Exploring the self-assembly of glycopeptides using a diphenylalanine scaffold

Article abstract of DOI:10.1039/c1ob05071k

Diphenylalanine, a key building block for organic nanotechnology, forms discrete, rigid and hollow nanotubes that are assembled spontaneously upon their dilution from organic phase into aqueous solution. Here we report the efficient preparation of several

Full text of DOI:10.1039/c1ob05071k

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PAPER
Exploring the self-assembly of glycopeptides using a diphenylalanine scaffold†
Rinat Roytman,‡a Lihi Adler-Abramovich,‡b K. S. Ajish Kumar,a Ting-Chun Kuan,c Chun-Cheng Lin,c Ehud
Gazit*b and Ashraf Brik*a
Received 13th January 2011, Accepted 17th May 2011
DOI: 10.1039/c1ob05071k
Diphenylalanine, a key building block for organic nanotechnology, forms discrete, rigid and hollow
nanotubes that are assembled spontaneously upon their dilution from organic phase into aqueous
solution. Here we report the efficient preparation of several S-linked glycosylated diphenylalanine
analogues bearing different monosaccharide, di-saccharide and sialic acid residues. The self-assembly
studies revealed that these glycopeptides adopted various structures and glycosylation could be a tool
to manipulate the self-assembly process. Moreover, the solubility of these analogues was found to be
much greater than diphenylalanine, which could open new applications based on these nanostructures.
Introduction
The formation of ordered nanostructures by simple organic build-
ing blocks is a new and promising direction in nanotechnology.1
The diphenylalanine peptide 1 (Fig. 1), is considered as a
key structural element as this small building block and its
derivatives form spherical and tubular nanostructures that can
be deposited using various techniques.2 Moreover, it was also
demonstrated that the addition of a hydrophobic group such
as the 9-fluorenylmethoxycarbonyl (Fmoc), to the N-terminus of
diphenylalanine produced more rigid and branched fibrils that are
similar to the amyloid fibrils in longer polypeptides.3 In addition,
Fmoc-diphenylalanine creates material with the characteristic of
a hydrogel with remarkable mechanical rigidity for the potential
use in various biotechnological applications.4 Nevertheless, the
Fig. 1 The diphenylalanine scaffold and its glycosylated analogues used
in this study.
low solubility of diphenylalanine and its analogues in aqueous
media has hampered several potential applications and studies
aimed at understanding the mechanism of the self-assembly
at the molecular level. Inspired by the role of glycosylation
ample, current data suggest that O-linked-b-N-acetylglucosamine
(O-GlcNAc) has a significant role in normal function and the
pathology of neurodegenerative disorders. In this regard, it has
in the physiochemical and biological function of various bio-
been reported that tau protein is extensively O-GlcNAcylated, in
macromolecules e.g. lipids, peptides and proteins,5 we reasoned
the healthy adult brain.6 On the other hand, the phosophorylated
that glycosylation of diphenylalanine could have an impact on its
structure of tau causes it to aggregate into paired helical filaments
solubility and self-assembly properties.
in the cytoplasm, which is one of the hallmarks of Alzheimer’s
disease.6 In another example, it has been found that O-linked
The effect of glycosylation on the self-assembly of peptides and
proteins involved in health and diseases has been reported. For ex-
monosaccharide can affect the coil to b-structural transition of
the prion peptide.7 While the O-linked a-N-acetylgalactosamine,
aBen-Gurion University of the Negev, Department of Chemistry, Beer Sheva,
(a-GalNAc) at Ser 135, was found to suppress amyloid formation
of the prion peptide, PrP(108-144), the same sugar at Ser132 shows
Israel. E-mail: abrik@bgu.ac.il; Fax: (+972) 8-647-2943; Tel: (+972) 8-
646-1195
bTel-Aviv University, Department of Molecular Microbiology and Biotech-
nology, Tel Aviv, Israel. E-mail: ehudg@post.tau.ac.il; Fax: (+972) 3-640-
8475; Tel: (+972) 3-640-9030
opposite effect. Moreover, replacement of the a-GalNAc to b-
GlcNAc did not lead to the same results, indicating that this effect
is sugar specific.
The study of the self-assembly properties of amphiphilic systems
based on lipid and peptides has also been investigated.8 For
example, it has been recently reported that glycosylation of fatty
acids comprised of 18 carbons with a variety of monosaccharides
cDepartment of Chemistry, National Tsing Huan University, Hsinchu, Taiwan
† Electronic supplementary information (ESI) available: Synthesis of pep-
tides, HPLC, full mass spectrometry analysis of precursors and products
and NMR analysis of all building blocks. See DOI: 10.1039/c1ob05071k
‡ Authors contributed equally.
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(b-D-glucose, b-D-galactose and b-D-mannose) promotes the
formation of lipid nanotubes. Notably, only the b-D-glucose
has triggered the self-assembly process.9 In another interesting
study, it was found that carbohydrate (b-D-galactose and a-D-
mannose) conjugate Rod-Coil amphiphiles were found to self-
assemble in different supramolecular structures depending on the
molecular architecture.10 In a related study, glycosylation was
also reported to alter the aggregation kinetics of an amyloid-
forming model peptide in a site-specific manner.11 Together, these
important studies support an important role for glycosylation
in self-assembly processes. However, most of these studies were
limited to probe the effect of monosaccharide on a particular
scaffold and the use of diverse carbohydrate structures to examine
their influence on the self-assembly and solubility properties has
not been fully addressed. Herein, we report the design, synthesis
and characterization of different S-linked glycosylated dipheny-
lalanine analogues bearing carbohydrates of varying length and
complexity (Fig. 1) and the influence of these saccharides on the
self-assembly, morphology and the solubility of diphenylalanine
peptides.
to nucleophilic substitution reaction with b-GlcNAc thiol12 to
provide corresponding S-linked glycopeptides in the protected
form.13 Subsequent treatment with hydrazine in DMF to remove
the acetyl protecting groups followed by cleavage from resin using
TFA yielded the S-b-GlcNAc diphenylalanine 2 (Scheme 1A).
Similar procedure was then adopted, however using a-GlcNAc
thiol14 as a nucleophile to generate S-a-GlcNAc diphenylalanine
3 (Scheme 1B). Both glycopeptides were purified by using reverse
phase HPLC to give, after lyophilization, the desired products in
27–30% isolated yield, which were further analyzed for structure
integrity by mass spectrometry (Fig. 2) and NMR (ESI†).
Results and discussion
The low solubility of the diphenylalanine and its analogues
in aqueous media has hampered several potential applications
and studies aiming at understanding the mechanism of the self-
assembly at the molecular level. Thus, we wanted to examine
whether we could enhance diphenylalanine solubility while re-
taining its self-assembly properties. Our endeavor to explore the
effect of glycosylation on the diphenylalanine self-assembly and
solubility started with the introduction of the monosaccharide,
b-GlcNAc, to generate glycosylated diphenylalanine 2. To achieve
this goal we prepared diphenylalanine using Fmoc-solid phase
peptide synthesis (Fmoc-SPPS) on 2-chlorotrityl resin (Scheme
1). To allow for a straightforward N-terminus glycosylation,
the diphenylalanine was condensed with bromoacetic acid using
DIC. The resultant bromo-dipeptide derivative was then subjected
Fig.
2
Analytical HPLC traces and ESMS spectra of the pure
S-glycopeptides (A) S-b-GlcNAc diphenylalanine (B) S-a-GlcNAc
diphenylalanine (C) S-a-glucopyranosyl-(1→3)-D-glucosamine dipheny-
lalanine (D) S-Neu5Ac diphenylalanine. FF stands for diphenylalanine.
Diphenylalanine often requires the addition of organic solvent
or heating in order to dissolve it in aqueous media.1,2 The
fusion of b-GlcNAc to the diphenylalanine peptide to generate
glycosylated diphenylalanine 2 enabled the glycopeptide to be
Scheme 1 Synthesis of S-glycopeptides 2–6. The synthesis of glycopeptide
4 was carried out in a similar manner to glycopeptide 3, however using
Rink amide resin.
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a slightly more water soluble than the parent diphenylalanine
peptide, however this glycopeptide also produced insoluble self-
assembled structures after a short period of incubation. Notably,
the morphology of the nanostructures in a saturated glycosylated
diphenylalanine 2 solution in water is different from the assemblies
formed by the diphenylalanine peptide. While diphenylalanine
1 self-assembles into discrete tubular structures2 (Fig. 3a–b),
glycosylated diphenylalanine 2 forms bundles of fibers as was
found by Transition electron microscopy (TEM) (Fig. 3c) as well
as by Scanning electron microscopy (SEM) micrographs (Fig. 3d).
Glycosylated diphenylalanine 2 was also dissolved in dimethyl
sulfoxide (DMSO) then diluted in water, nevertheless, no ordered
nanostructures were observed as analyzed by electron microscopy.
saturated water solution of glycosylated diphenylalanine 3 enabled
the formation of similar elongated discrete assemblies (Fig. 3g–h).
The replacement of the C-terminal acid in amide derivative 4,
did not lead to changes in the morphology of the self-assembled
structure.
One possible explanation of the differences of self-assembly
structures between the S-a-GlcNAc diphenylalanine 3 and S-b-
GlcNAc diphenylalanine 2 could be attributed to the position
occupied by the sugar unit in the supramolecular structure. In
glycopeptide 3, it is possible that the sugar unit is pointing towards
the channel that could be formed in a similar manner to the
diphenylalanine case,15 thus leaving the interaction between the
aromatic residues intact and hence the ability to self assemble. On
the other hand, the change in the configuration in glycopeptide
2 could place the sugar unit in a different position, which could
interfere with the intramolecular aromatic stacking and disrupts
self-assembly.
Our results on the self-assembly studies with S-b-GlcNAc glyco-
sylated diphenylalanine 2 and S-a-GlcNAc glycosylated dipheny-
lalanine 3 triggered us to prepare a glycosylated diphenylalanine
analogue with a disaccharide unit bearing the a-GlcNAc, as we
found that the a-configuration is preferential for the self-assembly
process. This would give us the opportunity to examine how a more
complex glycan would affect diphenylalanine self-assembly and
solubility. For this goal, we synthesized a novel a-glucopyranosyl-
(1→3)-D-glucosamine thiol 10 from corresponding acceptor 13
and donor 14. The acceptor, 2-azo-4,6-O-benzylidene-2-deoxy-a-
D-glucopyranosyl triisopropylsilane 13 was synthesized from the
known precursor 12,16 while the donor 2,3,4,6-tetra-O-benzoyl-a-
D-glucopyranosyl trichloroacetimidate 14 was prepared according
to the reported synthesis from D-glucose.17 The acceptor and
the donor were subjected to Schmidt glycosylation18 to yield
the disaccharide that on global deprotection followed by per-
acetylation afforded the glycosyl azido-thioacetate 15. Reaction
of azido-thioacetate 15 with PPh3 promoted thiazoline forma-
tion by an aza-Wittig reaction followed by acidic hydrolysis to
furnish a-glucopyranosyl-(1→3)-D-glucosamine thiol 10 (Scheme
2). With 10 in hand, we then followed the reaction steps
shown in Scheme 1C to afford the S-a-glucopyranosyl-(1→3)-
D-glucosamine diphenylalanine 5 in 20% isolated yield.
Fig. 3 Self assembled nanostructures of diphenylalanine and glycopep-
tide 2 and 3 after two hours of incubation at concentration of 6 mM. a–b)
TEM and SEM micrograph respectively of diphenylalanine dissolved in
HFIP/water; c–d) TEM and SEM micrograph respectively of glycosylated
diphenylalanine 2 dissolved in water; e–f) TEM and SEM micrograph
respectively of glycosylated diphenylalanine 3 dissolved in DMSO and
water demonstrating the ordered nanostructures; g–h) TEM and SEM
micrograph respectively of glycosylated diphenylalanine 3 dissolved in
water demonstrating the ordered nanostructures.
Scheme 2 a) i. TIPS-SH, TMSOTf, CH2Cl2, Et2O, -20 to 23 ◦C, 5 h,
74%; ii. NaOMe, MeOH, 23 ◦C, 45 min, 85%; iii. PhCH(OMe)2, CSA,
THF, 60 ◦C, 4 h, 67%; b) i. TMSOTf, 13, CH2Cl2, -78 ◦C to r.t., 4 h, 91%;
ii. NaOH, MeOH, H2O, 23 ◦C, 18 h; iii. Ac2O, DMAP, pyr, 23 ◦C, 24 h,
57% over two steps; c) i. PPh3, THF, 23 ◦C, 20 h; ii. TFA, MeOH, H2O,
8 h, 0 ◦C, 66% over two steps.
Interestingly, the addition of a-GlcNAc thiol as a nucleophile to
generate S-a-GlcNAc diphenylalanine 3 enabled a self assembly
process and the formation of assemblies similar to those formed
by diphenylalanine peptide, however with a similar solubility
profile to glycopeptide 2. Electron microscopy micrographs depict
the elongated, discrete, rigid tubular structures (Fig. 3e–f). A
We next examined the solubility and self-assembly properties of
glycosylated diphenylalanine 5. Notably, this glycopeptide did not
require heating or the addition of any organic solvent and gave
a very clear solution, even after 24 h of incubation, due to the
high water-solubility of this glycopeptide. On the other hand, this
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glycopeptide self-assembles to form elongated fibrils. However, the
diameter of these nanostructures is approximately 10 nm, lower
than those formed by glycosylated diphenylalanine 3, as was found
in the electron microscopy images (Fig. 4a).
to present, multivalently, the sialic acid containing glycan.21,22
Thus, the introduction of a new simpler scaffold that could be
potentially used to present sialic acid containing glycan could be
beneficial in various applications.
Recently, we have developed a S-alkylation strategy to synthe-
size S-linked a-(2→9)-octasialic acid with exclusive a-S-glycosidic
bond formation.23 By in situ selective deprotection of S-acetate
in compound 1124 followed by S-alkylation, the epimerization of
anomeric sulfur could be avoided. Thus, to achieve the synthesis
of the diphenylalanine sialic acid derivative we used compound
11 for the S-alkylation step, in the presence of diethylamine
(Scheme 1D). Subsequently, the fully protected glycopeptide was
cleaved from resin using TFA followed by saponification to
afforded S-Neu5Ac-diphenylalanine 6. With glycopeptide 6 in
hand, we then focused on the self-assembly analysis. Similar
to 5, glycosylated diphenylalanine 6 was also highly water-
soluble. However, after its dissolution in water, glycosylated
diphenylalanine 6 self assembled to form elongated and curved
discrete nanostructures, the diameter of the fibrils is approximately
100 nm, as can be seen by TEM and SEM analysis (Fig. 5a–b). The
nanostructure obtained in this case resembled the ones obtained
by diphenylalanine 1 and the a-glycosylated diphenylalanine 3
in their discrete nature. Contrary to glycopeptide 1 and 3, this
glycopeptide was highly soluble and gave a clear solution even after
24 h.
Fig. 4 Glycosylated diphenylalanine 5 self-assembles into ordered
nano-structures after two hours of incubation at concentration of 6 mM.
a. TEM micrograph of the assemblies. b. FTIR analysis of the assemblies
indicating a b-sheet conformation with a major minimum peak at 1639
cm-1.
Next we examined the nature of the secondary structures of
the nano-assemblies using FTIR spectroscopy. The wavenumber
amide I bands of diphenylalanine 5 were located with a major
minimum peak at 1639 cm-1 (Fig. 4b). This vibrational peak is
consistent with supramolecular organization of a peptide in a
secondary structure of b-sheet conformation and consistent with
the electron microscopy data described above.19 These assemblies
show ultrastructural similarity to b-sheet rich amyloidal fibrillar
structures. The well-studied amyloidal assemblies indeed appear
as thin entangled nano-fibrils with a diameter of 7–10 nm rich
with b-sheet secondary structures as observed by IR spectroscopy.
Considering the biological importance of sialic acid, its unique
chemical structure and its potential uses in drug development, we
decided to study the effect of this sugar unit on the self-assembly
and solubility of diphenylalanine. Sialic acid is a monosaccharide
with nine-carbon backbone and negatively charged carboxylate
group and typically is linked to the outermost unit of the
glycoproteins. It plays vital roles in many physiological and
pathological processes and has been used in the design of various
carbohydrate-based antigens for vaccine development and various
enzyme inhibitors.20 In this regard, several templates were designed
Fig. 5 Glycosylated diphenylalanine 6 self-assembles into ordered
nano-structures after two hours of incubation at concentration of 6 mM.
a–b. TEM and SEM micrograph respectively of the nanostructures.
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Bromoacetylation
Conclusions
A preactivated solution of bromoacetic acid (278 mg, 2 mmol)
with DIC (0.37 mL, 2.4 mmol) in DMF (1.70 mL) for 15 min at 0
◦C was added to the resin (77 mg, 0.1 mmol) with dipeptide and
shaken well for 1 h. Resin was washed with DMF (¥3).
We have shown that the introduction of different carbohydrate
units into the diphenylalanine motif led to changes in the morphol-
ogy of the self-assembled nanostructures. While a-glycosylated
diphenylalanine 3 enabled the formation of assemblies simi-
lar to those formed by diphenylalanine 1, the b-glycosylated
diphenylalanine 2 gave no ordered nanostructures. On the other
hand, the glycopeptide bearing disaccharide unit self-assembles
to form elongated ordered nanostructure, however with different
diameter than the diphenylalanine 1, a-glycosylated peptide 3
and S-Neu5Ac-diphenylalanine 6. We have also found that the
addition of the carbohydrate moiety to the diphenylalanine
building block, in particular the disaccharide and sialic acid
units, have significantly increased the solubility of this peptide
in water to initiate the self-assembly process. To best of our
knowledge, this is the first example that explores variations of
different carbohydrate units on the self-assembly of a peptidic
scaffold. Taken together, our results could pave the way for
new biotechnological applications based on the diphenylalanine
motif and for better understanding of the self-assembly pro-
cess at the molecular level. For example, these glycopeptide
derivatives could be applied in those cases in which the use of
organic solvents should be avoided such as drug delivery nano-
assemblies, imaging agents encapsulating ferromagnetic elements,
hydrogels for slow drug release and tissue engineering, antibac-
terial activity,25 and as scaffold to present multivalently complex
carbohydrates.
General procedure for glycosylation of 8, 9, 10
The 1-thiosaccharide (5 equiv) was dissolved in DMF (4 mL), and
was added to the resin (77 mg, 0.1 mmol) followed by the addition
of TEA (133 mL, 9 mmol). The resin was shaken for 5 h and
washed with DMF (¥3).
Acetyl deprotection
The resin was treated with a solution of 4 mL of DMF/hydrazine
(6 : 1) for 2.5 h, and washed with DMF (¥3). The procedure was
repeated twice.
Cleavage
A mixture of TFA, triisopropylsilane and water (95 : 2.5 : 2.5) was
added to the dried peptide-resin and the reaction mixture was
shaken for 1.5 h at r.t. The resin was removed by filtration and
was washed with TFA (2 ¥ 2 mL). TFA was evaporated and the
residue was dissolved in 25% acetonitrile–water and lyophilized.
Peptide analysis and purification of 2, 3, 5
Analytical RP-HPLC was performed on a Thermo instrument
(Spectra System p4000) using an analytical column (Jupiter 5
micron, C18, 300A 150 ¥ 4.6 mm) and a flow rate of 1.2 mL min-1.
Preparative RP-HPLC was performed on an ECOM instrument
using a preparative column (Jupiter 5 micron, C18, 300A, 250 ¥
10 mm) and a flow rate of 25 mL min-1. Commercial peptide
reagents were used without further purification. Buffer A: 99.9%
water + 0.1% TFA, Buffer B: 99.9% ACN + 0.1% TFA.
Experimental
1H and 13C NMR spectra were recorded on a Bruker AMX-
500 MHz spectrometer. An internal standard, and J values are
given in Hz. Mass of the materials was analyzed with LCQ
Fleet Ion Trap (Thermo Scientific). Flash column chromatography
was performed with silica gel (60–100 mesh). The reactions
were carried out in oven-dried glassware under dry argon.
Dichloromethane, diethyl ether, methanol and THF were dried
before use. Fmoc-Phe-OH was purchased from Novabiochem and
coupling reagents from Luxembourg Biotechnologies. Analytical
thin-layer chromatography (TLC) was performed using thin layer
chromatography on pre-coated plates (0.25 mm, silica gel 60 F254).
Compound spots were visualized by UV light (254 nm) and were
stained with phosphomolybdic acid.
S-b-GlcNAc diphenylalanine (2)
1H NMR (500 MHz, DMSO-D6, 10% TFA-D) d 8.36 (d, J = 7.8
Hz, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.71 (d, J = 9.2 Hz, 1H), 7.23–
7.10 (m, 10 H), 4.50 (dd, J = 9.3, 4.6 Hz, 1H), 4.40 (dd, J = 8.7, 5.4
Hz, 1H), 4.31 (d, J = 10.3 Hz, 1H), 3.60 (dd, J = 11.9, 1.6 Hz, 1H),
3.48 (t, J = 10.3 Hz, 1H), 3.37 (dd, J = 11.9, 5.6 Hz, 1H), 3.24 (d,
J = 14.4 Hz, 1H), 3.20–3.16 (m, 2H), 3.02 (m, 3H), 2.94 (dd,
J = 13.9, 4.6 Hz, 1H), 2.88 (dd, J = 13.8, 8.7 Hz, 1H), 2.70 (dd, J =
13.8, 9.3 Hz, 1H), 1.75 (s, 3H); 13C NMR (126 MHz, DMSO-D6,
10% TFA-D) d 172.8, 171.3, 169.6, 168.9, 137.8, 137.6, 129.5,
129.4, 128.5, 128.3, 126.7, 126.5, 83.8, 81.4, 75.4, 70.6, 61.4,
54.6, 53.8, 53.7, 37.9, 36.9, 32.9, 23.0; ESI-MS: Calculated for
C28H35N3O9S: 589.21 Da [M+], Observed: 589.1 Da [M+H].
SPPS
Solid-phase chemistry was carried out in syringes, equipped with
teflon filters purchased from Torviq according to the Fmoc-
strategy (Fmoc-SPPS) on 2-chlorotrityl resin (loading 1.3). The
resin was pre-swollen in DCM followed by coupling Fmoc-Phe-
OH to 2-chlorotrityl chloride resin using diisopropylethylamine
(DIEA) in dichloromethane. The Fmoc group was removed with
20% piperidine in DMF. Peptide bond with second Fmoc-Phe-OH
was formed using DIEA in the presence of 1-hydroxybenzotriazole
(HOBt) and O-benzotriazole-N,N,N¢,N¢-tetramethyl-uronium-
hexafluoro-phosphate (HBTU).
S-a-GlcNAc diphenylalanine (3)
1H NMR (500 MHz, DMSO-D6, 10% TFA-D) d 8.33 (d, J =
6.6 Hz, 1H), 8.02 (d, J = 8.6 Hz, 1H), 7.88 (d, J = 5.3 Hz, 1H),
7.24–7.11 (m, 10H), 5.36 (d, J = 5.1 Hz, 1H), 4.50 (dd, J = 9.0, 4.7
Hz, 1H), 4.40 (dd, J = 8.6, 5.4 Hz, 1H), 3.81 (dd, J = 10.6, 5.1 Hz,
1H), 3.67–3.63 (m, 1H), 3.62–3.59 (m, 1H), 3.46 (dd, J = 11.9, 5.6
Hz, 1H), 3.42–3.38 (m, 1H), 3.13 (t, J = 8.8 Hz, 1H), 3.07–3.01
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(m, 3H), 2.93 (dd, J = 13.8, 4.6 Hz, 1H), 2.88 (dd, J = 13.9, 8.6
Hz, 1H), 2.67 (dd, J = 14.0, 9.0 Hz, 1H), 1.79 (s, 3H); 13C NMR
(126 MHz, DMSO-D6, 10% TFA-D) d 173.0, 171.2, 170.2, 168.6,
137.9, 137.8, 129.7, 129.5, 128.6, 128.4, 126.8, 126.6, 83.1, 73.9,
71.1, 70.9, 61.0, 54.3, 54.0, 53.8, 38.1, 37.0, 32.3, 22.7; ESI-MS:
Calculated for C28H35N3O9S: 589.21 Da [M+], Observed: 589.1 Da
[M+H].
2, 3, were also dissolved in DMSO at a concentration of 60 mM
then diluted in pure water at a concentration of 6 mM.
Transmission Electron Microscopy: After two hours and 24 h,
10 mL the nanostructures solutions were loaded on carbon coated
copper grid, dried and stained with uranyl acetate, followed by
imaging.
Scanning Electron Microscopy: After two hours and 24 h, 10
mL of the nanostructures solutions were deposited on slides, and
then coated with gold. SEM measurements were performed on a
JSM JEOL 6300 SEM operating at 5 kV.
S-a-glucopyranosyl-(1→3)-D-glucosamine diphenylalanine (5)
1H NMR (500 MHz, DMSO-D6, 10% TFA-D) d 8.35 (d, J =
7.8 Hz, 1H), 8.04 (d, J = 8.5 Hz, 1H), 7.63 (d, J = 6.9 Hz, 1H),
7.23–7.15 (m, 10H), 5.39 (d, J = 5.3 Hz, 1H), 4.49–4.45 (m, 1H),
4.39–4.33 (m, 2H), 3.91 (dt, J = 11.0, 5.6 Hz, 1H), 3.67–3.63 (m,
2H), 3.45 (dd, J = 11.9, 5.4 Hz, 1H), 3.33 (t, J = 9.5, 1H), 3.28–
3.24 (m, 1H), 3.17–3.13 (m, 1H), 3.12–2.88 (m, 9 H), 2.85 (dd, J =
Fourier Transform Infrared Spectroscopy: infrared spectra were
recorded using Nicolet Nexus 470 FT-IR spectrometer with DTGS
detector. Nanostructure solution sample were dried on polyethy-
lene card to form thin film. The nanostructures deposits were
resuspended with D2O and dried. The resuspension procedure
was repeated twice to ensure maximal hydrogen to deuterium
exchange. The measurements were taken using a 4 cm-1 resolution
and 1000 scans averaging. The transmittance minima values were
determined by OMNIC analysis program (Nicolet).
13.9, 8.9 Hz, 1H), 2.64 (dd, J = 13.8, 9.0 Hz, 1H), 1.75 (s, 3H); 13
C
NMR (126 MHz, DMSO-D6) d 171.6, 169.8, 168.8, 166.9, 136.5,
136.4, 128.2, 128.0, 127.1, 126.9, 125.4, 125.1, 101.5, 81.4, 79.2,
76.1, 75.4, 72.2, 68.9, 67.6, 60.0, 59.3, 52.6, 52.5, 51.3, 40.6, 36.6,
35.5, 30.6, 21.5; ESI-MS: Calculated for C34H45N3O14S: 751.80 Da
[M+], Observed: 751.2 Da [M+H].
Acknowledgements
The work was supported by the Edmond J. Safra Foundation
(A.B).
Synthesis of glycopeptide 6
Procedure for glycosylation of 11. Protected Neu5Ac-thiol
(309 mg, 0.56 mmol) was dissolved in DMF (1.17 mL) and was
added to the resin (230 mg, 0.30 mmol). To this, freshly distilled
diethylamine (350 mL, 3.37 mmol) was added, shaken for 5 h and
washed with DMF (¥3).
Notes and references
1 (a) X. Yan, P. Zhu and J. Li, Chem. Soc. Rev., 2010, 39, 1877; (b) S.
Zhang, Nat. Biotechnol., 2003, 21, 1171; (c) M. R. Ghadiri, J. R. Granja,
R. A. Milligan, D. E. McRee and N. Khazanovich, Nature, 1993, 366,
324; (d) X. Gao and H. Matsui, Adv. Mater., 2005, 17, 2037; (e) G.
A. Silva, C. Czeisler, K. L. Niece, E. Beniash, D. A. Harrington, J. A.
Kessler and S. I. Stupp, Science, 2004, 303, 1352.
Acetyl and methylester hydrolysis. After the cleavage step the
crude product was dissolved in methanol and the pH was adjusted
to ~12 by the addition of 1 M NaOH (aq), stirred for 24 h at
ambient temperature. After completion of the reaction on HPLC,
0.5 M HCl was added dropwise to adjust the pH to 4. The reaction
mixture was concentrated, the crude product was dissolved in
acetonitrile–water and HPLC purification as described above
afforded the glycopeptide 6.
2 (a) M. Reches and E. Gazit, Science, 2003, 300, 625; (b) Y. Song, S. R.
Challa, C. J. Medforth, Y. Qiu, R. K. Watt, D. Pena, J. E. Miller, F.
van Swol and J. A. Shelnutt, Chem. Commun., 2004, (9), 1044; (c) L.
Adler-Abramovich, D. Aronov, P. Beker, M. Yevnin, S. Stempler, L.
Buzhansky, G. Rosenman and E. Gazit, Nat. Nanotechnol., 2009, 4,
849; (d) M. Reches and E. Gazit, Nano Lett., 2004, 4, 581; (e) L.
Adler-Abramovich and E. Gazit, J. Pept. Sci., 2008, 14, 217; (f) L.
Adler-Abramovich, D. Aronov, E. Gazit and G. Rosenman, J. Nanosci.
Nanotechnol., 2008, 8, 1.
3 M. Reches and E. Gazit, Isr. J. Chem., 2005, 45, 363.
1H NMR (500 MHz, DMSO-D6, 10% TFA-D) d 8.40 (d, J =
2.8 Hz, 1H), 8.39 (d, J = 3.5 Hz, 1H), 8.13 (d, J = 7.8 Hz, 1H),
7.35–7.22 (m, 10H), 4.55 (dd, J = 9.9, 4.4 Hz, 1H), 4.49 (dd, J =
8.7, 5.4 Hz, 1H), 3.72–3.59 (m, 4H), 3.53–3.47 (m, 2H), 3.41 (dd,
J = 8.7, 1.7 Hz, 1H), 3.36–3.31 (m, 2H), 3.13 (dd, J = 13.9, 5.3 Hz,
1H), 3.03 (dd, J = 14.4, 4.8 Hz, 1H), 2.98 (dd, J = 14.0, 8.8 Hz, 1H),
2.78 (dd, J = 13.8, 9.8 Hz, 1H), 2.63 (dd, J = 12.5, 4.2 Hz, 1H),1.94
(d, J = 0.8 Hz, 3H), 1.67 (dd, J = 12.3, 10.9 Hz, 1H); 13C NMR
(126 MHz, DMSO-D6, 10% TFA-D) d 172.7, 171.9, 171.3, 171.1,
171.0, 167.0, 137.8, 137.5, 129.4, 129.3, 128.4, 128.2, 126.6, 126.4,
81.8, 76.1, 68.5, 62.9, 54.1, 53.9, 53.6, 53.5, 52.3, 52.2, 41.0, 37.5,
36.7, 32.7, 22.7; ESI-MS: Calculated for C31H39N3O12S: 677.72 Da
[M+], Observed: 678.1 Da [M+H].
4 (a) A. Mahler, M. Reches, M. Rechter, S. Cohen and E. Gazit, Adv.
Mater., 2006, 18, 1365; (b) S. Toledano, R. J. Williams, V. Jayawarna
and R. V. Ulijn, J. Am. Chem. Soc., 2006, 128, 1070; (c) R. Orbach,
L. Adler-Abramovich, S. Zigerson, I. Mironi-Harpaz, D. Seliktar and
E. Gazit, Biomacromolecules, 2009, 10, 2646; (d) N. Kol, L. Adler-
Abramovich, D. Barlam, R. Z. Shneck, E. Gazit and I. Rousso, Nano
Lett., 2005, 5, 1343; (e) L. Adler-Abramovich, N. Kol, I. Yanai, D.
Barlam, R. Z. Shneck, E. Gazit and I. Rousso, Angew. Chem., Int. Ed.,
2010, 49, 9939.
5 (a) B. G. Davis, Chem. Rev., 2002, 102, 579; (b) M. J. Grogan, M. R.
Pratt, L. A. Marcaurelle and C. R. Bertozzi, Annu. Rev. Biochem., 2002,
71, 593; (c) O. Seitz, ChemBioChem, 2000, 1, 214; (d) A. Brik, S. Ficht
and C. –H. Wong, Curr. Opin. Chem. Biol., 2006, 10, 638.
6 G. W. Hart, M. P. Housley and C. Slawson, Nature, 2007, 446, 1017.
7 P. Y. Chen, C. C. Lin, Y. T. Chang, S. C. Lin and S. I. Chan, Proc. Natl.
Acad. Sci. U. S. A., 2002, 99, 12633.
8 (a) J.-H. Fuhrhop, P. Schnieder, J. Rosenberg and E. Boekema, J. Am.
Chem. Soc., 1987, 109, 3387; (b) S. Kimuraa, Y. Murajia, J. Sugiyamab,
K. Fujitaa and Y. Imanishic, J. Colloid Interface Sci., 2000, 222, 265.
9 G. John, M. Mason, P. M. Ajayan and J. S. Dordick, J. Am. Chem. Soc.,
2004, 126, 15012.
10 B. S. Kim, D. J. Hong, J. Bae and M. Lee, J. Am. Chem. Soc., 2005,
127, 16333.
Nanostructure sample preparation and imaging
Nanostructure preparation: the lyophilized powder of each of the
synthesized glycopeptides, diphenylalanine 2, 3, 4, 5, 6, were dis-
solved in pure water at a concentration of 6 mM, diphenylalanine
2 was incubated at 40 ◦C for 30 min. Glycosylated diphenylalanine
11 M. Broncel, J. A. Falenski, S. C. Wagner, C. P. R. Hackenberger and B.
Koksch, Chem.–Eur. J., 2010, 16, 7881.
5760 | Org. Biomol. Chem., 2011, 9, 5755–5761
This journal is
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View Article Online
12 D. P. Gamblin, P. Garnier, S. van Kasteren, N. J. Oldham, A.
J. Fairbanks and B. G. Davis, Angew. Chem., Int. Ed., 2004, 43,
828.
13 X. Zhu and R. R. Schmidt, Chem.–Eur. J., 2004, 10, 875. The S-linked
when compared to O-linked usually exhibits more stability in vivo.
Moreover, S-alklyation is advantageous as the stereochemistry of the
anomeric center is fixed before alkylation, thus yielding only the desired
anomer.
14 S. Knapp and D. S. Myers, J. Org. Chem., 2001, 66, 3636.
15 C. H. Go¨rbitz, Chem. Commun., 2006, (22), 2332.
16 S. M. Rele, S. S. Iyer, S. Baskaran and E. L. Chaikof, J. Org. Chem.,
2004, 69, 9159.
A. Ermeydan, F. Dumoulin, V. Ahsen, H. Savoie and R. W. Boyle,
Photochem. Photobiol. Sci., 2009, 8, 312.
18 B. Wegmann and R. R. Schmidt, J. Carbohydr. Chem., 1987, 6, 357.
19 J. Kong and S. Yu, Acta Biochim. Biophys. Sin., 2007, 39, 549.
20 S. J. Gamblin and J. J. Skehel, J. Biol. Chem., 2010, 285, 28403.
21 R. Roy, Curr. Opin. Struct. Biol., 1996, 6, 692.
22 S. A. Kalovidouris, O. Blixt, A. Nelson, S. Vidal, W. B. Turnbull, J. C.
Paulson and J. F. Stoddart, J. Org. Chem., 2003, 68, 8485.
23 C.-F. Liang, M.-C. Yan, T.-C. Chang and C.-C. Lin, J. Am. Chem. Soc.,
2009, 131, 3138.
24 A. Hasegawa, J. Nakamura and M. J. Kiso, J. Carbohydr. Chem., 1986,
5, 11.
17 (a) M. A. Brimble, R. Kowalczyk, P. W. R. Harris, P. R. Dunbar
and V. J. Muir, Org. Biomol. Chem., 2008, 6, 112; (b) Y. Zorlu, M.
25 L. Motiei, S. Rahimipour, D. A. Thayer, C.-H. Wong and M. R.
Ghadiri, Chem. Commun., 2009, (25), 3693.
This journal is
The Royal Society of Chemistry 2011
Org. Biomol. Chem., 2011, 9, 5755–5761 | 5761
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