Direct and Regioselective Di-α-fucosylation on the Secondary Rim of β-Cyclodextrin

Article abstract of DOI:10.1002/chem.201806090

A straightforward glycosylation method is described to regio- and stereoselectively introduce two α-l-fucose moieties directly to the secondary rim of β-cyclodextrin. Using NMR and MS fragmentation studies, the nonasaccharide structure was determined, which was also visualized using molecular dynamics simulations. The reported glycosylation method proved to be robust on gram-scale, and may be generally applied to directly glycosylate β-cyclodextrins to make well-defined multivalent glycoclusters.

Full text of DOI:10.1002/chem.201806090

A Journal of
Accepted Article
Title: Direct and Regioselective Di-α-Fucosylation on the Secondary
Rim of β-Cyclodextrin
Authors: Stella A. Verkhnyatskaya, Alex H. de Vries, Elmatine Douma-
de Vries, Renze J.L. Sneep, and Marthe Walvoort
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content of this Accepted Article.
To be cited as: Chem. Eur. J. 10.1002/chem.201806090
Link to VoR: http://dx.doi.org/10.1002/chem.201806090
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Direct and Regioselective Di--Fucosylation on the Secondary
Rim of -Cyclodextrin
[a]
[b]
[a]
[a]
Stella A. Verkhnyatskaya, Alex H. de Vries, Elmatine Douma-de Vries, Renze J.L. Sneep, and
Marthe T.C. Walvoort*[a]
Abstract: A straightforward glycosylation method is described to
regio- and stereoselectively introduce two -L-fucose moieties directly
to the secondary rim of -cyclodextrin. Using NMR and MS
fragmentation studies, the nonasaccharide structure was determined,
which was also visualized using molecular dynamics simulations. Our
glycosylation method proved to be robust on gram-scale, and may be
generally applied to directly glycosylate -cyclodextrins to make well-
defined multivalent glycoclusters.
Scheme 1. -CD cone representation and schematic depiction of secondary rim
-fucosylation
Introduction
generally adopt the shape of a truncated cone, which has the C-
hydroxyls on the top (primary rim) and the C-2 and C-3
hydroxyls on the bottom of the cone (secondary rim, Scheme 1).
6
Carbohydrate-protein
interactions are central to many biological processes in all
domains of life, including glycoprotein folding, cell signaling,
immunomodulation, and host-pathogen binding. [1] Generally,
these interactions are highly multivalent in character, and distinct
and
carbohydrate-carbohydrate
[
9]
Most examples of chemically synthesized glycoclusters display
the glycans on the primary rim, [
10-17]
and only a few examples are
reported of direct glycosylation of the hydroxyls. [18-21] Alternatively,
enzymatic transglycosylation strategies resulted in the formation
carbohydrate moieties spaced at specific distances are essential
for productive binding. [2] This ‘multivalency effect’ has inspired the
of a mixture of multiply glycosylated CDs, bearing -galactosyl
units on the C-2 or C-6 position of the glucosides. [
22, 23]
When two
[3]
design of glycoconjugate clusters based on polymers,
nanoparticles, [4] cyclodextrins [5, 6] and calixarenes, [7] amongst
or more glycoside units were introduced, low regioselectivity was
observed. Thus, application of chemical modifications allows for
better control over the substitution pattern of CD.
others, to which a plethora of different glycans are attached. [8]
Interestingly, amongst the various attachment methods and
linkers, the natural glycosidic linkage is virtually absent. Instead,
glycans are often attached through a peptide, thioester or triazole
moiety.
In our on-going program to generate purely carbohydrate-
based inhibitors of host-pathogen interactions for clinical and food
applications, we set out to glycosylate cyclodextrins (CDs) directly
with L-fucose moieties. Cyclodextrins are cyclic oligosaccharides
consisting of six, seven, or eight α-(1→4)-D-glucoside moieties, to
give the so-called -, -, and -CDs, respectively. Owing to their
non-toxic nature, CDs are generally regarded as safe (GRAS) and
have received widespread attention in food, agriculture,
cosmetics and pharmacy. As depicted in Scheme 1, CDs
Interestingly, due to the primary alcohols, the primary rim is
considered conformationally more flexible, resulting in a reduced
effective diameter. In contrast, the alcohols on the secondary rim
are sterically more constrained and less flexible, positioning the
substituents in a well-defined manner. For the generation of
multivalent CD-based glycoclusters, it is highly desirable to have
a method to directly glycosylate the secondary rim. The main
challenges associated with directly glycosylating cyclodextrins
arise from the lack of discrimination between the chemically
similar hydroxyls on both rims, and the complicated
characterization of the resulting products. Illustrative of the first
challenge are the seminal reports from Sollogoub and co-workers
that deal with the discrimination between the primary hydroxyls to
install different functionalities. [24] Also, several methods exist to
regioselectively introduce or remove one [25] or two[26-32] protecting
groups on the primary rim. To the best of our knowledge, methods
[
a]
S.A. Verkhnyatskaya, E.Douma-de Vries, R.J.L. Sneep, Dr. M.T.C.
Walvoort
Stratingh Institute for Chemistry, University of Groningen
Nijenborgh 7, 9747 AG Groningen, the Netherlands
E-mail: m.t.c.walvoort@rug.nl
to regioselectively liberate hydroxyls on the secondary rim are
scarce, with one example reported for permethylated CD. [33]
Herein, we report the stereoselective di-α-fucosylation of β-CD on
two C-3 positions of the secondary rim. L-Fucose is selected as
multivalent CD decoration for its central role in biological glycans,
[
b]
A.H. de Vries
Groningen Biomolecular Sciences and Biotechnology Institute,
University of Groningen
Nijenborgh 7, 9747 AG Groningen, the Netherlands
[34-36]
where it is attached to blood group antigens, the complex N-
glycan of glycoproteins, and human milk oligosaccharides,
amongst others. Fucosylated glycans have shown to block the
intestinal adhesion of pathogenic bacteria such as Salmonella
Supporting information for this article is given via a link at the end of
the document
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Chemistry - A European Journal
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enterica [37] and Escherichia coli, [38] and this makes multivalent
fucosylated scaffolds interesting targets for novel antimicrobial
therapies.
Results and Discussion
Our method makes use of a β-CD ring (compound 4) that has the
C-3 hydroxyl groups of each glucoside unit available. The
synthesis of the acceptors started with perbenzylation of
commercially available α- and β-CD, providing the protected CDs
[
29]
1
and 2 in high yield (Scheme 2).
These were then subjected
[
39, 40]
3 2
to Et SiH/I
to regioselectively liberate all C-3 hydroxyls to
give acceptors 3 and 4, having six and seven free hydroxyls
respectively. For the -fucosylation of these acceptors, reported
thio-fucoside donor
5 was utilized (Scheme 3). While the
stereoselective introduction of multiple -fucosidic bonds
simultaneously remains a challenging endeavor, this donor has
proven to provide robust -fucosylation in other glycan syntheses.
Scheme 3. Fucosylation of CD acceptors 3 and 4, and synthesis of 2Fuc--CD
nonasaccharide 10 (numbering of Glc residues is given in blue)
[
41]
The first glycosylation experiment was performed using 1.15
equivalents of donor 5 per hydroxyl (total: 6.9 eq for -CD, 8.05
eq for -CD), which was treated with pre-activation conditions
(
To elucidate the structure of the major nonasaccharide, three
different regioisomers were considered (Figure S1B): fucosides
on the 3-OH positions of neighbouring glucoside units (termed
2 2
Ph SO/Tf
O) at -80°C (Scheme 3). [ After complete donor
42]
activation, the CD acceptor was added and the reaction was left
to proceed at -80°C overnight, after which time the products were
analyzed by UPLC-UV/MS (C4 column). To our surprise, the
mixture of products from the α-CD acceptor 3 contained more
than 55% of the twice fucosylated octasaccharide, albeit a mixture
of three isomers (peak f=2, Figure 1A, and Figure S1A). The
coupling constants of the anomeric fucose H1 signals indicated
that the fucosidic linkage was formed stereoselectively,
eliminating the possibility that the mixture of octasaccharides
resulted from erosion of stereoselectivity (Figure S2). In addition,
a significant amount of the (1→1’)-fucosyl disaccharide was
isolated, probably resulting from donor hydrolysis and subsequent
glycosylation, highlighting the poor reactivity of the CD acceptor.
Interestingly, similar experimental conditions with the β-CD
acceptor 4 also gave the twice fucosylated nonasaccharide 9 as
the major product (50%), but it appeared as a single peak in the
UPLC chromatogram, suggesting the formation of one major
nonasaccharide isomer (Figure 1B). The -stereoselectivity of the
3A,3B), two Fuc-Glc motifs separated by one unmodified
glucoside (3A,3C), and two Fuc-Glc motifs separated by two
unmodified glucose residues (3A,3D). We commenced our
characterization efforts with NMR on the purified sample. Signals
arising from H-1, H-2 and H-3 within the ring of several Glc and
Fuc units were identified with 1H NMR, COSY and TOCSY
experiments for each residue (see supporting information). HSQC
in combination with HMBC experiments provided the clear
identification of the C-3 signals of the two Glc units to which a
fucoside was attached. The latter signals shifted significantly
downfield in HSQC as compared to the non-modified Glc-C3
signals (from 73.2-72.6 ppm to 77.4 and 77.2 ppm, Figure S11)
and had a cross-peak with H-1 of fucoses in HMBC spectrum
(
Figure 2B, cross-peak A).
1
newly formed glycosidic bonds was confirmed by H NMR
(J1-2= 4.0 Hz). To simplify the characterization of the
nonasaccharide product, compound 9 was isolated by preparative
HPLC (20% isolated yield), and all benzyl protecting groups were
2
removed by hydrogenolysis using Pd/C in THF/H O to give
product 10 (80%).
Scheme 2. Synthesis of acceptors 3 and 4.
Figure 1. UPLC-UV traces of fucosylated CDs after size-exclusion
chromatography: A) Fuc-α-CDs, B) Fuc-β-CDs.
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then undergo cross-ring fragmentations (Figure 3A). [47, 48] The
observation of the consecutive 2,4A fragments 11, 13, 15, 17, 19
and 21 confirms the presence of two glucoside units between the
fucosylated residues, as in the 3A,3D type of modification (Figure
3
B). Additionally, the m/z values corresponding to the 0,2A and 0,2
X
fragments 23, 25, 26, 27 and 28 support the 3A,3D substitution
pattern by demonstrating evidence of three glucosides between
the Glc-Fuc moieties (see SI for more details). Specific fragments
corresponding to the 3A,3C substitution pattern were not
observed.
The clear regioselectivity of fucosylation on β-CD
encouraged the optimization of the reaction conditions to increase
the yield of the twice fucosylated product 9. The reaction times
and temperatures were varied, while keeping the equivalents of
2 2
donor 5, acceptor 4, Tf O, Ph SO and TTBP unchanged (Table
S1). First, conditions were changed towards a shorter reaction
time and higher temperature: -70°C for 3 hours (compared
to -80°C overnight). These conditions provided
a mixture
containing 33% of the di-fucosylated product. However, the
formation of higher fucosylated compounds, i.e. tetra- and
pentafucosylation, had significantly increased. Next, the reaction
was started at -80°C followed by warming the mixture to different
temperatures (-70°C, -60°C and -50°C), resulting in short reaction
times (10-30 minutes). Quenching the reaction at -70°C resulted
in 35% of the nonasaccharide, while a significant amount of
unreacted acceptor was still present (19%) together with
1
monofucosylated Fuc -β-CD product (24%). Increasing the
Figure 2. A) Fragment of the structure of compound 10with the identified cross-
temperature to -60°C provided 57% of the desired di-fucosylated
product, while quenching the reaction at -50°C led to a reduced
contribution of difucosylated product (35%). As a result, the
highest yield of nonasaccharide 9 was observed when the
reaction was neutralized at -60°C, and these conditions were
used to α-fucosylate the -cyclodextrin acceptor on a large scale
peaks H1Fuc
→ C3Glc (A, green bond), H3Glc→C4Glc (B, red bond), and
C4Glc→2xH1Glc’ (C, blue bond); B) Fragment of the HMBC NMR of 10.
Also using HMBC analysis, we identified the C4 signals of the
modified Glc residues (Figure 2, bond and cross-peak B).
Interestingly, these C4 signals had a cross-peak to H1 of a non-
modified glucose residue (Figure 2A, bond C) indicating the
presence of a non-fucosylated Glc residue linked to each of the
Fuc-(1→3)-Glc motifs, eliminating the possibility of the 3A,3B
regioisomer (see SI for a detailed explanation). However, further
proof to determine the relative positions of the other unmodified
Glc residues remained difficult to obtain, since many Glc signals
overlapped. Also comparing it to the singly fucosylated -CD,
(
1 g, 0.42 mmol, Table S1, entry 6).
isolated from HPLC (BnFuc
1
-CD, see SI), did not aid in the
characterization.
Next, we turned our attention to mass spectrometry
fragmentation studies. [43] Ionization of fucosylated glycans in
positive mode is notoriously prone to allow migration of the
fucosides to neighbouring residues upon MS analysis of intact
protonated glycan ions, as recently highlighted by Seeberger and
co-workers. [44] In contrast, the analysis of the deprotonated
species formed with negative-mode ionization reduces the
[
45,
46]
occurrence of side-reactions.
Therefore, HILIC
chromatography in combination with MS in negative ion-mode
Figure 3. A) Fragmentation pattern of two possible parent ions; B) Low-
resolution MS/MS spectrum of nonasaccharide 10 (region from m/z 500 to 1150)
measured on LCQ Fleet mass spectrometer (Thermo Fisher Scientific) in
combination with an ion-trap.
was applied to fucosylated cyclodextrin 10, and parent masses of
-
2-
m/z 1425 [M-H] and 712 [M-2H] were observed (Figure S3).
Next, fragmentation of the double charged ion was optimized,
resulting in cross-ring fragmentations predominantly. In general,
the CD ring is first opened to form linear parent ions, which will
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These optimized conditions provided a mixture of fucosylated
CDs in 95% yield containing 61% of nonasaccharide, which could
be purified by preparative HPLC (purity >90%), or by regular flash
column chromatography, providing nonasaccharide 9 in 67%
isolated yield and in 69% purity. After global deprotection, 275 mg
of compound 10 was obtained (75%).
Structural information of the fucoside positioning in space is
important to rationalize future carbohydrate-lectin binding, so in
an effort to understand the spatial arrangement of di-fucosylated
This is apparent from the tumbling of glucoside units, which bend
outwards and distort the conical shape and the hydrogen-bonding
network. [ Indeed, our analysis reveals that specifically the
fucosylated glucoside units show enhanced rotation and
increased chair flexibility. These tilted states are not frequent, but
are stable over several nanoseconds (Figure S4). The tilting of
the cyclodextrin ring sugars is more pronounced upon
fucosylation (Figure 4), which can be seen from the enhanced
probability of finding the dihedral angles connecting Glc units G-
A (blue dashed line) and C-D (orange dashed line) at values of
60-90 degrees away from the main peak. Both force fields show
this effect, but the Q4MD force field shows it more strongly, which
is a reflection of the larger conformational flexibility found using
this force field. This flexibility is also apparent from the wider
distributions of the dihedral angles (Figure 4C), and is discussed
in more detail in the SI. The conformations visited during the
simulations were grouped into distinguishable conformational
states (so-called clusters) by cluster analysis (for details, see SI).
Considering the most abundant conformations (Figure S5), the
fucosides are attached to a canonical CD ring and prefer to be
slightly tilted inward or outward with respect to the CD cavity. The
distance between the two centers of the fucosides is peaked at
49]

-CD 10, molecular dynamics (MD) simulations were performed.
Since a recent comprehensive study of native CDs in water
showed that structural and dynamic properties may differ
depending on the choice of force field, [49] two different force fields,
i.e. GROMOS 53A6 [50] and Q4MD, [51] were used in combination
with the GROMACS simulation package. [52] In contrast to what is
commonly assumed, the cyclodextrin cone is quite flexible,
especially in protic solvents. [53]
~
~
7 Å, but the groups can occasionally be considerably closer at
5 Å, or more distant depending on the tumbling of sugar rings
and rotations of the fucoses with respect to the −CD barrel wall
Figure S6).
In an effort to confirm the structural representation from MD
simulations with NMR analysis, NOE intensities were calculated
see the supporting information for a detailed description). The
(
(
MD simulations suggested the presence of weak NOE signals that
reflect the conformational flexibility of nonasaccharide 10, in
particular regarding the different orientations of the fucosides. The
H-atoms on the fucose C5 and C6 positions (H5FucA,D and
H6FucA,D) are relatively close to the H-atom on C3 of the upstream
glucoside (H3GG,C) in many of the most populated clusters
(
Figures S7 and S8). The closest distance between H5FucA,D and
H3GG,C is ~1.9 Å in the most populated cluster, and the closest
distance between the H6FucA,D and H3GG,C is ~2 Å. The H-atoms
involved in these expected NOE signals are highlighted in the
representative structure of the most populated conformational
cluster in Figure 5A. Excitingly, these NOE cross-peaks were
indeed observed experimentally in the NOESY spectrum as
shown in Figure 5B, supporting the dynamical ensemble
observed in the MD simulations. The MD analysis also suggests
that the bending of the Fuc rings away from the CD barrel is
associated with an enhanced chair flexibility of the substituted Glc
3
rings (residues A and D). The relatively low J couplings of 8.3 Hz
measured for these Glc H3 hydrogens in compound 10, as
compared to 9.5 Hz for the H-3 signals in natural -CD, are
consistent with the MD simulation, and support the contribution of
Figure 4. Comparison of the distribution of dihedral angles between
two neighboring Glc residues within the β-CD rings between β-CD
and nonasaccharide 10. (A) The O5-C1-C4-C5 dihedral angles
connecting the ring Glc residues. Dihedral angle distributions for
natural β-CD are depicted with solid lines and fucosylated β-CD 10
with dashed lines, using both the GROMOS force field (B) and the
Q4MD force field (C)
1
[54]
the unfavored C
4
conformer (see supporting information).
The
MD analysis shown here should be taken as qualitative: the
dynamics of the conformational changes is slow on the MD time-
scale, and it is not possible to extract converged thermodynamic
(
equilibrium) data from the extensive simulations of
4
microseconds.
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Figure 5. A) Characteristic conformation of 10 with H-5Fuc, H-6Fuc and H-3Glc highlighted as black spheres for close contact between fucose on Glc(A) and Glc(G)
fucoside C-atoms are shown in dark blue); B) Observed NOE of H-5Fuc(A,D) → H-3Glc (G,C)
(
Conclusions
We acknowledge Dr. Johan Kemmink for his help with measuring
the NMR spectra, and Dr. Sander van Leeuwen for his assistance
in characterizing the products. M.T.C.W. acknowledges financial
support from the European Union through the Rosalind Franklin
Fellowship COFUND project 600211.
This report presents the regioselective 3A,3D-fucosylation
of the secondary rim of semi-protected -CD, which proved to be
robust, reproducible, and scalable. In natural glycans, such as
HMOs, the fucosides are often separated by non-fucosylated
carbohydrate residues, making this 3A,3D pattern a highly
relevant mimic. While the regioselective 6A,6D substitution of
CDs is well-developed for decoration of the primary rim, [29, 55] only
limited examples of di-substitutions on the secondary rim exist, [56]
and none of these examples covers a direct glycosylation reaction.
We propose that the regioselectivity observed here arises from
the kinetic conditions employed in the glycosylation reaction, i.e.
low temperatures and short reaction times. This may drive the
reaction to yield the least sterically hindered 3A,3D
nonasaccharide as the kinetic product of the reaction. We
hypothesize that the remarkable regioselectivity observed for the
β-, but not for the α-CD, is likely to depend on the size of the CD
ring. MD simulations to corroborate this hypothesis are currently
underway.
Keywords: -cyclodextrin • fucosylation • regioselective • mass
fragmentation • MD simulation
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This article is protected by copyright. All rights reserved.

Chemistry - A European Journal
10.1002/chem.201806090
FULL PAPER
Entry for the Table of Contents
FULL PAPER
A robust method to decorate -
cyclodextrin with two fucoses
through direct glycosylation is
developed. Using kinetic reaction
conditions, the regioselectivity was
steered towards the 3A,3D-
fucosylated nonasaccharide. The
structure was elucidated using a
combination of HPLC, NMR and MS
fragmentation, and visualized using
MD simulations.
S.A. Verkhnyatskaya, A.H. de Vries,
E.Douma-de Vries, R.J.L. Sneep,
M.T.C. Walvoort*
Page No. – Page No.
Direct and Regioselective Di--
Fucosylation on the Secondary Rim
of -Cyclodextrin
This article is protected by copyright. All rights reserved.