Self-assembly of supramolecular polymers from β-strand peptidomimetic-poly(ethylene oxide) hybrids

Article abstract of DOI:10.1021/ma100444b

The use of hydrogen-bonding β-strand peptidomimetics for the preparation of supramolecular polymers is described here. The β-strand mimics were selected for their ability to form hydrogen bonds on only one face of the strand, allowing for controlled assem

Full text of DOI:10.1021/ma100444b

Macromolecules 2010, 43, 4453–4459 4453
DOI: 10.1021/ma100444b
Self-Assembly of Supramolecular Polymers from β-Strand
Peptidomimetic-Poly(ethylene oxide) Hybrids
Tayirjan T. Isimjan,† John R. de Bruyn,‡ and Elizabeth R. Gillies*,†,§
§
†Department of Chemistry, ‡Department of Physics and Astronomy, and Department of Chemical and
Biochemical Engineering, The University of Western Ontario, 1151 Richmond St., London, Canada N6A 5B7
Received May 27, 2009; Revised Manuscript Received April 14, 2010
ABSTRACT: The use of hydrogen-bonding β-strand peptidomimetics for the preparation of supramole-
cular polymers is described here. The β-strand mimics were selected for their ability to form hydrogen bonds
on only one face of the strand, allowing for controlled assembly into linear polymers. Alkyne-functionalized
peptidomimetics with the capacity to form either four, six, or eight self-complementary hydrogen bonds were
synthesized and conjugated to both termini of low molecular weight (MW) R,ω-diazidopoly(ethylene glycol).
The assembly of these polymers into higher MW supramolecular polymers was investigated by multiangle
light scattering, dynamic light scattering, and circular dichroism. It was found that the eight-hydrogen-
bonding system was required for the significant formation of high MW assemblies and that the degree of
assembly was dependent on the polymer concentration as well as the solvent. Thus, these peptidomimetics
provide a new platform for the development of supramolecular polymers with the promise to tune their
properties using functionalities on the amino acid side chains.
Introduction
based on amyloid-β,37 silk-based,38 or de novo amino acid
sequences.39-42 These structures have generally been demon-
In natural systems, noncovalent intramolecular and intermo-
lecular interactions are exploited to impart biomaterials with
exceptional properties. For example, in dragline silk relatively
weak intermolecular forces drive the assembly of nanocomposites
composed of β-sheet nanocrystals embedded in an amorphous
matrix, imparting silk with exceptional strength, toughness,
and elasticity.1 In recent years there have been significant ad-
vances in the development of synthetic polymer systems that
exploit noncovalent interactions in order to modulate the
properties of materials.2,3 Noncovalent interactions such as
metal-ligand coordination,4-8 hydrogen bonding,9-13 π-π
strated to assemble into fibrils based on β-sheets. Most amino
acid sequences are not amenable to the preparation of supramo-
lecular linear polymers due to their propensity to hydrogen bond
on both edges of the strand, leading to fibers or uncontrolled
aggregation.
With the aim of forming high MW linear polymers in solution,
we report here for the first time the use of a β-strand peptidomi-
metic that is designed to undergo hydrogen bonding on only one
edge. Oligomers based on alternating R-amino acids and azacy-
clohexenone (Ach) units, developed and termed @-tides by
Bartlett and co-workers, were selected as the hydrogen-bonding
β-strand peptidomimetics for this work.43 The replacement of
alternating amino acids with the Ach units provides conforma-
tional restriction that favors elongated conformations, and the
tertiary amide limits hydrogen bonding to one edge of the
strand. Phillips et al. have reported detailed studies to demon-
strate that @-tides of various lengths and compositions assemble
in organic andaqueous solutionstoform β-sheets.43,44 The design
strategy for this work involved the coupling of two @-tide
molecules to the termini of relatively low MW (∼3400 g/mol)
PEO in order to induce the formation of higher MW polymers as
shown inFigure 1. PEO was selected dueto the ease withwhich its
end groups can be derivatized as well as its high solubility in a
wide range of aqueous and organic systems. In contrast to the
more conventional hydrogen-bonding systems that have pre-
viously been used for the preparation of linear supramolecular
polymers,9-13 this system provides the advantage that the length of
the peptidomimetic can readily be tuned to control the strength of
the association, and the amino acid side chains have the potential
to later introduce chemical functionalities to the polymers for a
diverse range of properties and applications. Furthermore, the use
of peptide based hydrogen-bonding moieties provides the potential
for the eventual formation of supramolecular polymers in water.
The syntheses of polymers comprising varying numbers of hydro-
gen-bonding groups, and the characterization of their assemblies
stacking,14-17 inclusion complexes,18-22 or
a combination
of these interactions23-28 have all been explored for the develop-
ment of supramolecular polymers. Through this work, useful pro-
perties such as temperature-dependent physical properties11,29,30
and self-healing capabilities31,32 have been introduced to synthetic
polymer systems. In biomedical applications, supramolecular
polymers have been used to form hydrogels that effectively direct
the growth of cells,33,34 strong sacs for the encapsulation of human
stem cells for cell therapy,35 and tissue engineering scaffolds with
tunable mechanical and cell adhesive properties.36
Of the available noncovalent interactions used in nature,
hydrogen bonding is of particular interest for the development
supramolecular polymers. It combines reversibility, directiona-
lity, excellent strength depending on the surrounding environ-
ment and can be readily tuned by altering the number of bonds
involved. While the development of synthetic hydrogen-bonding
systems that operate in aqueous solution is still a challenge,
hydrogen-bonding systems are common in nature, existing in
DNA as well as in protein β-sheets. Despite this, there are
relatively few examples involving the use of β-sheets and their
peptidomimetics in supramolecular polymers. Several poly-
(ethylene oxide) (PEO)-peptide hybrids have been developed
*Corresponding author. E-mail: egillie@uwo.ca.
r 2010 American Chemical Society
Published on Web 04/29/2010
pubs.acs.org/Macromolecules

4454 Macromolecules, Vol. 43, No. 10, 2010
Isimjan et al.
Figure 1. Schematic of supramolecular polymer formation.
by static and dynamic light scattering as well as circular dichroism
(CD) spectroscopy is described here.
Dimer Methyl Ester (3). Compound 2 (1.0 g, 2.9 mmol, 1.0
equiv) was dissolved in 80 mL of THF, and 8 mL of H2O was
added. The solution was adjusted to pH 7 with 8 mL of 20%
Cs2CO3 and evaporated to dryness to give the cesium salt of
the acid. This salt was then stirred with methyl iodide (0.13 mL,
3.5 mmol, 1.2 equiv) in 25 mL of DMF for 1 h. The solvent was
then removed, and the resulting mixture was purified by silica
gel chromatography using EtOAc/hexanes 2/1 as an eluent to
provide 0.95 g (93%) of 3 as a white solid. 1H NMR (400 MHz,
CDCl3): δ 0.90 (d, J = 6.8, 3H), 0.95 (d, J = 6.8, 3H), 2.07-2.15
(m, 1H) 3.68 (s, 3H), 3.84 (dd, J = 8.1, 6.0, 1H), 4.01 (dd, J =
16.0, 32, 1H), 4.28 (dd, J = 16.2, 40, 1H), 5.09 (s, 2H), 5.16 (s,
1H), 6.45 (br s, 1H), 7.21-7.32 (m, 5H). 13C NMR (100 MHz,
CDCl3): δ 18.6, 31.1, 44.1, 50.6, 52.3, 60.9, 67.7, 95.3, 127.8,
128.5, 135.9, 154.9, 161.2, 171.6, 191.0. MS calcd for [M]þ
(C19H24N2O5): 360.2. Found (ESþ): 359.9.
Experimental Section
Materials and General Procedures. Solvents were purchased
from Caledon Laboratories (Georgetown, ON). All other che-
micals were purchased from Sigma-Aldrich (Milwaukee, WI).
Unless noted otherwise, all chemicals were used as received.
All dry solvents were obtained from a solvent purification
system. Column chromatography was performed using silica
gel (0.063-0.200 mm particle size, 70-230 mesh). 1H NMR
data were obtained at 400 or 600 MHz, and 13C NMR data were
obtained at 100 or 150 MHz. All chemical shifts are reported in
ppm and are calibrated against residual solvent signals of CDCl3
(δ 7.26, 77.2) or CD3OD (δ 3.31, 48.9). All coupling constants
(J) are reported in hertz. Mass spectrometry data were ob-
tained using a Finnigan MAT 8200 instrument in TOF ESþ
mode. HPLC was performed at a flow rate of 1 mL/min on a
Waters 2695 separations module with a Waters 2998 photodiode
array detector at a wavelength of 285 nm. A Luna C18 3 μm
(150 mm ꢀ 4.6 mm) column from Phenomenex equipped with
the corresponding guard column was used. The HPLC gra-
dient was linear from either 20/80 or 30/70 MeCN/H2O to 95/5
MeCN/H2O over 20 min. Solvent mixtures for all chromato-
graphic analyses contained 0.1% TFA. Circular dichroism (CD)
spectroscopy was performed at a concentration of 0.25 mM
using a Jasco J-810 spectrometer.
Dimer Amine (4). To a solution of compound 3 (0.40 g,
1.1 mmol) in 20 mL of MeOH was added 10 wt % Pd/C (40 mg).
The mixture was stirred under H2 in a Parr shaker apparatus at a
pressure of 30 psi overnight. The resulting mixture was filtered
through Celite, and the filtrate was concentrated in vacuo to
provide a quantitative recovery of the product, which was taken
to the next step without further purification.
Tetramer (5). The amine 4 (65 mg, 0.29 mmol. 1.0 equiv), the
acid 2 (0.11 g, 0.32 mmol, 1.1 equiv), O-(7-azabenzotriazol-1-yl)-
N,N,N0,N0-tetramethyluronium hexafluorophosphate (HATU)
(0.12 g, 0.32 mmol, 1.1 equiv), and diisopropylethylamine
(DIPEA) (60 μL, 0.35 mmol, 1.2 equiv) were dissolved in 3 mL
of DMF, and the reaction mixture was stirred at room tempera-
ture overnight. The reaction mixture was then diluted with
H2O and was extracted with EtOAc. The organic layers were
combined, dried over MgSO4, and evaporated in vacuo. The
resulting mixture was purified by silica gel chromatography
using a gradient from CH2Cl2/MeOH 96/4 to CH2Cl2/MeOH
94/6 as an eluent to provide 0.13 g (81%) of 5 as a pale yellow
solid. 1H NMR (400 MHz, CDCl3): δ 0.86-1.02 (m, 12H),
2.01-2.06 (m, 1H), 2.10-2.21 (m, 1H), 3.77 (s, 3H), 3.85-
4.39 (m, 8H), 4.65 (d, J = 8.0, 1H), 4.85 (d, J = 8.0, 1H), 5.16
(s, 1H), 5.17 (s, 2H), 5.39 (s, 1H), 6.29 (br s, 1H), 6.99 (br s, 1H),
7.27-7.37 (m, 5H). 13C NMR (100 MHz, CDCl3): δ 17.91, 18.6,
18.7, 19.1 31.3, 32.5, 42.9, 44.4, 52.4, 54.8, 56.6, 61.1, 61.3, 67.7,
95.0, 94.7, 127.7, 127.9, 128.2, 128.5, 135.8, 155.1, 162.0, 162.1,
170.1, 171.6, 189.3, 191.2. MS calcd for [M]þ (C29H38N4O7):
554.3. Found (ESþ): 554.3. HPLC tR 8.7 min (gradient from
20/80 MeCN/H2O).
Dimer Acid (2). Compound 145 (1.0 g, 4.1 mmol, 1.0 equiv) and
L-valine (0.54, 4.1 mmol, 1.0 mmol) were dissolved in 40 mL of
MeOH, and the reaction mixture was heated at 60 °C overnight.
The solution was then concentrated, redissolved in EtOAc/
MeOH, and basified with 1 M NaOH (20 mL). The organic layer
was separated and washed with 1 M NaOH (3 ꢀ 10 mL). The
aqueous layers were combined and acidified with 1 M KHSO4.
The resulting solution was extracted with EtOAc. The organic
layers were combined, dried over MgSO4, and concentrated in vacuo
1
generating 0.80 g (57%) of 2 as a pale yellow solid. H NMR
(400 MHz, CDCl3/CD3OD (2/1)): δ 0.97 (dd, 6H, J = 13.1, 6.5),
2.06-2.20 (m, 1H), 3.67-3.73 (m, 1H), 3.94-4.12 (m, 2H),
4.23-4.43 (m, 2H), 5.11 (s, 2H), 5.15 (s, 1H), 7.26-7.40 (m,
5H). 13C NMR (100 MHz, CDCl3): δ 18.4, 18.7, 30.5, 43.9, 50.0,
61.4, 67.8, 94.5, 127.8, 128.2, 128.4, 135.7, 154.8, 163.0, 173.2,
192.1. MS calcd for [M]þ (C18H22N2O5): 346.2. Found (ESþ):
345.9. HPLC tR 4.2 min (gradient from 30/70 MeCN/H2O).

Article
Tetramer Acid (6). Tetramer 5 (0.10 g, 0.18 mmol, 1.0 equiv)
was dissolved in 2 mL of THF/H2O 1/1, and LiOH (15 mg,
0.36 mmol, 2.0 equiv) was added. The reaction mixture was
stirred at room temperature for 1 h, then was diluted with 1 M
KHSO4, and extracted with EtOAc. The organic layer was dried
over MgSO4, filtered, and concentrated in vacuo to provide
82 mg (82.0%) of 6 as a glassy solid. This product was taken to
the next step without further purification.
Macromolecules, Vol. 43, No. 10, 2010 4455
in vacuo. The product was purified by silica gel chromatography
using a gradient from CH2Cl2/MeOH 95/5 to CH2Cl2/MeOH
90/10 to provide 0.11 g (77% yield) of 12 as a pale yellow
solid. NMR spectra are complicated by rotamers. 1H NMR: δ
0.77-1.09 (m, 24H), 1.96-2.22 (m, 4H), 3.62-4.72 (m, 21H),
4.95-5.82 (m, 8H), 7.05 (br s, 1H), 7.21-7.37 (m, 5H), 7.45 (br s,
1H), 7.63 (br s, 2H). MS calcd for [M þ Na]þ (C49H66N8NaO11):
965.5. Found (ESþ): 965.5. HPLC tR 8.5 min (gradient from
20/80 MeCN/H2O).
Tetramer Propargylamide (7). The acid 6 (0.21 g, 0.39 mmol,
1.0 equiv), propargylamine hydrochloride (33 mg, 0.78 mmol,
2.0 equiv), HATU (0.27 g, 0.78 mmol, 2.0 equiv), and DIPEA
(0.14 mL, 0.78 mmol, 2.0 equiv) were dissolved in 10 mL of
DMF, and the reaction mixture was stirred at 0 °C for 3 h. The
reaction mixture was then diluted with H2O (60 mL) and was
extracted with EtOAc. The organic layers were combined, dried
over MgSO4, filtered, and evaporated in vacuo. The product
was purified by silica gel chromatography using EtOAc/MeOH
95/5 as the eluent to provide 0.12 g (54%) of 7 as a pale yellow
solid. NMR spectra are complicated by rotamers. 1H NMR
(400 MHz, CDCl3): δ 0.75-1.10 (m, 12H), 1.85-2.25 (m, 3H),
3.75-5.20 (m, 14H), 5.70-5.95 (m, 2H), 7.10-7.40 (m, 5H),
8.42 (br s, 1H), 8.64 (br s, 1H). MS calcd for [M þ H]þ (C31H40-
N5O6): 578.3. Found (ESþ): 578.4. HPLC tR 7.9 min (gradient
from 30/70 MeCN/H2O).
Octamer Acid (13). The same procedure as described above
for the preparation of the tetramer acid 6 was used to provide a
64% recovery of the product, which was taken to the next step
without further purification.
Octamer Propargylamide (14). The acid 13 (45 mg, 50 μmol,
1.0 equiv), propargylamine hydrochloride (6.0 mg, 0.14 mmol,
2.8 equiv), HATU (26 mg, 75 μmol, 1.5 equiv), and DIPEA
(30 μL, 0.15 mmol, 3.0 equiv) were dissolved in 2 mL of DMF,
and the reaction mixture was stirred at 0 °C for 3 h. The reaction
mixture was then diluted with H2O and was extracted with
EtOAc. The organic layers were combined, dried over MgSO4,
filtered, and evaporated in vacuo. The product was purified
by silica gel chromatography using a gradient from CH2Cl2/
MeOH 95/5 to CH2Cl2/MeOH 90/10 to provide 40 mg (88%) of
14 as a pale yellow solid. NMR spectra are complicated by
rotamers. MS calcd for [M þ Na]þ (C51H67N9NaO10): 988.5.
Found (ESþ): 989.5. HPLC tR 6.2 min (gradient from 30/70
MeCN/H2O).
Tetramer-PEO-Tetramer (15). R,ω-Diazidopoly(ethylene
glycol),46 MW ∼3400 g/mol (0.14 g, 41 μmol, 1.0 equiv), and
the alkyne 7 (58 mg, 0.10 mmol, 2.4 equiv) were dissolved in
2 mL of tBuOH/H20 1/1, and the solution was degassed using
the freeze-pump-thaw technique. To the solution were added
sodium ascorbate (2.0 mg, 10 μmol, 0.25 equiv) and CuSO4
(1.2 mg, 7.5 μmol, 0.18 equiv), and the reaction mixture was
stirred under nitrogen at room temperature overnight. 0.2 mL of
28% aqueous NH4OH was added, then the solution was eva-
porated in vacuo. The product was purified by silica gel chro-
matography using a gradient from EtOAc/MeOH 95/5 to
CH2Cl2/MeOH 90/10 to CH2Cl2/MeOH 50/50 as the eluent to
provide 0.13 g (74%) of 15 as a white solid. MS calcd for [M þ
Na]þ based on conjugation to the starting polymer N3CH2-
(CH2OCH2)nCH2N3 with a peak MW of 3370 g/mol (n = 74):
4548. Found (MALDI-TOFþ): 4550. HPLC tR 6.9 min
(gradient from 30/70 MeCN/H2O).
Hexamer-PEO-Hexamer (16). The same procedure as de-
scribed above for the preparation of polymer 15 was used to
provide 70 mg (75%) of polymer 16 as a white solid. MS calcd
for [M þ Na]þ based on conjugation to the starting polymer
N3CH2(CH2OCH2)nCH2N3 with a peak MW of 3370 g/mol
(n = 74): 4936. Found (MALDI-TOFþ): 4937. HPLC tR
6.8 min (gradient from 30/70 MeCN/H2O).
Octamer-PEO-Octamer (17). The same procedure as de-
scribed above for the preparation of polymer 15 was used to
provide 50 mg (60%) of polymer 17 as a white solid. MS calcd
for [M þ Na]þ based on conjugation to the starting polymer
N3CH2(CH2OCH2)nCH2N3 with a peak MW of 3370 g/mol
(n = 74): 5324. Found (MALDI-TOFþ): 5327. HPLC tR
6.8 min (gradient from 30/70 MeCN/H2O).
Tetramer Amine (8). The same procedure as described above
for the preparation of the dimer amine 4 was used to provide a
quantitative recovery of the product, which was taken to the
next step without further purification.
Hexamer (9). The amine 8 (50 mg, 0.12 mmol, 1.0 equiv), the
acid 2 (39 mg, 0.16 mmol, 1.5 equiv), HATU (56 mg, 0.16 mmol,
1.5 equiv), and DIPEA (50 μL, 0.24 mmol, 2.0 equiv) were
dissolved in 2 mL of DMF, and the reaction mixture was stirred
at room temperature overnight. The reaction mixture was then
diluted with H2O and was extracted with EtOAc. The organic
layers were combined, dried over MgSO4, filtered, and evapo-
rated. The product was purified by silica gel chromatography
using a gradient from CH2Cl2/MeOH 95/5 to CH2Cl2/MeOH 90/
10toprovide 54 mg(70%) of9 as a yellowsolid. NMRspectra are
complicated by rotamers. 1H NMR: δ 0.76-1.05 (m, 18H),
1.95-2.20 (m, 3H), 3.65-4.60 (m, 18H), 4.90-5.55 (m, 5H),
7.11 (br s, 1H), 7.22-7.28 (m, 5H), 7.52 (br s, 1H), 7.61 (br s, 1H).
MS calcd for [M þ H]þ (C39H53N6O9): 749.4. Found (ESþ):
750.4. HPLC tR 6.8 min (gradient from 30/70 MeCN/H2O).
Hexamer Acid (10). The same procedure as described above
for the preparation of the tetramer acid 6 was used to provide a
quantitative recovery of the product, which was taken to the
next step without further purification.
Hexamer Propargylamide (11). The acid 10 (50 mg, 80 μmol,
1.0 equiv), propargylamine hydrochloride (10 mg, 0.24 mmol,
3.0 equiv), HATU (42 mg, 0.12 mmol, 1.5 equiv), and DIPEA
(0.05 mL, 0.24 mmol, 3.0 equiv) were dissolved in 2 mL of DMF,
and the reaction mixture was stirred at 0 °C for 3 h. The reaction
mixture was then diluted with H2O and was extracted with
EtOAc. The organic layers were combined, dried over MgSO4,
filtered, and evaporated in vacuo. The product was purified by
silica gel chromatography using a gradient from CH2Cl2/MeOH
95/5 to CH2Cl2/MeOH 90/10 as the eluent to provide 37 mg
(70%) of 11 as a pale yellow solid. NMR spectra are complicated
1
Multiangle Light Scattering Analyses. Polymers 15, 16, and 17
were dissolved in spectral grade CHCl3 at a concentration of
1 mM. Polymer 17 was also analyzed at 0.5 mM. The samples
were filtered through a 0.2 μm PTFE filter into an injection loop
of ∼0.9 mL, and care was taken to void the loop of air bubbles.
The samples were then injected at a flow rate of 1 mL/min
through a miniDAWN Treos detector (Wyatt Technology,
Santa Barbara, CA). Each sample was injected at least three
times. The data were analyzed using Astra software (Wyatt)
using the following equation47
by rotamers. H NMR: δ 0.76-1.11 (m, 18H), 1.91-2.20 (m,
4H), 3.74-5.22 (m, 19H), 5.60-6.02 (m, 3H), 7.14-7.31 (m,
5H), 7.41 (br s, 1H), 7.61 (br s, 1H), 7.75 (br s, 1H). MS calcd for
[M þ H]þ (C41H54N7O8): 772.4. Found (ESþ): 772.4. HPLC tR
6.2 min (gradient from 20/80 MeCN/H2O).
Octamer (12). The amine 8 (82 mg, 0.20 mmol, 1.1 equiv), acid
6 (95 mg, 0.18 mmol, 1.0 equiv), HATU (85 g, 0.22 mmol, 1.2
equiv), and DIPEA (40 μL, 0.22 mmol, 1.2 equiv) were dissolved
in 3 mL of DMF, and the reaction mixture was stirred at room
temperature overnight. The reaction mixture was then diluted
with H2O and was extracted with EtOAc. The organic layers
were combined, dried over MgSO4, filtered, and evaporated
ꢁ
RðθÞ ¼ K McPðθÞ½1 - 2A2McPðθÞꢂ
ð1Þ

4456 Macromolecules, Vol. 43, No. 10, 2010
Isimjan et al.
a regularized inverse Laplace transform method,48 as discussed
in more detail below. The autocorrelation functions are shown
in Figure S21 to illustrate the reproducibility of the measure-
ments and the differences in correlation times between the
polymers. An example illustrating the quality of the fits to the
data produced by the data analysis is shown in Figure S22.
where R(θ)=excess Rayleigh ratio, the ratio of scattered and
incident light intensity (corrected for scattering volume
and distance from scattering volume), K*=(4πn0
4
2/NAλ0 )(dn/
dc)2, n0 =solvent refractive index, NA =Avogadro’s number,
λ0 = vacuum wavelength of incident light, dn/dc = specific
refractive index of the material, M = molar mass, c = solute
concentration (g/mL), P(θ)=scattering function which relates
the angular variation in scattering intensity to the mean square
radius of the particle, and A2 = second viral coefficient which is
a measure of solute-solvent interactions (usually very small).
A dn/dc of 0.135 for PEO in CHCl3 was used, and the reported
errors on the measurements were generated in the software
based on the fits. The Debye plots are shown in Figures 2,
S18, S19, and S20.
Results and Discussion
Syntheses of @-Tide-PEO Hybrids. In order to conjugate
the @-tides to the termini of PEO in high yield, a Cu(I)-
catalyzed “click” cycloaddition reaction was selected. Thus,
R,ω-diazidopoly(ethylene glycol) was prepared as previously
reported.46 It was determined by matrix-assisted laser de-
sorption ionization (MALDI) MS that this material had a
peak MW of 3370 g/mol. @-Tides with carboxy-terminal
propargylamides were prepared as the complementary click
reaction partners. L-Valine was selected as the amino acid
component of the @-tides due to its propensity to favor
β-sheet formation,49 and the target molecules were prepared
by a convergent solution phase synthesis based on modifica-
tions to the previously reported @-tide synthesis.45
As shown in Scheme 1, the previously reported benzyl
carbamate (Cbz) protected @-tide unit 1 was reacted with
L-valine to provide the corresponding condensation pro-
duct 2 which will be referred to as a dimer. This dimer
was converted to the corresponding methyl ester 3 by reac-
tion with Cs2CO3, followed by methyl iodide. After removal
of the Cbz group by catalytic hydrogenolysis in methanol,
the resulting amine 4 was coupled with the acid 2 using
O-(7-azabenzotriazol-1-yl)-N,N,N0,N0-tetramethyluronium
hexafluorophosphate (HATU) in diisopropylethylamine
(DIPEA) and DMF to provide the tetramer 5. The methyl
ester of 5 could be hydrolyzed to the acid 6 by treatment with
LiOH, followed by coupling with propargylamine hydro-
chloride to provide the amide 7. Alternatively, the Cbz group
could be removed by catalytic hydrogenolysis to give the
amine 8. For the preparation of longer hydrogen-bonding
oligomers, as shown in Scheme 2, the tetramer amine 8 was
coupled to the dimer acid 2 to provide the hexamer, while
coupling of 8 with the tetramer acid 6 gave the octamer. The
hexamer and octamer were functionalized to form the re-
spective propargylamides 11 and 14 using the same proce-
dures described above.
Dynamic Light Scattering Analyses. Polymers 16 and 17 were
dissolved in 2 mL of spectral grade CHCl3 at a concentration of
2 mM and were filtered through a 0.2 μm filter into a quartz
cuvette. The measurements were taken using a Zetasizer Nano
ZS from Malvern Instruments. Multiple runs on each sample
were performed to check the reproducibility of the measure-
ments. To investigate the effect of MeOH on the hydrodynamic
diameter, aliquots of filtered MeOH were added to provide
solutions containing 2 or 5% MeOH in CHCl3. The autocorre-
lation functions measured by the Zetasizer were analyzed using
Figure 2. Multiangle light scattering Debye plot for polymer 17 at a
concentration of 1 mM in CHCl3 (K*, c, θ, and R(θ) are defined in the
Experimental Section).
Molecules up to the tetramer length (5) were characterized
by the standard methods used for small molecules, including
Scheme 1

Article
Macromolecules, Vol. 43, No. 10, 2010 4457
Scheme 2
Figure 3. Mean hydrodynamic radii of scatterers determined from
dynamic light scattering measurements on polymers 16 and 17 in CHCl3
and polymer 17 in CHCl3 þ MeOH. The error bars indicate the
standard deviation of the peak in the distribution due to the larger
scatterers and give a measure of polydispersity. The corresponding
width for the smaller scatterers is approximately the size of the symbols.
Scheme 3
associated with the low scattering intensities, but these
measurements indicate that there was not a significant degree
of supramolecular assembly of these polymers. In contrast,
at 1 mM, polymer 17 exhibited much greater scattered light
intensities, resulting in much greater signal-to-noise, and an
Mw of 61 200 ( 1300 g/mol was calculated. The Debye plot
for polymer 17 is shown in Figure 2. This indicates that
polymer 17, containing octameric @-tides with the capacity
to form eight hydrogen bonds, is capable of assembling to
form higher MW polymers. In addition, when MALS ex-
periments were performed on polymer 17 at a concentration
of 0.5 mM, a decreased Mw of 33 500 ( 1800 g/mol was
observed. This result indicates that the polymerization is
concentration dependent, a result that is expected for an
assembly based on noncovalent intermolecular bonding
between the monomers.3
1H NMR, 13C NMR, high performance liquid chromatog-
raphy (HPLC), and mass spectrometry (HRMS). All char-
acterization data were consistent with the proposed struc-
tures and high purities. Because of the increasing broadness
and complexity of the NMR spectra with increasing length
and solubility constraints, the propargyl amides as well as the
hexamers and octamers were characterized mainly by HPLC
and MS, techniques that are standard for the characteriza-
tion of oligopeptides, along with 1H NMR spectroscopy.
As shown in Scheme 3, the propargylamide-functionalized
@-tides 7, 11, and 14 were then coupled to the PEO under
click chemistry conditions consisting of CuSO4 and sodium
ascorbate in a mixture of THF/H2O to provide the corre-
sponding @-tide functionalized PEOs 15, 16, and 17. The
purity of these final @-tide-PEO hybrids was critical to
their abilities to assemble as free @-tide impurities would
inhibit the assembly, while monofunctionalized PEOs would
provide end-caps, thus reducing the MWs of the assemblies.
Therefore, following purification, HPLC was used to con-
firm the absence of excess @-tide or other impurities. In
addition, MALDI MS was used to investigate the MWs of
the hybrids, and it was found that the peaks corresponding to
the mass of the starting PEO with two @-tide units added
were found in each case.
Characterization of the Assemblies by Light Scattering.
The assembly of polymers based on the tetramer, hexamer,
and octamer (15, 16, and 17, respectively) into higher MW
polymers via hydrogen bonding of the @-tide units into
β-sheet mimics was investigated by light scattering. Multi-
angle light scattering (MALS) was used to determine the
weight-average molecular weights (Mws) for the polymers in
CHCl3. At a concentration of 1 mM, polymers 15 and 16
exhibited low scattered light intensities, and Mws of 11 610 (
8700 and 7100 ( 2200 g/mol were calculated.47 The errors on
these measurements are high due to the low signal-to-noise
The assembly of polymers 16 and 17 was also investigated
by dynamic light scattering (DLS) at a concentration of
2 mM in CHCl3. The Zetasizer measures the intensity auto-
correlation function g(2)(τ) as a function of the delay time τ.
These data were converted to the field autocorrelation func-
tion g(1)(τ) using the Siegert relation.50 g(1)(τ) decays with
time as a result of the diffusion of the scatterers in the solvent.
We determined the distribution of hydrodynamic radii of the
scatterers using an implementation of the “CONTIN”
method48,51 written in MATLAB.52 A typical size distribu-
tion is shown in Figure S23. In all cases, the distributions
displayed two prominent peaks, indicating the presence of
two populations of scatterers with different characteristic
sizes. Figure 3 shows the mean hydrodynamic radii of the
scatterers and the widths (standard deviations) of the peaks
determined for the two polymers. For both polymers, the
field autocorrelation function had a rapidly decaying com-
ponent corresponding to scatterers with a hydrodynamic
radius r1 of ∼3.4 ( 1.2 nm, likely corresponding to unas-
sembled polymer molecules, and a slower component of the
decay due to larger particles which we assume to be supra-
molecular assemblies. These larger scatterers had a hydro-
dynamic radius r2 of about 40 ( 13 nm. For polymer 16, the
two peaks in the size distribution are similar in strength, but
the large-radius peak is much stronger for polymer 17. The
magnitude of the scattered electric field increases as the cube
of the size of the scatterer. Taking the ratio of the areas of
the peaks in the size distribution and dividing by (r2/r1)3 thus
gives an estimate of the relative number of scatterers of each
size. This quantity is plotted in Figure 4, which shows that,
while there is a small number of higher MW assemblies
formed through hydrogen-bond-mediated assembly of poly-
mer 16, the relative number of large assemblies is about
20 times higher in polymer 17. Assuming that the larger
scatterers are uniform-density assemblies of the individual

4458 Macromolecules, Vol. 43, No. 10, 2010
Isimjan et al.
Figure 4. Ratio N2/N1 of number of large and small scatterers, respec-
tively, estimated from the DLS data. Data are for polymers 16 and 17 in
CHCl3 and polymer 17 in CHCl3 þ MeOH.
polymer molecules, our results suggest that roughly 98% of
the molecules of 17 are assembled into larger aggregates,
compared to only 45% for 16. Although a hydrodynamic
radius around 40 nm still seems somewhat large for the Mw
of 61 200 ( 1300 g/mol measured by MALS, these DLS
experiments were carried out at a 2-fold higher concentra-
tion, which would be expected to lead to higher MW
assemblies due to the concentration-dependent nature of
the assembly.
The effect of methanol addition on the assemblies formed
by polymer 17 was also investigated by dynamic light scatter-
ing. When 2% or 5% methanol was added to the chloroform
solution, two populations of scatterers were still observed,
as indicated in Figure 3. The hydrodynamic radii of these
scatterers remained approximately the same as in the absence
of methanol, but the relative number of large assemblies
decreased substantially, as seen in Figure 4. These data are
consistent with the dissociation of the high MW polymer
assemblies due to the fact that MeOH is a competitive
hydrogen bond donor and acceptor.
Characterization of the Assemblies by Circular Dichroism
Spectroscopy. In order to further probe the nature of the
polymer assemblies, circular dichroism (CD) spectroscopy
was performed on polymers 16 and 17. It has previously been
demonstrated by Bartlett et al. that the assembly of @-tides
into β-sheet-like structures led to a characteristic signal in the
CD spectrum near 280 nm, attributable to the vinylogous
amide absorbance.44 The addition of methanol to @-tide
solutions led to a significant attenuation of this signal as the
oligomers became unstructured. As shown in Figure 5, at a
concentration of 0.25 mM in CHCl3, polymer 16 provided a
weak signal near 280 nm. Upon the addition of 5% MeOH,
the signal was attenuated to only a small degree. This result
supports the light scattering data above, suggesting that this
polymer does not assemble to a great extent. In contrast, at
the same concentration polymer 17 exhibited a stronger peak
near 280 nm, and this peak was significantly attenuated when
5% MeOH was added. These results are important as they
suggest that the assemblies observed by light scattering were
not randomly structured aggregates, but rather polymer
assemblies formed at least predominantly by well-defined
and structured β-sheet mimics. While the formation of
branched structures formed by imperfect hydrogen bonding
cannot be excluded, it is unlikely due to the short lengths of
the @-tides and their relatively rigid structures.
Figure 5. CD spectra of polymers 16 and 17 in pure CHCl3 or 5%
MeOH in CHCl3.
werereported to have Kds on the order of 10-4 M in CDCl3, so
only a small degree of association was expected. This is
consistent with the light scattering and CD spectroscopy
results. On the other hand, @-tides capable of forming eight
hydrogen bonds had Kds too large to measure in pure CHCl3
but were reported to assemble with Kds on the order of
10-3 M, even in 15% CD3OH in CDCl3. Therefore, as
expected, the minimum @-tide length required for a signifi-
cant degree of assembly in this study was the octamer. Itis also
possible that the PEO chains inhibit the degree of assembly to
some degree due to their ability to backfold and stabilize the
unimers via competing intramolecular hydrogen bonding.53
In order to obtain assemblies at lower concentrations or to
obtain higher MW assemblies, it will be necessary to prepare
and conjugate longer @-tides. In addition, the effects of the
methanol addition on the assemblies indicate that to obtain
stableassemblies in water for biomedicalapplications itwillbe
necessary to protect the hydrogen-bonding units from water
by encapsulating them within the environment of a hydro-
phobic polymer, an approach that has previously been suc-
cessful with supramolecular polymers based on the ureido-
pyrimidinone unit.36
Conclusions
In conclusion, alkyne-functionalized @-tide β-strand peptido-
mimetics of varying lengths were successfully synthesized and
conjugated to PEO functionalized with azides at each terminus.
Unlike previous examples of PEO-β-strand hybrids, hydrogen
bonding in the @-tides was limited to one edge of the strand,
making the formation of linear polymers rather than fibrillar
structures possible. The assembly of the resulting low MW
polymers into higher MW polymers was investigated by MALS,
DLS, and CD spectroscopy. It was found that @-tides capable of
forming a minimum of eight hydrogen bonds were necessary for
the significant formation of high MW polymers in CHCl3, and
the sizes of the assemblies was sensitive to both concentration and
the presence of MeOH in the solution. CD spectroscopy results
supported the proposed mode of assembly via the formation of
β-sheet mimics from the @-tide β-strand mimics. This study thus
provides the first groundwork for the use of β-strand peptido-
mimetics for the development of supramolecular linear polymers
and suggests that the strength of the association can be tuned by
the length of the @-tide, an aspect that is not readily accessible in
other hydrogen-bonding moieties. Upon further development,
the potential utility of the amino acid side chains to introduce
Comparison of the Results with @-Tide Dissociation Con-
stants. The results observed for the assembly of polymers 15,
16, and 17 can be compared with the dissociation constants
previously reported for the @-tide oligomers.43 @-Tides
capable of forming only four hydrogen bonds were reported
to exhibit dissociation constants (Kds) on the order of 10-2
M
in 1% CD3OH in CDCl3, so the formation of supramolecular
assemblies was not expected for polymer 15 at the concentra-
tions tested. Systems capable of forming six hydrogen bonds

Article
Macromolecules, Vol. 43, No. 10, 2010 4459
€
chemically and biologically active functionalities makes these
materials highly promising for the development of new functional
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Acknowledgment. The authors thank the Natural Sciences
and Engineering Research Council of Canada and the Canada
Research Chairs Program for support of the research.
Supporting Information Available: HPLC chromatograms/
NMR spectra of compounds 2, 3, 5, 7, 9, 11, 12, 14, 15, 16, and 17
as proof of purity, Debye plots for polymers 15, 16, and 17,
correlograms for polymers 16 and 17, fit of eq 1 to DLS data.
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