Folding control of a non-natural glycopeptide using saccharide-coded structural information for polypeptides

Article abstract of DOI:10.1039/c9cc10030j

We synthesized "glyco-arylopeptides", whose folding structure significantly changes depending on the kind of saccharide in their side chain. The saccharide moiety interacts with the main chain via hydrogen bonding, and the non-natural polypeptides form two well-defined architectures - (P)-3₁- and (M)-4₁-helices - depending on the length of the saccharide chains and even the configuration of a single stereo-genic center in the epimers.

Full text of DOI:10.1039/c9cc10030j

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DOI: 10.1039/C9CC10030J
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Folding Control of Non-natural Glycopeptide Using Saccharide-
coded Structural Information for Polypeptide
Yuki Ishidoa, Naoya Kanbayashi,*a Naoka Fujii,b Taka-aki Okamura,a Takeharu Hainob and Kiyotaka
Received 00th January 20xx,
Accepted 00th January 20xx
Onitsuka*a
DOI: 10.1039/x0xx00000x
We synthesized “glyco-arylopeptide”, whose folding structure molecular system that recognizes slight structural differences in
significantly changes depending on the kind of saccharide in side saccharides.
chain. the saccharide moiety interacts with the main chain via
Recently, we reported an aromatic polyamide peptide,
hydrogen bonding, and the non-natural polypeptide form two well- poly-“arylopeptide”, which comprises aromatic amide spacers
defined architectures—(P)-31- and (M)-41-helices—depending on between each residue.8 Arylopeptides show structural changes
the length of the saccharide chains and even the configuration of a depending on their chemical structures. Therefore, we
single stereo-genic center in the epimers.
surmised that the arylopeptide might be able to extract the
folding information from saccharide substituents similar to
natural glycopeptide. Herein, we present a glycosylated
arylopeptide called “glyco-arylopeptide”. This arylopeptide
could recognize the saccharide moieties depending on the
length of the saccharide chain and the epimers with the slight
differences in the configuration of a single stereogenic center to
form discrete well-defined structures (Scheme 1c). In addition,
the folding information varied reversibly/irreversibly because of
the chemical modification same as in nature and the recognition
of other molecules.
Saccharides are the foundation of living systems, and
glycosylated polypeptide (glycopeptide) plays a unique role in a
series of biological events. The saccharide moieties not only
determine the physicochemical properties, such as solubility
and stability of polypeptides, but also provide significant
information about the folding/unfolding of polypeptides and
molecular recognition involving other molecules (Scheme 1a).1
These functions have inspired chemists to utilize saccharide
units in artificial polymers, including the peptide backbone.2 In
general, synthetic glycopolymers are known to show enhanced
interactions with cells and have potential applications in cell
sensing,3 drug delivery,4 molecular recognition,5 etc. (Scheme 1-
b).6 However, there are only a few studies that have
investigated the role of saccharide structures in the folding
behavior and stability of glycopolymers and extracted
information related to folding,7 although lectins rigorously
distinguish the slight differences between monosaccharides
and disaccharides in saccharide chains in vivo. The reason is that
the saccharides have similar hydrophilic properties and may
only differ in the number of OH groups and configuration of a
single or a few stereogenic centers from the standpoint of
organic chemistry. Therefore, it is difficult to design an artificial Scheme 1. Schematic diagram of (a) glycoprotein and (b) glycopolymer. (c) This
work; Chemical structure of glycol-arylopeptide and folding control using
saccharide-coded folding information.
a. Department of Macromolecular Science Graduate School of Science
As shown in Scheme 2, we synthesized glycoarylopeptides
Osaka University, Toyonaka, Osaka 560-0043, Japan.
bearing various saccharide derivatives (poly-1a–d). All reactions
were done by using the optimized procedures,9 and they
proceeded quantitatively. Experimental details and full
characterization of the products are presented in the
E-mail: naokou@chem.sci.osaka-u.ac.jp, onitsuka@chem.sci.osaka-u.ac.jp
b. Department of Chemistry, Graduate School of Science Hiroshima University,
1-3-1, Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8526, Japan.
†Electronic Supplementary Information (ESI) available: Material, Experimental
details, spectroscopic data (CD/UV, NMR and IR spectra) and computational study.
See DOI: 10.1039/x0xx00000x
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Supporting Information (SI). Poly-1a–d are soluble in ordinary corresponded to the (M)-twisted conformation (Fig. 1b). The CD
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solvents, such as THF, acetonitrile, ethanol, and water.10
spectra obtained from CD simulation of oligomers (n = 2) in
water and THF by using TD-DFT were very similar to the
observed spectra for poly-1a. The major difference between the
optimized structure of (P)-twist and (M)-twist is the reversal in
the sign of dihedral angles (,  in the local structures and the
formation of cyclic intramolecular C6-OH···O=C hydrogen bonds
in the (M)-twist (Fig. 1b and Fig. S47d).
DOI: 10.1039/C9CC10030J
To study the role of saccharide structure, CD spectra of the
analog were measured in water. The CD spectrum of poly-1b
without C6-OH showed ordinary (P)-conformation (Fig. 2a). It is
surprising that poly-1c having D-cellobiose, a disaccharide of
glucose, also formed the (P)-conformation, although the D-
glucose moiety is connected to the main chain in the same
manner as that in poly-1a. The C6-OH of D-cellobiose in the
optimized structure was easily incorporated into the hydrogen
bond with the adjacent pyranose ring and could not interact
with the main chain (Fig. S53). Interestingly, poly-1d also
formed the (P)-conformation with the epimer of D-glucose at
the C2-position. The axial C2-OH of mannose and its hydration
probably disrupts the preferred conformation of carbohydrate
for (M)-helix (Fig. S54).11 In glycopeptide, a few saccharides near
the glycosylated site are known to determine the stability of the
peptide backbone via hydrogen bonding between the
saccharide and the carbonyl group on the main chain.12
Therefore, the intramolecular hydrogen bonding of D-glucose in
the glycoarylopeptide would also play an important role in the
folding behavior similar to that observed in glycopeptides. In
other words, the backbone of arylopeptide identifies the length
of the saccharide-chains and the epimer and hence can receive
distinct folding information from each saccharide.
Scheme 2. Synthetic pathway of glycoarylopeptides
The above experimental results and computational
simulations lead us to propose two global conformations (Figs.
2b and 2c)—(P)-helix and (M)-helix. The (P)-helix is 31-like
helical structure formed by successive formation of a (P)-twist
with a pitch of 5.1 Å. The (M)-helix is more contracted 41-like
helix with a pitch of 3.0 Å. The structural study using atomic
force microscopy (AFM) revealed detailed insights the
glycopeptide.13 The AFM images of poly-1a film, cast using
pyridine/THF solution on mica, exhibited a height of 0.9 ± 0.1
nm (Fig. 2d). This matched well with the height of the proposed
structure of poly-1a, supporting the estimated structures of Fig.
To investigate the global conformation of poly-1, circular
dichroism (CD) spectroscopy was conducted using water as the
solvent. As shown in Fig. 1a, CD spectra of poly-1a in organic
solvents showed a plus to minus bisignate Cotton effects,
indicating the formation of (P)-helix caused by the asymmetric
carbon (S) on the main chain. This is in accordance with our
previous work.9 In water, however, the CD spectrum of poly-1a
presented the opposite bisignate Cotton effect between 240
and 259 nm (Fig. 1a, blue line). The computational analysis of
the oligomers suggested that the minus to plus Cotton effect
Fig.1 (a) Solvent effect of CD spectra of poly-1a in 1% (v/v) DMSO/(solvent) at 298 K. Solvent; THF (pink), acetonitrile (orange), ethanol (green) and H2O (blue).
2 | J. Name., 2012, 00, 1-3
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Concentration: [poly-1a] = 0.30-0.32 mM. (b) Simulated Structures and CD spectra of oligomer (n = 2), (P)-twist and (M)-twist by TD-DFT calculation (B3LYP/6-
31**) in THF (pink curve) and water (blue curve) and their local structures.
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2b and Fig. 2c. The AFM images of poly-1a cast using other molecules and saccharide moieties. The OH groups of
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DOI: 10.1039/C9CC10030J
pyridine/H2O solution showed nanoparticles, therefore no saccharides are frequently acetylated to change the chemical
useful information was from these data.
and biological properties in vivo.17 Thus, acetylation and
deacetylation of the OH groups on poly-1a were carried out.
The CD spectrum of the acetylated poly-1a showed a plus to
minus Cotton effect originating from the (P)-helix (Fig. 3b). The
other acetylated glycoarylopeptides (poly-1b-d) showed no
inversion in Cotton effect, implying that the acetyl groups
disrupted the delicate energy balance of the (M)-helix in poly-
1a, and thus, the folding information for (M)-helix was
manipulated. Subsequently, the structural transitions caused by
molecular recognition were also investigated. Boronic acid
reagent is known to show favorable interactions with
saccharides.18 Adding the 4-carboxy-phenylboronic acid to the
poly-1a in aqueous solution resulted in a drastic change
(consistent with the helix-to-helix transition) from the
contracted (M)-41-helix to the extended (P)-31-helix (Fig. 3c).
The CD spectrum changes presented in Figs. 3a and 3c show an
isodichroic point, indicating the existence of mainly two kinds
of chiral architectures; (P)-helix and (M)-helix.
Fig. 2 (a) CD spectra of poly-1a-d in 1% (v/v) DMSO/H2O at 25 ºC. Concentration:
[poly-1a] = 0.31 mM, [poly-1b] = 0.30 mM, [poly-1c] = 0.37 mM, and [poly-1d] =
0.38 mM. Two plausible 3D helical conformations of poly-1a (n = 17) in (b) THF
and (c) water. (d) AFM image of poly-1a (3.0 um ×3.0 um, topography) and the
height profiles.
L-Amino acid residues induce only a right-handed -helix;
however, the residues of glycoarylopeptide form helices with
both screw-senses.14 Thus, the helical stability of poly-1a was
investigated in detail. The molecular weight-dependences of
the helical formation were examined by CD measurement in
THF and H2O. The CD intensity of poly-1a increased nonlinearly
as a function of the degree of polymerization (n) and plateaued
at higher n values (Fig. S44a). The results suggest that a long
polymer chain fixes the (P)- and (M)-conformation.15 The
temperature dependence of the helices indicated that the (M)-
helix diastereomer is a more unstable than the (P)-helix
diastereomer (Fig. S44b).16 Actually, when THF was added to the
solution of poly-1a in H2O, (M)-helix changed suddenly to (P)-
helix (Fig 3a). On the other hand, the addition of H2O to (P)-helix
in THF did not adopt the (M)-helix (Fig. S40). The dynamic
behavior of glycoarylopeptide including helical inversion is a
unique character that is not seen in previously reported
arylopeptides.8a
Fig. 3 CD spectra of (a) poly-1a in the mixture of H2O and THF, and (b) poly-1a with
acetylation and deacetylation. ([poly-1a] = 0.31 mM, 1%DMSO/H2O solution, 298
K). (c) CD titration experiment of poly-1a in H2O at 298 K in the presence of 0, 4,
8,…, 60 equiv of 4-carboxyphenylboronic acid. (Initial concentration: [poly-1a] =
0.25 mM, 1%DMSO/ 10 mM NH3 solution, pH = 10.5). (d) The image picture of
helix to helix transition of poly-1a. The chain-length from (M)-41-helix to (P)-31-
helix extend about 1.7 times of its length (pitch per residue; [(P)-31-helix] 5.1 Å
and [(M)-41-helix] 3.0 Å). Side chains are omitted for clarity.
In conclusion, we succeeded in establishing a folding
control in a glycoarylopeptide using saccharide-coded folding
information for
distinguished the configuration of a single stereogenic center in
the epimers—D-glucose and D-mannose—and formed
a peptide backbone. The arylopeptide
a
completely distinct and well-defined architecture ((P)-31-helix
or (M)-41-helix) in an aqueous solution. Similar to the recently
reported mechanism for glycopeptides,11 the saccharides near
the glycosylated site would determine the properties of the
peptide backbone. (P/M)-helix was easily converted to the
other structure via simple chemical modification
(acetylation/de-acetylation) or by using additives. These results
indicate that the properties of completely artificial polypeptide
Natural glycopeptides change their folding structures and
functions through various modifications or interactions with
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
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Oomens and J. P. Simons, J. Am. Chem. Soc., 2005, 127,
molecules are also dominated by the type of saccharides; this is
analogous to the behavior of natural polypeptides. Further
studies are in progress for establishing an elaborated
relationship between peptide backbone and the saccharides in
terms of chiral information and hydration.
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11414
DOI: 10.1039/C9CC10030J
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This work was supported by a grant-in-aid for JSPS KAKENHI
Grant JP19K05582, JSPS Research Fellowships for Young
Scientists, and the financial support from Ogasawara
Foundation. We thank the group of Prof. Takahiro Sato (Osaka
University) for CD measurements.
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15 CD spectra of oligomer (n = 1, 2) in THF and H2O show similar
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aggregation of the sample in the solid state.
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