Photoinduced Thiol-ene Chemistry Applied to the Synthesis of Self-Assembling Elastin-Inspired Glycopeptides

Article abstract of DOI:10.1002/chem.201604831

Synthetic (glyco)peptides inspired by proteins able to self-assemble are appealing biomaterials in the field of tissue engineering and regenerative medicine. Herein, for the first time, taking advantage of thiol-ene chemistry coupled to solid-phase peptide synthesis, a self-assembling peptide inspired by elastin protein was bioconjugated to three carbohydrates in order to obtain the corresponding glycopeptides. They were studied at the molecular and supramolecular level. The results show that the carbohydrate influences the molecular conformation of the glycopeptide and its self-aggregation properties as well. As future perspective, the results could enable us to tune the final self-aggregation properties of the glycopeptide by changing the sugar moiety.

Full text of DOI:10.1002/chem.201604831

A Journal of
Accepted Article
Title: Photoinduced Thiol-ene chemistry applied to the synthesis of
elastin-inspired self-assembling glycopeptides
Authors: Germano Piccirillo, Antonietta Pepe, Emiliano Bedini, and
Brigida Bochicchio
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201604831
Link to VoR: http://dx.doi.org/10.1002/chem.201604831
Supported by

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Photoinduced Thiol-ene chemistry applied to the synthesis of
self-assembling elastin-inspired glycopeptides
Germano Piccirillo,#[a] Antonietta Pepe, #[a] Emiliano Bedini,[b] and Brigida Bochicchio*[a]
Abstract: Synthetic (glyco-)peptides inspired by proteins able to
self-assemble are appealing biomaterials in the field of tissue
engineering and regenerative medicine. Herein for the first time,
taking advantage of thiol-ene chemistry coupled to Solid Phase
Peptide Synthesis, a self-assembling peptide inspired by elastin
protein was bioconjugated to three carbohydrates in order to obtain
the corresponding glycopeptides. They were studied at molecular
and supramolecular level. The results show that the carbohydrate
influences the molecular conformation of the glycopeptide and its
self-aggregation properties as well. As future perspective, the results
could enable us to tune the final self-aggregation properties of the
glycopeptide by changing the sugar moiety.
engineering and regenerative medicine for their ability in
directing cells to normal development and tissue repair[5].
Besides, some peptide-carbohydrate conjugates able to self-
assemble and to form nanoaggregates in the form of fibers,
micelles and spherical particles represent an added value for
various applications, such as bacterial agglutination, drug-
delivery, and vaccines[6]. While the synthesis of standard
peptides has become routine and can be readily accomplished
via automated methods, the synthesis of glycopeptides can be
significantly more challenging[7] Generally, the preparation of
glycopeptides has been realized by attaching carbohydrates to
side-chains of amino acid residues such as lysine, cysteine,
serine and threonine. Conjugation in solution can be performed
by chemoselective ligation techniques involving the formation of
disulfide, thioether, thioester, oxime, hydrazone linkages[8].
However, some ligation techniques - e.g. oxime bond forming
ones - can suffer from low yields, lower purity, dimerization
byproducts, and acid lability, among other drawbacks[9]. Click
chemistry has gained high attention for the synthesis of
biomaterials thanks to the efficiency of click couplings in mild
conditions and at low concentrations. Sharpless introduced the
concept of click chemistry referred to reactions carried out on
Introduction
Elastin is the protein responsible for the elasticity of organs and
tissues. It is a cross-linked polymer whose soluble precursor is
tropoelastin exerting its elasticity in the Extracellular Matrix
(ECM) together with other components, such as microfibril-
associated glycoproteins (MAGPs), fibulins, and fibrillins. All of
them are present in the form of fibers considered crucial for the
exerted biological role[1]. Elastin is widely considered as an
inspiring protein for the design of biomaterials due to its poor
immunogenicity and high biocompatibility[2]. Furthermore, elastin
is characterized by repetitive motifs thoroughly repeated along
the primary structure and considered significant for its function.
Short elastin-inspired peptides constituted of these repetitive
motifs are an interesting class of self-assembling materials able
to give rise to fibrils despite of their small size[3]. In that context,
glycine-rich sequences where the XGGZG motif (X, Z = V, L) is
three-fold repeated were demonstrated to be prone to self-
aggregation[4]. Among them, (LGGVG)3 self-aggregated into
intertwined fibers with a regular pitch of the helix[3a]. That finding
lets us consider it as a model peptide for useful applications in
the nanotechnology field.
aerobic and atmosferic pressure conditions[10]
. Thiol-ene click
chemistry (TEC) represents an emergent ligation strategy[11]
based on the photo-induced coupling of an olefin with a
sulfhydryl group in mild conditions[12]. TEC methodology is
essentially based on the alkene hydrothiolation reaction that
occurs by
a
radical mechanism with anti-Markovnikov
regioselective orientation[13]. Some attempts were made to
investigate TEC methodology in order to prepare glycosylated
amino acids, peptides and proteins in solution [12, 14]
.
Here, we describe the synthesis of three glycosylated amino
acids obtained by coupling different O-allyl carbohydrates with
N-9-Fluorenil-methoxy-carbonyl-L-cysteine
(Fmoc-L-cysteine)
via TEC. These derivatives were then conjugated via Solid
Phase Peptide Synthesis (SPPS) to a self-assembling elastin-
derived peptide. SPPS represents
a versatile and clean
Glycosylated peptides are gaining increasing interest in tissue
synthetic methodology that also allowed us to avoid the early
undesired aggregation of the peptidic moiety. Concerning
carbohydrates, our attention was focused on three -glycosides
[a]
[b]
[#]Mr. G.Piccirillo, [#]Prof. A. Pepe, Prof. B. Bochicchio
Departmentof Science
(L-rhamnopyranoside,
D-galactopyranoside,
and
D-
University of Basilicata
Via Ateneo Lucano, 10
mannopyranoside) because of their appealing biological
properties [15]. For example, L-rhamnose has been recently
evidenced to be involved in recognition events by human
immune system[16] as already known for mannose and galactose
oligosaccharides. Furthermore, galactose has been widely
employed in tissue engineering application, because it supports
85100 Potenza, Italy. [#] The authors equally contributed to the work
E-mail: brigida bochicchio@unibas.it
Dr. E. Bedini
Department of Chemical Sciences
University of Naples Federico II
Complesso Universitario Monte S.Angelo,
Via Cintia, 4
hepatocyte adhesion and enhances cell functions as
a
consequence of the interactions with asialoglycoprotein
80126 Naples, Italy
Supporting information for this article is given via a link at the end of
the document.
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Table 1. Chemical structure of the synthesized glycopeptides 9a-c
Carbohydrate
derivative [a]
Chemical Structure
MW (Da)
1531.77
-L-rhamno-
pyranosyl
-D-galacto-
pyranosyl
1547.77
-D-manno-
pyranosyl
1547.77
receptors.[17] The three synthesized bioconjugates are shown in
Table 1. All of them share the amino acid sequence
GLGGVGLGGVGLGGVG inspired by the elastin-derived peptide
(LGGVG)3 [3a]. However, the original sequence has been
modified through the addition at N-terminus of a glycine as a
spacer. Circular dichroism (CD), nuclear magnetic resonance
(NMR) and Fourier transform-infrared (FT-IR) spectroscopies
were used in order to assess the effects of carbohydrate
modifications on the molecular conformation of the
glycopeptides. The self-assembling properties were assessed by
turbidimetry and the aggregates characterized by transmission
electron (TEM), scanning electron (SEM) and atomic force
(AFM) microscopies.
72% yield. Since chromatographic attempts to separate the two
anomers by silica gel chromatography failed, a per-O-acetylation
of the mixture was performed, followed by purification of the
2,3,4-tri-O-acetylated allyl -glycoside 2a-(86%, Scheme
1).Cleavage of acetyl groups by Zémplen transesterification then
gave allyl--L-rhamnopyranoside 1a-α (96%) in pure form.
Results and Discussion
Synthesis of the allyl glycoside of L-rhamnose. The
synthesis of the allyl glycoside of L-rhamnose 1a (Scheme 1)
was conducted by treating it in refluxing allyl alcohol with
Scheme 1. Synthesis of allyl rhamnoside 1a-α
concentrated sulfuric acid, as already reported[18]
.
An
anomeric mixture with a marked predominance of the
thermodynamically preferred -glycoside (12:1) was obtained in
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Scheme 2. Synthesis of glycopeptide 4.
Synthesis of elastin-inspired glycopeptides. The first attempt
to synthesize an elastin-inspired glycopeptide by thiol-ene
reaction was performed in solution by reacting peptide 3 with the
to take advantage of SPPS in order to easily manage a product
prone to self-aggregation. Furthermore, the acetylation of
hydroxyl groups of allyl carbohydrates 1b,c (Scheme 3) was
carried out in order to avoid undesired side-chain reactions. The
allyl
rhamnoside
1a-.
(Scheme
2).
Peptide 3, synthesized by solid-phase peptide synthesis (SPPS), incorporation of cysteine derivatives during SPPS is well-known
was photochemically coupled through the side-chain free thiol
group of cysteine to allyl -L-rhamnopyranoside 1a- by adding
catalytic amounts of 2,2-dimethoxy-2-phenylacetophenone
(DPAP) as initiator in methanol/water (1:2, v/v). The desired
glycopeptide 4 was obtained after purification in low amount
(26%). Therefore, we explored as alternative strategy the
synthesis of the glyco-conjugated cysteine intermediates 6a,b,c
(Scheme 3) eligible to be coupled by SPPS to peptide-resin 7 in
order to obtain glycopeptides 9a,b,c (Scheme 4). The idea was
for its high propensity to racemization under standard coupling
conditions[19]. In order to limit epimerization the coupling of the
glyco-conjugated cysteine intermediates 6a,b,c was conducted
manually in conditions that ensure a safe incorporation with
minimal racemization (Scheme 4). In particular, the pre-
activation step was avoided, the amount of base was reduced
and a less polar solvent mixture (DMF/DCM; 1/1, v/v) was
employed[19]
.
Scheme 3. Synthesis of the glyco-conjugated cysteine intermediates 6a,b,c
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Scheme 4. Synthesis of the glycopeptides 9a,b,c.
Finally, acetyl groups in glycopeptides 8a,b,c were removed in
order to obtain glycopeptides 9a,b,c (Scheme 4) in a 3:2 ratio
with the non-conjugated GLGGVGLGGVGLGGVG-NH2 peptide.
The final products 9a,b,c were recovered after purification by
RP-HPLC in yields of 56, 58 and 55%, respectively.
bioconjugation of the peptide with the carbohydrate both in the
pre-aggregated and in the post-aggregated state (Table 2,
Figures S23-S32 in Supporting Information). Additionally, in
order to have useful insights on the conformation of the pre-
aggregated state, CD analysis was carried out in aqueous and
fluorinated solvents and as a function of the temperature as well.
The CD spectrum of glycopeptides 9a is shown in Figure 1a. In
aqueous solution the CD spectrum at 273 K shows a strong
negative band centred at 195 nm and a small positive one at
Spectroscopy studies. FT-IR studies provided insightful
information concerning the effects of modification on the
secondary structure of the peptide triggered by the
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Figure 1. Temperature–dependent CD spectra. CD spectra recorded at 273 K (blue), °C (green), 310 K (magenta) and 333 K (red) in aqueous (solid line) and
TFE solution (dashed line) of (a) glycopeptide 9a, (b) glycopeptide 9b, (c) glycopeptide 9c, (d) peptide 3 and (e) peptide 7a.
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Table 2. Assignments and relative areas of amide I components of FT-IR spectra in the pre-aggregated and post-aggregated state.
3
7a
9a
9b
9c
Assignments
A
A
A
A
A





(cm-1)
(cm-1)
(cm-1)
(cm-1)
(cm-1)
(%)
(%)
(%)
(%)
(%)
1631
1650
1668
1681
11.9
22.6
25.4
23.1
17.0
1635
1652
1674
29.3
33.9
28.2
-sheet
random coil
PPII
-turn
antip.  -sheet
1642
1671
40.8
40.8
1646
1667
1681
1701
29.8
30.3
26.4
13.5
1645
1679
47.9
52.1
1697
18.4
1699
1696
8.7
1614
1630
19.3
41.5
Cross-
-sheet
-sheet
random coil
loops
PPII
-turn
antip. -sheet
1631
1652
1675
1696
51.7
26.7
16.4
5.1
1630
1660
1695
41.5
29.7
28.7
1630
1662
1698
44.3
31.5
24.2
1630
1637
29.6
29.9
1650
1671
1695
19.5
12.5
8.2
1661
17.4
1679
1698
13.6
9.5
[a] samples were analyzed after freeze-drying. [b] samples were analyzed after turbidimetry studies; the components between 1610-1600 cm-1, arising
from side-chain vibrations, are not included in the calculations, as well as the component at 1740-1745 cm-1, arising from C-terminal COOH.
212 nm. The negative maximum near 197 nm is characteristic of
the CD spectra of unordered peptides, but the poly-L-proline II
(PPII) conformation also gives such band[20].The presence of
PPII is also suggested by the positive band at 212 nm that is a
CD spectra with positive bands in the range 195-210 nm that
could be responsible for the low intensity of the observed
negative band occurring in the same wavelength range. In TFE,
the CD spectra dramatically change. At 273 K it shows a strong
positive band at 190 nm and two smaller negative bands at 201
and 222 nm indicating the presence of a type I -turn. At higher
temperatures, the strong positive band strongly decreases while
the negative band at 201 nm slightly increases suggesting the
destabilization of -turn conformation. Conformational analysis
by NMR of the glycopeptide 9b, recorded in TFE-d3/H2O (80/20,
v/v), confirmed the presence of a turn structure spanning GVGL
amino acidic residues. Discernible NMR features of the -turn
are dN(i,i+2) and dNN(i,i+2) medium range NOEs involving V and
L residues. Given the presence of the three-fold repeated
LGGVG motif along the sequence and the close similarity of the
chemical shifts of amide and H protons of the residues
belonging to repeated sequences, the exact localization of the
turn at G5VGL8 or G10VGL13 is not possible. Consequently, a
precise molecular model of the glycopeptide 9b in fluorinated
solvent is not achievable. Interestingly, the GVGL turn was not
previously detected in the pristine peptide devoid of the
carbohydrate[3a]. Therefore, it has been demonstrated that the
bioconjugation with the galactoside moiety induces the adoption
of specific conformations for the peptide chain.
distinctive feature of the CD spectra of PPII conformation[20a]
.
However, the value for []195 (-10,000 deg cm2dmol-1) is
compatible only with a small fraction of PPII[21]. The increase of
the temperature to 298, 310 and 333 K induces the loss of the
positive band and the gradual decrease of the negative one
together with a slight red-shift suggesting the progressive
destabilization of PPII conformation as a function of the
temperature and the increase of the contribution from -turn and
-sheet. The CD spectrum at 273 K in TFE shows two small
positive bands centred at 197 and 209 nm that suggest the
tendency by the glycol-conjugate to adopt a type II -turn[22]. The
positive bands disappear at 298 and 310 K while at 333 K a
small negative band, centred at 202 nm, appears thus
suggesting the destabilization of the turn. The FT-IR data
confirm the presence of multiple conformations (Table 2).
The CD spectrum of glycopeptide 9b is shown in Fig. 1b. In
aqueous solution the CD spectrum at 273 K is characterized by
a weak negative band at 194 nm decreasing on increasing the
temperature. The small intensity of the negative band, compared
to a CD negative band expected for a fully unordered peptide,
suggests the co-presence of -turn and -sheet in equilibrium
with random coil conformation. FT-IR data confirm this finding,
showing that glycopeptide 9b is the only compound with a
discrete percentage of -sheet before aggregating (Table 2).
Actually, -turn and -sheet conformations are characterized by
The CD spectrum of glycopeptide 9c is shown in Fig.1c. The CD
spectrum at 273 K shows a strong negative band at 194 nm and
a small positive one at 214 nm. Both bands decrease on
increasing the temperature. These spectral findings are
indicative of the presence of a small amount of PPII
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Figure 2. Turbidimetric determination of the self-assembly of a) glycopeptide 9a; b) glycopeptide 9c; c) peptide 3; d) peptide 7a
conformation, together with random coil, proved by the
persistence of the positive band even at 333 K. In TFE, the CD
spectrum at 273 K shows a small negative band at 201 nm
decreasing with increasing temperature. Such spectral features
are indicative of the presence of turn structures together with
extended conformations, such as PPII, and random coil.
According to CD analysis, the decomposition of the FT-IR
spectrum of the pre-aggregated state of glycopeptide 9c shows
an high percentage area assigned to -turn and random coil
conformations (Table 2).
The CD spectrum of peptide 3 is shown in Fig. 1d. In aqueous
solution the CD spectra at 273 and 298 K are characterized by a
negative band at 191 nm and a small positive band at 215 nm
suggesting the presence of PPII conformation with a certain
percentage of random coil and -turns. The increase of the
temperature to 310 and 333 K triggers the decrease of both
bands suggesting the destabilization of PPII and the stabilization
of -turns. In TFE, the CD spectrum at 273 K shows a small
negative band at 199 nm gradually decreasing at higher
temperatures. These spectral findings are assigned to presence
of turns together with random coil conformations. FT-IR analysis
shows for peptide 3 in the pre-aggregated state a discrete
percentage of -sheet, as expected for FTIR in solid state, with
the co-presence of random coil, PPII and -turn conformations.
The CD spectrum of peptide 7a, lacking cysteine residue, is
shown in Figure 1e. In aqueous solution the CD spectrum at 273
K is characterized by a weak negative band at 192 nm and a
small positive band at 214 nm. The CD spectra at 298 and
show the increase of the negative band and the loss of the
positive one. At 333 K the negative band further decreases.
These spectra suggest the presence of PPII conformation,
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Figure 3. AFM images of the aggregates formed after turbidimetry assay by glycopeptide 9a (a, b); glycopeptide 9b (c, d); glycopeptide 9c (e, f). The bars in
panel a, c, e represent 2 m while in panel b, d, f 0.5m.
stable at 273 K, together with unordered conformations
prevalent at high temperatures. In TFE, the CD spectrum at 273
K shows a weak positive band at 198 nm even decreasing with
the temperature and a weak shoulder at 210 nm. The CD
features are indicative of the presence of small amounts of -
turn and -sheet together with unordered conformations. Finally,
the CD spectrum at 333 K shows a small negative band at 200
nm and a tendency toward a positive one at 212 nm, probably
due to a small contribution of PPII conformation. FT-IR data
confirm the presence of PPII helix in the pre-aggregated state
while -sheets prevail in the post-aggregated one.
FT-IR in the solid state of the post-aggregated phase (Table 2),
shows for all compounds huge amounts of -sheet conformation,
as expected for the strongly hydrogen bonded aggregated
samples. Interestingly, glycopeptide 9a shows some percentage
of cross- conformation.
able to trigger the aggregation, like pH, temperature, and
solvent[23]. Additionally, the presence of conjugated molecules
may exert an important influence on the aggregation process.
For example, hydrophobic tails present in the amphiphilic
peptides or aromatic groups, such as 9-fluorenylmethoxy moiety,
in Fmoc-peptides drive the assembly in fibrous hydrogels by
hydrophobic and  interactions, respectively[24]
.
In order to define the role of the specific glycans in the self-
assembly of glycoconjugates, we performed the aggregation
studies of the glycopeptides by turbidimetry assay and
compared the results with the non-decorated peptides 3 and 7a.
Turbidimetric measurements can be used to monitor
aggregation in a simple way, giving us qualitative information on
how the aggregation process evolves with time. The intensity of
turbidimetry on apparent absorbance (TAA) depends mainly on
the size and the number of aggregates in suspension. As shown
in Figure 2, all the peptides show a typical sigmoidal curve.
A remarkable difference among the graphs is observed in the
lag phase and in the TAA values reached at the plateau. The
glycopeptide most prone to aggregation is glycopeptide 9b,
aggregating in few minutes (data not shown), followed by
glycopeptide 9c and peptide 3, showing the highest TAA values,
1.7 and 1.2, respectively. The model peptide 7a requires more
than 3 hrs to aggregate (Figure 2d), while glycopeptide 9a
Supramolecular studies
Self-assembling peptides are able to form assorted
supramolecular structures ranging from nanofibers, sheets and
tapes to spheres and networks. The morphology of the
aggregates is strongly influenced by the sequence of the peptide
and the environmental conditions. Furthermore, many self-
assembling peptides respond to external environmental stimuli
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Figure 4: AFM images of the aggregates formed after turbidimetry assay by peptide 3 (a, b) and peptide 7a (c, d). The scale bars in panel a and c indicate 2 m;
in panel b and d the scale bars indicate 0.5 m.
requires more than 24 hrs, with a final TAA value of 0.32 (Figure
2a). Thus we conclude that the glycopeptide 9a is the one with
lower tendency to aggregate under the used experimental
conditions. Taken together, our data show that TAA values at
the plateau are inversely correlated with the aggregation lag
phase.
In order to have a deeper insight into the supramolecular
structures of the aggregates, we performed studies by AFM,
TEM and SEM microscopies as well.
AFM is widely used in order to analyse the morphology of
nanostructures formed by self-assembling molecules[25]. Small
aliquots of the samples after aggregation were deposited on
silicon wafers and after air-drying analysed by AFM. In Figure 3
the formed aggregates after turbidimetry analysis by
glycopeptides 9a, 9b, and 9c are shown. AFM data show that
the morphology is significantly dependent on the nature of the
carbohydrate. Glycopeptide 9a shows the formation of entangled
flake-like structures (Figure 3a,b), while glycopeptide 9b shows
the formation of fibrils prone to evolve in fibers (Figure 3c,d).
Glycopeptide 9c presents a carpet of globular structures (Figure
3 e,f).
The nanostructures formed by the model peptide 3 and 7a,
without the carbohydrate group, are shown in Figure 4.
Peptide 7a evidences a morphology very similar to those
observed for glycopeptide 9a, with entangled flake-like
structures (Figure 4c, d), while peptide 3, containing an N-
terminal cysteine shows highly peculiar structures (Figure 4a, b).
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Figure 5: TEM images of a) glycopeptide 9a; b) glycopeptide 9b; c) peptide 3; and d) peptide 7a. Scale bars indicate 200 nm.
The film visible in Figure 4a contains basket-like structures,
particularly evident at higher magnification (Figure 4b). The
observed structures point to the formation of hollow
images for mannose-peptide 9c lack evidence of higher-order
assembled structures. The TEM grid was mostly blank with the
occasional formation of amorphous aggregates (data not shown).
nanospheres, that with air-drying could give rise to open vesicles. By comparing AFM and TEM images of the nanostructures of
The same samples were studied also by TEM in order to have
deeper insights into the fine supramolecular structure.
the glycopeptides 9a, 9b, 9c and peptide 3 and 7a, we observe
striking differences in the morphology of the nanostructures
mainly for peptide 3. While in AFM a film with vesicle-like
structures is observed (Figure 4a and b), in TEM a wrapped film,
with some protofibrils, is evident (Figure 5c). The high
dehydration encountered during the observation by TEM
microscopy has likely altered the morphology of the weak
vesicular nanostructures formed by peptide 3. The aggregates
were analysed also by SEM, where the nanostructures were
coated with a subtle film of gold prior to dehydration and
observation, and globular structures are confirmed (Figure S34
in Supporting Information).
All the peptides display considerable self-aggregation forming
nanostructures, whose morphological features are characterized
by a mixture of fibrils and large clumps of protofibrillar
aggregates, particularly evident for glycopeptide 9a (Figure 5a)
and for peptide 7a (Figure 5d). Consistent with the reduced lag
phase observed in turbidimetry experiments implying a higher
aggregation propensity, glycopeptide 9b shows the presence of
straight ribbons (Figure 5b), that at higher magnification, result
from lateral association of fibres (Figure S33 in Supporting
Information). In the background, evolving protofibrils, previously
observed in AFM images, are also discernible. By contrast, TEM
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
General Experimental Methods. Nuclear magnetic resonance (NMR)
spectra were acquired at room temperature on a Varian Inova 500
spectrometer operating at 500 MHz for 1H nuclei and 125 MHz for 13C
nuclei or on a Bruker DRX-400 (1H: 400 MHz, 13C: 100 MHz). Reference
Summarizing, clear differences at molecular level detected by
CD spectroscopy are evident for the three glyco-conjugates
(Figure 1). A dominant type I -turn is adopted only by
glycopeptide 9b while glycopeptides 9a and 9c take a mixture of
random coil, -turn and PPII conformations. At supramolecular
level, slight variations are evident for the glycopeptide
aggregates (Figures 3 and 5). With carbohydrate introduction,
additional hydrogen bonding interactions involving hydroxyl
groups of sugar moieties and peptide amide groups occur.
These modifications also affects the supramolecular interactions
in the aggregated state. According to our results, the pattern of
hydrogen bonding is dependent on the stereochemistry of the
1
peaks for H and 13C spectra were respectively set to  7.26 ppm and 
77.0 ppm for CDCl3,  2.49 ppm and  39.5 ppm for DMSO-d6, and 
4.79 ppm for 1H spectra in D2O. NMR data were reported in values of
chemical shift as parts per million (ppm) on the  scale, and coupling
constants in hertz (Hz). The conformational analysis by 1H-NMR
spectroscopy of galacto-peptide 9b in TFE-d3/H2O (80/20, v/v) is
described in Supporting Information (SI). Before characterization the
(glyco-)peptides were purified by semi-preparative reversed phase high-
performance liquid chromatography on a Shimadzu automated HPLC
system supplied with
a
semipreparative Jupiter C18 column
carbohydrate.
Galacto-
and
manno-pyranose
are
(Phenomenex, 250 x10 mm, 5 micron). The eluents were A (0.1% TFA in
H2O) and B (0.1% TFA in CH3CN). A binary gradient from 5 to 60% B
was used. ESI Mass spectrometry analysis was performed on the mass
spectrometer Quattro Micro (Waters), equipped with a triple quadrupole
and an electrospray source. MALDI-TOF spectrometry analysis was
performed by CEINGE (Naples, Italy). Thin layer chromatography (TLC)
was performed on Merck Kieselgel F254 200 m silica plates. Ultraviolet
light at 254 nm was used as a visualizing agent. Alternatively, the plates
were developed with 5% H2SO4 ethanolic solution and then heating to
230°C. Merck Silica gel60 (0.063-0.200 mm) was used for column
chromatography. All UV reactions were carried out in a Photochemical
Multirays Reactor (Helios-Italquartz, Milan, Italy) equipped with ten 15 W
lamp whose output was centered at 366 nm.
diastereoisomers belonging to aldohexose series and differ for
their configurations at C-2 and C-4. The peculiar orientation in
the space could be responsible for the observed differences.
Moreover, for a deoxyhexose such as rhamnopyranose an
additional variable to be taken into account is the smaller
number of hydroxyl groups limiting the number of hydrogen
bonds.
Conclusions
Herein, the chemical synthesis of three glyco-conjugated elastin-
inspired peptides was accomplished through thiol-ene photo-
induced coupling between per-O-acetylated allyl glycosides and
Fmoc-cysteine, followed by SPPS. It has been demonstrated,
by spectroscopy and microscopy studies, that the insertion of
the carbohydrate has a significant effect on the specific
conformation adopted and consequently on the self-assembly
propensity of the bioconjugated molecules. The differences
could be ascribed to the different chemical structure and
stereochemistry of each carbohydrate. As future perspective,
deeper insights into the self-aggregation mechanism could be
given by further studies involving computational methodologies
for Molecular Modeling and Dynamics. Moreover, we would like
to demonstrate that the functionality of the sugar moiety is
maintained in the here described glyco-conjugates[26] in order to
Allyl -L-rhamnopyranoside (1a-):
A suspension of L-rhamnose monohydrate (1.035 g, 5.683 mmol) in allyl
alcohol (13 mL) was refluxed at 100°C. After 10 min stirring a clear
solution was obtained. It was cooled to room temperature, then treated
quickly with 97% sulfuric acid (100L) and heated again to 100°C. After
30 min stirring at this temperature, TLC analysis (5:2 v/v ethyl acetate-
ethanol) showed complete consumption of the starting compound. The
reaction was worked-up by cooling to room temperature and then
neutralizing by addition of anhydrous potassium carbonate. The mixture
was filtrated and concentrated to give a residue, that, after a column
chromatography (18:1 to 10:1 v/v chloroform-methanol), afforded a 12:1
/ anomeric mixture of allyl L-rhamnopyranoside 1a (830 mg, 72%) as a
colourless oil. This mixture (819 mg, 4.017 mmol) was dissolved in 1:1
v/v pyridine/acetic anhydride (8 mL) and the solution was stirred at rt
overnight. TLC analysis (5:2 v/v toluene-ethyl acetate) showed complete
consumption of the starting compound. The solution was diluted with
CH2Cl2 (100 mL) and washed with 0.5 M HCl (100 mL). The organic
phase was collected, dried over anhydrous Na2SO4, filtered and
be recognized by specific cellular receptors[17b, 27]
.
concentrated. The obtained residue was subjected to
chromatography (6:1 to 4:1 v/v toluene-ethyl acetate) to give pure allyl
2,3,4-tri-O-acetyl--L-rhamnopyranoside (2a-, 1.135 g, 86%) as
colourless oil. 1H-NMR spectroscopic analysis of 2a- returned signals in
full agreement with literature reference[28]
a column
Experimental Section
a
Materials. All solvents and reagents were used as supplied. HPLC grade
ACN, DMF were purchased from Romil Ltd. (Cambridge, UK).
.
Diisopropilethylamine
(DIEA),
1-hydroxybenxotriazol
hydrate
(HOBT·H2O), DCM, piperidine, pyridine (pyr), acetic anhydride (Ac2O),
trifluoroacetic acid (TFA), dimethylaminopiridine (DMAP), 2,2-dimethoxy-
2-phenylacetophenone (DPAP), triisopropylsilane (TIS), allyl -D-
galactopyranoside and allyl -D-mannopyranoside were purchased from
Compound 2a- (926.6 mg, 2.808 mmol) was dissolved in methanol (12
mL) and treated with a 1.77 M methanolic solution of sodium methoxide
(950 L, 1.682 mmol). After 60 min stirring at rt, TLC analysis (5:2 v/v
ethyl acetate-ethanol) showed complete consumption of the starting
compound. The reaction mixture was neutralized with Amberlyst® 15 (H+
form), filtered and concentrated. The obtained residue was subjected to
column chromatography (15:1 to 12:1 v/v chloroform-methanol) to give
pure 1a- (548 mg, 96%) as a colourless oil. 1H-NMR spectroscopic
analysis of 1a-α returned signals in full agreement with literature
Sigma-Aldrich
(Germany).
2-[(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU), (benzotriazol-1
yloxy)tripyrrolidinophosphonium hexafluorophosphate (PyBOP), Wang
resin and Sieber Amide resin were purchased from Novabiochem
(Darmstadt, Germany). Fmoc-amino acids were obtained from Inbios
(Pozzuoli, Naples, Italy). Tris(2-Carboxyethyl)-Phosphine hydrochloride
(TCEP) was purchased from Pierce .
reference[29]
.
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
1H-NMR (500 MHz, DMSO-d6):  7.90 (d, 2H, J = 7.8 Hz); 7.70 (d, 2H, J = 7.6
Hz); 7.20 (t, 2H, J = 7.6 Hz); 7.15 (t, 2H, J = 7.8 Hz); 5.06 (m, 1H); 5.03 (m,
1H), 4.85(t, 1H, J =10.0 Hz); 4.78 (m, 1H); 4.40-4.20 (m, 4H); 4.08 (td, 1H, J=
9.1, 4.4 Hz); 3.79 (dq, 1H, J =10.0, 6.4 Hz); 3.67 (m. 1H); 2.91 (dd, 1H, J =
13.8, 4.6 Hz); 2.73 (dd, 1H, J =13.8, 9.6 Hz); 2.57 (dt, 2H, J = 7.0, 3.1 Hz);
2.07 (s, 3H); 1.99 (s, 3H); 1.90 (s, 3H); 1.79 (m, 2H); 1.08 (d, 3H, J = 6.4 Hz).
13C{1H}NMR (125 MHz, DMSO-d6):  172.89; 170.22; 170.20; 170.15; 156.47;
144.25; 141.17; 128.13; 127.54; 125.71; 120.56; 97.01; 70.56; 69.50; 69.16;
66.32; 66.18; 66.1; 54.71; 47.09; 33.26; 29.1; 28.55; 21.04; 20.92; 20.87;
17.64.
Peptide CGLGGVGLGGVGLGGVG-OH (3): SPPS synthesis was
accomplished as described in Supporting Information.
-L-Rhamnopyranosyl-conjugated peptide (4): Peptide 3 (30 mg, 22.6
mol) and allyl -L-rhamnopyranoside 1a- (5 mg, 24 mol) were
dissolved in MilliQ water (30 mL). Then DPAP (0.8 mg, 3 mmol) was
added. The reaction mixture was poured in quartz tubes and irradiated
for 1 h under stirring at 366 nm in a photochemical reactor. After
lyophilization, the reaction mixture was purified by RP-HPLC obtaining
pure compound 4 as fluffy solid (9 mg, 26%). 1H-NMR (500 MHz, D2O): 
4.72 (overlap with HOD signal); 4.26 (m, 2H); 4.14 (t, 1H, J = 6.8 Hz);
4.09 (d, 2H, J =7.1 Hz); 3.98-3.80 (m, 20H); 3.46 (m, 1H); 3.33 (t, 1H, J =
9.8 Hz); 3.03 (dd, 1H, J= 6.0,14.5 Hz) 2.93 (dd, 1H, J= 7.4,14.5 Hz);
2.60 (m, 2H); 2.01 (m, 3H); 1.81 (m, 2H); 1.52 (m, 9H); 1.08 (d, 3H, J =
6.4 Hz); 0.88-0.70 (m, 36H). MS-MALDI (m/z calcd. for C65H114N17O23S
[M+H]+ 1532.799) (m/z found [M+H]+ 1532.83, [M+Na]+ 1554.81, [M+K]+
1570.79) analysis.
(R)-2-[[(9H-fluoren-9-yl)methoxy]carbonylamino]-3-[[3-(2,3,4,6-tetra-
O-acetyl--D-galactopyranosyl]oxypropylthio]propanoic acid(6b). 6b
was prepared from 2b (320 mg, 0.95 mmol) and 5 (357 mg, 0.95 mmol)
according to the procedure for 6a and recovered as a white solid (365 mg,
53% yield). 1H-NMR (500 MHz, CDCl3, TMS):  7.68 (d, 2H, J = 7.8 Hz);
7.56 (d, 2H, J = 7.6 Hz); 7.32 (t, 2H, J = 7.2 Hz); 7.21 (t, 2H, J = 7.5 Hz);
5.45 (d, 1H); 5.30 (dd, 1H, J = 10.3, 2.1 Hz); 5.07 (dd, 1H, J = 10.6, 2.1
Hz), 5.01 (d, 1H, J = 1.6 Hz); 4.50-4.00 (m, 7H); 3.60 (m, 1H); 3.20 (m,
1H); 3.03 (t, 1H); 2.94 (t, 1H); 2.60 (m, 2H), 2.10 (s, 3H); 1.94 (s, 9H);
1.80 (m, 2H).13C{1H}NMR (125 MHz, CDCl3):  172.99; 170.39; 170.30;
170.21; 169.97; 156.17; 143.94; 133.15; 129.42; 128.22; 126.25; 118.02;
95.26; 73.41; 68.73; 68.12; 68.07; 67.59; 66.33; 56.80; 47.09; 33.26;
29.10; 28.55; 20.75; 20.65; 20.62;20.60.
Allyl 2,3,4,5-tetra-O-acetyl--D-galactopyranoside (2b)
Allyl -D-galactopyranoside 1b (220 mg, 1 mmol) was treated with acetic
anhydride (1.5 mL) and pyridine (0.2 mL), followed by stirring at room
temperature overnight. The reaction was slowly poured into DCM (10
mL) and then extracted with 1M HCl (3x 4 mL). The organic layer was
washed with saturated NaHCO3 until the evolution of gasses ceased (2 x
4 mL), and then with water (1 x 4 mL), and brine (1 x 4 mL). The organic
phase was dried over MgSO4 and condensed 2b as a white solid (360
(R)-2-[[(9H-fluoren-9-yl)methoxy]carbonylamino]-3-[[3-(2,3,4,6-tetra-
O-acetyl--D-mannopyranosyl]oxypropylthio]propanoic acid (6c). 6c
was prepared from 2c (180 mg, 0.50 mmol) and 5 (170 mg, 0.5 mmol)
according to the procedure for 6a and recovered as a white solid (280 mg,
1
mg, 94 %). H-NMR spectroscopic analysis of 2b returned signals in full
agreement with literature reference[30]
.
1
76% yield). H-NMR (500 MHz, CDCl3, TMS):  7.68 (d, 2H, J = 7.5 Hz);
7.55 (d, 2H, J = 7.3 Hz); 7.32 (t, 2H, , J = 7.5 Hz); 7.21 (t, 2H, J = 7.6 Hz);
5.25 (m br, 1H); 5.23 (m,br, 1H); 5.19 (t, 1H, J = 2.4 Hz), 4.71 (m, 1H);
4.50-4.10 (m, 6H); 4.00 (dd, 1H, J = 12.5, 2.2 Hz 3.78 (m, 1H); 3.50 (m,
1H); 3.03 (dd, 1H, J = 14.3, 3.3 Hz); 2.88 (dd, 1H, J = 14.3, 5.4 Hz); 2.55
(m, 2H), 2.10 (s, 3H); 2.03 (s, 3H); 1.98 (s, 3H); 1.94 (s, 3H); 1.76 (m,
2H). 13C{1H}NMR (125 MHz, CDCl3  172.85; 170.92; 170.27; 170.27;
169.83; 156.92; 143.82; 143.73; 127.65; 127.03; 125.17; 119.87; 95.42;
69.49; 69.27; 68.34; 67.17; 65.96; 62.96; 55.38; 46.95; 34.29; 29.10;
28.87; 20.81; 20.66; 20.62; 20.61.
Allyl 2,3,4,5-tetra-O-acetyl--D-mannopyranoside (2c)
2c was prepared from allyl -D-mannopyranoside 1c (110 mg, 0.5 mmol)
according to the procedure for 2b (180 mg, 94 % yield). 1H-NMR
spectroscopic analysis of 2c returned signals in full agreement with
literature reference [31]
.
N-Fmoc-L-cysteine-OH (5). Compound 5 was obtained from N-Fmoc-L-
cysteine(Trt)-OH following reported procedure[32]
Briefly, N-Fmoc-L-
.
cysteine(Trt)-OH (1.0 g, 1.7 mmol) was dissolved in CH2Cl2 (50 mL). TFA
(1.5 mL, 19.6 mmol) and TIS (0.75 mL) were added to the mixture kept
under stirring for 2 hours at room temperature. The solution was basified
to pH=9 with Na2CO3, washed with EtOAc and separated. The aqueous
solution was acidified with 10M HCl, extracted with EtOAc and
concentrated under vacuum. The final product was recovered as an oil
and dissolved in CH3CN:H2O (1:4) mixture and lyophilized. The final
product was recovered as a white powder (424 mg, 70%). The product
was used with no further purification. 1H-NMR spectroscopic analysis of 5
Peptide-resin GLGGVGLGGVGLGGVG-OH (7) and peptide Ac-
GLGGVGLGGVGLGGVG-OH
(7a):
SPPS
syntheses
were
accomplished as described in Supporting Information.
Example procedure for preparation of glycosyl-conjugated peptides
9a,b,c.
a) General procedure for manual coupling of cysteine derivatives
returned signals in full agreement with literature reference[32]
.
The coupling protocol to the peptide-resin 7 was accomplished by using
cysteine derivative 6/PyBOP/HOBt·H2O/DIPEA (2:2:2:2) with respect to
peptide-resin in DCM:DMF (1:1, v/v) without preactivation. The reaction
was carried out for 24 hours at room temperature under continuous
shaking. The completeness of amino acid coupling was monitored
through the Kaiser testkit (Fluka Analytical, Switzerland). The resin was
finally filtrated and washed (3x15mL DMF, 3x20mL DCM). The N-
terminal Fmoc protecting group was removed from the decorated peptide
by 20% piperidine in DMF for three hours.
Example Procedure for Thiol-ene Coupling to prepare Glyco-Cys
Conjugates(6a-c). N-Fmoc-L-cysteine 5 (340 mg, 1 mmol), 2a- (360
mg, 1 mmol) and DPAP (26 mg, 0.1 equiv) were dissolved in AcOEt (15
mL). The reaction mixture was transferred into quartz tubes and then
irradiated under stirring with a Photochemical Multirays Reactor (Helios-
Italquartz, Milan, Italy) equipped with ten 15 W lamp whose output was
centered at 366 nm for 4 hours. The crude reaction mixture was
concentrated under vacuum. The resulting residue was eluted from a
column of silica gel (DCM:MeOH, 95/5 v/v) obtaining 6a as a white solid
(200 mg, 30%).
(R)-2-[[(9H-fluoren-9-yl)methoxy]carbonylamino]-3-[[3-(2,3,4-tri-O-
acetyl--L-rhamnopyranosyl]oxypropylthio]propanoic acid(6a).
b) General Procedure for peptide cleavage from Sieber amide resin:
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
The dry resin was swelled with DCM (10 mL) for 20 minutes in a sealable
sintered glass funnel and then the excess of DCM removed. TFA (1%
v/v) in dry DCM was added, the funnel was sealed and shaken for 2
minutes. The solution was filtered into a flask containing pyridine in
methanol (10% v/v). The filtration was repeated up to 10 times, the resin
was washed with DCM (3 x 30 mL) and MeOH. The filtrates were
combined and evaporated under reduced pressure to 5% of the initial
volume. Diethyl ether was added to the flask in order to aid the
precipitation of the peptide. The precipitate was recovered by
centrifugation and dissolved in MilliQ water and finally lyophilized
obtaining a white fluffy powder.
(m, 1H); 2.60 (t, 2H); 2.01 (m, 3H); 1.79 (m, 2H); 1.52 (m, 9H); 1.18 (d,
3H); 0.90-0.72 (m, 36H). MS-MALDI (m/z calcd. for C65H115N18O22S
[M+H]+ 1531.81; m/z found [M+H]+ 1531.72; [M+Na]+ 1553.69; [M+K]+
1569.67) analysis.
9b (21 mg, 0.015 mmol) 1H-NMR (500 MHz, D2O):  4.86 (overlap with
HOD signal); 4.81 (overlap with HOD signal); 4.59 (overlap with HOD
signal); 4.25 (m, 3H); 4.03 (m, 3H); 3.94-3.76 (m, 21H); 3.75-3.65 (m,
4H); 3.61 (m, 1H); 3.47 (m, 1H); 2.81 (m, 2H); 2.56 (t, 2H); 1.96 (m, 3H);
1.79 (m, 2H); 1,50 (m, 9H); 0.88-0.70 (m, 36H). MS-MALDI (m/z calcd.
for C65H115N18O23S 1547.81) (m/z found [M+H]+ 1547.85, [M+Na]+
1569.00 [M+K]+ 1585.81) analysis.
8a was prepared according to procedures a) and b) from peptide resin 7
(390 mg, 0.145 mmol) and cysteine derivative 6a (200 mg, 0.29 mmol).
1H-NMR analysis evidenced that the coupling efficiency was 67%. Small
aliquot (5 mg) of crude mixture was purified by RP-HPLC obtaining of
pure 8a (2 mg). 1H-NMR: (500 MHz, DMSO-d6):  8.76 (t, 1H); 8.32-8.10
(m, 7H); 8.03-7.90 (m, 5H); 7.88-7.80 (m, 3H), 7.17 (s, br, 1H); 7.02 (s, br,
1H); 5.06 (d, 1H); 5.03 (dd, 1H), 4.87 (t, 1H); 4.78 (d, 1H); 4.40-4.05 (m,
6H); 4.05-3.60 (m, 22H); 2.99 (m, 2H); 2.61 (t, 2H); 2.09 (s, 3H); 2.02 (s,
3H); 1.92 (s, 3H); 1.97(m, 3H) 1.83 (m, 2H); 1.57 (m, 3H); 1.46 (m, 6H),
1
9c (15 mg, 0.010 mmol): H-NMR (500 MHz, D2O):  4.25 (m, 3H); 4.12
(m, 1H); 4.03 (m, 4H); 3.94 (m, 3H); 3.90-3.75 (m, 18H); 3.66 (m, 2H);
3.50 (m, 2H); 3.02 (m, 1H); 2.92 (m, 1H); 2.60 (t, 2H); 1.99 (m, 3H); 1.80
(m, 2H); 1,51 (m, 9H); 0.86-0.72 (m, 36H). MS-MALDI (m/z calcd. for
C65H115N18O23S 1547.81) (m/z found [M+H]+ 1547.93 [M+Na]+ 1569.69
[M+K]+ 1585.88) analysis.
CD Spectroscopy
1.08 (t, 3H); 0.85 (m, 36H). MS-ESI (m/z calcd. for C71H121N18O25
[M+H]+ 1657.339) (m/z found [M+H]+ 1657.884) analysis
S
Circular dichroism spectra were recorded on
a JASCO J-815
Spectropolarimeter (JASCO, Milan, Italy), equipped with HAAKE
temperature controller. Samples were dissolved at a concentration of 0.1
8b was prepared according to procedures a) and b) from peptide resin 7
(180 mg, 0.065 mmol) and cysteine derivative 6b (95 mg, 0.23 mmol).
1H-NMR analysis evidenced that the coupling efficiency was 60%. Small
aliquot (5 mg) of crude mixture was purified by RP-HPLC obtaining pure
8b (1.5 mg): 1H-NMR (500 MHz, DMSO-d6):  8.76 (t, 1H); 8.32-8.20 (m,
5H); 8.17 (m, 2H); 8.03-7.90 (m, 5H); 7.88-7.80 (m, 3H), 7.17 (s, br, 1H);
7.02 (s, br, 1H); 5.36 (d, 1H); 5.20 (dd, 1H), 5.07 (d, 1H); 4.96 (dd, 1H);
4.40-3.90 (m, 10H); 3.90-3.65 (m, 20H); 3.64 (m, 2H); 2.96 dd, 1H); 2.80
(dd, 1H); 2.63 (t, 2H); 2.12 (s, 3H); 2.03 (s, 3H); 2.00 (s, 3H); 1.97(m, 3H)
1.94 (s, 3H); 1.83 (m, 2H); 1.59 (m, 3H); 1.46 (m, 6H), 0.85 (m, 36H).
MS-MALDI (m/z calcd. for C73H123N18O27S [M+H]+ 1715.853) (m/z found
[M+H]+ 1715.891, [M+Na]+ 1737.855) analysis
mg/mL in H2O and 2,2,2-Trifuoroethanol (TFE)
and loaded into
cylindrical quartz cells with 1 mm pathlength. Spectra were acquired in a
wavelength range of 190–250 nm at 0, 25, 37 and 60 °C, with a scan
speed of 20 nm/min and a band width of 1 nm. CD spectra represented
the average of 9 scans. The CD spectra were processed using the
JASCO Spectral analysis software®. After background subtraction, a
FFT filtering algorithm was applied for smoothing, and data expressed as
mean residue molar ellipticity []MRW deg cm2 dmol-1 residue-1. CD
spectra of peptide 3 were carried out in presence of reducing agent 1 mM
tris(2-carboxyethyl)phosphine hydrochloride (TCEP).
Turbidimetry
8c was prepared according to procedures a) and b) from peptide-resin 7
(510 mg, 0.190 mmol) and cysteine derivative 6c (280 mg, 0.38 mmol).
1H-NMR analysis evidenced that the coupling efficiency was 65%. Small
aliquot of crude mixture was purified by RP-HPLC obtaining pure 8c (3
mg). 1H-NMR: (500 MHz, H2O/D2O, 9/1, DSS):  8.76 (t, 1H); 8.45-8.35
(m, 6H); 8.20 (d, 1H); 8.15-8.05 (m, 5H); 7.95 (d, 1H), 7.90 (d, 1H),
7.88(d, 1H), 7.29 (s, br, 1H); 6.95 (s, br, 1H); 5.25-5.10 (m, 3H); 4.85 (m,
2H),4.40-4.20 (m, 3H); 4.16-3.90 (m, 8H); 3.88-3.70 20H); 3.55 (m, 2H);
2.99 (m, 2H); 2.63 (t, 3H); 2.09 (s, 3H); 2.02 (s, 3H); 2.01-1.95 (m, 6H);
1.91 (s, 3H); 1.83 (m, 2H); 1.51 (m, 9H); 0.86-0.72 (m, 36H). MS-MALDI
(m/z calcd. for C73H123N18O27S [M+H]+ 1715.853) (m/z found [M+H]+
1715.958, [M+Na]+ 1737.945; [M+K]+ 1753.925) analysis
A time coarse measurement was carried out by UV spectroscopy on a
Cary 60 UV spectrophotometer (Agilent) equipped with
a Peltier
temperature controller. The absorbance is expressed as TAA (Turbidity
on Apparent Absorbance) as a function of time. The peptides were
solubilized in PBS buffer [Phosphate (50 mM), (pH 7.0)] to yield a final
concentration of 1 mM, transferred in one cm quartz cuvette kept under
stirring at the constant temperature of 37°C. At the end of the turbidity
assay, the obtained suspension was centrifuged and the supernatant
was removed from the pellet. The pellet was washed with MilliQ water,
centrifuged at 13000 rpm for 10 minutes in a HERMLE Z323K and the
supernatant removed. The process was repeated twice in order to
remove salts. The pellet was suspended in 1 mL of MilliQ water. The
suspension was used for AFM, TEM, SEM microscopies and FT-IR
spectroscopy, as described in the respective paragraphs.
c) Procedure for deacetylation
Crude glyco-conjugated peptides 8a-c (25 mg of 8a; 32 mg of 8b or 62
mg of 8c) were dissolved in 3-5 mL of a freshly prepared 0.1 M solution
of K2CO3 in MeOH. The mixture was allowed to react at room
temperature under magnetic stirring for 72 hours. The solution was then
concentrated under vacuum and the pH was neutralized using HCl 0.1M.
The water was finally removed by freeze-drying. The crude
glycoconjugated peptides 9a-c were purified by RP-HPLC.
FT-IR spectroscopy
The samples for FT-IR spectroscopy were analyzed either as lyophilized
powder in the pre-aggregation state or recovered from the pellet after
turbidimetry studies in the post-aggregation state. The lyophilized
peptides and the pellets were mixed with KBr to a final concentration of
approximately 1% (w/w). IR spectra were recorded on a Jasco FT-IR-460
spectrometer. Each spectrum is the result of signal-averaging of 256
scans at a resolution of 2 cm-1 . All spectra are absorbance spectra after
background subtraction. Smoothing of spectra was carried out with a
1
9a (12 mg, 0.008 mmol): H-NMR (500 MHz, D2O):  4.66 (overlap with
HOD signal); 4.26 (m, 3H); 4.04 (m, 4H); 3.94 (m, 1H) 3.90-3.76 (m,
18H); 3.72-3.53 (m, 2H); 3.47 (m, 1H); 3.33 (m, 1H); 3.01 (m, 1H); 2.92
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
step of 11 or 13 data points, using the Savitzky–Golay function[33]
.
[4] P. Moscarelli, F. Boraldi, B. Bochicchio, A. Pepe, A. M. Salvi and D.
Quaglino, Matrix Biol 2014, 36, 15-27.
Second derivatives of the spectra were obtained using a step of 13
datapoints to identify discrete absorption bands that make up the
complex amide profiles. Quantitative analysis of the individual component
bands of amide I was achieved by using the peak fitting module
implemented in the Origins ® Software (Microcalc Inc.). In the curve
fitting procedure, the Voigt peak shape has been used for all peaks. The
Voigt shape is a combination of the Gaussian and Lorentzian peak
shapes and accounts for the broadening present in the FT-IR spectrum.
[5] O. Seitz, Chembiochem 2000, 1, 214-246.
[6] a) J. Huang and A. Heise, Chem Soc Rev 2013, 42, 7373-7390; b) M.
Delbianco, P. Bharate, S. Varela-Aramburu and P. H. Seeberger, Chem
Rev 2016, 116, 1693-1752; c) M. Y. Mistou, I. C. Sutcliffe and N. M.
van Sorge, FEMS Microbiol Rev 2016, 40, 464-479.
[7] a) T. Buskas, S. Ingale and G. J. Boons, Glycobiology 2006, 16, 113R-
136R; b) H. Hojo and Y. Nakahara, Biopolymers 2007, 88, 308-324.
[8] a) Q. Y. Hu, F. Berti and R. Adamo, Chem Soc Rev 2016, 45, 1691-1719;
b) J. M. Langenhan and J. S. Thorson, Current Organic Synthesis 2005,
2, 59-81.
Atomic force microscopy (AFM)
[9] a) S. Ramesh, P. Cherkupally, T. Govender, H. G. Kruger, F. Albericio and
B. G. de la Torre, Org Lett 2015, 17, 464-467; b) J. Kalia and R. T.
Raines, Angew Chem Int Ed Engl 2008, 47, 7523-7526.
[10] H. C. Kolb, M. G. Finn and K. B. Sharpless, Angew Chem Int Ed Engl
2001, 40, 2004-2021.
Ten microliters of the suspension were deposited on Silicon (100) wafer
substrate (Aldrich, Saint Louis, Mo, USA). The silicon wafers were
cleaned by using a two-solvent method consisting in the immersion of Si
wafer in warm acetone bath for a period of 10 min. Then a methanol bath
for a period of 5 min immediately followed with MilliQ water rinses.
Samples were stored in a Petri dish and air-dried overnight at room
temperature. Each sample was observed at room temperature by Atomic
Force Microscope (Park System XE-120). Data acquisition was carried
out in intermittent contact mode at scan rates between 0.2 and 1 Hz,
using rectangular Si cantilevers (TAP300Al-G, Budget Sensors) having
the radius of curvature less than 10 nm and with the nominal resonance
frequency and force constant of 300 kHz and 40 N m-1, respectively.
[11] L. M. Campos, K. L. Killops, R. Sakai, J. M. J. Paulusse, D. Damiron, E.
Drockenmuller, B. W. Messmore and C. J. Hawker, Macromolecules
2008, 41, 7063−7070.
[12] A. Dondoni, A. Massi, P. Nanni and A. Roda, Chemistry 2009, 15, 11444-
11449.
[13] K. Griesbaum, Angew Chem Int Ed Engl 1970, 9, 273-287.
[14] J. R. Kramer and T. J. Deming, J Am Chem Soc 2012, 134, 4112-4115.
[15] a) W. Chen, L. Gu, W. Zhang, E. Motari, L. Cai, T. J. Styslinger and P. G.
Wang, ACS Chem Biol 2011, 6, 185-191; b) S. Sarkar, S. A. Lombardo,
D. N. Herner, R. S. Talan, K. A. Wall and S. J. Sucheck, J Am Chem
Soc 2010, 132, 17236-17246; c) S. Sarkar, A. C. Salyer, K. A. Wall and
S. J. Sucheck, Bioconjug Chem 2013, 24, 363-375; d) P. Karmakar, K.
Lee, S. Sarkar, K. A. Wall and S. J. Sucheck, Bioconjug Chem 2016, 27,
110-120.
Transmission Electron Microscopy (TEM)
Twenty microliters of the suspension were deposited onto carbon coated
copper grids. Negative staining was performed by applying few drops of
1% uranyl acetate in MilliQ water used to increase the contrast and the
electron density of the samples. After air-drying, grids were observed by
transmission electron microscopy (Fei Tecnai G2 20 Twin) operating at
100 kV.
[16] S. Balzaretti, V. Taverniti, S. Guglielmetti, W. Fiore, M. Minuzzo, H. N.
Ngo, J. B. Ngere, S. Sadiq, P. N. Humphreys and A. P. Laws, Appl
Environ Microbiol 2016.
[17] a) L. G. Griffith and S. Lopina, Biomaterials 1998, 19, 979-986; b) L.
Russo and L. Cipolla, Chemistry 2016, 22, 13380-13388.
[18] J. Zhang, J. Mao, H. Chen and M. Cai, Tetrahedron: Asymmetry 1994, 5,
2283-2290.
Scanning Electron Microscopy (SEM)
Twenty microliters of the suspension were deposited on Silicon (100)
wafer substrate (Aldrich, Saint Louis, Mo, USA). Prior to imaging the
sample was freeze-fractured after immersion in liquid nitrogen and then
mounted using carbon tape on aluminum SEM stubs and sputtered with
a thin gold layer. The sample was observed by a scanning electron
microscope (Philips-Fei ESEM XL30-LaB6) operating at 30 kV.
[19] a) Y. M. Angell, J. Alsina, G. Barany and F. Albericio, The Journal of
Peptide Research 2002, 60, 292-299; b) Y. Han, F. Albericio and G.
Barany, J Org Chem 1997, 62, 4307-4312.
[20] a) A. F. Drake, G. Siligardi and W. A. Gibbons, Biophys. Chem. 1988, 31,
143-146; b) G. Siligardi and A. F. Drake, Biopolymers 1995, 37, 281-
292; c) B. Bochicchio and A. M. Tamburro, Chirality 2002, 14, 782-792.
[21] E. A. Bienkiewicz, A. Moon Woody and R. W. Woody, J Mol Biol 2000,
297, 119-133.
Acknowledgements
[22] A. Perczel, M. Hollosi, P. Sandor and G. D. Fasman, Int J Pept Protein
Res 1993, 41, 223-236.
[23] a) K. Chockalingam, M. Blenner and S. Banta, Protein Eng Des Sel 2007,
20, 155-161; b) D. E. Meyer and A. Chilkoti, Nat Biotechnol 1999, 17,
1112-1115.
This work was supported by the following grants: MIUR (PRIN
2010LSH3K). We thank and acknowledge from University of
Basilicata, Dr. Maria Antonietta Crudele for technical assistance,
Mr. Alessandro Laurita and Mr. Antonio V. Romano (ex-CIGAS)
for TEM and AFM images, respectively.
[24] a) H. Cui, M. J. Webber and S. I. Stupp, Biopolymers 2010, 94, 1-18; b) R.
Orbach, L. Adler-Abramovich, S. Zigerson, I. Mironi-Harpaz, D. Seliktar
and E. Gazit, Biomacromolecules 2009, 10, 2646-2651; c) C. Tang, R.
V. Ulijn and A. Saiani, Langmuir 2011, 27, 14438-14449.
[25] R. Mammadov, A. B. Tekinay, A. Dana and M. O. Guler, Micron 2012, 43,
69-84.
Keywords: Glycopeptides • Elastin • Atomic Force Microscopy •
Circular dichroism • Self-assembly
[26] L. Russo, A. Gautieri, M. Raspanti, F. Taraballi, F. Nicotra, S. Vesentini
and L. Cipolla, Carbohydr Res 2014, 389, 12-17.
[1] J. E. Wagenseil and R. P. Mecham, Birth Defects Res C Embryo Today
2007, 81, 229-240.
[27] D. A. Grant and N. Kaderbhai, Biochem J 1986, 234, 131-137.
[28] J. Srivastava, A. Khare and N. K. Khare, Carbohydr Res 2008, 343, 2822-
2825.
[2] B. Bochicchio, A. Bracalello and A. Pepe, Chirality 2016, 28, 606-611.
[3] a) B. Bochicchio, A. Pepe, M. Crudele, N. Belloy, S. Baud and M. Dauchez,
Soft Matter 2015, 11, 3385-3395; b) L. Gotte, M. G. Giro, D. Volpin and
R. W. Horne, J Ultrastruct Res 1974, 46, 23-33.
[29] B. M. Pinto, D. G. Morissette and D. R. Bundle, Journal of the Chemical
Society, Perkin Transactions 1 1987, 9-14.
[30] B. Meng, Z. Zhu and D. C. Baker, Org Biomol Chem 2014, 12, 5182-5191.
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
[31] J. P. Utille and B. Priem, Carbohydr Res 2000, 329, 431-439.
[32] T. H. Wright, A. E. Brooks, A. J. Didsbury, J. D. MacIntosh, G. M. Williams,
P. W. Harris, P. R. Dunbar and M. A. Brimble, Angew Chem Int Ed Engl
2013, 52, 10616-10619.
[33] A. Savitzky and M. J. E. Golay, Anal. Chem. 1964, 36, 1627-1639.
This article is protected by copyright. All rights reserved.

10.1002/chem.201604831
Chemistry - A European Journal
FULL PAPER
Entry for the Table of Contents (Please choose one layout)
FULL PAPER
Turn-on light and be sweet.
Glycopeptides synthesis still
G. Piccirillo, A. Pepe, E. Bedini, and
B.Bochicchio* Page No. – Page No.
presents important challenges. The
first example of the bioconjugation
of a self-assembling elastin-inspired
peptide and three carbohydrates
has been realized exploiting photo-
induced thiol-ene chemistry coupled
to Solid Phase Peptide Synthesis.
The self-assembly of glycopeptides
triggered nanoaggregate formation
as shown by AFM and TEM
Photoinduced Thiol-ene chemistry
applied to the synthesis of self-
assembling elastin-inspired
glycopeptides
microscopies.
This article is protected by copyright. All rights reserved.