Bioinspired modular synthesis of elastin-mimic polymers to probe the mechanism of elastin elasticity

Article abstract of DOI:10.1021/ja9104446

Bioinspired modular synthesis of elastin-mimic polymers (EMPs) is achieved via Cu-catalyzed alkyne-azide cyclization (CuAAC). By changing the module, EMPs with different secondary structures determined by circular dichroism (CD) spectra in trifluoroethanol (TFE) solution are obtained. The EMPs are characterized by measuring the lower critical solution temperatures (LSCTs) and the bulk mechanic properties under the conditions of both dry and hydrated forms. The unique molecular design enables us to probe mechanistic questions and assess the structure-property relationship of the EMPs. Our results indicate that, instead of a highly organized secondary structure, hydrophobic hydration is critical for the elasticity of EMPs.

Full text of DOI:10.1021/ja9104446

Published on Web 03/17/2010
Bioinspired Modular Synthesis of Elastin-Mimic Polymers To Probe the
Mechanism of Elastin Elasticity
Yulin Chen and Zhibin Guan*
Department of Chemistry, UniVersity of California, 1102 Natural Sciences 2, IrVine, California 92697-2025
Received December 10, 2009; E-mail: zguan@uci.edu
Elastin is an important extracellular matrix protein conferring
elasticity to tissues and organs.1 The native elastin isolated from
animal tissues has two domains, the repeating hydrophobic domain
and the alanine-rich cross-linking region.1 It is widely accepted
that the hydrophobic domain contributes to the elasticity of elastin.2
Because of its remarkable elasticity1 and stimuli-responsive proper-
ties,3 recently elastin and elastin-like polypeptides (ELPs) have been
actively pursued as biomaterials for various biomedical applications4
including protein purification,4 drug delivery,5 and tissue engineer-
ing.6 For many applications, it is critical to develop efficient and
scalable synthesis for ELPs.4,7,8 Herein we report a bioinspired
modular synthesis of elastin-mimic polymers (EMPs) via “click”
polymerization (Figure 1).
Taking advantage of the versatility of our modular synthesis,
Figure 1. Bioinspired modular synthesis of elastin-mimic polymers via
we further proposed to permute the peptide sequence of the module
to probe the mechanism of elasticity of elastin (Figure 1). Despite
many elegant studies, the mechanism of elastin elasticity remains
a subject of scientific debate with respect to the origin of elastomeric
storing force.2,9 Previous mechanistic studies of elastin elasticity
primarily focused on direct investigation of native elastin structures
or protein-based polypeptides by employing various physiochemical
methods.9 With the flexibility of our modular synthesis, we
envisioned a different approach to gain mechanistic understanding
through structural permutation and structure-property correlation.
A number of mechanisms have been proposed to account for
elastin elasticity. First, the “random” model9a treats elastin as a
random coil polymer with simple entropic elasticity like classical
rubbers. However, this model could not explain why elastin only
shows elasticity in its hydrated form. Second, the two-phase models,
including “liquid-drop”9b and “oiled-coil” models,9c propose that
elastin is composed of hydrophobic domains of protein with aqueous
diluents confined between them. However, this model is not
consistent with the highly dynamic nature of elastin chains.10
Finally, Urry et al. proposed a molecular model in which the
elasticity of elastin is attributed to a special “ꢀ-spiral” secondary
structure formed as a continuous coil of type II ꢀ-turns.11
Nevertheless, a number of studies have indicated that this model
has significantly overestimated the content of ꢀ-turns and long-
range order of elastin.12
“click” chemistry (top). Structures of pentapeptide monomers 1 (VPGVG),
2 (GVGVP), and 3 (VDPGVG) with the corresponding elastin-mimic
polymers P-1, P-2, and P-3 (bottom).
Figure 2. CD spectra of elastin-mimic polymers in trifluoroethanol (TFE)
at 25 °C: P-1, black; P-2, red; P-3, green.
to adopt similar local conformation (type II ꢀ-turns) as the natural
sequence. In P-2, the pentapeptide in the repeat unit is scrambled
to GVGVP. Based on previous studies, the VPG triad is the core
of the ꢀ-turn conformation.11a,b By disrupting the VPG triad
sequence, the local ꢀ-turn conformation should be perturbed. In
P-3, the D-proline residues are chosen to replace the L-proline
residues in P-1. It is well-known that D-proline can be used as a
turn-inducing moiety in peptides.13 The peptide sequence with
D-proline has the tendency to form a type II′ ꢀ-turn which results
from the restricted ꢁ (+60° ( 20°) of the D-proline residue.14
We choose Cu-catalyzed alkyne azide cyclization (CuAAC)
chemistry for polymerization because of its high efficiency and
excellent tolerance of functional groups.15 The pentapeptides were
capped with azide and alkyne terminals, which enables us to
efficiently make peptide polymers via CuAAC chemistry (Figure
1 and see Supporting Information for the procedure to synthesize
the monomers and polymers). The click polymerization was very
Given the highly dynamic nature10 and lack of long-range order12
in elastin, we hypothesize that it may not be critical to have a
continuous peptide sequence in the bioinspired design of EMPs.
Therefore, we propose a bioinspired modular synthesis of EMPs
in which short elastin repeating peptides (elastic motifs) are
efficiently polymerized by “click” chemistry (Figure 1). Specifically,
we design three EMPs, P-1, P-2 and P-3, inspired by one of the
most abundant repetitive sequences of elastin hydrophobic domain,
(VPGVG)n (Figure 1).2 The three polymers share an identical
chemical composition but have different peptide sequences or D/L-
proline stereochemistry. In P-1, the pentapeptide in the repeat unit
is identical to the canonical elastic module, VPGVG, and is expected
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C O M M U N I C A T I O N S
Figure 3. LCSTs of elastin-mimic polymers at a concentration of 3.0 mg/
mL in water: P-1, black; P-2, red; P-3, green.
Figure 4. True stress-strain curves for the elastin-mimic polymers in dry
and hydrated forms. The inset at the upper-right corner shows the curves
for dry films: (a) black, dry P-1; (b) red, dry P-2; (c) green, dry P-3. For
hydrated films: (d) blue, P-1 with 13% water; (e) cyan, P-2 with 13% water;
(f) magenta, P-3 with 13% water. (Percentage of water is defined by the
weight of absorbed water to the weight of the dry film).
efficient, affording polymers in high yields (93%-96%). As
measured by GPC using PEG standards, the polymers are of high
molecular weight with Mn ) 43, 26, and 21 kDa, for P-1, P-2, and
P-3, respectively. The chemical structures of the polymers were
1
confirmed by H and 13C NMR.
ELPs.3,12b Apparently, the introduction of triazole linkages onto
the peptide polymer backbone does not destroy the LCST behavior.
Compared to polypentapeptide (VPGVG)n, the slight decrease in
LCST transition temperature for P-1 and P-2 can be attributed to
the increased hydrophobicity due to the nonpeptido linkages. The
difference in LCST transition temperature for P-1, P-2, and P-3
suggests that the local peptide conformation can perturb their
interactions with water molecules.
To gain information of local secondary structures of the polymers,
circular dichroism (CD) spectra are collected for the three polymers
(Figure 2) and compared with the CD spectrum of polypentapeptide
(VPGVG)n reported in literature, which shows a positive π-π*
band at 206 nm and negative n-π* band at 224 nm in trifluoro-
ethanol (TFE) solution.16 Polymer P-1 shows a very similar CD
spectrum to that of the native polypentapeptide (VPGVG)n, implying
that polymer P-1 has similar local secondary structures to natural
(VPGVG)n. Thus the introduction of the triazole ring apparently
does not interrupt the inherent local conformation of the VPGVG
pentapeptide. This is not surprising because both molecular dynamic
simulation and the proposed ꢀ-spiral model indicate that each repeat
unit forms a ꢀ-turn independently with no specific interaction
between adjacent repeat units.11 Therefore, individual VPGVG
pentapeptide units in P-1 can still adopt a similar conformation as
in the natural system. In contrast, scrambling the pentapeptide
sequence to GVGVP results in a dramatic change in the CD
spectrum for polymer P-2. The CD spectrum for P-2 is nonchar-
acteristic with the negative peak at ∼195 nm suggesting a random
coil conformation in solution.17 This agrees with our hypothesis
that breaking the VPG triad core should disrupt the ꢀ-turn
conformation for the pentapeptide. A noticeable change is also seen
in the spectrum for polymer P-3 compared to P-1, but the curve
for P-3 still keeps the feature of a ꢀ-type secondary structure.
One important characteristic of elastin and ELPs is their
temperature responsive behavior in aqueous solution. For example,
polypentapeptide (VPGVG)n (n ) ∼200) has an LCST of ca. 25
°C at a concentration of ∼5 mg/mL in water.18 Since both the LCST
behavior and elasticity of elastin share the same entropic driving
force from interactions of water molecules with the hydrophobic
side chains,12 it would be important to find out if the bioinspired
polymers preserve the LCST behavior in water. We measured the
temperature responsive behavior for the EMPs in water (Figure 3);
excitingly, all polymers show elastin-like LCST behavior with
transition temperatures of 12, 24, and 32 °C for P-1, P-2, and P-3,
respectively. The LCST behavior for EMPs is due to temperature-
dependent water hydration of hydrophobic side chains. Below
LCST, water molecules form ordered clathrate-like structures around
the hydrophobic side chains. Above LCST, expulsion of water
molecules associated with the hydrophobic side chains is thermo-
dynamically favorable due to entropic gain, leading to hydrophobic
collapse and precipitation in a process analogous to that for other
One of the most important properties of elastin is its excellent
elasticity for fulfilling its biological functions.1 Despite great efforts
to elucidate the link between structure and property for elastin,9,11
the molecular mechanism for its elasticity remains controversial.
One major disagreement is about the contribution of the specific
peptide secondary structure to the macroscopic elasticity. Another
important factor is the role that water molecules play in the
elasticity. Recently, a number of new studies, including NMR
spectroscopy,12a,19 molecular dynamics simulations,12c and single
molecule force spectroscopy,20 have revealed the significance of
“hydrophobic hydration” for elastin elasticity.9d Indeed, dehydrated
elastin has the properties of a brittle polymer, while the hydrated
form is highly elastic, pointing to the profound importance of water
to elastin’s mechanical response.1b
With our EMPs sharing identical chemical composition but
having subtle differences in secondary structures, we reason that
investigation of their bulk mechanical properties should provide
useful insight to the origin of elasticity of elastin. Particularly, we
want to examine the importance of two factors, i.e., peptide local
conformation and hydration, on the polymer elasticity. For this
purpose, films were cast from the solution in methanol for P-1,
P-2, and P-3 and their tensile mechanical properties were tested in
both dry and hydrated forms. The dry films were immediately
subjected to mechanical testing after vacuum drying, while the
hydrated films were prepared by equilibrating the films in a closed
chamber saturated with water vapor for a controlled period before
testing immediately. Figure 4 shows the true stress-strain curves
of the elastin-mimic polymers in dry and hydrated forms. In the
dry form the films are brittle with relatively high moduli but very
small extensibility (inset in Figure 4). For example, the Young’s
moduli for dry samples are 2.80, 1.53, and 1.95 GPa for P-1, P-2,
and P-3, respectively, while the maximal strains are only ∼4%,
∼6%, and ∼4% for P-1, P-2, and P-3, respectively. Hydration of
the samples results in dramatic changes in mechanical properties.
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C O M M U N I C A T I O N S
The 13% hydrated films (w/w, weight of absorbed water to weight
of the dry film) clearly show that the hydrated films are much more
extensible. Young’s modulus decreases significantly with hydration:
∼200 MPa for polymers P-1 and P-2 with 13% of hydration.
Polymer P-3 is much softer after hydration. Young’s modulus is
∼50 MPa for the hydrated films of P-3. After yielding at 4-5%
strain, the hydrated samples undergo a large deformation. The films
can be pulled up to 3 times its length before rupturing for the
hydrated films of P-1 and P-3 (curves d and f in Figure 4). While
these samples are not recoverable because of the lack of cross-
links like those found in the noncross-linking ELPs, the transition
from brittle to highly extensible behavior is what one would expect
for a noncross-linked elastomeric polymer, as reported for native
and synthetic ELPs.21
While the EMPs in dry form are very stiff and nonextensible,
partial hydration (13 wt %) significantly lowers Young’s modulus
and dramatically increases their extensibility. Due to the lack of
any cross-linking, further hydration makes the samples too soft for
stress-strain analysis. Instead, the fully hydrated P-1 was charac-
terized by rheological analysis. The results show that it has typical
viscoelastic properties at 40 °C. Its storage modulus (G′ ≈ 5 MPa)
is close to the value of native elastin in fully hydrated form.22 The
relatively small tan δ value (∼0.1) for P-1 also strongly supports
that the gel has good elastomeric properties (see Figure S4 in
Supporting Information).
Two important observations are worth noting in the physical
property studies of the EMPs. First, despite their dramatic difference
in local secondary structure in solution (Figure 2), P-1, P-2, and
P-3 display similar behavior in LCST and mechanical performance,
suggesting that local secondary structure is not essential for elasticity
in EMPs. This agrees with the highly dynamic nature and lack of
long-range order for elastin. A recent investigation of (VPGVG)3
peptides by solid state NMR confirmed that an ensemble of dynamic
conformations coexists in solid state with only a minor fraction
existing in a compact ꢀ-turn conformation.12a Second, our results
confirm that hydration plays a critical role in the elasticity of elastin.
Similar to natural elastin, simple hydration converts P-1, P-2, and
P-3 from very brittle into very ductile. Presumably, solvation of
backbone amide bonds by water reduces main chain/main chain
hydrogen bonds, hence, making the polymer chains more dynamic.
In the meantime, the interaction of water molecules with hydro-
phobic side chains, i.e., hydrophobic hydration, provides the entropic
driving force for both the elasticity and LCST behavior observed.12,19
In summary, we have demonstrated a novel bioinspired synthesis
of EMPs. The unique molecular design enables us to probe
important mechanistic questions and assess the structure-property
relationship of EMPs. Our results indicate that polymer conforma-
tion is not essential for the elasticity of EMPs. Instead, our data
confirm that hydrophobic hydration, as opposed to an organized
secondary structure, plays a critical role for the elasticity. The
bioinspired polymers can be conveniently prepared through a
modular approach using “click chemistry”. Despite the introduction
of nonpeptido linkages, the bioinspired EMPs fully preserve critical
features of native elastin: the LCST behavior in aqueous solution
and high elasticity in bulk. The simple modular synthesis provides
an efficient approach to access a broad range of elastin-mimic
polymers for many potential biomaterials applications.
Acknowledgment. We thank the Department of Energy - Basic
Energy Sciences (DE-FG02-04ER46162) for the generous financial
support. We thank Prof. A. Summers for the use of the mechanical
test instrument, Dr. P. Dennison for the assistance with NMR
experiments, Dr. J. Greave for mass spectroscopy, Dr. W. van der
Veer for the help with CD and LCST experiments, and Aaron
Kushner for helpful discussions and manuscript revision.
Supporting Information Available: Experimental details of syn-
thesis and characterization of monomers and polymers. This material
is available free of charge via the Internet at http://pubs.acs.org.
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