Glycopeptide self-assembly modulated by glycan stereochemistry through glycan-aromatic interactions

Article abstract of DOI:10.1021/jacs.0c06360

Carbohydrates are often utilized to provide hydrophilicity and hydroxyl-based hydrogen bonds in self-assembling glycopeptides, affording versatile scaffolds with wide applicability in biomedical research. However, how stereochemistry of carbohydrates impacts the self-assembly process remains unclear. Here we have established a dimeric tyrosine-rich glycopeptide system for probing the corresponding hydrogelating behavior under the influence of site- and stereospecific glycosylations. Comparison of 18 glycoforms bearing monosaccharides at Tyr4 and Tyr4' shows that the glycopeptides with either α- or β-anomers exhibit contrary gelating abilities, when the glycan moieties contain axial hydroxyl groups. A high-resolution X-ray crystallographic structure of the β-galactose-containing gelator, along with other results from spectroscopic, microscopic, and rheological experiments, indicate an unusual carbohydrate-aromatic CH-π bonding that promotes glycopeptide self-assembly. These mechanistic findings, particularly evidence obtained at the angstrom scale, illuminate an unconventional role that carbohydrates can play in building supramolecules. Potential biomaterials exploiting the CH-π bond-based stabilization, as exemplified by an enzymeresistant hydrogel, may thus be developed.

Full text of DOI:10.1021/jacs.0c06360

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Article
Glycopeptide Self-Assembly Modulated by Glycan
Stereochemistry through Glycan-Aromatic Interactions
Changdong He, Shuang Wu, Dangliang Liu, Changbiao
Chi, Weilin Zhang, Ming Ma, Luhua Lai, and Suwei Dong
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.0c06360 • Publication Date (Web): 18 Sep 2020
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Glycopeptide Self-Assembly Modulated by Glycan Stereochemistry
through Glycan-Aromatic Interactions
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Changdong He1,2, Shuang Wu1,2, Dangliang Liu1,2, Changbiao Chi1,3, Weilin Zhang4, Ming Ma1,3,
Luhua Lai4, and Suwei Dong1,2*
1State Key Laboratory of Natural and Biomimetic Drugs, 2Department of Chemical Biology, 3Department of Natural
Medicines at School of Pharmaceutical Sciences, 4BNLMS, State Key Laboratory for Structural Chemistry of Unsta-
ble and Stable Species, Peking-Tsinghua Center for Life Sciences at College of Chemistry and Molecular Engineer-
ing, Peking University, Beijing, China.
ABSTRACT: Carbohydrates are often utilized to provide hydrophilicity and hydroxyl-based hydrogen bonds in self-assem-
bling glycopeptides, affording versatile scaffolds with wide applicability in biomedical research. However, how stereochem-
istry of carbohydrates impacts the self-assembling process remains unclear. Here we have established a dimeric tyrosine-
rich glycopeptide system for probing the corresponding hydrogelating behavior under the influence of site- and stereo-
specific glycosylations. Comparison of eighteen glycoforms bearing monosaccharides at Tyr4 and Tyr4’ shows that the gly-
copeptides with either α- or β-anomers exhibit contrary gelating abilities, when the glycan moieties contain axial hydroxyl
groups. A high-resolution X-ray crystallographic structure of the β-galactose-containing gelator, along with other results
from spectroscopic, microscopic and rheological experiments, indicate an unusual carbohydrate-aromatic CH-π bonding
that promotes glycopeptide self-assembly. These mechanistic findings, particularly evidences obtained at the angstrom
scale, illuminate an unconventional role that carbohydrates can play in building supramolecules. Potential biomaterials
exploiting the CH-π bond-based stabilization, as exemplified by an enzyme-resistant hydrogel, may thus be developed.
lacking covalent bonds, it is challenging to predict the or-
ganization of monomers, and the design of peptide self-as-
1. INTRODUCTION
sembly still is mostly empirical.15 For example, to mimic the
modification by glycans of eukaryotic proteins and im-
prove their biocompatibility, glycosylation of peptide skel-
etons has been achieved, and is generally considered as a
hydrophilic poly-hydroxyl motif.16, 17 Evidence has been col-
lected showing that the size and structure18, 19 of glycans
can influence the self-assembling ability of peptides, as
Self-assembly is an important process in living organisms
that builds up polymeric structures from repetitive “build-
ing blocks”, often through non-covalent interactions, in-
troducing increased diversity and complexity.1 Tubulins,
for example self-assemble dynamically to form microtu-
bules as long chains or filaments which serve as an essen-
tial component of the cytoskeleton.2 Inspired by microtu-
bules and other natural systems, such as actin filaments,
clathrin and ribosomes, the use of self-assembly of proteins
or peptides to produce novel materials has emerged as a
fast-growing research area.3 Particularly, because readily
accessible peptide monomers could assemble to form var-
ious nanostructures including spheres, fibers or tubes,4, 5, 6
peptide-based systems with useful properties have at-
tracted significant attention, and been used in cancer ther-
apy,7 tissue engineering,8 drug delivery,9 as well as devel-
opment of novel catalysts,10 pigments,11 semiconductors,12
and so on.13, 14
well as the chirality and micromorphology of the resulting
21
supramolecules.20,
The carbohydrate components in
these systems also provide O–H based hydrogen bonding
along with steric effects that can interfere with other hy-
drophobic forces such as π-π interactions.22, 23 This ration-
alization however, mostly neglects the stereochemistry of
carbohydrates and lacks a structural basis to support the
hypothetical properties. Thus it could not fully explain the
distinct impacts glycosylation can generate in comparison
to other hydroxyl-containing structures or hydrophilic
modifications, such as phosphorylation.
Unlike synthetic polymers with fixed bond formation,
self-assembling structures built with non-covalent interac-
tions offer the potential to dynamically control polymeri-
zation and de-polymerization. However, with assemblies
Despite the challenges in development of glycopeptide-
based supramolecular assemblies, recent examples of uti-
lizing such biomimetic scaffolds for cell adhesion and pro-
liferation,24 human mesenchymal cell therapy,25 immuno-
suppression26 and DNA delivery27 illustrate their potential,
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and emphasize the need for studies leading to an under- imental results suggest that the glycan moiety with suita-
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4
standing of the irreplaceable roles of glycans. Here, we de-
scribe our research on the self-assembling behavior and
hydrogel forming ability under the influence of different
monosaccharides of a glycopeptide sequence. These exper-
ble stereochemistry may provide crucial stabilization to
the supramolecular assembly through a distinct carbohy-
drate-aromatic interaction (Figure 1).
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Figure 1. Glycopeptides may self-assemble to form hydrogels depending on the stereochemistry of contained mono-
saccharides. The self-assembly is presumably stabilized by carbohydrate-aromatic interactions.
Tyr-Cys-Tyr(β-Gal)-Tyr]2 (4), was found to self-assemble
under a heating/cooling regimen, forming a white opaque
hydrogel in phosphate buffer (Figure 2a). In contrast, the
glycopeptides 2, 3 and 5 generated only a white suspension.
2. RESULTS AND DISCUSSION
Experimental design and sequence screen. In order
to explore the potential multifaceted functions of carbohy-
drates in a peptide self-assembly system, especially taking
This suggests that a glycosylation site is crucial for the self-
stereochemistry into account, a peptide sequence that
assembly of peptide derivatives. To investigate the require-
shows self-assembling ability would be preferred as a start
point. Such peptide could then be site-specifically modi-
fied using various monosaccharides with different stereo-
chemistry of chiral hydroxyl groups and anomeric posi-
tions, and the properties of generated glycopeptides would
be evaluated. As the tyrosine residue contains an aromatic
ments of amino acid residues, an alanine scan of the se-
quence was carried out (Table 1, entries 6-10), which
showed that peptide 10 could also form a hydrogel while
peptides 6 and 8 generated white suspension and peptides
7 and 9 afforded a clear solution (Figure S1). These results
suggest that tyrosine at sites 2, 4, and 5 play important
moiety that is widely utilized in numerous self-assembling
roles in maintaining the self-assembling ability of the gly-
systems,28 and a phenolic group that represents a common
copeptides. Notably, peptide 8 with a thioether linkage
post-translation modification site for O-glycosylation,29
a
does not support any organized self-assembly, indicating
that the disulfide is required to construct the dimer with a
preferred orientation. It is also noteworthy that the unex-
pected gel-forming properties of glycopeptides 4 and 10 of-
fered additional perspectives for studying their supramo-
lecular self-assembly, because gelation could be easily eval-
uated through unequivocal manifestation such as vial in-
version, and quantitative indicators such as rheology or
melting point. The broad applications of hydrogels in
many fields, in particular biomaterials,32 prompted us to
further examine the hydrogel derived from glycopeptide 4.
Its critical gelation concentration, pH tolerance, and ther-
mostability (Figure S2) were all considered, and the pep-
tide sequence of 4 was used in studies of other glycopep-
tides bearing various monosaccharides.
tyrosine-containing sequence could be used.
With these considerations in mind, we initiated our
study on a sequence, Tyr-Tyr-Ala-Tyr-Tyr, which has pre-
viously been utilized in assembling supramolecular struc-
tures under UV irradiation.30 We found that once a β-D-
galactose (β-Gal) was appended to this pentamer peptide,
the glycopeptide 1 (Table 1, entry 1) has good solubility in
water and phosphate buffer (See Figure S1 in the Supple-
mentary Information). Drawing inspirations from a recent
report from Lee et al., where a disulfide bond-linked pep-
tide dimer, [Tyr-Tyr-Cys-Tyr-Tyr]2, can enjoy two-dimen-
sional self-assembly,31 a cysteine was introduced to derivat-
ize peptide 1 in hope of altering its solubility while the size
increases. After a survey of disulfide-linked dimers 2-5 de-
rived from sequence Tyr-Tyr-Cys-Tyr-Tyr with every pos-
sible glycosylation site (entries 2-5), the glycopeptide [Tyr-
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Table 1. Screen of Glycopeptide Sequences
(11-17) with various glycosylation patterns were prepared,
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8
including those containing readily available D-glucose
(Glc), D-mannose (Man), and D-xylose (Xyl). In its most
stable chair conformation, galactose has an axial 4-hy-
droxyl. Glc and Xyl however have no axial hydroxyls, and
Man possesses an axial 2-hydroxyl group (Figure 2b). Fol-
lowing the hydrogel formation procedure, it was observed
that with the exception of α-galactose- and α-mannose-
modified peptides (11, 15 respectively), all these dimeric
glycopeptides form white opaque hydrogels (Figure 2a). It
appears that the stereochemistry of the glycosidic linkage
influences the self-assembling capability when the glycan
contains an axial -OH group. However, in the cases of
monosaccharides that have all equatorial hydroxyl groups,
both α- and β-anomers were capable of gelation.
Entry
Sequences
Resultsa
1
2
Tyr-Tyr-Ala-Tyr(β-Gal)-Tyr (1)
[Tyr(β-Gal)-Tyr-Cys-Tyr-Tyr]2 (2)
[Tyr-Tyr(β-Gal)-Cys-Tyr-Tyr]2 (3)
[Tyr-Tyr-Cys-Tyr(β-Gal)-Tyr]2 (4)
[Tyr-Tyr-Cys-Tyr-Tyr(β-Gal)]2 (5)
[Tyr-Tyr-Cys-Tyr(β-Gal)-Ala]2 (6)
[Tyr-Tyr-Cys-Ser(β-Gal)-Tyr]2 (7)
[Tyr-Tyr-Xaa-Tyr(β-Gal)-Tyr]2 (8)
[Tyr-Ala-Cys-Tyr(β-Gal)-Tyr]2 (9)
[Ala-Tyr-Cys-Tyr(β-Gal)-Tyr]2 (10)
clear solution
white suspension
white suspension
hydrogel
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4
5
white suspension
white suspension
clear solution
white suspension
clear solution
hydrogel
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8b
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10
To evaluate the gelating abilities of glycopeptides bear-
ing more diverse glycans with varied stereochemistry, in-
cluding D-N-Acetylgalactosamine (D-GalNAc), L-Arabi-
nose (L-Ara), D- Fucose (D-Fuc), L-Rhamnose (L-Rha) and
L-Fucose (L-Fuc), compounds 18-27 were synthesized. In all
these cases only the β-anomer-modified peptide formed
hydrogel under the gelation conditions. These results fur-
ther demonstrate the impacts generated from the axial -
OH group in the sugar moiety. Moreover, the example of
GalNAcylated peptide proved that the 2-acetylamino
group does not affect the hydrogel formation.
aVisualizable results after a heating-cooling operation (85 °C
to r.t.) of glycopeptides in phosphate buffer (0.25% w/w). bXaa
= L-lanthionine.
Evaluation of glycopeptides with different gly-
coforms and characterization of generated hydrogels.
Besides the galactosylated peptide 4, seven glycopeptides
Figure 2. Gelation ability of glycopeptides with different glycan modifications. (a) Macroscopic images of hydrogels formed from
self-assembling glycopeptides (top left) and precipitation (bottom left) in phosphate buffer (0.25%, w/w), and schematic repre-
sentation of glycopeptide sequences evaluated (right). (b) Chemical structures of monosaccharides in evaluated glycopeptides that
either form hydrogels (displayed in the orange box), or precipitate from the solution (displayed in the blue box). The axial hydrox-
yls are highlighted in red.
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The morphology of hydrogels formed in this way was ex- trast, glycopeptides 11 and 15 only produced irregular ag-
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amined using transmission electron microscopy (TEM). As
shown in Figure 3, under aqueous conditions glycopep-
tides 4, 12, 13, 14, 16, and 17 assembled to nanofibers that
intertwined to form nanobundles, which trap water inside
a 3D network thus generating viscous gels. Interestingly,
the nanobundles derived from 13 displayed a very orga-
nized bamboo-like morphology (Figure 3d), but in con-
gregates in phosphate buffer, and no self-assembled
nanostructures were observed. Similar results could also be
observed for glycopeptides 18-27 (Figure S3). It is notewor-
thy that some short nanofibers could be observed for the
sample prepared from glycopepitde 23 with α-D-Fuc mod-
ification, but no hydrogel was formed (Figure S3f).
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Figure 3. TEM images of glycopeptides 4 (a), 11 (b), 12 (c), 13 (d), 14 (e), 15 (f), 16 (g), and 17 (h) in phosphate buffer after a
heating-cooling procedure. (a), (c), (d), (e), (g) and (h) show the nanofiber structures in consistence with their gel-forming
ability. (b) and (f) show the irregular aggregates generated from glycopeptide 11 and 15. Scale bars: 100 nm
Mechanistic investigations. In an attempt to rational-
ize the self-assembly process, a series of measurements
were made of the representative glycopeptide 4 to obtain
its structural information. ThT stain experiments showed
that the fluorescence intensity increased when the sample
became more concentrated in solution (Figure 4a), indi-
cating that the peptides in the assembly may adopt a β-
sheet structure.33 The FTIR spectra of 4 (Figure 4b) display
a shift of Amide I absorbance from 1645 cm-1 (dry powder)
to 1628 cm-1 (D2O hydrogel), suggesting the presence of β-
sheet structures, possibly promoted by water,34 in contrast
to a reported case in which the addition of water results in
a weakened signal of the β-sheet.35 Unfortunately, neither
the variable temperature CD measurements (Figure S4),
nor the NMR study could provide more detailed structural
information of the assembly in solution, but a close prox-
imity between carbohydrates and two tyrosines, Tyr1 and
Tyr2, as suggested by the NOESY experiments using deu-
terated DMSO as the solvent (Figures S5-S11), was ob-
served.
number of conditions commonly used in protein crystalli-
zation. Fortunately, we were able to obtain a single-crystal
of glycopeptide 4, and subsequently solved its X-ray struc-
ture in a high resolution (1.6 Å ). The crystallographic data
(see Table S1 in the Supplementary Information; PDB code:
7C0N) shows that the dimeric molecule adopts a C-2 sym-
metric conformation and the two β-sheet pentapeptides
adopt an anti-parallel conformation (Figure 4c). By exam-
ining the structure seen through the disulfide bonds, it
could be noticed that the side chains of four tyrosines (Tyr1,
Tyr1′, Tyr5 and Tyr5′) at both peptide termini are on one side,
and the aromatic rings of Tyr4 and Tyr4′ with the glycans
attached are positioned on the opposite side. Such an am-
phiphilic arrangement represents the basic unit of the as-
sembly. Moreover, these glycopeptides stack in a left-hand
helical direction to form cross-β amyloid-like structure,
and the rotation of two adjacent molecules leads to a ~45°
angle (Figure 4d). Notably, the diameter of the helical as-
sembly in the crystal structure (ca. 2.49 nm) corresponds
well with the average width of a single fibril in the hydrogel
derived from 4 which, according to the TEM data is 2.31 ±
0.34 nm (Figure S12, Table S2). This suggests that the self-
assembling mechanism for generating nanofibers in hydro-
gel may be similar to that observed in the crystal structure.
In light of the previous reports using single-crystal X-ray
diffraction to probe the mechanism of self-assembly,36, 37, 38
and in particular, studying the ability of protein low-com-
plexity domains (LCDs) to form gel-like networks based on
a potential correlation between crystalline LCD segments
and the primary fibrils formed in their assembly,39, 40 we at-
tempted to grow crystals of glycopeptide 4. Despite the
challenges in obtaining suitable crystals of glycoconjugates
due to the flexible carbohydrate moiety,41 we tested a large
Further examination of both the intra- and inter-molec-
ular interactions between each glycosylated pentapeptide
in the crystal structure, showed that not only the hydrogen
bonds between the β-sheet peptides are crucial for the self-
assembling process (Figure 4e), but the galactose units in
each assembling unit are in close proximity to the aromatic
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through intra- or intermolecular backbone hydrogen bonding
rings in the adjacent glycopeptide dimer (Figure 4d). On
1
2
3
4
5
6
7
8
in the crystal structure. (f) Calculated data based on crystal
structure of 4, characterizing H1, H3 and H5 of galactose that
interact with the aryl ring. Parameters used to identify CH−π
interactions43, 44 include CH−π angle (θ, ≤ 40°), CH−π dis-
tance (dCH−π, ≤ 4.0 Å ), C-projection distance (doffset ≤ 2.0 Å
for Tyr).
the contrary, no obvious aromatic-aromatic interactions
were observed in this system. When a three-parameter op-
erational definition was used to characterize the potential
interactions,42, 43, 44 the C1 axial hydrogen (H1) located on
the α-face of Gal lies in the range where interaction with
the aromatic π system (Figure 4f) is possible. Based on
these observations, as well as the distinct gelating proper-
ties of glycopeptide 4 compared to the non-glycosylated di-
meric peptide reported previously,31 we suspected that the
carbohydrate C-H/aromatic π bonds might play essential
roles in stabilization of the cross-β fibrils. This prompted
us to further investigate the potential impacts of such in-
teractions on gel formation.
Carbohydrate-aromatic interactions and the influ-
ence from the glycan stereochemistry on the proper-
ties of hydrogels. Besides the frequently observed π-π in-
teractions, aromatic rings can also participate in other
types of non-covalent forces, such as CH-π interactions,
which in the carbohydrate-aromatic system often exist be-
tween two or three methine hydrogens of the pyranose and
the π electrons of the aromatic ring.45 Although CH-π in-
teractions are considered less common than hydroxyl-
based hydrogen bonding in carbohydrates, studies have
shown that such non-covalent bonds exist in many related
protein-carbohydrate systems. The preference of forming
CH-π bonds is associated with the stereochemistry of car-
bohydrates, concerning the distribution of axial C-Hs and
orientation of glycosidic bonds.46 For instance, Kiessling
and Woolfson et al. discovered that the existed CH-π inter-
actions between glycans and proximate aromatics favored
the α-faces of Gal and Man more than their β-faces regard-
less of the anomeric structures, while for Glc or Xyl, both
faces could form CH-π bonds depending on the appropri-
ate anomeric configuration.43 In our study, the gelating
abilities of glycopeptides seemed to be correlated well with
such preferences. Specifically, in the cases of the galacto-
sylated peptides 4 and 11, only the former with fully ex-
posed α-faces of Gals could effectively form bonds with ty-
rosine side chains, significantly stabilizing the self-assem-
bly. In contrast, 11 could not produce the crucial CH-π
bonds from the disfavored β-faces of Gals, because the α-
glycosidic linkages block the preferred bonding sides (Ta-
ble 2, Figures S13). In comparison, the glucosylated pep-
tides 12 and 13 could both generate stable CH-π bonds from
the monosaccharides regardless of the configurations of
glycosidic bonds, as in either case there is a suitable open
face with axial C–H bonds able to interact with the aro-
matic rings (Table 2). The performance of other glycopep-
tides 14-27 in hydrogel formation experiments can also be
rationalized in terms of the CH-π interactions.
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To further prove the hypothesis that strong CH-π inter-
actions in this glycopeptide system could stabilize the nan-
ofibril formation and promote the self-assembly, we mod-
ified the substituents on the side chains of involved tyro-
sines, and evaluated the properties of hydrogels produced
from the resulting galactosylated peptides. Four glycopep-
tides analogous to 4 were prepared, with –H, –F, –CN, and
Figure 4. Representative spectroscopic data of self-assem-
bling glycopeptide 4 and mechanistic insights related to CH-
π interactions. (a) Emission intensities of ThT (20 μM, λex
=
440 nm) for 4 at various concentrations. (b) FT-IR spectra of
4 in dry powder (dotted line) and hydrogel states (solid line).
(c) Crystal structure of 4 in assembly viewed perpendicular to
the fibril axes (left) and viewed down the fibril axes (right). (d)
Cartoons showing an elongated structure of 4 assembling to
form a fibril viewed perpendicular to the axes (left), and the
enlarged views rendering the close proximity of carbohydrate
and tyrosine residues between two molecules of 4 (right) in
the crystal structure. (e) Cartoons showing the β sheet pep-
tides of 4 (carbohydrate omitted) side-by-side stacking
–NO2 replacing the –OH on the aryl rings of Tyr2 and Tyr2
.
′
Under the gelating conditions, all four glycopeptides were
able to form hydrogels with a storage modulus (G′) that
could be measured using rheology experiments. A negative
correlation was found to exist between G′ and the elec-
trostatic surface potential values,47 indicating that the stor-
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age modulus of hydrogels decreases as the side chain aro-
matics of Tyr2 and Tyr2′ become more electron-poor (Fig-
ure S14). Since CH-π interactions are weaker with more
electron-poor aromatics,43 the obtained results support the
idea that the non-covalent CH-π bonds may play essential
stabilizing roles in this glycosylated pentapeptide dimer-
derived self-assembly.
1
2
3
4
Table 2. Explanation of the relationship between glycan stereochemistry and glycopeptide self-assembly.
5
6
7
8
9
Glycoform
Modela
β-Gal
α-Gal
β-Glu
α-Glu
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Preferred face for
α
α
α
β
α
α
β
β
CH-π interaction43
Exposed face
a Cartoons rendering the interactions between the aromatic ring and β-Gal at the α-face of glycan based on crystal structure of 4,
and working models in the cases of α-Gal 11, β-Glc 12, and α-Glc 13 representing preferred CH−π interactions (highlighted in yellow)
and interaction unlikely to be formed (highlighted in grey) with the aromatic rings according to literature precedent43 and the
exposed faces of monosaccharides opposite to the ones with glycosidic linkages to aromatics. Carbon atoms are colored cyan,
nitrogen is blue, oxygen is red, sulfur is yellow orange, hydrogen is white, and the pyranose rings are highlighted in salmon, unless
otherwise noted.
Next, we further investigated whether different mono-
saccharides in the pentapeptide dimer would influence the
properties of the formed hydrogels. The rheological exper-
iments indicated that the storage modulus at angular fre-
quency (ω=1) of six hydrogel samples were ranging from
~10 Pa to >1000 Pa, depending on different types of mono-
saccharide (Figures S15, S16a). The melting points of the
hydrogels (Figures S16b), as well as the vertical and hori-
zontal distances (Figures S18) of the formed nanobundles
determined by atomic force microscopy (AFM), (Figures
S17), showed that measurable changes could result from
adjustment of the stereochemistry of glycans in the self-
assembling glycopeptides. However, no obvious trends
could be found to quantitatively correlate the glycoforms
and their physical properties, further underscoring the
complexity of this supramolecular system. Nevertheless,
our data demonstrate that the stereochemistry of carbohy-
drates could be an important factor in modulation of gly-
copeptide-derived self-assembly.
tion.48 However for glycopeptides, the approach of mirror-
ing both the peptide and the glycan may result in difficul-
ties to obtain the requisite compounds, since the enantio-
mers of common D-glucose, D-mannose, D-galactose, etc.
might not be readily available. In addition, it has been dif-
ficult to predict whether the combination of unnatural D-
peptides and common sugars would result structures with
self-assembling ability similar to those with known peptide
motifs. Thus, what we have discovered in the study of gly-
copeptide systems may provide a feasible solution. For ex-
ample, to predict the self-assembling ability of [yycy(β-D-
Glc)y]2 (28), its mirror-image form, [YYCY(β-L-Glc)Y]2 (28’),
could be of reference (Figure 5a) as they should have iden-
tical physical properties. Because β-L-Glc possesses three
axial C–Hs on its α face that may facilitate effective CH-π
hydrogen bonds in the [YYCYY]2 system (Figure 5b), it
would be anticipated that glycopeptide 28’ should be a hy-
drogelator. Therefore, D-peptide-derived sequence 28
should have the ability to self-assemble and form a hydro-
gel.
Development of an enzyme-resistant hydrogel from
a self-assembling glycosylated D-peptide. While pep-
tide hydrogels have shown significant potential in the de-
velopment of biomaterials, a major problem is that pep-
tides derived from natural amino acids are susceptible to
enzymatic degradation. In the cases of materials requiring
durability under physiological conditions, the short half-
life of L-peptides becomes a serious issue and the use of
mirrored D-peptides has been shown to be a viable solu-
Experimentally, the synthesized β-D-Glc-D-peptide 28
successfully forms a hydrogel at a concentration of 0.5 %
(w/w), as expected, and the TEM image clearly indicates
that 28 self-assembles to nanofibers (Figure 5c). A prote-
ase assay was conducted to further evaluate the properties
of two hydrogels prepared from glycopeptides 12 and 28,
and demonstrated that the D-peptide-derived hydrogel
was much more resistant to proteinase K, no obvious deg-
radation being observed after five days. In contrast, severe
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decomposition was seen for the other hydrogel that were In this supramolecular system, hydrogen bonding and
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produced from glycopeptide 12 after two days (Figure S19).
These findings also suggest that in the supramolecular sys-
tems involving essential carbohydrate-aromatic interac-
tions, poorly available and rare sugars may be replaced by
more easily accessible carbohydrates that can meet the re-
quirements for generating CH-π bonds.
solvophobic effects are certainly among the major driving
forces for the self-assembly. Surprisingly, π–π interactions
that are often found to be crucial in many other self-assem-
bling structures were not obvious in the crystal structure
that was obtained, although the sequence contains a high
content of tyrosine residues. A rational explanation would
be that the CH-π bonds afford essential stabilization to the
nanostructures. This is supported by experiments involv-
ing different monosaccharides that apparently possess var-
ied CH-π bond-forming capability, and also electronic var-
iations of the aromatic π bond donors. Furthermore, the
experimental results indicate that tuning the strength of
CH-π interactions may contribute to the change of glyco-
peptide assemblies in terms of micromorphology, as well
as the mechanical property and thermostability of the hy-
drogels that are formed.
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In conclusion, the self-assembling tyrosine-rich glyco-
peptide reported here represents a novel example of am-
phiphilic structure that contains aromatics closely inter-
acting with the carbohydrate moiety. This is distinct from
previous strategies that recommended more distant sepa-
ration of hydrophobic and hydrophilic termini.16, 17 On the
basis of these results, several new pointers for designing
self-assembling glycopeptides could be added to the exist-
ing empirical rules. A carbohydrate could be used as a CH-
π hydrogen bond donor, which may play crucial roles be-
yond the commonly observed O-H hydrogen bonding. By
changing the stereochemistry in the carbohydrate motif,
the strength of CH-π bond could be finely tuned, which
may influence the self-assembling process and the proper-
ties of the formed nanostructure. We have also demon-
strated that such glycopeptide self-assembly could be ex-
panded to D-peptide-derived structures, and along this line,
we believe that more gelators and applications may be dis-
covered using properly designed glycopeptides.
Figure 5. Design of D-glucosyl-D-peptide hydrogel. (a) Graph-
ical presentation of mirroring glycopeptide 28. (b) A plausible
working model showing the interaction between L-glucose on
Tyr4 and the Tyr2 residue, where three axial hydrogens at the
exposed α-face of glucose may facilitate the CH-π interactions.
(c) TEM image and gelating test of hydrogel prepared from the
glycosylated D-peptide 28 in 10 mM phosphate buffer (0.5%,
w/w).
3. CONCLUSIONS
Carbohydrates usually possess complicated stereochem-
ical information originating from the chiral centers in each
monosaccharide unit. These stereogenic centers not only
provide various chiral alcohols for hydrogen bonding,
which has been extensively studied, but also afford me-
thines that may engage interactions with aromatics via
CH-π hydrogen bonding.46 Although a number of studies
have revealed the critical roles of carbohydate-aromatic
CH-π bonds in biological processes including protein fold-
ing49 and protein-glycan recognition,43 very few mechanis-
tic investigations have been conducted on carbohydrate-
related self-assembling systems, perhaps due to the chal-
lenges in finding convincing structural evidences. Based on
a fluorenylmethyloxycarbonyl (Fmoc)-protected amino
sugar system, Ulijn et al. pioneered the study of CH-π in-
teractions as a key driving force in the self-assembly utiliz-
ing spectroscopic methods and molecular simulations.50
However, such non-covalent bonding in supramolecular
assembly involving more complexed glycopeptide struc-
tures still needed to be discovered and understood. Our X-
ray diffraction data of a single crystal from the tyrosine-
rich glycopeptide 4 has provided information demonstrat-
ing that CH-π interactions may be a potential link between
the stereochemistry of glycans and its impact on glycopep-
tide self-assembly.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI:
Supporting tables and graphics as well as experi-
mental procedures and spectroscopic data for all re-
actions and compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
* E-mail: dongsw@pku.edu.cn
Author Contributions
C.H. designed the research, performed the synthetic experi-
ments, carried out characterization of hydrogels, analyzed
data and wrote the manuscript, S.W. performed the synthetic
experiments and carried out crystallization experiment, D.L.
performed the synthetic experiments, C.C. analyzed the crys-
tallization data, W.Z. performed the NMR simulation experi-
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(14) Zozulia, O.; Dolan, M. A.; Korendovych, I. V. Catalytic pep-
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search, S.D. designed and supervised the research, discussed
the results and wrote the manuscript.
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Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors are grateful for financial support from the Na-
tional Key R&D Program of China (2018YFA0507602,
2019ZX09301-106), National Natural Science Foundation of
China (21822701), Beijing National Science Foundation
(JQ18024), and State Key Laboratory of Natural and Biomi-
metic Drugs. We thank Professors Jianjun Wang (Chinese
Academy of Sciences) and Shenlin Wang (Peking University)
for helpful discussions, Zhichao Liu (Peking University),
Qingrui Fan and Han Xue (Chinese Academy of Sciences) for
experimental assistance, Zenghui Wang (Peking University)
for manuscript preparation, and Drs. Yuan Wang, Xiaohui
Zhang, Qin Li, Fen Liu and Wei Pan (Peking University) for
spectroscopic assistance.
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