Bioorganic Chemistry 141 (2023) 106913

Contents lists available at ScienceDirect

Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg

Expanding the synthesis of a library of potent glucuronic acid

glycodendrons for Dengue virus inhibition
Pedro Ramírez-López, Carlos Martínez, Alejandro Merchán, Almudena Perona,
María J. Hernaiz *
Departamento de Química en Ciencias Farmacéuticas, Facultad de Farmacia, Universidad Complutense de Madrid, Plz. Ramón y Cajal s/n, Madrid C.P. 28040, Spain

A R T I C L E I N F O A B S T R A C T

Keywords: Multivalent glycodendrons are valuable tools to mimic many structural and functional features of cell-surface
Glycodendrons glycoconjugates and its focal position scaffolds represent important components to increase specificity and af­
Glycosaminoglycan mimetics finity. Previous work in our group described the preparation of a tetravalent glucuronic acid dendron that binds
Glucuronic acid
with good affinity to Dengue virus envelope protein (KD = 22 μM). Herein, the chemical synthesis and binding
Click chemistry
Dengue virus
analysis of a new library of potent glucuronic acid dendrons bearing different functional group at the focal
Carbohydrate-Protein interactions position and different level of multivalency are described. Their chemical synthesis was performed sequentially
Multivalency in three stages and with good yields. Namely a) the chemical synthesis of the oligo and polyalkynyl scaffolds, b)
assembling with fully protected glucuronic acid-based azide units by using a microwave assisted copper-
catalysed azide-alkyne cycloaddition reaction and c) sequential deprotection of hydroxyl and carboxylic acid
groups.
Surface Plasmon Resonance studies have demonstrated that the valency and the focal position functional
group exert influence on the interaction with Dengue virus envelope protein. Molecular modelling studies were
carried out in order to understand the binding observed. This work reports an efficient glycodendrons chemical
synthesis that provides appropriate focal position functional group and multivalence, that offer an easy and
versatile strategy to find new active compounds against Dengue virus.

1. Introduction macromolecules. [4,7,14] While glycodendrimers consist of a central

Carbohydrates are key players in the molecular recognition pro­
cesses that take place in viral infection events due to their interaction
with viral surface proteins. In general, carbohydrates are displayed in
the form of multivalent conjugates on the surface of cells. The actual
structural requirements for carbohydrate–protein interactions are not
known in full detail. However it is accepted that these interactions are
highly specificity and weak forces that enable them are compensated by
multivalent presentation of the ligand. [1–6] In order to investigate
multivalency in carbohydrate recognition, numerous artificial multiva­
lent glycostructures have been reported that try to mimic the structural
and functional features of glycoconjugates present in the cell membrane,
which will inhibit the adhesion of viruses. [5,7–13] In this context,
glycodendrimers and glycodendrons are architectures based on oligo
and polyvalent scaffolds to which multiple identical sugar units are
attached to form a chemically well-defined glycosylated
scaffold with branched chains attached to it repetitively bearing termi­
nal carbohydrate on the surface and forming spherical macromolecules,
dendrons typically contain a branched fragment of a whole dendrimer
with an orthogonal functional group at the focal point. [7,15]
In recent years, the use of glycodendrimers has proven to be a ver­
satile strategy to multivalent viral entry inhibition suggesting its great
potential in glycobiology. However there are very few examples of
glycodendrons in the literature. Different chemical strategies have been
used to synthesize glycodendrimers [4,6,14,16–19] and glycodendrons,
[20–23,24,25] being copper(I)-catalysed azide-alkyne cycloaddition
(CuAAC) reaction one of the most commonly employed. [26–30]
To our knowledge, there are not previous published reports of the
biological activity of glycodendrons against the Dengue virus (DENV).
Vargas et al [31] have designed and synthesized different mannose-
based glycodendrimers as DC-SIGN ligands [31] and studied their
properties as antivirals against DENV infection. These studies have

* Corresponding author.
E-mail address: mjhernai@ucm.es (M.J. Hernaiz).

https://doi.org/10.1016/j.bioorg.2023.106913
Received 14 August 2023; Received in revised form 28 September 2023; Accepted 8 October 2023
Available online 10 October 2023
0045-2068/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
P. Ramírez-López et al.
shown that binding depends on the type of ligand, valency and scaffold,
being an hexavalent mannose-based glycodendrimers the compound
that bound most efficiently to DC-SING in a micro molar range and
showing greater antiviral activity against DENV.
Considering that the adhesion of viruses, as well as other types of
biological recognition processes, are dependent on multivalent presen­
tation, molecular structure and spatial organization, the synthesis of
new glycodendrons bearing functional groups of different nature at the
focal position can be used to mimic many structural and functional
features of cell-surface glycoconjugates. These structures can be also
used to interfere with biological interactions and block viral adhesion
and entry into host cells as an effective strategy to inhibit viral in­
fections. [7,24].
Heparan sulphate (HS) is a glycosaminoglycan (GAG) that plays a
major role in Dengue virus infection where glucuronic acid (GlcA) a key
residue in their structure. [32,33] Previous studies have demonstrated
that a highly charged heparan sulphate found on the cell surface, serves
as a receptor for DENV by binding to its envelope protein. The main
driving force for the interactions between the surface GAGs, on the host
cell and envelope protein of DENV, and other pathogenic flaviviruses are
electrostatic interactions between the negative charge of GAGs and
positively charged regions on the envelope proteins. [34] Based on that,
different carbohydrate derivatives have been described as inhibitors of
virus entry. Most of them are soluble GAGs or anionic polysaccharides
such as heparin, [34–37] chondroin sulphate, [38] fucoidan, [39]
carrageenan, [40] pentosan polysulfate, [41] sulfonated and carbox­
ymethylated β–glucan derivatives [42] and highly charged oligosac­
charides or polyanions such heparin decasaccharide, [34] suramin, [43]
and methyl-α-sulfated GlcA. [44] With these results and supporting the
hypothesis that GlcA is a key residue in the structure of GAGs [32,33],
our group has previously described the synthesis of a tetravalent GlcA
dendron that mimic the terminal moiety of the natural HS glycan, a
major binding determinant of the DENV envelope protein. This glyco­
dendron binds with good affinity to DENV envelope protein (KD = 22
μM). This dendron was composed of an aromatic methyl 3,5-dihydroxy­
benzoate scaffold, bearing four copies of GlcA ligand and a methyl ester
on the focal position. [24,29] To explain the interaction observed with
the tetravalent glycodendron, molecular modelling studies were carried
out to characterise DENV-glycodendron complex. These studies showed
that the ligand was anchored by electrostatic interactions, namely
multiple hydrogen bonding and van der Waals, but the main charge-
charge interactions took place between the carboxylate groups of the
dendrimer and the key residues involved in heparin binding. [24] As a
result of this work, it was confirmed that the GlcA glycodendron could
be a versatile candidate to find new active compounds against DENV.
Bearing this in mind and aiming at developing a dendron with higher
affinity for the DENV protein a series of glycodendrons have been pre­
pared, where the functional group of the focal position and the degree of
valence have been modified.
2. Experimental section
2.1. General Methods
Unless noted otherwise, all manipulations were carried out under an
argon atmosphere. All reagents were obtained from commercial sources
and used without further purification. Solvents were HPLC or synthesis
grade and were used without further purification. A Monowave 50, 315
W, Anton Paar Microwave was employed for the microwave assisted
CuAAC Procedures. 1H and 13C NMR spectra were recorded at 250, 300,
500 or 700 MHz (1H NMR) and at 62.5, 75, 100 or 175 MHz (13C NMR)
using CDCl3, D2O as solvent with the residual solvent signal as internal
reference (CDCl3, 7.26 and 77.0 ppm) and (D2O, 4.79 ppm). The
following abbreviations are used to describe peak patterns when
appropriate: s (singlet), d (doublet), t (triplet), q (quadruplet), m
(multiplet), and br (broad). Complete signal assignments from 1D and
2
Bioorganic Chemistry 141 (2023) 106913
2D NMR spectroscopy were based on COSY, HSQC, and HMBC corre­
lation experiments. Thin layer chromatography (TLC) was carried out on
aluminium sheets coated with silica gel 60 F254 (Merck). TLC plates
were inspected under UV light (λ = 254 nm) and developed by treatment
with sulfuric acid in methanol (10 %) or by KMnO4/H2O solution, fol­
lowed by heating. Flash column chromatography was performed using
silica gel (230–400 mesh). Mass spectra were recorded using electro­
spray (ES) chemical ionization techniques in its positive mode, unless
noted otherwise.
DENV envelope protein 2 (DENV2) was purchased from The Native
Antigen Company. SPR sensor chips CM4 (carboxymethylated dextran)
and other reagents used in SPR experiments were purchased from GE
Healthcare. SPR experiments were performed with a Bia core 3000.
2.2. General procedure for microwave assisted CuAAC reaction: synthesis
of compounds 5a-c, 5e, 6a-b, 6e, 7a-b, 7e, 14b, 14e, 17a-b and 17e
CuSO4⋅5H2O (0.20 equiv/alkyne) and sodium ascorbate (0.35–0.4
equiv/alkyne) were sequentially added to a solution of the appropriate
alkyne (1.0 equiv) and GlcA-derived azide 1 (1.15–1.5 equiv/alkyne) in
appropriate solvent mixture (DMF/H2O 98:2; DMSO/H2O 5:1, details
are described in Supporting Information) (2 mL) at room temperature.
The resulting mixture was placed in a Monowave 50 microwave (Anton
Paar) at 315 W and irradiated at 65 ◦ C until completion of the reaction in
short reaction times, TLC analysis (30–240 min, details of each glyco­
dendrimer are specified in supporting information). After cooling to
room temperature, the reaction mixture was diluted with AcOEt (20 mL)
and washed with a solution of Na2EDTA/NaOH aq (2 × 10 mL) and brine
(2 × 10 mL). The combined organic layers were dried over anhydrous
Na2SO4, filtered, and concentrated under vacuum. The residue was
purified by flash chromatography on silica gel (hexanes/AcOEt 1:1 to
DCM/MeOH 10:1 or AcOEt/MeOH 10:1, details are described in Sup­
porting Information) affording the corresponding acetyl-protected
dendrons 5a-c, 5e, 6a-b, 6e, 7a-b, 7e, 14b, 14e, 17a-b and 17e.
2.2.1. Dendron octamer 17a
Following the general procedure A, a mixture of azide 1 (116 mg,
0.278 mmol, 12.0 equiv), Octaalkyne 16a (23 mg, 0.023 mmol, 1.0
equiv), sodium (L)-ascorbate (13 mg, 0.065 mmol, 2.8 equiv) and
CuSO4⋅5H2O (12 mg, 0.0465 mmol, 2.0 equiv) in DMF/H2O 98:2 (2 mL)
was irradiated at 65 ◦ C for 240 min. The resulting residue was purified
(SiO2, hexanes/AcOEt 1:2 to DCM/MeOH 10:1) to yield 17a as a white
foam solid (71.4 mg, 69 %). MS(MALDI-TOF): [M þ H]þ = 4542.5043.
1
H NMR (CDCl3, 250 MHz): δ = 7.76 (br s, 8H, H-25), 7.25 (over­
lapped with CHCl3) (H-2, H-6), 6.80 (t, J = 2.3 Hz, 1H, H-4), 6.71–6.64
(m, 12H, H-11, H-15, H-18, H-22), 6.59 (t, J = 2.3 Hz, 4H, H-20), 6.53 (t,
J = 2.3 Hz, 2H, H-13), 5.25 (t, J = 9.4 Hz, 8H, H-3′), 5.17 (t, J = 9.6 Hz,
8H, H-4′), 5.15 (br s, 16H, H-23), 5.07–4.92 (m, 20H, H-9, H-16, H-2′),
5.01 (br s, 4H, H-9, H-16), 4.53 (d, J = 7.7 Hz, 8H, H-1′), 4.51–4.33 (m,
16H, H-26), 4.01 (d, J = 9.4 Hz, 8H, H-5′), 3.88 (s, 3H, H-8), 3.87–3.77
(m, 8H, H-28a), 3.68 (s, 24H, H-7′), 3.57–3.44 (m, 8H, H-28b) 2.22–2.19
(m, 16H, H-27), 2.06 (s, 24H, OAc), 2.01 (s, 24H, OAc), 2.00 (s, 24H,
OAc).
13
C NMR (CDCl3, 62.5 MHz): δ = 170.1 (CH3CO), 169.6 (CH3CO),
169.5 (CH3CO), 167.3 (CO2CH3, C-7, C-6′), 160.1 (CAr, C-5, C-3), 159.7
(CAr, C-12, C-14, C-19, C-21), 143.6 (CTriazole, C-24), 139.4 (CAr, C-17),
139.0 (CAr, C-10), 132.1 (CAr, C-1), 124.0 (CHTriazole, C-25), 108.5 (CAr,
C-2, C-6), 106.6 (CAr, C-4, C-11, C-15, C-18, C-22), 101.5 (CAr, C-13, C-
20), 100.5 (CH, C-1′), 72.3 (CH, C-5′), 72.0 (CH, C-3′), 71.1 (CH, C-2′),
69.9 (CH2, C-9, C-16), 69.4 (CH, C-4′), 65.9 (CH2, C-28), 62.0 (CH2, C-
23), 53.0 (OCH3, C-7′), 52.4 (OCH3, C-8), 46.6 (CH2, C-26), 30.2 (CH2, C-
27), 20.8 (CH3CO), 20.7 (CH3CO), 20.6 (CH3CO).
2.2.2. Dendron octamer 17b
Following the general procedure B, a mixture of azide 1 (169 mg,
P. Ramírez-López et al.
0.406 mmol, 9.6 equiv), Octaalkyne 16b (50 mg, 0.042 mmol, 1 equiv),
sodium (L)-ascorbate (28 mg, 0.142 mmol, 3.2 equiv) and CuSO4⋅5H2O
(18 mg, 0.071 mmol, 1.6 equiv) in DMSO/H2O (5:1) (2 mL) was irra­
diated at 65 ◦ C for 90 min. The resulting residue was purified (SiO2,
3
Bioorganic Chemistry 141 (2023) 106913
hexanes/AcOEt 1:2 to AcOEt/MeOH 10:1) to yield 17b as a white foam
solid (165 mg, 83 %). MS(MALDI-TOF): [M þ H]þ = 4514.5096.
1
H NMR (CDCl3, 250 MHz): δ = 7.77 (s, 8H, H-24), 6.66 (d, J = 2.3
Hz, 8H, H-17, H-21), 6.62 (d, J = 2.2 Hz, 4H, H-10, H-14), 6.57 (s br, 6H,
P. Ramírez-López et al.
H-12, H-19), 6.49 (t, J = 2.2 Hz, 2H, H-2, H-6), 6.46 (t, J = 2.1 Hz, 1H,
H-4), 5.32–5.08 (m, 32H, H-22, H-3′, H-4′), 5.06–4.90 (m, 20H, H-2′, H-
8, H-15), 4.60 (s, 2H, H-7), 4.52 (d, J = 7.7 Hz, 8H, H-1′), 4.49–4.30 (m,
16H, H-25), 4.00 (d, J = 9.5 Hz, 8H, H-5′), 3.87–3.74 (m, 8H, H-27a),
3.66 (s, 24H, H-7′), 3.54–3.41 (m, 8H, H-27b), 2.19–2.09 (m, 16H, H-
16), 2.05 (s, 24H, OAc), 2.00 (s, 24H, OAc), 1.99 (s, 24H, OAc).
13
C NMR (CDCl3, 62.5 MHz): δ = 170.1 (CH3CO), 169.6 (CH3CO),
169.5 (CH3CO), 167.3 (CO2CH3, C-6′), 160.0 (CH, C-3, C-5), 159.7 (C-
11, C-13, C-18, C-20), 143.6 (CTriazole, C-23), 139.6 (C, C-16), 139.5 (C,
C-1, C-9), 124.0 (CHTriazole, C-24), 106.6 (CH, C-17, C-21), 106.5 (CH, C-
10, C-14), 106.3 (CH, C-12, C-19), 105.7 (CH, C4), 101.5 (CH, C-2, C-6),
100.6 (CH, C-1′), 72.3 (CH, C-5′), 72.0 (CH, C-3′), 71.2 (CH, C-2′), 69.9
(CH2, C-22), 69.5 (CH, C-4′), 65.9 (CH2, C-27), 64.7 (CH2, C-7), 61.9
(CH2, C-8, C-15), 53.0 (OCH3, C-7′), 46.6 (CH2, C-25), 30.2 (CH2, C-26),
20.8 (CH3CO), 20.7 (CH3CO), 20.6 (CH3CO).
2.2.3. Dendron octamer 17e
Following the general procedure B, a mixture of azide 1 (132 mg,
0.315 mmol, 9.6 equiv), Octaalkyne 16d (39 mg, 0.0327 mmol, 1
equiv), sodium (L)-ascorbate (21 mg, 0.105 mmol, 3.2 equiv) and
CuSO4⋅5H2O (13 mg, 0.052 mmol, 1.6 equiv) in DMSO/H2O (5:1) (2 mL)
was irradiated at 65 ◦ C for 60 min. After that time, sodium azide (5 mg,
0.065 mmol, 2 equiv) was added to the mixture, also being irradiated at
65 ◦ C for 30 min. The resulting residue was purified (SiO2, hexanes/
AcOEt 1:2 to AcOEt/MeOH 10:1) to yield 17e as a white foam solid (103
mg, 69 %). MS(MALDI-TOF): [M þ H]þ = 4539.5163.
1
H NMR (CDCl3, 250 MHz): δ = 7.76 (s, 8H, H-24), 6.69–6.62 (m,
12H, H-10, H-14, H-17, H-21), 6.60–6.55 (m, 6H, H-12, H-19),
6.54–6.47 (m, 3H, H-2, H-4, H-6), 5.26–5.11 (m, 16H, H-3′, H-4′), 5.14
(s, 16H, H-22), 5.07–4.88 (m, 20H, H-2′, H-8, H-15), 4.52 (d, J = 7.7 Hz,
8H, H-1′), 4.48–4.29 (m, 16H, H-25), 4.25 (s, 2H, H-7), 4.00 (d, J = 9.4
Hz, 8H, H-5′), 3.87–3.76 (m, 8H, H-27a), 3.66 (s, 24H, H-7′), 3.55–3.40
(m, 8H, H-27b), 2.18–2.09 (m, 16H, H-26), 2.04 (s, 24H, OAc), 1.99 (s,
24H, OAc), 1.99 (s, 24H, OAc).
13
C NMR (CDCl3, 62.5 MHz): δ = 170.1 (CH3CO), 169.6 (CH3CO),
169.5 (CH3CO), 167.3 (CO2CH3, C-6′), 160.1 (C-3, C-5), 159.7 (C-11, C-
13, C-18, C-20), 143.6 (CTriazole, C-23), 139.4 (C, C-16), 139.2 (C, C-1, C-
9), 124.0 (CHTriazole, C-24), 107.4 (CH, C-12, C-19), 106.6 (CH, C-17, C-
21), 106.5 (CH, C-10, C-14), 101.6 (CH, C-4), 101.5 (CH, C-2, C-6),
100.6 (CH, C-1′), 72.3 (CH, C-5′), 72.0 (CH, C-3′), 71.2 (CH, C-2′), 69.9
(CH2, C-22), 69.4 (CH, C-4′), 65.9 (CH2, C-27), 62.0 (CH2, C-8, C-15),
53.55 (CH2, C-7), 53.0 (OCH3, C-7′), 46.6 (CH2, C-25), 30.2 (CH2, C-26),
20.8 (CH3CO), 20.7 (CH3CO), 20.6 (CH3CO).
4
Bioorganic Chemistry 141 (2023) 106913
2.3. General method for acetal and methyl ester deprotection: Synthesis of
compounds 8a-c, 8e, 9a-b, 9e, 10a-b, 10e, 15b, 15e, 18a-b and 18e
To a solution of the corresponding protected dendrimer in anhydrous
MeOH a 0.5 N NaOMe/MeOH solution was added (1.5 equiv NaOMe/
AcO) and the resulting mixture was stirred for 4 h. Then, a 0.2 M NaOH
aq solution was added (1.5 equiv/CO2Me) and the mixture was stirred
overnight. Amberlyst® 15 was added at room temperature until pH = 5
and the mixture was filtered, the solvent removed under vacuum
affording the corresponding unprotected DNs 8a-c, 8e, 9a-b, 9e, 10a-b,
10e, 15b, 15e, 18a-b and 18e.
2.3.1. Dendron octamer 18a
Following the general procedure a solution of protected Dendron
octamer 17a (70 mg, 0.0154 mmol) in 2 mL of anhydrous MeOH was
treated sequentially with solution 0.5 N NaOMe/MeOH solution (36
equiv) and 0.2 M NaOH aq solution (13.5 equiv) to afford glycodendron
18a in quantitative yields as a white powder. MS(MALDI-TOF): [M þ
2Na]2þ = 1726.5406.
1
H NMR (D2O, 700 MHz): δ = 7.76 (s, 8H, H-24), 6.77–5.95 (m, 21H,
H-2, H-4, H-6, H-10, H-12, H-14, H-17, H-19, H-21), 4.78–4.65 (m, 8H
H-15), 4.58–4.06 (m, 28H, H-8, H-25, H-1′), 4.00–3.16 (m, 70H, H-8, H-
15, H-27, H-2′, H-3′, H-4′, H-5′), 2.17–1.74 (m, 16H, H-26).
13
C NMR (D2O, 176 MHz): δ = 171.8 (CO2H, C-6′), 165.5 (CO2H, C-
7), 158.8 (C, C-3, C-5, C-11, C-13, C-18, C-20), 142.7 (CTriazole, C-23),
139.2 (C, C-1, C-9, C-16), 124.6 (CHTriazole, C-24), 106.3 (CH, C-2, C-6,
C-10, C-14, C-17, C-21), 102.2 (CH, C-1′), 99.6 (CH, C-4, C-12, C-19),
75.1 (CH, C-3′ or C-4′), 74.3 (CH, C-5′), 72.5 (CH, C-2′), 71.1 (CH, C-3′ or
C-4′), 68.9 (CH2, C-22), 66.3 (CH2, C-27), 60.7 (CH2, C-8, C-15), 47.0
(CH2, C-25), 29.4 (CH2, C-26).
2.3.2. Dendron octamer 18b
Following the general procedure a solution of protected Dendron
octamer 17b (119 mg, 0.026 mmol) in 2 mL of anhydrous MeOH was
treated sequentially with solution 0.5 N NaOMe/MeOH solution (36
equiv) and 0.2 M NaOH aq solution (12 equiv) to afford glycodendron
18b in quantitative yields as a white powder. MS(MALDI-TOF): [M þ
2Na]2þ = 1719.5510.
1
H NMR (D2O, 700 MHz): δ = 7.71 (s, 8H, H-24), 6.71–5.74 (m, 21H,
H-2, H-4, H-6, H-10, H-12, H-14, H-17, H-19, H-21), 4.72–4.02 (m, 42H,
H-7, H-22, H-25, H-1′), 3.95–3.01 (m, 70H, H-8, H-15, H-27, H-2′, H-3′,
H-4′, H-5′), 2.15–1.53 (m, 16H, H-26).
13
C NMR (D2O, 176 MHz): δ = 172.0 (CO2H, C-6′), 159.3 (C, C-3, C-
5), 158.9 (C, C-11, C-13, C-18, C-20), 142.8 (CTriazole, C-23), 139.3 (C, C-
1, C-9, C-16), 124.8 (CHTriazole, C-24), 106.5 (CH, C-2, C-6, C-10, C-14,
P. Ramírez-López et al.
C-17, C-21), 102.3 (CH, C-1′), 100.7 (CH, C-4, C-12, C-19), 75.2 (CH, C-3′
or C-4′), 74.4 (CH, C-5′), 72.6 (CH, C-2′), 71.2 (CH, C-3′ or C-4′), 69.0
(CH2, C-22), 66.4 (CH2, C-27), 60.8 (CH2, C-8, C-15), 47.0 (CH2, C-25),
29.5 (CH2, C-26).
2.3.3. Dendron octamer 18e
Following the general procedure, a solution of protected Dendron
octamer 17e (108 mg, 0.023 mmol) in 2 mL of anhydrous MeOH was
treated sequentially with solution 0.5 N NaOMe/MeOH solution (36
equiv) and 0.2 M NaOH aq solution (12 equiv) to afford glycodendron
18e in quantitative yields as a white powder. MS(MALDI-TOF): [M þ
2Na]2þ = 1732.0546.
1 washed with 1 M NaCl to remove the bound HS. Sensor chip flow cell 2
H NMR (D2O, 700 MHz): δ = 7.76 (s, 8H, H-24), 6.73–5.79 (m, 21H,
H-2, H-4, H-6, H-10, H-12, H-14, H-17, H-19, H-21), 4.70–3.98 (m, 42H,
H-7, H-22, H-25, H-1′), 3.98–3.07 (m, 70H, H-8, H-15, H-27, H-2′, H-3′,
H-4′, H-5′), 2.20–1.68 (m, 16H, H-26).
13
C NMR (D2O, 176 MHz): δ = 171.8 (CO2H, C-6′), 158.9 (C, C-3, C-
5, C-11, C-13, C-18, C-20), 142.6 (CTriazole, C-23), 139.3 (C, C-1, C-16),
130.2 (C, C-9), 124.8 (CHTriazole, C-24), 106.4 (CH, C-2, C-6, C-10, C-14,
C-17, C-21), 102.2 (CH, C-1′), 100.7 (CH, C-4, C-12, C-19), 75.1 (CH, C-3′
or C-4′), 74.4 (CH, C-5′), 72.6 (CH, C-2′), 71.2 (CH, C-3′ or C-4′), 69.1
(CH2, C-22), 66.4 (CH2, C-27), 60.8 (CH2, C-8, C-15), 47.2 (CH2, C-25),
29.5 (CH2, C-26).
5
Bioorganic Chemistry 141 (2023) 106913
2.4. Surface resonance plasmon studies
SPR experiments were performed at 25 ◦ C 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 ex­
periments. A solution with DENV2 was adjusted to 20 μg/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 HS, for 30 min at
4 ◦ C, to protect the GAG-binding domains of the envelope protein.
Immobilization response was 2000 RU and then the sensor surface
was activated blocked and used as a reference surface. Blank samples
and concentration series were injected on CM4 chip at a flow rate of 40
μL/min for 180 s and dissociation was registered for 180 s.
Initially compounds 8a-c, 8e, 9a-b, 9e, 10a-b, 10e, 15b, 15e, 18a-b
and 18e were flowed on the immobilized DENV2 at a 100 μM concen­
tration. Compound 1 (GlcA derived azide) was used as monovalent li­
gands. Then chip CM4 concentration series were 5–125 μM of the DNs
18a, 18b and 18e. 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 χ2, and values of less than 10 indicated
P. Ramírez-López et al.
good fit.
2.5. Molecular modelling studies
2.5.1. Protein structure
We used the Cartesian coordinates for a 3D apo form of envelope
protein (E) DENV 2 Thailand 16681/84 with PDB accession code 3C5X
[45]. The protocol for protein preparation for MD simulations was the
same as we have described in previous works. [24].
2.5.2. Docking
Ligand preparation. 3D structures for compounds 18a, 18b and 18e
were built using the interactive molecular graphics program PyMOL
[46] (URL: https://pymol.org/2/) and the carbohydrate builder web
server glycam (URL: https://glycam.org/). The ground state geometry of
the ligand was optimized using the program Gaussian 16 (URL: https
://gaussian.com/gaussian16/) [47] and fitted to the atoms as AMBER
atom types and RESP charges using the program antechamber
(AmberTools 20, URL: https://ambermd.org/) [48].
We used AutoDock Vina (URL: http://vina.scripps.edu) [49] as
docking program, adopting a basic docking protocol (flexible ligand and
rigid receptor) to predict the 3D geometry of the complexes formed by
the DENV2 with dendrimers. Octadendrons 18a, 18b, 18e were docked
in the domain III (DIII) of the protein (residues 292–395). [50] The most
favorable poses were selected according to its predicted docking energy
and visual inspection. The resultant complexes were used as the starting
point for molecular dynamics studies.
2.5.3. Molecular dynamics simulation
The selected complexes for 18a, 18b and 18e were immersed in
cubic boxes of TIP3P water molecules [51] large enough to guarantee
that the shortest distance between the solute and the edge of the box was
greater than 15 Å. Counterions were also added to maintain electro
neutrality. We performed molecular dynamics simulations using AMBER
20 package. [48] The starting structures were simulated in the NPT
ensemble with the periodic boundary conditions and particle mesh
Ewald method to treat long-range electrostatic effects. [52] The protocol
was as follows: three consecutive minimizations were performed: (i)
involving only hydrogen atoms, (ii) involving only the water molecules
and ions, and (iii) involving the entire system. The system was then
heated and equilibrated in two steps: (i) 20 ps of MD heating the whole
system from 100 to 300 K and (ii) equilibration of the entire system
during 100 ps at 300 K. The equilibrated structure was the starting
points for 50 ns of MD simulations carried out using the pmemd_cuda.
SPFP at constant temperature (300 K) and pressure (1 atm) and the
standard ff14SB force field parameter. The constraint algorithm SHAKE
[53] was used to keep bonds involving H atoms at their equilibrium
length, allowing a 2 fs time step for the integration of Newton’s equa­
tions of motion. The cpptraj [54] module in AMBER20 was employed for
data processing and geometry analysis of the calculated trajectories.
2.5.4. Analysis of MD trajectories
The stability of the complexes (DENV2-18a, DENV2-18b and
DENV2-18e) were evaluated by calculating the root-mean-square-
deviation (RMSD) of the Cα atoms along the trajectories, using their
starting structures as reference. Additionally, the root-mean-square-
fluctuation (RMSF) of each residue, relative to the corresponding
average value, was calculated once each snapshot had been fitted to its
initial structure. The effective binding free energies between the ligands
and the more relevant residues in the binding site were qualitatively
estimated using the MM/GBSA. [55] MM/GBSA is a popular approach to
estimate the free energy of the binding of small ligands to biological
macromolecules. MM/GBSA takes into account a MM interaction term, a
solvation contribution through a generalized born (GB) model, and a
surface area (SA) contribution to account for the non-polar part of
desolvation. [56,57] The MM part estimates the enthalpic contributions
6
Bioorganic Chemistry 141 (2023) 106913
for the protein–ligand interactions (bonded, electrostatic, van der
Waals). The polar solvation energy represents the electrostatic interac­
tion between the solute and the continuum solvent. In addition, three
non-polar solvation terms include cavitation, dispersion, and repulsion
energies, representing the cost of making a cavity in the solvent, as well
as the attractive and repulsive parts of the van der Waals interactions
between the solute and the solvent. All these three non-polar solvation
terms are free energies and in particular the cavitation energy should
have important entropic components, representing the reorganization of
the solvent around the solute. In summary, in MM/GBSA, the free energy
of a state is estimated from the following sum: G = Ebnd + Eel + EwdW
+ Gpol + Gnp - TS. A 12–6 Lennard-Jones term was used to model de
MM contribution. For GB, the solute dielectric constant was set to 4
while that of the solvent was set to 80, and the dielectric boundary was
calculated using a solvent probe radius of 1.4 Å. [56,58] The polar
contribution is calculated using GB, and the non-polar energy is esti­
mated by solvent accessible surface area (SASA).
3. Results and discussion
3.1. Synthesis of glucuronic acid based-dendrons
Several research groups have recently reported the preparation of
glycodendrons based on alkyne-functionalized platforms linked to car­
bohydrate derived azides via copper(I)-catalyzed azide alkyne cyclo­
addition (CuAAC). [24,26,59].
Based on our previously reported results and with the intention of
increasing the affinity of the glycodendrons towards the DENV envelope
protein, [24] an optimized family of GlcA-based dendrons bearing
different levels of multivalency, polarity and structural features were
designed for affinity binding assays. Their chemical synthesis was per­
formed sequentially in three stages, as follows: a) the chemical synthesis
of the oligo and polyalkynyl scaffolds, b) assembling with fully protected
GlcA-based azide units by using microwave (MW) assisted CuAAC pro­
tocol and c) fully deprotection of hydroxyl and carboxylic acid groups
(Scheme 1).
GlcA-derived azide (1) was prepared using a reported procedure [24]
and the different oligo and polyalkynyl scaffolds as indicated in sup­
porting information.
3.2. Synthesis of di, tri and tetravalent glucuronic acid-based dendrons
A first set containing di, tri and tetravalent GlcA-based dendrons
(GlcA DNn) was envisioned. These aromatic dendritic scaffolds will
allow us to study the influence of low valency (n = 2, 3, 4) as well as the
substitution at the focal position of the core in the molecular recognition
of these structures as a crucial part of oligovalent GlcA-based dendrons.
Firstly, di and tetravalent GlcA-based dendrons were performed starting
from commercially available aromatic compounds such as methyl 3,5-
dihydroxybenzoate to prepare bisalkynyl scaffolds 2a-d, and tetrava­
lent alkynyl cores 4a-b and 4d. In parallel, this set was complemented
with the synthesis of trialkynyl scaffolds (3a-b and 3d) starting from
readily available methyl gallate. Standard propargylation strategies
were used in all cases (see Supporting Information).
The aromatic dendritic alkynyl scaffold structures (2a-d, 3a-b, 3d,
4a-b and 4d) were assembled with protected acid glucuronic-based
azide units (1) by using microwave (MW) assisted CuAAC protocol fol­
lowed by full deprotection of hydroxyl and carboxylic acid groups as
shown in Scheme 2. Protected GlcA DNn 5a-c, 5e, 6a-b, 6e, 7a-b and 7e
were obtained in high yields after microwave irradiation at 65 ◦ C until
completion of the reaction (60–180 min, depending on the valency of
scaffold, see details in supporting information). The complete absence of
the terminal alkyne proton resonance (δ 3.6–2.2 ppm) and appearance
of a new singlet (δ 8.0–7.7 ppm) attributable to the triazole moiety
suggested successful completion of the reaction. It is worth mentioning
that to prepare GlcA DNn 5e, 6e and 7e bearing an azidomethyl group at
P. Ramírez-López et al.
Scheme 1. Schematic synthesis of GlcA based dendrons.
Scheme 2. Preparation of di, tri and tetravalent GlcA acid based dendrons (GlcA DNs).
the focal position a one-pot click azidation/reaction sequence was
developed obtaining the corresponding azide derivatives with satisfac­
tory yields in two steps. Sequential deprotection of hydroxyl and car­
boxylic acid groups using a firstly MeONa/MeOH and then NaOH
furnished water-soluble GlcA DNs 8a-c, 8e, 9a-b, 9e, 10a-b and 10e in
quantitative yields.
3.3. Synthesis of hexavalent glucuronic acid-based dendrons
In order to examine intermediate valence GlcA-based dendrons (n =
6) hexaalkynyl scaffold 13b bearing a hydroxylmethyl group at the focal
position was prepared starting from 3,5-dihydroxybenzyl alcohol (11)
and 2.5 equiv of the benzylic bromide 12 in the presence of K2CO3 and
18-crown-6 in acetone at reflux. Then, treatment of hexaalkyne 13b
with SOCl2 in DCM gave rise to chlorinated core 13d with excellent
yields. Subsequent coupling of alkynyl scaffolds (13b and 13d) with
glucuronic acid-derived azide 1 through CuAAC and CuAAC/azidation
followed by full deprotection of hydroxyl and carboxylic acid groups
7
Bioorganic Chemistry 141 (2023) 106913
furnished water-soluble GluA dendrons 15b and 15e, respectively
(Scheme 3).
3.4. Synthesis of multivalent glucuronic acid-based dendrons
Once low and intermediate valence GlcA-based dendrons were pre­
pared, we tackled the synthesis of GlcA DNs with higher valency in order
to improve the protein binding. Octaalkynyl scaffolds 16a-b and 16d
were synthesised using similar strategies as those used for the prepara­
tion of analogous oligoalkynyl core structures with lower valency (see
details in supporting information). Water-soluble octameric GlcA DNs
18a-b and 18e were obtained via CuAAC and CuAAC/azidation onepot
sequence, respectively, followed by sequential deprotection as shown in
Scheme 4.
3.5. Surface resonance plasmon studies
In order to evaluate the ability of the deprotected GlcA GDNs to
P. Ramírez-López et al.
Scheme 3. Synthetic route for the preparation of hexavalent GlcA based dendrons.
interact with DENV2 a SPR binding study was carried out. As mentioned
above, previous studies have demonstrated that a tetravalent GlcA
dendron has shown efficient binding to DENV envelope protein with a
KD value of 22 μM.
Compounds 8a-c, 8e, 9a-b, 9e, 10a-b, 10e, 15b, 15e, 18a-b and
18e were flowed on immobilized DENV2 at a 100 μM concentration
(Fig. 1). Compound 1 (GlcA-derived azide) was used as monovalent
ligand. As can be seen in Fig. 1, the GDNs binding to DENV2 depends on
the valency and the functional group in focal position. No binding was
observed for the monomeric ligand 1. GlcA compounds 8a-c, 8e, 9a-b
and 9e (di and trivalent derivatives) showed negligible binding response
with DENV2 envelope protein, GDNs 10a-b and 10e (tetravalent de­
rivative) exhibited good binding, whereas compounds 15b and 15e,
(hexavalent derivatives) displayed a higher response, which indicates
strong binding. More significant was the effect due to the GlcA DNs with
higher valency (octavalent derivatives, compounds 18a-b and 18e).
Compounds, with eight GlcA acid blocks (18a-b and 18e), showed a
substantial increase in the interaction, supporting the positive correla­
tion between valency and interaction intensity (Fig. 1).
All of these GlcA DNs have a specific functional group (–COOH,
–CH2OH, -CH2OMe and –CH2N3) at the focal position and as can be seen
in Fig. 1 this group also exerts a great influence on the interaction with
Dengue virus envelope protein.
Thus, compounds with an azidomethyl group at the focal position
and bearing six (compound 15e) or eight GlcAs (compound 18e),
showed stronger binding than the GlcA DNs with a hydroxylmethyl
group at the same position and equal valency (compounds 15b and 18b
respectivelly), supporting the hypothesis about the importance of the
focal position functional group in the arrangement of sugars for their
molecular recognition. Thus, we observe a clear trend between the
8
Bioorganic Chemistry 141 (2023) 106913
functional group in focal position and valency, indicating that not only
valency but also the functional group in focal position exert influence on
the interaction of this DNs (hexavalent and octavalent) and DENV2
envelope protein (Fig. 1).
Then, different concentrations of GlcA DNs 18a, 18b and 18e were
flowed over the chip containing immobilized DENV2. Sensograms ob­
tained showed increased interaction profiles with the DN 18a (Fig. 2A),
DN 18b (Fig. 3A) and DN 18e (Fig. 4A). Using steady-state analysis of
SPR measurements for the interaction between DNs (18a, 18b and 18e)
and DENV2, the dissociation constants (KD) were found to be KD =
1.093 μM, 0.798 μM and 0.638 μM respectively (Fig. 2B, Fig. 3B and
Fig. 4B). Our results indicated a strong interaction between DENV2 and
DNs 18b and 18e, which are 28 and 34 times better, respectively, than
the one obtained in previous studies with a tetravalent GlcA dendron
with a methyl ester at the focal position (KD = 22 μM).
The high interaction obtained for the DNs having eight GlcAs and an
azidomethyl group at the focal position (KD = 0.638 μM, compound
18e), compared to compound bearing also eight GlcAs and a carboxylic
acid group at the focal position (KD = 1.093 μM, 18a), suggested that
this group has a great influence on the multivalent presentation of the
GlcA, favouring the molecular recognition of these GlcA derivatives by
DENV2.
3.6. Molecular modelling
In order to explain the difference interaction observed in the binding
of DENV2 to the GlcA DNs 18a, 18b and 18e, a closer look at DENV2-
NDs complex was carried out using molecular modelling studies.
A structural rationale for tetravalent GlcA DNs and DENV2 in­
teractions has already been published. [24] Briefly, after docking and
P. Ramírez-López et al. Bioorganic Chemistry 141 (2023) 106913

Scheme 4. Preparation of octavalent GlcA acid based dendrons.

Fig. 1. SPR measurement of GlcA GDNs binding to immobilized DENV2 at 100 μM.

molecular dynamics (MD) simulation of tetravalent GlcA dendron in a
3D validated model of DENV2 protein we conclude that the main in­
teractions were due to electrostatic interactions between the carboxylate
groups of GlcA-GD and LYS-299 and LYS-295 in the GAG-binding region
of DENV2, which are the key residues involved in the heparin binding
region. [24]
In the present work, in order to try to explain the results obtained in
SPR experiments, we performed docking and MD simulations of the GlcA
DNs (18a, 18b and 18e) with the best interactions values in GAG-
binding region of DENV2.
Based on our previous work [24] the DNs 18a, 18b and 18e were
docked in GAG-binding region of DENV2, specifically on the cavity

9
P. Ramírez-López et al. Bioorganic Chemistry 141 (2023) 106913

Fig. 2. A. Binding responses of different concentrations of GDN 18a with DENV2 immobilized on the CM4 chip, showing association and dissociation phases.
Responses were reference subtracted and blank corrected; B. Steady state affinity study of the interaction between GDN 18a and DENV2 immobilized on a CM4 chip.

Fig. 3. A. Binding responses of different concentrations of GDN 18b with DENV2 immobilized on the CM4 chip, showing association and dissociation phases.
Responses were reference subtracted and blank corrected. B. Steady state affinity study of the interaction between GDN 18b and DENV2 immobilized on a CM4 chip.

Fig. 4. A. Binding responses of different concentrations of GDN 18e with DENV2 immobilized on the CM4 chip, showing association and dissociation phases.
Responses were reference subtracted and blank corrected. B. Steady state affinity study of the interaction between GDN 18e and DENV2 immobilized on a CM4 chip.

formed by the sequence 294DKLQLKGMSYSM305 that contains LYS-295
and LYS-299 responsible of GAG interactions (Fig. S1 in Supporting
information). After docking, the best fitting poses of DNs 18a, 18b and
18e, in DENV2 cavity were chosen for MD simulation, taking in
consideration that the key residues in this cavity are LYS-295 and LYS-
299. The three dendrons place one of the GlcA moieties in the cavity
where LYS-299 is located, forming strong electrostatic interactions with
it, and place another GlcA moiety near LYS-295, enabling the interac­
tion. Compounds 18a and 18e place the central core of the dendron in
front of MET-301, forming van der Waals interactions, while central core
of DN 18b forms van der Waals interactions with PRO-336 (Fig. 5).
The predicted interaction observed in docking studies were validated

10
P. Ramírez-López et al. Bioorganic Chemistry 141 (2023) 106913

Fig. 5. The most likely binding conformation of 18b (a), 18e (b) and 18a (c) in DENV2 cavity. The protein is represented as white cartoon, LYS-295 and LYS − 299
side chain as yellow sticks representation and the DNs are shown as cyan sticks and central core as pink sticks. (For interpretation of the references to colour in this
figure legend, the reader is referred to the web version of this article.)

with the simulation results, once the complexes were selected, we used
Table 1
molecular dynamics simulations in water during 50 ns to study their
Calculated total interaction energies and component contributions.
structural stability. The time evolution plots of the RMSD values are
shown in Supplementary Information (Fig. S2-S4 in Supporting Infor­ Interaction Energies 18a 18b 18e

mation). The three complexes, after maintaining convergence at
approximately 2–5 ns and were structurally and energetically stable
throughout the MD simulations (Fig. 6). It should be noted that for
DENV2-18b complex, a slight fluctuation of the RMSD values of DENV2
at the end of the dynamics is noteworthy, reaching values of 4 Å, while
18b, after the rearrangement at the beginning of the simulations,
maintains the RMSD value (~8 Å) during the 50 ns of the simulation.
RMSD analysis of the simulated DENV2-18a shows values no higher
than 2 Å for DENV2 and values around 8 Å for the DN 18a, with some
fluctuation during the first 20 ns. The data for the DENV2-18e complex
also reveal fluctuations during the first 20 ns for protein and ligand, after
this time period both remain stable with values around 4 Å and 9 Å
respectively.
Protein–ligand interaction energies measured during the simulations
remained nearly constant, with high interaction energy values (18a ≈
− 130 kcal/mol 18b ≈ − 150 kcal/mol; 18e ≈ − 90 kcal/mol), indicating
the poses of the dendrons in the protein were energetically favourable
(Fig. S5-S6 in Supporting Information). The calculated binding energies
are dominated by electrostatic interactions (charged-charged in­
teractions and hydrogen bond) involving the afore mentioned amino
acids GAG-binding region of DENV2 (Table 1).
It can be seen that there is a slight difference between the calculated
binding free energy for the three DNs and the SPR results. DN 18e was
the dendron with the best interaction according to SPR results, however,
it is not the one with the best interaction energy (Table 1). Therefore, to
better understand the interaction between the dendrons and the protein,
the binding free energy was decomposed into ligand-residue pairs using
Van der Waals contribution − 160.22 − 165.84 − 131.03
Electrostatic contribution − 528.37 − 775.74 − 553.38
Desolvation contribution 556.85 785.37 597.54
ΔG TOTAL (kcal/mol) ¡131.74 ¡156.21 ¡86.87
the MM/GBSA approach.
In the three dendrons evaluated (18a, 18b and 18e), the main
interaction takes place between one of the glucuronic acid residues and
LYS-299 of DENV2, as we predicted in docking experiments, but also
other interactions take place with THR-180, TYR-182, TYR-303, VAL-
358, ASN-359, ILE-361 and THR-363. Besides the interactions that DNs
18a, 18b and 18e have in common, each one interacts in a different area
of the protein surface, in DENV2-18b complex highlight the interaction
with residues LYS-40, ASN-41 and LYS-42. Whereas DENV2-DN-18a
complex the dendron interacts with residues HIS-153, ASN-157 and LYS-
161, however 18e interacts with LYS-295, ASN-359, THR-363 and GLU-
364 (Fig. 7). All other interactions are listed in Table S1 in Supporting
Information.
The energy contribution of each residue of GAG-binding region of
DENV2 with 18a, 18b and 18e are shown in Fig. 8. Analizing the results
shown, it can be seen that 18e is the only dendron that interact with the
two specific lysines (LYS-295 and LYS-299), that are key residues
involved in heparin binding. [50] Monitoring the interaction with the
two lysines throughout the 50 ns of simulation, we observed that 18e
interacts during all simulation time (Fig. S7 in Supporting Information).
Hence, the higher inhibitory capacity of 18e may be due to the fact that
it is the only dendrons that interacts with LYS-295 (Fig. 7).

Fig. 6. APBS electrostatic surface representation of DENV2 in complex with 18a (a), 18b (b) and 18e (c) after 50 ns of simulation.

11
P. Ramírez-López et al. Bioorganic Chemistry 141 (2023) 106913

Fig. 7. Final poses of DENV2 bound to the dendrons 18a (a), 18b (b) and 18e (c) after 50 ns of simulation. Protein is shown in cartoon mode grey with the relevant
residues shown as yellow sticks. Dendrons are represented as green sticks, and the electrostatic interactions are represented as magenta dash lines. (For interpretation
of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 8. Calculated contribution of 18a (orange), 18b (blue) and 18e (grey) with individual residues of GAG-binding region of DENV2 after 50 ns of simulation. (For
interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Glycodendron 18b interacts with LYS-295, but only during a short
period of the simulation, specifically between 20 and 30 ns (Fig. S7 in
Supporting Information), the rest of the time the interaction is null. In
the case of the complex DENV2-18a no interaction with LYS-295 was
observed during the simulation (Fig. S7 in Supporting Information).
In conclusion and after visual inspection of the final structures of the
complexes can be concluded that the dendron 18b does not adopt the
best pose for interaction with the GAG-binding region of DENV2. It
forms strong bonds with the protein, especially with LYS-299, which
explains its high interaction energy values, and the results in SPR ex­
periments. However, the dendron 18e, with lower calculated interaction
energy values, is positioned on the protein with a good orientation for
the interaction with the GAG region allowing a better inhibition.
4. Conclusions
We have designed and synthesized a new family of GlcA-based DNs
bearing different functional group at the focal position and different
level of multivalency. CuAAC click chemistry was used for conjugation
and it was optimized to synthesize di, tri, tetra, hexa and octavalent
glycodendrons containing different functional group at the focal posi­
tion (–COOH, –CH2OH, -CH2OMe and –CH2N3).
Their chemical synthesis was performed sequentially in three stages:
a) the chemical synthesis of the alkynyl scaffolds, b) assembling with
fully protected GlcA-based azide units by using a MW assisted CuAAC
protocol and c) sequential deprotection of hydroxyl and carboxylic acid
groups. The CuAAC coupling conditions were optimized with these
scaffolds and maximum conversions were obtained at short reaction
times (87–45 % in 60–180 min).
GlcA DNs were evaluated in their ability to interact with DENV2
using Surface Plasmon Resonance technology. The binding capacity of
compounds 8a-c, 8e, 9b-c and 9e (di and trivalent derivatives) is rela­
tively low compared to GlcA DNs 10a-b, 10e (tetravalent derivatives).
More significant is the binding capacity of GlcA DNs 15b, 15e, 18a-b
and 18e with higher valency (hexa and octavalent derivatives), which
confirms the positive correlation between valency and interaction
intensity.
At the same time, the focal position functional group is another
important factor in the interaction between GlcA DNs to DENV2, higher
binding was observed for the DNs 18e bearing an azidomethyl group at
the focal position and eight GlcAs (KD = 0.638 μM) compared to octa­
valent GlcA DNs 18a (KD = 1.093 μM and 18b (KD = 0.798 μM), having
a carboxylic acid and hydroxymethyl groups at the focal position,
respectively. These results confirm the hypothesis about the importance
of the focal position functional group in the arrangement of sugars for
their molecular recognition by DENV2.
Molecular modeling provided an explanation for the difference in
binding observed between GlcA DNs (18a, 18b and 18e) 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 GlcA DNs (18a, 18b and 18e) and LYS-299 and
LYS-295 in the GAG-binding region of DENV2. [24] This computational
study showed that compound 18e is the only DN that interact with the
two specific lysines (LYS-299 and LYS-295) during all simulation time
and adopt a good orientation for the interaction with the GAG region
allowing a better inhibition. These results reasonably explain the strong
binding of GlcA DN 18e, bearing high valency an azidomethyl group at
the focal position, with DENV2. The analysis of molecular dynamics

12
P. Ramírez-López et al.
simulations on the docking complexes reinforces the conclusion about
that not only valency but also the functional group in focal position exert
influence on the interaction of compound 18e and DENV2 envelope
protein.
In summary, these results can contribute to design more specific and
potent therapeutic glycodendrons and help us to get closer toward
multivalent systems against bacterial and viral infections.
Declaration of Competing Interest
The authors declare the following financial interests/personal re­
lationships which may be considered as potential competing interests:
María J Hernáiz reports financial support was provided by Complutense
University of Madrid. María J Hernáiz reports a relationship with
Complutense University of Madrid that includes: employment. No con­
flict of interest.
Data availability
Data will be made available on request.
Acknowledgments
The 1H-NMR and 13C-NMR spectra were carried out at CAI Unidad de
Resonancia Magnética (UCM). The authors gratefully acknowledge
financial support provided by the Spanish Ministerio de Ciencia e
Innovación, Grants RTI2018-096037B-I00, TED2021-130430B-C21 and
PDC2022-133817-I00.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2023.106913.
References
[1] C.R. Bertozzi, L.L. Kiessling, Chemical glycobiology, Science 291 (5512) (2001)
2357–2364.
[2] A. Varki, Biological Roles of Oligosaccharides - All of the Theories Are Correct,
Glycobiology 3 (2) (1993) 97–130.
[3] Y.C. Lee, R.T. Lee, Carbohydrate-Protein Interactions - Basis of Glycobiology,
Accounts Chem. Res. 28 (8) (1995) 321–327.
[4] A. Bernardi, J. Jiménez-Barbero, A. Casnati, C. De Castro, T. Darbre, F. Fieschi,
J. Finne, H. Funken, K.E. Jaeger, M. Lahmann, T.K. Lindhorst, M. Marradi,
P. Messner, A. Molinaro, P.V. Murphy, C. Nativi, S. Oscarson, S. Penades, F. Peri, R.
J. Pieters, O. Renaudet, J.L. Reymond, B. Richichi, J. Rojo, F. Sansone, C. Schaffer,
W.B. Turnbull, T. Velasco-Torrijos, S. Vidal, S. Vincent, T. Wennekes, H. Zuilhof,
A. Imberty, Multivalent glycoconjugates as anti-pathogenic agents, Chem. Soc.
Rev. 42 (11) (2013) 4709–4727.
[5] M. Marradi, F. Chiodo, I. García, S. Penades, Glyconanoparticles as multifunctional
and multimodal carbohydrate systems, Chem. Soc. Rev. 42 (11) (2013)
4728–4745.
[6] Y.M. Chabre, R. Roy, Multivalent glycoconjugate syntheses and applications using
aromatic scaffolds, Chem. Soc. Rev. 42 (11) (2013) 4657–4708.
[7] P. Hoyos, A. Perona, O. Juanes, A. Rumbero, M.J. Hernáiz, Synthesis of
Glycodendrimers with Antiviral and Antibacterial Activity, Chem.-Eur. J. 27 (28)
(2021) 7593–7624.
[8] W.J. Lu, R.J. Pieters, Carbohydrate-protein interactions and multivalency:
implications for the inhibition of influenza A virus infections, Expert Opin. Drug
Dis 14 (4) (2019) 387–395.
[9] A.K. Adak, B.Y. Li, C.C. Lin, Advances in multifunctional glycosylated
nanomaterials: preparation and applications in glycoscience, Carbohyd. Res. 405
(2015) 2–12.
[10] N.P. Pera, R.J. Peters, Towards bacterial adhesion-based therapeutics and
detection methods, Medchemcomm. 5 (8) (2014) 1027–1035.
[11] J.L.J. Blanco, C.O. Mellet, J.M.G. Fernández, Multivalency in heterogeneous
glycoenvironments: hetero-glycoclusters, -glycopolymers and -glycoassemblies,
Chem. Soc. Rev. 42 (11) (2013) 4518–4531.
[12] P. Hoyos, A. Perona, T. Bavaro, F. Berini, F. Marinelli, M. Terreni, M.J. Hernáiz,
Biocatalyzed Synthesis of Glycostructures with Anti-infective Activity, Accounts
Chem Res 55 (17) (2022) 2409–2424.
[13] N. Varga, I. Sutkeviciute, R. Ribeiro-Viana, A. Berzi, R. Ramdasi, A. Daghetti,
G. Vettoretti, A. Amara, M. Clerici, J. Rojo, F. Fieschi, A. Bernardi, A multivalent
inhibitor of the DC-SIGN dependent uptake of HIV-1 and Dengue virus,
Biomaterials 35 (13) (2014) 4175–4184.
13
Bioorganic Chemistry 141 (2023) 106913
[14] R. Roy, T.C. Shiao, K. Rittenhouse-Olson, Glycodendrimers: versatile tools for
nanotechnology, Braz. J. Pharm. Sci. 49 (2013) 85–108.
[15] T.K. Lindhorst, K. Elsner, Postsynthetic functionalization of glycodendrons at the
focal point, Beilstein J. Org. Chem. 10 (2014) 1482–1487.
[16] G. Goti, C. Colombo, S. Achilli, C. Vives, M. Thepaut, J. Luczkowiak, N. Labiod,
R. Delgado, F. Fieschi, A. Bernardi, Precision Glycodendrimers for DC-SIGN
Targeting, Eur. J. Org. Chem. 2022 (29) (2022).
[17] A. Imberty, Y.M. Chabre, R. Roy, Glycomimetics and glycodendrimers as high
affinity microbial anti-adhesins, Chem.-Eur. J. 14 (25) (2008) 7490–7499.
[18] S. Ordanini, G. Goti, A. Bernardi, From optimized monovalent ligands to size-
controlled dendrimers: an efficient strategy towards high-activity DC-SIGN
antagonists, Can. J. Chem. 95 (9) (2017) 881–890.
[19] R. Sharma, N. Kottari, Y.M. Chabre, L. Abbassi, T.C. Shiao, R. Roy, A highly
versatile convergent/divergent “onion peel” synthetic strategy toward potent
multivalent glycodendrimers, Chem. Commun. 50 (87) (2014) 13300–13303.
[20] C.D. Goncalves, R.C. Figueiredo, A. Giani, D. Collado, E. Perez-Inestrosa, J. Rojo, C.
C. Figueredo, Detection of glycidic receptors in microalgae using glycodendrons as
probes: a new tool for studies on cell surface interactions, J. Appl. Phycol. 31 (1)
(2019) 211–221.
[21] C.D. Heidecke, T.K. Lindhorst, Iterative synthesis of spacered glycodendrons as
oligomannoside mimetics and evaluation of their antiadhesive properties, Chem.-
Eur. J. 13 (32) (2007) 9056–9067.
[22] S. Munneke, K. Kodar, G.F. Painter, B.L. Stocker, M.S.M. Timmer, The modular
synthesis of multivalent functionalised glycodendrons for the detection of lectins
including DC-SIGN, RSC Adv. 7 (72) (2017) 45260–45268.
[23] P. Krist, L. Vannucci, M. Kuzma, P. Man, K. Sadalapure, A. Patel, K. Bezouska,
M. Pospisil, L. Petrus, T.K. Lindhorst, V. Kren, Fluorescent labelled thiourea-
bridged glycodendrons, Chembiochem 5 (4) (2004) 445–452.
[24] C. García-Oliva, A.H. Cabanillas, A. Perona, P. Hoyos, A. Rumbero, M.J. Hernáiz,
Efficient Synthesis of Muramic and Glucuronic Acid Glycodendrimers as Dengue
Virus Antagonists, Chem-Eur J 26 (7) (2020) 1588–1596.
[25] L. Mousavifar, R. Roy, Design, Synthetic Strategies, and Therapeutic Applications
of Heterofunctional Glycodendrimers, Molecules 26 (9) (2021).
[26] C. Bayon, N. He, M. Deir-Kaspar, P. Blasco, S. Andre, H.J. Gabius, A. Rumbero,
J. Jiménez-Barbero, W.D. Fessner, M.J. Hernáiz, Direct Enzymatic Branch-End
Extension of Glycocluster-Presented Glycans: An Effective Strategy for
Programming Glycan Bioactivity, Chem.-Eur. J. 23 (7) (2017) 1623–1633.
[27] R. Ribeiro-Viana, J.J. García-Vallejo, D. Collado, E. Pérez-Inestrosa, K. Bloern,
Y. van Kooyk, J. Rojo, BODIPY-Labeled DC-SIGN-Targeting Glycodendrons
Efficiently Internalize and Route to Lysosomes in Human Dendritic Cells,
Biomacromolecules 13 (10) (2012) 3209–3219.
[28] A.K. Agrahari, P. Bose, M.K. Jaiswal, S. Rajkhowa, A.S. Singh, S. Hotha, N. Mishra,
V.K. Tiwari, Cu(I)-Catalyzed Click Chemistry in Glycoscience and Their Diverse
Applications, Chem. Rev. 121 (13) (2021) 7638–7955.
[29] C. García-Oliva, A. Merchán, A. Perona, P. Hoyos, A. Rumbero, M.J. Hernáiz,
Development of sustainable synthesis of glucuronic acid glycodendrimers using
ball milling and microwave-assisted CuAAC reaction, New J. Chem. 46 (14) (2022)
6389–6393.
[30] A.K. Agrahari, A. Mishra, V.K. Tiwari, CuAAC in Carbohydrate Conjugation, Sci.
Synth. (2022) 135–179.
[31] N. Varga, I. Sutkeviciute, C. Guzzi, J. McGeagh, I. Petit-Haertlein, S. Gugliotta,
J. Weiser, J. Angulo, F. Fieschi, A. Bernardi, Selective Targeting of Dendritic Cell-
Specific Intercellular Adhesion Molecule-3-Grabbing Nonintegrin (DC-SIGN) with
Mannose-Based Glycomimetics: Synthesis and Interaction Studies of Bis
(benzylamide) Derivatives of a Pseudomannobioside, Chem.-Eur. J. 19 (15) (2013)
4786–4797.
[32] A. Jinno, P.W. Park, Role of Glycosaminoglycans in Infectious Disease,
Glycosaminoglycans, Chem. Biol. 1229 (2015) 567–585.
[33] R.J. Linhardt, T. Toida, Role of glycosaminoglycans in cellular communication,
Accounts Chem. Res. 37 (7) (2004) 431–438.
[34] Y.P. Chen, T. Maguire, R.E. Hileman, J.R. Fromm, J.D. Esko, R.J. Linhardt, R.
M. Marks, Dengue virus infectivity depends on envelope protein binding to target
cell heparan sulfate, Nat. Med. 3 (8) (1997) 866–871.
[35] R. Germi, J.M. Crance, D. Garin, J. Guimet, H. Lortat-Jacob, R.W.H. Ruigrok, J.
P. Zarski, E. Drouet, Heparan sulfate-mediated binding of infectious dengue virus
type 2 and yellow fever virus, Virology 292 (1) (2002) 162–168.
[36] P. Hilgard, R. Stockert, Heparan sulfate proteoglycans initiate dengue virus
infection of hepatocytes, Hepatology 32 (5) (2000) 1069–1077.
[37] Y.L. Lin, H.Y. Leib, Y.S. Lin, T.M. Yeh, S.H. Chen, H.S. Liu, Heparin inhibits dengue-
2 virus infection of five human liver cell lines, Antivir. Res. 56 (1) (2002) 93–96.
[38] D. Kato, S. Era, I. Watanabe, M. Arihara, N. Sugiura, K. Kimata, Y. Suzuki,
K. Morita, K.I.P.J. Hidari, T. Suzuki, Antiviral activity of chondroitin sulphate E
targeting dengue virus envelope protein, Antivir. Res. 88 (2) (2010) 236–243.
[39] K.I.P.J. Hidari, N. Takahashi, M. Arihara, M. Nagaoka, K. Morita, T. Suzuki,
Structure and anti-dengue virus activity of sulfated polysaccharide from a marine
alga, Biochem. Bioph. Res. Co. 376 (1) (2008) 91–95.
[40] L.B. Talarico, E.B. Damonte, Interference in dengue virus adsorption and uncoating
by carrageenans, Virology 363 (2) (2007) 473–485.
[41] E. Lee, M. Pavy, N. Young, C. Freeman, M. Lobigs, Antiviral effect of the heparan
sulfate mimetic, PI-88, against dengue and encephalitic flaviviruses, Antivir. Res.
69 (1) (2006) 31–38.
[42] J.L. Lopes, V.S.T. Quinteiro, J. Wouk, M.L. Darido, R.F.H. Dekker, A.M. Barbosa-
Dekker, V. Vetvicka, M.A.A. Cunha, L.C. Faccin-Galhardi, A. Orsato, Sulfonated
and Carboxymethylated beta-Glucan Derivatives with Inhibitory Activity against
Herpes and Dengue Viruses, Int. J. Mol. Sci. 22 (20) (2021).
P. Ramírez-López et al.
[43] R.M. Marks, H. Lu, R. Sundaresan, T. Toida, A. Suzuki, T. Imanari, M.J. Hernáiz, R.
J. Linhardt, Probing the interaction of dengue virus envelope protein with heparin:
Assessment of glycosaminoglycan-derived inhibitors, J. Med. Chem. 44 (13) (2001)
2178–2187.
[44] K.I.P.J. Hidari, K. Ikeda, I. Watanabe, T. Abe, A. Sando, Y. Itoh, H. Tokiwa,
K. Morita, T. Suzuki, 3-O-sulfated glucuronide derivative as a potential anti-dengue
virus agent, Biochem. Bioph. Res. Co. 424 (3) (2012) 573–578.
[45] L. Li, S.M. Lok, I.M. Yu, Y. Zhang, R.J. Kuhn, J. Chen, M.G. Rossmann, The
flavivirus precursor membrane-envelope protein complex: Structure and
maturation, Science 319 (5871) (2008) 1830–1834.
[46] L. Schrödinger, The PyMOL Molecular Graphics System, (2.3.1) (2013).
[47] R.A. Gaussian09, 1, mj frisch, gw trucks, hb schlegel, ge scuseria, ma robb, jr
cheeseman, g. Scalmani, v. Barone, b. Mennucci, ga petersson et al., gaussian, Inc.,
Wallingford CT 121 (2009) 150-166.
[48] D. Case, K. Belfon, I. Ben-Shalom, S. Brozell, D. Cerutti, T. Cheatham, V.C. III, T.
Darden, R. Duke, G. Giambasu, AMBER2020, university of California, San
Fransisco, J. Amer. Chem. Soc 142 (2020) 3823-3835.
[49] O. Trott, A.J. Olson, Software News and Update AutoDock Vina: Improving the
Speed and Accuracy of Docking with a New Scoring Function, Efficient
Optimization, and Multithreading, Journal of Computational Chemistry 31 (2)
(2010) 455–461.
[50] D. Watterson, B. Kobe, P.R. Young, Residues in domain III of the dengue virus
envelope glycoprotein involved in cell-surface glycosaminoglycan binding, J. Gen.
Virol. 93 (2012) 72–82.
14
Bioorganic Chemistry 141 (2023) 106913
[51] W.L. Jorgensen, J. Chandrasekhar, J.D. Madura, R.W. Impey, M.L. Klein,
Comparison of Simple Potential Functions for Simulating Liquid Water, J. Chem.
Phys. 79 (2) (1983) 926–935.
[52] T. Darden, D. York, L. Pedersen, Particle mesh Ewald: An N⋅ log (N) method for
Ewald sums in large systems, J. Chem. Phys. 98 (12) (1993) 10089–10092.
[53] J.-P. Ryckaert, G. Ciccotti, H.J. Berendsen, Numerical integration of the cartesian
equations of motion of a system with constraints: molecular dynamics of n-alkanes,
J. Comput. Phys. 23 (3) (1977) 327–341.
[54] D.R. Roe, T.E. Cheatham III, PTRAJ and CPPTRAJ: Software for Processing and
Analysis of Molecular Dynamics Trajectory Data, J. Chem. Theory Comput. 9 (7)
(2013) 3084–3095.
[55] S. Genheden, U. Ryde, The MM/PBSA and MM/GBSA methods to estimate ligand-
binding affinities, Expert Opin. Drug Discov. 10 (5) (2015) 449–461.
[56] W.C. Still, A. Tempczyk, R.C. Hawley, T. Hendrickson, Semianalytical Treatment of
Solvation for Molecular Mechanics and Dynamics, J. Am. Chem. Soc. 112 (16)
(1990) 6127–6129.
[57] J. Srinivasan, M.W. Trevathan, P. Beroza, D.A. Case, Application of a pairwise
generalized Born model to proteins and nucleic acids: inclusion of salt effects,
Theor. Chem. Acc. 101 (6) (1999) 426–434.
[58] B.R. Miller, T.D. McGee, J.M. Swails, N. Homeyer, H. Gohlke, A.E. Roitberg,
MMPBSA.py: An Efficient Program for End-State Free Energy Calculations,
J. Chem. Theory Comput. 8 (9) (2012) 3314–3321.
[59] L. Rodríguez-Pérez, J. Ramos-Soriano, A. Pérez-Sánchez, B.M. Illescas, A. Muñoz,
J. Luczkowiak, F. Lasala, J. Rojo, R. Delgado, N. Martín, Nanocarbon-Based
Glycoconjugates as Multivalent Inhibitors of Ebola Virus Infection, J. Am. Chem.
Soc. 140 (31) (2018) 9891–9898.