Aqueous Self-Assembly of Unsymmetric Peptide Bolaamphiphiles into Nanofibers with Hydrophilic Cores and Surfaces

Article abstract of DOI:10.1021/ja035882r

Unsymmetric peptide bolaamphiphiles that incorporate (L-glutamyl)₃glycine at one terminus and either tetraethylene glycol or aspartic acid at the other were found to form hydrogels at low wt %, presumably by self-assembling into nanofibers pres

Full text of DOI:10.1021/ja035882r

Published on Web 09/27/2003
Aqueous Self-Assembly of Unsymmetric Peptide Bolaamphiphiles into
Nanofibers with Hydrophilic Cores and Surfaces
Randal C. Claussen, Bryan M. Rabatic, and Samuel I. Stupp*
Department of Chemistry, Department of Materials Science and Engineering and the Feinberg School of Medicine,
Northwestern UniVersity, EVanston, Illinois 60208
Received April 30, 2003; E-mail: s-stupp@northwestern.edu
Natural proteins and peptides adopt well-defined secondary
structures such as R-helices and â-sheets through an interplay of
hydrophobic collapse, hydrogen bonding, and electrostatic and van
der Waal interactions.1 Using these same forces, artificial peptides2-4
and peptide amphiphiles5-7 (PAs) have been used to form novel
nanostructures. For example, peptide nanotubes are formed through
sheetlike hydrogen bonding between cyclic D,L-peptides.2 PAs with
lipid tails have been shown to induce formation of stable triple-
helices7 and also nanofibers containing parallel â-sheets5,6 and
hydrophobic cores. Bolaamphiphiles, molecules containing two
hydrophilic headgroups linked by a hydrophobic spacer, are
receiving increased attention as building blocks for structures such
as membranes, fibers, tubes, ribbons, and ropes.8-12 Several groups
have reported the self-assembly of unsymmetric bolaamphiphiles.11,12
Figure 1. Structure of peptide bolaamphiphiles 1 and 2 and control PA 3.
However, these molecules typically form lamellar or tubular
structures in water, and in one instance they create monolayer
Scheme 1. Synthesis of Amphiphile Building Blocks 6, 9, and 10
rods.12c We report here the aqueous self-assembly of unsymmetric
peptide bolaamphiphiles 1 and 2 (Figure 1) to give nanofibers with
hydrophilic cores and surfaces, in our efforts to control the core
chemistry within nanofibers.
Bolaamphiphiles 1 and 2 contain headgroups (L-glutamyl)3glycine
and tetraethylene glycol (EO4) or succinyl-L-aspartic acid separated
by a hydrophobic segment based on â-alanine (â-Ala), p-amino-
benzoic acid (PABA), 6-aminohexanoic acid, and L-leucine. Control
PA 3 is similar to 1 but lacks the second headgroup. These
amphiphiles were synthesized by Fmoc solid phase peptide synthesis
from commercially available amino acids with the exception of
building blocks 6, 9, and 10, which were prepared as outlined in
Scheme 1. Dipeptide 6 is a semirigid â-peptide-like building block
that combines the stiffness of PABA with the reactivity of the
aliphatic amino group of â-alanine and can be readily prepared
through the temporary silyl ester protection of PABA.
of 1 negatively stained with a 2 wt % aqueous solution of
phosphotungstic acid shows one-dimensional objects that aggregate
along their long axis.14 Individual fiber components of these bundles
Self-assembly of the peptide amphiphiles was achieved by
are up to 5 nm in width, with lengths in excess of a micrometer.
exposing 1 wt % solutions in 0.1 N KOH or NH4OH to HCl vapors,
By preferentially staining the carboxylic acid groups of the
resulting in the formation of translucent, birefringent, self-supporting
(L-glutamyl)3glycine with 2 wt % uranyl acetate, TEM shows that
gels. The peptides require a base to be soluble in water, and gelation
the outer edge of individual fibers has higher contrast than the
occurs upon acidification to a pH of ca. 2.13 Self-healing and self-
interior (Figure 2a). This contrast is quantified by line profiles
supporting gels were formed from solutions of 1 as dilute as 0.5
wt %, whereas 2 and 3 gave weak gels at 0.5 wt %, and 3 partially
precipitated. The solutions of 1-3 at a pH of 8 and their gels at a
pH of 1 were analyzed by circular dichroism (CD) to show random
structure in the solutions becoming â-sheet structure after gelation.14
The solid-state FTIR amide I region of lyophylized 1 wt % gels
showed predominately â-sheet character with peaks at 1634-1641
cm-1, with possible random content at 1653-1662 cm-1. The
similarities between the amide I regions of 1 and 3 likely indicate
the adoption of similar conformations.14
(Figure 2a, inset) taken normal to the long axis of individual fibers.
Tilting experiments on the TEM samples support a fiber, not a flat
ribbon, morphology. Negatively stained TEM samples of 2 and 3
also show fiber morphologies, with diameters of 5 nm and 6-8
nm, respectively, where 3 gives shorter fibers than 1 or 2.14 Positive
staining of 2 with uranyl acetate for 1 h shows evidence for staining
at the cores and the peripheries of the fibers (Figure 2b).
FTIR and CD data indicate â-sheet formation among peptide
segments of the nanofibers. Because peptide segments can only be
parallel to each other along the fiber axis, the fiber axis must lie
within the plane of the â-sheets. Within these â-sheets, parallel
alignment of molecules is promoted by designing two hydrogen-
Self-assembled gels of 1 wt % were analyzed by transmission
electron microscopy (TEM). The bright field image of a gel sample
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J. AM. CHEM. SOC. 2003, 125, 12680-12681
10.1021/ja035882r CCC: $25.00 © 2003 American Chemical Society

C O M M U N I C A T I O N S
Figure 2. The TEM image of 1 positively stained with uranyl acetate (a) shows the preferential staining of fiber peripheries, with line profile inset, and the
TEM image of 2 positively stained with uranyl acetate (b) shows staining at both cores and peripheries, with line profile and high magnification insets. The
molecular graphics rendition of the cross section of the nanofibers of 1 (c) illustrates hydrophilic domains A and C separated by the hydrophobic section B.
Supporting Information Available: CD spectra for 1-3 in solution
and as gelled. Solid-state FTIR amide I and II spectra for 1-3. TEM
images of negatively stained 1-3. An additional molecular graphics
image. Experimental procedures for compounds 1-10 (PDF). This
material is available free of charge via the Internet at http://pubs.acs.org.
bonding patterns into the hydrophobic midsection. These patterns
lead to selective intermolecular hydrogen bonding of the R-amino
acid regions and the â-Ala-PABA regions, resulting in parallel
alignment of molecules along the length of the fiber. Even though
the wedge shape of the molecule may favor nanofiber formation,
we believe the driving force for self-assembly is â-sheet formation.
In Figure 2c, we schematically show the proposed arrangement of
amphiphiles viewed along the axis of the â-sheets. Additionally,
this representation illustrates the fact that the hydrophilic peptide
headgroup (L-glutamyl)3glycine is at the outer periphery and the
hydrophilic EO4 segment of 1 (or the aspartic acid of 2) is confined
to the core of the fiber.
These results show the self-assembly of unsymmetric peptide
bolaamphiphiles into cylindrical micelles that presumably bury one
headgroup in their core and present the other at the surface. We
believe this self-assembly is largely driven by the hydrogen-bonding
patterns that lead to sheet formation along the axis of the fiber.
Whereas we previously showed that the surface chemistry of the
nanofiber can be varied by using hydrophilic peptide epitope
sequences,5 here we demonstrate peptide-based bolaamphiphiles
self-assemble in water to form nanofibers with hydrophilic cores
as well as hydrophilic surfaces. These nanofibers could be used as
both bioactive structures as well as ion channels in biomedical
applications. Further research on their functionality is currently in
progress.
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Acknowledgment. This manuscript is based upon work sup-
ported by the U.S. Department of Energy (DOE) under Award No.
DE-FG02-00ER45810. We thank the Electron Probe Instrumenta-
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