Synthesis of self-assembling cyclic peptide-polymer conjugates using click chemistry

Article abstract of DOI:10.1071/CH10128

Self-assembling cyclic peptide-polymer conjugates were prepared by 'clicking' polymers (prepared by RAFT polymerization) to an azide functionalized d-alt-l cyclic octapeptide via the Huisgen 1,3-dipolar cycloaddition reaction. Due to the high graft density, the efficiency of the click chemistry conjugation reaction was found to be highly dependent on the size of the polymer. At relatively low molecular weights, as many as four polymer chains could be grafted to each 8 residue cyclic peptide ring. Evidence for the self assembly of the conjugates into peptide-polymer nanotubes was observed by TEM and IR. CSIRO 2010.

Full text of DOI:10.1071/CH10128

RESEARCH FRONT
Communication
CSIRO PUBLISHING
www.publish.csiro.au/journals/ajc
Aust. J. Chem. 2010, 63, 1169–1172
Synthesis of Self-assembling Cyclic Peptide-polymer
Conjugates using Click Chemistry
Robert Chapman,A,B Katrina A. Jolliffe, and Sébastien PerrierA
A
AKey Centre for Polymers & Colloids, School of Chemistry, The University of Sydney,
NSW 2006, Australia.
B
Corresponding author. Email: r.chapman@chem.usyd.edu.au
Self-assembling cyclic peptide-polymer conjugates were prepared by ‘clicking’ polymers (prepared by RAFT polymer-
ization) to an azide functionalized d-alt-l cyclic octapeptide via the Huisgen 1,3-dipolar cycloaddition reaction. Due to
the high graft density, the efficiency of the click chemistry conjugation reaction was found to be highly dependent on the
size of the polymer. At relatively low molecular weights, as many as four polymer chains could be grafted to each 8 residue
cyclic peptide ring. Evidence for the self assembly of the conjugates into peptide-polymer nanotubes was observed by
TEM and IR.
Manuscript received: 18 March 2010.
Manuscript accepted: 30 June 2010.
Modern techniques of polymerisation have revolutionalized the
chemists’ ability to design functional materials with tailored
nanotubes. Furthermore, click reactions allow access to high
graft densities that are not possible with other approaches. In
the present work we show that the click approach enables the
conjugation of polymeric chains to cyclic peptides and that
the resulting conjugates show self-assembly as evidenced by
transmission electron microscopy (TEM) and IR.
[
1]
properties, but these synthetic capabilities are still far out-
stripped by nature’s.As scientists try to synthesize more complex
materials, the challenge to do so by traditional synthetic methods
increases significantly and recent research has taken inspiration
from the way that nature builds well defined and complex struc-
tures through the self assembly of smaller molecules.[ This
bottom-up approach has proved to be versatile and the most
promising route to design materials with structures controlled at
the nanoscale.
2]
Results and Discussion
Scheme 1 shows a summary of the synthetic approach used
to create cyclic peptide-polymer conjugates. A novel four-arm
azide functionalized cyclic peptide cyclo-[l-Lys(N )-d-Leu] 3
Perhaps the most interesting and well understood self-
assembling molecules are β-sheet peptides, which can assemble
into tapes, ribbons, fibres, and tubes.[3–6] This range of nano-
structures can be exploited by conjugating polymers to such
3
4
was prepared by cyclization of the linear precursor 2, which
was synthesized by standard Fmoc SPPS protocols (HBTU
coupling).[ The precursor amino acid, azido Fmoc-l-Lysine
1 was synthesized from Fmoc-l-Lysine using the diazo trans-
fer agent developed by Stick and coworkers (see supplementary
information),[ which was preferred over the more dangerous
procedure using triflyl azide by which azido lysine is more com-
monly made. Polymers of hydroxy ethyl acrylate (HEA) were
18]
peptides, and using the peptide to drive the self-assembly of
the conjugate into a nanostructure.[
7–10]
With the advance of
19]
modern polymer chemistry, and in particular living radical poly-
merization (LPR) techniques, there exists a variety of routes
through which to conjugate polymeric chains to peptide seg-
ments. Approaches are generally divided between divergent
prepared via reversible addition fragmentation chain transfer
[
11–15]
[20,21]
synthesis,
which fully exploits the versatility of LRP by
(RAFT) polymerization,
then attached to the peptide using
growingapolymericchainfromapeptideusedaspolymerization
initiator, and convergent synthesis,[16,17] in which the peptide
is coupled to a pre-made polymer. The self-assembly of cyclic
peptide-polymer conjugates into nanotubes has been demon-
strated with selected systems and offers access to well defined
polymeric nanotubes. Most examples of polymer peptide nan-
otubesintheliteraturearesynthesizedbythedivergentapproach,
click chemistry. HEA was chosen in order to improve the sol-
ubility of the conjugate, and the resulting nanotubes, in polar
solvents.Alkyne functionality was introduced to the polymers by
using an alkyne functionalized trithiocarbonate RAFT agent 4
(see supplementary information) to polymerize the monomer,
since trithiocarbonates are stable at the reaction temperatures.[
Protection of the alkyne group (such as with TMS) during
polymerization was found to be unnecessary at the times and
temperatures used.
22]
growing polymeric chains from an ATRP initiator modified
cyclic peptide, either before or after self-assembly.[
12–15]
While
grafting to a cyclic peptide before self-assembly has been shown
to work in principle through a condensation reaction,[ click
chemistry offers the ability to conjugate almost any polymer
to the cyclic peptide and then self-assemble it into polymer
The unconjugated cyclic peptide 3 was found to be remark-
ably insoluble, even in very polar solvents such as DMF and
DMSO, presumably due to strong self-assembly properties.
While trifluoroacetic acid (TFA) could be used to break the self
17]
©
CSIRO 2010
10.1071/CH10128
0004-9425/10/081169

RESEARCH FRONT
1
170
R. Chapman, K. A. Jolliffe, and S. Perrier
NH2
N3
1
(
i)
ϩ
Fmoc
O
Fmoc
N
H
C4H9
C4H9
S
Fmoc
N
H
CO H
S
2
N
H
CO H
O
2
S
S
Fmoc-d-Leu on
S
S
2-chlorotrityl chloride resin
O
O
n
n
(ii), (iii), (iv)
N3
O
O
O
O
OH
HO
O
O
N
N3
N3
O
H
N
O
N
N3
N3
N
N
NH
N
N
HN
O
O
O
H
N
O
N3
O
O
O
(v)
NH
HN
NH
H
N
H
N
H
N
O
O
H
N
HN
O
7
8
n ϭ 12
n ϭ 34
H N
N
H
N
H
N3
HN
NH
2
N
H
OH
O
O
NH
O
O
O
HN
O
O
O
NH
N
H
(
vii)
HN
3
O
O
N
H
O
O
2
N
N
N
N
N3
N
N
O
O
O
HO
OH
O
O
O
S
S
O
O
(vi)
n
S
S
C H
n
O
C H
O
4
9
O
O
4
9
n
S
S
O
O
S
S
S
S
4
5
6
n ϭ 12
n ϭ 34
S
S
C4H9
C4H9
OH
Scheme 1. Synthesis of cyclic peptide-polymer conjugates; (i) Fmoc-l-Lys-OH, Im-SO2-N3, CuSO4, NaHCO3; (ii) Fmoc-l-Lys(N3)-OH/Fmoc-d-Leu-OH,
HBTU, Hünig’s base, DMF; (iii) 20% Piperidine, DMF; (iv) TFA, TIPS, thioanisole, H2O; (v) HOBt, HBTU, Hünig’s base, DMF; (vi) HEA, AIBN, t-butanol;
(
vii) DMSO, PMDETA, Cu(I)Br.
assembly and solubilize the peptide,[17] the presence of TFA in
the click reaction was found to interfere with ligation of the
copper. As a result, the conjugation was carried out in a hetero-
geneous solution of DMSO prepared by sonication for 10 min.
which can be attributed to significant steric hindrance, consis-
tent with a structure in which 3 or 4 bulky polymer chains are
attached to a single, small peptide centre.
The presence of azide functionality in the final conjugates
demonstrates that the reaction is not hindered by the destruc-
tion of the azide groups, and the fact that the entire mixture is
soluble at the end of the click reaction rules out the possibility
that the unconverted azide results from a small fraction of cyclic
peptides that have not reacted at all. An analogous reaction car-
ꢀ
A two-fold excess of CuBr and 2,2 -pentamethyldiethylene-
triamine (PMDETA) with respect to the cyclic peptide was used
due to the large degree of binding expected between copper
[
23]
and peptide.
As the reaction proceeded, the insoluble cyclic
peptide was found to dissolve, leading to a homogeneous mix-
ture, indicating that the attachment of polymer dramatically
improved the solubility of the peptide, pushing the reaction
towards the product. The product 6 was purified by precipitation
from aqueous EDTA (0.065 M), and subsequent washing with
water. Click reactions were carried out with short (60% conver-
◦
ried out at 40 C over 6 days gave similar results, indicating that
changing the temperature does not affect the efficiency of the
reaction. It is therefore apparent that blocking of the reactive
sites by the polymer and/or aggregation of the peptide rings are
the most likely explanations for the lack of complete conversion.
In order to investigate these steric effects further, the molec-
ular weight of the polymer clicked to the cyclic peptide was
−
1
sion, Mn = 1700 g mol , PDI 1.10) and long (35% conversion,
−1
Mn = 4000 g mol , PDI 1.13) polymers.
−
1
The conjugation of short polymers to the cyclic peptide was
increased. Increasing the weight of the polymer to 4000 g mol
very efficient, proceeding to near full conversion in 3 days at
dramatically reduced the efficiency of the click reaction, as can
be seen by SEC (Fig. 1c). The size of the resulting conjugate
was nearly equal to that of the shorter chain conjugate despite
the increase in molecular weight of the polymer, and a very
strong azide signal was observed in the IR spectrum. Increas-
ing the time of reaction to 6 days yielded some, although not
significant, improvement in conversion. This demonstrates that
steric effects play a significant role in the conjugation of poly-
mers to cyclic peptides, especially to those with such a high
tendency to aggregate. While high conversions can be reached
with low molecular weight polymers, harsher conditions would
be required to achieve the same efficiency of conjugation with
higher molecular weight polymers.
◦
6
0 C. Despite the persistence of an azide signal in the IR spec-
trum (Fig. 1a), indicating some unreacted sites on the cyclic
peptide, a clear shift in the molecular weight was seen in the
size exclusion chromatography (SEC) trace of the click prod-
uct (Fig. 1b). The high molecular weight shoulder in the SEC
trace of the conjugate suggests some small scale self-assembly
of two or more cyclic peptide conjugates in DMF. While the
molecular weight of such aggregates would be double or triple
that of a single conjugate, the molecular weight observed by
SEC only increases slightly, because the effect of aggregation
on hydrodynamic volume is only slight. The conversion of the
1
reaction was estimated to be ∼70–90% by H-NMR spectro-
metry (Fig. S1), by integration of the signals corresponding to
the triazole proton and the CH2 groups α to the triazole relative
to the Leu-CH3 groups. No improvement in conversion was seen
after 6 days.The signal corresponding to the O-CH2-triazole pro-
tons is notable, as it appears as a doublet of doublets rather than
as a singlet as is more commonly reported for similar molecules
To investigate the self-assembly of the conjugates, we pre-
pared aggregates by dilution of a concentrated DMF solution
of the conjugates in methanol, as previously described for sim-
ilar systems.[ The TEM images of the short chain polymer
17]
nanotubes in Fig. 2 show the tubes of ∼15 nm in diameter, in
the range that is expected from such conjugates.[
13,15]
While
[
24]
in d6-DMSO. This indicates reduced rotation about this bond,
no spectra of the unassembled peptide could be taken due to

RESEARCH FRONT
Synthesis of Self-assembling Cyclic Peptide-polymer Conjugates
1171
(
a)
Cyclic peptide
Short polymer-peptide
conjugate (3 days)
Long polymer-peptide
conjugate (3 days)
N3
2
500 2400 2300 2200 2100 2000 1900 1800 1700 1600 1500 1400 1300
Ϫ1
Wavenumbers [cm
]
(
b) Short polymer chains
Ϫ1
(1700 gmol
)
pHEA
Conjugate (3 days)
50 nm
(
c) Long polymer chains
Ϫ1
(
4000 gmol
)
pHEA
Conjugate (3 days)
100 nm
1
000
10000
Ϫ1
Molecular weight, Mn [gmol
]
Fig. 2. TEM images of the low molecular weight peptide-polymer conju-
Fig. 1. (a) ATR-FTIR of the cyclic peptide and both the short
gate cast from methanol.
− −1
1
(
1700 g mol ) and long (4000 g mol ) chain polymer-peptide conjugates;
SEC traces (DMF + 0.3% LiBr) for (b) the low molecular weight poly-
mer and its conjugate and (c) the high molecular weight polymer and its
conjugate.
reduced pressure to ∼3 mL, and precipitated from methanol
(
∼30 mL). The cyclic peptide 3 (0.036 g, 46%) was recovered
as a white powder after further washing with methanol (×2).
−
1
νmax (ATR)/cm 3275 (NH), 2092 (N3), 1623 (Amide-I), 1536
Amide-II). δH (300 MHz, d-TFA) 0.97 (br s, 24H, Leu CH3),
the low solubility and high tendency to aggregate, IR spectra
of methanol cast films of both the cyclic peptide and the
peptide-polymer conjugate show the presence of amide-I bands
(
1
4
1
.34–2.21 (m, 36H, Lys & Leu), 3.35 (m, 8H, 4 N3-CH2), 4.60–
+
.97 (m, 8H, 8 α-CH). m/z (APCI) 1069.53 [M + H] , calc.
−
1
−1
at 1628 cm and amide-II bands at 1541 cm , suggestive of
069.69.
[25,26]
the formation of β-sheet structure.
General synthesis of polymer-peptide conjugate: Click
In summary, we have shown that by using click reactions it
is possible to graft as many as four polymer chains to a cyclic
peptide, and that such a high graft density does not prevent the
natural self-assembly process of the peptide into nanotubes. In
principle, such an approach enables access to high graft den-
sity nanotubes from almost any polymer. These click reactions
are highly dependent on steric bulk, and increasing the molec-
ular weight of the polymer decreases the efficiency of reaction
significantly.
[23]
reactions were performed in DMSO with PMDETA/Cu(I)Br.
The cyclic peptide 3 (15 mg, 0.014 mmol) was suspended in
DMSO (2.2 mL) with the polymer (1.5 equiv/site) by sonication
for 10 min. In separate vials, CuBr (4.0 mg, 0.028 mmol), and a
solution of PMDETA (4.9 mg, 0.028 mmol) in DMSO (2.5 mL)
were prepared. All three vials were degassed with N2 for 30 min.
The PMDETA solution was then transferred into the CuBr vial
by cannula, and then this solution was added to the peptide and
◦
polymer solution by cannula, and allowed to react at 60 C under
an atmosphere of nitrogen. The crude mixture was precipitated
out of an aqueous EDTA solution (0.065 M) at 0 C to remove the
Experimental
◦
Synthesis of cyclo[l-Lys(N3)-d-Leu]4 (3): The linear precur-
sor peptide H2N-(l-Lys(N3)-d-Leu)4-OH 2 was assembled on
copper and any unreacted pHEA, and the resulting solid washed
with ice-cold water (2 × 10 mL).
2
-chlorotrityl chloride resin via standard SPPS techniques in
Short polymer-peptide conjugate (7): Conjugation of the
−
1
DMF (HBTU/Hünig’s base as the coupling agents). The lin-
ear peptide 2 (0.08 g, 0.074 mmol) was dissolved in DMSO
short pHEA chain 5 (Mn = 1700 g mol , PDI 1.10) to the
cyclic peptide 3 was performed according to the general
−
1
(
20 mL) in a sonic bath, and the resulting solution diluted
synthesis above. νmax (ATR)/cm
3273 (NH), 2092 (N3),
◦
to 50 mL with DMF. The mixture was cooled to 0 C in an
ice bath and HBTU (1.5 equiv, 0.042 g, 0.110 mmol), HOBt
1728 (C=O), 1628 (Amide-I), 1541 (Amide-II). δH (300 MHz,
d6-DMSO + 5% D2O) (see supplementary information for full
spectrum): 0.77 (m, 24H, Leu CH3), 0.87 (t, J 7.6, 9H RAFT
CH3), 1.01(m, 9H, RAFTCH3), 1.05–2.00(m, pHEAbackbone,
Leu CH, Leu CH2, Lys CH2), 3.36 (t, 9H, J 7.2, RAFT CH2),
(
0
1.5 equiv, 0.015 g, 0.110 mmol) and Hünig’s base (3 equiv,
.029 g, 0.221 mmol) were added portion-wise. The mixture
was stirred at room temperature for 6 h, concentrated under

RESEARCH FRONT
1
172
R. Chapman, K. A. Jolliffe, and S. Perrier
3
(
.53 (m, ∼84H, pHEA CH2), 3.99 (m, ∼84H, pHEA CH2), 4.73
m, 14H, peptide backbone + ε-Lys), 4.64 (s, unclicked polymer
CH2), 4.74 (s, 3H, polymer CH-S), 4.87 (s, polymer OH), 5.01–
.13 (dd, J 12.0, 25.7, 8H, Lys ε-CH2), 8.03 (s, 3H, triazole CH),
.12 (br s, NH).
[9] H. A. Klok, Macromolecules 2009, 42, 7990. doi:10.1021/
MA901561T
10] J. C. M. Van Hest, Pol. Rev. 2007, 47, 63. doi:10.1080/
[
[
[
15583720601109578
5
8
11] Y. L. Zhao, S. Perrier, Chem. Commun. 2007, 4294. doi:10.1039/
B708293B
12] J. Couet, J. D. Jeyaprakash, S. Samuel, A. Kopyshev, S. Santer,
M. Biesalski, Angew. Chem. Int. Ed. 2005, 44, 3297. doi:10.1002/
ANIE.200462993
Accessory Publication
Details of the synthesis of the cyclic peptide precursors and
the RAFT agent, as well as characterisation data for the poly-
mers and polymer-peptide conjugate are given in the accessory
publication, which is available on the journal’s website.
[
[
[
[
[
[
13] S. Loschonsky, J. Couet, M. Biesalski, Macromol. Rapid Commun.
2008, 29, 309. doi:10.1002/MARC.200700700
14] J. Couet, M. Biesalski, Macromolecules 2006, 39, 7258. doi:10.1021/
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Acknowledgements
R.C. thankstheAustralianGovernmentforanAustralianPostgraduateAward
scholarship, and the University of Sydney for funding to attend PPC11.
We are grateful to Dominik Konkolewicz for experimental assistance and
Polymer Laboratories for SEC equipment and technical support.
39, 7831. doi:10.1021/MA061738P
18] Abbreviations: 1-hydroxy-l-azabenzotriazoleuronium (HBTU),
ꢀ
ꢀ
O-benzotriazole-l-yl-N,N,N ,N -tetramethyluroniumhexafluorophos-
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