Synthesis of β-galactosylamides as ligands of the peanut lectin. Insights into the recognition process

Article abstract of DOI:10.1016/j.carres.2017.03.018

The synthesis of mono and divalent β-galactosylamides linked to a hydroxylated chain having a C2 symmetry axis derived from L-tartaric anhydride is reported. Reference compounds devoid of hydroxyl groups in the linker were also prepared from β-galactosylamine and succinic anhydride. After functionalization with an alkynyl residue, the resulting building blocks were grafted onto different azide-equipped scaffolds through the copper catalyzed azide-alkyne cycloaddition. Thus, a family of structurally related mono and divalent β-N-galactopyranosylamides was obtained and fully characterized. The binding affinities of the ligands towards the model lectin PNA were measured by the enzyme-linked lectin assay (ELLA). The IC₅₀ values were significantly higher than that of galactose but the presence of hydroxyl groups in the aglycone chain improved lectin recognition. Docking and molecular dynamics experiments were in accordance with the hypothesis that a hydroxyl group properly disposed in the linker could mimic the Glc O3 in the recognition process. On the other hand, divalent presentation of the ligands led to lectin affinity enhancements.

Full text of DOI:10.1016/j.carres.2017.03.018

Accepted Manuscript
Synthesis of β-galactosylamides as ligands of the peanut lectin. Insights into the
recognition process
María Emilia Cano, Oscar Varela, María Isabel García-Moreno, José Manuel García
Fernández, José Kovensky, María Laura Uhrig
PII:
S0008-6215(16)30674-7
10.1016/j.carres.2017.03.018
CAR 7347
DOI:
Reference:
To appear in: Carbohydrate Research
Received Date: 29 December 2016
Revised Date: 16 March 2017
Accepted Date: 22 March 2017
Please cite this article as: M.E. Cano, O. Varela, M.I. García-Moreno, J.M. García Fernández, J.
Kovensky, M.L. Uhrig, Synthesis of β-galactosylamides as ligands of the peanut lectin. Insights into the
recognition process, Carbohydrate Research (2017), doi: 10.1016/j.carres.2017.03.018.
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ACCEPTED MANUSCRIPT
UNIVERSIDAD DE BUENOS AIRES
FACULTAD DE CIENCIAS EXACTAS Y NATURALES
DEPARTAMENTO DE QUIMICA ORGANICA
Pabellón 2-Ciudad Universitaria-C1428EHA-Buenos Aires
TEL/FAX: 5411-4576-3346/ 5411-4576-3352
Synthesis of
β
-Galactosylamides as ligands of the Peanut lectin. Insights
into the recognition process.
María Emilia Canoa, Oscar Varelaa, María Isabel García-Morenob, José Manuel García Fernándezc, José
Kovenskyd and María Laura Uhriga*
Ser211
Asp80
Asp83
Leu212
HO
Ile101
O
OH
O
HO
HO
Tyr125
H
OH
O
N
N
N
N
H
N
N
N
OH
N
scaffold
N
O
OH
N
H
HO
O
H
OH
HO
OH
O
OH
O
OAc
AcO
AcO
O
+
NH2
OAc
+
H2N
O
+
N3
sc
N3
AcO
affold
AcO
O

ACCEPTED MANUSCRIPT
Synthesis of β-Galactosylamides as ligands of the
Peanut lectin. Insights into the recognition process.
María Emilia Canoa, Oscar Varelaa, María Isabel García-Morenob, José Manuel García
Fernándezc, José Kovenskyd and María Laura Uhriga*
aUniversidad de Buenos Aires. CONICET. Centro de Investigación en Hidratos de
Carbono (CIHIDECAR). Facultad de Ciencias Exactas y Naturales. Departamento de
Química Orgánica. Buenos Aires, Argentina.
bDepartamento de Química Orgánica, Facultad de Química, Universidad de Sevilla, c/
Profesor García González 1, 41012, Sevilla, Spain.
cInstituto de Investigaciones Químicas (IIQ), CSIC - Universidad de Sevilla, Avda.
Américo Vespucio 49, 41092, Sevilla, Spain.
dLaboratoire de Glycochimie, des Antimicrobiens et des Agroressources (LG2A)-CNRS
UMR 7378, Université de Picardie Jules Verne, 33 rue Saint Leu, 80039 Amiens Cedex,
France.
*Corresponding author: E-mail: mluhrig@qo.fcen.uba.ar; CIHIDECAR-CONICET,
Departamento de Química Orgánica, Facultad de Ciencias Exactas y Naturales,
Universidad de Buenos Aires, Pabellón 2, Ciudad Universitaria, 1428 Buenos Aires,
Argentina.

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Abstract
The synthesis of mono and divalent β-galactosylamides linked to a hydroxylated chain
having a C2 symmetry axis derived from -tartaric anhydride is reported. Reference
L
compounds devoid of hydroxyl groups in the linker were also prepared from β-
galactosylamine and succinic anhydride. After functionalization with an alkynyl
residue, the resulting building blocks were grafted onto different azide-equipped
scaffolds through the copper catalyzed azide-alkyne cycloaddition. Thus, a family of
structurally related mono and divalent β-N-galactopyranosylamides was obtained and
fully characterized. The binding affinities of the ligands towards the model lectin PNA
were measured by the enzyme-linked lectin assay (ELLA). The IC50 values were
significantly higher than that of galactose but the presence of hydroxyl groups in the
aglycone chain improved lectin recognition. Docking and molecular dynamics
experiments were in accordance with the hypothesis that a hydroxyl group properly
disposed in the linker could mimic the Glc O3 in the recognition process. On the other
hand, divalent presentation of the ligands led to lectin affinity enhancements.
Keywords: Peanut agglutinin, β-galactosylamides, divalent ligands, ELLA assay,
Molecular Dynamics.

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1. Introduction
A number of biological events are triggered by the recognition of a glycosidic
fragment by a complementary protein, such as a carbohydrate processing enzyme, a
lectin or an antibody.1 This process is highly specific and depends strongly on the
configurational pattern of the sugar ligand.2,3 Legume lectins provide excellent models
for the study of the recognition process. Among the lectins, the Arachis hypogaea lectin
(peanut agglutinin, PNA) is a relevant one, because of its specificity for β-galactosides
over other monosaccharides, showing high affinity for the disaccharides N-
acetyllactosamine and lactose,4,5 being the highest affinity ligand for the disaccharide β-
D
-Galp-(1→3)-D
-GalNAc,6 known as the Thomsen−Friedenreich (TF) antigen. The
PNA lectin has been extensively used in the glycobiology field on inhibition studies,
including our own, of a variety of β-galactoside and β-lactoside ligands.6-11
The recognition of carbohydrates by PNA has been the subject of varied
experimental and theoretical studies. Thus, crystallographic and computational
investigations revealed that the PNA-lactose complex is stabilized by interaction of the
Gal O3 with Asp83, Gly104 and Asn127, while the Gal O4 and O6 interact with Asp83
and Asp80, respectively. Gal O4 and Gal O5 also interact with Ser211 and the side
chain of the aromatic residue Tyr125 presents CH/π stacking interactions with the more
hydrophobic β-face of the Gal ring. In addition, the Glc O3 exhibits hydrogen bonding
with Ser211, Gly213 and Leu212, interactions that explain the lower affinity of β-
galactosides when compared to lactosides.3,6,7,10,12 In fact, we have previously shown
that the affinity of 3-deoxylactoside ligands are c.a. 15 times lower than the
corresponding lactosides, due to the lack of Glc O3, which participates in the
recognition process.9 The conformation of the Gal residue in the TF antigen is identical
to that of lactose, but the orientation of the reducing end with respect to the non-
reducing Galp differ in these disaccharides.6c However, the hydroxyl groups vicinal to
the glycosidic linkages (4-OH axial in the TF antigen and 3-OH equatorial in lactose)
occupy the same polar region defined by Ser211, Gly213 and Leu212.6d This brings up
the question of whether the integrity of the glucose residue is required for this additional
interaction, or if a hydroxyl group properly positioned in the aglycone or spacer linker
could mimic this OH. One possibility is to use a flexible glycosidic bond as connection
to the hydroxylated spacer, which should be able to adopt a suitable conformation that
must facilitate the interaction with the lectin. However, it has been reported that the

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flexibility of the aglycone may be detrimental for the affinity. For example, the affinity
of lactitol for several human galectins drops substantially compared to that of
lactose.13,14 The higher flexibility of the sorbitol moiety should also impact on the
hydration of this residue, an a higher interaction with the solvent would account for the
lower affinity.15 Thus, a balance in the rigidity imparted by the chemical bonds seems to
be crucial for the activity. Therefore, we speculate that the rather flexible disposition of
a hydroxyl group in the linker should be compensated by a fragment imparting a
conformational restriction to the glycosidic bond. In this context, compounds bearing an
amide function linked to the anomeric position could satisfy this requirement. The
conformational restrictions imposed by the high sp2-hybridation character of amide-type
nitrogen atoms16 and the hydrogen bonding donor and acceptor capabilities of the amide
group can potentially be exploited to modulate the lectin binding properties.
So far, the rather few studies performed on mono and divalent N-conjugates
have evidenced a different behavior in their interactions with the lectin, in comparison
with those of O- or S-glycosides.7-9,17-20 In fact, is has been shown that the type of
linkage can strongly affect the mobility and orientation of the putative sugar ligands
about the glycosidic linkage.21
On the other hand, the affinity of
be enhanced by means of their multivalent presentation onto a suitable platform. The
mechanisms governing the cluster effect in the binding of both -galactose and lactose
D-galactopyranosides for the PNA lectin may
D
glycotopes to PNA have been previously studied.22 With respect to multivalency, the
glycoside cluster effect clearly depends on the number of copies (valency) of the
carbohydrate residues but their spatial disposition is also a determinant feature.23 Thus,
the topology of the scaffold and the flexibility of the spacer segments can play a
decisive role in the recognition process.24-28
As part of our ongoing research project on the synthesis of multivalent ligands
with modified glycosidic bonds, we report here the synthesis of mono and divalent β-N-
galactosylamides linked through hydroxylated and non hydroxylated flexible linkers to
scaffolds differing in their rigidity. Their affinities toward PNA lectin were determined
by enzyme-linked lectin assay (ELLA). Structurally related mono and divalent N-
lactosides were also tested for comparative purposes. Furthermore, initial docking
studies and molecular dynamics simulations were performed in order to shed some light
on the interactions involved in the carbohydrate recognition domain and to explain the
differences in the affinities observed.

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2. Results and discussion
2.1. Synthesis
Taking into consideration the interactions that take place in the carbohydrate
recognition domain of PNA lectin with disaccharide lactose6,7 and in an attempt to
validate our hypothesis on the role of an hydroxyl group adequately placed in the
proximities of the Gal residue mimicking the Glc O3 as discussed above, the structure A
(Fig. 1c) was designed to be used in preliminary modelling studies. Model compound A
arises from the retrosynthetic analysis shown in Fig. 1b, where an anomeric amide
function was selected for the linkage between the Gal residue and the flexible linker. It
should be noted that there is a range of methodologies to efficiently form amide or
pseudoamide functionalities compatible with multiconjugation strategies. Tartaric
anhydride was chosen as the source of hydroxyl groups, in a sequence that resulted
compatible with previous synthetic methodologies developed in our group.7-9 The
carboxylic acid released by ring-opening of tartaric anhydride may be condensed with
propargyl amine for further multivalent conjugation with azide scaffolds. The analogous
compound B (synthesized for comparative purposes), lacking the hydroxyl groups in the
linker can be obtained by a similar route from succinic anhydride. The structures A and
B were employed for docking studies, taking into account that the distal triazol ring
might provide extra contacts with the protein as previously shown in other sistems.29
Fig. 1. (a) Representation of PNA−lactose complex. Hydrogen-bond interactions are depicted
with dotted lines.7 (b) Retrosynthetic analysis of the proposed structure. (c) Model compounds
A
and
B for docking studies.

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Thus, the major conformers of A and B (obtained as explained in the Materials
and methods section, Fig. S1), were docked into the binding site of peanut agglutinin
using the program AUTODOCK 4.2.30 The best docking poses of model compounds A
and B bound to PNA are shown in Fig. 2 and S2. It was observed that for both model
compounds, the Gal residues are surrounded by Asp83, Gly104 and Asn127 similar to
the Gal residue in the lactose-PNA complex. The position of the Tyr125 was compatible
with the stabilizing CH/π stacking interactions (Fig. S2). Regarding the aglycone linker,
docking experiments show a similar disposition of the tartaramidyl chain in A, with
respect to that of the succinimidyl in B. Interestingly, as expected, one of the hydroxyl
groups in A is located in the polar environment defined by Ser211, Leu212 and Gly213
(Fig. 2a).
Leu212
Ser211
Ser211
Gly213
Gly213
(a)
(b)
Fig. 2.
Docking results of PNA with (a) compound A, (b) compound B. The lectin is shown
in NewCartoon representation with key side chains in licorice. The images were prepared by
using the VMD program.
The auspicious docking results prompted us to synthesize ligands containing
these structural motifs, according to the retrosynthetic analysis depicted in Figure 1b.
The synthesis of the pivotal alkynyl precursor 5 was readily accomplished in two steps
starting from 2,3,4,6-tetra-O-acetyl-β-
quantitatively obtained by catalytic hydrogenation of the corresponding β-
galactopyranosyl azide (1).31 As 2 was unstable in solution, it was immediately treated
D-galactopyranosylamine (2), which was
D
-
with di-O-acetyl-L-tartaric anhydride (3) to give the acid derivative 4 in 76% yield
(Scheme 1).

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Scheme 1.
Synthesis of alkynyl precursor 5
Compound 4 was obtained as a single stereoisomer, as a result of the C2
symmetry of the anhydride 3. Compound 5 was obtained by condensation of 4 with
propargylamine, promoted by dicyclohexylcarbodiimide (DCC). The alkynyl derivative
5 was properly functionalized for the copper(I)-catalyzed azide-alkyne cycloaddition
(CuAAC), a ligation reaction broadly employed in the glycosciences,24,32 which leads to
the formation of 1,4-disubstituted 1,2,3-triazole rings. The succinic acid diamide
analogue 6,33 previously obtained in our laboratory, was used as control ligand.
1
The H NMR spectra of 4, 5 and 6 showed the signals of the amide protons directly
linked to the sugar residue as doublets at δ 7.00 ppm (J ≈ 9.2 Hz). The anomeric proton
was also coupled to the axial H-2, and appeared as a triplet in the range 5.13-5.10 ppm,
shielded by the proximity of the amide nitrogen atom.
As azide counterparts for click reactions we selected a monoazide carbohyhdrate
platform as a precursor of monovalent species, and three diazide scaffolds that would
give rise to divalent compounds, having similar intersaccharide distances. We and
others demonstrated the suitability of sugars as scaffolds for multivalent ligands.7-9,22,34-
37
Methyl 6-azido-2,3,4-tri-O-acetyl-β-D-glucopyranoside (7) and 6,6’-diazido-
2,3,4,2’,3’,4’-hexa-O-acetyl-α,α’-trehalose (8), were prepared as previously described.37
The diazides 9 or 10 derived respectively from isomannide38 or diethylenglycol, were
prepared by tosylation of the precursor diol followed by displacement of the tosyl
groups with sodium azide (Figure 3).

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Fig. 3. Azide scaffolds, precursors of monovalent and divalent ligands
The typical CuAAC reaction conditions (CuSO4/sodium ascorbate), applied to
the alkyne-armed compound 5 and azide scaffolds 7-10, led to the corresponding O-
1
protected triazol adducts 11, 14, 17 and 21 (Figure 4). The H-NMR spectrum of 11
showed the signals corresponding to the galactosyl and glucosyl groups. The diagnostic
signal of the triazole proton at δ 7.66 ppm and the two signals corresponding to the
amide NH groups at δ 6.91 ppm (d, J = 8.9 Hz) and 6.83 ppm (t, J = 5.7 Hz) were also
observed. The small J value (2.4 Hz) of the proton signals of the linker suggested a
planar zig-zag conformation for this segment. In the case of the divalent di-N-
1
galactopyranosyl derivatives 14, 17 and 21, the H and 13C NMR spectra were
consistent with the C2 symmetry of the molecules. The protected products were O-
deacetylated by treatment with triethylamine in aqueous methanol, then desalted with an
exchange resin and finally purified by reverse-phase chromatography to give the fully
unprotected monovalent (12) and divalent ligands (15, 18 and 22). In parallel series of
reactions, the succinyl diamide monovalent (13) and divalent (16, 20 and 24) analogues
were obtained. Compounds 13 and 16 had been previously reported and were
synthesized again for the purpose of this study,33 whereas compounds 20 and 24 were
obtained by click coupling of 9 and 6 (→19) or 10 and 6 (→23) and subsequent
deacetylation.
Fig. 4. β-N-galactosylamine glycoclusters synthesized

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For the free ligands 12, 15, 18, 20, 22 and 24, the signals of the anomeric
protons of the N-linked βGal residues appeared in a narrow region of the spectra (4.74-
4.98 ppm) as doublets with J ≈ 9.0 Hz, consistent with the β anomeric configuration.
The anomeric protons of the sugar scaffolds trehalose and glucose of 12 and 15
appeared at 4.73 and 4.54 ppm with a J value (≈ 4.0 Hz) characteristic of αGlc moieties.
The signals corresponding to the CHa─CHb tether of the tartaramide unit were observed
as doublets with JHa,Hb ≈ 1.7-2.0 Hz. In contrast, the CH2─CH2 system of succinic-
derived β-N-galactosides showed complex multiplets with J ≈ 5.00-7.00 Hz. The
averaged J values are indicative of a more flexible chain for the succinamide segment
compared with that of the tartaramide analogues.
For the purpose of our structure-PNA binding affinity relationship study, we
decided to test the affinity of the previously synthesized mono and divalent lactosyl
derivatives 25 and 26 (Fig. 5).33 Compounds 25 and 26 share the same sugar-derived
central scaffold as the galactosyl conjugates 13 and 16.
OH
OH
OH
HO
OH
OH
O
OH
O
HO
HO
OH
O
O
O
OH
OH
O
HO
O
OH
O
OH
O
OH
HO
HO
NH
OH
NH
O
OH
O
O
NH
OH
O
HN
HN
O
O
N
N
N
N
N
HN
N
O
N
N
HO
HO
O
HO
HO
HO
N
O
HO
OMe
OH
O
HO
OH
25
26
Fig. 5. Mono and divalent N-lactose derivatives 25 and 26.
2.2. PNA binding affinity studies
The relative binding affinities of the N-galactosyl (12, 13, 15, 16, 18, 20, 22 and
24) and N-lactosyl (25 and 26) conjugates for PNA were assessed by a competitive
enzyme-linked lectin assay (ELLA).39 This assay measures the ability of the synthetic
ligands to inhibit the association of the peanut lectin (labeled with horseradish
peroxidase, HRP-PNA) to a polymeric ligand that is used as a coating material at the
surface of a well.40 The IC50 values were assumed to be proportional to the

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corresponding binding affinities. The experiments were reproduced three times for each
ligand and the individual values did not differ by more than 15%. The corresponding
inhibition plots and IC50 values are collected in Figure 6 and Table 1. As compound 13
showed the highest value of IC50, it was taken as reference, and its relative potency was
defined as 1.
A
B
β-N-Galactosyl ligand (μM)
β-N-Lactosyl ligand (μM)
Fig. 6. Inhibition curves obtained from ELLA experiments for: (A) β-N-galactosyl
compounds 12, 13, 15, 16, 18, 20, 22 and 24; (B) β-N-lactosyl derivatives 25 and 26.

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Table 1. Inhibition of lactose glycopolymer − PNA binding by mono and divalent galactosyl and lactosyl ligands determined by ELLA.
Compound
Compound
Compound
OH
HO
OH
OH
HO
OH
O
O
HO
HO
Val.a
IC50 (mM)b
Rel. Pot.c
Val.a IC50 (mM)b
Rel. Pot.c
Val.a
IC50 (mM)b
Rel. Pot.c
NH
NH
O
OH
O
O
HO
O
HN
1
2
2
2
7.39±0.07
2.19±0.06
0.87±0.03
0.82±0.03
1.2
4.1
1
2
2
2
9.02±0.10
4.90±0.06
1.39±0.05
1.17±0.05
1
1
2
3.37±0.05
0.69±0.02
2.6
12
13
16
20
24
25
26
1.8
6.5
7.7
13.1
15
18
22
10.4
11.0
aValency. bThe IC50 values are expressed as mean values ± SD obtained from at least three independent determinations. cRelative
values are compared to the monovalent compound 13.

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Lactose (IC50 1.05 mM) and galactose (IC50 1.60 mM) were used in the ELLA
experiments as control compounds. As a general trend, compounds incorporating the L-
tartaric acid diamide segment (15, 18 and 22) behaved as better PNA ligands than the
homologous succinic acid diamide derivatives (16, 20 and 24). For monovalent ligands
(12 and 13), despite the geometric constraints provided by the anomeric amide linkage,
which seems to perturb in some way the lectin recognition (IC50 for galactose: 1.60
mM), these results are in accordance with our initial hypothesis that a properly
positioned hydroxyl group in the aglycone moiety would improve the binding affinities.
On the other hand, as can be deduced from the results indicated in Table 1, PNA
binding affinity increased after presenting the galactosyl motifs in divalent form. For
example, the affinities of 15, 18 and 22 resulted higher than that of 12. The same was
observed with the succinic acid diamide derivatives 16, 20 and 24 with respect to 13.
Remarkably, the relative affinity enhancements were strongly dependent on the
structure of the linker, and, again, compounds incorporating the L-tartaric acid diamide
segment (15, 18, 22) were better PNA ligands than the homologous succinic acid
diamide derivatives (16, 20, 24).
The divalent compounds also experienced a significant cluster effect when
referred to the corresponding monovalent control 12 or 13. Within each series, the
cluster effect increased on going from derivatives built on the α,α’-trehalose scaffold
(15, 16) to the isomannide (18, 20) and diethylenglycol centered representatives (22,
24). Thus, compound 22 (IC50 0.82 mM), combining the tartaric acid and
diethylenglycol structural elements in the connector, was 9-fold (11.0/1.2) a better
ligand for PNA than the monovalent counterpart 12 (IC50 7.39 mM) meaning that each
galactosylamide moiety is recognized with a 4.5-fold higher efficiency. Indeed,
compounds 18 and 22 presented approximately 2-fold higher affinity than galactose
itself, showing that divalent presentation can overcome the initial low affinity of a given
carbohydrate motif. The preference of the lectin for lactosyl over galactosyl epitopes is
observed when comparing the mono- and di-N-galactopyranosyl conjugates 13 and 16
with the homologous mono- and di-N-lactosyl derivatives 25 and 26, respectively.
2.3 Molecular Dynamics Simulations
To get further insight into the dynamics and energetics of the PNA-sugar
systems, we performed 100 ns long Molecular Dynamics (MD) simulations of PNA in

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complex with either lactose, 12 or 13, and analyzed the resulting protein-ligand
interactions.41 The results show that PNA interacts predominantly through Asp80,
Asp83 and Ser211, with the Gal O3, Gal O4 and Gal O6, which retains this
monosaccharide tightly in place. On the contrary, the Glc residue -in the case of lactose-
and the linkers for 12 and 13 are much more loosely bound, and show key differences in
their interactions (Fig. 7 and Fig. S3). As observed in the preliminary docking studies,
the OH group of the linker vicinal to the galactosyl-amide in 12, shows a hydrogen
bond with Leu212 amide (and with Ile101 carbonyl oxygen to a lesser extent). This
interaction should be responsible for the increased affinity of the ligand 12 respect to
13, which lacks of such a hydroxyl group (Fig. S4). Hence, the absence of this
interaction, results in a softer binding and higher mobility even for the bound
monosaccharide (the Gal residue). The linker in 13 is shown to be highly flexible and
stretches out to the solvent (Fig. S3).
A
B
C
Fig. 7. Hydrogen bond analysis which characterize the protein-ligand
contacts in: (A) lactose-PNA complex; (B) compound 12-PNA complex
and (C) compound 13-PNA complex.41

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3. Conclusion
In the present work, we try to establish if the whole glucose residue is required
for the stabilizing interaction provided by the Glc O3 in the complex of lactose with the
PNA lectin, or if a hydroxyl group properly positioned in the aglycone or spacer linker
could mimic the Glc O3. We took into account that the flexibility of the hydroxylated
linker should be compensated by a function imparting a conformational restriction to
the glycosidic bond, as highly flexible ligands show decreased affinity by their receptor
proteins in other systems. We speculate that galactosides with an amide group
connecting the anomeric position and a hydroxylated chain could satisfy such
requirements. Therefore, we have (a) designed hydroxylated amide-linked galactosides,
(b) performed preliminary docking studies on model structures, (c) synthesized mono
and divalent ligands grafted on different scaffolds, (d) determined their affinities toward
PNA lectin by ELLA, and finally, (e) accomplished molecular dynamic simulations to
rationalize the obtained results from a structural dynamics viewpoint.
Globally analyzed these results are in accordance with our initial hypothesis that
a hydroxyl group properly disposed could mimic the Glc O3 in the recognition process.
Regarding the multivalent effect, divalent species showed an increased affinity with
respect to their monomeric counterparts. Even though bidendate binding is not possible
as the linkers are not long enough to span the distance between two binding sites in the
tetrameric lectin (57-79 Å),12,42 the most interesting result is the notable cluster effect
observed for some of them. Probably, for divalent compounds, the “bind and recapture”
mechanism could be operating.1-3
4. Experimental
4.1. General methods
Analytical thin layer chromatography (TLC) was performed on Silica Gel 60 F254
aluminum supported plates (layer thickness 0.2 mm) with solvent systems given in the
text. Visualization of the spots was effected by exposure to UV light and charring with a
solution of 5% (v/v) sulfuric acid in EtOH, containing 0.5% p-anisaldehyde. Column
chromatography was carried out with Silica Gel 60 (230-400 mesh). Optical rotations
were measured at 20 °C in a 1 dm cell with a Perkin-Elmer 343 polarimeter. Microwave
irradiation was carried out in a CEM Discover MW instrument with a System Internal

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IR probe type, at 70 ºC (power max 300 W). High resolution mass spectra (HRMS)
1
were obtained by Electrospray Ionization (ESI) and Q-TOF. H and 13C nuclear
magnetic resonance (NMR) spectra were recorded at 25 °C at 500 and 125.7 MHz,
1
13
respectively, using a Bruker Avance II 500 spectrometer. For H, C nuclear magnetic
resonance (NMR) spectra, chemical shifts are reported in parts per million relative to
13
tetramethylsilane or a residual solvent peak (CHCl3: 1H: δ 7.26 ppm, C: δ 77.2 ppm).
Assignments of 1H and 13C were assisted by 2D 1H–COSY and 2D 1H–13C experiments.
In the description of the spectra, the signals corresponding to the glucose or trehalose
moieties were labeled as “G” or “T”, respectively. Azide-sugar scaffolds 7 and 8, and
compounds 13, 16, 25 and 26 were prepared as previously reported.33
4.2. Synthesis of the precursors
4.2.1.
2,3-di-O-acetyl-N-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-L-tartaric
acid monoamide (4)
To a solution of 2,3,4,6-tetra-O-acetyl-β-D-galactosylamine (2) (389 mg, 1.12 mmol) in
anhydrous MeCN (1.3 mL), was added (R,R)-tartaric anhydride43 (1.34 mmol, 290 mg).
The reaction proceeded for 15 min, when TLC showed complete consumption of the
starting 2. The solution was evaporated and compound 4 was purified by column
chromatography, using Toluene : EtOAc (2 : 1) to AcOEt : MeOH (7 : 3) containing
20
1% AcOH as eluting solvents. Yield: 478 mg (76%); [α]D + 30.7 (c 0.5, CHCl3); Rf
1
0.24 (CHCl3 : MeOH 10:1); H NMR (CDCl3): δ 7.03 (d, 1H, J1,NH 9.2 Hz, NH), 6.28
(br s, 1H, OH), 5.70 (d, 1H, JCHa,CHb 2.6 Hz, CHa), 5.68 (d, 1H, JCHa,CHb 2.6 Hz, CHb),
5.45 (dd, 1H, J4,5 0.7, J3,4 3.0 Hz, H-4), 5.17-5.07 (m, 3H, H-1, H-2, H-3), 4.10-4.03 (m,
13
3H, H-5, H-6a, H-6b), 2.25, 2.15, 2.13, 2.02 (2x), 1.99 (6 s, 18 H, CH3CO). C NMR
(CDCl3): δ 172.2, 170.7, 170.2, 169.9, 169.5, 169.4, 169.3, 167.1 (8 × CO), 78.5 (C-1),
72.6 (C-5), 72.3 (CHb), 70.6 (CHa), 71.5 (C-3), 67.9 (C-2), 67.3 (C-4), 61.1 (C-6), 20.7
(3x), 20.6, 20.2 (CH3CO). HRMS (ESI): m/z [M+Na]+ calcd for C22H29NNaO16:
586.1379, found: 586.1401
4.2.2. 2,3-di-O-acetyl-N-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-N-propargyl-
L-tartaric diamide (5)
To a solution of 4 (0.85 mmol, 480 mg) in anh CH2Cl2, DCC (1.07 mmol, 220 mg) was
added under Ar atmosphere. After stirring for 20 min, propargylamine (1.02 mmol, 56.4
mg) was added and the mixture was stirred at room temperature for 24 h and then

ACCEPTED MANUSCRIPT
filtered. The solution was concentrated and the products purified by column
chromatography using CHCl3 : MeOH (50 : 1 to 35 : 1) as eluting solvents. Yield: 430
mg (84%); mp 107-111 ºC; [α]D20 +46.5 (c 1.0, CHCl3); Rf 0.46 (CHCl3 : MeOH 10:1);
1H NMR (CDCl3): δ 6.95 (d, 1H, J1,NH 9.2 Hz, NH), 6.49 (t, 1H, JCH2,NH 5.4 Hz, NH),
5.79 (d, 1H, JCHa,CHb 2.5 Hz, CHa), 5.62 (d, 1 H, JCHa,CHb 2.5 Hz, CHb), 5.43 (d, 1H, J3,4
3.2 Hz, H-4), 5.15 (dd, J3,4 3.4, J2,3 10.2 Hz, H-3), 5.13 (t, 1H, J1,2 = J1,NH 9.2 Hz, H-1),
5.06 (t, 1H, J1,2 = J2,3 9.7 Hz, H-2), 4.17 (ddd, 1H, JCH2, C≡CH 2.5, JCH2,NH2 6.2, Jgem 17.5
Hz, CH2N), 4.09-4.02 (m, 3H, H-5, H-6a, H-6b), 3.92 (ddd, 1H, JCH2,C≡CH 2.5, JCH2,NH2
4.7, Jgem 17.5 Hz , CH2N), 2.25 (t, 1H, JCH2,C≡CH 2.5 Hz, C≡CH), 2.20, 2.15, 2.13, 2.02,
13
2.00, 1.98 (6 s, 18H, CH3CO). C NMR (CDCl3): δ 172.2, 170.6, 170.0, 169.8, 169.2,
168.7, 167.1, 165.6 (8 × CO), 79.0 (HC≡C), 78.3 (C-1), 73.0 (CHa), 72.5 (C-5), 72.1
(HC≡C), 72.0 (CHb), 70.5 (C-3), 67.8 (C-2), 67.3 (C-4), 61.1 (C-6), 29.2 (CH2N), 20.7
(3x), 20.6 (2x), 20.5 (CH3CO). HRMS (ESI): m/z [M+H]+ calcd for C25H33N2O15:
601.1875, found: 601.1877.
4.3. General Procedure for the Click Reaction
The click reaction was conducted under the conditions previously described.33 The
azide/derivatives 7, 8, 9 or 10 (0.20 mmol) and the alkynyl derivatives 5 or 6 (0.20
mmol per mole of reacting azide) were dissolved in a dioxane/H2O mixture (8 : 2, 2.5
mL). Copper sulfate (0.05 mmol per mole of reacting azide) and sodium ascorbate (0.10
mmol per mole of azide group) were added, and the mixture was stirred at 70 ºC under
microwave irradiation during 40 min. The mixture was then poured into a 1:1
NH4Cl/H2O solution (20 mL) and extracted with EtOAc (4 x 15 mL). The organic layer
was dried (Na2SO4) and filtered, and the solvent was evaporated under reduced
pressure. The residue was purified by flash chromatography, using the solvent systems
indicated in each case.
4.3.1. Methyl 2,3,4-tri-O-acetyl-6-deoxy-6-{4-[2,3-di-O-acetyl-N’-(2,3,4,6-tetra-O-
acetyl-β-
D
-galactopyranosyl)-L-tartaramidoyl-N-methyl]-1,2,3-triazol-1-yl}
α-D-
glucopyranoside (11).
Compound 11 was obtained by reaction of 5 and 7. Column solvent system: CHCl3 :
MeOH (50 : 1 to 35 : 1), 176 mg (93%); mp 124-126 ºC; [α]D20 +76.9 (c 1.0, CHCl3); Rf
1
0.53 (CHCl3 : MeOH 9:1); H NMR (CDCl3): δ 7.66 (s, 1H, H-triazole), 6.91 (d, 1H,

ACCEPTED MANUSCRIPT
J1,NH 8.9 Hz, NH), 6.83 (t, 1H, JCH2,NH2 5.7 Hz, NH), 5.75 (d, 1H, JCHa,CHb 2.4 Hz, CHa),
5.60 (d, 1H, JCHa,CHb 2.4 Hz, CHb), 5.47 (dd, 1H, J3G,4G 9.3, J2G,3G 10.2 Hz, H-3G), 5.44
(dd, 1H, J4,5 0.8, J3,4 3.5 Hz, H-4), 5.13 (dd, 1H, J3,4 3.5, J2,3 10.3 Hz, H-3), 5.12 (t, 1H,
J1,2 = J1,NH 8.8 Hz, H-1), 5.07 (dd, 1H, J1,2 9.3, J2,3 10.0 Hz, H-2), 4.92 (d, 1H, J1G,2G 3.6
Hz, H-1G), 4.83 (dd, 1H, J1G,2G 3.7, J2G,3G 10.3 Hz, H-2G), 4.78 (dd, 1H, J3G,4G 9.3,
J4G,5G 10.2 Hz, H-4G), 4.54 (dd, 1H, JCH2,NH 5.9, Jgem 15.2 Hz, CH2N), 4.53 (dd, 1H,
J5,6aG 2.4, J6aG,6bG 14.4 Hz, H-6aG), 4.49 (dd, 1H, JCH2,NH 5.9, Jgem 15.3 Hz, CH2N), 4.41
(dd, 1H, J5,6bG 7.8, J6aG,6bG 14.4 Hz, H-6bG), 4.16 (ddd, 1H, J5G,6aG 2.4, J5G,6bG 7.9,
J4G,5G 10.2 Hz, H-5G), 4.10 (dd, 1H, J5,6a 8.8, J6a,6b 12.8 Hz, H-6a), 4.04 (dd, 1H, J5,6b
6.1, J6a,6b 12.8 Hz, H-6b), 4.03 (ddd, 1H, J4,5 1.0, J5,6b 6.1, J5,6a 8.2 Hz, H-5), 3.17 (s,
13
3H, OCH3), 2.15, 2.14, 2.12, 2.10, 2.06, 2.03, 2.00 (2x), 1.99 (9 s, 27H, CH3CO). C
NMR (CDCl3): δ 172.0, 170.4, 170.1, 169.9 (2x), 169.7, 169.6, 169.0, 168.6, 167.0,
165.9 (CO), 143.9 (C-4 triazole), 123.7 (C-5 triazole), 96.7 (C-1G), 78.3 (C-1), 72.7
(CHa), 72.5 (C-5), 72.1 (CHb), 70.5 (C-2G), 70.4 (C-3G), 69.8, 69.7 (C-3, C-4G), 67.7
(2x) (C-2, C-5G), 67.1 (C-4), 60.9 (C-6), 55.6 (OCH3), 50.7 (C-6G), 34.8 (CH2N), 20.7
(2x), 20.6 (4x), 20.5, 20.4 (2x) (CH3CO). HRMS (ESI): m/z [M+Na]+ calcd for
C38H51N5NaO23: 968.2873, found: 968.2849.
4.3.2.
(2,3,4,6-tetra-O-acetyl-β-
triazol-1-yl} α,α’-trehalose (14).
2,2’,3,3’,4,4’-Hexa-O-acetyl-6,6’-dideoxy-6,6’-bis-{4-[2,3-di-O-acetyl-N’-
D
-galactopyranosyl)- -tartaramidoyl-N-methyl]-1,2,3-
L
Compound 14 was obtained by reaction of 5 and 8. Column solvent system: CHCl3 :
20
MeOH (50 : 1 to 20 : 1), 170 mg (46%); mp 141-143 ºC; [α]D + 46.2 (c 1.0, CHCl3);
1
Rf 0.52 (CHCl3 : MeOH 9:1); H NMR (CDCl3): δ 7.63 (s, 1H, H-triazole), 7.03-7.01
(m, 2H, 2 × NH), 5.68 (d, 1H, JCHa,CHb 1.8 Hz, CHa), 5.60 (d, 1H, JCHa,CHb 1.9 Hz, CHb),
5.44 (d, 1H, J3,4 2.2 Hz, H-4), 5.42 (t, 1H, J3T,4T = J2T,3T 10.0 Hz, H-3T), 5.17 (dd, 1H,
J3,4 3.6, J2,3 10.6 Hz, H-3), 5.16 (dd, 1H, J1,2 9.3, J2,3 10.6 Hz, H-2), 5.11 (t, 1H, J1,2 9.7
Hz, H-1), 5.06 (dd, 1H, J1T,2T 3.5, J2T,3T 10.5 Hz, H-2T), 4.97 (t, 1H, J3T,4T = J4T,5T 9.8
Hz, H-4T), 4.92 (d, 1H, J1T,2T 3.4 Hz, H-1T), 4.59−4.49 (m, 3H, CH2N, H-6aT), 4.29
(dd, 1H, J5T,6bT 9.1, J6aT,6bT 14.3 Hz, H-6bT), 4.07−4.00 (m, 4H, H-5T + H-5 + H-6a +
13
H-6b), 2.21, 2.14 (2x), 2.11, 2.01 (2x), 2.00, 1.99, 1.96 (9 s, 27H, CH3CO). C NMR
(CDCl3): δ 172.2, 170.5, 170.3, 170.1, 169.9 (2x), 169.8, 169.5, 169.4, 169.3, 165.8
(CO), 144.8 (C-4 triazole); 123.7 (C-5 triazole), 91.6 (C-1T), 78.5 (C-1), 72.9 (CHb),
72.4 (C-5), 72.2 (CHa), 70.5 (C-3), 69.7 (C-4T), 69.6 (C-5T), 69.5 (C-3T), 69.1 (C-2T),

ACCEPTED MANUSCRIPT
67.9 (C-2), 67.2 (C-4), 60.9 (C-6), 50.7 (C-6T), 35.2 (CH2N), 20.9, 20.8, 20.7 (6x), 20.6
(COCH3). HRMS (ESI) m/z [M + Na]+ calcd for C74H96N10NaO45 1867.5429, found
1867.5436.
4.3.3. 1,4:3,6-Dianhydro-2,5-dideoxy-2,5-bis-{4-[2,3-di-O-acetyl-N-(2,3,4,6-tetra-O-
acetyl-β-D-galactopyranosyl)-L-tartaramidoyl-N-methyl]-1,2,3-triazol-1-yl}-L-iditol
(17).
Compound 17 was obtained by reaction of 5 and 9. Column solvent system: CHCl3 :
MeOH (50 : 1 to 25 : 1), 151 mg (54%); mp 156−158 ºC; [α]D20 + 63.2 (c 1.0, CHCl3);
Rf 0.27 (CHCl3 : MeOH 9:1); 1H NMR (CDCl3): δ 7.68 (s, 1H, H-triazole), 7.10 (t, 1H,
JCH2,NH 5.9 Hz, NH), 7.04 (d, 1H, J1, NH 9.1 Hz, NH), 5.71 (d, 1H, JCHa,CHb 2.5 Hz, CHa),
5.61 (d, 1H, JCHa,CHb 2.5 Hz, CHb), 5.44 (d, 1H, J3,4 3.4 Hz, H-4), 5.22 (dd, 1 H, J2´, 3a´
2.7, J2´,3b´ 5.3 Hz, H-2´), 5.18 (dd, 1H, J3,4 3.4, J2,3 10.2 Hz, H-3), 5.14 (t, 1H, J1,2
=
J1,NH 9.2 Hz, H-1), 5.13 (s, 1H, H-1´), 5.08 (dd, 1H, J1,2 9.2, J2,3 10.2 Hz, H-2), 4.53 (dd,
1H, JCH2,NH2 6.0, Jgem 15.3 Hz, CH2N), 4.41 (dd, 1H, JCH2,NH2 5.9, Jgem 15.3 Hz, CH2N),
4.38 (dd, 1H, J2´,3a´ 5.4, J3a´,3b´ 10.4 Hz, H-3a´), 4.29 (dd, 1H, J2´,3b´ 2.6, J3a´,3b´ 10.4 Hz,
H-3b´), 4.09–4.03 (m, 3H, H-5, H-6a, H-6b), 2.14, 2.13, 2.08, 2.02, 2.00, 1.99 (6 s,
18H, CH3CO). 13C NMR (CDCl3): δ 172.0, 170.4, 169.9, 169.7, 169.1, 168.8, 167.1,
166.1 (CO), 144.6 (C-4 triazole), 122.0 (C-5 triazole), 87.5 (C-1´), 78.3 (C-1), 72.6,
72.5, 72.4, 72.1 (C-5, C-3´,CHa, CHb), 70.4 (C-3), 67.8 (C-2), 67.1 (C-4), 65.7 (C-2´),
60.9 (C-6), 34.7 (CH2N), 20.6 (3x), 20.5, 20.4 (2x) (CH3CO). HRMS (ESI): m/z
[M+Na]+ calcd for C56H72N10NaO32: 1419.4206, found: 1419.4158.
4.3.4.
1,4:3,6-dianhydro-2,5-dideoxy-2,5-bis-{4-[N-(2,3,4,6-tetra-O-acetyl-β-
D-
galactopyranosyl)-succinamoyl-N-methyl]-1,2,3-triazol-1-yl}-
L-iditol (19)
Compound 19 was obtained by reaction of 6 and 9. Column solvent system: CHCl3 :
MeOH (50 : 1 to 20 : 1), 182 mg (78%); mp 136−138 ºC; [α]D20 + 56.7 (c 1.0, CHCl3);
Rf 0.33 (CHCl3 : MeOH 9:1); 1H NMR (CDCl3): δ 7.66 (s, 1H, H-triazole), 6.78 (m, 2H,
2 × NH), 5.44 (d, 1H, J3,4 3.4 Hz, H-4), 5.29 (t, 1H, J1,2 = J1,NH 9.3 Hz, H-1), 5.29 (dd,
1H, J2´,3a´ 2.3, J2´,3b´ 5.6 Hz, H-2´), 5.23 (dd, 1H, J3,4 3.4, J2,3 10.2 Hz, H-3), 5.12 (t, 1H,
J1,2 = J2,3 9.3 Hz , H-2), 5.09 (s, 1H, H-1´), 4.54 (dd, 1H, JCH2,NH 6.0, Jgem 15.4 Hz,
CH2N), 4.41 (dd, 1H, JCH2,NH2 5.6, Jgem 15.2 Hz, CH2N), 4.40 (dd, 1H, J2´,3a´ 5.2, J3a´,3b´
10.4 Hz, H-3a´), 4.30 (dd, 1H, J2´,3b´ 2.2, J3a´,3b´ 10.4 Hz, H-3b´), 4.11–4.09 (m, 3H, H-5,
H-6a, H-6b), 2.55−2.45 (m, 4H, CH2-CH2), 2.14, 2.04, 2.02, 1.98 (4 s, 12H, CH3CO).
13C NMR (CDCl3): δ 172.6, 171.8, 171.3, 170.5, 170.1, 169.8 (CO), 145.8 (C-4

ACCEPTED MANUSCRIPT
triazole), 121.3 (C-5 triazole), 87.5 (C-1´), 78.3 (C-1), 72.5, 72.2 (C-5, C-3´), 70.8 (C-
3), 68.3 (C-2), 67.3 (C-4), 65.7 (C-2´), 61.2 (C-6), 35.0 (CH2N), 31.2, 30.5 (CH2–CH2),
20.7 (2x), 20.6 (2x) (CH3CO). HRMS (ESI): m/z [M+Na]+ calcd for C48H64N10NaO24:
1187.3993, found: 1187.4023.
4.3.5. 2,2’-bis-{4-[2,3-di-O-acetyl-N-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-
L-tartaramidoyl-N-methyl]-1,2,3-triazol-1-yl}-diethylether (21).
Compound 21 was obtained by reaction of 5 and 10. Column solvent system: EtOAc :
MeOH (98 : 2 to 90 : 10), 120 mg (44%); mp 136−138 ºC; [α]D20 + 35.7 (c 0.3, CHCl3);
Rf 0.13 (EtAcO : MeOH 9:1); 1H NMR (CDCl3): δ 7.83 (t, 1H, JCH2,NH 5.8 Hz, NH),
7.39 (s, 1H, H-triazole), 7.10 (d, 1H, J1,NH 9.5 Hz, NH), 5.77 (d, 1H, JCHa,CHb 2.4 Hz,
CHa), 5.72 (d, 1H, JCHa,CHb 2.5 Hz, CHb), 5.44 (d, 1H, J3,4 3.1 Hz, H-4), 5.17 (dd, 1H,
J3, 4 4.4, J2, 3 9.4 Hz, H-3), 5.16 (t, 1H, J1,2 = J1,NH 9.4 Hz, H-1), 5.10 (dd, 1H, J1,2 9.5,
J2,3 9.8 Hz , H-2), 4.51 (dd, 2H, JCH2,NH 2.1, Jgem 5.5 Hz, CH2N), 4.43 (m, 2H, CH2Ar),
4.11−4.00 (m, 3H, H-5, H-6a, H-6b), 3.77 (t, 2H, J 4.8 Hz, CH2O), 2.16, 2.14, 2.08,
13
2.01 2(x), 1.99 (6 s, 18H, CH3CO). C NMR (CDCl3): δ 172.0, 170.5, 170.1, 169.9,
169.7, 169.5, 167.6, 166.3 (CO), 144.3 (C-4 triazole), 123.8 (C-5 triazole), 78.4 (C-1),
72.9 (CHb), 72.4 (C-5), 72.3 (CHa), 70.6 (C-3), 69.2 (CH2O), 67.9 (C-2), 67.2 (C-4),
60.9 (C-6), 50.2 (CH2Ar), 35.0 (CH2N), 20.8 (3x), 20.7, 20.6 (2x) (CH3CO). HRMS
(ESI): m/z [M+Na]+calcd for C54H72N10NaO31: 1379.4263, found: 1379.4243.
4.3.6. 2,2’-bis-{4-[N-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-succinamoyl-N-
methyl]-1,2,3-triazol-1-yl}-diethylether (23).
Compound 23 was obtained by reaction of 6 and 10. Column solvent system: EtOAc to
EtOAc : MeOH (80 : 20), 135 mg (60%); [α]D20 + 20.3 (c 0.3, CHCl3); Rf 0.12 (EtOAc :
1
MeOH 9:1); H NMR (CDCl3): δ 7.69 (br s, 1H, H-triazole), 7.47 (br s, 1H, NH), 7.04
(d, 1H, J1,NH 9.2 Hz, NH), 5.42 (d, 1H, J3,4 1.8 Hz, H-4), 5.28 (t, 1H, J1,2 = J1,NH 9.0 Hz,
H-1), 5.16 (dd, 1H, J3,4 1.9, J2,3 10.4 Hz, H-3), 5.11 (t, 1H, J1,2 = J2,3 9.7 Hz , H-2),
4.58–4.32 (m, 4H, CH2N, CH2Ar), 4.11−4.02 (m, 3H, H-5, H-6a, H-6b), 3.84−3.72 (m,
2H, CH2O), 2.66-2.46 (m, 4H, CH2-CH2), 2.12, 2.02, 2.00, 1.96 (4 s, 12H, CH3CO). 13C
NMR (CDCl3): δ 172.9, 172.1, 171.1, 170.5, 170.2, 169.9 (CO), 78.5 (C-1), 72.4 (C-5),
71.1, (C-3), 69.2 (CH2O), 68.3 (C-2), 67.3 (C-4), 61.2 (C-6), 50.3 (CH2Ar), 35.1
(CH2N), 31.3, 30.6 (CH2−CH2), 20.8 (2x), 20.7, 20.6 (CH3CO). HRMS (ESI): m/z
[M+Na]+ calcd for C46H64N10NaO23: 1147.4043, found: 1147.4001.

ACCEPTED MANUSCRIPT
4.4. General procedure for the O-deacetylation
Compounds 11, 14, 17, 19, 21 and 23 were suspended in a mixture of MeOH : Et3N :
H2O 4:1:5 (3 mL/0.10 mmol of starting acetylated precursor) and stirred at room
temperature. After 1 h, TLC (EtOAc or EtOAc : MeOH, 9:1) showed complete
consumption of the starting material. The solution was concentrated and the residue was
dissolved in water (1 mL) and passed through a column filled with Dowex MR-3C
mixed bed ion-exchange resin. The eluate was concentrated and further purified by
filtration through an Octadecyl C18 minicolumn. Evaporation of the solvent afforded
the free product, which showed a single spot by TLC (n-BuOH : EtOH : H2O, 1:1:1)
whose Rf are indicated in each case.
4.4.1. Methyl 6-deoxy-6-[4-(N’-β-
D-galactopyranosyl-L-tartaramidoyl-N-methyl)
1,2,3-triazol-1-yl]-α- -glucopyranose (12).
D
20
Yield: 61 mg (85%), obtained from 11 (120 mg, 0.127 mmol); [α]D + 110.9 (c 0.7,
H2O); Rf 0.53; 1H NMR (D2O): δ 7.97 (s, 1H, H-triazole), 4.98 (d, 1H, J1,2 8.8 Hz, H-1),
4.84−4.79 (m, H-6aG, under the signal of HDO), 4.73 (d, 1H, J1G,2G 3.8 Hz, H-1G), 4.65
(d, 1H, JCHa,CHb 1.8 Hz, CHa), 4.62 (d, 1H, JCHa,CHb 2.0 Hz, CHb), 4.61 (dd, 1H, J5G,6bG
8.0, J6aG,6bG 14.6 Hz, H-6bG), 4.55 (s, 2H, CH2N), 3.98 (d, 1H, J3,4 2.1 Hz, H-4), 3.92
(ddd, 1H, J5G,6aG 2.3, J5G,6bG 8.1, J4G,5G 10.2 Hz, H-5G), 3.81 (t, 1H, J5,6a = J5,6b 6.2 Hz),
3.77−3.71 (m, 4H, H-2, H-3, H-6a, H-6b), 3.65 (t, 1H, J2G,3G = J3G,4G 9.5 Hz, H-3G),
3.53 (dd, 1H, J1G,2G 3.8, J2G,3G 9.8 Hz, H-2G), 3.22 (dd, 1H, J3G,4G 9.2, J4G,5G 9.9 Hz, H-
13
4G), 3.12 (s, 3H, CH3O). C NMR (D2O): δ 175.3, 173.5 (CO), 144.6 (C-4 triazole),
124.8 (C-5 triazole), 99.1 (C-1G), 79.7 (C-1), 76.9 (C-5), 73.3 (C-3), 73.0 (C-3G), 72.5
(2x) (CHa, CHb), 71.0 (C-2G), 70.8 (C-4G), 69.9 (C-5G), 69.2 (C-2), 68.7 (C-4), 61.0
(C-6), 54.7 (-OCH3), 50.9 (C-6G), 34.3 (CH2N). HRMS (ESI): m/z [M+Na]+ calcd for
C20H33N5NaO14: 590.1922, found: 590.1948.
4.4.2.
6,6’-dideoxy-6,6’-bis-[4-(N’-β-
D
-galactopyranosyl-L-tartaramidoyl-N-
methyl)-1,2,3-triazol-1-yl]-α,α’-trehalose (15).
20
Yield: 43 mg (87%) obtained from 14 (84 mg, 0.046 mmol); [α]D + 112.4 (c 0.7,
H2O); Rf 0.38; 1H NMR (D2O) δ 7.91 (s, 1H, H-triazole), 4.98 (d, 1H, J1,2 8.9 Hz, H-1),
4.80 (dd, 1H, J5T,6aT 2.2, J6aT,6bT 14.5 Hz, H-6aT), 4.67 (d, 1H, JCHa,CHb 1.8 Hz, CHa),
4.62 (d, 1H, JCHa,CHb 1.8 Hz, CHb), 4.57 (dd, 1H, J5T,6bT 8.4, J6aT,6bT 14.9 Hz, H-6bT),
4.55 (s, 2H, CH2N), 4.54 (d, 1H, J1T,2T 4.1 Hz, H-1T), 4.01 (ddd, 1H, J5T,6aT 2.3, J5T,6bT

ACCEPTED MANUSCRIPT
8.4, J4T,5T 10.3 Hz, H-5T), 4,03 (dd, 1H, J4,5 0.7, J3,4 2.9 Hz, H-4), 3.81 (ddd, 1H, J4,5
0.7, J5,6a 6.0, J5,6b 6.9 Hz, H-5), 3.77−3.72 (m, 5H, H-2, H-3, H-3T, H-6a, H-6b), 3.49
(dd, 1H, J1T,2T 3.9, J2T,3T = 9.9 Hz, H-2T), 3.25 (dd, 1H, J3T,4T 9.1, J4T,5T 10.0 Hz, H-4T).
13C NMR (D2O): δ 175.3, 173.5 (CO), 144.7 (C-4 triazole), 124.7 (C-5 triazole), 93.2
(C-1T), 79.8 (C-1), 76.9 (C-5), 73.3 (C-3), 72.6 (2x) (C-3T, CHa), 72.5 (CHb), 70.9 (C-
4T), 70.6, 70.5 (C-2T, C-5T), 69.2 (C-2), 68.7 (C-4), 61.0 (C-6), 50.9 (C-6T), 34.3
(CH2N). HRMS (ESI) m/z [M + H]+ calcd for C38H61N10O27 1089.3702, found
1089.3695.
4.4.3.
1,4:3,6-dianhydro-2,5-dideoxy-2,5-bis-[4-(N-β-D-galactopyranosyl-L-
tartaramidoyl-N-methyl)-1,2,3-triazol-1-yl]-L-iditol (18)
Yield: 49 mg (78%) obtained from 17 (96 mg, 0.07 mmol); [α]D20 + 111.3 (c 0.5, H2O);
1
Rf 0.40; H NMR (D2O): δ 7.79 (s, 1H, H-triazole), 5.28 (dd, 1H, J2´,3b´ 2.2, J2´,3a´ 5.1
Hz, H-2´), 5.01 (s, 1H, H-1´), 4.82 (d, 1H, J1,2 8.9 Hz, H-1), 4.48 (d, 1H, JCHa,CHb 2.0
Hz, CHa), 4.47 (d, 1H, JCHa,CHb 2.0 Hz, CHb), 4.42 (d, 1H, Jgem 15.7 Hz, CH2N), 4.37
(d, 1H, Jgem 15.7 Hz, CH2N), 4.29 (dd, 1H, J2´,3a´ 5.2, J3a´,3b´ 10.9 Hz, H-3a´), 4.21 (dd,
1H, J2´,3b´ 2.2, J3a´,3b´ 10.8 Hz, H-3b´), 3.81 (dd, 1H, J4,5 0.7, J3,4 2.8 Hz, H-4), 3.59 (ddd,
13
1H, J4,5 0.6, J5,6a 6.2, J5,6b 6.5 Hz, H-5), 3.61−3.56 (m, 4H, H-2, H-3, H-6a, H-6b). C
NMR (D2O): δ 175.3, 173.6 (CO), 144.9 (C-4 triazole), 123.0 (C-5 triazole), 87.2 (C-
1´), 79.7 (C-1), 77.0 (C-5), 73.3, (C-3), 72.5 (2x) (CHa, CHb), 72.1 (C-3´), 69.2 (C-2),
68.7 (C-4), 65.7 (C-2´), 61.0 (C-6), 34.3 (CH2N). HRMS (ESI): m/z [M+H]+ calcd for
C32H49N10O20: 893.3119, found: 893.3113.
4.4.4.
1,4:3,6-dianhydro-2,5-dideoxy-2,5-bis-[4-(N-β-
D
-galactopyranosyl-
succinamoyl-N-methyl)-1,2,3-triazol-1-yl]-
L-iditol (20)
20
Yield: 150 mg (86%) obtained from 19 (245 mg, 0.21 mmol); [α]D + 46.9 (c 0.9,
H2O); Rf 0.47; 1H NMR (D2O): δ 7.78 (s, 1H, H-triazole), 5.28 (dd, 1H, J2´,3b´ 1.9, J2´,3a´
5.1 Hz, H-2´), 5.04 (s, 1H, H-1´), 4.74 (d, 1H, J1,2 9.0 Hz, H-1), 4.33−4.26 (m, 3H, H-
3a´, CH2N), 4.24 (dd, 1H, J2´,3b´ 1.9, J3a´,3b´ 10.8 Hz, H-3b´), 3.81 (dd, 1H, J4,5 0.6, J3,4
3.3 Hz, H-4), 3.59 (ddd, 1H, J4,5 0.8, J5,6a 6.0, J5,6b 6.8 Hz, H-5), 3.58−3.52 (m, 3H, H-3,
H-6a, H-6b), 3.46 (t, 1H, J1,2 = J2,3 9.6 Hz, H-2), 2.53−2.40 (m, 4H, CH2-CH2). 13C
NMR (D2O): δ 176.0, 174.8 (CO), 145.1 (C-4 triazole), 123.0 (C-5 triazole), 87.2 (C-
1´), 79.7 (C-1), 76.7 (C-5), 73.3, (C-3), 72.1 (C-3´), 69.3 (C-2), 68.6 (C-4), 65.7 (C-2´),
60.9 (C-6), 34.5 (CH2N), 30.7, 30.3 (CH2-CH2). HRMS (ESI): m/z [M+H]+ calcd for
C32H49N10O16: 829.3323, found: 829.3348.

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4.4.5. 2,2’-bis-[4-(N-β-D-galactopyranosyl-L-tartaramidoyl-N-methyl)-1,2,3-triazol-
1-yl] diethylether (22).
Yield: 25 mg (73%) obtained from 21 (50 mg, 0.04); [α]D20 + 73.0 (c 1.1, H2O); Rf 0.41;
1H NMR (D2O): δ 7.73 (s, 1H, H-triazole), 4.97 (d, 1H, J1,2 8.5 Hz, H-1), 4.66 (d, 1H,
JCHa,CHb 1.8 Hz, CHa), 4.64 (d, 1H, JCHa,CHb 1.7 Hz, CHb), 4.54 (s, 2H, CH2N), 4.51 (t,
2H, JCH2Ar,CH2O 4.9 Hz, CH2Ar), 3.98 (d, 1H, J3,4 2.0 Hz, H-4), 3.87 (t, 2H, JCH2Ar,CH2O
4.9 Hz, CH2O), 3.81 (t, 1H, J5,6a = J5,6b 6.0 Hz, H-5), 3.75–3.72 (m, 4H, H-2, H-3, H-6a,
H-6b). 13C NMR (D2O): δ 175.3, 173.6 (CO), 144.3 (C-4 triazole), 124.0 (C-5 triazole),
79.7 (C-1), 76.9 (C-5), 73.3 (C-3), 72.5 (2x) (CHa, CHb), 69.2 (C-2), 68.7 (C-4), 68.5
(CH2O), 61.0 (C-6), 49.9 (CH2Ar), 34.3 (CH2N). HRMS (ESI): m/z [M+Na]+ calcd for
C30H48N10NaO19: 875.2995, found: 875.2965.
4.4.6. 2,2’-bis-4-[(N-β-D-galactopyranosyl-succinammoyl-N-methyl)-1,2,3-triazol-1-
yl]-diethylether (24).
Yield: 62 mg (76%), obtained from 23 (116 mg, 0.10 mmol); [α]D20 + 7.5 (c 1.0, H2O);
1
Rf 0.36; H NMR (D2O): δ 7.71 (s, 1H, H-triazole), 4.89 (d, 1H, J1,2 9.0 Hz, H-1), 4.51
(t, 2H, JCH2Ar,CH2O 5.0 Hz, CH2Ar), 4.42 (s, 2H, CH2N), 3.97 (dd, 1H, J4,5 0.4, J3,4 3.2
Hz, H-4), 3.87 (t, 2H, JCH2Ar,CH2O 5.0 Hz, CH2O), 3.76 (ddd, 1H, J4,5 0.5, J5,6a 6.0, J5,6b
6.5 Hz, H-5), 3.71–3.70 (m, 2H, H-6a, H-6b), 3.70 (dd, 1H, J3,4 3.4, J2,3 9.7 Hz, H-3),
13
3.62 (t, 1H, J1,2 = J2,3 9.5 Hz, H-2), 2.66–2.58 (m, 4H, CH2−CH2). C NMR (D2O): δ
176.0, 174.6 (CO), 144.5 (C-4 triazole), 123.9 (C-5 triazole), 79.7 (C-1), 76.7 (C-5),
73.3 (C-3), 69.3 (C-2), 68.6, 68.5 (CH2O, C-4), 60.9 (C-6), 49.9 (CH2Ar), 34.5 (CH2N),
30.7, 30.3 (CH2−CH2). HRMS (ESI): m/z [M+Na]+ calcd for C30H48N10NaO15:
811.3198, found: 811.3185.
4.5. Enzyme Linked Lectin Assay (ELLA)
Nunc-Inmuno™ plates (MaxiSorp™) were coated overnight with click lactose-
polystyrene glycopolymer44 at 100 ꢀL/well diluted from a stock solution of 10 ꢀg·mL-1
in 0.01 m phosphate buffer saline (PBS, pH 7.3 containing 0.1 mm Ca2+ and 0.1 mm
Mn2+) at room temperature. The wells were then washed three times with 300 µL of
washing buffer (containing 0.05% (v/v) Tween 20) (PBST). The washing procedure was
repeated after each of the incubations throughout the assay. The wells were then
blocked with 150 ꢀL/well of 1% BSA/PBS for 1 h at 37 ºC. After washing, the wells
were filled with 100 µL of serial dilutions of horseradish peroxidase labelled peanut

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(Arachis hypogaea) lectin (PNA-HRP) from 10-1 to 10-5 mg mL-1 in PBS, and incubated
at 37 ºC for 1 h. The plates were washed and 50 µL/well of 2,2'-azinobis-(3-
ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) (0.25 mg·mL-1) in
citrate buffer (0.2 m, pH 4.0 with 0.015% H2O2) was added. The reaction was stopped
after 20 min by adding 50 ꢀL/well of 1 m H2SO4 and the absorbances were measured at
415 nm. Blank wells contained citrate-phosphate buffer. The concentration of lectin-
enzyme conjugate that displayed an absorbance between 0.8 and 1.0 was used for
inhibition experiments. ELLA is considered to provide information on the intrinsic
multivalent effect, devoid of aggregation phenomena, since the presence of the
voluminous HRP enzyme prevents cross-linking the lectin unless very long spacer arms
are incorporated in the divalent ligand.12,23
In order to carry out the inhibition experiments, each inhibitor was added in a
serial of 2-fold dilutions (60 ꢀL/well) in PBS with 60 ꢀL of the desired PNA-peroxidase
conjugate concentration on Nunclon™ (Delta) microtiter plates and incubated for 1 h at
37 ºC. The above solutions (100 ꢀL) were then transferred to the lactose polymer-coated
microplates, which were incubated for 1 h at 37 ºC. The plates were washed and the
ABTS substrate was added (50 µL/well). Color development was stopped after 20 min
and the absorbances were measured. The percent of inhibition was calculated as
follows: % Inhibition = (A(no inhibitor)- A(with inhibitor))/A(no inhibitor) × 100. The IC50 values
corresponding to lactose and galactose were determined in the same conditions. A
positive control of a high affinity multivalent lactosylated cyclodextrin (valency: 21)
ligand was also tested to validate the methodology. The IC50 value obtained for this
ligand was 25 ± 2 ꢀM (Lit.: 21 ± 2 ꢀM).45
Results in triplicate were used for plotting the inhibition curves for each
individual ELLA experiment. Typically, the IC50 values (concentration required for
50% inhibition of the Con A-coating lactose polymer association) obtained from several
independently performed tests were in the range of ± 15%. Nevertheless, the relative
inhibition values calculated from independent series of data were highly reproducible.
4.6. Docking and Molecular Dynamics calculations
Dockign and Molecular Dynamics (MD) calculations were performed as in previous
works41 using modified AUTODOCK30 version for carbohydrates and AMBER MD
package (Ref amber). Briefly, minimized structures of lactose, and compounds A and B

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(represented in Fig. S1), were docked into the carbohydrate-binding site of the PNA
lectin (PDBid 1CR7) using previously reported parameters. The available X-ray PNA-
Lactose structure was used as a positive control. For MD simulations Amberff99SB
force field was used for the protein and Glycam-04 plus GAFF (Refs Glycam y Gaff)
for the ligands. Production simulations were run for 100 ns and analyzed with VMD
1.8.7 program (Fig. 2a and S2b, Fig. S3a and S3b).
Acknowledgements Support for this work from the National Agency for Promotion of
Science and Technology, ANPCyT, Project PICT Bicentenario 2010 – No. 1080, the
National Research Council CONICET, the University of Buenos Aires (UBACyT 2013-
2016 GC, 20020120100101BA), Argentina, the Centre National de la Recherche
Scientifique (CNRS), France, the Spanish MINECO (SAF2013-44021-R), the Junta de
Andalucía (FQM-1467) and the European Regional and Social Founds (FEDER and
FSE) is gratefully acknowledged. We deeply thank to Dr. Lautaro Álvarez for his kind
help with the modelling experiments, Dr. David Lesur for conducting some of the
HMRS-ESI determinations and Aurélie Vallin for technical assistance. MEC is a fellow
from CONICET. MLU and OV are research members of CONICET.
References
1.
(a) Essentials of Glycobiology, 2nd ed.; Varki, A.; Cummings, R. D.; Esko, J. D.; Freeze,
H. H.; Stanley, P.; Bertozzi, C. R.; Hart, G. W.; Etzler, M. E. Eds.; Cold Spring Harbor
Laboratory Press, New York, 1999; (b) Lee, Y. C.; Lee, R. T. Acc. Chem. Res. 1995, 28,
321-327; (c) The sugar code, Fundamentals of glycosciences, Gabius, H.- J. Ed.; Wiley-
Blackwell, 2009; (d) Jiménez Blanco, J. L.; Ortiz Mellet C.; García Fernández, J. M.
Chem. Soc. Rev. 2013, 42, 4518-4531.
2.
3.
4.
Fernández-Alonso, M. C.; Díaz, D.; Berbis, M. A.; Marcelo, F.; Cañada, J.; Jiménez-
Barbero, J. Curr. Prot. Pept. Sci. 2012, 13, 816-830.
Asensio, J. J.; Ardá, A.; Cañada, F. J.; Jiménez-Barbero, J. Acc. Chem. Res. 2013, 46,
946-954.
Sharma, V.; Srinivas, V. R.; Adhikari, P.; Vijayan, M.; Surolia, A. Glycobiology 1998, 8,
1007-1012.
5.
6.
Srinivas, O.; Mitra, N.; Surolia, A.; Jayaraman, N. Glycobiology 2005, 15, 861-873.
(a) Swamy, M. J.; Gupta, D.; Mahanta, S. K.; Surohat, A. Carbohydr. Res. 1991, 213, 59-
67; (b) Lotan, R.; Skutelsky, E.; Danon, D.; Sharon, N. J. Biol. Chem. 1975, 250, 8518-
8523; (c) Sharma, V.; Vijayan, M.; Surolia,V. J. Biol. Chem. 1996, 271, 21209–21213;
(d) Ravishankar, R., Ravindran, M., Suguna, K., Surolia, A., Vijayan, M. Current Science
1997, 72, 855-861.
7.
8.
Cagnoni, A. J.; Kovensky, J.; Uhrig, M. L. J. Org. Chem. 2014, 79, 6456-6467.
Cagnoni, A. J.; Varela, O.; Kovensky, J.; Uhrig, M. L. Eur. J. Org. Chem. 2013, 972-983.

ACCEPTED MANUSCRIPT
9.
Cagnoni, A. J.; Varela, O.; Kovensky, J.; Uhrig, M. L. Org. Biomol. Chem. 2013, 11,
5500-5511.
10. Ravishankar, R.; Thomas, V; Suguna, V; Surolia, V; Vijayan, V. Proteins 2001, 43, 260-
270.
11. Chandrasekaran, E.V.; Xue, J.; Xia, J.; Khaja, S. D.; Piskorz, C. F.; Locke, R. D.;
Neelamegham, S.; Matta, K. L. Glycoconj. J. 2016, 33, 819-836.
12. Banerjee, R.; Das, K.; Ravishankar, R.; Suguna, K.; Surolia, A.; Vijayan, M. J. Mol. Biol.
1996, 259, 281-296.
13. Mossine, V. V.; Glinsky, V. V.; Mawhinney, T. P. Food-related carbohydrate ligands for
Galectins. In Galectins, Klyosov, A. A., Witczak, Z. J. and Platt, D. Eds., John Wiley and
Sons, Hoboken, New Jersey, 2008, pp 235-270.
14. Ideo, H.; Seko, A.; Ohkura, T.; Matta, K. L.; Yamashita, K. Glycobiology 2002, 12, 199-
208.
15. D’Amelio, N.; Esteban, C.; Coslovi, A.; Feruglio, L.; Uggeri, F.; Villegas, M.; Benegas,
J.; Paoletti, S.; Donati, I. J. Phys. Chem. B 2013, 117, 13578-13587.
16. Avalos, M.; Babiano, R.; Carretero, M. J.; Cintas, P.; Higes, F. J.; Jiménez, J. L.;
Palacios, J. C. Tetrahedron 1998, 54, 615-628.
17. Masaka, R.; Ogata, M.; Misawa, Y.; Yano, M.; Hashimoto, C.; Murata, T.; Kawagishi,
H.; Usui, T. Bioorg. Med. Chem. 2010, 18, 621-629.
18. Vrasidas, I.; André, S.; Valentini, P.; Böck, C.; Lensch, M.; Kaltner, H.; Liskamp, R. M.
J.; Gabius, H. J.; Pieters, R. J. Org. Biomol. Chem. 2003, 1, 803-810.
19. Baussanne, I.; Benito, J. M.; Ortiz Mellet, C.; García Fernández, J. M.; Defaye, J.
ChemBioChem, 2001, 2, 777-783.
20. André, S.; Jarikote, D. V.; Yan, D.; Vincenz, L.; Wang, G.-N.; Kaltner, H.; Murphy, P.
V.; Gabius, H. J. Bioorg. Med. Chem. Lett. 2012, 22, 313-318.
21. (a) Espinosa, J. F.; Cañada, F. J.; Asensio, J. L.; Martín-Pastor, M.; Dietrich, H.; Martín-
Lomas, M.; Schmidt, R. R.; Jiménez-Barbero, J. J. Am. Chem. Soc. 1996, 118, 10862-
10871; (b) Ravishankar, R.; Surolia, A.; Vijayan, M.; Lim, S.; Kishi, Y. J. Am. Chem.
Soc. 1998, 120, 11297-11303; (c) Espinosa, J. F.; Bruix, M.; Jarreton, O.; Skrydstrup, T.;
Beau, J. M.; Jiménez-Barbero, J. Chem. Eur. J. 1999, 5, 442-448; (d) Jiménez-Barbero,
J.; Espinosa, J. F.; Asensio, J. L.; Cañada, F. J.; Poveda, A. Adv. Carbohydr. Chem.
Biochem. 2000, 56, 235-284; (e) García-Herrero, A.; Montero, E.; Muñoz, J. L.;
Espinosa, J. F.; Vián, A.; García, J. L.; Asensio, J. L.; Cañada, F. J.; Jiménez-Barbero, J.
J. Am. Chem. Soc. 2002, 124, 4804-4810.
22. Almant, M.; Mastouri, A.; Gallego-Yerga, V.; García Fernandez, J. M.; Ortiz Mellet, C.;
Kovensky, J.; Morandat, S.; El Kirat, K.; Gouin, S. G. Chem. Eur. J. 2013, 19, 729-738.
23. Lundquist, J. I.; Toone, E. J. Chem. Rev. 2002, 102, 555-578.
24. Chabre, Y. M.; Roy, R. Adv. Carbohydr. Chem. Biochem. 2010, 63, 165-393.
25. Gingras, M.; Chabre, Y. M.; Roy, M.; Roy, R. Chem. Soc. Rev. 2013, 42, 4823-4841.
26. Bhatia, S.; Dimde, M.; Haag, R. Med. Chem. Commun. 2014, 5, 862-878.
27. Cecioni, S.; Praly, J. P.; Matthews, S. E.; Wimmerov, M.; Imberty, A.; Vidal, S. Chem.
Eur. J. 2012, 18, 6250-6263.
28. (a) Sansone, F.; Casnati, A. Chem. Soc. Rev. 2013, 42, 4623-4639; (b) Martínez, A.; Ortiz
Mellet, C.; García Fernández, J. M. Chem. Soc. Rev. 2013, 42, 4746-4773; (c) Chabre, Y.
M.; Roy, R. Chem. Soc. Rev. 2013, 42, 4657-4708.
29. Zhou C. H., Wang Y. Curr. Med. Chem. 2012, 19, 239-280.
30. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639-1662.
31. Tropper, F. D.; Andersson, F. O.; Braun, S.; Roy, R. Synthesis 1992, 618-620.
32. (a) Ortiz Mellet, C.; Méndez-Ardoy, A.; García Fernández, J. M. Click multivalent
glycomaterials: glycoclusters, glycopolymers, hybrid glycomaterials and glycosurfaces.
In Click Chemistry in Glycoscience: New Developments and Strategies, Witczak, Z. J.;
Bielski, R. Eds.; Wiley: New Jersey, 2013, pp 143-182; (b) Uhrig, M. L.; Kovensky, J.
Clicking sugars onto sugars: oligosaccharide analogues and glycoclusters on carbohydrate

ACCEPTED MANUSCRIPT
scaffolds. In Click Chemistry in Glycoscience: New Developments and Strategies,
Witczak, Z. J.; Bielski, R. Eds.; Wiley: New Jersey, 2013, pp 107-142.
33. Cano, M. E.; Agusti, R.; Cagnoni, A. J.; Tesoriero, M. F.; Kovensky, J.; Uhrig, M. L.;
Lederkremer, R. M. Beilstein J. Org. Chem. 2014, 10, 3073-3086.
34. Gouin, S. G.; Vanquelef, E.; García Fernandez, J. M.; Ortíz Mellet, C.; Dupradeau, F.-Y.;
Kovensky, J. J. Org. Chem. 2007, 72, 9032-9045.
35. Gouin, S. G.; Wellens, A.; Bouckaert, J.; Kovensky, J. ChemMedChem 2009, 4, 749-755.
36. Köhn, M.; Benito, J. M.; Ortíz Mellet, C.; Lindhorst, T. K.; García Fernández, J. M.
ChemBioChem 2004, 5, 771-777.
37. Cagnoni, A. J.; Varela, O.; Gouin, S. G.; Kovensky, J; Uhrig, M. L. J. Org. Chem. 2011,
76, 3064-3077.
38. Besset, C.; Pascault, J. P.; Fleury, E.; Drockenmuller, E.; Bernard, J. Biomacromolecules
2010, 11, 2797-2803.
39. Rodríguez-Lavado, J.; de La Mata, M.; Jiménez-Blanco, J. L.; García-Moreno, M. I.;
Benito, J. M.; Díaz-Quintana, A.; Sánchez-Alcázar, J. A.; Higaki, K.; Nanba, E.; Ohno,
K.; Suzuki, Y.; Ortiz Mellet, C.; García Fernández, J. M. Org. Biomol. Chem. 2014, 12,
2289-2301.
40. Gómez García, M.; Benito, J. M.; Butera, A. P.; Ortiz Mellet, C.; García Fernández, J.
M.; Jiménez Blanco, J. L. J. Org. Chem. 2012, 77, 1273-1288.
41. (a) Gauto, D. F.; Di Lella, S.; Estrin, D. A.; Monaco, H. L.; Martí, M. A. Carbohydr. Res.
2011, 346, 939-948; (b) Gauto, D. F.; Petruk, A. A.; Modenutti, C. P.; Blanco, J. I.; Di
Lella, S.; Martí, M. A. Glycobiology 2013, 23, 241-258; (c) Modenutti, C. P.; Gauto, D.
F.; Radusky L.; Blanco, J. I.; Turjanski A.; Hajos, S.; Marti, M. A. Glycobiology 2015,
25, 181-96.
42. Wittmann, V.; Pieters, R. J. Chem. Soc. Rev. 2013, 42, 4492-4503.
43. Shriner, R. L., Furrow, C. L., Leonard, N. J., Sauers, R. R. Org. Synth. 1955, 35, 49.
44. Ortega-Muñoz, M., Morales-Sanfrutos, J., Perez-Balderas, F., Hernández-Mateo, F.,
Girón-González, M. D., Sevillano-Tripero, N., Salto-González, R., Santoyo-González, F.
Org. Biomol. Chem. 2007, 5, 2291-2301.
45. Gómez García, M., Benito, J. M., Butera, A. P., Ortiz Mellet, C., García Fernández, J. M.,
Jiménez Blanco, J. L. J. Org. Chem. 2012, 77, 1273-1288.

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UNIVERSIDAD DE BUENOS AIRES
FACULTAD DE CIENCIAS EXACTAS Y NATURALES
DEPARTAMENTO DE QUIMICA ORGANICA
Pabellón 2-Ciudad Universitaria-C1428EHA-Buenos Aires
TEL/FAX: 5411-4576-3346/ 5411-4576-3352
Title: “Synthesis of β-Galactosylamides as ligands of the Peanut lectin. Insights into the recognition
process.”
Authors: María Emilia Cano, Oscar Varela, María Isabel García-Moreno, José Manuel García
Fernández, José Kovensky and María Laura Uhrig
Highlights
β-Galactosylamine was coupled to L-tartaric anhydride as a precursor of a hydroxylated linker.
An analogous succinimidyl β-galactosylamine moiety was synthesized for comparative purposes.
Mono and divalent ligands were synthesized by click chemistry.
The affinity of the compounds obtained for the PNA lectin was determined by ELLA.
Docking and molecular dynamics calculations were in agreement with affinities observed.