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PeptideЦPolymer Hybrid Nanotubes.

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Angewandte
Chemie
Nanotubes
Peptide–Polymer Hybrid Nanotubes**
Julien Couet, J. D. Jeyaprakash S. Samuel,
Alexey Kopyshev, Svetlana Santer, and
Markus Biesalski*
Since the discovery of carbon nanotubes,[1] there has been a
growing interest in organic tubular structures with very small
dimensions. The number of possible applications of these
microstructures has also grown, particularly in the fields of
microelectronics,[2, 3] separation technology,[4] and biomedical
technology.[5] There is an increased use of so-called bottom-up
strategies for the design of organic nanotubes and nanofibers.[6] Some promising examples of this include the selfassembly of amphiphilic molecules (small lipids,[7] di-[8] and
triblock,[9] and coil-ring-coil[10] block copolymers), polymeric
foldamers,[11] as well as the self-assembly of cyclic peptides to
form peptide nanotubes.[12–14]
Cyclic peptides that consist of an even number of d- and
l-amino acids exhibit a pronounced tendency to self-assemble
into tubular structures in which a large number of peptide
subunits are held in place by intermolecular hydrogen
bonds.[12–14] Through their design principle, these cyclic
molecules maintain a flat conformation in which the amide
functional groups lie perpendicular to the plane of the
molecular ring. This allows the formation of an extended
network of hydrogen bonds, preferably in an antiparallel bsheet structure.[15] As a result of the peptide configuration, all
the side-chain functional groups are located on the ring
periphery. This imparts the nanotubes with a defined hollow
core and enables simple access to the functional groups for
modification of the surface chemistry. The side-chain functional groups of the peptide can have an influence on the
[*] J. Couet, A. Kopyshev, Dr. S. Santer,+ Dr. M. Biesalski
Institute for Microsystem Technology (IMTEK)
University of Freiburg
Georges-Khler-Allee 103, 79110 Freiburg (Germany)
Fax: (+ 49) 761-203-7163
E-mail: biesalsk@imtek.de
Dr. J. D. J. S. Samuel
IBM Almaden Research Center
San Jose, CA95120 (USA)
[+] Previous surname: Prokhorova
[**] We thank the following people for their assistance with the analysis
of cyclic peptides and polymer hybrid materials: Dr. J. Wrth and C.
Warth (ESI MS and LC–MS), Dr. M. Ade (X-ray diffraction), Dr. R.
Thomann and A. Neub (TEM), and K. Shroff (automated peptide
synthesis). Financial support came from the Deutsche Forschungsgemeinschaft (Emmy Noether Programm), the Landesstiftung Baden-Wrttemberg (Elitefrderprogramm Nachwuchswissenschaftler), and the Fonds der Chemischen Industrie. J.C. thanks
the DAAD for a fellowship with the postgraduate student exchange
program. We thank Prof. J. Rhe and Dr. O. Prucker for valuable
support and stimulating discussions.
Supporting information for this article is available on the WWW
under http://www.angewandte.org or from the author.
Angew. Chem. Int. Ed. 2005, 44, 3297 –3301
formation of either single-peptide nanotubes, or higher-order
3D aggregates.[16]
Since the pioneering work by Ghadiri and co-workers,[12]
such self-assembled peptide nanotubes have been applied as
novel antibiotics[17] and as artificial ion channels.[18] The range
of application of peptide nanotubes is determined mainly by
their surface chemistry. As shown by Ghadiri and co-workers,[12–14] the surface chemistry can be tuned by individually
synthesizing cyclic peptides with different primary sequences.
An alternative way to adjust the surface chemistry is through
the attachment of synthetic macromolecules to the surface of
the structure after self-assembly, thus embedding the peptide
nanotube in a polymeric shell. In this case, the structure of the
resulting hybrid material is governed by both the peptide
nanotube core as well as the polymeric shell, and the surface
chemistry is determined by the choice of grafted synthetic
macromolecules.
The assembly of cyclic peptide precursors into nanotubes
with subsequent surface-induced polymerization around the
nanotubes offers a novel approach toward the preparation of
a large number of shape-persistent hybrid materials that are
not easily accessed by any other technique. An interesting
aspect of this strategy is that defined structural information
can be transferred from a biologically derived module to a
synthetic polymer (and vice versa).
Recently, it was shown that synthetic peptides can be
modified with polymerization initiators, which can subsequently initiate reactions that link peptides into covalent
polymers.[19–21] For example, such modified peptides have
been used as initiators for nitroxide-mediated controlled
radical polymerizations[19] and atom-transfer radical polymerizations (ATRP).[20, 21]
In our work, we have taken advantage of the ability of
distinct cyclic peptides to self-organize into defined tubular
structures, as discussed above. In particular, we have used
peptide nanotubes as structurally defined templates to
prepare nanometer-sized peptide–polymer hybrid nanotubes.
Figure 1 schematically outlines this approach. Cyclic peptides
composed of eight amino acids that are arranged with
alternating d- and l-configurations are modified with certain
chemical groups at distinct side-chain positions. These groups
can serve as initiation sites for controlled free-radical
polymerization by the ATRP technique. The cyclic-peptide
initiators self-assemble into peptide nanotubes in a fashion
analogous to that described above. A subsequent polymerization reaction of N-isopropylacrylamide (NIPAM) in aqueous dispersion is triggered by the surface-attached initiator
groups to yield a novel class of polymeric hybrid nanostructures in which the peptide core is covalently attached to a
functional polymeric shell.
The process begins with a solution of the cyclic-peptide
initiator in pure trifluoroacetic acid (TFA). The precursors
slowly self-assemble and precipitate from solution in the form
of nanotubes upon a gradual increase in the amount of water
added to the TFA phase. The nanotubes can be isolated
through a simple centrifugation procedure, followed by
rinsing with water. The products are then characterized with
respect to chemical identity, surface chemistry, internal
structure, and morphology. Analysis by FTIR spectroscopy
DOI: 10.1002/anie.200462993
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3297
Communications
Figure 1. Schematic outline of the synthesis of peptide–polymer hybrid nanotubes. A cyclic peptide with polymerization initiator groups at distinct
side chains self-assembles to form a peptide nanotube that has the initiator groups exclusively at the outer surface. A subsequent surface-initiated
polymerization in the presence of NIPAM monomer coats the peptide core with a covalently bound PNIPAM polymer shell.
shows the presence of closely packed peptides that are
stacked in a b-sheet structure held together through intermolecular hydrogen bonds.[22] Furthermore, X-ray photoelectron spectroscopy proves the presence of the bromine groups
that are required for the nanotube surface-initiated polymerization.[22] The morphology of the cyclic-peptide nanotubes
was studied by AFM and TEM. A representative AFM image
of nanotubes that were drop cast from an aqueous dispersion
onto a silicon wafer is shown in Figure 2. Both single
nanotubes and 2D aggregates were observed on the surface.
The average height of a cyclic-peptide nanotube, determined
by statistical analysis of AFM cross-section data, is 1.7 0.2 nm (Figure 2 c). The length of the peptide nanotubes
ranges between 100 and 500 nm. Both values are consistent
with experimental and theoretical studies of single-peptide
nanotubes consisting of eight-membered rings of d- and lamino acids.[16] The height of the 2D fibrous aggregates,
shown in Figure 2, is equal to those of single-peptide nanotubes which implies that the aggregates are most likely
formed during the surface drying procedure. Clear experimental support for this is still pending, however.
3298
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
The cyclic-peptide initiator nanotubes were dispersed into
a mixture of water, monomer, and polymerization additives
by which a surface-induced ATRP reaction was initiated at
room temperature. NIPAM was chosen as the monomer, as it
yields peptide–polymer nanotubes with a nontoxic polymer
(PNIPAM) coating and displays interesting physical properties, such as a lower critical solution temperature. After a
defined polymerization time, the cyclic-peptide–PNIPAM
nanotubes were washed extensively with water to remove any
physisorbed polymer generated from the transmission of
surface-bound radicals to NIPAM monomers in solution.
Figure 3 b shows AFM images of the cyclic-peptide–
PNIPAM nanotubes adsorbed to a silicon wafer (the sample
has been drop cast from aqueous solution). Notably, 2D
aggregates are no longer observed; the nanotubes are present
as distinct rod-shaped structures that are 80 20 nm in length
and 12 3 nm in height as determined by the statistical
analysis of more than 400 nanotubes (Figures 3 c and d). A
cross-section of a single polymer–peptide nanotube (Figures 3 e and f) shows that the polymer shell surrounds the
peptide core in a very homogeneous manner. The presence of
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Angew. Chem. Int. Ed. 2005, 44, 3297 –3301
Angewandte
Chemie
Figure 2. Analysis of the self-assembled cyclic-peptide initiator nanotubes: a) AFM image of peptide nanotubes adsorbed on a silicon
wafer (scale: 2 mm; height: 5 nm); b) cross-section of the marked positions in part a); c) height distribution from the statistical analysis of
adsorbed nanotubes (relative abundance A as a function of measured
height h).
the PNIPAM polymer is proven by FTIR[22] spectroscopy and
NMR spectroscopy. Furthermore, FTIR analysis of the cyclicpeptide–PNIPAM nanotubes suggests that the internal peptide assembly is an intact b-sheet structure.[22] Finally, as can
be observed in the AFM phase image, a black halo surrounds
each rodlike structure, indicative of a softer polymeric shell
covering the peptide nanotube core (Figure 3 a).
To demonstrate that the polymer is covalently bound to
the peptides, control experiments were performed by mixing
free PNIPAM polymer in solution with similar peptide
nanotubes that lack initiator groups at the surface (Figure 4 a). In this case the purely physisorbed macromolecules
can be completely removed through simple solvent extraction. AFM analysis demonstrates that the unmodified peptide
nanotubes can be recovered after solvent extraction. This
indicates that the nanotubes described above consist of an
assembly of covalently linked peptide–polymer hybrid molecules.
In summary, the strategy introduced herein for the design
and preparation of functional peptide–polymer hybrid nanostructures allows the preparation of a novel class of nanotubes
whose structure is governed primarily through the biomolecular component (peptide), and whose surface chemistry can
be adjusted by the grafted synthetic polymer. AFM analysis
demonstrates that the PNIPAM polymeric shell coats the
peptide nanotube core in a highly homogeneous manner, and,
as it is covalently attached to the cyclic peptide subunits, it
cannot be removed by simple solvent extraction. Beside the
peptide core, the internal structure of the polymeric shell is an
Angew. Chem. Int. Ed. 2005, 44, 3297 –3301
Figure 3. Analysis of the peptide–PNIPAM nanotubes: a) AFM phase
image of cyclic-peptide nanotubes adsorbed on a silicon wafer (scale:
2 mm); an enlarged area of the phase image is shown (scale: 0.8 mm);
b) AFM image (scale: 2 mm; height 50 nm); c) relative abundance A as
a function of the length l and d) height h as determined by statistical
analysis of the of the nanotubes; e) AFM image (scale: 0.5 mm, height:
25 nm) and f) cross-section analysis along the marked positions in
part e) of a cyclic-peptide–PNIPAM nanotube.
important factor that governs the structure of this novel class
of polymeric hybrid materials. Presumably, the molar mass
and the conformation of the peptide-bound polymer chains
can be controlled by adjusting the appropriate polymerization
conditions. Additionally, the graft density may be controlled
www.angewandte.org
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
3299
Communications
Figure 4. a) Schematic image of a cyclic peptide (CPB) that was synthesized for control experiments. It was allowed to self-assemble to form similar peptide nanotubes, which were then mixed with free PNIPAM polymer, and adsorbed to the surface of a silicon wafer, and analyzed by AFM
both before and after extraction with water. b) AFM image (size: 5 mm, height: 35 nm) and cross-section analysis of polymer–CPB nanotubes
before and c) after extraction with water.
by the dilution of initiator sites; systematic studies of these
issues will be reported in forthcoming communications.
Notably, one would expect neighboring chains to repel each
other more strongly with an increase in the molecular mass
and graft density of the polymers. If such forces were to
overcome those that hold the cyclic-peptide subunits
together, the point at which self-assembled peptide–polymer
nanotubes break up into shorter aggregates may be determined. This may become a way to gain control of the length of
such hybrid nanotubes.
determined by HPLC, was 0.7. At this point, the polymer-modified
peptide nanotubes were still in a dispersed phase (the solution
maintained a hazy appearance); workup could therefore be done by
repeated centrifugations and extensive washings with cold water,
which ensures the removal of all physisorbed macromolecules from
the polymer–peptide hybrids.
The control experiment was carried out with peptide nanotubes
generated from a cyclic peptide that lacks polymerization initiation
groups at the surface. These reference nanotubes were mixed with
free PNIPAM polymer in aqueous dispersion and analyzed by AFM
without further solvent extraction. The aggregates were then
repeatedly washed with water, and analyzed again by AFM for any
remaining polymer physisorbed to the peptide nanotubes.
Experimental Section
Received: December 20, 2004
Published online: April 14, 2005
All cyclic peptides were prepared with standard Fmoc (Fmoc = 9fluorenylmethoxycarbonyl) chemistry protocols for solid-phase peptide synthesis. Cyclization of the linear peptide (HOOC-l-Asp(OAll)-[d-Ala-l-Lys(Mtt)]-d-Ala-NH2) and the attachment of functional groups (the ATRP initiators) were carried out directly on the
solid phase (All = allyl, Mtt = 4-methyltrityl). The cyclic peptides
were cleaved from the resin and purified by recrystallization from
TFA/water and preparative reversed-phase HPLC (C18, CH3CN/H2O
elution gradient). Characterization of the cyclic-peptide precursor
product was carried out with ESI MS, NMR spectroscopy, and FTIR
spectroscopy.[22]
Peptide nanotube surface-initiated ATRP reactions were carried
out in dispersed aqueous phase similar to a recently published
protocol by Kizhakkedathu and co-workers.[23] In brief, the monomer,
solvent, and HMTETA (HMTETA = 1,1,4,7,10,10-hexamethyltriethylenetetramine) were mixed and degassed by repeated freeze/
thaw cycles under vacuum. Subsequently, CuI, CuII, and Cu0 were
added under frozen conditions and the reaction mixture was allowed
to slowly warm to room temperature. The peptide initiator nanotubes
were dispersed in an aqueous solution and added to a polymerization
chamber. Polymerization was carried out at 20 1.0 8C under an
atmosphere of inert gas. After 90 min, the reaction was quenched by
addition of excess CuII and dilution with excess water. The molar ratio
of monomer/initiator was 200:1, and the monomer conversion, as
3300
2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
.
Keywords: nanotechnology · nanotubes · peptides · polymers ·
supramolecular chemistry
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