Controlling morphology of peptide-based soft structures by covalent modifications

Article abstract of DOI:10.1002/psc.2411

Control of gross morphology of soft matter remains an area of continued interest. Towards this goal, this paper describes conjugation of mannose residues and introduction of thiol functionalities to diphenylalanine (FF) dipeptide, a fibrillating motif from amyloid-β peptide, as covalent modifiers of its solution-phase self-assembly process. It was found that covalent attachment of a single mannose residue to FF leads to the retention of tubular structures, whereas the conjugation of two mannose units, linked through a Lys residue, resulted in a dramatic change from tubular morphology to spherical structures. However, a similar switch to spherical objects could be achieved by introducing a thiol residue in the mono-mannosylated FF dipeptide. Interestingly, these glycopeptides also exhibited interaction with concanavalin A, thereby providing an indirect evidence for the availability of mannose units for the process of lectin-carbohydrate interaction in the self-organized state.

Full text of DOI:10.1002/psc.2411

Research Article
Received: 11 January 2012
Revised: 3 March 2012
Accepted: 8 March 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/psc.2411
Controlling morphology of peptide-
based soft structures by covalent
modifications
Nidhi Gour,a,c Apurba K. Barmana and Sandeep Vermaa,b*
Control of gross morphology of soft matter remains an area of continued interest. Towards this goal, this paper describes
conjugation of mannose residues and introduction of thiol functionalities to diphenylalanine (FF) dipeptide, a fibrillating
motif from amyloid-b peptide, as covalent modifiers of its solution-phase self-assembly process. It was found that covalent
attachment of a single mannose residue to FF leads to the retention of tubular structures, whereas the conjugation of two
mannose units, linked through a Lys residue, resulted in a dramatic change from tubular morphology to spherical structures.
However, a similar switch to spherical objects could be achieved by introducing a thiol residue in the mono-mannosylated FF
dipeptide. Interestingly, these glycopeptides also exhibited interaction with concanavalin A, thereby providing an indirect
evidence for the availability of mannose units for the process of lectin-carbohydrate interaction in the self-organized state.
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: diphenylalanine; mannose; self-assembly; vesicles; nanotubes; lectin
a stable, left-handed helix, via intramolecular hydrogen bonding,
which contributed to the aggregation process to reveal a tooth-like
Introduction
Defined self-assembly of molecular components, based on the
principles of non-covalent stabilization, could give rise to a
variety of structures of different shapes such as spherical micelles,
vesicles, nanofibers, toroids, and tubes in the nanoscale dimen-
sion [1–4]. Such processes are critically supported by interactions
such as hydrogen bonds, electrostatic interactions, hydrophobic
interactions, p–p stacking, and cation–p interactions, to name a
few, which offer sufficient stabilization to hold a soft structure
together [5–9]. Having achieved considerable success in
designing soft structures, efforts are being invested to gain
control over morphological transformations at the level of
supramolecular assembly. Success in such endeavors require the
ability to controllably manipulate self-assembly process in
solution or in solid state [10–13]. In the context of bio-inspired
self-organization, native or synthetic peptides offer significant
prospect in achieving size and shape control because of the
possibility of chemical modifications and side-chain functional
diversity, which directly impacts peptide conformation and
self-assembly [14–17].
gross morphology [22]. In another example, Zhang and coworkers
described self-assembled structures originating from a cone-shaped
peptide [23]. The latter self-assembled to afford spherical micelles,
which through fusion or elongation process lead to the formation
of nanopipe-like structures. These nanopipes gave rise to nanodonut
architectures upon bending, thereby providing a good example of
change in morphology.
A simple Phe-Phe (FF) dipeptide has interesting structural
manifestations in solution-phase self-assembly. It forms stiff
self-assembled nanotubes [24–28], could be aligned to grow
unidirectionally with the help of ferrofluid coating [29], and
undergoes a morphological change from tubular shape to
spherical architectures due to an introduction of a thiol group [30].
Given our interest in peptide-based soft structures [31–34] and our
experience in triggering morphological changes via chemical
approaches [34], we became motivated to investigate the effect of
carbohydrate conjugation to FF dipeptide. This conjugation was also
expected to impart water solubility. This paper describes our
Shape-selective organization of peptide or peptide conjugates
could be manipulated by the introduction of specific secondary
structural signatures, such as a-helices and b-pleated sheets
in peptide segments, or by employing unnatural building
blocks or linker units. The former gives an entry into preferred
three-dimensional orientations, whereas the latter affords
hybrid structures where a given unnatural constituent could exert
control over the gross architecture [15,18–21]. In a recent example,
Lee and coworkers described hybrids of ‘foldamer’ and ‘architecture’
to introduce a new shape-specific control process which is governed
by foldamer self-assembly. They demonstrated that a protected
hexamer of 2-aminocyclopentanecarboxylic acid was able to adopt
*
Correspondence to: Sandeep Verma, DST Unit of Excellence in Soft Nanofabrica-
tion, Indian Institute of Technology Kanpur, Kanpur-208 016 (UP), India. E-mail:
sverma@iitk.ac.in
a Department of Chemistry, Indian Institute of Technology Kanpur, Kanpur-208
016 UP, India
b DST Unit of Excellence in Soft Nanofabrication, Indian Institute of Technology
Kanpur, Kanpur-208 016 UP, India
c
Department of Inorganic, Analytical and Applied Chemistry, University of
Geneva–Sciences II, 30, quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland
J. Pept. Sci. (2012)
Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.

GOUR, BARMAN AND VERMA
synthetic efforts in conjugating mannose residues to FF dipeptide
and studies related to its effect on the gross morphology of the
dipeptide in solution.
128.2, 129.1, 136.9, 172.0, 172.5, 173.0. HRMS [M + Na]+ for
C27H34N2NaO9S calculated 585.1883, found 585.1886.
Diphenylalanine bis-mannose conjugate (3)
Material and Methods
A solution of acetylated Lys bismannose [35,36] (1 g, 1.09 mmol)
in dry DCM (20 ml) and DMF (10 ml) was cooled to 0 ꢀC, and
1-hydroxybenzotriazole (0.16 g, 1.18 mmol) was added, followed
by dicyclohexylcarbodimide (0.25 g, 1.21 mmol). After 1 h, TFA
salt of Phe-Phe methyl ester (0.51 g, 1.15 mmol) was added to
the reaction mixture, followed by triethylamine (0.2 ml,
1.42 mmol). After stirring for 12 h at room temperature, precipi-
tated dicyclohexylurea was filtered off. Filtrate was evaporated
under reduced pressure; the crude compound was dissolved in
100 ml DCM, washed with 1N HCl (2 Â 50 ml), 10% aqueous
NaHCO3 solution (3 Â 50 ml) and saturated brine solution
(2 Â 50 ml), followed by drying of the organic layer over anhy-
drous sodium sulfate. DCM was evaporated to obtain the crude
compound, which was further purified with silica gel column
(2%–4% MeOH gradient in DCM) to give protected derivative
of 3c, as a white solid. Yield 0.68 g (48%). This compound (0.2 g,
0.16 mmol) was dissolved in 10 ml of MeOH. Sodium methoxide
(0.09 g, 1.66 mmol) was added, and the reaction mixture was
stirred for 1 h at room temperature under nitrogen atmosphere.
The resulting solution was neutralized by cation-exchange resin
column that was activated prior to use by 1N HCl. The resulting
solution was then concentrated to dryness under reduced pres-
sure to yield acetyl deprotected conjugate. This conjugate
(0.14 g, 0.15 mmol) was then dissolved in a minimum volume
of methanol and NaOH (8 mg, 0.20 mmol) as its 1N aqueous
solution was added and was stirred for 12 h. The resulting
solution was neutralized by cation-exchange resin column that was
activated prior to use by 1N HCl. The neutralized solution was then
concentrated to dryness, washed with diethyl ether, and finally dried
in vacuum to yield diphenylalanine bis-mannose conjugate 3 as
a white solid. Yield 110 mg (73%). mp 74–77 ꢀC [a]D25 = +184.5º
General
Dichloromethane (DCM), N, N′-dimethylformamide (DMF), triethyla-
mine, and methanol were purchased from s. d. fine-chem ltd.
(Mumbai, India) and distilled following standard procedures prior
to use. N, N′-dicyclohexylcarbodiimide, 1-hydroxybenzotriazole, amino
acids, boron trifluoride ethyl etherate, mannose, perchloric acid, acetic
anhydride, and acetic acid were purchased from SISCO Research lab-
oratories Pvt. Ltd. (Mumbai, India), s.d. fine-chem ltd. (Mumbai, India),
Spectrochem Pvt. Ltd, (Mumbai, India), respectively, and used without
further purification. Concanavalin A (Con A) was purchased from
Sigma Aldrich (Steinheim, Germany). H and 13C (JEOL Ltd., Tokyo,
1
Japan) NMR spectra were recorded on a JEOL-JNM LAMBDA 400
model (JEOL Ltd., Tokyo, Japan) operating at 400 and 100 MHz,
respectively, and JEOL ECX-500 model (JEOL Ltd., Tokyo, Japan)
operating at 500 and 125 MHz, respectively. High resolution mass
spectra (HRMS) were recorded on Waters Q-Tof Premier Micromass
HAB 213 mass spectrometer (Waters Ltd., Manchester, U. K), using a
capillary voltage of 2.6–3.2 kV.
Synthesis and Characterization
Diphenylalanine mono-mannose conjugate (2)
N-hydroxy-succinimide ester of tetra-acetyl mannose mercapto
propionic acid (1.0 g, 1.87 mmol) [35] was dissolved in dry DCM
(15 ml). TFA salt of Phe-Phe methyl ester (0.84 g, 1.9 mmol)
dissolved in dry DMF (5 ml) was then added followed by addition
of triethylamine (0.3 ml, 2.2 mmol). Reaction mixture was stirred
for 12 h under nitrogen atmosphere and then evaporated to
dryness. Residue obtained was dissolved in DCM (100 ml) and
washed with 1N HCl (2 Â 50 ml) and saturated aqueous sodium
bicarbonate solution (2 Â 75 ml). Organic layer was dried over an-
hydrous sodium sulfate and evaporated to obtain a yellow oil,
which was purified using column chromatography on silica gel
(2%–5% MeOH gradient in DCM) to give protected derivative of
2b, as white solid. Yield 0.56 g (40%). This compound (0.5 g,
0.67 mmol) was dissolved in 15 ml of MeOH. Sodium methoxide
(0.37 g, 6.9 mmol) was added, and the reaction mixture was
stirred for 1 h at room temperature under nitrogen atmosphere.
The resulting solution was neutralized by cation-exchange resin
column that was activated prior to use by 1N HCl. The solution
was then concentrated to dryness under reduced pressure to
yield acetyl deprotected conjugate. This conjugate (0.3 g,
0.53 mmol) was then dissolved in a minimum volume of metha-
nol and NaOH (0.025 g, 0.63 mmol) as its 1N aqueous solution
was added. After stirring for 12 h, the reaction mixture was neu-
tralized by cation-exchange resin, concentrated to dryness,
washed with diethyl ether, and finally dried in vacuum to yield
diphenylalanine mono-mannose conjugate 2 as white fluffy hy-
groscopic solid. Yield 0.2 g (54%), [a]2D7 = +45.2º (c 0.1, CH3OH).
1H NMR (500 MHz, CD3OD, 25 ꢀC, TMS) d(ppm) 2.44–2.48(m,2H);
2.64–2.71(m,1H); 2.74–2.83(m, 2H); 2.90–3.00(m, 1H), 3 .05–3.11
(m, 1H); 3.12–3.19(m, 1H); 3.42–3.45(m, 1H); 3.56–3.58(dd, 2H,
J = 3.05 Hz, 8.40 Hz); 3.65–3.70(dt, 1H, J = 6.85 Hz, 12.25 Hz);
3.81–3.85(m, 2H); 4.60–4.65(m, 2H); 5.18–5.19(d, 1H, J = 3.05 Hz);
1
(c 0.2, methanol). H NMR (500 MHz, CD3OD, 25 ꢀC, TMS) d (ppm)
1.24–1.53(m, 6H), 2.46–2.53(m, 2H), 2.55–2.65(m, 2H), 2.78–2.88
(m, 2H), 2.90–3.01(m, 4H), 3.08–3.18(m, 4H), 3.57–3.61(m, 4H),
3.68–3.72(m, 2H), 3.87–3.93(m, 6H), 4.22–425(dd, 1H, J = 5.35 Hz,
9.20 Hz), 4.59–4.60(m, 2H), 5.25–5.26(d, 2H, J = 2.65 Hz), 7.15–7.26
13
(m, 10H). C NMR (125 MHz, CD3OD, 25 ꢀC, TMS) d (ppm) 23.0,
27.6, 28.7, 30.8, 31.3, 36.0, 36.1, 37.0, 37.5, 38.9, 53.5, 53.8, 54.2,
61.7, 67.7, 71.8, 72.4, 73.8, 86.2, 86.4, 126.4, 126.5, 128.1, 128.2,
129.1, 136.9, 171.8, 172.6, 172.85, 173.0. HRMS [M+ Na]+ for
C42H60N4NaO16S2 calculated 963.3343, found 963.3346.
Diphenylalanine thiolated mono-mannose conjugate (4)
A solution of tetraacetylated diphenylalanine mono-mannose
conjugate (0.8 g, 1.09 mmol) [35] in dry DCM (30 ml) and DMF
(5 ml) was cooled to 0 ꢀC, and 1-hydroxybenzotriazole (0.16 g,
1.18 mmol) was added, followed by dicyclohexylcarbodiimide
(0.2 5 g, 1.21 mmol). After 1 h, N-(3-aminopropyl)-3-(tritylthio)
propanamide (0.44 g, 1.09 mmol) was added, followed by addi-
tion of triethylamine (0.18 ml, 1.28 mmol). After the reaction
mixture was stirred for 12 h at room temperature, precipitated dicy-
clohexylurea was filtered off. Filtrate was evaporated under reduced
pressure; the crude compound was dissolved in 100 ml ethyl acetate
and washed with 1N HCl and (2 Â 50 ml), 10% aqueous NaHCO3
(3 Â 50 ml), and saturated brine solution (2 Â 50 ml), followed by
drying of the organic layer over anhydrous sodium sulfate. Ethyl
acetate was evaporated to obtain the crude compound, which was
13
7.08–7.25(m, 10H). C NMR (500 MHz, CD3OD, 25 ꢀC, TMS) d(ppm)
26.8, 35.9, 37.1, 53.6, 54.5, 61.5, 67.6, 71.9, 72.5, 73.8, 85.7, 126.4,
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J. Pept. Sci. (2012)

PEPTIDE-BASED SOFT STRUCTURES
further purified with a silica gel column (0.5%–3% MeOH gradient in
DCM) to give protected derivative of 4d (yield, 0.73 g, 60%). This
compound (0.2 g, 0.17 mmol) was dissolved in methanol (10 ml).
Sodium methoxide (0.09 g, 1.7 mmol) was added, and after 1 h of
stirring at room temperature under nitrogen atmosphere, reaction
was neutralized by cation-exchange resin. The resulting solution
was concentrated to dryness under reduced pressure to yield acetyl
deprotected conjugate. This compound (150 mg, 0.15 mmol) was
dissolved in 75% trifluoroacetic acid–DCM (5 ml) and stirred for 1 h
under N2 atmosphere. Solvent was evaporated at room temperature
by using vacuum, washed with diethyl ether (3 Â 10 ml). The solid
obtained was dissolved in methanol and passed through strong
anion-exchange resin; the solvent was evaporated under vac-
uum to obtain white solid product 4. Yield 102 mg (84%). mp
90–92 ꢀC; [a]2D5 = À105º (c 0.3, methanol). 1H NMR (500 MHz,
CD3OD, 25 ꢀC, TMS) d(ppm) 1H 1.53–1.56(t, 2H, J = 6.85 Hz),
2.44–2.51(m, 4H), 2.70–2.73(m, 2H), 2.78–2.84(m, 2H),
2.90–2.94(dd, 1H, J = 8.05 Hz, 13.75 Hz), 3.01–3.13(m, 7H),
3.57–3.65(m, 3H), 3.67–3.71(dd, 1H, J = 6.5 Hz, 11.45 Hz),
3.84–3.87(m, 2H), 4.47–4.5(t, 1H, J = 7.3 Hz), 4.53–4.56(dd, 1H,
J = 6.10 Hz, 8.80 Hz), 5.22(s, brd, 1H), 7.15–7.25(m, 10H). 13C
NMR (125 MHz, CD3OD, 25 ꢀC, TMS) d (ppm) 19.8, 26.9, 28.7, 35.8,
36.3, 36.4, 37.2, 37.5, 39.7, 54.9, 55.1, 61.6, 67.7, 71.8, 72.3, 73.8, 85.8,
126.5, 126.6, 128.2, 128.9, 129.1, 137.0, 171.7, 172.0, 172.6, 173.0.
HRMS [M + H]+ for C33H47N4O9S2 calculated 707.2784, found
707.2782.
illuminator and a rhodamine filter (540/625 nm). This filter
optimized visualization of rhodamine-treated (positive resolution)
compared with untreated (negative resolution) spherical structures
that are virtually invisible to this light. 10 mM rhodamine B dye solu-
tion in water was added directly to glycopeptide conjugate (1 mM)
and co-incubated for 12–16 h. 20 ml of this solution was spread on
a glass slide, dried at room temperature, and imaged under a
fluorescence microscope.
Congo red staining and birefringence
A 1ml stock solution of 2 was made in double distilled water at a
final concentration of 2 mM and incubated for 1 h. To this sample,
100 ml solution of 80% ethanol saturated with Congo red and
NaCl was added, and peptide solution was further incubated for
6 h. Birefringence was determined with a SZX-12 stereoscope
(Olympus, Hamburg, Germany) equipped with a polarizing stage.
Turbidimetric assay with Con A
Time-dependent turbidimetric assay were performed in a quartz
cell (1 cm  1 cm). Aliquots of Con A solution (300 ml, l.0 mg mlÀ1
)
dissolved in HEPES [4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid] buffer [10 mM, pH 7.4, containing CaCl2 (1 mM), MnCl2
(1 mM)] were diluted with the same HEPES buffer (1125 ml). Aque-
ous solutions of 1, 2, 3, and 4 (1 mM) were incubated for 12 h at
37 ꢀC. 10 ml of the pre-incubated solutions were added to Con A
solution, and UV–vis spectra of the mixture were recorded after
every 2 min. The absorbance of mixture was monitored by scan-
ning samples in a range of 250–600 nm and was recorded in
UV–vis spectrometer Varian Cary 100 Bio UV-Visible spectropho-
tometer(Palo Alto, California, USA).
Atomic force microscopy
Atomic force microscopy (AFM) was carried out in air with the use
of an Agilent Technologies AFM (5500 AFM/SPM) (Santa Clara,
California, USA) operating under the Acoustic AC mode. The sample
was mounted on the XY stage of the AFM, and the integral video
camera (NAVITAR, Model N9451A-USO6310233 with the Fibre-light
source, MI-150 high intensity illuminator from Dolan-Jenner Indus-
tries) (Boxborough, Massachusetts, USA) was used to locate the
regions of interest. Silicon nitride cantilevers with resonant fre-
quency of 150 kHz were used. The average dimension thickness,
width, and length of cantilever were approximately 2.0, 51, and
446 mm, respectively. The scanner model N9524A-USO7480132.
xml/N9520A-USO7480152.xml (Agilent Technologies, California,
USA) was calibrated and used for imaging. The images were taken
in air at room temperature, with the scan speed of 1.5–2.2 lines/s.
Data acquisition and analysis was carried out using Pico ViewW
1.4 and Pico ImageW Basic software, respectively. Fresh solutions
of glycopeptides (1mM) in appropriate solvents were prepared
and incubated at 37 ꢀC for appropriate time, prior to microscopy
investigations. A sample of 20ml aliquot was transferred onto
freshly cleaved mica surface or glass and was dried at room
temperature, followed by AFM imaging.
Fourier transform infrared spectroscopy
Samples for Fourier transform infrared (FTIR) analysis were pre-
pared by dissolving 3 in water at a final concentration of 1 mM.
FTIR spectra were recorded for fresh sample and sample after in-
cubation for 12 h at 37°C respectively after lyophilization. Lyoph-
ilized powder were examined as and as KBr pellets on a Bruker
Vector 22 FT IR spectrophotometer (Billerica, Massachusetts,
USA) operating from 400 to 4000 cm-1.
Thermogravimetric analysis
Solutions of 3 and 4 (5 mg mlÀ1) in 50% aqueous methanol were
aged for 12 h at 37 ꢀC and lyophilized. This sample was analyzed
in a Perkin-Elmer Pyris6 thermogravimetric analyzer (Waltham,
Massachusetts, USA). The rate of heating was 10 ꢀC minÀ1 under
nitrogen atmosphere.
Results and Discussion
Scanning electron microscopy
Lack of water solubility limits potential applications of FF
nanotubes. As a first step, we decided to modify this hydrophobic
dipeptide with carbohydrate moieties to improve its aqueous
solubility, as it has been demonstrated before that sugars have
the ability to solubilize lipophilic and nonpolar compounds, such
as naphthalene and biphenyl, in water [37,38]. Thus, FF (1) was
conjugated to mannose residue(s) (Scheme 1), which expectedly
rendered the conjugates water soluble with interesting
consequences on the self-assembled morphology. Mannose
residue was chosen for conjugation as a 2.6 Å resolution crystal
structure of a pea lectin-trimannoside complex suggested beneficial
A 20 ml aliquot of the glycopeptides solution (1 mM) was deposited
on copper stubs and dried at room temperature, which was then
gold-coated. Scanning electron microscopy (SEM) images were
recorded on FEI QUANTA 200 microscope (FEI Company, Hillsboro,
Oregon, USA) equipped with a tungsten filament gun, operating at
WD 10.6 mm and 20 kV.
Fluorescence microscopy
Dye-stained structures were examined under
a fluorescent
microscope (Leica DM2500M), provisioned with a fluorescence
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GOUR, BARMAN AND VERMA
Scheme 1. Methodology for synthesis of various diphenylalanine conjugates.
interactions between Phe-123 and C5 and C6 carbon atoms of a
mannose residue, through van der Waals and stacking interactions,
as being crucial for the recognition process [39].
Standard synthetic protocols were used to conjugate tetra-
acetylated mannose-1-mercaptopropionic acid to 1 to obtain
water-soluble diphenylalanine mono-mannose conjugate (2).
Figure 1. (a) SEM micrograph of FF (1) (1 mM, 50% aqueous methanol). Microscopy images of 2 (1 mM, water): (b) SEM image displaying fibrillar
structures on copper surface; (c) AFM micrograph on mica surface; and (d) Congo red-stained fibrillar structures showing greenish birefringence.
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PEPTIDE-BASED SOFT STRUCTURES
Dissolution of 2 in water exhibited a strong tendency for instan-
taneous gelation (5 mg mlÀ1, 8.9 mM). This gel when diluted with
water to a final concentration of 1 mM and incubated at 37 ꢀC for
24 h afforded long tubular structures as observed in SEM, AFM,
and optical microscopy (Figure 1(b–d)). It is likely that the man-
nose units help provide water solubility and remain decorated
at the outer walls of fibrillar structures for optimal interaction
with the aqueous medium. There was a distinct change in
morphology going from rigid tubular structures of 1 to fibrillar
aggregates observed for conjugate 2, which is reflective of subtle
interactions between phenylalanine side chain and mannose
residue which are able to morph the structures from tubular to
fibrillar aggregates.
This discernible change in morphology provided impetus to
explore the effect of conjugation of two mannose residues to
FF dipeptide. This synthesis was achieved by first appending
two mannose units to the a-amino and e-amino groups of Lys,
followed by conjugation with FF affording diphenylalanine
bis-mannose conjugate 3 (Scheme 1). A remarkable change in
the morphology was observed when 3 (1 mM in water) was
incubated for 12 h at 37 ꢀC. SEM micrographs revealed the forma-
tion of soft spherical structures with a diameter of 500–700 nm
(Figure 2(a)). These structures were also studied with fluo-
rescence microscopy after staining with rhodamine B and by AFM
(Figure 2(b and c)). However, self-assembly of Lys-Phe-Phe (KFF)
tripeptide alone (1 mM in water) revealed indiscrete structures
suggesting that conjugation of Lys alone has perhaps no role in
triggering such a morphological change [35].
Figure 4. Turbidimetric assay for 1 (black trace), 2 (blue trace), 3 (red
trace) and 4 (green trace) with Con A. Increase in absorbance at 400 nm
with time shows binding of Con A with different FF conjugates.
by carbohydrate-aromatic amino acid and/or carbohydrate-
carbohydrate interactions [36]. The latter interpretation was
supported by FTIR analysis of 3 before and after aggregation,
where the O–H stretching frequency shifted from 3384 to a more
broadened peak at 3300 cmÀ1, whereas the peak of O–H bending
in COOH also shifted from 1455 to a more broad peak at 1421 cm-1,
suggesting the presence of hydrogen bonding network in self-
assembled 3 [35].
Such a change in self-assembly of diphenylalanine bis-
mannose conjugate 3 might be governed by at least two factors:
first, destabilization of aromatic stacking in 1 and second,
An earlier report had suggested that conjugation of a
Cys residue to dipeptide 1 changes tubular morphology to
spherical structures. It was reasoned that Cys thiol side chain
Figure 2. Microscopic images of 3 (1 mM, water). (a) SEM image; (b) fluorescence micrograph stained with rhodamine B; and (c) AFM micrograph on
mica: topographic image (inset shows height profile of spherical aggregates).
Figure 3. Microscopic images of 4 (1 mM, 50% aqueous methanol). (a) AFM micrograph on glass surface; (b) SEM image after 12-h incubation display-
ing spherical structures; and (c) fluorescence micrograph of 4 stained with rhodamine B.
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GOUR, BARMAN AND VERMA
biomedical applications such as inhibition of cell–cell or patho-
gen–cell adhesion, gene delivery, drug delivery, glycan
microarrays, and cellular imaging [4,42–48]. In addition, many
groups have reported that carbohydrate-coated architectures
and microarrays show strong interaction with proteins and
lectins [49–55] and Escherichia coli type 1 pili [56].
Some of the mannose residues in all the mannosylated
derivatives of FF were expected to project outwardly from the
self-assembled structures because of their hydrophilic nature.
We decided to confirm whether mannose appendages are
indeed projected outwards through an assay exploiting carbohy-
drate-lectin interactions. Con A, a lectin, is known to selectively
recognize a-mannopyranoside and a-glucopyranoside at its four
binding sites. As mannose-coated ligands lead to aggregation of
Con A enhancing solution turbidity [45,48,49], we decided to use this
assay to study the interaction of 2, 3, and 4 with Con A to verify the
presence and availability of mannose residues on the surface of soft
structures. Increase in the absorbance, with respect to time, was
observed in cases of 2, 3, and 4 at 400 nm, indicating that self-
assembled tubes and spheres have mannose residues displayed at
the exterior of soft structures (Figure 4). It was also interesting to note
that conjugates 3 and 4 caused lectin aggregation to a similar level
as judged by the turbidity. This suggests that both 3 and 4 have sim-
ilar affinity, whereas 2 showed a higher turbidity providing an indi-
rect proof that mannose-coated fibers caused more aggregation of
Con A as compared with the spherical aggregates. In a control exper-
iment, FF alone was added to Con A, and no increase in the absor-
bance was observed, with respect to time, confirming that Con A
selectively interacts with mannose-containing self-assembled FF
structures.
Figure 5. Thermogravimetric analysis (TGA) of self-assembled structures
of 3 and 4.
gets oxidized to form a disulfide bridge, which facilitates fur-
ther closure of two-dimensional layers in nanotubes to form
compact spherical structures [30]. We decided to use this
strategy to exert morphological control in fibrillar aggregates
of 2 by introducing a thiol group in diphenylalanine mono-
mannose conjugate to afford thiolated derivative 4 (Scheme 1).
Interestingly, the self-assembly of 4 (1 mM, 50% aqueous
methanol) revealed a switch from fibrillar structures to spher-
ical aggregates, as confirmed by AFM, SEM, and fluorescence
microscopy (Figure 3(a–c)).
There is considerable interest and scope for designing objects
that display multiple carbohydrate residues to invoke multivalent
interactions [40–42]. Self-assembled carbohydrate display
systems could also find use in certain niche areas concerning
Thermogravimetric analysis of spheres of 3 and 4 showed that
spheres formed by 3 are more stable than 4. It indicated that
whereas spheres formed by 4, thiolated peptide having one
mannose residue, showed continuous weight loss and are stable
Scheme 2. Overview of morphological transformations in diphenylalanine nanotubes subsequent to chemical modifications.
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J. Pept. Sci. (2012)

PEPTIDE-BASED SOFT STRUCTURES
till ~230 ꢀC, spheres of 3, containing two mannose residues,
showed only ~10% weight loss till ~240 ꢀC and continuous
weight loss thereafter (Figure 5).
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Conclusions
In conclusion, we have conferred water solubility to FF dipeptide by
mannose conjugation, and with this approach, we were also able to
exert morphological changes in the self-assembled structures
(Scheme 2). Furthermore, the display of mannose units and their
interaction with lectin Con A raises the possibility of invoking spe-
cific recognition at the surface of these soft structures. Future
endeavors will include further refinement in the design of these
self-assembled structures to utilize them as delivery vehicles and
as soluble glycan arrays for glycomics applications.
Acknowledgements
NG thanks IIT Kanpur for a pre-doctoral fellowship, and AKB
thanks CSIR for a senior research fellowship. This work is sup-
ported by Department of Science and Technology, India, (SV)
and by DST Unit of Excellence on Soft Nanofabrication at IIT
Kanpur.
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