Uniting polypeptides with sequence-designed peptides: Synthesis and assembly of poly(γ-benzyl L-glutamate)-b-coiled-coil peptide copolymers

Article abstract of DOI:10.1021/ja909540a

A new class of peptide has been created, polypeptide-b-designed peptides, which unites the useful qualities of the two constituent peptide types. We demonstrate the synthesis and self-assembly possibilities of this class of peptide chimera with a series of amphiphilic polypeptide-b-designed peptides in which the hydrophobic block is poly(γ-benzyl L-glutamate) (PBLG) and the hydrophilic block is a coiledcoil forming peptide (denoted E). The synthetic approach was to synthesize the coiled-coil forming peptide on the solid phase, followed by the ring-opening polymerization of γ-benzyl L-glutamate AAcarboxyanhydride, initiated from the N-terminal amine of the peptide E on the solid support. The polypeptide-b-peptide was then cleaved from the resin, requiring no further purification. Peptide E contains 22 amino acids, while the average length of the PBLG block ranged from 36 to 250 residues. This new class of peptide was applied to create a modular system, which relied on juxtaposing the properties of the component peptide types, namely the broad size range and structure-inducing characteristics of the polypeptide PBLG blocks, and the complex functionality of the sequence-designed peptide. Specifically, the different PBLG block lengths could be connected noncovalently with various hydrophilic blocks via the specific coiled-coil folding of E with K or K-poly(ethylene glycol), where K is a peptide of complementary amino acid sequence to E. In this way, nanostructures could be formed in water at neutral pH over the entire compositional range, which has not been demonstrated previously with such large PBLG blocks. It was found that the size, morphology (polymersomes or bicelles), and surface functionality could be specified by combining the appropriate modular building blocks. The self-assembled structures were characterized by dynamic light scattering, circular dichroism, scanning electron microscopy, cryogenic-transmission electron microscopy, fluorescence spectroscopy, and zeta-potential measurements. Finally, as the structures are able to encapsulate watersoluble compounds, and the surfaces are easily functionalized via the coiled-coil binding, it is expected that these peptide-based nanocapsules will be able to act as delivery vehicles to specific targets in the body.

Full text of DOI:10.1021/ja909540a

Published on Web 01/28/2010
Uniting Polypeptides with Sequence-Designed Peptides:
Synthesis and Assembly of Poly(γ-benzyl
L-glutamate)-b-Coiled-Coil Peptide Copolymers
†
‡
§
Hana Robson Marsden, Jan-Willem Handgraaf, Fabio Nudelman,
§
,†
Nico A. J. M. Sommerdijk, and Alexander Kros*
Department of Soft Matter Chemistry, Leiden Institute of Chemistry, Leiden UniVersity, P.O. Box
9
502, 2300 RA Leiden, The Netherlands, Culgi B.V., P.O. Box 252, 2300 AG Leiden, The
Netherlands, and Soft Matter cryoTEM Research Unit, Department of Biomedical Engineering,
EindhoVen UniVersity of Technology, P.O. Box 513, 5600 MB EindhoVen, The Netherlands
Received November 10, 2009; E-mail: a.kros@chem.leidenuniv.nl
Abstract: A new class of peptide has been created, polypeptide-b-designed peptides, which unites the
useful qualities of the two constituent peptide types. We demonstrate the synthesis and self-assembly
possibilities of this class of peptide chimera with a series of amphiphilic polypeptide-b-designed peptides
in which the hydrophobic block is poly(γ-benzyl L-glutamate) (PBLG) and the hydrophilic block is a coiled-
coil forming peptide (denoted E). The synthetic approach was to synthesize the coiled-coil forming peptide
on the solid phase, followed by the ring-opening polymerization of γ-benzyl L-glutamate N-carboxyanhydride,
initiated from the N-terminal amine of the peptide E on the solid support. The polypeptide-b-peptide was
then cleaved from the resin, requiring no further purification. Peptide E contains 22 amino acids, while the
average length of the PBLG block ranged from 36 to 250 residues. This new class of peptide was applied
to create a modular system, which relied on juxtaposing the properties of the component peptide types,
namely the broad size range and structure-inducing characteristics of the polypeptide PBLG blocks, and
the complex functionality of the sequence-designed peptide. Specifically, the different PBLG block lengths
could be connected noncovalently with various hydrophilic blocks via the specific coiled-coil folding of E
with K or K-poly(ethylene glycol), where K is a peptide of complementary amino acid sequence to E. In
this way, nanostructures could be formed in water at neutral pH over the entire compositional range, which
has not been demonstrated previously with such large PBLG blocks. It was found that the size, morphology
(
polymersomes or bicelles), and surface functionality could be specified by combining the appropriate
modular building blocks. The self-assembled structures were characterized by dynamic light scattering,
circular dichroism, scanning electron microscopy, cryogenic-transmission electron microscopy, fluorescence
spectroscopy, and zeta-potential measurements. Finally, as the structures are able to encapsulate water-
soluble compounds, and the surfaces are easily functionalized via the coiled-coil binding, it is expected
that these peptide-based nanocapsules will be able to act as delivery vehicles to specific targets in the
body.
Introduction
drides (NCAs) and solid-phase synthesis. The ROP of NCAs
is the most common method of synthesizing polypeptides
For materials scientists, peptides are a fertile area for
investigation as they can be programmed with the ability to
adopt specific intra- and intermolecular conformations, which
may allow heightened levels of control over the morphologies
3
,4
containing a single amino acid residue. The polymers can be
readily prepared and have no detectable racemization at the
5
chiral centers. Blocks based on glutamic acid (γ-benzyl
1
and properties of the self-assembled structures. The structure
L-glutamate) have been commonly synthesized as their polym-
erization is known to be the best controlled, and because they
form well-defined rod-like R-helical secondary structures both
and functional properties of proteins and peptides are determined
by their primary sequence of amino acids. Materials scientists
are still unable to design the complex structures found in nature.
Yet there has been some progress, particularly in understanding
6
in the solid-state and in solution. They have been initiated from
7
8,9
traditional linear coil polymers, polymer dendrimers, modi-
2
the folding of silks, elastins, collagens, and coiled-coil motifs.
Two methods for the synthesis of peptides are the ring-
opening polymerization (ROP) of amino acid N-carboxyanhy-
(
(
3) Smeenk, J. M.; Ayres, L.; Stunnenberg, H. G.; van Hest, J. C. M.
Macromol. Symp. 2005, 225, 1–8.
4) Marsden, H. R.; Kros, A. Macromol. Biosci. 2009, 9, 939–951.
†
Leiden University.
Culgi B.V.
Eindhoven University of Technology.
(5) Deming, T. J. AdV. Polym. Sci. 2006, 202, 1–18.
(6) Gallot, B. Prog. Polym. Sci. 1996, 21, 1035–1088.
(7) Caillol, S.; Lecommandoux, S.; Mingotaud, A. F.; Schappacher, M.;
Soum, A.; Bryson, N.; Meyrueix, R. Macromolecules 2003, 36, 1118–
1124.
‡
§
(
(
1) Cavalli, S.; Kros, A. AdV. Mater. 2008, 20, 627–631.
2) van Hest, J. C. M.; Tirrell, D. A. Chem. Commun. 2001, 1897–1904.
2
370 9 J. AM. CHEM. SOC. 2010, 132, 2370–2377
10.1021/ja909540a  2010 American Chemical Society

Noncovalent Rod-Rod Peptosomes
A R T I C L E S
Table 1. Molecular Characteristics of the Compounds Used in This Study
c
name
structure
yield (%)
M
n
(g/mol)
PDI
a
K
E
Ac-(K I A A L K E)
3
G-NH
-NH
G-PEG77
2
∼40
∼40
∼10
28
30
56
2378.0
2380.6
5832
10 230
14 396
19 877
24 262
57 148
a
Ac-G(E I A A L E K)
Ac-(K I A A L K E)
3
2
a
,
b
a
PEG-K
3
1.05
b
b
b
b
b
,
,
,
,
,
c
c
c
c
c
PBLG36-E
PBLG55-E
PBLG80-E
PBLG100-E
PBLG250-E
PBLG36-G(E I A A L E K)
PBLG55-G(E I A A L E K)
PBLG80-G(E I A A L E K)
PBLG100-G(E I A A L E K)
PBLG250-G(E I A A L E K)
3
-NH
3
-NH
3
-NH
2
2
2
1.1
1.3
1.4
1.4
1.7
3
-NH
-NH
2
69
74
3
2
a
Obtained from MALDI-TOF MS. Based on a comparison of 1H NMR peaks. GPC calibrated with polystyrene standards. PEG, poly(ethylene
b
c
glycol); PBLG, poly(γ-benzyl L-glutamate); amino acids in the designed peptides are represented by their one letter codes, Ac: acetyl.
1
0
fied lipids, and polypeptides themselves synthesized by the
The potential of this novel combination of peptide classes is
demonstrated by the creation of a modular system with which
one can mix-and-match hydrophobic and hydrophilic peptide
blocks, with resultant control over the size, morphology, and
surface functionality of the structures that self-assemble in
aqueous solution.
11
ROP of NCAs. Most commonly initiators with primary amine
end-groups are used, although the polymerization can also be
initiated with transition metal-amine-functionalized polymers.
Block copolymers have also been synthesized in the reverse
manner, that is, the ROP of NCA, followed by polymerization
5
12
1
3,14
of another monomer from the polypeptide.
The ROP of
In the polypeptide-b-peptides presented here, the hydrophobic
block is composed of PBLG, and the second block is composed
of the hydrophilic coiled-coil forming designed peptide E (Table
1). The block copolymers are made by a two-step process.
Initially peptide E was synthesized using solid-phase synthesis
protocols. While still anchored to the resin, the amine terminus
was used to initiate the ROP of γ-benzyl L-glutamate N-
carboxyanhydride. This gives access to polypeptide-b-peptides
with well-defined block sizes and functionalities. Additionally,
it overcomes one of the main disadvantages of NCA polymer-
ization, as any PBLG homopolymer that is formed as a side
product can be readily washed away from the resin. A series of
PBLG-E block copolymers were synthesized with hydrophobic
block lengths ranging from tens to hundreds of monomers, and
cleaved from the resin, requiring no further purification.
Peptide E was chosen as the sequence-specific block because
it folds into a stable coiled-coil dimer with peptide, K, due to
their amino acid sequence designs, and the E/K pair has been
shown to retain its coiled-coil binding upon conjugation of
BLG-NCAs has the disadvantage of multiple side-reactions and
termination reactions, resulting in polypeptides with a wide
range of polymer lengths. To reduce the range of lengths, which
are likely to have different self-assembly properties, fractionation
5
is often applied. Additionally, the abundance of side-reactions
leads to contamination with homopolymers, which have to be
5
separated from the desired block copolymers.
Using solid-phase peptide synthesis (SPPS) protocols, pep-
tides are designed with an exact molecular weight and specific
amino acid sequence, which can result in a well-defined shape
and functionality. Importantly, the functionality can be more
complex as compared to NCA-based peptides; hence they are
very interesting for incorporation into block-copolymers. The
class of coiled-coil forming peptides, for which the design rules
for the sequence-to-structure relationships are relatively well
1
5
understood, are accessible by solid-phase synthesis and are
of particular interest. Coiled-coil forming peptides are strongly
complementary in spatial packing and interpeptide electrostatic
interactions and form highly specific noncovalent complexes
in aqueous solution. By designing the amino acid sequence
appropriately, many aspects of coiled-coil binding can be
specified, such as the oligomerization state, complex size,
orientation of binding, homo- or heterobinding, and stability.
In this Article, we combine for the first time solid-phase
synthesis with ROP to build up polypeptide-b-peptides. This
new synthetic approach gives access to peptides that are not
possible to synthesize using only solid-phase synthesis or only
ROP. The well-defined shape and large size range of polypep-
tides can be united with the complex functionality of sequence-
specific peptides, leading to peptides with an increased range
of properties.
1
6-18
macromolecules to E and/or K.
Of particular relevance is
our previous coupling of a short polystyrene chain with an
average length of 9 monomers to the N-terminus of peptide E
1
7
(denoted PS -E). PS -E forms spherical micelles in aqueous
9
9
solution, and via coiled-coil folding with peptide K or K-
poly(ethylene glycol)77 the corona can be altered, resulting in
larger spherical micelles or rod-like micelles. Furthermore, the
E/K complex was shown to reversibly dissociate upon raising
the temperature, again leading to a change in the self-assembled
structures. Thus, by utilizing a coiled-coil forming peptide as
the hydrophilic block of a block copolymer, one can obtain a
high degree of control over the hydrophilic block(s) and hence
the nanostructures that form. However, the self-assembly of
amphiphilic block copolymers depends not only on the corona
chains, but also to a large extent on the hydrophobic blocks.
As of yet, the conjugation of a range of hydrophobic block
lengths to designed peptides has not been demonstrated. By
combining polypeptides with sequence-designed peptides as
presented here, we are able to create a modular system in which
(
(
8) Huang, H.; Dong, C. M.; Wei, Y. Comb. Chem. High Throughput
Screen. 2007, 10, 368–376.
9) Higashi, N.; Koga, T.; Niwa, M. Langmuir 2000, 16, 3482–3486.
(
(
(
(
10) Dimitrov, I. V.; Berlinova, I. V.; Iliev, P. V.; Vladimirov, N. G.
Macromolecules 2008, 41, 1045–1049.
11) Sun, J.; Chen, X. S.; Lu, T. C.; Liu, S.; Tian, H. Y.; Guo, Z. P.; Jing,
X. B. Langmuir 2008, 24, 10099–10106.
12) Brzezinska, K. R.; Deming, T. J. Macromolecules 2001, 34, 4348–
4
354.
(16) Yano, Y.; Yano, A.; Oishi, S.; Sugimoto, Y.; Tsujimoto, G.; Fujii,
N.; Matsuzaki, K. ACS Chem. Biol. 2008, 3, 341–345.
13) Kros, A.; Jesse, W.; Metselaar, G. A.; Cornelissen, J. J. L. M. Angew.
Chem., Int. Ed. 2005, 44, 4349–4352.
(17) Robson Marsden, H.; Korobko, A. V.; van Leeuwen, E. N. M.; Pouget,
E. M.; Veen, S. J.; Sommerdijk, N. A. J. M.; Kros, A. J. Am. Chem.
Soc. 2008, 130, 9386–9393.
(
(
14) Imanishi, Y. J. Macromol. Sci., Chem. 1984, A21, 1137–1163.
15) Woolfson, D. N. Fibrous Proteins: Coiled-Coils, Collagen And
Elastomers; Elsevier Academic Press Inc.: San Diego, CA, 2005; Vol.
(18) Robson Marsden, H.; Elbers, N. A.; Bomans, P. H. H.; Sommerdijk,
N. A. J. M.; Kros, A. Angew. Chem., Int. Ed. 2009, 48, 2330–2333.
7
0, p 79.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 7, 2010 2371

A R T I C L E S
Marsden et al.
the hydrophobic PBLG length is readily controlled by ROP,
and the hydrophilic block(s) are readily controlled by coiled-
coil folding, allowing one to piece together different components
to access a range of self-assembled structures.
yields ranging from 28% for the block copolymer with the shortest
average PBLG length, which does not sediment well in cold
methanol, to 74% for the block copolymer with longest PBLG
block, which is the most hydrophobic of the series (Table 1).
Characterization of the PBLG-E Polypeptide-b-Peptides.
Molecular weights and their distributions of the protected PBLG-E
polypeptide-b-peptides were determined using gel phase chroma-
tography (GPC). GPC was performed with a Shimadzu system
equipped with a refractive index detector. A Polymer Laboratories
column was used (3M-RESI-001-74, 7.5 mm diameter, 300 mm
length) with DMF as the eluent, at 60 °C, and a flow rate of 1 mL
Experimental Section
Materials. FMOC-protected amino acids were purchased from
Novabiochem. Tentagel PAP resin was purchased from Rapp
Polymere. All other reagents and solvents were obtained at the
highest purity available from Sigma-Aldrich or BioSolve Ltd. and
used without further purification. PBS: 50 mM phosphate, 100 mM
KCl, pH 7.0.
Solid-Phase Peptide Synthesis of the Coiled-Coil Forming
Peptides E, K, and K-PEG. The peptides E and K and the hybrid
K-PEG were prepared and characterized as described previously.
-1
min . Both the coiled-coil peptide and the PBLG are soluble in
DMF, and the runs were conducted at 60 °C to prevent aggregation.
The molecular weights were calibrated using polystyrene standards.
The purity and molecular weights of the deprotected polypeptide-
17
1
b-peptides were checked using H NMR spectra recorded on a
After the peptide E was prepared, the resin was removed from the
reaction vessel, swollen in 1:1 (v/v) DMF:NMP, and FMOC
deprotected. The amount of successfully synthesized E on a given
weight of peptide-resin was estimated using the mass added to the
resin during the synthesis of E, and by integration of HPLC peaks
from an LCMS run of a test cleavage of 10 mg of resin-bound
peptide.
Bruker AV-500 spectrometer and a Bruker DPX300 spectrometer
at room temperature. The residual proton resonance of deuterated
dichloromethane was used for calibration. To ensure that there were
no aggregation artifacts in the spectra that were analyzed for
1
molecular weight determination, a range of H NMR spectra of
the deprotected hybrids were recorded, from deuterated dichlo-
romethane to 1:1 (v/v) deuterated dichloromethane:trifluoroacetic
acid.
Synthesis of γ-Benzyl L-Glutamate N-Carboxyanhydride
(
BLG-NCA). A suspension of γ-benzyl L-glutamate (ca. 5.0 g, 21.1
The absolute masses of the polypeptide-b-peptides with shorter
PBLG blocks were determined using MALDI-TOF mass spectrom-
etry. Spectra were acquired using an Applied Biosystems Voyager
System 6069 MALDI-TOF spectrometer. Samples were dissolved
in 1:1 (v/v) 0.1% TFA in water:acetonitrile (TA), at concentrations
mmol) in anhydrous ethyl acetate was heated to reflux (120 °C)
under an argon atmosphere with vigorous stirring. Triphosgene (ca.
2
.1 g, 7.0 mmol) was added quickly, and stirring was continued
for 3 h, until the suspension became clear. If the suspension
remained turbid, a small quantity of triphosgene was added every
-1
of ∼3 mg mL . Solutions for spots consisted of (v/v) 1:10 sample
1
5 min. The solution was filtered and concentrated to one-third of
-1
solution: 10 mg mL R-cyano-4-hydroxycinnamic acid (ACH) in
the initial volume (oily yellow liquid). The product was transferred
TA.
to a glovebox under an argon atmosphere and precipitated in hexane,
1
The secondary structure of the polypeptide-b-peptides was
determined using FT-IR spectroscopy. FT-IR spectra were recorded
on a BIORAD FTS-60A instrument equipped with a deuterated-
filtered, recrystallized, and dried. H NMR (300 MHz, CDCl
3
, δ):
7
.3 (aromatic H, m), 5.1 (benzylic CH
CH , m), 4.4 (R-CH, t), 6.8 (N-H, br).
2
, s), 2.6 (γ-CH
2
, t), 2.2 (ꢀ-
2
-
1
triglycine-sulfate (DTGS) detector at a resolution of 20 cm . The
compounds were dried from dichloromethane onto an ATR ZnSe
crystal. A clean ATR ZnSe crystal was used as the background.
Preparation of PBLG-E Suspensions. 0.1 µmol of each
compound (PBLG-E, or PBLG-E and K, or PBLG-E and K-PEG)
was dissolved in 200 µL of tetrahydrofuran (THF). Two milliliters
Solid-Phase Synthesis of Poly(γ-benzyl L-glutamate)-
block-E (PBLG-E). Poly(γ-benzyl L-glutamate) was synthesized
via a one-pot NCA polymerization of γ-benzyl L-glutamate N-
carboxyanhydride, initiated from the amine at the N-terminus of
the peptide E while still on the resin. The resin-bound peptide was
dried under reduced pressure at 40 °C overnight, and then in argon
with reduced pressure for 5 h. Under an argon atmosphere, the
peptide-resin was swollen in DCM (2.5 wt % NCA to DCM), and
subsequently the appropriate weight of NCA (determined from the
mass loading and HPLC peak integration) was added. The flask
was shaken for 24-65 h. A small volume of DCM was drained
from the reaction vessel, and the contents were analyzed with FT-
IR spectroscopy, showing that no NCA monomer remained (as
determined by the absence of the carbonyl stretching absorption
of phosphate buffered saline (PBS, 50 mM PO
.0) was added, and the sample was immediately sonicated with
4
, 100 mM KCl, pH
7
an open lid for 2 h in a Branson 1510 bath sonicator with an output
of 70 W and 42 kHz. The final concentration of each polymer was
5
0 µM.
For the encapsulation of rhodamine B in the vesicles, the samples
were prepared as described above, with the addition of rhodamine
-1
B (0.2 mg mL , 0.418 mM) to the buffer. The unencapsulated
rhodamine B was removed over a fast protein liquid chromatog-
raphy (FPLC) column. FPLC was performed with an a¨ kta prime,
Amarsham Pharmacia Biotech apparatus with a Pharmacia XK 26
column (135 mm × 25 mm) packed with Sephadex G50-fine. PBS
-
1
band of the C
2
carbonyl at 2000-1800 cm , which is released as
CO during the reaction). The resin was drained and washed
2
profusely with DCM, NMP, DMF, and finally with DCM. The
initial DCM washes were dried to collect any homopolymer that
formed in solution. The yields of the resin-bound block copolypep-
tides were 85-92%.
The hybrid material was cleaved in the protected form from the
resin using 1:99 (v/v) TFA:DCM for 2 min, 10 times. Each cleavage
mixture was precipitated dropwise in cold methanol. The white
precipitate was compacted with centrifugation, and the supernatant
was removed. This was repeated three times with the addition of
fresh methanol. The pellets were vacuum-dried.
The OtBu and BOC protecting groups of the glutamic acid and
lysine residues of the E block were removed by stirring the block
copolymer in 47.5:47.5:2.5:2.5 (v/v) TFA:DCM:water:TIS for 1 h,
and the product was precipitated dropwise in cold methanol. The
white precipitate was compacted with centrifugation, and the
supernatant was removed. This was repeated three times with the
addition of fresh methanol. The pellets were vacuum-dried, with
-
1
was used as the eluent. The flow rate was 5 mL min , UV
sensitivity was set on 0.1 AU, 1%, the conductivity was set on
-1
1
5-20 mS cm , and the wavelength for UV recording was 254
nm.
Characterization of PBLG-E Suspensions. Experimental dif-
fusion coefficients, D, were measured at 25 °C by dynamic light
scattering (DLS) using a Malvern Zetasizer Nano ZS equipped with
a peltier-controlled thermostatic cell holder. The laser wavelength
was 633 nm, and the scattering angle was 173°. The Stokes-Einstein
relationship D ) k
radius, D . Here, k
viscosity.
b
T/3πηD
b
h
was used to estimate the hydrodynamic
is the Boltzmann constant, and η is the solvent
h
Zeta potentials were measured at 25 °C using the same
instrument. The laser wavelength was 633 nm, and the scattering
angle was 173°. A Malvern universal dip cell (ZEN1002) was used,
2
372 J. AM. CHEM. SOC. 9 VOL. 132, NO. 7, 2010

Noncovalent Rod-Rod Peptosomes
A R T I C L E S
Scheme 1. Synthesis of PBLG-E by Solid-Phase Peptide
Synthesis Followed by N-Carboxyanhydride Ring-Opening
Polymerization Initiated from the N-Terminal Amine of the
Resin-Bound Peptide
and the samples were diluted 10-fold with 1 mM NaCl to reduce
the ionic strength of the buffer.
Scanning electron microscopy (SEM) was conducted on a Nova
NanoSEM FEI instrument with an accelerating voltage of 10 kV
and spot size of 3.5. Samples for SEM were prepared by placing 5
µL of the solution on SEM stubs with a TEM grid on the carbon
tape. After 30 min, the excess buffer was removed. Samples were
coated with gold for 1 min, resulting in a layer ∼15 nm thick.
Samples for cryogenic TEM were concentrated by centrifuging
in Centricon centrifugal filter devices MWCO 3000 g mL- at 4
1
°
C. Sample stability was verified by DLS and TEM. The cryogenic
transmission microscopy measurements were performed on a FEI
Technai 20 (type Sphera) TEM or on a Titan Krios (FEI). A Gatan
cryo-holder operating at ∼ -170 °C was used for the cryo-TEM
6
measurements. The Technai 20 is equipped with a LaB filament
operating at 200 kV, and the images were recorded using a 1k ×
1
k Gatan CCD camera. The Titan Krios is equipped with a field
emission gun (FEG) operating at 300 kV. Images were recorded
using a 2k × 2k Gatan CCD camera equipped with a post column
Gatan energy filter (GIF). The sample vitrification procedure was
carried out using an automated vitrification robot: a FEI Vitrobot
Mark III. TEM grids, both 200 mesh carbon coated copper grids
and R2/2 Quantifoil Jena grids, were purchased from Aurion.
Copper grids bearing lacey carbon films were homemade using 200
mesh copper grids from Aurion. Grids were treated with a surface
plasma treatment using a Cressington 208 carbon coater operating
at 25 A for 40 s prior to the vitrification procedure.
Circular dichroism (CD) spectra were obtained using a Jasco
J-815 spectropolarimeter equipped with a peltier-controlled ther-
mostatic cell holder. Spectra were recorded from 260 to 200 nm in
a 1 mm quartz cuvette at 25 °C. Data were collected at 0.5 nm
intervals with a 1 nm bandwidth and 1 s readings. Each spectrum
was the average of five scans. For analysis, each spectrum had the
appropriate background spectrum (buffer or buffer/THF) subtracted.
The amount of encapsulated Rhodamine B in each sample was
determined by fluorescence spectroscopy, with excitation at 555
nm, and emission monitored from 563-650 nm with 5 nm slits
using a Cary-50 spectrophotometer.
Results and Discussion
Synthesis and Characterization of the Protected PBLG-E
Block Copolymer Series. Poly(R-amino acid)s can be prepared
by the ROP of NCAs starting from nucleophilic attack of the
5
C carbonyl group of the NCA by an initiator such as amines,
1
9
alkoxide anions, alcohols, transitions metals, and water. In
our approach to synthesize polypeptide-b-peptides, we first
synthesized the coiled-coil peptide block E on a Sieber amide
resin using standard Fmoc solid-phase peptide protocols and
removed the N-terminal Fmoc group. ROP of the BLG-NCA
was then initiated by the deprotected N-terminal amine of
peptide E while still anchored to the resin (Scheme 1). The
polymerization was conducted by shaking the resin-bound
peptide with the NCA in DCM at room temperature under an
argon atmosphere for 1-3 days. When all of the BLG-NCA
monomer was consumed, the resin was drained and washed
thoroughly with DCM, NMP, and DMF. Typically 8% of the
NCA monomer formed short oligomers during the polymeri-
zation reaction, due to trace amounts of water. These impurities
were easily removed from the desired product by extensive
washing of the resin.
it was found that within each synthesis the longer PBLG-E
polypeptide-b-peptides were cleaved first from the resin, with
a progressive shortening of the PBLG chain with each fraction
collected, until finally peptide fragments from the solid-phase
peptide synthesis of E were cleaved. In this way, polypeptide-
b-peptides with a lower polydispersity index (PDI) can be
obtained by selecting which fractions to combine. Because of
the washing away of homo-PBLG while the block copolymer
is still attached to the resin, and the cleavage of peptide
fragments from the resin only after the bulk of PBLG-E
molecules have been cleaved, no further purification was
necessary. The GPC chromatographs of the PBLG-E series are
shown in Figure 1. Peaks are monomodal, and the PDIs range
from 1.1 for the hybrid with the shortest PBLG block to 1.7
for the hybrid with the longest PBLG block.
The protected polypeptide-b-peptides were released from the
solid support by shaking 10 times (2 min each) in 99:1 (v/v)
DCM:TFA, with subsequent precipitation in cold methanol. The
purity of each fraction was ascertained with GPC, from which
Synthesis and Characterization of the PBLG-E Block
Copolymer Series. The protecting groups from the glutamic acid
and lysine residues of peptide E were selectively removed using
(
19) Blout, E. R.; Karlson, R. H. J. Am. Chem. Soc. 1956, 78, 941–946.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 7, 2010 2373

A R T I C L E S
Marsden et al.
residues, while the hydrophobic PBLG block ranges from 36
2
3
to 250 benzyl glutamate residues.
Modular Assembly of the Polypeptide-b-Peptides and
Polymer Mixtures in Aqueous Solution. The synthesis and self-
assembly of rod-rod block copolymers is an emerging topic
in polymer chemistry, and the well-defined shapes of rod
2
4
polymers lead to novel self-assembly properties. PBLG is
hydrophobic and with a degree of polymerization (n) larger than
2
5
1
0 has an R-helical secondary structure, resulting in a rod-
like molecular shape. The length of PBLG R-helices is n × 1.5
2
6
27
nm Å while the diameter is ∼2 nm, hence in the R-helical
configuration the PBLG rod-like blocks in this study range in
average length from 5.4 to 37.5 nm long. The hydrophilic block,
peptide E, forms an R-helical coiled-coil dimer with K, a peptide
Figure 1. GPC chromatographs of the coiled-coil forming peptide E (not
purified) and PBLG-E polypeptide-b-peptides in the protected form. The
numbers above each trace refer to the degree of polymerization of the PBLG
block.
1
7,28
with a complementary amino acid sequence (Figure S1).
E
and K form complexes with a well-defined rod-like geometry
of cylinders 3.5 nm long with approximately the same diameter
2
9
as PBLG rods. Poly(ethylene glycol) is a hydrophilic coil
polymer, and the PEG used in this study, with an average of
3
0
7
7 monomers, has an average diameter of approximately 5 nm,
1
7
although it is more compact when attached to peptide K. The
peptides K and the hybrid K-PEG are predominantly hydrophilic
3
1
and do not aggregate in aqueous solutions.
Because of the amphiphilic nature of the rod-rod PBLG-E
polypeptide-b-peptides and the noncovalent complexes PBLG-
E/K and PBLG-E/K-PEG, we expected that the PBLG and
hydrophilic blocks would phase separate in aqueous solution.
The self-assembling characteristics of the PBLG-E series, both
in isolation and with equimolar amounts of K or K-PEG, were
studied in phosphate buffered saline solution (PBS) at pH 7.0.
The PBLG-E polypeptide-b-peptides, having large hydrophobic
PBLG blocks, were not soluble in aqueous solutions. The
Figure 2. 1H NMR spectrum of PBLG36-E in 7:3 (v/v) DCM-d
the proton assignments. X is from DCM.
3
:TFA with
32
standard methods for inducing self-assembly, film hydration,
or solvent injection followed by dialysis, were ineffective as
the PBLG-E block copolymers precipitated. Better results were
achieved by injecting PBS into a tetrahydrofuran (THF) solution
of the polymer with sonication. PBS is selective for the
hydrophilic E, E/K, and E/K-PEG blocks and induces aggrega-
tion of the PBLG blocks. The initial ratio of THF to PBS was
a mixture of TFA, DCM, water, and TIS, while retaining the
benzyl protecting groups of the PBLG block, and the am-
phiphilic polypeptide-b-peptide was precipitated in cold metha-
nol. The complete removal of the protecting groups was
confirmed by the disappearance of the peak from the methyl
1
groups of the OtBu and Boc moieties at 1.5 ppm from H NMR
n
spectra. To determine the degree of polymerization and M of
(
(
23) See the Supporting Information.
24) Scherf, U.; Adamczyk, S.; Gutacker, A.; Koenen, N. Macromol. Rapid
Commun. 2009, 30, 1059–1065.
the PBLG blocks, spectra were obtained for each compound in
deuterated dichloromethane with increasing amounts of trifluo-
roacetic acid, ensuring the absence of aggregation of the
amphiphilic block copolymer and hence that accurate peak
(25) Rinaudo, M.; Domard, A. Biopolymers 1976, 15, 2185–2199.
(
26) Murthy, N. S.; Knox, J. R.; Samulski, E. T. J. Chem. Phys. 1976, 65,
4835–4839.
2
0-22
comparisons between E and PBLG blocks could be made.
(
27) Chang, Y. C.; Frank, C. W. Langmuir 1996, 12, 5824–5829.
The peak arising from the leucine and isoleucine methyl protons
of the E block was compared to the peak arising from the benzyl
protons of the PBLG block (Figure 2). The degree of polym-
(28) The secondary and quaternary structures of the peptides E and K in
PBS were evaluated by circular dichroism spectroscopy. Peptide E
adopts a predominantly random coil conformation, while K exhibits
a predominantly R-helical spectrum. Both peptides are in the mono-
meric state as indicated by the observed ellipticity ratios ([θ]222/
[θ]208) of 0.59 and 0.74, respectively. When peptides E and K were
combined in an equimolar ratio, denoted E/K, a typical R-helical
spectrum is exhibited, with minima at 208 and 222 nm. The ellipticity
ratio was determined to be 1.00, consistent with interacting R-helices.
This clearly shows that E and K specifically interact to form a hetero-
R-helical coiled coil. The formation of the dimeric species was
confirmed by determining the molecular weights using sedimentation
equilibrium, revealing that separate solutions of E and K are purely
monomeric while the mixture of E/K exists as dimers.
1
erization of the PBLG blocks determined using H NMR
spectroscopy matched the data obtained by GPC, indicating that
the polystyrene standards used for GPC molecular weight
comparison are reliable for these hybrids. The molecular
characteristics of the compounds used in this study are shown
in Table 1. The hydrophilic peptide E has 22 amino acid
(
20) Note that when PBLG-E is in the R-helical conformation, for example,
in DCM or DMSO, the R-H resonance peak is at 4.0 ppm, and by
adding TFA the peak position is shifted low-field to 4.7 ppm, indicating
that the hybrids have random coil conformation in this solvent mixture
and are not aggregated.
(29) Lindhout, D. A.; Litowski, J. R.; Mercier, P.; Hodges, R. S.; Sykes,
B. D. Biopolymers 2004, 75, 367–375.
(30) The hydrodynamic diameter of the PEG block was determine by
DLS.
(
21) Higashi, N.; Kawahara, J.; Niwa, M. J. Colloid Interface Sci. 2005,
(31) Kaiser, E.; Colescot, R.; Bossinge, C.; Cook, P. I. Anal. Biochem.
1970, 34, 595–598.
2
88, 83–87.
(22) Bradbury, E. M.; Cranerob, C.; Goldman, H.; Rattle, H. W. E. Nature
968, 217, 812–816.
(32) Kita-Tokarczyk, K.; Grumelard, J.; Haefele, T.; Meier, W. Polymer
2005, 46, 3540–3563.
1
2
374 J. AM. CHEM. SOC. 9 VOL. 132, NO. 7, 2010

Noncovalent Rod-Rod Peptosomes
A R T I C L E S
Figure 3. Typical hydrodynamic diameters of the self-assembled structures
formed by the polypeptide-b-peptides and noncovalent complexes ([total
peptide] ) 50 µM, PBS, 25 °C).
such that the E/K coiled coil is stable (Figure S2), and the THF
was subsequently removed by continuous sonication for 2 h in
an open vessel. An example of circular dichroism spectra of
the polymers and polymer mixtures is given in the Supporting
Information (Figure S3).
Figure 4. Cryo-TEM images of (A) PBLG36E, (B) PBLG36E/K (with
multilamellar vesicle inset), (C) PBLG36E/K-PEG (with bilamellar vesicle
and bicelles inset), and (D) PBLG100E/K-PEG. Scale bars ) 100 nm ([total
peptide] ) ∼1500 µM, PBS).
Particle Sizes. The aggregation of the individual polypeptide-
b-peptides (PBLG-E) was investigated with dynamic light
scattering (DLS), as well as with the addition of equimolar
amounts of the K or K-PEG components. It was found that when
the PBLG block length was 80 monomers or shorter, each of
the three permutations had a suitable balance of hydrophobicity
and hydrophilicity to form ordered supramolecular structures
in aqueous solution. For PLBG100-E, complexation with K or
K-PEG resulted in a noncovalent block copolymer with a corona
sufficient to form ordered structures. The amphiphilic block
copolymer with the longest hydrophobic block, PBLG250-E,
required association with K-PEG to have a large enough corona
to aggregate in an ordered manner. The average hydrodynamic
diameter of the structures that self-assembled from the polymers
and polymer mixtures ranged from 100 to 400 nm and were
significantly larger than the calculated sizes of spherical micelles
3
5
hydrophobic chains is minimal (lipid tails are stretched, and
the PBLG rods have a well-defined structure and size with no
3
6
change in configuration expected upon aggregation ), hence
the role of deformation of the core block on the free-energy of
the structure is reduced.
Particle Morphology. Information about the morphology of
the supramolecular structures that formed was obtained by
electron microscopy. The effects of PBLG chain length and the
relative size of the corona on the ability of the molecules to
controllably self-assemble as observed by scanning electron
microscopy (SEM) were consistent with the DLS observations
(Figure S4).
Combining the shortest PBLG length with an E or E/K
hydrophilic block results in spherical objects assembling in PBS,
which collapse when dried (Figure S4). PBLG36-E/K-PEG has
the same hydrophobic block length, but forms smaller structures,
as explained in the DLS section, which are stable upon drying.
This sample also contained disk-like aggregates, vide infra. To
obtain further insight into the internal structure of the assemblies,
cryo-TEM studies were performed for a selection of the self-
assembled structures (Figure 4). The shortest polypeptide-b-
peptide, PBLG36-E (with a low PDI of 1.1), self-assembles into
vesicles with rather uniform membrane thicknesses, which seem
to be independent of the vesicle diameter. The thicknesses
observed increase slightly with increasing size of the hydrophilic
block(s): 17.2 ( 2.6 nm for PBLG -E, 18.5 ( 2.4 nm for
3
3
(
10.8-85 nm ). All size distributions were monomodal, and
the polydispersity index (PDI) of the samples was 0.35 or less.
As shown in Figure 3, increasing the PBLG block length
resulted in larger aggregates. Additionally, for a particular PBLG
block length, increasing the size of the headgroup (going from
E to E/K and E/K-PEG) leads to a smaller hydrodynamic
diameter of the particles. These trends can both be explained
by classical packing parameter considerations: the larger the
headgroup is in comparison to the hydrophobic PBLG, the
greater is the curvature of the aggregate surface, and hence the
3
4
36
particle size decreases. The packing parameter was originally
designed to predict the morphology and size of nanostructures
formed from lipids, and this approach is not always suited to
block copolymers because it does not take into account the
complexity of the interfacial energy and interaction free-energies
PBLG36-E/K, and 21.5 ( 2.2 nm for PBLG36-E/K-PEG (Figure
A-C). The observed membrane thicknesses are in remarkably
close accordance with the calculated bilayer thicknesses, as seen
in Table 2. These results show that the rigid hydrophobic PBLG
rods are able to assemble into very well-defined bilayers through
coupling to the water-soluble peptide rods. In contrast to other
4
1
7
of the blocks. That being said, it is sufficient to explain the
trends observed in the assembly of this system. This may be
because in the case of both lipid structures and structures formed
from the PLBG-E series the influence of stretching of the
3
7
block copolymer vesicles, there does not appear to be any
interdigitation of the two layers of the hydrophobic block,
(
35) Opsteen, J. A.; Cornelissen, J. J. L. M.; van Hest, J. C. M. Pure Appl.
Chem. 2004, 76, 1309–1319.
(
(
33) Based on PBLG length, n × 0.15 nm; E and E/K length, 3.5 nm;
PEG length, 3 nm.
34) Israelachvili, J. N.; Mitchell, D. J.; Ninham, B. W. J. Chem. Soc.,
Faraday Trans. 2 1976, 72, 1525–1568.
(36) Halperin, A. Macromolecules 1990, 23, 2724–2731.
(37) Srinivas, G.; Discher, D. E.; Klein, M. L. Nat. Mater. 2004, 3, 638–
644.
J. AM. CHEM. SOC. 9 VOL. 132, NO. 7, 2010 2375

A R T I C L E S
Marsden et al.
Table 2. Experimental and Theoretical Membrane Thicknesses of
the Vesicles
as in this geometry the rods pack well together, and large coil
blocks are less affected by confinement free energy.
36
membrane thickness
For the polypeptide-b-peptides and noncovalent complexes
with longer PBLG lengths, the SEM images exclusively show
spherical objects that withstand the drying process. Cryo-TEM
was also conducted on the structures assembled from the
complex PBLG100-E/K-PEG. As seen in Figure 4D, the spherical
particles were observed to contain aqueous interiors and have
very bulky membranes of variable thickness (∼68 ( 22 nm).
The range of membrane thicknesses likely arises from the
polydispersity of molecular lengths in this sample. GPC of the
protected polypeptide-b-peptide yielded a PDI of 1.4, and it was
estimated that 95% of the PBLG100-E lengths are between 9
and 80 nm when in the R-helical conformation.
a
b
sample
experimental (nm)
theoretical (nm)
PBLG36-E
PBLG36-E/K
PBLG36-E/K-PEG
17.2 ( 2.6 nm
18.5 ( 2.4 nm
21.5 ( 2.2 nm
18
18
24
a
b
26
Determined by cryo-TEM. PBLG length, n × 1.5 Å; E and E/K
2
9
17
length, 3.5 nm; PEG length, ∼3 nm.
An advantage of block copolymer capsules over liposomes
is that their membrane thickness varies depending on the
composition, molecular weight, and degree of stretching of the
blocks. The hydrophobic core of lipid bilayers is always
approximately 3-4 nm thick, regardless of the lipid composi-
4
1
tion, while the membrane thickness of polymersomes ranges
4
2
43
from 3-5 nm to 200 nm (in 1:1 v/v TFA/DCM). In the
present series, the nature of the membrane can be dictated by
the modular choice of hydrophobic and hydrophilic compo-
nent(s), and as far as we are aware the breadth of the membrane
of PBLG100-E/K-PEG aggregates is the largest reported for
polymersomes in aqueous solutions.
Figure 5. The space-filling model of the PBLG36-E/K-PEG complex
illustrates that without chain stretching PEG readily extends the length of
the coiled-coil peptides and the PBLG block, as is proposed to occur at the
edges of the polymeric bicelles. The E/K dimer structure (red and blue
2
9
helices) is based on the work of Litowski and Hodges, the PBLG adopts
an R-helix (green), and the PEG has a random coil conformation (orange).
3
9
The PBLG36-E/K-PEG model was built with Molden version 4.6, and
4
0
the image was generated with VMD version 1.8.6.
Encapsulation, Stability, and Surface Functionality. To
demonstrate the ability of the supramolecular structures to
encapsulate water-soluble compounds, samples were prepared
in the presence of the water-soluble fluorescent dye rhodamine
B. Following sonication, the unencapuslated rhodamine B was
removed by FPLC. The samples for which DLS indicated
ordered structures (monomodal intensity distributions and PDI
< 0.35) exhibited rhodamine B fluorescence (Figure S5),
confirming that the polypeptide-b-peptides and noncovalent
complexes had a suitable balance of the hydrophilic block size
to the hydrophobic PBLG block to lead to controlled assembly,
and that these self-assembled structures had aqueous interiors.
As expected, the samples that did not show well-defined
assembly by DLS (PBLG100-E, PBLG250-E, and PBLG250-E/K)
contained insignificant amounts of rhodamine B, as verified by
fluorescence spectroscopy (Figure S5). The hydrodynamic
diameter of the nanocapsules did not change for at least 10-20
months at 4 °C as determined by DLS, showing the extensive
presumably due to the rod-like structure of the PBLG and the
comparable diameter of the hydrophobic and corona rods.
In addition to polymersomes, bicelles, disks of uniform height
and density, were observed in the SEM and cryo-TEM images
of PBLG36E/K-PEG (arrow in Figure S4C, and Figure 4C; note
that the bicelles were predominantly situated on the carbon film
of the TEM grids, as shown in the inset of Figure 4C). This is
the sample with the shortest PBLG block and the longest
hydrophilic component in comparison to the PBLG block.
Presumably polymeric bicelles are observed only for this
noncovalent block-copolymer because the lengths of the PBLG
blocks are short enough that the hydrophilic PEG block is able
to fold over any exposed PBLG on the sides of the discs,
3
8
shielding it from the aqueous buffer. This eliminates the
energetic impetus for the bilayers to close the hydrophobic sides
by curving to form vesicles. This possibility was tested with
computer modeling, which confirmed that PEG is able to cover
the average length of the extended R-helical PBLG block
without any chain stretching, that is, while still in the random
coil configuration (Figure 5). A theoretical study has found that
for rod-coil block copolymers the only stable micellar form
has disk-like cores and relatively large corona thicknesses. The
disk-like core reduces the core-corona interfacial free energy,
4
4
stability of the samples. As well as the particle size and
morphology, the surface functionality is also determined by
which molecules are chosen to complex with the E block and
was likewise retained over the same measurement period, with
PBLG-E samples (having a net charge per molecule of -3)
displaying a negative zeta potential, and PBLG-E/K and PBLG-
(
39) Schaftenaar, G.; Noordik, J. H. J. Comput.-Aided Mol. Des. 2000,
4, 123–134.
1
(
38) There is likely to be sorting within the bicelles such that the polymer-
b-peptides with shorter hydrophobic blocks are located at the rim. The
shortest PBLG length in the PBLG36-E polymer-b-peptide is 23
monomers (as determined by MALDI-TOF MS), which corresponds
to an R-helical length of ∼3.5 nm. The persistence length of bulk
PBLG is ∼2 nm and increases when under thermodynamic confine-
ment, as is the case in this nanophase separated system. Floudas, G.;
Speiss, H. W. Macromol. Rapid Commun. 2009, 30, 278–298. It is
possible that the PBLG rods are kinked, further reducing/eliminating
the area of solvent-exposed PBLG.
(40) Humphrey, W.; Dalke, A.; Schulten, K. J. Mol. Graph. 1996, 14, 33–
38.
(41) Discher, B. M.; Hammer, D. A.; Bates, F. S.; Discher, D. E. Curr.
Opin. Colloid Interface Sci. 2000, 5, 125–131.
(42) Schillen, K.; Bryskhe, K.; Mel’nikova, Y. S. Macromolecules 1999,
32, 6885–6888.
(43) Jenekhe, S. A.; Chen, X. L. Science 1998, 279, 1903–1907.
(44) The hydrodynamic diameter of PBLG36-E, PBLG36-E/K, and PBLG36
-
E/K-PEG was monitored over 10 months, while the remainder of the
samples were monitored (and were stable) over 20 months.
2
376 J. AM. CHEM. SOC. 9 VOL. 132, NO. 7, 2010

Noncovalent Rod-Rod Peptosomes
A R T I C L E S
E/K-PEG samples (with no net charge per complex) having zeta
potentials closer to zero (Table S1).
the hybrid K-PEG. Polymersomes with a range of sizes,
membrane thicknesses, and surface functionality were formed,
as well as disk-like micelles. The structures were characterized
by means of CD, DLS, SEM, cryo-TEM, fluorescence spec-
troscopy, and zeta potential measurements. It was found that
the diameters of the structures increased with decreasing
hydrophilic to hydrophobic block size ratios, as is typical for
traditional surfactants such as lipids. The form of the polymer-
some membrane depended on the length of the PBLG block,
with the shortest PBLG length forming very well-defined
membranes that were twice the calculated molecular length. The
structures were demonstrated to encapsulate water-soluble
compounds, hence there is potential for the use of these materials
as drug delivery devices, and through E/K complexation the
outside of the polymersomes can be decorated with targeting/
stealth molecules.
Conclusions
A new class of peptides, polypeptide-b-peptides, has been
synthesized by a straightforward synthetic approach. The new
peptide class is achieved by initiating ring-opening polymeri-
zation of NCAs from a designed peptide. The concept is
demonstrated by the solid-supported polymerization of γ-benzyl
L-glutamate NCA from the coiled-coil forming peptide E, which
was synthesized using standard solid-phase peptide synthesis
protocols. Cleavage of the diblock from the resin and precipita-
tion yielded block copolymers requiring no further purification.
The polypeptide-b-peptides had PBLG block lengths ranging
from 36 to 250 monomers. The chemical structure of the
polypeptide-b-peptides was confirmed by GPC, NMR, FTIR,
and MALDI-TOF MS. This new class of peptides will broaden
the scope of peptide-based nanostructures, as the combination
of polypeptides with designed peptides enables control over both
the peptide size and the functionality, which are both determin-
ing factors in molecular self-assembly. This potential is
investigated by selecting four molecular building blocks and
showing that a variety of structures could be accessed, relying
on the size of the PBLG block and the noncovalent coiled-coil
interaction of peptide E with the complementary peptide K or
Supporting Information Available: Characterization of the
compounds. Circular dichroism spectra of the coiled-coil
forming peptides in PBS and THF. Circular dichroism spectra,
SEM images, fluorescence spectra, and zeta potential measure-
ments of the self-assembled nanostructures. This material is
available free of charge via the Internet at http://pubs.acs.org.
JA909540A
J. AM. CHEM. SOC. 9 VOL. 132, NO. 7, 2010 2377