Synthesis and single-molecule studies of a well-defined biomimetic modular multidomain polymer using a peptidomimetic β-sheet module

Article abstract of DOI:10.1021/ja0448871

In the pursuit of advanced biomaterials with combined strength, toughness, and elasticity, a new class of well-defined modular polymers has been synthesized, and their nanomechanical properties have been studied using atomic force microscopy. These polymers are based on a peptidomimetic β-sheet-based double-closed loop (DCL) module, which was designed to overcome the limitation of the modular polymers we reported previously (J. Am. Chem. Soc. 2004, 126, 2059). Single-molecule force-extension experiments revealed the sequential unfolding of these modules as the polymer is stretched, resulting in more regular sawtooth-patterned curves similar to those seen in titin and other biopolymers. The single-molecule data agreed well with computer modeling, which suggested that hydrogen bonding and π-stacking are both involved in the formation of small DCL clusters along the polymer chain. Copyright

Full text of DOI:10.1021/ja0448871

Published on Web 10/19/2004
Synthesis and Single-Molecule Studies of a Well-Defined Biomimetic Modular
Multidomain Polymer Using a Peptidomimetic â-Sheet Module
Jason T. Roland and Zhibin Guan*
Department of Chemistry, UniVersity of California at IrVine, 516 Rowland Hall, IrVine, California 92697-2025
Received August 24, 2004; E-mail: zguan@uci.edu
One fundamental challenge in polymer science is to design
polymers that have a combination of mechanical strength, fracture
toughness, and elasticity.1 These properties require different mo-
lecular mechanisms and, hence, are generally considered exclusive
to each other.2 Nature, on the other hand, has evolved many
biopolymers, such as the skeletal muscle protein titin, cell adhesion
Figure 1. Schematic representation of domain unfolding of a modular
polymer composed of double-closed loop monomers.
proteins, and connective proteins in both soft and hard tissues, which
Scheme 1. Synthesis of the DCL Monomer by RCM
have a remarkable combination of these three important properties.3-7
Single-molecule nanomechanical studies have revealed that their
combined mechanical properties originate from their unique modular
multidomain structure, which appears to be a general mechanism
used in nature to achieve advanced materials.7 Inspired by nature,
we intend to mimic this modular domain design in the pursuit of
synthetic biomaterials with advanced properties. We have previously
reported the design of polymers containing folded loops held
by the strong hydrogen-bonding (H-bonding) motif, 2-ureido-4-
pyrimidone (UPy).8 Single-molecule force-extension studies re-
vealed the sequential unfolding of loops as a polymer chain was
stretched. The excellent correlation between the single-molecule
and the bulk properties demonstrated our biomimetic concept of
using a modular domain structure to achieve advanced polymer
properties.
While demonstrating our biomimetic concept, the UPy system
had a few limitations. The structure of the polymer had nonuni-
formity that arose from the polydispersed poly(tetramethylene
oxide) loop and the different enchainment of the UPy units (head-
to-head, head-to-tail, and tail-to-tail).8 The UPy units could also
Scheme 2. Structure of the DCL Modular Polymer
randomly bind to each other within a chain or between different
chains. Finally, the binding strength of UPy is not tunable. For
further exploration of modular biomimetic materials with high
strength and toughness, more uniform and higher-ordered polymer
systems would be desirable. Herein, we report the use of a
peptidomimetic â-sheet motif to construct a modular polymer that
has a better-defined structure (Figure 1). The module in this system
is composed of a â-sheet-like duplex that is connected at both ends
with hydrocarbon loops. As a module is stretched, the force will
shear the hydrogen bonds in the duplex, and the loops will be
extended. After releasing the force, the double-closed loop topology
should ensure the strands rebind to their original pairs.
We envision that this system should overcome the limitations
of our previously reported UPy system. First, the double-closed
loop (DCL) topology will enhance the possibility for each H-
bonding unit to bind to its original counterpart, therefore, minimiz-
ing dimerization of nonadjacent units on the same chain or from
different chains. Second, by choosing a monodispersed alkyl linker
in the monomer synthesis, the loop size is monodispersed. Last,
by choosing the sequence and adjusting the length, the binding
strength between â-sheets can be easily tuned.
tion of the DCL monomer began with the synthsis of the
self-complementary H-bonding dimer 1.11 Covalent capture of the
H-bonded dimer via ring-closing metathesis (RCM) provided the
protected DCL monomer as a mixture of E/Z double bond isomers.12
Hydrogenation reduced the double bonds and removed the benzyl
protecting groups simultaneously, affording the DCL monomer 2
(Scheme 1). Polymerization reactions were performed by mixing
equimolar amounts of the DCL monomer and 4,4’-methylenebis-
(phenyl-isocyanate) (MDI) in chloroform at 45 °C. GPC analysis
showed number average molecular weights for the DCL polymers
as high as 89 000 g/mol (Scheme 2).
The DCL polymers were subjected to single-molecule force-
extension experiments using atomic force microscopy (AFM)
following the literature protocols.8,13 Sawtooth patterns were
consistently observed in the force-extension curves and are similar
to those seen in both natural and synthetic modular polymers (Figure
Among the many peptidomimetic â-sheets reported in the
literature,9 we chose the quadruple H-bonding duplex developed
by Gong and co-workers for its simplicity in synthesis.10 Construc-
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C O M M U N I C A T I O N S
π-stacking interactions have been observed in other aromatic
oligomers and polymers.15-17 The association energy between the
four phenyl rings on two DCL modules is comparable to that for
the four hydrogen bonds within each DCL module. As the force
builds to the threshold, both the π-stacking and hydrogen bonding
within each cluster unfold simultaneously. Clusters with varying
number of DCL modules will gain different length upon unfolding,
resulting in a distribution of ∆L. Computer modeling also shows
that the large alkyl loops on both sides of a DCL module repel
each other due to sterics as DCL modules π-stack with each other,
making the smallest cluster formed between two DCL modules most
favorable (see Supporting Information). The unfolding of the
smallest cluster containing two DCL modules should gain 12 nm
in contour length, which agrees well with the most probable ∆L
value observed experimentally (Figure 2).
Figure 2. AFM single-molecule force-extension curve for DCL modular
polymer. The solid line is the fitting with WLC model at a 0.55 nm
persistence length (L is the contour length during stretching).
In conclusion, a new class of well-defined modular polymers
has been synthesized using a double-closed-loop peptidomimetic
â-sheet module. Single-molecule nanomechanical studies on these
polymers show more uniform sawtooth patterns, suggesting that
the DCL modular polymer undergoes sequential unfolding upon
stretching. Further studies toward the bulk mechanical properties
and the design of well-defined modules having varying H-binding
strength are currently underway in our laboratory.
2). The patterns in the force-extension curves were more uniform
for the DCL modular polymer than for the UPy system, which was
attributed to its more uniform structure. The number of peaks in
the stretching curves ranges from 2 to 7 with the most probable
range being 3-5. The chain detaches from the surface typically
after 60-120 nm stretching. The most probable peak force for
unfolding each module is ∼ 50 pN (see the histograms in the
Supporting Information). This force value is lower than that of our
UPy modular polymers which had an unfolding force of ∼100-
200 pN. This is consistent with the binding strengths of the two
modules: the dimerization constants (Kdim) measured in chloroform
for the UPy and the current peptidomimetic â-sheet units are ∼107
and 104, respectively.10,14
The consistent sawtooth pattern in the single-molecule force-
extension curves suggests the DCL monomer units unfold sequen-
tially as the polymer is stretched. As the AFM tip picks up a single
chain and is retracted from the surface, tension builds up on the
bridging polymer chain. Once the force builds up enough to unfold
one of the DCL units, a rupturing event occurs and the force drops.
This sequence of events can happen repeatedly as the polymer chain
is stretched, resulting in the sawtooth pattern seen in Figure 2.
Further evidence for single-chain stretching comes from fitting the
single-molecule data to the wormlike chain (WLC) model,5 which
is used to predict the relationship between the extension of a
polymer chain and its entropic restoring force. Our force-extension
data fit nicely to this model with a persistence length (b) of 0.55
nm (Figure 2). This value is reasonable as compared to the 0.4 nm
values for proteins.4 The persistence length for DCL polymer is
larger than for the UPy polymer or natural proteins because the
DCL polymer has a more rigid backbone than these systems.
Biological modular polymers such as titin show a constant
increase in contour length (∆L) between consecutive peaks in their
force-extension curves due to the sequential unfolding of identical
modules as the polymer is stretched.4 For DCL polymers, the ∆L
ranges from 5 to 21 nm with the most probable value of 12.5 nm
(see the histograms in the Supporting Information). We propose
that this nonconstant peak spacing is due to the formation of tertiary
structures by π-π stacking between adjacent DCL modules.
Computer modeling shows that the four phenyl rings on each DCL
module are coplanar. Strong π-π-stacking interactions between
phenyl rings on adjacent DCL modules drive them to form small
clusters along polymer chains (see Supporting Information). Similar
Acknowledgment. We are thankful for the financial support
from the Arnold and Mabel Beckman Foundation. We thank
Professor James Nowick for helpful discussion and Jane Bai for
the help on AFM experiments and WLC modeling.
Supporting Information Available: Synthesis and characterization.
This material is available free of charge via the Internet at http://
www.pubs.acs.org.
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