Hydrogelation and self-assembly of Fmoc-tripeptides: Unexpected influence of sequence on self-assembled fibril structure, and hydrogel modulus and anisotropy

Article abstract of DOI:10.1021/la903678e

The self-assembly and hydrogelation properties of two Fmoc-tripeptides [Fmoc = N-(fluorenyl-9-methoxycarbonyl)] are investigated, in borate buffer and other basic solutions. A remarkable difference in self-assembly properties is observed comparing Fmoc-VLK(Boc) with Fmoc-K(Boc)LV, both containing K protected by N^ε-tert-butyloxycarbonate (Boc). In borate buffer, the former peptide forms highly anisotropic fibrils which show local alignment, and the hydrogels show flow-aligning properties. In contrast, Fmoc-K(Boc)LV forms highly branched fibrils that produce isotropic hydrogels with a much higher modulus (G′ > 10⁴ Pa), and lower concentration for hydrogel formation. The distinct self-assembled structures are ascribed to conformational differences, as revealed by secondary structure probes (CD, FTIR, Raman spectroscopy) and X-ray diffraction. Fmoc-VLK(Boc) forms well-defined β-sheets with a cross-β X-ray diffraction pattern, whereas Fmoc-KLV(Boc) forms unoriented assemblies with multiple stacked sheets. Interchange of the K and V residues when inverting the tripeptide sequence thus leads to substantial differences in self-assembled structures, suggesting a promising approach to control hydrogel properties.

Full text of DOI:10.1021/la903678e

pubs.acs.org/Langmuir
©
2010 American Chemical Society
Hydrogelation and Self-Assembly of Fmoc-Tripeptides: Unexpected
Influence of Sequence on Self-Assembled Fibril Structure, and
Hydrogel Modulus and Anisotropy
G. Cheng, V. Castelletto, C. M. Moulton, G. E. Newby, and I. W. Hamley*
Department of Chemistry, University of Reading, Reading RG6 6AD, United Kingdom
Received September 29, 2009. Revised Manuscript Received December 1, 2009
The self-assembly and hydrogelation properties of two Fmoc-tripeptides [Fmoc=N-(fluorenyl-9-methoxycarbonyl)]
are investigated, in borate buffer and other basic solutions. A remarkable difference in self-assembly properties is
ε
observed comparing Fmoc-VLK(Boc) with Fmoc-K(Boc)LV, both containing K protected by N -tert-butyloxycarbo-
nate (Boc). In borate buffer, the former peptide forms highly anisotropic fibrils which show local alignment, and the
hydrogels show flow-aligning properties. In contrast, Fmoc-K(Boc)LV forms highly branched fibrils that produce
0
4
isotropic hydrogels with a much higher modulus (G > 10 Pa), and lower concentration for hydrogel formation.
The distinct self-assembled structures are ascribed to conformational differences, as revealed by secondary structure
probes (CD, FTIR, Raman spectroscopy) and X-ray diffraction. Fmoc-VLK(Boc) forms well-defined β-sheets with a
cross-β X-ray diffraction pattern, whereas Fmoc-KLV(Boc) forms unoriented assemblies with multiple stacked sheets.
Interchange of the K and V residues when inverting the tripeptide sequence thus leads to substantial differences in self-
assembled structures, suggesting a promising approach to control hydrogel properties.
1
3
Introduction
(vanocomycin) binding. Fmoc-dipeptides containing non-
natural residues have recently been investigated by the Xu group
due to their enhanced biostability, that is, potential for use in
Short peptides are attracting great interest as low molecular
1
,2
weight gelators, in particular hydrogelators. Molecules such as
dipeptide derivatives are attractive for the development of novel
biomaterials, and have been demonstrated to be usable as scaf-
folds for cell growth with potential applications in tissue engi-
1
4
controlled drug release. Enzyme-catalyzed formation of hydro-
9
,15
gels based on Fmoc-peptides has been reported.
The forma-
tion of hydrogels from other short peptides with bulky aromatic
end units (e.g., naphthalene) in response to enzyme has recently
been investigated. The peptide was a tripeptide comprising a
sequence of two β-amino acids (providing enhanced biostability)
and an R-amino acid (tyrosine phosphate), responsive to a phos-
phatase enzyme. An assay for enzyme inhibitors based on a
smaller Fmoc-amino acid (Fmoc-tyrosine phosphate) was also
reported, relying on hydrogelation.
the Fmoc-peptides studied by Xu and co-workers are based on
superhelical arrangements of the aromatic residues, possibly
1
,3-6
neering.
The self-assembly and hydrogelation of Fmoc-dipeptides
Fmoc = N-(fluorenyl-9-methoxycarbonyl)] and dipeptides con-
jugated to other bulky aromatic units such as naphthalene have
been investigated in detail by the groups of Xu, Ulijn,
[
1
6
7
,8
2,4-6,9
1
0
11
Gazit, and others. Even Fmoc-amino acids such as Fmoc-
phenylalanine or Fmoc-tyrosine are able to form hydrogels in
7
,17
The hydrogels formed by
1
2
appropriate acidic media. Responsive materials have been
developed that undergo sol-gel transitions in response to ligand
8
,15
driven by π-π* stacking interactions.
Ulijn and co-workers
have, in a similar vein, investigated the enzymatic hydrogelation
and dehydrogelation of Fmoc-dipeptides using subtilisin to
hydrolyze C-terminal esters and thermolysin to induce reverse
hydrolysis (linking an Fmoc-amino acid with amino acid methyl
esters).
There are few studies on Fmoc-tripeptides. Ulijn and co-
*
To whom correspondence should be addressed. E-mail: I.W.Hamley@
reading.ac.uk. Also at Diamond Light Source, Harwell Science and Innova-
tion Campus, Didcot OX11 0DE, U.K.
(
1) Xu, B. Langmuir 2009, 25(15), 8375–8377.
(
2) Mart, R. J.; Osborne, R. D.; Stevens, M. M.; Ulijn, R. V. Soft Matter 2006, 2,
1
8
8
22–835.
3) Yang, Z.; Xu, K.; Wang, L.; Gu, H.; Wei, H.; Zhang, M.; Xu, B. Chem.
Commun. 2005, 4414–4416.
4) Zhou, M.; Smith, A. M.; Das, A. K.; Hodson, N. W.; Collins, R. F.; Ulijn,
R. V.; Gough, J. E. Biomaterials 2009, 30(13), 2523–2530.
5) Jayawarna, V.; Ali, M.; Jowitt, T. A.; Miller, A. F.; Saiani, A.; Gough, J. E.;
Ulijn, R. V. Adv. Mater. 2006, 18, 611–614.
6) Jayawarna, V.; Richardson, S. M.; Hirst, A. R.; Hodson, N. W.; Saiani, A.;
Gough, J. E.; Ulijn, R. V. Acta Biomater. 2009, 5(3), 934–943.
(
workers prepared tripeptides Fmoc-X-F (X = G, A, V, F, P, L)
(
2
9
by reverse hydrolysis, using thermolysin as described above.
(
Banerjee and co-workers have investigated the self-assembly into
1
9
20
(
fibrils or nanotubes of certain non-Fmoc-tripeptides and have
examined organogelation of peptides protected at the N terminus
(
7) Yang, Z.; Xu, B. Chem. Commun. 2004, 2424–2425.
(
8) Yang, Z.; Liang, G.; Ma, M.; Gao, Y.; Xu, B. J. Mater. Chem. 2007, 17, 850–
8
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54.
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T.; Lu, W. W.; Xu, B. Langmuir 2009, 25(15), 8419–8422.
(15) Yang, Z.; Gu, H.; Fu, D.; Gao, P.; Lam, J. K. W.; Xu, B. Adv. Mater. 2004,
16(16), 1440–1444.
(16) Yang, Z.; Liang, G.; Ma, M.; Gao, Y.; Xu, B. Small 2007, 3(4), 558–562.
(17) Yang, Z.; Xu, B. Adv. Mater. 2006, 18, 3043–3046.
(18) Das, A. K.; Collins, R.; Ulijn, R. V. Small 2008, 2, 279–287.
(19) Naskar, J.; Drew, M. G. B.; Deb, I.; Das, S.; Banerjee, A. Org. Lett. 2008, 10
(13), 2625–2628.
(
9) Toledano, S.; Williams, R. J.; Jayawarna, V.; Ulijn, R. V. J. Am. Chem. Soc.
006, 128(4), 1070–1071.
(
10) Mahler, A.; Reches, M.; Rechter, M.; Cohen, S.; Gazit, E. Adv. Mater.
006, 18, 1365–1370.
11) Adams, D. J.; Butler, M. F.; Frith, W. J.; Kirkland, M.; Mullen, L.;
Sanderson, P. Soft Matter 2009, 5(9), 1856–1862.
12) Sutton, S.; Campbell, N. L.; Cooper, A. I.; Kirkland, M.; Frith, W. J.;
Adams, D. J. Langmuir 2009, 25(17), 10285–10291.
13) Zhang, Y.; Gu, H.; Yang, Z.; Xu, B. J. Am. Chem. Soc. 2003, 125, 13680–
3681.
(
(
(
(20) Ray, S.; Haldar, D.; Drew, M. G. B.; Banerjee, A. Org. Lett. 2004, 6, 4463–
4465.
1
4
990 DOI: 10.1021/la903678e
Published on Web 01/14/2010
Langmuir 2010, 26(7), 4990–4998

Cheng et al.
Article
ε
ε
Scheme 1. Synthesis of Fmoc-Tripeptides Fmoc-VLK (N -Boc) and Fmoc-K(N -Boc)LV
ε
with aromatic units such as N -tert-butyloxycarbonate (Boc) or
rheometry. An unexpected difference in gel dynamic mechanical
and structural properties is revealed. In borate buffer, Fmoc-
VLK(Boc) exhibits nematic-like ordering of highly anisotropic
and aligned fibrils, whereas Fmoc-K(Boc)LV shows a dense
branched fibrillar network structure which produces isotropic
hydrogels with high rigidity. These gels have numerous potential
applications in nanobiotechnology, such as cell growth media,
controlled release drug delivery media, enzyme responsive sys-
tems, and antimicrobial gels.
2
1
carbobenzyloxy (Cbz) units. We have recently been investigat-
ing the self-assembly and hydrogelation of peptides containing
sequences from the amyloid β (Aβ) peptide core sequence Aβ-
2
2-24
(
16-20), i.e. KLVFF.
These sequences do not contain an
Fmoc terminus. Without the Fmoc terminus, previous work
suggests that KLVFF is the shortest peptide based on a sequence
from this region of Aβ capable of self-assembling into β-sheets
The dipeptide FF (within KLVFF)
can self-assemble into nanotubes in water,
within the walls of these is not based on a β-sheet motif. We have
been interested to investigate whether the self-assembly of shorter
sequences can be induced by attachment to Fmoc which leads to
aromatic stacking interactions that can drive fibrillization. The
present Article describes our initial studies in this direction.
We report on the self-assembly and hydrogelation of Fmoc-
K(Boc)LV, which contains the KLV sequence, part of KLVFF,
as studied previously by our group and others. The self-assembly
and hydrogelation of Fmoc-K(Boc)LV is compared to that of a
peptide containing the inverse sequence, Fmoc-VLK(Boc), that
is, one in which K(Boc) and V residues are interchanged. For
convenience, in the following, the samples are denoted Fmoc-
K(Boc)LV and Fmoc-VLK(Boc). The Boc group is retained in
order to investigate the role of hydrophobic versus electrostatic
interactions; in future work, we will present our ongoing studies
on Fmoc-K(Boc)LV and Fmoc-VLK(Boc) with deprotected K
groups, and also Fmoc protected K groups. Here, we present a
comprehensive study on self-assembly comparing the two Fmoc-
tripeptides, studying fibril formation via cryo-transmission elec-
tron microscopy (cryo-TEM) and small-angle X-ray scattering
2
4-26
and forming hydrogels.
2
7,28
but the structure
2
9
Experimental Section
Materials. Amino acids [Fmoc-lysine(Boc)-OH, Fmoc-leucine-
OH, Fmoc-valine-OH, Fmoc-phenylalanine-OH], HOBt (1-hydro-
xybenzotriazole), and HBTU [2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate] were purchased from
Novabiochem (United Kingdom). N-Hydroxysuccinimide (HOSu),
1,3-dicyclohexylcarbodiimide (DCC), N-methylmorpholine (NMM),
anhydrous dimethoxyethane (DME), anhydrous N,N-dimethyl-
formaimde (DMF), glucono-δ-lactone (GdL), Congo Red, and
citric acid were purchased from Sigma-Aldrich (United Kingdom);
sodium bicarbonate, tetahydrofuran (THF), ethyl acetate (EtOAc),
and borate buffer (pH = 10) were purchased from Fisher
Scientific (United Kingdom). Scheme 1 describes the route to
synthesize Fmoc-tripeptides Fmoc-VLK(Boc) and Fmoc-
K(Boc)LV.
1
0
Synthesis of Fmoc-L-Val-OSu. N-(Fluorenyl-9-meth-
oxycarbonyl) -Val (5.09 g, 15 mmol) and N-hydroxysuccinimide
L
(1.73 g, 15.00 mmol) were dissolved in 80 mL of anhydrous
dimethoxyethane. The solution was cooled in ice-water, and a
solution of DCC (3.40 g, 16.50 mmol) in 20 mL of anhydrous
dimethoxyethane was dropwise added to the solution which
became cloudy white. The mixture was stirred for 24 h at room
temperature. The urea precipitate was filtered out, and the filtrate
was dried by evaporation of the solvent. The resulting product
was redissolved ina minimal amount of acetone and then kept ina
fridge for 4 h. The small amount of urea precipitate was filtered
out, and the filtratewasconcentratedto drynessbyevaporationof
the solvent. The white foam was dissolved in EtOAc and pre-
(
SAXS). The secondary structure is probed via X-ray diffraction,
circular dichroism (CD), Fourier transform infrared (FTIR) and
Raman spectroscopy. Gel properties are probed via SAXS and
(
21) Das, A. K.; Bose, P. P.; Drew, M. G. B.; Banerjee, A. Tetrahedron 2007, 63
31), 7432–7442.
22) Krysmann, M. J.; Castelletto, V.; Kelarakis, A.; Hamley, I. W.; Hule, R. A.;
Pochan, D. J. Biochemistry 2008, 47, 4597–4605.
(
(
cipitated in petroleum ether (40-60 °C) to give a white powder
1
(6.10 g, 93%). H NMR (250 MHz, DMSO-d
(
5.
(
23) Castelletto, V.; Hamley, I. W.; Harris, P. J. F. Biophys. Chem. 2008, 139, 29–
6
) δ (ppm): 8.18 (d,
3
J=10.0 Hz, 1H), 7.92 (d, J=7.5 Hz, 2H), 7.74 (t, J=7.5 Hz, 2H),
.42 (t, J=7.5 Hz, 2H), 7.33 (t, J=7.5 Hz, 2H), 4.33 (m, 4H), 2.82
s, 4H), 2.09 (m, 1H), 1.01 (d, J=7.5 Hz, 6H).
Synthesis of Fmoc- -Val- -Leu. N-(Fluorenyl-9-meth-
oxycarbonyl) -Leu (1.69 g, 12.91 mmol) was dissolved in 200
mL of cosolvent of THF/water (1:1) containing sodium bicarbo-
nate (2.17 g, 25.82 mmol). To this solution, a solution of Fmoc-
24) Castelletto, V.; Hamley, I. W.; Hule, R. A.; Pochan, D. J. Angew. Chem.,
7
(
Int. Ed. Engl. 2009, 48, 2317–2320.
25) Tjernberg, L. O.; Callaway, D. J. E.; Tjernberg, A.; Hahne, S.; Lillieh o€ o€ k,
C.; Terenius, L.; Thyberg, J.; Nordstedt, C. J. Biol. Chem. 1999, 274(18), 12619–
2625.
(
L
L
1
L
(
(
(
(
26) Gordon, D. J.; Tappe, R.; Meredith, S. C. J. Pept. Res. 2002, 60, 37–55.
27) Reches, M.; Gazit, E. Science 2003, 300, 625–627.
28) Reches, M.; Gazit, E. Isr. J. Chem. 2005, 45, 363–371.
29) G o€ rbitz, C. H. Chem. Commun. 2006, 2332–2334.
L-
Val-OSu (5.63 g, 12.91 mmol) in 20 mL of acetone was added
Langmuir 2010, 26(7), 4990–4998
DOI: 10.1021/la903678e 4991

Article
Cheng et al.
dropwise at room temperature. After addition, it was stirred for
borate buffer with a concentration of 1% wt. The
mixture was sonicated in an ultrasonic bath with shak-
ing at 40-50 °C for 5-10 min. Upon cooling to room
temperature, gelation was observed by the inverted vial
method.
4
8 h and the solvent was concentrated to dryness in vacuo. The
crude product was dissolved in 200 mL of EtOAc, washed with
0% aq citric acid and water, and then dried (MgSO ). It was
1
4
filtered, and the filtrate was concentrated to dryness in vacuo to
give a white powder 4.44 g (76%). HNMR (250 MHz, DMSO-
1
-3
B. Each Fmoc-tripeptide (6.2 mg, 9.1ꢀ 10 mmol) was
d6) δ (ppm): 8.13 (d, J=7.5 Hz, 1H), 7.90 (d, J=7.5 Hz, 2H), 7.74
first dispersed in 1.5 mL of water by sonication for
5 min, followed by careful addition of aq NaOH (1 M)
to dissolve the Fmoc-tripeptide via vortexing so that a
clear solution was obtained. To the basic peptide solu-
(
3
m, 3H), 7.44 (m, 2H), 7.32 (t, J=7.5 Hz, 2H), 4.33-4.12 (m, 4H),
.91(m, 1H), 2.02 (m, 1H), 1.68-1.40 (m, 3H), 0.88 (m, 12H). MS:
þ
þ
calcd [M ] = 452, obsvd [Mþ1 ] =453.
Synthesis of Fmoc- -Val- -Leu-
-Val- -Leu (4.00 g, 8.84 mmol) and N-hydroxysuccinimide
1.02 g, 8.84 mmol) were dissolved in 40 mL of anhydrous
1
1
tion was added 15 mg of GdL sonicated for 5 min at
room temperature and then vortex mixed. Homoge-
neous hydrogels were formed in 15 min.
L
L
L-Lys(Boc). Fmoc-
L
L
(
dimethoxyethane. The solution was cooled in ice-water, and a
solution of DCC (2.00 g, 9.72 mmol) in 10 mL of anhydrous
dimethoxyethane was added dropwise to the solution which
became cloudy white. The mixture was stirred for 24 h at room
temperature. The urea precipitate was filtered out, and the filtrate
was concentrated to dryness by evaporation of the solvent. The
resulting product was redissolved in a minimal amount of acetone
and then kept in a fridge for 4 h. The small amount of urea
precipitate was filtered out, and the filtrate was concentrated to
Congo Red Staining and Birefringence Monitored by
Polarized Optical Microscopy. Fresh Congo Red solution
was made as follows: Sodium chloride was added in excess to an
80% ethanol solution, vortexed, and thenfiltered. CongoRedwas
added in excess to the saturated NaCl ethanol solution, and the
3
2
resulting solution was vortexed and filtered. Congo Red solu-
tion was pipetted onto a glass microscope slide, and then the
peptide gel was placed underneath the surface of the Congo Red
solution and stained for approximately 1 min. After the excess
Congo Red solution was removed by blotting, images of the
sample placed between crossed polarizers were obtained with an
Olympus CX-41 microscope.
dryness by evaporation of the solvent to give white powder Fmoc-L-
1
Val-
L
-Leu-OSu. H NMR (250 MHz, DMSO-d) δ (ppm): 8.65 (d,
J=7.5 Hz, 1H), 7.91 (d, J=7.5 Hz, 2H), 7.74 (d, J=7.5 Hz, 2H),
7.51 (d, J = 7.5 Hz, 1H), 7.42 (t, J = 7.5 Hz, 2H), 7.32 (t, J =
7.5 Hz, 2H), 4.65 (m, 1H), 4.23 (m, 3H), 3.92 (t, J=7.5 Hz, 1H),
2.80 (s, 4H), 2.02 (m, 1H), 1.71 (m, 3H), 0.88 (m, 12H).
Circular Dichroism (CD). The CD spectra were recorded
using a Chirascan spectropolarimeter (Applied Photophysics,
U.K.). Solutions of Fmoc-tripeptide in borate buffer (pH = 10)
with a range of concentrations (0.1, 0.5, 1 wt.%) were loaded into
a 0.01 mm quartz cell (Hellma 0.01 mm quartz Suprasil). Spectra
were obtained from 200 to 280 nm with a 0.5 nm step and 2.5 s
collection time per step at 25 °C, taking four averages.
Fourier Transform Infrared (FTIR) and Raman Spectro-
scopy. Spectra were recorded using a Nexus-FTIR spectrometer
equipped with a DTGS detector and a multiple reflection atte-
nuated total reflectance (ATR) system. Solutions of Fmoc-tripep-
tide in borate buffer (pH = 10) with a concentration of 1 wt %
incubated for 16 h were sandwiched in ring spacers between two
To the solution of Fmoc-L-Val-L-Leu-OSu in 60 mL of DMF
was added dropwise a solution of N-(fluorenyl-9-methoxycarbo-
nyl) L-Lys(N -Boc) (2.18 g, 8.84 mmol) in 100 mL of cosolvent of
ε
THF/water (3:2) containing 2.3 mL of NMM at 0 °C. After
addition, the reaction mixture became clear, and it was stirred for
4
8 h. It was filtered, and the filtrate was concentrated by removing
THF in vacuo. The resulting mixture was diluted with 250 mL of
EtOAc, washed with 10% aq citric acid, and dried (MgSO ). It
4
was filtered and dried by evaporation of the solvent in vacuo. The
crude product was purified by column chromatography using
silica gel as the stationary phase with a gradient eluent system
(
DCM) = 0.45), followed by crystallization from EtOAc to give
f
from DCM to 10% MeOH in DCM, R (10% MeOH in
CaF plate windows (spacer 0.006 mm). A spectrum for each
2
sample was also measured for a film dried from a 1 wt % solution
1
a white powder (4.33 g, 72%). H NMR (250 MHz, DMSO-d
6
) δ
onto a CaF
range of 4000-950 cm
2
plate. All spectra were scanned 128 times over the
(
ppm): 8.05 (d, J=5.0 Hz, 1H), 7.91 (t, J=7.5 Hz, 3H), 7.73 (m,
-1
.
2
5
1
H), 7.42 (t, J=5.0 Hz, 2H), 7.32 (t, J=7.5 Hz, 2H), 6.79 (t, J=
.0 Hz, 1H), 4.30 (m, 5H), 3.87 (t, J=7.5 Hz, 1H), 2.86 (m, 2H),
.99 (m, 1H), 1.65-1.20 (m, 19H), 0.83 (m, 12H). MS: calcd
Raman Spectroscopy. Raman spectra were recorded using a
Renishaw inVia Raman microscope. The light source was a
multiline laser, such that the experiments were performed using
the λ=785 nm edge. Experiments were made on stalks prepared
by drying filaments of the peptide obtained from 2 wt % Fmoc-
VLK(Boc) and 2 wt % Fmoc-KLV(Boc) gels in borate buffer.
The stalks were focused byusing a 20ꢀ magnification lens. Spectra
þ
þ
[
M ]=680, obsvd [Mþ1 ]=681.
Fmoc- -Lys(Boc)- -Leu- -Val was synthesized starting form
N-(fluorenyl-9-methoxycarbonyl) -Lys (Boc) according to the
L
L
L
L
above method in the route shown in Scheme 1.
1
Fmoc-L-Lys(Boc)-OSu. H NMR (250 MHz, DMSO-d ) δ
-1
6
were obtained in the interval 100-3000 cm , using a 20 s
(
2
4
ppm): 8.11 (d, J= 7.5 Hz, 1H), 7.91 (d, J= 5.0 Hz, 2H), 7.70 (m,
H), 7.42 (t, J=7.5 Hz, 2H), 7.33 (t, J=7.5 Hz, 2H), 6.79 (m, 1H),
.33 (m, 4H), 2.91 (m, 2H), 2.82 (s, 4H), 1.79 (m, 2H), 1.37 (m, 13H).
collection time with 10% laser power and taking two averages.
Cryogenic-Transmission Electron Microscopy (Cryo-
TEM). Experiments were performed at Unilever Research,
Colworth, Bedford, U.K. Sample preparation was carried out
using a CryoPlunge 3 unit (Gatan Instruments) employing a
double blot technique. The amount of 3 μL of sample in borate
buffer was pipetted onto a plasma etched (15 s) 400 mesh holey
carbon grid (Agar Scientific) held in the plunge chamber at
approximately 90% humidity. The samples were blotted, from
both sides, for 0.5, 0.8, or 1.0 s depending on sample viscosity.
The samples were then plunged into liquid ethane at a tem-
perature of -170 °C. The grids were blotted to remove excess
ethane and then transferred, under liquid nitrogen to the cryo-
TEM specimen holder (Gatan 626 cryo holder) at -170 °C.
Samples were examined using a Jeol 2100 transmission electron
microscope operated at 200 kV and imaged using a Gatan
Ultrascan 4000 camera, and images were captured using Digi-
talMicrograph software (Gatan).
1
Fmoc-L-Lys(Boc)-L-Leu. H NMR (250 MHz, DMSO-d
ppm): 8.06 (d, J=7.5 Hz, 1H), 7.91 (d, J=7.5 Hz, 2H), 7.74 (m,
H), 7.42 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 6.78
6
) δ
(
2
(
1
m, 1H), 4.24 (m, 4H), 3.99 (m, 1H), 2.89 (m, 2H), 1.66-1.20 (m,
þ
8H), 0.86 (q, J = 7.5 Hz, 6H). MS: calcd [M ] = 581, obsvd
þ
[Mþ1 ]=582.
ε
Fmoc-L-Lys(N -Boc)-L-Leu-L-Val. Rf (10% MeOH in
1
DCM) = 0.57; H NMR (250 MHz, DMSO-d
6
) δ (ppm): 7.97
(
2
d, J=7.5 Hz, 1H), 7.91(d, J=7.5 Hz, 2H), 7.88(m, 1H), 7.78 (m,
H), 7.50-7.30 (m, 4H), 6.77 (m, 1H), 4.39-4.11 (m, 5H), 3.99
(
1
m, 1H), 2.89 (m, 2H), 2.02 (m, 1H), 1.70-1.20 (m, 19H), 0.86 (m,
þ
þ
2H). MS: calcd [M ] =680, obsvd [Mþ1 ]=681.
Hydrogel Formation
A. Standard pH-adjusting method: Each Fmoc-tripeptide
was added into a vial, followed by addition of 0.5 mL of
4
992 DOI: 10.1021/la903678e
Langmuir 2010, 26(7), 4990–4998

Cheng et al.
Article
Fiber X-ray Diffraction (XRD). X-ray diffraction was
performed on the same stalks used for Raman experiments. The
stalks were mounted (vertically) onto the four axis goniometer of
a RAXIS IVþþ X-ray diffractometer (Rigaku) equipped with a
rotating anode generator. The XRD data were collected using a
Saturn 992 CCD camera.
Small-Angle X-ray Scattering (SAXS). SAXS was perfor-
med on beamline I22 at Diamond Light Source, Harwell Science
and InnovationCampus, U.K. The energyusedwas12.4keV with
a 2.2 m camera length and beamstop in the middle of the RAPID
2
D detector. The peptide gels were loaded into a liquid cell, placed
between two mica windows, and then mounted directly into the
sample position on the beamline. A total of six frames of 10s were
collected. The same was also done for backgrounds.
Light Scattering. Static light scattering was performed using
3
0
a custom built instrument described elsewhere. Briefly, it
comprises a He-Ne laser, an optics train, a sample contained in
a Linkam CSS 450 shear cell, a diffuser plate, and a CCD camera
to detect images. The sample was subjected to a range of shear
rates;however, nosignificantorientation wasobserved, andsothe
two-dimensional patterns obtained were integrated to one-dimen-
sional profiles of intensity versus scattering angle and hence
wavenumber q (calibrated using a diffraction grating).
Figure 1. Self-supporting hydrogels of (left) Fmoc-VLK(Boc)
and (right) Fmoc-K(Boc)LV in (a) borate buffer (pH = 10) and
(b) GdL (pH ≈ 4).
Rheology. The rheological properties of the systems were
determined using a controlled stress AR-2000 rheometer from
TA Instruments. A cone and plate geometry (20 mm diameter, 1°
angle) was used for all samples. Frequency sweeps were per-
formed in the angular frequency (ω) range 0.1-600 rad/s with the
instrument in oscillatory mode at 25 °C. After an initial frequency
sweep, another was carried out including a preshear (controlled
-1
stress mode) at 50 s for 2 min. Further frequency sweeps were
then performed at regular intervals to investigate the
recovery of the gel. To ensure that the moduli were independent
of strain, preliminary strain sweeps were performed for each
sample.
Figure 2. Polarized optical microscopyimage ofFmoc-tripeptides
stained with Congo Red: (a) Fmoc-VLK(Boc) and (b) Fmoc-
K(Boc)LV . Scale bar indicates 1 mm.
Results
b. Secondary Structure. Congo Red staining experiments
showed strong green birefringence in the polarized optical micro-
scope for Fmoc-VLK(Boc) over regions approximately 0.15 mm
in size (Figure 2), suggesting amyloid formation. The yellow-
green birefringence of sample Fmoc-KLV(BOC) is less pro-
nounced, but there are clear areas with yellowish color, consistent
a. Synthesis and Hydrogelation. Fmoc-VLK(Boc) and
Fmoc-K(Boc)LV (Scheme 1) were synthesized by peptide bond
formation via aminolysis of N-hydroxysuccinimidyl esters in
1
0,31
aqueous/organic solvent.
(
Since tripeptides Fmoc-VLK
Boc) and Fmoc-K(Boc)LV are soluble in basic media but
insoluble in acid or neutral water, hydrogelation was initially
3
1
with uptake of the dye. The uptake of Congo Red is usually
considered to be a robust diagnostic of amyloid fibril formation,
and this dye is taken up by fibrils and oligomers of many peptides
investigated using basic solution. Fmoc-tripeptide (6.2 mg, 9.1ꢀ
-3
1
0
mmol) was first dispersed in 1.5 mL of water by sonication,
3
2,33
followed by careful addition of aq NaOH (1 M) to dissolve the
Fmoc-tripeptides with vortex mixing to obtain a clear solution.
After 10 min, gelation took place, producing a transparent uni-
form gel with pH 12-13. In 1 h, it gradually became turbid,
followed by collapse of the whole network. In contrast, a stable gel
was achieved when borate buffer (pH = 10) was used as a
and proteins forming cross-β structures.
However, its modeof
3
3
binding does depend on β-sheet structure, and as discussed
below the reduced binding to fibrils of Fmoc-KLV(Boc) may
reflect differences in molecular packing within the fibrils.
The secondary structure of Fmoc-tripeptides was assessed
using circular dichroism (CD) (Figure 3), measured for 1 wt %
samples in pH 10 borate buffer. The peaks observed in two ranges
of 210-220 nm and 240-260 nm showed features due to nfπ*
and πfπ* transitions. Spectra with peaks in these regions have
previously been reported for Fmoc-peptides such as Fmoc-
3
1
solvent. However, the resulting gel was inhomogeneous with
some denser regions in the network, probably due to the slower
1
1
kinetics of mixing than that of gelation. Adams et al. reported a
new approach to prepare homogeneous hydrogels using GdL to
control the acidic pH of the obtained hydrogels. With addition of
GdL, we were able to prepare homogeneous transparent hydro-
gels based on tripeptides Fmoc-VLK(Boc) and Fmoc-K(Boc)LV,
confirmed by the inverted vial method as shown in Figure 1.
However, the focus of the remainder of this paper is on hydrogels
in basic media, that is, in borate buffer.
7
,15
tyrosine derivative.
Fmoc-FF exhibits positive peaks in the
CD spectrumat 200-210 and 260 nm, with a minimum at218 nm,
3
4
ascribed to β-sheet structure. A β-sheet minimum at 215 nm is
observed for Fmoc-K(Boc)LV (Figure 3) but not for Fmoc-
VLK(Boc), for which a large positive maximum is observed at
220 nm. A detailed molecular interpretation of CD spectra for
short peptides such as Fmoc-derivatives is still lacking and
(30) Castelletto, V.; Hamley, I. W. Polym. Adv. Technol. 2006, 17(3), 137–144.
(31) Teng, P. K.; Eisenberg, D. Protein Eng., Des. Sel. 2009, 22(8), 531–536.
(32) Lendel, C.; Bertoncini, C. W.; Cremades, N.; Waudby, C. A.; Vendruscolo,
(33) Childers, W. S.; Mehta, A. K.; Lu, K.; Lynn, D. G. J. Am. Chem. Soc. 2009,
131(29), 10165–10172.
M.; Dobson, C. M.; Schenk, D.; Christodoulou, J.; Toth, G. Biochemistry 2009, 48
35), 8322–8334.
(34) Smith, A. M.; Williams, R. J.; Tang, C.; Coppo, P.; Collins, R. F.; Turner,
M. L.; Saiani, A.; Ulijn, R. V. Adv. Mater. 2008, 20, 37–41.
(
Langmuir 2010, 26(7), 4990–4998
DOI: 10.1021/la903678e 4993

Article
Cheng et al.
Figure 3. Circular dichroism spectra of Fmoc-VLK(Boc) and
Fmoc-K(Boc)LV tripeptides (1 wt % in pH 10 borate buffer).
Figure 5. Cryo-TEM images of (a,b) Fmoc-VLK(Boc) and (c)
Fmoc-K(Boc)LV. Scale bar in (a) represents 200 nm, and in (b,c) it
represents 100 nm.
-1
1
685 cm for the gels or dried gels (Figure 4a). The latter may be
Figure 4. (a) FTIR spectra and (b) Raman spectra of the amide I
region of Fmoc-VLK(Boc) and Fmoc-K(Boc)LV tripeptides
(dried films from gels with a concentration of 1 wt % in pH 10
borate buffer).
assigned to β-sheet structure. Deconvolution of the broad peaks
was not attempted; their width suggests the presence of elements
-1
of β-sheet structure (1625-1640 cm ) and random coil (1640-
-
1
1
650 cm ) structures (the peptides are too short to form
2
2,34
prohibits further detailed interpretation. It can be noted that the
spectrum obtained for Fmoc-K(Boc)LV is notably less intense
R-helices).
Similar spectra were reported for Fmoc-TL and
1
8
Fmoc-TF dipeptides. Raman spectroscopy can provide com-
plementary information, and spectra were measured using stalks
prepared from the two Fmoc-tripeptide gels (Figure 4b). Typi-
(
lower molar ellipticity) than that for Fmoc-VLK(Boc), pointing
to lower chiral ordering in the former system. This may be related
to the nature of the fibrils, which are quite straight for Fmoc-
VLK(Boc) but highly branched for Fmoc-K(Boc)LV, as will be
discussed shortly.
cally, astrongbandat1610-1625 cm- and a weak one at 1690 cm
1
-1
were observed and associated with antiparallel β-sheet structure
2
2
-1
for both gels. A band centered at 1652 cm (quite strong for
Fmoc-VLK(Boc)) indicated the presence of non-β-sheet struc-
ture. As with circular dichroism, detailed molecular interpretation
of FTIR spectra from short peptides is a current theoretical
As the amide I band is sensitive to secondary structure, FTIR
spectra of the two Fmoc tripeptides were recorded. Broad peaks
-1
centered at 1650 cm were observed, with a shoulder around
4
994 DOI: 10.1021/la903678e
Langmuir 2010, 26(7), 4990–4998

Cheng et al.
Article
challenge. We believe that interpretation of such spectra based on
secondary structural elements from large proteins may not always
be fully applicable.
c. Fibril Morphology and Superstructure: Electron Mi-
croscopy, Light Scattering, and X-ray Scattering. Cryo-
TEM confirms that both Fmoc-VLK(Boc) and Fmoc-
K(Boc)LV form fibrillar structures in 0.2 wt % borate buffer
solution (Figure 5). The fibrils showed a high degree of align-
ment locally for the former sample, whereas for the latter
sample mainly dense clusters of branched fibrils are observed,
coexisting with a small number of quite straight fibrils, which
are wider than the branched fibrils in the network clusters.
The fibril diameter estimated from the cryo-TEM images is
8
nm for Fmoc-VLK(Boc) and 7 nm for the fine branched
fibrils in Fmoc-K(Boc)LV, with 9 nm diameter larger straight
fibrils. The dark blobs in the cryo-TEM images are ice
crystals, and white bubbles are regions where evaporation
has occurred due to exposure to the electron beam. These
artefacts do not affect the imaging of the fibrils. Static light
scattering confirmed a more branched structure for Fmoc-K
Figure 6. Static light scattering data presented in Kratky-plot
format.
cell used for SAXS experiments. In contrast, orientation was not
observed in SAXS patterns for Fmoc-K(Boc)LV.
(
Boc)LV. A Kratky plot of the scattered intensity (Figure 6)
Fiber X-ray diffraction was performed on dried stalks to obtain
information on molecular packing. Figure 9 compares XRD
patterns for the two compounds. Some features are common to
the data for both systems; in particular, a series of reflections can
be ascribed to the cross-β pattern observed for β-sheet fibrils. At
higher angle, sharper reflections are observed that are ascribed to
the borate buffer (a separate XRD pattern was obtained on a
dried borate buffer solution, data not shown, strong reflections
listed in Table 1).
shows a more pronounced maximum for this peptide, con-
sistent with a more branched structure.
2
2,35
SAXS also confirmed that both samples self-assemble into
fibrils in pH 10 borate buffer solution. Figure 7 shows intensity
profiles with fits for the lower concentration data (1 wt %; this is
higher than cryo-TEM or CD data in order to achieve good
scattering data, i.e. sufficient signal/noise ratio) for Fmoc-VLK-
3
9
(
Boc) to a model form factor for a polydisperse uniform cylin-
3
6
der. The fit indicates a diameter D = 4.2 ( 0.3 nm, which is
lower than the fibril diameter obtained from cryo-TEM (8 nm);
however, a hydrated boundary layer may not be detectable by
SAXS. The data for Fmoc-K(Boc)LV could not be fitted to a
simple polydisperse cylinder form factor (the slope of the intensity
profile at low q is approximately q , whereas q is expected for
cylinders). Instead, we were able to fit the data to the structure
factor for a mass fractal object, with an exponential cutoff, using
The fiber diffraction pattern for Fmoc-VLK(Boc) shows a high
˚
degree of alignment. There are meridional 4.7 A reflections and
series of equatorial reflections, of which the strongest corresponds
˚
to a spacing of 21.5 A. The high degree of alignment of the
diffraction pattern reflects the local orientation of fibrils observed
by cryo-TEM (Figure 5a,b) and the macroscopic alignment in the
SAXS pattern (Figure 8a). Much sharper reflections are observed
at higher angle, and since the peak width is different from the
cross-β pattern peaks, these are associated with features not
related to β-sheets. We believe they result from the buffer. All
reflections are listed in Table 1. The reflections observed for
Fmoc-VLK(Boc) may be indexed to an orthorhombic unit cell.
The X-ray diffraction pattern for Fmoc-K(Boc)LV (Figure 9b)
shows very little orientation, consistent with the observed en-
tangled nature of the fibrils observed for this sample (Figure 5c).
Peaks at low angle (Table 1) can be assigned to an unoriented
cross-β structure. A larger number of strong reflections are
observed at low angle than those for Fmoc-VLK(Boc). Since
these peaks are associated with stacking of β-sheets, this may
indicate more ordered β-sheet stacks within the Fmoc-K(Boc)LV
-2.5
-1
3
7
the freely available software SASfit. This structure factor
captures the branched nature of the fibrillar network. The
corresponding structure factor intensity is given by
3
8
h
i
-
1
sin ðD - 1Þ tan ðqξÞ
IðqÞ ¼
ð1Þ
2
2
ðD - 1Þ=2
ðD - 1Þqξð1 þ q ξ Þ
2
2
g
Here, D is the fractal dimension, ξ =2R /D(D þ 1), and R is the
g
radius of gyration of the fractal aggregate. From the fit, we
obtained D=2.5 and R =10.4 nm for the Fmoc-K(Boc)LV data
(
g
1 wt %).
The 2 wt % gel of Fmoc-K(Boc)LV shows a broad structure
˚
fibrils. The d-spacing at 4.88 A is somewhat larger than that
-1
˚
factor peak, centered at q* = 0.071 A , corresponding to a
structure with a domain size d = 89 A. This is in very close
observed for Fmoc-VLK(Boc) for which the corresponding
˚
˚
strong meridional reflection is observed at 4.74 A; however, it is
4
0
agreement with the fibril diameter obtained from the cryo-TEM
image (Figure 5c) and suggests close-packing of the fibrils. The
alignment of fibrils observed by cryo-TEM for gels formed by
Fmoc-VLK(Boc) is also reflected in anisotropic SAXS patterns;
see Figure 8 for example. The anisotropy results from sponta-
neous macroscopic alignment of the sample when poured into the
within the range observed for β-sheets. The difference in this
interstrand spacing may reflect differences in molecular confor-
mation or packing motif within the sheets. For Fmoc-K(Boc)LV,
a series of sharp reflections are again observed at higher angle,
˚
with the principal one being at 3.2 A, as for Fmoc-VLK(Boc),
which is ascribed to the buffer (Table 1). A slightly larger spacing
˚
of 3.4 A was ascribed to phosphate buffer in XRD data presented
for Fmoc-LLL, while an Fmoc stacking distance was observed
(
35) Pallitto, M. M.; Ghanta, J.; Heinzelman, P.; Kiessling, L. L.; Murphy,
R. M. Biochemistry 1999, 38, 3570–3578.
36) Krysmann, M. J.; Castelletto, V.; Hamley, I. W. Soft Matter 2007, 3, 1401–
406.
37) http://kur.web.psi.ch/sans1/SANSSoft/sasfit.html (2009).
38) Sorensen, C. M.; Wang, G. M. Phys. Rev. E 1999, 60(6), 7143–7148.
(
1
(39) Hamley, I. W. Angew. Chem., Int. Ed. 2007, 46, 8128–8147.
(40) Nelson, R.; Sawaya, M. R.; Balbirnie, M.; Madsen, A. O.; Riekel, C.;
Grothe, R.; Eisenberg, D. Nature (London) 2005, 435, 773–778.
(
(
Langmuir 2010, 26(7), 4990–4998
DOI: 10.1021/la903678e 4995

Article
Cheng et al.
Figure 7. SAXS profiles (obtained by radial integration of 2-D patterns) for (a) Fmoc-VLK(Boc) and (b) Fmoc-K(Boc)LV. Fitted intensity
profiles are included, as discussed in the text.
Figure 8. SAXS patterns for 1 wt % solutions of (a) Fmoc-VLK(Boc) and (b) Fmoc-K(Boc)LV.
Figure 9. Fiber X-ray diffraction patterns for (a) Fmoc-VLK(Boc) and (b) Fmoc-K(Boc)LV.
4
1
˚
at 3.8 A. It is interesting that a strong peak at around this
distance is not observed for either of our samples, suggesting less
regular packing of the Fmoc groups, possibly due to greater
conformational flexibility of the attached tripeptide.
Figure 10 shows frequency sweep data. The two peptides exhibit
distinct behavior. Fmoc-VLK(Boc) forms a weak gel at 1 wt %,
0
characterized by a low value of G ∼ 100 Pa at low frequency. The
upturn in moduli at high frequency may be due to a thickening
instability. The 2 wt % gel of Fmoc-VLK(Boc) has a higher
d. Gel Rheology. Oscillatory shear rheometry was used to
probe the dynamic mechanical properties of hydrogels formed by
0
modulus G >4000 Pa. A pronounced shear thinning is observed
1
and 2 wt % gels of Fmoc-VLK(Boc) and Fmoc-K(Boc)LV.
after preshearing. Partial recovery of the moduli is observed after
several hours. Incomplete recovery is probably due to shear
alignment, as observed by SAXS (Figure 8a).
(
41) Xu, H.; Das, A. K.; Horie, M.; Shaik, M. S.; Smith, A. M.; Luo, Y.; Lu, X.;
Fmoc-K(Boc)LV forms stiff hydrogels at 1 and 2 wt %
Collins, R.; Liem, S. Y.; Song, A.; Popelier, P. L. A.; Turner, M. L.; Xiao, P.;
Kinloch, I. A.; Ulijn, R. V. Nanoscale 2010, submitted.
0
5
(Figure 10c,d), with a weakly frequency-dependent G > 10 Pa.
4
996 DOI: 10.1021/la903678e
Langmuir 2010, 26(7), 4990–4998

Cheng et al.
Table 1. Spacings (in A) from X-ray Diffraction Patterns
Article
˚
a
44-46
rapid self-assembly.
Substitution of one lysine with one
glutamic acid lead to more rapid self-assembly due to reduced
net charge per peptide, and a more branched hydrogel, as
b
Fmoc-VLK(Boc)
Fmoc-KLV(Boc)
21.1
borate buffer
4
6
2
1
1
9
1.4 (e)
7.3 (e)
3.8 (e)
.90 (m)
characterized by small-angle neutron scattering. The modulus
0
reported for this type of β-hairpin hydrogel ranges up to G =
13.4
9.46
1
000-4000 Pa depending on the peptide, its concentration, and
45,47-49
buffer.
In borate buffer under similar conditions to those
8
.33
6.76
.85
0
of our Fmoc-tripeptide hydrogels, a modulus G = 1000 Pa was
reported. The modulus of this type of peptide hydrogel can also
be tuned by addition of salt which causes electrostatic screening.
6.85 (e)
4
8
5
4
9
4
3
3
.74 (m)
.32 (e)
.20 (s)
4.88
4.26
3.22 (s)
2.88 (s)
2.27 (s)
Saiani and co-workers investigated the influence of sequence
design on self-assembly and hydrogelation for octapeptides
comprising alternating hydrophobic and charged residues, and
3.26 (s)
2.79 (s)
2.28 (s)
2.27 (s)
a
b
50
Key: e, equatorial; m, meridional; s, sharp. Strong peaks only.
hydrogels were observed for FEFEFKFK and FEFKFEFK. In
the case of our two peptides, the overall charge is the same;
Shear thinning is again observed after application of stress for
min; however, recovery is largely complete after 2 h (at high
frequency). This correlates to the fact that orientation of the gels is
not observed by SAXS.
therefore, in contrast to previous studies such as that on β-hairpin
2
46
peptides mentioned above, this cannot explain the difference in
branching in the fibrillar peptide networks. It is likely that the
distribution of charge is different due to conformational differ-
ences, in particular due to the cation-π interaction between
adjacent Fmoc and K residues in Fmoc-K(Boc)LV.
Summary and Discussion
It is remarkable that a small sequence difference, that is,
exchange of two residues, leads to dramatically different proper-
ties of Fmoc-VLK(Boc) and Fmoc-K(Boc)LV. A detailed mole-
cular level understanding of how differences in packing motif
influence self-assembly and ultimately macroscopic properties
such as gelation (and gel modulus) and flow alignment is still
lacking. Molecular modeling of isolated molecules suggested a
more compact conformation for Fmoc-K(Boc)LV with the lysine
side chain and Fmoc units forming two “arms” within the
molecule. Interaction between Fmoc and Boc-protected lysine
in Fmoc-K(Boc)LV may favor bending of the β-sheets and hence
less straight fibrils compared to Fmoc-VLK(Boc). This interac-
tion may be stabilized in part by hydrophobic interactions but
also by cation-π interactions, since the amide nitrogen has a
positive charge. The CD data show lower chiral ordering of
Fmoc-K(Boc)LV compared to Fmoc-VLK(Boc). Fuller model-
ing would require extensive simulation beyond the single molecule
level, considering different possible packing motifs and so on.
This is planned for future work.
To our knowledge, flow-induced orientational ordering of
Fmoc-peptide hydrogels has not previously been noted. We
checked birefringence of the gel formed by Fmoc-VLK(Boc),
and none was observed. This means that the alignment of fibrils
observed by cryo-TEM and SAXS is only local and there is no
long-range nematic order leading to birefringence. Nematic
ordering within gels has been reported for a de novo designed
5
1
peptide fragment. Nematic ordering within fluid peptide solu-
tions has also been reported for several natural and synthetic
peptides, as well as poly(ethylene glycol)/peptide conjugates as
5
2
summarized elsewhere. Observation of nematic ordering re-
quires sufficient fibril anisotropy, quantified in terms of diameter/
length at a given concentration, approximately according to the
4
2
53
Onsager criterion. It is an interesting question as to the condi-
tions which favor viscoelastic nematic fluid ordering as opposed
5
1
to nematic gel formation. Entanglements or branching effects
might predominate, depending on fibril persistence length.
The self-assembly motif may be compared to previous work on
4
1
Fmoc-tripeptides. In contrast to Fmoc-LLL, cryo-TEM reveals
that Fmoc-VLK(Boc) and Fmoc-K(Boc)LV form fibrils and not
nanotubes. Fmoc-K(Boc)LV is intriguing in that while it does not
take up Congo Red stain as readily as Fmoc-VLK(Boc), it does
show features in FTIR and Raman spectra associated with
β-sheet structure. In addition, the fiber X-ray diffraction pattern
reveals an interstrand spacing somewhat larger than usually
Other recent work on other self-assembling peptides has shown
that subtle sequence differences can substantially impact proper-
ties such as gel properties. The hydrogels of Fmoc-VLK(Boc)
0
4
have a modulus G >10 Pa. This is actually rather high compared
to previously reported values for Fmoc-peptide hydrogels and
0
other self-assembling peptide gels. Fmoc-FF hydrogels have G <
4
3
3
Pa (depending on frequency). The authors comment that
higher values observed previously in their group might be due to
kinetically trapped aggregates which reinforce the mechanical
6
(
44) Rajagopal, K.; Lamm, M. S.; Haines-Butterick, L. A.; Pochan, D. J.;
Schneider, J. P. Biomacromolecules 2009, 10(9), 2619–2625.
45) Hule, R. A.; Nagarkar, R. P.; Altunbas, A.; Ramay, H. R.; Branco, M. C.;
4
properties of the gels. However, a modulus>10 Pa for a 0.5 wt %
(
sample in water was reported in an earlier independent study of
Fmoc-FF self-assembly and hydrogelation. A modulus appro-
Schneider, J. P.; Pochan, D. J. Faraday Discuss. 2008, 139, 251–264.
(46) Branco, M. C.; Nettesheim, F.; Pochan, D. J.; Schneider, J. P.; Wagner,
N. J. Biomacromolecules 2009, 10(6), 1374–1380.
1
0
0
4
aching G = 10 Pa has been reported for an Fmoc-tyrosine
(
47) Schneider, J. P.; Pochan, D. J.; Ozbas, B.; Rajogopal, K.; Pakstis, L.;
7
,15
phosphatase hydrogel,
and naphthalene-dipeptide hydrogels
Kretsinger, J. J. Am. Chem. Soc. 2002, 124, 15030–15037.
(48) Pochan, D. J.; Schneider, J. P.; Kretsinger, J.; Ozbas, B.; Rajogopal, K.;
Haines, L. J. Am. Chem. Soc. 2003, 125, 11802–11803.
(49) Ozbas, B.; Kretsinger, J.; Rajogopal, K.; Schneider, J. P.; Pochan, D. J.
Macromolecules 2004, 37, 7331–7337.
(50) Saiani, A.; Mohammed, A.; Frielinghaus, H.; Collins, R.; Hodson, N.;
Kielty, C. M.; Sherratt, M. J.; Miller, A. F. Soft Matter 2009, 5(1), 193–202.
(51) Aggeli, A.; Nyrkova, I. A.; Bell, M.; Harding, R.; Carrick, L.; McLeish,
T. C. B.; Semenov, A. N.; Boden, N. Proc. Natl. Acad. Sci. U.S.A. 2001, 98(21),
8
have been reported with similar moduli. The influence of
sequence design within β- hairpin peptides (each strand compri-
sing a similar hydrophobic/polar alternating sequence with a
D
V PPT tetrapeptide β-turn) on hydrogel properties has also been
probed; in particular, substitution of a single lysine by other
residues changes the total charge on the peptide and enables more
11857–11862.
(
52) Hamley, I. W.; Castelletto, V.; Moulton, C. M.; Myatt, D.; Siligardi, G.;
Oliveira, C. L. P.; Pedersen, J. S.; Gavish, I.; Danino, D. Macromol. Biosci. 2010,
(
(
42) Ma, J. C.; Dougherty, D. A. Chem. Rev. 1997, 97, 1303–1324.
43) Tang, C.; Smith, A. M.; Collins, R. F.; Ulijn, R. V.; Saiani, A. Langmuir
10, 40-48.
(53) Hamley, I. W. Introduction to Soft Matter. Revised Edition; Wiley: Chichester,
2009, 25(16), 9447–9453.
2007.
Langmuir 2010, 26(7), 4990–4998
DOI: 10.1021/la903678e 4997

Article
Cheng et al.
0
00
-1
Figure 10. Dynamic shear moduli as a function of frequency: solid symbols, G ; open symbols, G . Black, as mounted; red, preshear of 50 s
for 2 min; green, 1 h after preshear; blue, 2 h after preshear. (a) 1 wt % Fmoc-VLK(Boc), (b) 2 wt % Fmoc-VLK(Boc), (c) 1 wt % Fmoc-
KLV(Boc), and (d) 2 wt % Fmoc-KLV(Boc).
1
observed. This points possibly to a distinct β-sheet packing struc-
ture instead of the conventional one, as exhibited, for example, by
Fmoc-VLK(Boc).
and medicine. The aligned fibrillar structure of Fmoc-VLK-
(Boc), and flow alignment properties, may be useful, for instance,
where an aligned medium is needed, for example, in controlling
5
4
In summary, our results show that sequence design, in parti-
stem cell morphology.
ε
cular use of (N -Boc)K, offers the ability to control intermolecular
Acknowledgment. This work was supported by EPSRC
Grants EP/F048114/1 and EP/G026203/1. We are grateful to
Steve Furzeland and Derek Atkins (Unilever, Colworth, U.K.)
for the cryo-TEM experiments.
interactions and hence ultimate hydrogel properties of simple
model tripeptides via differences in fibril self-assembly properties.
The tunable gel modulus may be useful in applications such as
regenerativemedicine, as it has previously been shown that Fmoc-
5
,6
peptides can serve as three-dimensional scaffolds for cell culture
among other applications as functional nanomaterials for biology
(
54) Mata, A.; Hsu, L.; Capito, R.; Aparicio, C.; Henrikson, K.; Stupp, S. I. Soft
Matter 2009, 5, 1228–1236.
4
998 DOI: 10.1021/la903678e
Langmuir 2010, 26(7), 4990–4998