Efficient Synthesis of Muramic and Glucuronic Acid Glycodendrimers as Dengue Virus Antagonists

Article abstract of DOI:10.1002/chem.201903788

Carbohydrates are involved in many important pathological processes, such as bacterial and viral infections, by means of carbohydrate-protein interactions. Glycoconjugates with multiple carbohydrates are involved in multivalent interactions, thus increasing their binding strengths to proteins. In this work, we report the efficient synthesis of novel muramic and glucuronic acid glycodendrimers as potential Dengue virus antagonists. Aromatic scaffolds functionalized with a terminal ethynyl groups were coupled to muramic and glucuronic acid azides by click chemistry through optimized synthetic strategies to afford the desired glycodendrimers with high yields. Surface Plasmon Resonance studies have demonstrated that the compounds reported bind efficiently to the Dengue virus envelope protein. Molecular modelling studies were carried out to simulate and explain the binding observed. These studies confirm that efficient chemical synthesis of glycodendrimers can be brought about easily offering a versatile strategy to find new active compounds against Dengue virus.

Full text of DOI:10.1002/chem.201903788

A Journal of
Accepted Article
Title: Efficient Synthesis of Muramic and Glucuronic Acid
Glycodendrimers as Dengue Virus Antagonist
Authors: Maria J. Hernaiz, Cecilia García-Oliva, Alfredo H. Cabanillas,
Almudena Perona, Pilar Hoyos, and Ángel Rumbero
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To be cited as: Chem. Eur. J. 10.1002/chem.201903788
Link to VoR: http://dx.doi.org/10.1002/chem.201903788
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Efficient Synthesis of Muramic and Glucuronic Acid
Glycodendrimers as Dengue Virus Antagonists
Cecilia García-Oliva,[a]† Alfredo H. Cabanillas,[b]† Almudena Perona,[a] Pilar Hoyos,[a] Ángel Rumbero,*[b]
and María J. Hernáiz*[a]
Abstract: Carbohydrates are involved in many important pathological
processes, such as bacterial and viral infections, by means of
carbohydrate-protein interactions. Glycoconjugates with multiple
carbohydrates are involved in multivalent interactions, thus
increasing their binding strengths to proteins. In this work, we report
the efficient synthesis of novel muramic and glucuronic acid
glycodendrimers as potential Dengue virus antagonists. Aromatic
scaffolds functionalized with a terminal ethynyl groups were coupled
to muramic and glucuronic acid azides by click chemistry through
optimized synthetic strategies to afford the desired glycodendrimers
with high yields. Surface Plasmon Resonance studies have
demonstrated that the compounds reported bind efficiently to the
Dengue virus envelope protein. Molecular modelling studies were
carried out to simulate and explain the binding observed. These
studies confirm that efficient chemical synthesis of glycodendrimers
can be brought about easily offering a versatile strategy to find new
active compounds against Dengue virus.
As mentioned above, the significance of glycoconjugates,
including glycolipids, glycoproteins and glycosaminoglycans
(GAGs) play a major role in bacterial and viral infection.[2c, 5] On
the one hand glucuronic acid (GlcA) plays an essential role in the
structure of GAGs, polysaccharide chains with variable degrees
of sulfation at different positions. Previous studies have
demonstrated that a highly charged heparan sulfate, a GAG found
on the cell surface, serves as a receptor for dengue virus by
binding to its envelope protein.[2d, 6] The main driving force for
interactions between the surface GAGs on the host cell and
envelope protein of DENV and other pathogenic flaviviruses is
electrostatic interaction between the negative charge of GAGs
and positive regions on the envelope proteins.[6a, 6e] Interventions
that disrupt this binding effectively inhibit infectivity.[7]
On the other hand, N-acetylmuramic acid (MurA) can be
found naturally as peptidoglycans, the repeating disaccharide
element β-D-GlcNAc-(1→4)-β-D-MurNAc composing bacterial
cell walls.[8] Both monosaccharides (GlcA and MurA) with an acid
group are involved in biological processes which are essential for
the infection and survival of the infective agents. However, these
structures are extremely difficult to synthesize, as can be seen in
the scarcity of studies on the topic. The availability of the
carbohydrates is a crucial step to simulate and understand their
implication in these processes. Monosaccharides may not be
enough by themselves to trigger a biological response due to the
small unit of interaction. This problem could be solved by the
preparation of dendrimers, consisting of a central core with a
variable number of branches, which can be decorated with
several saccharides, increasing the number of molecules
exposed.[9]
All pathogenic flaviviruses, such as dengue virus (DENV),[10]
yellow fever virus, West Nile virus, Japanese encephalitis virus,
and Zika virus, bind to GAGs through the putative GAG binding
sites within their envelope proteins to gain access to the surface
of host cells. DENV, the world’s most dangerous mosquito-borne
flavivirus disease, is classified in four different serotypes,[11]
places 2.5 billion at risk of infection and results in 20 million cases
each year in 100 countries and to date there is no completely
effective vaccine. Since a GAG is a putative receptor for DENV
envelope protein, soluble GAGs or other GlcA or MurA
glycodendrimers (monosaccharides with an acid group) could be
effective inhibitors of viral infection.
Introduction
Glycodendrimers are an important class of synthetic
macromolecules that can be used to mimic many structural and
functional features of cell surface glycoconjugates.[1]
Glycoconjugates perform key important functions in many
pathological processes, such as viral and bacterial infections,[2]
often regulated by carbohydrate-protein interactions. With
mounting evidence that not only the molecular structure but also
the valency and spatial organization of carbohydrate epitopes in
glycoconjugates influence the specificity and avidity of their
interactions with cognate receptors,[3] glycodendrimers have
increasingly served as a tool for probing the underlying
mechanisms of these events.[1a, 1b, 1e, 1f, 2a] In addition, there has
been much interest in using glycodendrimers mimetics of
glycoconjugates found on cell membrane to control the
presentation of carbohydrates in glycan microarrays,[4] which
have emerged as a powerful platform for interrogating ligand
specificities of carbohydrate-binding proteins.[4b-f]
[a]
C. García-Oliva, Dr. A. Perona, Dr. P. Hoyos and Dr. María J.
Hernáiz
Departamento de Química en Ciencias Farmacéuticas, Facultad de
Farmacia
Universidad Complutense de Madrid
E-mail: mjhernai@ucm.es
A. H. Cabanillas, Dr. A. Rumbero
Departamento de Química Orgánica
Universidad Autónoma de Madrid
Both authors contributed equally to this work
In this study, we present for the first time an efficient
synthesis of novel GlcA and MurA glycodendrimers as potential
active compounds in the treatment of Dengue virus. Surface
plasmon resonance (SPR) in conjunction with molecular
modelling were used to investigate the interaction of the MurA and
GlcA glycodendrimers with DENV envelope protein.
[b]
†
Supporting information for this article is given via a link at the end of
the document.
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10.1002/chem.201903788
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Results and Discussion
d) with (S)-2-bromopropanoic acid in the presence of sodium
hydride, resulting in the formation of different muramic acids 4,6-
O-protected and functionalized in the anomeric position 4(a-d).[16]
The (S)-2-bromopropanoic acid was obtained by diazotization
with sodium nitrite and KBr in acid pH and using natural L-Alanine
as starting material.[17]
Only compound 4d was purified and characterized, compounds 4
(a-c) were employed in the deprotection without further
purification. Then deprotected O-linked MurA -glycosides (5a-d)
were also prepared as monovalent ligands. The dimethyl acetal
was treated with Amberlyst in acid pH to provide the required
deprotected O-linked MurA -glycosides (5a-d).
Synthesis of O-linked MurA -glycosides
Four O-linked MurA -glycosides having different linkers
were prepared following a concise and efficient synthesis and
using commercially available D-GlcNAc as starting material
(Scheme 1). The synthetic strategy is shown in Scheme 1.
Synthesis of O-linked GlcA -glycosides
As illustrated in Scheme 2, a synthetic strategy was followed
to obtain the key intermediate 8 from commercial available
glucoronolactone (see Supporting information for Experimental
18]
section).[14b,
This was converted into the fully protected
glucopyranuronate 6 by reaction with NaOH in MeOH at room
temperature and acetic anhydride in pyridine from 0 ºC to room
temperature. The anomeric position protected with the acetyl
group could be deprotected using benzylamine to generate the
intermediate 7 as an - mixture. Activation of anomeric position
was carried out with trichloroacetonitrile and 1,8-diazabicyclo[5.4-
0]undec-7-ene (DBU) in THF to give the activated
trichloroacetimidate 8 that was efficiently glycosylated with
different alcohol to afford monosaccharides 9(a-c) that beared
different linkers in the anomeric position. Compound 9c was
treated with sodium azide to obtain compound 9d that displayed
a suitable linker for further conjugation with alkyne-terminated
dendritic cores.
Scheme 1. Synthesis of O-linked MurA -glycosides
In this strategy, first O-linked GlcNAc--glucopyranosides
(compounds 2a-c) via a furanosyl oxazoline 1 were synthesized
(see Supporting Information for Experimental section), and then
incorporated the acid moiety in C-3 (compounds 4a-d). For the
preparation of O-linked GlcNAc--glucopyranosides (2a-c) a
protocol developed by Bundle and co-workers was followed.[12]
The furanose Oxazoline 1 was prepared on a large scale from D-
GlcNAc in anhydrous acetone and dry FeCl3 as catalyst. The
crude reaction was used without further purification. The
1
presence of the product was confirmed by H-NMR, showing a
characteristic signal of anomeric carbon δ (ppm) 6.09 (d, 1H
[13]
J1,2=5 Hz).
Oxazoline 1 can be cleanly converted to the
(a-c) in
corresponding unprotected -glucopyranosides
2
quantitative yield. The ring expansion from furanose to pyranose
was detected at δ (ppm) 4.3 (d, 1H J1,2= 8.5 Hz) .[14] This strategy
simplifies the number of steps in comparison with other
glycosylation alternatives, such as the imidate formation, the use
of temporary protecting groups, or glycosylation of simple
alcohols under Koenings-Knorr conditions[15] (employing heavy
metal). Compound 2 (a-c) was obtained in high yield (80 %) by
simply evaporating the reaction mixture after the neutralization
with Et3N, followed by multiple washing of the residue with
CH2Cl2.[14b] Subsequent regioselective protection of the C-4 and
C-6 positions of the obtained -glucopyranoside was performed
using 2,2-dimethoxypropane and catalytic amount of camphor-
10-sulfonic acid (CSA) in anhydrous dimethylformamide (DMF) to
produce the partially unprotected saccharides 3 (a-c) with a free
3-hydroxyl group.[14a] In order to obtain the conveniently
functionalised carbohydrate ligand that displayed a suitable linker
for further conjugation with alkyne-terminated dendritic cores,
compound 3c was treated with sodium azide to afford
monosaccharide 3d. With this approach one can take advantage
of the presence of a single hydroxyl group at C-3 by coupling 3(a-
Scheme 2. Synthesis of O-linked GlcA -glycosides
Glycodendrimers synthesis
Several studies have reported the synthesis of
glycodendrimers with peripheral propargyl groups to enable a 1,3-
dipolar cycloaddition (“click”) reaction with carbohydrate derived
azides.[19] Three different cores (Scheme 3) bearing 2, 3 and 4
alkyne groups respectively (compounds 11, 12 and 13), were
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Scheme 3. Synthesis of glycodendrimers 14-25
quantitative yields (17, 21 and 24). All six individual deprotected
glycodendrimers were fully characterized by one- and two-
dimensional NMR spectroscopy (1H, 13C, HSQC) to verify their
structures as shown in Scheme 3.
selected for the conjugation step in order to study the influence of
valency on the molecular recognition of these multivalent
systems. Multivalent core scaffolds were prepared as previously
described in the literature from commercially available starting
20]
materials.[19a,
Once the ester-protected -glycosyl azides
Surface Resonance Plasmon studies
(compounds 4d and 9d) were obtained, preparation of the
corresponding glycodendrimers was undertaken (Scheme 3).
Cu (I) catalyzed cycloaddition reaction between fully
protected -glycosyl azides (compounds 4d and 9d) and the
dendritic cores (11, 12 and 13) was first performed. Global
deprotection would afford the final water-soluble MurA and GlcA
In order to evaluate the ability of the glycodendrimers 15,
17, 19, 21, 23 and 25 to interact with Dengue virus envelope
protein 2 (DENV2) a SPR binding study was carried out. As
mentioned above, previous studies have demonstrated that a
heparin-like glycosaminoglycan found on the cell surface, where
GlcA plays an essential role, serves as a receptor for Dengue
virus by binding to its envelope protein.[2d, 6a]
dendrimers.
We envisioned that the use of 4d and 9d , instead of the highly
polar unprotected monosaccharides (5d and 10d), would facilitate
the monitoring and the purification of the click chemistry reaction
products. Thus, 4d and 9d were coupled to compounds 11, 12
and 13 using CuSO4 as copper source and sodium ascorbate to
reduce in situ Cu (II) to Cu (I). Di, Tri and tetravalent
glycodendrimers (14, 16, 18, 20, 22 and 24) were obtained in
excellent yield after microwave heating at 80ºC for 45 min. The
compounds were purified by flash chromatography. The complete
absence of propargylic
appearance of a new triazole singlet  8.03 ppm suggested
successful completion of the reaction.
Deprotection of N-acetylmuramic glycodendrimers (14, 18
and 22) was then attempted by treatment with Amberlist H 15 in
a THF: H2O (1:1) mixture obtaining the desired compounds in
quantitative yields (15, 19 and 23). For the deprotection of
glucuronic acid derivatives (16, 20 and 24), the compounds were
treated with NaOMe/MeOH and the final products were
neutralized with amberlist H+ obtaining the desired compound in
Compounds 15, 17, 19, 21, 23 and 25 were flowed on the
immobilized DENV2 at a 175 m concentration (Fig 1). As can be
seen in Fig. 1, the glycodendrimers binding to DENV2 depends
on the type of carbohydrate presence and the valency. MurA
compounds 15 and 19 showed negligible binding response with
DENV2 envelope protein whereas compound 23 displayed a
higher response (45 RU).
On the other hand, a clear effect was observed with GlcA
compounds 21 and 25, especially with compound 25. Thus,
compounds with a GlcA showed stronger binding than the MurA
derivatives of equal core structure and valency. More significant
was the effect due to the GlcA glycodendrimer with higher valency,
compound 25. The four GlcA had a stronger interaction with
DENV2 than their corresponding MurA glycodendrimer 23.
Compound 25, with four GlcA acid blocks, showed a substantial
increase in the interaction, supporting the positive correlation
between valency and interaction intensity. Then glycodendrimer
25 was flowed over the chip containing immobilized DENV2.
Sensograms obtained showed increased interaction profiles with
1H resonance at  2.55 ppm and
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the GlcA tetravalent glycodendrimer (Fig. 2). Our results indicated
a strong interaction between DENV 2 and glycodendrimer 25 with
four GlcA with a KD value in M range. Using steady-state
analysis of SPR measurements for the interaction between 25
and DENV2, the dissociation constant (KD) was found to be 22
M (Fig S1 in Supporting Information). These results demonstrate
that the presentation of GlcA ligands on dendritic scaffolds
strongly increase their interaction with DENV2 due to the
multivalent effect. Previous studies showed that DENV2 bound to
heparin with a KD of 31 nM.[21] Our studies assessing the
interaction of the DENV2 with glycodendrimer 25 demonstrated
binding (KD of 22 M) that was weaker than that observed with
heparin. Nevertheless, high affinities can be achieved if the sugar
residues are presented with higher valency. This compound
represents an excellent candidate for structural optimization to
achieve higher affinity and to expect anti-adhesive properties
against Dengue virus.
Model web server, the 3D model was further minimized with the
same force field. 3D structure for dendrimer was built using
glycam carbohydrate builder web server and the interactive
molecular graphics program PyMOL.[23] Finally the 3D geometry
of the complex formed by the DENV2 with glycodendrimer 25 was
deduced by using AutoDock Vina.[24] Stability of the predicted
DENV2-glycodendrimer 25 complex was studied by MD
simulation (20ns). Structure stability of the complex was
evaluated by calculating the root-mean-square-deviation (RMSD)
of the Cα atoms along the trajectory. Global RMSD values of the
protein were lower than 3Å, indicating high stability of the protein
(Fig. S2 in Supporting Information). Regarding the ligand, after
the first 2ns of simulation it changed the pose with respect to the
docked structure, reaching values between 10-15 Å RMSD. This
new pose was very stable, and no changes were observed in
RMSD values after 8 ns of the MD, which means that the rest of
the simulation the ligand remained in that position. The effective
binding free energies between the glycodendrimer 25 and the
more relevant residues in the binding site were qualitatively
estimated using the program MM-ISMSA.[25]
DENV2-glycodendrimer 25 interaction energy was
measured during the simulation and it remained nearly constant
around -110 kcal/mol, indicating the pose of the dendrimer in the
protein was energetically favourable (Table 1).
Table 1. Description of the interactions in complex DENV2-glycodencrimer 25
E
Type of
Interaction*
DENV2
Glycodendrimer 25
(kcal/mol)
LYS-299
Glucuronic acid moiety
-12,85
qq+ HB+vdW
GLN-297
ASN-359
TYR-182
SER-302
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
Triazol and phenyl moieties
(aromatic rings)
Glucuronic acid and phenyl
(aromatic ring) moieties
Triazol moiety
-5,91
-5,38
-5,21
-5,09
HB+vdW
qq+ HB
qq
HB
GLY-181
THR-180
GLY-300
-5,05
-4,89
-4,63
HB
HB
HB
Figure 1. SPR measurement of MurA (15, 19 and 23) and GlcA (17, 21 and 25)
glycodendrimers binding to immobilized DENV2 at 175 m
(aromatic rings)
ILE-361
MET-301
LYS-295
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
Triazol and phenyl moieties
(aromatic rings)
Glucuronic acid moiety
Triazol moiety
-4,53
-4,36
-4,32
HB+vdW
HB
HB+vdW
TYR-303
SER-149
LYS-161
-3,91
-3,52
-3,33
vdW
HB+vdW
vdW
175 M
15 M
(aromatic rings)
LEU-298
LYS-42
Glucuronic acid moiety
-2,80
-2,14
-2,13
-2,07
-1,57
-1,54
-1,45
HB
qq+ HB
HB
qq+ HB
qq+ HB
HB
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
Triazol moiety
(aromatic rings)
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
Glucuronic acid moiety
GLY-183
LYS-164
LYS-338
PRO-360
THR-184
HB
Figure 2. Binding responses of different concentrations of glycodendrimers 25
with DENV2 immobilized on the CM4 chip, showing association and dissociation
phases. Responses were reference substracted and blank corrected.
THR-363
-1,45
vdW
GLU-151
LEU-179
HIP-148
LEU-296
THR-159
HIE-162
-1,31
-1,26
-1,24
-1,09
-1,07
-0,98
vdW
vdW
HB
HB
HB
Molecular modelling studies
In order to explain the strong interaction observed with the
tetravalent GlcA glycodendrimer (compound 25), we set out to
characterize properties of DENV2−glycodendrimer 25 complex
using molecular modeling. Crystal structure of DENV2 (PDB:
3C5X) was refined by homology modelling. Protein-
glycodendrimers 25 interactions were studied performing a
docking simulation in GAG-binding domains.
HB
ARG-292
Glucuronic acid moiety
-0,92
HB
*Type of interaction: charge-charge (qq), hydrogen bond (HB), van der Waals (vdW)
The ligand was anchored by important electrostatic interactions
including multiple hydrogen bonding and van der Waals (Figs. 3
and 4).
The Cartesian coordinates for a 3D model of the apo
form of envelope protein (E) Dengue Virus 2 Thailand 16681/84
were taken directly from the PDB (accession code 3C5X),[22]
internal missing string of six residues was modelled using Swiss-
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Figure 4. a) Final MD pose of GlcA glycodendrimer 25 in DENV2.
Dendrimer 25 is shown as green sticks and protein is shown as
electrostatic surface calculated by APBS (negative charged surface in
red; positive charged surface in blue; hydrophobic surface in white)
Figure 3. 2D free energy per-residue interaction plot of DENV2-glycodendrimer 25
complex. X axis shows the interaction energies, and Y axis the name and number
of DENV2 residues.
A)
B)
Figure 5. A) 3D view of the interaction between LYS-299 and final MD pose of GlcA glycodendrimer 25 in DENV2. B) 3D view of the interaction between LYS-295
and final MD pose of GlcA glycodendrimer 25 in DENV2. Dendrimer 25 is shown as green sticks and protein as white cartoon. Main residues of the protein are
highlighted in blue. Electrostatic interactions are depicted as black dash lines.
The main charge-charge interactions took place between the
carboxylate groups of dendrimer and LYS-299 (-13 kcal/mol) and
LYS-295 (-5 kcal/mol), that are the key residues involved in
Conclusions
heparin
binding[6d]
(Fig.
5)
(DENV2
294DKLQLKGMSYSMCTGKFKVVKEIAET319).[6d]
MurA and GlcA acids are both monosaccharides that play a
key role in biological processes such as bacterial or viral infection.
It is well known that Dengue virus binds to GAGs through the
putative GAG binding sites within their envelope proteins to gain
access to the surface of host cells. These interactions are
predominantly ionic where positively charged basic aminoacid
residues in the GAG binding site of DENV2 interact with
negatively charged residues in the cell receptor, a highly sulfated
form of the GAG heparan sulfate. Since heparan sulfate GAG is
a putative receptor for DENV2, one can envisage that a good
strategy to develop viral infectivity inhibitors is the use soluble
multivalent highly charged glycodendrimers. Multivalency is a
very attractive approach to improve the affinity of carbohydrates.
Therefore, conjugation of several copies of MurA or GlcA to a
multivalent scaffold will certainly result in new multivalent
glycostructures with good binding affinities to their receptors.
Due to the complexity of the synthetic route leading to MurA
and GlcA acid derivatives, new strategies have to be developed.
As can be seen in Table 1 most of the residues of the DENV2
GAG binding region (23 residues) provide hydrogen bond and
charge-charge interactions with the glucuronic acid moiety, while
only few provide van der Waals interactions with this moiety
(Glu151 and Leu179). No major contacts between the triazol and
phenyl moieties (aromatic rings) could be highlight only Gly181,
forms hydrogen bonds with this aromatic rings and Tyr303,
Lys161 and Thr363 show van der Waals interactions. Therefore
we can say that the core architecture of glycodendrimer 25 does
not have a significant effect on the binding affinity These results
are consistent with previous carbohydrate-protein studies carried
out with similar glycodendrimer where the carbohydrate region of
the ligand (sialic acid and lactose moieties) was in close contact
with the Viscumin protein and played a significant role in the
binding affinity. In the same study the effect of the triazol and
phenyl moieties was lower.[19a]
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assignments from 1D and 2D NMR spectroscopy were based on COSY,
HSQC, and HMBC correlation experiments. Mass spectra were recorded
with an Applied Biosystems QSTAR XL. Quantitative elemental analysis
by combustion of carbon, hydrogen, nitrogen and sulfur were carried out
in Servicio de Microanálisis Elemental from Universidad Complutense
(Madrid, Spain), using a Leco CHNS 932 Elemental Analyzer. The purity
of new compounds was assessed by CHNS elemental analysis, and all
values were verified to be within ±0.4% of the theoretical values. Thin layer
chromatography (TLC) was carried out on aluminum sheets coated with
silica gel 60 F254 (Merck). TLC plates were inspected under UV light
(l=254 nm) and developed by treatment with phosphomolybdic acid
hydrate in ethanol (10 %), followed by heating. Silica gel column
chromatography was performed with silica gel (230–400 mesh).
Dengue virus envelope protein 2 (DENV2) was purchased from The Native
Antigen Company.
A series of O-linked MurA and GlcA -glycosides functionalized
with a suitable linker for further conjugation with alkyne-
terminated scaffolds were prepared. These compounds are
readily obtained through a synthetic route that has been optimized
by choosing appropriate protecting groups and reaction
conditions. This methodology is mild and compatible with a range
of functional linkers and has become the method of choice in our
group.
MurA and GlcA derivatives bearing a short linker with a
terminal azide group were coupled with alkyne-terminated
scaffolds by using a simple, and efficient Cu(I) catalyzed
cycloaddition reaction resulting in the desired MurA and GlcA
glycodendrimers with excellent yields. The success in applying
this methodology to MurA and GlcA chemistry should provide
abundant opportunities to expand and apply these O-linked MurA
or GlcA glycosides bearing an azide group for preparation of
challenging new and different multivalent system related to
bacterial or viral infections and investigations into mechanism of
action.
SPR sensor chips CM4 (carboxymethylated dextran) and other reagents
used in SPR experiments were purchased from GE Healthcare. SPR
experiments were performed with a Biacore 3000.
General procedure for click reaction, synthesis of compounds 14,
16, 18, 20, 22 and 24
MurA and GlcA glycodendrimers were evaluated in their
ability to interact with DENV2 assayed. The binding capacity of
MurA glycodendrimers (compounds 15, 19 and 23) is relatively
low compared to GlcA glycodendrimers (compounds 17, 21 and
25) which confirms the selectivity of the DENV2 towards GAGs
receptors on the cell surface. At the same time, multivalency is
another important factor in the interactions between MurA and
GlcA glycodendrimers to DENV, no binding was observed for the
monovalent ligands, either MurA or GlcA. However,
glycodendrimers with higher valency show strong binding,
especially GlcA glycodendrimer 25 with four GlcA acid blocks.
Molecular modeling provided an explanation for the binding
observed between compound 25 and the GAG-binding region of
DENV2, which is a basic-residue rich region of DENV2. The main
type of interactions are electrostatic interactions between the
carboxylate groups of glycodendrimers 25 and Lys-299 and Lys-
295 in the GAG-binding region of DENV2, which are the key
residues involved in the heparin binding region. The evaluation of
the triazol and phenyl moieties showed that the core architecture
does not have a significant effect on the interactiob with DENV2.
Binding mode of compound 25 in the GAG-binding region of
DENV2 will form the basis for future structure-activity relationship
optimization.
Scaffold 11, 12 or 13 (1 equiv.) was dissolved in a DMF:H2O (4:1) mixture,
CuSO4·5H2O (0.05 equiv./alkyne), sodium ascorbate (0.1 equiv./alkyne)
and compound 4d or 9d (1.2eq/alkyne) were added to the solution. The
resulting mixture was stirred at 60 °C overnight.[26] Undissolved
compounds were removed by filtration and the solvent was evaporated
under vacuum. The residue was purified by flash chromatography (AcOEt:
MeOH: NH3 25 % 2:1:0.2).
The reaction mixture was carried out in a microwave reactor (CEM-
Discover), Scaffolds 11, 12 or 13 (1 equiv.) was dissolved in a DMF:H2O
(98:2) mixture, CuSO4·5H2O (0.2 equiv./alkyne), sodium ascorbate (0.3
equiv./alkyne) and compound 4d or 9d (1.2eq/alkyne) were added to the
solution. The resulting mixture was heated at 80°C for 60 min.[19a] After
cooling to RT, the mixture was dissolved in ethyl acetate and washed with
a solution of 1% EDTA in water (2x10 mL) and brine (2x10 mL). The
combined organic layers were dried over anhydrous magnesium sulfate,
filtered, and concentrated under reduced pressure. The residue was
purified by flash chromatography on silica (AcOEt: MeOH: NH3 25 %
2:1:0.2).
General method for acetal group deprotection: Synthesis of
compounds 15, 17, 19, 21, 23 and 25.
Compound 14, 18 and 22 (40 mg) was dissolved in 1 mL of a mixture
THF:H2O (1:1) and Amberlyst® 15 was added at room temperature to
adjust the solution to acid pH. The mixture was stirred overnight. The solid
of the mixture was removed by filtration and the solvent was evaporated
under reduced pressure affording 15, 19 and 23 as a white solids.
Peracetylated compounds (16, 20 and 24) was dissolved in anhydrous
methanol and NaOMe previously suspended in methanol was added
dropwise (4 equiv). The mixture was stirred at 0°C until the consumption
of the substrate. Then the reaction was quenched by the addition of
Amberlite IRA-15 to pH 3. The solution was filtered and concentrated
under vacuum. The residue was purified by silica gel column
chromatography (CH2Cl2/MeOH/NH3 (25%) 6:1:0,2) affording 17, 21 and
25 as a white solid.
This novel multivalent approach using
a
simple
monosaccharide (GlcA or MurA) as ligand has shown to be a
viable strategy to develop compounds that interact with DENV2
and work is in progress to further increase these valuable
properties.
Experimental Section
All reagents were commercially available and used without further
Compound 14 (82 % yield)
purification.
A CEM-Discover focused microwave synthesizer with
1H NMR (300 MHz, CD3OD)  (ppm): 8.03 (s, 2H, H-11), 7.22 (d, J=2.3 Hz,
2H; H-2, H-6), 6.90 (t, J=2.3 Hz, 1H, H-4), 5.17 (s, 4H, H-9), 4.51 – 4.40
(m, 4H, H-12), 4.36 (d, J=8.2 Hz, 2H, H-1’), 4.26 (q, J=6.8 Hz, 2H, H-12’),
3.86 (s, 3H, H-8), 3.84 – 3.74 (m, 4H, H-6’ax, H-6’eq), 3.74 – 3.71 (m, 2H,
H-14a), 3.71 – 3.65 (m, 2H, H-2’), 3.68 – 3.49 (m, 4H, H-4’, H-3’), 3.40 (dt,
J=10.0, 5.8 Hz, 2H, H-14b), 3.20 (ddd, J=9.6, 9.6 y 5.5 Hz, 2H, H-5’), 2.14
microwave power maximum level of 300 W and microwave frequency of
2455 MHz was employed for the microwave-assisted reactions,
NMR spectra were recorded at 293 K, with a 500 MHz spectrometer
(Bruker AC) and 300 MHz spectrometer (Bruker Avance). Shifts are
referenced relative to deuterated solvent residual peaks. Complete signal
This article is protected by copyright. All rights reserved.

10.1002/chem.201903788
Chemistry - A European Journal
FULL PAPER
– 2.03 (m, 4H, H-13), 1.99 (s, 6H, H-11’), 1.47 (s, 6H, H-8’), 1.33 (s, 6H,
H-9’), 1.29 (d, J=6.8 Hz, 6H, H-14’).
H-6’ax), 3.76 – 3.66 (m, 12H, H-2’, H-6’ec, H-28a), 3.65 – 3.56 (m, 4H, H-
4’), 3.55 – 3.44 (m, 4H, H-3’), 3.38 (dt, J=10.7, 5.5 Hz, 4H, H-28b), 3.18
(ddd, J=9.7, 9.7, 5.4 Hz, 4H, H-5’), 2.09 – 2.01 (m, 8H, H-27), 1.98 (s, 12H,
H-11’), 1.44 (s, 12H, H-8’), 1.31 (s, 12H, H-9’), 1.29 (d, J=7.3 Hz, 12H, H-
14’).
13C NMR (76 MHz, CD3OD)  (ppm): 180.5 (C-13’), 174.5 (C-10’), 167.9
(C-7), 160.9 (C-3, C-5), 160.9 (C-12, C-14, C-19, C-21), 144.6 (C-24),
140.8 (C-10, C-17), 133.1 (C-1), 125.6 (C-25), 109.5 (C-2, C-6), 107.9 (C-
4), 107.6 (C-11, C-15, C-18, C-22), 104.0 (C-1’), 102.6 (C-13, C-20), 100.5
(C-7’), 79.5 (C-3’), 79.3 (C-12’), 75.9 (C-4’), 70.8 (C-9, C-16), 68.5 (C-5),
66.7 (C-28), 63.1 (C-6’), 62.6 (C-23), 56.5 (C-2’), 52.9 (C-8), 48.0 (C-26),
31.2 (C-27), 29.5 (C-9’), 23.4 (C-11’), 20.0 (C-14’), 19.5 (C-8’).
13C NMR (75 MHz, CD3OD)  (ppm): 179.8 (C-13’), 174.5 (C-10’), 167.9
(C-7), 160.8 (C-3, C-5), 144.4 (C-10), 133.3 (C-1), 125.8 (C-11), 109.7 (C-
2,C-6), 108.1 (C-4), 104.0 (C-1’), 100.6 (C-7’), 79.3 (C-3’), 78.6 (C-12’),
76.2 (C-4’), 68.4 (C-5’), 66.6 (C-14), 63.1 (C-6’), 62.8 (C-9), 56.5 (C-2’),
52.9 (C-8), 48.0 (C-12), 31.2 (C-13), 29.5 (C-9’), 23.4 (C-11’), 19.8 (C-14’),
19.5 (C-8’).
Compound 15 (85 % yield)
1H NMR (300 MHz, CD3OD)  (ppm): 8.11 (s, 2H, H-11), 7.27 (d, J=2.3 Hz,
2H, H-2, H-6), 6.96 (t, J=2.3 Hz, 1H, H-4), 5.22 (s, 4H, H-9), 4.57 – 4.47
(m, 4H, H-12), 4.44 (q, J=6.8 Hz, 2H, H-9’), 4.33 (d, J=8.1 Hz, 2H, H-1’),
3.90 (s, 3H, H-8), 3.89 – 3.80 (m, 4H, H-6, H-6’a, H-14a), 3.72 – 3.66 (m,
4H, H-6’b, H-2’), 3.51 (t, J=9.6 Hz, 2H, H-3’), 3.48 – 3.40 (m, 4H, H-4’, H-
14b), 3.27 (ddd, J=9.5, 5.7, 2.3 Hz, 2H, H-5’), 2.16 – 2.08 (m, 4H, H-13),
1.94 (s, 6H, H-8’), 1.37 (d, J=6.8 Hz, 6H, H-11’).
Compound 23 (80 % yield).
1H NMR (300 MHz, CD3OD)  (ppm): 8.05 (s, 4H, H-25), 7.21 (d, J=2.4
Hz, 2H; H-2, H-6), 6.87 – 6.75 (m, 1H, H-4), 6.77 – 6.69 (m, 4H, H-11, H-
15, H-18, H-22), 6.64 (s, 2H; H-13, H-20), 5.16 (s, 8H, H-23), 5.04 (s, 4H,
H-9, H-16), 4.60 (q, J=6.8 Hz, 4H, H-9’), 4.56 – 4.44 (m, 8H, H-26), 4.41
(d, J=8.3 Hz, 4H, H-1’), 3.92 – 3.82 (m, 8H, H-6’a, H-28a), 3.76 – 3.66 (m,
8H, H-6’b, H-2’), 3.54 – 3.43 (m, 12H, H-3’, H-4’, H-28b), 3.31 – 3.24 (m,
4H, H-5’), 2.19 – 2.08 (m, 8H, H-27), 2.03 (s, 12H, H-8’), 1.39 (d, J=6.8 Hz,
12H, H-11’).
13C NMR (76 MHz, CD3OD)  (ppm): 176.4 (C-10’), 174.2 (C-7’), 168.0 (C-
7), 160.9 (C-3, C-5), 144.4 (C-10), 133.4 (C-1), 125.9 (C-11), 109.7 (C-
2,C-6), 108.1 (C-4), 103.4 (C-1’), 82.0 (C-3’), 77.9 (C-5’, C-9’), 71.7 (C-4’),
66.4 (C-14), 62.7 (C-9), 62.6 (C-6’), 55.7 (C-2’), 52.8 (C-8), 48.1 (C-12),
31.3 (C-13), 22.0 (C-8’), 19.8 (C-11’).
Anal. Calcd. for C42H60N8O20: C, 50.60; H, 6.07; N, 11.24. Found: C, 50.53;
H, 6.06; N, 11.20.
HRMS: m/z calcd for C42H60N8O20Na [M+Na]+ 1019.3821, found
1019.3847.
13C NMR (76 MHz, CD3OD)  (ppm): 175.8 (C-10’), 173.8 (C-7’), 168.0 (C-
7), 161.0 (C-3, C-5), 161.0 (C-12, C-14, C-19, C-21), 144.7 (C-10, C-17),
125.8 (C-25), 109.6 (C-2, C-6), 108.0 (C-4), 107.6 (C-11, C-15, C-18, C-
22), 102.8 (C-13, C-20), 102.6 (C-1’), 83.1 (C.3’), 77.8 (C-5’), 77.0 (C-9’),
72.3 (C-4’), 70.9 (C-9, C-16), 66.5 (C-28), 62.5 (C-23, C-6’), 56.2 (C-8),
52.9 (C-8), 48.1 (C-26), 31.3 (C-27), 23.4 (C-8’), 19.5 (C-11’).
Anal. Calcd. for C90H124N16O40: C, 52.22; H, 6.04; N, 10.83. Found: C,
52.03; H, 6.02; N, 10.79.
Compound 18 (90 % yield)
1H NMR (500 MHz, CD3OD)  (ppm): 8.01 (s, 3H, H-9), 6.31 (s, 3H, H-2,
H-4, H-6), 5.12 (s, 6H, H-7), 4.54 – 4.41 (m, 6H, H-10), 4.36 (d, J=8.3 Hz,
3H, H-1’), 4.28 (q, J=6.9 Hz, 3H, H-12’), 3.83 (dd, J=10.7, 5.4 Hz, 3H, H-
6’), 3.76 (m, 6H, H-6’ec, H-12a), 3.68 (dd, J=10.2, 8.3 Hz, 3H, H-2’), 3.64
(t, J=9.2 Hz, 3H, H-4’), 3.56 (dd, J=10.2, 8.9 Hz, 3H, H-3’), 3.43 – 3.36 (m,
3H, H-12b), 3.22 (ddd, J=9.9, 9.9, 5.4 Hz, 3H, H-5’), 2.10-1.9 (m, 6H, H-
11), 2.01 (s, 9H, H-11’), 1.49 (s, 9H, H-8’), 1.34 (s, 9H, H-9’), 1.30 (d, J=7.0
Hz, 9H, H-14’).
HRMS: m/z calcd for C90H124N16O40Na [M+Na]+ 2091.8058, found
2091.8032.
Compound 16. (85%).
1H-RMN (500 MHz, CDCl3)  (ppm): 7.77 (s br, 2H, H-11), 7.25 (t, J=2.2
Hz, 2H, H-2 and H-6), 6.81 (t, J=2.2 Hz, 1H, H-4), 5.26 (t, J=9.6 Hz, 2H,
H-3'), 5.24 (t, J=9.6 Hz, 2H, H-4'), 5.18 (s br, 4H, H-9), 4.99 (dd, J=9.6, 7.7
Hz, 2H, H-2'), 4.54 (d, J=7.7 Hz, 2H, H-1'), 4.51-4.46 (m, 2H, H-12a), 4.43-
4.37 (m, 2H, H-12b), 4.01 (d, J=9.6 Hz, 2H, H-5'), 3.87 (s, 3H, H-8), 3.82-
3.78 (m, 2H, H-14a), 3.67 (s, 6H, H-7'), 3.52-3.48 (m, 2H, H-14b), 2.18-
2.11 (m, 4H, H-13), 2.04 (s, 6H, OAc), 1.99 (s, 12H, OAc).
13C NMR (126 MHz, CD3OD)  (ppm): 180.0 (C-13’), 174.6 (C-10’), 161.6
(C-1, C-3, C-5), 144.7 (C-8), 125.7 (C-9), 104.1 (C-1’), 100.6 (C-7’), 96.3
(C-2, C-4, C-6), 79.1 (C-3’), 78.6 (C-12’), 76.4 (C-4’), 68.5 (C-5’), 66.6 (C-
12’), 63.2 (C-6’), 62.6 (C-7), 56.6 (C-2’), 48.0 (C-10), 31.2 (C-11), 29.5 (C-
9’), 23.4 (C-11’), 19.8 (C-14’), 19.5 (C-8’).
13C-RMN (125 MHz, CDCl3)  (ppm): 170.1 (CO), 169.5 (CO), 169.5 (CO),
167.4 (C-6'), 166.7 (C-7), 159.5 (C-3 and C-5), 143.4 (C-10), 132.2 (C-1),
124.1 (C-11), 108.7 (C-2 and C-6), 107.3 (C-4), 100.6 (C-1'), 72.4 (C-5'),
72.0 (C-3'), 71.3 (C-2'), 69.5 (C-4'), 65.8 (C-14), 62.2 (C-9), 53.0 (C-7'),
52.4 (C-8), 46.6 (C-12), 30.2 (C-13), 20.8 (AcO), 20.7 (AcO), 20.6 (AcO).
Anal. Calcd. for C46H58N6O24: C, 51.21; H, 5.42; N, 7.79. Found: C, 51.10;
H, 5.40; N, 7.77.
Compound 19 (95 % yield)
1H NMR (300 MHz, CD3
J=6.7 Hz, 3H, H-9), 6.33
(s, 3H, H-2, H-4, H-6), 5.18 (s, 6H, H-7), 4.59 – 4.47 (m, 9H, H-10, H-9’),
4.38 (d, J=8.1 Hz, 3H, H-1’), 3.87 – 3.75 (m, 6H, H-6’a, H-12a), 3.68 – 3.60
(m, 6H, H-6’b, H-2), 3.51 – 3.34 (m, 9H, H-12b, H-3, H-4), 3.25 – 3.20 (m,
3H, H-5’), 2.16 – 2.06 (m, 6H, H-11), 2.00 (d, J=7.3 Hz, 9H, H-8’), 1.31 (d,
J=6.8 Hz, 9H, H-11’).
13C NMR (76 MHz, CD3OD)  (ppm): 175.7 (C-10’), 174.3 (C-7’), 161.2 (C-
1, C-3, C-5), 143.6 (C-8), 126.7 (C-9), 103.2 (C-2, C-4, C-6), 102.38 (C-
1’), 83.3 (C-3’), 77.7 (C-5’), 77.1 (C-9’), 72.2 (C-4’), 66.6 (C-12), 62.3 (C-
6’), 61.8 (C-7), 56.3 (C-2’), 49.5 (C-10), 31.0 (C-11), 23.1 (C-8’), 19.4 (C-
11’).
Compound 17. (97%).
1H-RMN (500 MHz, CD3OD) δ (ppm): 8.20 (s br, 2H, H-11), 7.28 (t, J=2.2
Hz, 2H, H-2 an H-6), 6.96 (t, J=2.2 Hz, 1H, H-4), 5.24 (s br, 4H, H-9), 4.64
- 4.56 (m, 4H, H-12), 4.31 (d, J=7.7 Hz, 2H, H-1'), 3.91 (s, 3H, H-8), 3.87
(d, J=9.6 Hz, 2H, H-5'), 3.84-3.79 (m, 2H, H-14a), 3.55 (t, J=9.6 Hz, 2H,
H-4'), 3.56-3.51 (m, 2H, H-14b), 3.42 (t, J=9.6 Hz, 2H, H-3'), 3.28 (dd,
J=9.6, 7.7 Hz, 2H, H-2'), 2.24-2.16 (m, 4H, H-13).
Anal. Calcd. for C57H84N12O27: C, 50.00; H, 6.18; N, 12.27. Found: C,
49.84; H, 6.16; N, 12.25.
HRMS: m/z calcd for C57H84N12O27Na [M+Na]+ 1391.5466, found
1391.5453.
13C-RMN (125 MHz, CD3OD) δ (ppm): 170.1 (C-6'), 166.7 (C-7), 142.9 (C-
10), 159.5 (C-3 and C-5), 132.1 (C-1), 125.2 (C-11), 108.5 (C-2 and C-6),
106.9 (C-4), 103.2 (C-1'), 75.9 (C-3'), 75.3 (C-5'), 73.4 (C-2'), 71.9 (C-4'),
65.5 (C-14), 61.3 (C-9), 51.6 (C-8), 46.9 (C-12), 29.9 (C-13).
Anal. Calcd. for C32H42N6O18: C, 48.12; H, 5.30; N, 10.52. Found: C, 48.05;
H, 5.28; N, 10.50.
Compound 22 (75 %)
1H NMR (300 MHz, CD3OD)  (ppm): 7.94 (s, 4H, H-25), 7.13 (d, J=2.4 Hz,
2H; H-2, H-6), 6.75 (t, J=2.4 Hz, 1H, H-4), 6.66 (d, J=2.3 Hz, 4H; H-11, H-
15, H-18, H-22), 6.56 (t, J=2.4 Hz, 2H; H-13, H-20), 5.08 (s, 8H; H-23),
4.95 (s, 4H; H-9, H-16), 4.46 – 4.3 (m, 8H, H-26), 4.34 (d, J=8.1 Hz, 4H,
H-1’), 4.22 (q, J=7.3 Hz, 4H, H-12’), 3.82 (s, 3H, H-8), 3.80 – 3.76 (m, 4H,
HRMS: m/z calcd for C32H42N6O18Na [M+Na]+ 821.2453, found 821.2466.
Compound 20. (75%).
This article is protected by copyright. All rights reserved.

10.1002/chem.201903788
Chemistry - A European Journal
FULL PAPER
1H-RMN (500 MHz, CDCl3) δ (ppm): 7.76 (s br, 4H, H-25), 7.25 (d, J=2.3
Hz, 2H, H-2 and H-6), 6.77 (t, J=2.3 Hz, 1H, H-4), 6.69 (d, J=2.3 Hz, 4H,
H-11, H-15, H-18 and H-22), 6.61 (t, J=2.3 Hz, 2H, H-13 and H-20), 5.25
(t, J=9.6 Hz, 4H, H-3'), 5.19 (t, J=9.6 Hz, 4H, H-4'), 5.17 (s br, 8H, H-23),
5.02 (dd, J=9.6, 7.7 Hz, 4H, H-2'), 5.01 (s br, 4H, H-9 and H-16), 4.54 (d,
J=7.7 Hz, 4H, H-1'), 4.46-4.41 (m, 4H, H-26 a), 4.39-4.32 (m, 4H, H-26b),
4.02 (d, J=9.6 Hz, 4H, H-5'), 3.90 (s, 3H, H-8), 3.86-3.81 (m, 4H, H-28a),
3.68 (s, 12H, H-7'), 3.54-3.48 (m, 4H, H-28b), 2.22-2.12 (m, 8H, H-27),
2.06 (s, 12H, AcO), 2.01 (s, 12H, AcO), 2.00 (s, 12H, AcO).
13C-RMN (125 MHz, CDCl3) δ (ppm): 170.1 (CO), 169.6 (CO), 169.5 (CO),
167.3 (C-6'), 166.8 (C-7), 159.8 ( C-12, C-14, C-19 and C-21), 159.8 (C-5
and C-3), 143.8 (C-24), 139.2 (C-10 and C-17), 132.2 (C-1), 124.0 (C-25),
108.6 (C-2 and C-6), 107.3 (C-4), 106.7 (C-11, C-15, C-18 and C-22),
101.7 (C-13 and C-20), 100.6 (C-1'), 72.4 (C-5'), 72.1 (C-3'), 71.3 (C-2'),
70.1 (C-9 and C-16), 69.5 (C-4'), 65.9 (C-28), 62.1 (C-23), 53.0 (C-7'), 52.4
(C-8), 46.7 (C-26), 30.2 (C-27), 20.8 (AcO), 20.7 (AcO), 20.6 (AcO).
SPR experiments were performed at 258C with Biacore 3000 (GE
Healthcare). PBST (10 mm phosphate, pH 7.40, 150 mm NaCl, and
0.005% v/v surfactant P20) was used as running buffer for CM4
experiments. A solution with Dengue Virus serotype 2 protein (DENV-2)
was adjusted to 15 g/mL in 10 mm citrate pH 4.00 buffer and the protein
was immobilized in flow cell 1 of a CM4 sensor chip by following the amine
coupling method according to the manufacturer’s instructions. Prior to
injection over the sensor chip, DENV2 was mixed with a 4-fold molar
excess of the heparin, for 30 min at 4°C, to protect the glycosaminoglycan-
binding domains of the envelope protein. Immobilization response was
1800 RU and then the sensor surface washed with 1 M NaCl to remove
the bound heparin. Sensor chip flow cell 2 was activated, blocked and used
as a reference surface. Blank samples and concentration series were
injected on CM4 chip at a flow rate of 5 L/min for 180 s and dissociation
was registered for 180 s. Chip CM4 concentration series were 15-500 m
of the glycodendrimers (compound 14: 50-500 m; 18: 125-225 m; 22:
15-175 m; 12: 50-500 m: 16: 50-500 m; 20: 150-400 m). Data
processing and analysis were carried out with BiaEvaluation v.4.1.1 (GE
Healthcare). All signals were blank subtracted, reference corrected, and
globally adjusted to an adequate kinetic model to obtain binding
parameters. Goodness of fit was indicated by 2, and values of less than
10 indicated good fit.
Compound 21. (90%).
1H-RMN (500 MHz, CD3OD) δ (ppm): 8.29 (s br, 4H, H-25), 7.17 (d, J=2.3
Hz, 2H, H-2 and H-6), 6.77 (t, J=2.3 Hz, 1H, H-4), 6.72 (t, J=2.3 Hz, 4H,
H-11, H-15, H-18 and H-22), 6.65 (t, J=2.3 Hz, 2H, H-13 and H-20), 5.25 -
5.14 (m, 8H, H-23), 5.02 (s br, 4H, H-9 and H-16), 4.63 - 4.56 (m, 8H, H-
26), 4.25 (d, J=7.7 Hz, 4H, H-1'), 3.85 (s, 3H, H-8), 3.82 (d, J=9.6 Hz, 4H,
H-5'), 3.79 - 3.74 (m, 4H, H-28a), 3.53 - 3.46 (m, 4H, H-28b), 3.50 (t, J=9.6
Hz, 4H, H-4'), 3.38 (t, J=9.6 Hz, 4H, H-3'), 3.23 (dd, J=9.6, 7.7 Hz, 4H, H-
2'), 2.21 - 2.13 (m, 8H, H-27)
Acknowledgements
13C-RMN (125 MHz, CD3OD) δ (ppm): 171.4 (C-6'), 168.1 (C-7), 161.0
(C-5 and C-3), 160.8 (C-12, C-14, C-19 and C-21), 141.1 (C-24), 139.7 (C-
10 and C-17), 131.8 (C-1), 125.2 (C-25), 109.6 (C-2 and C-6), 108.1 (C-
4), 108.0 (C-11, C-15, C-18 and C-22), 104.5 (CH, C-1'), 102.9 (C-13 and
C-20), 77.2 (C-3'), 76.6 (C-5'), 74.7 (C-2'), 73.2 (C-4'), 70.9 (C-9 and C-
16), 66.9 (C-28), 62.0 (C-23), 53.0 (C-8), 48.7 (C-26), 30.1 (C-27).
Anal. Calcd. for C42H57N9O24: C, 47.06; H, 5.36; N, 11.76. Found: C, 46.90;
H, 5.34; N, 11.73.
The authors gratefully acknowledge financial support provided by
the Spanish Ministerio de Economía y Competitividad, Grants
CTQ2015-66206-C2-1-R and RTI2018-096037-B-I00.
Keywords: Muramic acid • glucuronic acid • Dengue virus •
dendrimers • binding
HRMS: m/z calcd for C42H57N9O24Na [M+Na]+ 1094.3414, found
1094.3426.
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Compound 24. (85%).
1H-RMN (500 MHz, CDCl3)  (ppm): 7.77 (s br, 3H, H-9), 6.27 (s, 3H, H-2,
H-4 and H-6), 5.25 (t, J=9.6 Hz, 3H, H-3'), 5.18 (t, J=9.6 Hz, 3H, H-4'), 5.12
(s br, 6H, H-7), 5.01 (dd, J=9.6, 7.7 Hz, 3H, H-2'), 4.55 (d, J=7.7 Hz, 3H,
H-1'), 4.52-4.46 (m, 3H, H-10a), 4.44-4.38 (m, 3H, H-10b), 4.02 (d, J=9.6
Hz, 3H, H-5'), 3.85-3.79 (m, 3H, H-12a), 3.75 (s, 9H, H-7'), 3.55-3.49 (m,
3H, H-12b), 2.21-2.11 (m, 6H, H-11), 2.06 (s, 9H, AcO), 2.01 (s, 18H, AcO).
13C-RMN (125 MHz, CDCl3) (ppm): 170.1 (CO), 169.6 (CO), 169.5 (CO),
167.4 (C-6'), 160.2 (C-1, C-3 and C-5), 143.7 (C-8), 124.1 (C-9), 100.6 (C-
1'), 95.2 (C-2, C-4 and C-6), 72.4 (C-5'), 72.0 (C-3'), 71.3 (C-2'), 69.5 (C-
4'), 65.9 (C-12), 61.9 (C-7), 53.0 (C-7'), 46.7 (C-10), 30.2 (C-11), 20.8
(AcO), 20.7 (AcO), 20.6 (AcO).
Compound 25 (95%).
1H-RMN (500 MHz, CD3OD) δ (ppm): 8.27 (s br, 3H, H-9), 6.36 ( s, 3H, H-
2, H-4 and H-6), 5.19 (s br, 6H, H-7), 4.65 - 4.60 (m, 6H, H-10), 4.30 (d,
J=7.7 Hz, 3H, H-1'), 3.87 (d, J=9.6 Hz, 3H, H-5'), 3.84 - 3.80 (m, 3H, H-
12a), 3.57 - 3.53 (m, 3H, H-12b), 3.55 (t, J=9.6 Hz, 3H, H-4'), 3.42 (t, J=9.6
Hz, 3H, H-3'), 3.28 (dd, J=9.6, 7.7 Hz, 3H, H-2'), 2.28 - 2.17 (m, 6H, H-11).
13C-RMN (125 MHz, CD3OD) δ (ppm): 171.4(C-6'), 161.4 (C-1, C-3 and C-
5), 140.3 (C-8), 125.2 (C-9), 104.2 (C-1'), 94.8 (C-2, C-4 and C-6), 77.0
(C-3'), 76.4 (C-5'), 74.4 (C-2'), 72.9 (C-4'), 66.6 (C-12), 61.9 (C-7), 48.3 (C-
10), 30.8 (C-11).
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Anal. Calcd. for C70H88N12O36: C, 50.24; H, 5.30; N, 10.04. Found: C,
50.09; H, 5.27; N, 10.01.
HRMS: m/z calcd for C70H88N12O36Na [M+Na]+ 1695.5322, found
1695.5307
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10.1002/chem.201903788
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FULL PAPER
FULL PAPER
Cecilia
García-Oliva,
Alfredo
H.
Cabanillas, Almudena Perona, Pilar
Hoyos, Ángel Rumbero,* and María J.
Hernáiz*
Binding
Studies
Page No. – Page No.
Efficient Synthesis of Muramic and
Glucuronic Acid Glycodendrimers as
Dengue Virus Antagonists
Glycodendrimers displaying muramic and glucuronic acids were efficiently
synthesized. This multivalent systems showed high binding affinities to Dengue virus
evelope protein.
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