g mol , PDI 1.14, PDS functionality =
−
4
1
9
0%) and thiol-terminated PEG (0.21 g, 1.9 × 10 mol, Mn ( H
1
−
NMR/GPC) 1100/1400 g mol , PDI 1.04) were dissolved in
acetonitrile (5 mL). The solution was purged with nitrogen for
[
25] C. Boyer, V. Bulmus, J. Liu, T. P. Davis, M. H. Stenzel,
C. Barner-Kowollik, J. Am. Chem. Soc. 2007, 129, 7145. doi:10.1021/
JA070956A
1
5 min and then stirred at room temperature for 14 h. The crude
product was analyzed by UV-vis spectroscopy, DMAc GPC, and
by H NMR spectrometry. The product was purified by dialysis
against water (membrane cut off 3500 Da) for 2 days. The prod-
uct was freeze-dried and analyzed by DMAc GPC and NMR
1
[
26] J. Liu, V. Bulmus, D. L. Herlambang, C. Barner-Kowollik,
M. H. Stenzel, T. P. Davis, Angew. Chem. Int. Ed. 2007, 46, 3099.
doi:10.1002/ANIE.200604922
(
yield calculated by UV-vis spectroscopy: 80%).
[27] Y. Zhao, S. Perrier, Chem. Commun. 2007, 4294. doi:10.1039/
B708293B
[
28] P. De, M. Li, S. R. Gondi, B. S. Sumerlin, J. Am. Chem. Soc. 2008,
30, 11288. doi:10.1021/JA804495V
Acknowledgements
1
We acknowledge theAustralian Research Council for the award of Discovery
Grants (to V.B. and T.P.D.). In addition, we acknowledge the receipt of a
Federation Fellowship (T.P.D.).
[29] M. Bathfield, F. D’Agosto, R. Spitz, M.-T. Charreyre, T. Delair, J. Am.
Chem. Soc. 2006, 128, 2546. doi:10.1021/JA057481C
[30] A. B. Lowe, R. Wang, V. Tiriveedhi, P. Butko, C. L. McCormick,
Macromol. Chem. Phys. 2007, 208, 2339. doi:10.1002/MACP.
2
00700280
References
[
31] A. W. York, C. W. Scales, F. Huang, C. L. McCormick, Biomacro-
molecules 2007, 8, 2337. doi:10.1021/BM700514Q
[
[
[
[
[
1] G. Moad, E. Rizzardo, S. H. Thang, Polymer 2008, 49, 1079.
doi:10.1016/J.POLYMER.2007.11.020
2] L. Barner, C. Barner-Kowollik, T. P. Davis, M. H. Stenzel, Aust. J.
Chem. 2004, 57, 19. doi:10.1071/CH03232
3] L. Barner, T. P. Davis, M. H. Stenzel, C. Barner-Kowollik, Macromol.
Rapid Commun. 2007, 28, 539. doi:10.1002/MARC.200600805
4] S. Perrier, P. Takolpuckdee, J. Polym. Sci., PartA: Polym. Chem. 2005,
[32] A. B. Lowe, C. L. McCormick, RAFT Polymerization in Homoge-
neous Aqueous Media: Initiation Systems, RAFT Agent Stability,
Monomers and Polymer Structures, in Handbook of RAFT Polymer-
ization (Ed. C. Barner-Kowollik) 2008, Ch 7, pp. 235–275 (Wiley:
Weinheim).
[33] C. L. McCormick, S. E. Kirkland, A. W. York, Polym. Rev. 2006, 46,
421. doi:10.1080/15583720600945428
[34] P. Vana, L. Albertin, L. Barner, T. P. Davis, C. Barner-Kowollik,
J. Polym. Sci., Part A: Polym. Chem. 2002, 40, 4032. doi:10.1002/
POLA.10500
4
3, 5347. doi:10.1002/POLA.20986
5] S. Perrier, P. Takolpuckdee, C. A. Mars, Macromolecules 2005, 38,
033. doi:10.1021/MA047611M
2
[
[
6] B. Boutevin, C. Boyer, G. David, P. Lutz, Macromol. Eng. 2007, 2, 775.
7] B. Boutevin, G. David, C. Boyer, Adv. Polym. Sci. 2007, 206, 31.
doi:10.1007/12_2006_101
[35] A. Postma,T. P. Davis, G. Moad, M. S. O’Shea, Macromolecules 2005,
38, 5371. doi:10.1021/MA050402X
[
8] A. B. Lowe, C. L. McCormick, Prog. Polym. Sci. 2007, 32, 283.
doi:10.1016/J.PROGPOLYMSCI.2006.11.003
9] A. W.York, S. E. Kirkland, C. L. McCormick,Adv. Drug Delivery Rev.
[36] M. R. Whittaker, Y.-K. Goh, H. Gemici, T. M. Legge, S. Perrier,
M. J. Monteiro, Macromolecules 2006, 39, 9028. doi:10.1021/
MA061070E
[
2
008, 60, 1018. doi:10.1016/J.ADDR.2008.02.006
[37] L. Wong, C. Boyer, Z. Jia, H. M. Zareie, T. P. Davis, V. Bulmus,
Biomacromolecules 2008, 9, 1934. doi:10.1021/BM800197V
[38] C. W. Scales, Y. A. Vasilieva, A. J. Convertine, A. B. Lowe,
C. L. McCormick, Biomacromolecules 2005, 6, 1846. doi:10.1021/
BM0503017
[
10] A. J. Convertine, B. S. Lokitz, Y. Vasileva, L. J. Myrick, C. W. Scales,
A. B. Lowe, C. L. McCormick, Macromolecules 2006, 39, 1724.
doi:10.1021/MA0523419
[
[
[
[
[
[
[
11] M. H. Stenzel, T. P. Davis, Aust. J. Chem. 2003, 56, 1035.
doi:10.1071/CH03124
12] M. H. Stenzel,T. P. Davis, C. Barner-Kowollik, Chem. Commun. 2004,
[39] M. Li, P. De, S. R. Gondi, B. S. Sumerlin, J. Polym. Sci., PartA: Polym.
Chem. 2008, 46, 5093. doi:10.1002/POLA.22837
[40] H. Gemici, T. M. Legge, M. Whittaker, M. J. Monteiro, S. Perrier,
J. Polym. Sci., Part A: Polym. Chem. 2007, 45, 2334. doi:10.1002/
POLA.21985
[41] X.-P. Qiu, F. M. Winnik, Macromolecules 2007, 40, 872. doi:10.1021/
MA062225L
[42] J. Xu, J. He, D. Fan, X. Wang, Y. Yang, Macromolecules 2006, 39,
8616. doi:10.1021/MA061961M
[43] X.-P. Qiu, F. M. Winnik, Macromol. Rapid Commun. 2006, 27, 1648.
doi:10.1002/MARC.200600436
[44] L. Zhang, Y. Chen, Polymer 2006, 47, 5259. doi:10.1016/
J.POLYMER.2006.05.052
[45] D. L. Patton, M. Mullings, T. Fulghum, R. C. Advincula, Macro-
molecules 2005, 38, 8597. doi:10.1021/MA051035S
[46] G. Moad,Y. K. Chong, A. Postma, E. Rizzardo, S. H. Thang, Polymer
2005, 46, 8458. doi:10.1016/J.POLYMER.2004.12.061
[47] V. Lima, X. Jiang, J. Brokken-Zijp, P. J. Schoenmakers,
B. Klumperman, R. Van Der Linde, J. Polym. Sci., Part A: Polym.
Chem. 2005, 43, 959. doi:10.1002/POLA.20558
[48] D. B. Thomas, A. J. Convertine, R. D. Hester, A. B. Lowe,
C. L. McCormick, Macromolecules 2004, 37, 1735. doi:10.1021/
MA035572T
1546. doi:10.1039/B404763J
13] M. H. Stenzel, T. P. Davis,A. G. Fane, J. Mater. Chem. 2003, 13, 2090.
doi:10.1039/B304204A
14] T. S. C. Pai, C. Barner-Kowollik, T. P. Davis, M. H. Stenzel, Polymer
2004, 45, 4383. doi:10.1016/J.POLYMER.2004.04.041
15] X. Hao, M. H. Stenzel, C. Barner-Kowollik, T. P. Davis, E. Evans,
Polymer 2004, 45, 7401. doi:10.1016/J.POLYMER.2004.08.068
16] M. Hales, C. Barner-Kowollik, T. P. Davis, M. H. Stenzel, Langmuir
2
004, 20, 10809. doi:10.1021/LA0484016
17] L. Albertin, M. H. Stenzel, C. Barner-Kowollik, L. J. R. Foster,
T. P. Davis, Macromolecules 2005, 38, 9075. doi:10.1021/
MA051310A
[
[
[
18] E. Min, K. H. Wong, M. H. Stenzel, Adv. Mater. 2008, 20, 3550.
doi:10.1002/ADMA.200800569
19] B. S. Sumerlin, A. B. Lowe, P. A. Stroud, P. Zhang, M. W. Urban,
C. L. McCormick, Langmuir 2003, 19, 5559. doi:10.1021/LA034459T
20] B. S. Lokitz, J. E. Stempka, A. W. York, Y. Li, H. K. Goel,
G. R. Bishop, C. L. McCormick, Aust. J. Chem. 2006, 59, 749.
doi:10.1071/CH06264
[
21] L. Barner, S. Perrier, Polymers with Well-defined End Groups via
RAFT—Synthesis, Applications and Postmodifications, in Handbook
of RAFT Polymerization (Ed. C. Barner-Kowollik) 2008, Ch 12, p. 455
[49] S. R. Gondi, A. P. Vogt, B. S. Sumerlin, Macromolecules 2007, 40,
474. doi:10.1021/MA061959V
(
Wiley: Weinheim).
[
22] K. Matyjaszewski, J. Xia, Chem. Rev. 2001, 101, 2921.
[50] C. Boyer, V. Bulmus, P. Priyanto, W. Y. Teoh, R. Amal, T. P. Davis,
J. Mater. Chem. 2009, 19, 111. doi:10.1039/B815202K
doi:10.1021/CR940534G

RESEARCH FRONT
RAFT Polymer End-Group Modification
847
[
[
[
[
[
[
[
51] C. Boyer, A. Granuille, T. P. Davis, V. Bulmus, J. Polym. Sci., Part A:
Polym. Chem. 2009, 47, 3773. doi:10.1002/POLA.23433
52] K. L. Killops, L. M. Campos, C. J. Hawker, J. Am. Chem. Soc. 2008,
[69] K. L. Heredia, T. H. Nguyen, C.-W. Chang, V. Bulmus, T. P. Davis,
H. D. Maynard, Chem. Commun. 2008, 28, 3245. doi:10.1039/
B804812F
[70] A. Dondoni, Angew. Chem. Int. Ed. 2008, 47, 8995. doi:10.1002/
ANIE.200802516
[71] D. Bontempo, K. L. Heredia, B. A. Fish, H. D. Maynard, J. Am. Chem.
Soc. 2004, 126, 15372. doi:10.1021/JA045063M
[72] C. Boyer, J. Liu, V. Bulmus, T. P. Davis, C. Barner-Kowollik,
M. H. Stenzel, Macromolecules 2008, 41, 5641. doi:10.1021/
MA800289U
[73] C. Boyer, J. Liu, L. Wong, M. Tippett, V. Bulmus, T. P. Davis, J. Polym.
Sci., PartA: Polym. Chem. 2008, 46, 7207. doi:10.1002/POLA.23028
[74] J. Xu, C. Boyer, V. Bulmus, T. P. Davis, J. Polym. Sci., Part A: Polym.
Chem. 2009, in press.
[75] T. R. Darling, T. P. Davis, M. Fryd, A. A. Gridnev, D. M. Haddleton,
S. D. Ittel, R. R. Matheson Jr, G. Moad, E. Rizzardo, J. Polym.
Sci., Part A: Polym. Chem. 2000, 38, 1706. doi:10.1002/(SICI)1099-
0518(20000515)38:10<1706::AID-POLA20>3.0.CO;2-5
[76] G.T. Zugates, D. G.Anderson, S. R. Little, I. E. B. Lawhorn, R. Langer,
J. Am. Chem. Soc. 2006, 128, 12726. doi:10.1021/JA061570N
[77] S. Harrisson, Macromolecules 2009, 42, 897. doi:10.1021/
MA900075V
[78] P. J. Roth, D. Kessler, R. Zentel, P. Theato, Macromolecules 2008, 41,
8316. doi:10.1021/MA801869Z
[79] K. L. Heredia, D. Bontempo, T. Ly, J. T. Byers, S. Halstenberg,
H. D. Maynard, J. Am. Chem. Soc. 2005, 127, 16955. doi:10.1021/
JA054482W
1
30, 5062. doi:10.1021/JA8006325
53] N. ten Brummelhuis, C. Diehl, H. Schlaad, Macromolecules 2008, 41,
946. doi:10.1021/MA802047W
9
54] C. Rissing, D. Y. Son, Organometallics 2008, 27, 5394. doi:10.1021/
OM8003527
55] G. Niu, H. Zhang, L. Song, X. Cui, H. Cao,Y. Zheng, S. Zhu, Z.Yang,
H.Yang, Biomacromolecules 2008, 9, 2621. doi:10.1021/BM800573E
56] S. Fan, F. Y. C. Boey, M. J. M. Abadie, Eur. Polym. J. 2008, 44, 2123.
doi:10.1016/J.EURPOLYMJ.2008.04.024
57] P. Jonkheijm, D. Weinrich, M. Koehn, H. Engelkamp, P. C. M.
Christianen, J. Kuhlmann, J. C. Maan, D. Nuesse, H. Schroeder,
R. Wacker, R. Breinbauer, C. M. Niemeyer, H. Waldmann, Angew.
Chem. Int. Ed. 2008, 47, 4421. doi:10.1002/ANIE.200800101
58] A. Pogantsch, S. Rentenberger, G. Langer, J. Keplinger, W. Kern,
E. Zojer, Adv. Funct. Mater. 2005, 15, 403. doi:10.1002/ADFM.
[
200305146
[
[
[
[
[
[
[
[
[
[
59] Z. Yang, D. A. Wicks, C. E. Hoyle, H. Pu, J. Yuan, D. Wan, Y. Liu,
Polymer 2009, 50, 1717. doi:10.1016/J.POLYMER.2008.12.018
60] G. Chen, S. Amajjahe, M. H. Stenzel, Chem. Commun. 2009, 1198.
doi:10.1039/B900215D
61] L. A. Connal, C. R. Kinnane, A. N. Zelikin, F. Caruso, Chem. Mater.
2009, 21, 576. doi:10.1021/CM803011W
62] C. Nilsson, E. Malmstroem, M. Johansson, S. M. Trey, J. Polym. Sci.,
Part A: Polym. Chem. 2009, 47, 589. doi:10.1002/POLA.23176
63] T. Clark, L. Kwisnek, C. E. Hoyle, S. Nazarenko, J. Polym. Sci., Part
A: Polym. Chem. 2009, 47, 14. doi:10.1002/POLA.23089
[80] G. J. L. Bernardes, D. P. Gamblin, B. G. Davis, Angew. Chem. Int. Ed.
2006, 45, 4007. doi:10.1002/ANIE.200600685
[81] J. T. Lai, D. Filla, R. Shea, Macromolecules 2002, 35, 6754.
doi:10.1021/MA020362M
[82] Y. Mitsukami, M. S. Donovan, A. B. Lowe, C. L. McCormick,
Macromolecules 2001, 34, 2248. doi:10.1021/MA0018087
[83] G. T. Hermanson, Bioconjugate Techniques 1996 (Academic Press:
New York, NY).
64] C. W. Scales, A. J. Convertine, C. L. McCormick, Biomacromolecules
2
006, 7, 1389. doi:10.1021/BM060192B
65] J. W. Chan, B. Yu, C. E. Hoyle, A. B. Lowe, Chem. Commun. 2008,
959. doi:10.1039/B813438C
66] C. Boyer, T. P. Davis, V. Bulmus, Macromol. Rapid Commun. 2009,
0, 493. doi:10.1002/MARC.200800708
4
3
67] N. Murthy, J. Campbell, N. Fausto, A. S. Hoffman, P. S. Stayton,
Bioconjug. Chem. 2003, 14, 412. doi:10.1021/BC020056D
68] V. Bulmus, M. Woodward, L. Lin, N. Murthy, P. Stayton, A.
S. Hoffman, J. Controlled Release 2003, 93, 105. doi:10.1016/
J.JCONREL.2003.06.001