Aggregation behaviour of peptide-polymer conjugates containing linear peptide backbones and multiple polymer side chains prepared by nitroxide-mediated radical polymerization

Article abstract of DOI:10.1039/c0ob01047b

A series of peptides with an alternating sequence of alkoxyamine conjugated lysine and glycine residues were synthesized by classical solution phase peptide coupling. The resulting peptides containing up to eight alkoxyamine moieties were used as initiato

Full text of DOI:10.1039/c0ob01047b

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PAPER
Aggregation behaviour of peptide–polymer conjugates containing linear
peptide backbones and multiple polymer side chains prepared by
nitroxide-mediated radical polymerization†‡
Michael Mo¨ller,a Carsten Hentschel,b Lifeng Chib and Armido Studer*a
Received 18th November 2010, Accepted 7th January 2011
DOI: 10.1039/c0ob01047b
A series of peptides with an alternating sequence of alkoxyamine conjugated lysine and glycine residues
were synthesized by classical solution phase peptide coupling. The resulting peptides containing up to
eight alkoxyamine moieties were used as initiators in nitroxide-mediated polymerization (NMP) to
obtain peptide–polymer conjugates with well defined linear peptide backbones and a defined number of
polymeric side chains. Polymerization of styrene and N-isopropylacrylamide (NIPAM) occurred in a
highly controlled fashion. Molecular weight and polydispersity index (PDI) were determined by gel
permeation chromatography (GPC). Aggregation behaviour of these hybrid materials was investigated
by dynamic light scattering (DLS) and atomic force microscopy (AFM). Depending on composition,
number and length of the polymer side chains, the conjugates aggregate to different topologies.
Whereas peptide–polystyrene conjugates may aggregate to so called honeycomb structures,
peptide–poly-N-isopropylacrylamide conjugates show differentiated aggregation behaviour.
been successfully used along this line.20 Especially the 1,3-dipolar
cycloaddition of azides to alkynes has been widely applied for this
Introduction
purpose.21–22 Disadvantages of using the “grafting to” strategy are
selectivity problems if the peptide contains more than one reactive
functionality. Moreover, full conversion in particular if more than
one polymer should be attached to the peptide, is difficult to
achieve. This method is only practical for conjugation of synthetic
polymers with small molecular weights (up to Mw ~ 5000).2
The second approach to the synthesis of peptide–polymer
conjugates is called the “grafting from” or polymerization
approach.12,16,23–24 A polymerization initiator is covalently attached
to a peptide. This peptide–initiator is then used for polymerization
to give the desired conjugate. This method allows the conjugation
of polymer moieties with much higher molecular weights. An
important issue for the applied polymerization technique is its
tolerance towards many functionalities as they occur in peptides.
Due to the great functional group tolerance of radical chemistry,
controlled radical polymerization techniques such as reversible
addition-fragmentation chain transfer (RAFT) and atom-transfer
radical polymerization (ATRP) have widely been used for the syn-
thesis of peptide–polymer conjugates.12,16,24–26 Nitroxide-mediated
polymerization (NMP) is also well established for the synthesis of
these complex materials.23,27–28 These methods lead to polymers
with a controlled chain length and narrow molecular weight
distribution.
Peptide–polymer conjugates belong to a very interesting class of
materials because they often show self-organizing properties and
biological activities.1–5 These hybrid materials offer the possibility
to build up defined nano- and microstructures due to the general
tendency of peptides to self-assemble.6–13 This biomimetic be-
haviour facilitates the design of complex molecular architectures.
Along with these properties, peptide–polymer conjugates can also
show enzymatic activity, alter the conductivity of a material and
spread or bound to surfaces they lead to bioactive surfaces.7,10,13,14
In addition, they are also able to control crystallization processes,
transport substances like dyes or drugs and are also used for
molecular targeting.15–17
On the one hand, the synthesis of peptide–polymer conjugates
can be accomplished by the “grafting to” or coupling strategy. In
that approach, one or several polymers are covalently bound to a
preformed peptide.3,7–11,18–19 In the past years “click” chemistry has
aOrganisch-Chemisches Institut and NRW Graduate School of Chemistry,
Westfa¨lische Wilhelms-Universita¨t Mu¨nster, Corrensstraße 40, 48149,
Mu¨nster, Germany. E-mail: studer@uni-muenster.de; Fax: +49-251-833-
6523; Tel: +49-251-833-3291
bPhysikalisches Institut, Westfa¨lische Wilhelms-Universita¨t Mu¨nster,
Wilhelm-Klemm-Straße 10, 48149, Mu¨nster, Germany. E-mail: chi@
uni-muenster.de; Fax: +49-251-833-3602; Tel: +49-251-833-3651
Whereas most approaches aimed for the conjugation of a single
polymer chain at one terminus of a peptide, examples of peptides
bearing polymeric side chains attached at the middle of the
† In memoriam Professor Athelstan L. J. Beckwith
‡ Electronic supplementary information (ESI) available. See DOI:
10.1039/c0ob01047b
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Scheme 1 Synthesis strategy for peptide–polymer conjugates 3.
peptide are rare. Even less investigated are peptides containing
Results and discussion
morethanone polymer side chain.12,29 Peptide–polymer conjugates
consisting of a well defined linear peptide backbone and a
multiple number of polymeric side chains have not been reported
to date.
The synthesis of all polymer conjugated peptides discussed herein
started with polymerization initiators like 1 which consists of an
alkoxyamine, covalently bound to a lysine–glycine dipeptide. This
modified dipeptide was homocoupled via conventional solution
phase peptide coupling procedures togive oligopeptides 2. Because
the number of initiator moieties doubles after each coupling
reaction, well defined peptides bearing multiple alkoxyamine
functionalities can be obtained in a few steps by this fragment
coupling strategy. These alkoxyamine derived peptides were then
used as initiators for NMP. As TEMPO-derived alkoxyamines
are only able to control polymerization of styrene or styrene
derivatives, synthesis of analogous alkoxyamine initiators bearing
a more sterically hindered nitroxide was necessary. The same
fragment coupling route was followed for immobilization of
a more sophisticated tetraethyl-substituted nitroxide into the
peptide.
Surface morphology of spin-coated polystyrene has been inten-
sively investigated. If unsubstituted linear polystyrene is spread in
solution on silicon substrates, it usually builds up droplet or net-
like structures after evaporation of the solvent.30–31 Franc¸ois et al.
first described the formation of so called honeycomb structures
by using the same approach with polystyrene-polyparaphenylene
copolymers in CS2.32 After publication of this initial paper,
numerous reports on honeycomb structures have appeared. Self-
assembly properties have been achieved by the use of specially
designed polymer architectures or by addition of polar additives.
In practice a special evaporation technique, the so called breath
figure method has mostly been used.33 Furthermore, carbohydrate-
polystyrene conjugates are also able to assemble in honeycomb
structures.34
Poly-N-isopropylacrylamide (PNIPAM) has attracted great in-
terests in the last years, because of its reversible thermoresponsive
solution behaviour in water. It is soluble in water below the lower
critical solution temperature (LCST) of 32 ◦C and it precipitates
at higher temperature. Hence, it is highly attractive to be used as a
component in biohybrid materials.12,35–36 Several studies on PNI-
PAM and its phase transition have been performed on covalently
bound PNIPAM-brushes and on spin-coated films. Homogeneous
films and beads of PNIPAM on silicon, gold and mica substrates
have been studied.37–38 Spin-coated copolymers of PNIPAM and
polyacrylic acid showed homogeneous structures with stick type
morphology whereas copolymers with polymethylmethacrylate
(PMMA) delivered honeycomb like structures.39–40
Herein we present the synthesis of peptide–polymer conjugates
containing well defined linear peptide backbones with a defined
number of polymeric side chains using NMP (Scheme 1). Our
report includes the synthesis of polymerization initiators, poly-
merization studies using styrene and NIPAM as monomers, DLS-
investigations on PNIPAM-conjugates and imaging of morpholo-
gies of the conjugates on silicon surfaces by AFM.
Initiator synthesis
The synthesis of the alkoxyamine moiety started with 1-
bromo-4-ethylbenzene (4) (Scheme 2). Radical bromination,
alkoxyamine formation under atom transfer conditions with
2,2,6,6-tetramethylpiperidin-N-oxyl radical (TEMPO) and in-
troduction of the carboxyl group by bromine-lithium-exchange
and subsequent addition of CO2 delivered 5 in a good over-
all yield. Reaction of double protected lysine 6 with glycin-
emethylester hydrochloride using 1-(3-dimethylaminopropyl)-3-
ethylcarbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole
(HOBt) and N-methylmorpholine (NMM) followed by depro-
tection of the side chain amino group led to dipeptide 7.
Hydrogenation of the benzylcarbamate group was conducted out
under slightly acidic conditions. The alkoxyamine functionality
was introduced by acylation of the lysine side chain of 7 with acid
5 using EDCI. Building block dipeptide 1 was obtained in 80%
yield.
Deprotection of the Boc-group was readily accomplished by
treatment of 1 with HCl in 1,4-dioxane (Scheme 3). Amine
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Scheme 2 Reagents and conditions: (i) Br2, CCl4, 50 ◦C, hn, 1 h;
(ii) TEMPO, Cu (powder), Cu(OTf)2, 4,4¢-di-tert-butyl-2,2¢-dipyridyl,
benzene, 75 ◦C, 16 h; (iii) t-Buli (2 eq.), CO2, THF, -78 ◦C → RT, 1 h; (iv)
HCl·H-Gly-OMe, EDCI, HOBt, NMM, CH2Cl2, RT, 18 h; (v) H2, (1 bar),
Pd/C, MeOH, HCl in MeOH (0.2 eq.), RT, 18 h, then HCl in MeOH (0.8
eq.); (vi) EDCI, HOBt, NMM, CH2Cl2, RT, 16 h.
hydrochloride 8a was isolated in a quantitative yield. Ester
hydrolysis under basic conditions in a mixture of water and
methanol afforded the free acid 8b in excellent yield. Coupling
of the dipeptide 8a with 8b was achieved with EDCI to give
tetrapeptide 9, containing two alkoxyamine moieties.
Removal of the Boc-group in 9 was performed with HCl in
methanol. Deprotection of the C-terminus was achieved applying
the same conditions used for ester hydrolysis of 1. The coupling
reaction between tetrapeptides 10a and 10b was again carried out
with EDCI, HOBt and NMM. Due to solubility problems, this
coupling reaction was conducted in DMF. The resulting octapep-
tide 11 was isolated by reversed-phase flash chromatography in
70% yield.
Selective deprotection of octapeptide 11 turned out to be
very challenging. After careful optimization we found that Boc-
cleavage was best conducted at 0.05 mmol scale with 12.5 equiv-
alents of HCl in 5.5 mL methanol for 48 h at room temperature
(Scheme 4). Reaction conditions had to be strictly followed in
order to achieve full conversion and to avoid methanolysis of
the peptide backbone. For the deprotection of the C-terminus,
peptide 11 was dissolved in methanol (0.167 mM) and 20 mL of
NaOH (1 M aq.) was added. High dilution was necessary due to
low solubility of the octapeptide 11. Full conversion for that ester
hydrolysis was achieved after 40 h.
Scheme 3 Reagents and conditions: (a) HCl (4 M in 1,4-dioxane), 1 h; (b)
NaOH (0.25 M, aq.), MeOH, 15 h; (c) EDCI, HOBt, NMM, CH2Cl2; (d)
HCl (1.25 M in MeOH), 16 h; (e) NaOH (0.25 M aq.), MeOH; (f) EDCI,
HOBt, NMM, DMF.
For the coupling reaction of peptide 10a with 12b, 2-
(7-aza-1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium-
hexafluorophosphate (HATU) was chosen as coupling reagent.
The reaction was performed in DMF as solvent and the targeted
peptide 13 was precipitated directly from the reaction mixture
by addition of methanol and acetone. The same procedure
was used also for coupling of octapeptides 12a and 12b using
N-methylpyrrolidone as solvent.
As mentioned in the introduction, TEMPO-derived
alkoxyamines are only suited for the controlled polymerization
of styrene.41 To enlarge the range of possible monomers which
can be polymerized in a controlled way, we also synthesized
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Polymerization studies with styrene as monomer
Polymerization of styrene was ◦conducted at initiator loadings
from 0.125 to 1.0 mol% at 125 C in neat solution under argon
atmosphere (Table 1). Excess of unreacted monomer was removed
in a vacuum drying cabinet at 60 ◦C for at least 18 h and conversion
was determined gravimetrically. Experimental molecular weights
(Mn,exp) and polydispersity indices (PDI) were measured by
gel-permeation chromatography (GPC). Styrene polymerization
proceeded highly controlled with alkoxyamines 1, 9, 11, 13. Best
results for polymerizations conducted with 1 were obtained with
initiator concentrations of 0.5 or 1.0 mol% (Table 1, entries 1–8).
PDIs were in most cases below 1.20 and tended to decrease at
longer polymerization time. Molecular weight distributions were
narrow if polymerization was allowed to proceed for 3 h up to
24 h. Lower initiator loadings led to larger polymers and to larger
PDIs.
Under analogous conditions peptide initiator 9 delivered poly-
mers with roughly doubled molecular weights as compared to
those synthesized with initiator 1 (entries 6–8, 9–12). This indicates
that both alkoxyamine moieties in 9 regulate NMP independently
from each other. Aberration of this relationship may occur up
to 15%, due to experimental and analytical variation. Pleasingly,
polymerization also took place in a highly controlled fashion with
alkoxyamine functionalized peptides 9 and 11 bearing up to four
initiator residues. In these cases, polymerizations were conducted
for up to 24 h. Because of statistical effects, PDIs decreased with
increasing number of polymer chains. When alkoxyamine hexamer
13 was used as a regulator at loadings of 0.083 or 0.167 mol%,
polymerization had to be stopped after 12 h (entries 18–20). If
reactions were allowed to run for longer times, bimodal molecular
weight distributions were observed. We assume this is a result of
dimerization of polymeric radicals, because the size of polymers
with higher molecular weight distribution is roughly about twice
the size of the smaller targeted ones. Due to the higher number
of potential radicals per molecule in polymers bearing multiple
alkoxyamines, dimerization is more likely.
Scheme 4 Reagents and conditions: (a) HCl (1.25 M in MeOH, 12.5 eq.),
MeOH (0.01 M solution of 11), 48 h; (b) NaOH (1 M, aq., 400 eq.), MeOH
(0.167 mM solution of 11), 40 h; (c) HATU, HOAt, DIPEA, DMF; (d)
HATU, HOAt, DIPEA, N-methylpyrrolidone.
the corresponding peptides bearing tetraethyl-substituted
alkoxyamines. To this end, TEMPO was replaced by nitroxide
15 (Scheme 5).42 The synthesis of 15-containing peptides were
performed in analogy to those used for the preparation of the
TEMPO-conjugated peptides. In most cases the yields were
slightly lower (see the ESI‡).
This problem was even more pronounced when hexadecapeptide
14, containing eight alkoxyamine functionalities, was used as a
polymerization initiator. Bimodal molecular weight distributions
resulted at initiator loadings of 0.0625 and 0.125 mol% even for
short polymerization times. However, this problem was solved by
further decreasing of initiator loading. Monomodal size distri-
butions of polymers were obtained at concentrations from 0.008
to 0.03 mol% (entries 21–25). However, the resulting polymers
showed larger PDIs. Nevertheless, we were able to synthesize
peptide–polystyrene conjugates with up to eight polymer side
chains and molecular weights with up to 300.000 g mol-1 with
narrow size distributions (see ESI‡ for additional polymerization
experiments).
Polymerization studies with NIPAM as monomer
Polymerization studies with NIPAM were conducted using peptide
initiators bearing the more sophisticated nitroxide 15. Experi-
ments were conducted in 1.78 M solution of NIPAM in benzene
at 125 ◦C in sealed tubes under argon atmosphere. The resulting
PNIPAM was separated from non reacted monomer by precip-
itation from an acetone solution upon addition of diethyl ether.
Scheme 5 Polymerization initiators derived from nitroxide 15.
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Table 1 Polymerization of styrene at 125 ◦C, neat, sealed tube
Entry
Initiator
Conc. (%mol)
Time (h)
Conversion (%)
Mn,th. (g mol-1)
Mn,exp. (g mol-1)
PDI
1
2
3
4
5
6
7
8
1
1
0.125
0.25
1.0
0.5
1.0
1.0
0.5
1.0
0.5
1
1
3
6
6
12
24
24
3
12
24
24
3
10
6
9300
2900
2200
4000
3000
4800
13800
6400
4900
9800
10100
2600
2400
5700
3200
5000
11400
5000
5000
8800
1.20
1.20
1.11
1.19
1.15
1.09
1.10
1.11
1.13
1.18
1.12
1.16
1.08
1.08
1.08
1.07
1.09
1.07
1.06
1.06
1.30
1.18
1.14
1.15
1.19
1
15
16
24
40
64
56
19
42
72
62
16
42
59
64
62
27
51
43
22
9
1
1
1
1
1
9
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
9
0.5
0.25
0.5
9
31000
13900
8700
25000
11200
8100
9
11
11
11
11
11
13
13
13
14
14
14
14
14
0.25
0.125
0.25
0.125
0.25
0.167
0.083
0.167
0.008
0.016
0.016
0.016
0.031
9
36900
26500
55200
22600
20000
65600
29800
294200
62000
103700
150600
28800
32000
24900
31200
22900
17000
95200
33100
297500
64300
87100
70700
30300
12
24
24
3
12
12
2
0.5
1
3
15
22
7
0.5
Conversion was determined gravimetrically, Mn,exp and PDIs were
determined by GPC.
Tetraalkoxyamine-substituted peptide initiator 18 also allowed
controlled NIPAM polymerization (entries 11–15).
Polymerization of NIPAM regulated by 16 occurred well con-
trolled with initiator loadings of 0.5 and 1.0 mol% (Table 2, entries
1–5). Low PDIs (<1.20) were obtained for these polymerizations.
Similar results were observed using tetrapeptide 17. As for the
styrene polymerizations, a doubling of the molecular weight
was achieved by switching from 16 to 17 containing two active
initiator moieties under analogous conditions (entries 6–10). The
polydispersities measured indicated a controlled polymerization.
Polymerization of NIPAM initiated with octaalkoxyamine 19
was difficult to control. Polymerizations occurred fast and side
reactions were observed. Acceptable results were obtained by
stopping the reactions at low conversions (reaction time < 6 h).
In contrast to the styrene polymerizations, we did not observe
bimodal molecular weight distributions for the polymerization of
NIPAM conducted by octaalkoxyamine substituted peptide 19
(see ESI‡ for additional polymerization results).
Table 2 Polymerization of NIPAM at 125 ◦C, 1.78 M in benzene, sealed tube
Entry
Initiator
Conc. (mol%)
Time (h)
Conversion (%)
Mn,th. (g mol-1)
Mn,exp. (g mol-1)
PDI
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
16
16
16
16
16
17
17
17
17
17
18
18
18
18
18
19
19
19
19
19
19
0.5
1.0
1.0
0.5
1.0
0.5
0.5
0.5
0.25
0.5
0.25
0.25
0.25
0.125
0.25
0.125
0.125
0.125
0.125
0.063
0.125
6
6
12
24
24
3
24
15
38
56
76
16
21
37
64
71
25
29
46
57
74
33
44
71
78
85
87
6100
2500
5000
13500
9300
5000
10000
6900
7400
20200
12600
5900
1.21
1.17
1.18
1.12
1.20
1.20
1.18
1.17
1.14
1.14
1.17
1.10
1.11
1.09
1.11
1.24
1.18
1.20
1.20
2.40
1.78
6
6200
9700
8200
12
24
24
3
10300
26500
19500
12100
21200
23800
45500
33600
36600
39000
43400
43400
149300
85900
30200
17400
13600
15700
23100
54300
35700
35500
40000
70000
76400
160100
84500
6
12
24
24
1
2
6
12
24
24
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Table 3 Hydrodynamic diameters of peptide–polystyrene conjugates in THF, c = 1 mg mL-1, T = 25 ◦C
Entry
Entry in Table 1
Initiator
Mn,exp (g mol-1)
Diameter (nm)
1
2
3
4
5
6
7
6
1
5000
25000
31200
17000
95200
30300
297500
1.74
7.24
6.88
6.30
10.55
8.43
11
16
18
19
25
21
9
11
13
13
14
14
27.27
DLS-analysis of conjugates
Investigations of peptide–polystyrene conjugates via DLS-
measurements revealed that no aggregation of these conjugates
occurred in THF or dichloromethane under the tested conditions.
Variation of concentration and temperature, as well as equili-
bration of the samples for several hours did not lead to any
aggregation. We were able to determine hydrodynamic diameters
in THF. Measurements were performed at room temperature in
THF (c = 1 mg mL-1). Diameters correlate roughly with molecular
weights determined by GPC (Table 3, entries 1, 2, 5, 7).
DLS-measurements turned out to be very useful to investigate
phase transition of peptide–PNIPAM conjugates in water. We
studied aggregation behaviour as a function of temperature
with peptide–PNIPAM conjugates bearing one, two, four and
eight PNIPAM side chains. These four samples derived from
polymerizations conducted under analogous conditions (1.0 mol%
with respect to alkoxyamine moieties, concentration of PNIPAM
= 1.78 M in benzene, 125 ◦C, 6 h, Table 2, entries 2, 7, 12,
18). Particle size distributions were analyzed either by their
volume maxima (vmax) or their number (nmax) maxima (Fig. 1,
Fig. 2). At 10 ◦C all samples were well dissolved and particle
sizes represent monomeric conjugates and correlate with their
molecular weights. By increasing the temperature aggregation of
the conjugates was observed. In comparison to unsubstituted
PNIPAM, which precipitates at a temperature of 32 ◦C, our
conjugates generally showed a lower phase transition temperature.
The monopolymer substituted conjugate started to precipitate at
Fig. 2 Size distribution maxima by number of peptide–PNIPAM conju-
gate particles (nmax), determined by DLS-measurement in water (c = 1 mg
mL-1). n indicates the number of attached polymers.
The conjugate containing two PNIPAM-chains, which can be
regarded as a linear PNIPAM polymer containing a peptide spacer,
showed a similar aggregation behaviour. Phase transition started
at 20 ◦C (vmax = 905 nm, T = 34 ◦C; nmax = 768 nm, T = 34 ◦C). For
linear peptide–polymer conjugates a steady increase of the particle
size was observed upon increasing the temperature.
Comb type peptide–PNIPAM conjugates showed a different
aggregation behaviour. A smooth phase transition over a large
temperature range was observed. In addition, aggregation to large
particles did not occur at higher temperature. The conjugate
with four PNIPAM-chains started to aggregate at the lowest
temperature (T ~ 14 ◦C). Particle size maxima did not exceed
220 nm (vmax = 219 nm, T = 28 ◦C; nmax = 182 nm, T = 26 ◦C).
The conjugate containing eight polymer moieties showed a similar
aggregation behaviour, but interestingly, phase transition started
at a higher temperature (T ~ 23 ◦C) and the maximal particle
diameter was far smaller (vmax = 130 nm, T = 28 ◦C; nmax = 110 nm,
T = 28 ◦C).
◦
23 C. Phase transition occurred in a narrow temperature range
and particles formed grew with increasing temperature to large
aggregates (vmax = 708 nm, T = 34 ◦C; nmax = 387 nm, T = 30 ◦C).
Aggregation behaviour on surfaces
We first studied aggregation behaviour at surfaces using the non
polymerized peptide initiators. To this end, a drop of a solution of
the peptide initiator in DMF (c = 1 mg mL-1) was spin-coated on
a silicon wafer. After evaporation of the solvent AFM-images of
the resulting surfaces were recorded.
TEMPO-substituted peptides 1, 9 and 11 did not show any
regular structures. After one week equilibration of the peptide
solution, peptide 13 bearing six alkoxyamine moieties formed
sharp fibres with heights of about 2–4 nm, widths of 20–40 nm
Fig. 1 Size distribution maxima by volume of peptide–PNIPAM conju-
gate particles (vmax), determined by DLS-measurement in water (c = 1 mg
mL-1). n indicates the number of attached polymers.
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and lengths of 100–1000 nm, which aggregated to fingerprint type
like structures at the silicon surface (Fig. 3, left side).
Conjugate 22 (Table 1, entry 29) was able to assemble to a
honeycomb structure containing up to three layers with a regular
hole distribution (Fig. 5, left side). Layer thickness was about
4 nm and diameters of the holes showed sizes from 20 up to 40 nm.
Surprisingly, conjugate 23 assembled after spin-coating completely
different than 22. The structures have a coral like morphology
(Fig. 5, right side). Its height is about 4 nm.
Fig. 3 AFM-images of peptides 13 (left) and 19 (right) after spin-coating
in DMF on silicon wafer.
Hexadecapeptide 19, bearing eight alkoxyamine functionalities
was dissolved in DMF and the solution was spread on a silicon
substrate. AFM-imaging after evaporation of the solvent revealed
a net-like structure consisting of peptide fibres. In comparison to
13, these fibres exhibit larger diameters of about 100 nm (Fig. 3,
right side).
These initial results encouraged us to study peptide–polystyrene
conjugates in the same manner. Instead of DMF we used THF
as solvent for spin-coating of the hybrids. AFM-measurement
revealed formation of so called honeycomb structures. Conju-
gate 20 (Table 1, entry 18), obtained from polymerization with
tetraalkoxyamine functionalized peptide 11, showed first tenden-
cies toward layered structures with holes (Fig. 4, left side), but
the layer is cracked and the holes have irregular distributions and
diameters. Conjugate 21 bearing six polystyrene chains (Table 1,
entry 24) showed a more defined morphology (Fig. 4, right side).
There were fewer but more regular holes in these layers. Layer
thickness varied from about 5 to 10 nm in both cases.
Fig. 5 AFM-images of conjugates 22 (left) and 23 (right) after spin-coat-
ing in THF a silicon wafer.
A possible reason for this behaviour could be that the long non-
polar polystyrene side chains hinder the polar peptide moieties
aggregating over large areas. Then the conjugates may only form
small aggregates which potentially show these structures. This
explanation is plausible considering that literature reports polar
additives or moieties in conjugates to be responsible for the
formation of the holes in honeycomb structures under humid
conditions.
We next investigated the surface self-organizing behaviour of
peptide–PNIPAM conjugates. First spin-coating attempts were
performed with solutions of the conjugates in acetone (c = 1 mg
mL-1). Conjugate 24 (Table 2, entry 24), bearing a hexadecapeptide
chain with eight PNIPAM side chains attached, formed ring
shaped structures (Fig. 6, left side). These larger aggregates also
showed internal void areas. Outer rings of these morphologies
were about 10 nm high whereas core rings could overtop the rings
with around 20 nm height. Diameters of these rings vary in size
up to 1 mm and thicknesses of the rings were approximately all
around 100 nm. These structures were observed over large surface
areas of about 250 mm2 (Fig. 6, right side).
By increasing the length of the PNIPAM polymers, morphology
of the aggregates changed. Conjugate 25 (Table 2, entry 26)
aggregated to large objects (Fig. 7, left side).
These structures with diameters of 200–500 nm, did not show
any inner void space. This morphology is also regular over large
areas (Fig. 7, right side). Size and morphology of these structures
are similar to those observed for PNIPAM-polyacrylic acid
copolymers.39 Obviously surface behaviour of peptide–PNIPAM
conjugates switches to properties of unsubstituted PNIPAM if
chain length increases. The influence of the peptide backbones
regarding aggregation behaviour then seems to fade.
Fig. 4 AFM-images of peptide–polystyrene conjugates 20 (left) and 21
(right) after spin-coating in THF on a silicon wafer.
Peptide conjugates with eight polystyrene chains showed dif-
ferent aggregation architectures. If the attached polymer chains
are short, formation of honeycomb structures was still favoured.
We were also interested in the aggregation behaviour of peptide–
PNIPAM conjugates in water. This was studied at the interface
between an aqueous solution and the silicon surface by coating. To
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Fig. 6 AFM-images of peptide–PNIPAM conjugate 24 after spin-coating
Fig. 8 AFM-images of conjugate 25 after spin-coating in water on a
in acetone on a silicon wafer.
silicon wafer.
and polymerization times. DLS-measurements revealed no aggre-
gation of peptide–polystyrene conjugates in solution. Aggregation
of peptide–PNIPAM conjugates in water at their critical solution
temperature was observed. Linear conjugates formed much larger
aggregates and showed sharp phase transitions whereas comb
type conjugates precipitated to particles with limited sizes. AFM-
imaging on silicon surfaces showed tendencies of peptide initiators
to build up fibre like structures. Peptide–polystyrene conjugates
bearing small polymer chains aggregated to so called honeycomb
structures and showed coral like morphologies for systems bear-
ing longer side chains. Spin-coated from acetone on a silicon
wafer, peptide–PNIPAM conjugates aggregated to ring shaped
topologies if PNIPAM chains were short. Conjugates containing
longer PNIPAM chains showed typical PNIPAM-like aggregation
behaviour. In water these conjugates accumulated to spherical
beads.
Fig. 7 AFM-images of conjugate 25 after spin-coating in acetone on a
silicon wafer.
this end, several conjugates were dissolved in water in an ice bath
(c = 1 mg mL-1). Evaporation of the solvent was conducted at 6 ◦C
to avoid precipitation of PNIPAM conjugates from solution. We
chose again conjugates 24 and 25 for these studies and we obtained
for both samples homogeneous structured surfaces coated with
spherical beads that accumulate to clusters (Fig. 8). These two
conjugates showed in water in contrast to acetone very similar
aggregation behaviour. Diameters of the observed beads varied
between 100 and 150 nm. As it is known that PNIPAM can
aggregate to beads38 and because peptide chain length does not
seem to have any influence on the morphology, our conclusion
is that bead formation of these conjugates is fully driven by the
PNIPAM residues and the peptide backbones do not exert a large
effect on the self-assembly process.
Acknowledgements
We thank the NRW Graduate School of Chemistry and the SFB-
Transregio 61 for funding.
Notes and references
1 H. G. Bo¨rner and H. Schlaad, Soft Matter, 2007, 3, 94.
2 J. Hentschel, K. Bleek, O. Ernst, J.-F. Lutz and H. G. Bo¨rner,
Macromolecules, 2008, 41, 1073.
3 H. G. Bo¨rner, B. M. Smarsly, J. Hentschel, A. Rank, R. Schubert, Y.
Geng, D. E. Discher, T. Hellweg and A. Brandt, Macromolecules, 2008,
41, 1430.
4 (a) H.-A. Klok, J. Polym. Sci., Part A: Polym. Chem., 2005, 43, 1;
(b) H.-A. Klok, Macromolecules, 2009, 42, 7990.
5 J. C. M. van Hest, Polym. Rev., 2007, 47, 63.
6 (a) G. M. Whitesides, Small, 2005, 1, 172; (b) S. Fo¨rster and T.
Plantenberg, Angew. Chem., 2002, 114, 712, (Angew. Chem., Int. Ed.,
2002, 41, 688); (c) R. V. Ulijn and A. M. Smith, Chem. Soc. Rev., 2008,
37, 664.
Conclusions
7 H. Ku¨hnle and H. G. Bo¨rner, Angew. Chem., 2009, 121, 6552, (Angew.
Chem., Int. Ed., 2009, 48, 6431).
8 G. W. M. Vandermeulen, C. Tziatzios and H.-A. Klok, Macromolecules,
2003, 36, 4107.
9 J. M. Smeek, M. B. J. Otten, J. Thies, D. A. Tirrell, H. G. Stunnenberg
and J. C. M. van Hest, Angew. Chem., 2005, 117, 2004, (Angew. Chem.,
Int. Ed., 2005, 44, 1968).
10 (a) H. Frauenrath and E. Jahnke, Chem.–Eur. J., 2008, 14, 2942; (b) E.
Jahnke, I. Lieberwirth, N. Severin, J. P. Rabe and H. Frauenrath, Angew.
In this article we presented the synthesis of peptide–polymer
conjugates composed of well defined linear peptide backbones
and multiple defined numbers of polystyrene or PNIPAM side
chains via NMP and studies on their aggregation behaviour.
We showed that NMP is a reliable method to attach up to
eight polymer chains onto a peptide backbone. Polymerizations
occurred well controlled over a wide range of initiator loadings
2410 | Org. Biomol. Chem., 2011, 9, 2403–2412
This journal is
The Royal Society of Chemistry 2011
©

View Article Online
Chem., 2006, 118, 5510, (Angew. Chem., Int. Ed., 2006, 45, 5383); (c) E.
Jahnke, N. Severin, P. Kreutzkamp, J. P. Rabe and H. Frauenrath, Adv.
Mater., 2008, 20, 409.
768, ed. K. Matyjaszewski, American Chemical Society, Washington,
DC, 2000, p. 278; (b) J. Chiefari, Y. K. Chong, F. Ercole, J. Kristina,
J. Jeffrey, T. P. T. Le, R. T. A. Mayadunne, G. F. Meijs, C. L. Moad,
E. Rizzardo and S. H. Thang, Macromolecules, 1998, 31, 5559; (c) A.
Favier and M.-T. Charreyre, Macromol. Rapid Commun., 2006, 27, 653.
26 (a) K. Matyjaszewski and J. Xia, Chem. Rev., 2001, 101, 2921; (b) M.
Kamigaito, T. Ando and M. Sawamoto, Chem. Rev., 2001, 101, 3689;
(c) J. S. Wang and K. Matyjaszewski, Macromolecules, 1995, 28, 7901;
(d) J. S. Wang and K. Matyjaszewski, J. Am. Chem. Soc., 1995, 117,
5614; (e) M. Kato, M. Kamigaito, M. Sawamoto and T. Higashimura,
Macromolecules, 1995, 28, 1721.
27 (a) Reviews: C. J. Hawker, A. W. Bosman and E. Harth, Chem. Rev.,
2001, 101, 3661; (b) A. Studer, Chem.–Eur. J., 2001, 7, 1159; (c) A.
Studer, Chem. Soc. Rev., 2004, 33, 267; (d) A. Studer and T. Schulte,
Chem. Rec., 2005, 5, 27; (e) M. K. Brinks and A. Studer, Macromol.
Rapid Commun., 2009, 30, 1043.
28 (a) E. Rizzardo and D. H. Solomon, Polym. Bull., 1979, 1, 529; (b) G.
Moad, E. Rizzardo and D. H. Solomon, Macromolecules, 1982, 15,
909; (c) M. K. Georges, R. P. N. Veregin, P. M. Kazmaier and G. K.
Hamer, Macromolecules, 1993, 26, 2987; (d) C. J. Hawker, G. G.
Barclay, A. Orella, J. Dao and W. Devonport, Macromolecules, 1996, 29,
5245; (e) E. Malmstrom, R. D. Miller and C. J. Hawker, Tetrahedron,
1997, 53, 15225; (f) C. J. Hawker, Acc. Chem. Res., 1997, 30, 373;
(g) M. K. Georges, R. P. N. Veregin, P. M. Kazmaier and G. K. Hamer,
Macromolecules, 1993, 26, 2987; (h) R. P. N. Veregin, M. K. Georges,
P. M. Kazmaier and G. K. Hamer, Macromolecules, 1993, 26, 5316;
(i) M. K. Georges, R. P. N. Veregin, P. M. Kazmaier, G. K. Hamer and
M. Sabau, Macromolecules, 1994, 27, 7228; (j) C. J. Hawker, J. Am.
Chem. Soc., 1994, 116, 11185.
11 J. Hentschel and H. G. Bo¨rner, J. Am. Chem. Soc., 2006, 128, 14142.
12 (a) J. Couet, J. D. J. S. Samuel, A. Kopyshev, S. Santer and M.
Biesalski, Angew. Chem., 2005, 117, 3361, (Angew. Chem., Int. Ed.,
2005, 44, 3297); (b) J. Couet and M. Biesalski, Macromolecules, 2006,
39, 7258; (c) S. Loschonsky, J. Couet and M. Biesalski, Macromol.
Rapid Commun., 2008, 29, 309; (d) M. G. J. ten Cate, N. Severin and
H. G. Bo¨rner, Macromolecules, 2006, 39, 7831; (e) J. Hentschel, M. G. J.
ten Cate and H. G. Bo¨rner, Macromolecules, 2007, 40, 9224.
13 G. W. M. Vandermeulen and H.-A. Klok, Macromol. Biosci., 2004, 4,
383.
14 E. K. Schillinger, E. Mena-Osteritz, J. Hentschel and H. G. Bo¨rner,
Adv. Mater., 2009, 21, 1562.
15 (a) S. Kessel, A. Thomas and H. G. Bo¨rner, Angew. Chem., 2007, 119,
9181, (Angew. Chem., Int. Ed., 2007, 46, 9023); (b) M. G. Page, N.
Nassif, H. G. Bo¨rner, M. Antonietti and H. Co¨lfen, Cryst. Growth
Des., 2008, 8, 1792.
16 T. K. Paira, S. Banerjee, M. Raula, A. Kotal, S. Si and T. K. Mandal,
Macromolecules, 2010, 43, 4050.
17 (a) B. R. Line, A. Mitra, A. Nan and H. Ghandehari, J. Nucl. Med.,
2005, 46, 1552; (b) H.-J. Wester and H. Kessler, J. Nucl. Med., 2005, 46,
1940.
18 (a) H. G. Bo¨rner, Macromol. Chem. Phys., 2007, 208, 124; (b) J. Nicolas,
G. Mantovani and D. M. Haddleton, Macromol. Rapid Commun., 2007,
28, 1083; (c) I. C. Reynhout, D. W. P. M. Lo¨wik, J. C. M. van Hest,
J. J. L. M. Cornelissen and R. J. M. Nolte, Chem. Commun., 2005,
602; (d) B. Le Droumaguet, G. Mantovani, D. M. Haddleton and
K. Velonia, J. Mater. Chem., 2007, 17, 1916; (e) N. Rosenberger, A.
Studer, N. Takatani, H. Nakajima and Y. Watanabe, Angew. Chem.,
2009, 121, 1979, (Angew. Chem., Int. Ed., 2009, 48, 1946); (f) A. C.
Engler, H.-i. Lee and P. T. Hammond, Angew. Chem., 2009, 121, 9498,
(Angew. Chem., Int. Ed., 2009, 48, 9334); (g) J. Y. Shu, C. Tan, W. F.
DeGrado and T. Xu, Biomacromolecules, 2008, 9, 2111; (h) P. Jing,
J. S. Rudra, A. B. Herr and J. H. Collier, Biomacromolecules, 2008, 9,
2438; (i) J. Y. Shu, Y.-J. Huang, C. Tan, A. D. Presley, J. Chang and T.
Xu, Biomacromolecules, 2010, 11, 1443; (j) K. Matyjaszewski and A. J.
Russell, Biomacromolecules, 2005, 6, 3380.
19 M. A. Gauthier and H.-A. Klok, Chem. Commun., 2008, 2591.
20 H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew. Chem., 2001, 113,
2056, (Angew. Chem., Int. Ed., 2001, 40, 2004).
21 (a) R. Huisgen, Angew. Chem., 1963, 75, 604; (b) V. V. Rostovtsev, L. G.
Green, V. V. Fokin and K. B. Sharpless, Angew. Chem., 2002, 114,
2708, (Angew. Chem., Int. Ed., 2002, 41, 2596); (c) C. W. Tornoe, C.
Christensen and M. Meldal, J. Org. Chem., 2002, 67, 3057; (d) W. H.
Binder and R. Sachsenhofer, Macromol. Rapid Commun., 2007, 28, 15;
(e) J.-F. Lutz, Angew. Chem., 2007, 119, 1036.
22 (a) A. J. T. Dirks, S. S. van Berkel, N. S. Hatzakis, J. A. Opsteen,
F. L. van Delft, J. J. L. M. Cornelissen, A. E. Rowan, J. C. M. van
Hest, F. P. J. T. Rutjes and R. J. M. Nolte, Chem. Commun., 2005,
4172; (b) J.-F. Lutz, H. G. Bo¨rner and K. Weichenhan, Macromol.
Rapid Commun., 2005, 26, 514; (c) J.-F. Lutz, H. G. Bo¨rner and K.
Weichenhan, Macromolecules, 2006, 39, 6376; (d) D. T. S. Rijkers, G. W.
van Esse, R. Merkx, A. J. Brouwer, H. J. F. Jacobs and R. J. Pieters,
Chem. Commun., 2005, 4581; (e) B. Parrish, R. Breitenkamp and T.
Emrick, J. Am. Chem. Soc., 2005, 127, 7404; (f) A. C. Engler, H.-i. Lee
and P. T. Hammond, Angew. Chem., 2009, 121, 9498, (Angew. Chem.,
Int. Ed., 2009, 48, 9334).
23 (a) K. Molawi and A. Studer, Chem. Commun., 2007, 5173; (b) M. L.
Becker, J. Liu and K. L. Woodley, Chem. Commun., 2003, 180; (c) M. L.
Becker, J. Liu and K. L. Woodley, Biomacromolecules, 2005, 6, 220;
(d) T. Trimaille, K. Mabrouk, V. Monnier, L. Charles, D. Bertin and D.
Gigmes, Macromolecules, 2010, 43, 4864.
24 (a) M. G. J. ten Cate, H. Retting, K. Bernhardt and H. G. Bo¨rner,
Macromolecules, 2005, 38, 10643; (b) S. Venkataraman and K. L.
Woodley, Macromolecules, 2006, 39, 9661; (c) M. G. J. ten Cate and
H. G. Bo¨rner, Macromol. Chem. Phys., 2007, 208, 1437; (d) R. M.
Boyer, G. M. Quaker and H. D. Maynard, J. Am. Chem. Soc., 2008,
130, 1041; (e) G. J. M. Habkraken, C. E. Koning and A. Heise, J. Polym.
Sci., Part A: Polym. Chem., 2009, 47, 6883; (f) Y. Mei, K. L. Beers, H. C.
Michelle Byrd, D. L. van der Hart and N. R. Washburn, J. Am. Chem.
Soc., 2004, 126, 3472.
29 S. VandeVondele, J. Vo¨ro¨s and J. A. Hubbell, Biotechnol. Bioeng., 2003,
82, 784.
30 T. G. Stange, R. Mathew and D. F. Evans, Langmuir, 1992, 8, 920.
31 L. Ciu, X. Li and Y Han, Appl. Surf. Sci., 2006, 252, 8156.
32 G. Widawski, M. Rawiso and B. Franc¸ois, Nature, 1994, 369, 387.
33 (a) B. Franc¸ois, O. Pitois and J. Franc¸ois, Adv. Mater., 1995, 7, 1041;
(b) S. A. Jenekhe and X. L. Chen, Science, 1999, 283, 372; (c) O.
Karthaus, N. Maruyama, X. Cieren, M. Shimomura, H. Hasegawa
and T. Hashimoto, Langmuir, 2000, 16, 6071; (d) M. Srinivasarao, D.
Collings, A. Philips and S. Patel, Science, 2001, 292, 79; (e) M. H.
Stenzel-Rosenbaum, T. P. Davis, A. G. Fane and V. Chen, Angew.
Chem., 2001, 113, 3536, (Angew. Chem., Int. Ed., 2001, 40, 3428); (f) H.
Yabu, Y. Hirai, M. Kojima and M. Shimomura, Chem. Mater., 2009,
21, 1787; (g) W. Dong, Y. Zhou, D. Yan, Y. Mai, L. He and C. Jin,
Langmuir, 2009, 25, 173; (h) L. Billon, M. Manguian, V. Pellerin, M.
Joubert, O. Eterradossi and H. Garay, Macromolecules, 2009, 42, 345;
(i) F. Galeotti, V. Calabrese, M. Cavazzini, S. Quici, C. Poleunis, S.
Yunus and A. Bolognesi, Chem. Mater., 2010, 22, 2764; (j) L. Li, J.
Li, Y. Zhong, C. Chen, Y. Ben, J. Gong and Z. Ma, J. Mater. Chem.,
2010, 20, 5446; (k) T. Nakanishi, Y. Hirai, M. Kojima, H. Yabu and
M. Shimomura, J. Mater. Chem., 2010, 20, 6741; (l) E. Nomura, A.
Hosoda, M. Takagaki, H. Mori, Y. Miyake, M. Shibakami and H.
Taniguchi, Langmuir, 2010, 26, 10266; (m) B. S. Kim, C. Basavaraja,
E. A. Jo, D. G. Kim and D. S. Huh, Polymer, 2010, 51, 3365; (n) L.
Li, C. Chen, A. Zhang, X. Liu, K. Cui, J. Huang, Z. Ma and Z. Han,
J. Colloid Interface Sci., 2009, 331, 446.
34 M. H. Stenzel, T. P. Davis and A. G. Fane, J. Mater. Chem., 2003, 13,
2090.
35 (a) M. Heskins and J. E. Guillet, J. Macromol. Sci., Part A: Pure Appl.
Chem., 1968, 2, 1441; (b) H. G. Schild, Prog. Polym. Sci., 1992, 17,
163; (c) E. S. Gil and S. M. Hudson, Prog. Polym. Sci., 2004, 29, 1173;
(d) C. de la Heras, S. Pennadam and C. Alexander, Chem. Soc. Rev.,
2005, 34, 276; (e) P. S. Stayton, T. Shimoboji, C. Long, A. Chilkoti,
G. H. Chen, Z. L. Ding, C. J. Long, P. S. Stayton and A. S. Hoffmann,
Bioconjugate Chem., 2000, 11, 78; (f) Z. L. Ding, R. B. Fing, C. J. Long,
P. S. Stayton and A. S. Hoffmann, Nature, 2001, 411, 59; (g) K. L.
Heredia, D. Bontempo, T. Ly, J. T. Byers, S. Halstenberg and H. D.
Maynard, J. Am. Chem. Soc., 2005, 127, 16955; (h) D. Bontempo, R. C.
Li, T. Ly, C. E. Brubaker and H. D. Maynard, Chem. Commun., 2005,
4702.
36 (a) T. Sonoda, T. Nogami, J. Oishi, M. Murata, T. Niidome and
Y. Katayama, Bioconjugate Chem., 2005, 16, 1542; (b) C. Zhao, X.
Zhuang, C. He, X. Chen and X. Jing, Macromol. Rapid Commun.,
2008, 29, 1810; (c) C. He, C. Zhao, X. Guo, Z. Guo, X. Chen, X.
Zhuang, S. Liu and X. Jing, J. Polym. Sci., Part A: Polym. Chem., 2008,
46, 4140.
25 (a) E. Rizzardo, J. Chiefari, R. T. A. Mayadunne, G. Moad and S. H.
Thang, in Controlled/Living Radical Polymerization: ACS Symp. Ser.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 2403–2412 | 2411
©

View Article Online
37 (a) N. Ishida and S. Biggs, Langmuir, 2007, 23, 11083; (b) W. Wang,
K. Troll, G. Kaune, E. Metwalli, M. Ruderer, K. Skrabania, A.
Laschewsky, S. V. Roth, C. M. Papadakis and P. Mu¨ller-Buschbaun,
Macromolecules, 2008, 41, 3209; (c) N. C. Estillore, J. Y. Park and R. C.
Advincula, Macromolecules, 2010, 43, 6588; (d) N. Ishida and S. Biggs,
Macromolecules, 2010, 43, 7269.
38 A. Pelah, T. M. Jovin and I. Szleifer, Colloids Surf., A, 2007,
299, 1.
39 S. Schmidt, H. Motschmann, T. Hellweg and R. von Klitzing, Polymer,
2008, 49, 749.
40 J. Yin, Z. Ge, H. Liu and S. Liu, J. Polym. Sci., Part A: Polym. Chem.,
2009, 47, 2608.
41 (a) C. A. Knoop and A. Studer, J. Am. Chem. Soc., 2003, 125, 16327;
(b) A. Studer, K. Harms, C. A. Knoop, C. Mu¨ller and T. Schulte,
Macromolecules, 2004, 37, 27; (c) C. Wetter, J. Gierlich, C. A. Knoop,
C. Mu¨ller, T. Schulte and A. Studer, Chem.–Eur. J., 2004, 10, 1156;
(d) T. Schulte, K. O. Siegenthaler, H. Luftmann, M. Letzel and A.
Studer, Macromolecules, 2005, 38, 6833; (e) K. O. Siegenthaler and A.
Studer, Macromolecules, 2006, 39, 1347; (f) C.-C. Chang and A. Studer,
Macromolecules, 2006, 39, 4062; (g) C.-C. Chang, K. O. Siegenthaler
and A. Studer, Helv. Chim. Acta, 2006, 89, 2200; (h) M. K. Brinks, M.
Hirtz, L. Chi, H. Fuchs and A. Studer, Angew. Chem., 2007, 119, 5324,
(Angew. Chem., Int. Ed., 2007, 46, 5231); (i) D. Benoit, V. Chaplinksi,
R. Braslau and C. J. Hawker, J. Am. Chem. Soc., 1999, 121, 3904; (j) S.
Grimaldi, J.-P. Finet, A. Zeghdaoui, P. Tordo, D. Benoit, Y. Gnanou,
M. Fontanille, P. Nicol and J.-F. Pierson, Polym. Prepr. (Am. Chem.
Soc., Div. Polym. Chem.), 1997, 38, 651; D. Benoit, S. Grimaldi, S.
Robin, J.-P. Finet, P. Tordo and Y. Gnanou, J. Am. Chem. Soc., 2000,
122, 5929.
42 (a) S. Miele, P. Nesvadba and A. Studer, Macromolecules, 2009, 42,
2419, Application in synthesis; (b) I. C. Wienho¨fer, A. Studer, Md. T.
Rahman, T. Fukuyama and I. Ryu, Org. Lett., 2009, 11, 2457.
2412 | Org. Biomol. Chem., 2011, 9, 2403–2412
This journal is
The Royal Society of Chemistry 2011
©