Cycloaddition-promoted self-assembly of a polymer into well-defined β sheets and hierarchical nanofibrils

Article abstract of DOI:10.1002/anie.200805009

(Chemical Equation Presented) "Click" to fold: Cu ^I-catalyzed azide-alkyne cycloaddition polymerization of a peptide monomer induced folding of the resultant polymer into well-defined β sheets, which further self-assemble into hierarchical nanofibrils. The antiparallel β-sheet structure was confirmed by several techniques. Scanning probe micrographs confirm the formation of hierarchical amyloid-like nanofibrils.

Full text of DOI:10.1002/anie.200805009

Angewandte
Chemie
DOI: 10.1002/anie.200805009
Cycloaddition Polymerization
Cycloaddition-Promoted Self-Assembly of a Polymer into Well-
Defined b Sheets and Hierarchical Nanofibrils**
Ting-Bin Yu, Jane Z. Bai, and Zhibin Guan*
Whereas biopolymers, such as proteins, are ubiquitous for
well-defined secondary, tertiary, and quaternary structures,[1]
it remains a fundamental challenge to design synthetic
polymers that can fold into predictable higher structures.
One major area of research in our laboratory is aimed at
incorporating weak forces into polymers to guide their
assembly into well-defined molecular structures and nano-
structures.[2] We reported biomimetic multidomain polymers
following modular design of titin.[3] In this study, our attention
was drawn to b-sheet polymers. Although significant progress
has been made in the area of designing discrete short
peptidomimetic oligomers with b-sheet structures,[4] the
design of synthetic high polymers that can fold into well-
defined b sheets and hierarchical nanostructures remains
largely illusive to materials chemists. The b sheet is not only
a basic secondary structure in proteins, but also an important
structural motif in many fibril biomaterials, such as amyloids[5]
and silks.[6] Their hierarchical nanostructures and excellent
mechanical properties have inspired biomimetic material
designs.[7] Even though a number of peptide-related systems
were reported to form b-sheet-based fibrils, most of them are
short peptides[8] or peptide–polymer conjugates.[9] The self-
assembly in these systems usually proceeds intermolecularly,
forming relatively weak structures. Genetically engineered
polypeptides, synthesized by recombinant DNA technology,
were reported to form b sheets and various other nano-
structures.[10] However, the efficiency and versatility of such
species are limited by the biosynthetic pathway. Both for
fundamental interest and for the design of advanced materi-
als, it is highly desirable to develop efficient syntheses to
access well-defined covalently bonded b-sheet polymers.
Herein we describe a new strategy to construct covalent
synthetic polymers that fold into well-defined b sheets and
further assemble into hierarchical nanofibrils (Figure 1).
Figure 1. Cycloaddition-induced folding and self-assembly: a) [2+3]
cycloaddition polymerization of a protected peptide monomer;
b) upon deprotection, polypeptides fold into well-defined antiparallel
b strands; c) self-assembly of multiple b sheets forms hierarchical
nanofibrils.
To this end, we employed copper(I)-catalyzed azide–
alkyne cycloaddition (CuAAC, “click” chemistry) for poly-
merization of a peptide monomer (Figure 1). CuAAC is a
versatile methodology because of its efficiency, functional-
group tolerance, and applicability to a wide range of
substrates.[11] Since initial reports of this method,[11a,b] the
reaction has been employed in a wide range of applications,
including selective ligation,[12] bioconjugation,[13] molecular
recognition,[14] and material and polymer synthesis.[15,16] Based
on structural similarities, 1,4- and 1,5-disubstituted 1,2,3-
triazole rings formed by azide–alkyne cycloaddition have
been used as biomimetic peptide surrogates[17] in a-helical
coils,[18] b strands,[19] b-turn mimics,[20,21] and prosthetic pro-
teins.[22] Despite these developments, it should be noted that
the work described here represents the first example of
applying this chemistry to induce high-order structure for-
mation in synthetic high polymers.
Our design is based on a convergent b-turn mimic that our
group recently developed, based on the CuAAC reaction. We
have shown that cycloaddition between two short peptide
strands terminated with azide and alkyne groups forms a 1,4-
disubstituted 1,2,3-triazole ring that induces b-turn forma-
tion.[21] 1H NMR and FTIR spectroscopies and molecular
mechanics calculations revealed that three-carbon linkers for
1,4-disubstituted triazole are optimal for the formation of the
b-turn structure in nonprotic media. We reasoned that, if an
AB peptide monomer was prepared (A = azide, B = acety-
lene), [2+3] dipolar cycloaddition would not only effect
efficient polymerization of the monomer, but should also
[*] T. Yu, J. Z. Bai, Prof. Dr. Z. Guan
Department of Chemistry, University of California, Irvine
Irvine, CA 92697-2025 (USA)
Fax: (+1)949-824-2210
E-mail: zguan@uci.edu
Homepage: http://chem.ps.uci.edu/~zguan
[**] We acknowledge financial support from the National Institutes of
Health (R01EB004936) and the Department of Energy (DE-FG02-
04ER46162). We thank Prof. Alexander McPherson in the Depart-
ment of Molecular Biology & Biochemistry for assistance with AFM,
Prof. James Nowick in the Department of Chemistry for assistance
with HPLC, Dr. Jian-Guo Zheng at the Materials Characterization
Center for assistance with TEM, and Dr. Wytze Van der Veer, Dr Phil
Dennison, and Dr. John Greaves in the Department of Chemistry for
assistance with CD, NMR spectroscopy, and MS. We also thank Dr.
Youli Li at the Materials Research Lab of UCSB for WXRD
instrument assistance.
Supporting Information for this article (including the syntheses and
characterization, NMR, HRMS, GPC, FTIR, CD, WXRD, TEM, and
AFM experiments) is available on the WWW under http://dx.doi.
org/10.1002/anie.200805009.
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Communications
form b turns to induce folding into
antiparallel b strands. Essentially,
the cycloaddition polymerization
induces folding of an encoded poly-
mer into extensive b sheets which
further self-assemble into nanofi-
brils (Figure 1).
For our initial concept demon-
stration, we installed alkyne and
azide moieties onto a simple alanyl-
glycine (AG)3 hexapeptide as the
monomer unit (6, Scheme 1)
because repeated AG units are a
common motif for antiparallel b-
sheet formation in silk[6] and biosyn-
thetic polypeptides.[10a,c] Based on
our previous model studies,[21]
three-carbon linkers were chosen to
attach alkyne and azide to the C and
N termini of the monomer 6 to
maximize b-turn formation. One
challenge in the synthesis and study
of well-defined b-sheet systems is
their generally poor solubility. Pro-
tecting strategies and switch-peptide
concepts have been utilized to cir-
cumvent this problem.[9a] We intro-
duced an acid-cleavable 2,4-dime-
thoxybenzyl (DMB) protecting
group onto one amide group, to
prevent premature aggregation
during the polymerization and facil-
itate polymer processing and char-
acterizations. DMB has widely been
used in peptide synthesis to inhibit
excessive hydrogen bonding.[23]
Removal of the DMB protecting
Scheme 1. Synthesis of the b-sheet mimic polypeptide 8. Reaction conditions: a) Phthalimide,
K2CO3, DMF, 708C, 24 h, (96%); b) hydrazine hydrate, EtOH, 708C, 2 h, (74%); c) Boc–glycine,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), 1-hydroxybenzotriazole
(HOBt), iPr2EtN, CH2Cl2, room temperature, 12 h, (98%); d) trifluoroacetic acid (TFA), CH2Cl2,
room temperature, 3 h; e) Boc–alanine, EDC, HOBt, iPr2EtN, CH2Cl2, room temperature, 12 h,
(90%); f) TFA, CH2Cl2, room temperature, 3 h; g) 2,4-dimethoxybenzaldehyde, NaCNBH3, MeOH,
room temperature, 12 h, (83%); (h) 7, HATU, iPr2EtN, 95:5 DMF/DMSO, 48 h, (46%); i) CuOAc
(2 mol%), DMF, 808C, 2 h, (85%); j) TFA, CH2Cl2, 2 h, room temperature (86%).
group triggers intramolecular folding and intermolecular
self-assembly (Figure 1b).
Monomer 6 was then subjected to [2+3] cycloaddition
polymerization using a modified procedure[16] to afford
The peptide monomer was prepared by combining
standard solution- and solid-phase peptide-coupling reactions
(Scheme 1). The preparation of the acetylene half of the
monomer (4) began with Gabriel synthesis of 5-amino-1-
pentyne followed by solution coupling to Boc-glycine (Boc =
tert-butoxycarbonyl), to give Boc-Gly-pentyne 2. Boc-depro-
tection of 2 followed by coupling with Boc-alanine afforded 3.
Following Boc removal, a DMB protecting group was
installed by reductive amination to give the DMB-protected
Ala-Gly-pentyne 4. The azide half of the monomer (azido-
Ala-Gly-Ala-Gly-OH, 5) was prepared by solid-phase pep-
tide synthesis using 2-chlorotrityl chloride resin and standard
Fmoc protocols.[24] The final step, coupling of the secondary
amine in 4 with the terminal carboxylic acid in 5, was effected
by using o-(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetramethyl-
uronium hexafluorophosphate (HATU) as the coupling
reagent in 95:5 DMF/DMSO (Scheme 1). Monomer 6 was
purified by column chromatography and characterized by
1H NMR, 13C NMR, and FTIR spectroscopy, HRMS, and
analytical HPLC.
polymer
7
with an Mn of 11500 gmolÀ1, Mw of
21800 gmolÀ1, and polydispersity index (PDI) of 1.89, deter-
mined by gel permeation chromatography (GPC) using
poly(ethylene glycol) (PEG) as a molecular-weight standard.
The structure of polymer 7 was confirmed by NMR and FTIR
spectroscopy. The FTIR spectrum of 7 showed a sharp signal
at around 2100 cmÀ1, corresponding to the azide functionality,
indicating that polymer 7 still carries active azide end groups,
amenable for further reaction. DMB protecting groups
successfully prevent premature aggregation to render poly-
mer 7 fully soluble in polar organic solvents, such as DMFand
2,2,2-trifluoroethanol (TFE), and enable the preparation of a
soluble and processable pre-polymer with encoded informa-
tion for subsequent folding and self-assembly. Upon complete
removal of the DMB groups in a 1:5 (v/v) TFA/methanol
1
mixture (confirmed by H NMR spectroscopy, see Figure S1
in the Supporting Information), the resulting polymer 8
folded and self-assembled into nanofibrils (Figure 1).
The b-sheet structure of polymer 8 was subsequently
confirmed by FTIR and far-UV circular dichroism (CD)
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spectroscopy, and wide-angle powder X-ray diffraction
(WXRD) (Figure 2). The FTIR spectrum (Figure 2a) of the
protected polymer 7 shows a strong amide I band at around
Figure 2. a) FTIR spectra of 7 (left) and 8 (right) in amide I,II band
region; b) wide-angle powder X-ray diffraction pattern of polymer 8,
with d spacings of major reflections highlighted.
1655 cmÀ1 and amide II band at around 1541 cmÀ1, character-
istic of a random coil conformation.[25] Upon removal of the
DMB protecting groups and the resultant nanofibril forma-
tion, polypeptide 8 exhibits a significant enhancement of the
amide I band at around 1628 cmÀ1 and amide II band at
around 1533 cmÀ1, indicating a b-sheet conformation.[25]
Additionally, a weak component at around 1695 cmÀ1 is
indicative of antiparallel b-sheet structures.[25] The CD
spectrum of 8 in hexafluoroisopropanol (HFIP) solution
exhibits a minimum at 206 nm and a maximum at 195 nm (see
Figure S2 in the Supporting Information), confirming the b-
sheet conformation, as b-sheet-forming poly(AG)3YG or
poly(AG)3HG (YG: tyrosine-glycine, HG: histidine-glycine),
made by genetic processes, gave rise to similar CD spectra.[10]
In addition, the wide-angle powder X-ray diffraction
(WXRD) pattern of polymer 8 reveals major reflections at
d spacings of 4.57, 4.22, and 3.64 ꢀ (see Figure 2b), which are
similar to those observed for the antiparallel b-sheet d spac-
ings reported for unoriented silk fibroin film[26] and biosyn-
thetic poly(AG).[10a]
Transmission electron microscopy (TEM) and atomic
force microscopy (AFM) were used to directly visualize the
self-assembled nanostructures. For TEM studies, polymer 8
nanofibril suspensions were deposited on a carbon-coated
grid, and a 2% uranyl acetate stain was used to increase edge
contrast of the nanofibrils. Indeed, TEM images show
assemblies of polymer 8 in long linear nanofibrils (Figure 3c).
Examination of the micrograph at higher magnification
indicates that the nanofibrils are composed of stacks of
molecular fibrils formed from the b-sheet polymer. The
average width of the molecular fibril was determined to be
(3.8 Æ 0.4) nm (see Figure S4 in the Supporting Information),
which is in good agreement with the estimated width of a
single b sheet (Figure 4). The average width of the nanofibrils
was found to be around 32 nm, indicating an average of
approximately eight b sheets stacked laterally in one nano-
fibril. To investigate further the fibrillar morphology and
texture of b-sheet fibrils, we analyzed the fibril dimensions by
AFM for samples spin-coated on mica surfaces. AFM images
Figure 3. a) A representative AFM micrograph for nanofibrils of 8;
b) height profile for five nanofibrils across the white line in (a); c) a
representative TEM image of nanofibrils of 8; d) a high-magnification
view of one nanofibril.
Figure 4. Proposed model for hierarchical self-assembly of 8 to form
nanofibrils: a) folding in an antiparallel single b sheet; b) face-to-face
stacking of a double layer of two b sheets; c) a single b sheet; d) face-
to-face stacked double layer of two b sheets; e) stacking of many
double layers forms the hierarchical nanofibrils.
(Figure 3a and S6 in Supporting Information) revealed a
similar morphology for the nanofibrils as shown by TEM. In
addition, AFM analysis indicated the height of the nanofibrils
in the range of 1.7–7 nm, with the most probable height (5.0 Æ
0.5) nm (Figure 3b and Figure S8 in the Supporting Informa-
tion). Both TEM and AFM images clearly demonstrated that
the folded b sheets of polymer 8 further self-assemble
intermolecularly into amyloid-like nanofibrils.
On the basis of the TEM and AFM results and previous
models,[10a,c] we propose a model for the hierarchical self-
assembly of polymer 8 into extensive b sheets and nanofibrils
(Figure 4). In previous studies of poly(AG) systems,[10a,c] all
alanine methyl groups were oriented towards the same face
and the b sheets were arranged in such a way that two alike
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A. F. M. Kilbinger, Angew. Chem. 2007, 119, 8484 – 8490; Angew.
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surfaces were in contact. We assume that our system adopts
the same conformation. The polymer 8 first folds into
individual antiparallel b sheets (Figure 4a,c), which then
stack face-to-face into bilayers (Figure 4b,d), which assemble
both horizontally and vertically into nanofibrils (Figure 4e).
The b strands run perpendicular to the fibril axis, resembling
the cross-b structure of amyloid fibrils.[5] This model agrees
with our experimental data. The width of individual molec-
ular fibrils, (3.8 Æ 0.4) nm, is consistent with the width of the
b strand estimated from the model (approximately 3.7 nm).
Nanofibril heights, measured by AFM, fell within the range
1.7–7 nm (in agreement with the stacking of one to four layers
of b-sheet bilayers), with the most probable height at (5.0 Æ
0.5) nm (three layers). Owing to the polydispersity of polymer
8, the longitudinal length of b sheets varies, resulting in
interdigitation of different b-sheet bilayers (Figure 4d,e).
In summary, we have described herein the first example of
a synthetic polymer that can fold into well-defined b sheets
and further self-assemble into hierarchical nanostructures.
The DMB-protected polymer was efficiently synthesized by
copper(I)-catalyzed azide–alkyne cycloaddition. Upon
removal of DMB, the polymer 8 folded into a well-defined
b-sheet structure, confirmed by FTIR and CD spectroscopies
and WXRD data. TEM and AFM images show that the
b sheets further assemble into hierarchical amyloid-like
nanofibrils. A key design element here is that the [2+3]
dipolar cycloaddition not merely serves as the polymerization
method, but also induces folding and self-assembly of the
resultant polymer, demonstrating a unique example in which
polymerization leads to intramolecular folding to form a
secondary structure (b sheet) and further intermolecular
organization into hierarchical nanostructures. The efficiency
and versatility of the click chemistry should allow for further
design of more complex polymer materials. Studies are under
way to combine the b-sheet motif with other folding motifs for
the design of novel hierarchical biomaterials with advanced
physical properties and specific functions on the nanometer
scale.
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Received: October 13, 2008
Published online: December 29, 2008
Keywords: cycloaddition · nanostructures · peptides · polymers ·
.
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