Exploring calixarene-based clusters for efficient functional presentation of Streptococcus pneumoniae saccharides

Article abstract of DOI:10.1016/j.bioorg.2019.103305

Calixarenes are promising scaffolds for an efficient clustered exposition of multiple saccharide antigenic units. Herein we report the synthesis and biological evaluation of a calix[6]arene functionalized with six copies of the trisaccharide repeating uni

Full text of DOI:10.1016/j.bioorg.2019.103305

BioorganicChemistry93(2019)103305
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Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Exploring calixarene-based clusters for efficient functional presentation of
Streptococcus pneumoniae saccharides
Marta Giuliania, Federica Faroldia, Laura Morellib, Enza Torrec, Grazia Lombardic,
⁎
⁎
⁎
Silvia Fallarinic, , Francesco Sansonea, , Federica Compostellab,
a Department of Chemistry, Life Sciences and Environmental Sustainability, University of Parma, Parco Area delle Scienze 17/A, 43124 Parma, Italy
b Department of Medical Biotechnology and Translational Medicine, University of Milan, Via Saldini 50, 20133 Milano, Italy
c Department of Pharmaceutical Sciences, University of “Piemonte Orientale”, Largo Donegani 2, 28100 Novara, Italy
A R T I C L E I N F O
A B S T R A C T
Keywords:
Calixarenes are promising scaffolds for an efficient clustered exposition of multiple saccharide antigenic units.
Herein we report the synthesis and biological evaluation of a calix[6]arene functionalized with six copies of the
trisaccharide repeating unit of Streptococcus pneumoniae (SP) serotype 19F. This system has demonstrated its
ability to efficiently inhibit the binding between the native 19F capsular polysaccharide and anti-19F antibodies,
despite a low number of exposed saccharide antigens, well mimicking the epitope presentations in the poly-
saccharide. The calix[6]arene mobile scaffold has been selected for functionalization with SP 19F repeating unit
after a preliminary screening of four model glycocalixarenes, functionalized with N-acetyl mannosamine, and
differing in the valency and/or conformational properties. This work is a step forward towards the development
of new fully synthetic calixarenes comprising small carbohydrate antigens as potential carbohydrate-based
vaccine scaffolds.
Saccharide antigens
Serotype 19F
Streptococcus pneumoniae
Calixarenes
Multivalent ligands
1. Introduction
molecular mass repetitive polysaccharides are able to simultaneously
display a greater number of epitopes capable to effectively interact with
Encapsulated bacterial infections are still one of the most prevalent
causes of serious disease in humans, especially in young children.
Immunization is the most appropriate way to prevent bacterial infec-
tions [1]. In the early stages of microbiology, capsular polysaccharides
(CPSs) were recognized as relevant virulence factors, and found to be
able to stimulate protective immunity against infections, laying the
bases for the development of current antibacterial vaccines [2].
Nevertheless, the study of the chemical determinants of im-
munogenicity to CPSs still requires detailed molecular insights. CPSs
are cell-surface polymers consisting of oligosaccharide repeating units,
characterized by a huge number of possible structural modifications
and linkage combinations and associated to a relative high degree of
flexibility. Typically, anti-carbohydrate antibodies show affinities in
their recognition that are several factors lower than those observed for
antibodies specific for proteins or peptide antigens. This peculiar
weakness is partially compensated by the multivalent nature of such
antibodies, that face in a clustered form a multiple number of densely
displayed antigen molecules to increase the effect of the response [3].
This can explain the existing relationship between molecular weight
and antigenicity, established for polysaccharide antigens. In fact, high
specific antibodies. This kind of model where the binding of a glycan-
binding protein to a clustered saccharide patch enhances the overall
affinity of the interaction has been widely described [4], for instance
between tumor associated saccharide antigens and lectins. Lectin-
glycan interactions are stabilized by weak hydrogen bonding and van
der Waals intermolecular forces, and the multivalent presentation is the
basis for a biologically relevant binding. There are several examples
demonstrating that lectins bind their targets only when glycans are
clustered at high density in a multivalent glycoconjugate backbone [5].
This concept has been exploited for instance in the field of cancer
vaccine development. For example, the Tn tumor associated antigen is
overexpressed on the surface of tumor cells. It is exposed on the Mucine
surface in clusters of 2–5 units. Data show that at least two contiguous
antigen molecules are crucial for the binding of the anti-Tn monoclonal
antibodies, with an even greater affinity in the presence of three re-
sidues [6]. Different studies have been oriented to the selection of the
proper scaffold for the presentation of multiple copies of the Tn anti-
genic unit to generate an anticancer vaccine [7–9]. The choice of the
scaffold for multivalent presentation of glycans to target proteins is
crucial in determining the biological activity [10–13]. In this context,
⁎ Corresponding authors.
E-mail addresses: silvia.fallarini@uniupo.it (S. Fallarini), francesco.sansone@unipr.it (F. Sansone), federica.compostella@unimi.it (F. Compostella).
https://doi.org/10.1016/j.bioorg.2019.103305
Received 18 July 2019; Received in revised form 17 September 2019; Accepted 19 September 2019
Availableonline19September2019
0045-2068/©2019ElsevierInc.Allrightsreserved.

M. Giuliani, et al.
BioorganicChemistry93(2019)103305
the use of calixarenes represents a viable approach explored in the
literature for the display of multiple glycan units [14]. This type of
macrocycle in fact, due to peculiar structural properties and selective
synthetic procedures [15], allows the achievement of small libraries of
potential multivalent ligands bearing the same epitopes but with a
controlled modulation of their number (valency) and orientation in the
space. The conformational behavior of the different possible types of
calixarenes strongly impacts on the presentation mode of the con-
jugated saccharide moieties [14,16], which can be exploited to increase
the avidity of the biological recognition. This versatility can make
possible the comparison of subtly modified parameters and their effects
on selectivity and efficiency in biological activities like interaction with
carbohydrate recognition proteins [14]. A calix[4]arene in the so called
cone geometry conjugated to LacNAc fragments, for example, was
found extremely selective in the inhibition of galectin-3 while totally
unable to bind to galectin-1 [17]. Calixarenes functionalized with lac-
tose units showed inhibition activity towards human galectins char-
acterized, in some cases, by an impressive selectivity strictly dependent
on their conformational properties and related arrangement of the
epitopes [18]. Analogously, different sized and conformationally fea-
tured calixarenes exposing galactose units resulted in different effi-
ciency in the binding to Pseudomonas aeurginosa lectin A (PA-IL) [19].
Ten years ago, a calix[4]arene decorated with four Tn units has indeed
been described as a promising synthetic multivalent vaccine candidate
[20]. In this framework, we have decided to explore the potential of
calixarenes as scaffolds for the multipresentation of bacterial CPS
fragments. Our hypothesis was to verify if such macrocyclic scaffold,
presenting multiple, but however limited copies of short CPS fragments,
is able to gain affinity and potency towards the natural antibodies ap-
proaching those observed for the natural polysaccharide. In principle, a
positive assessment would open the possibility to work with shorter
saccharide fragments, which can be obtained by chemical synthesis,
instead of the longer oligosaccharides, used in vaccine formulations,
obtained by size-reduced purified natural polysaccharides [21]. More-
over, the use of calixarenes and short saccharide fragments would have
the advantage of giving structurally well-defined and easily re-
producible systems.
effectively inhibit the binding between the native 19F CPS and the anti-
19F antibodies.
2. Results and discussion
2.1. Synthesis
We report the synthesis of a small library of glycocalixarenes
(compounds 1a-d and 2a-b, Fig. 2) functionalized at the upper rim with
saccharide units present in the CPS structure of SP19F. To this aim, both
calix[4]- and calix[6]arene were selected as scaffolds in order to have a
modulation of the valency. Moreover, the calix[4]arene based deriva-
tives were designed with three different conformational features, one
conformationally mobile, one blocked in the cone geometry, one
blocked in the 1,3-alternate one, in order to compare a different spatial
arrangement of the epitope units. Also the calix[6]arene based gly-
coclusters are conformationally mobile. In addition, a monovalent
acyclic analogue was planned to have a reference for verifying the role
of the multivalency in the interaction with the antibodies.
In a first step of the project, all the selected scaffolds were func-
tionalized with the N-acetyl-β-D-mannosamine residue (compounds 1a-
d and 1-MON, Fig. 2), while subsequently, based on a preliminary set of
inhibition studies, only the calixarene scaffold of the best inhibitor of
the binding between the 19F polysaccharide and the corresponding
anti-19F antibodies was functionalized with the SP19F trisaccharide
repeating unit. Thus, two additional glycocalixarenes (2a and 2b,
Fig. 2), were obtained, with increased similarity with respect to the
natural structure. This two-steps approach was pursued because of the
demanding synthesis of the trisaccharide and the consequent difficulty
in producing a sufficient amount for the functionalization of all the
available structures.
In order to have an efficient and clean reaction for the coupling
between the calixarene scaffolds and the sugar units, we decided to
exploit the amine-isothiocyanate condensation. To this aim, the calix-
arene isothiocyanates 4a-d and the sugar unit 6, functionalized at the
reducing end with an aminopropyl linker, were synthesized (Scheme 1).
Compounds 4a-d were prepared by treatment of the corresponding,
already known aminocalixarenes 3a-d [25–27] with thiophosgene
(caution) in the presence of a base, the cone isomer following a pre-
viously reported procedure [28] and the others through equivalent
strategies. It may be interesting to underline that for the con-
formationally mobile tetraisothiocyanate calixarene 4a the 1H NMR
spectrum in CDCl3 highlighted the presence of two conformers, iden-
tified as the cone and the partial cone, in equilibrium and in slow ex-
change on the NMR timescale, in a 1:4 ratio.
We focused on the gram positive bacterium Streptococcus pneumo-
niae (SP) which is among the major responsible of severe forms of
bacterial infectious diseases [1,22–24]. In particular, serotype 19F
(SP19F) is one of the most common causes of invasive pneumococcal
disease in children, and included in the current commercial pneumo-
coccal conjugate vaccines. Its CPS is a linear polymer made up of β-D-
ManpNAc-(1 → 4)-α-D-Glcp-(1 → 2)-α-L-Rha trisaccharide repeating
units, linked through phosphodiester bridges (Fig. 1). Herein, we report
on the preparation and biological evaluation of a family of calixarenes
functionalized with saccharide fragments related to the trisaccharide
repeating unit of SP19F with the aim to explore the effect of the mul-
tipresentation of the ligand mediated by calixarenes on the binding to
the antibodies. We found that a calix[6]arene, functionalized with six
copies of the trisaccharide repeating unit of SP19F, is capable to
On the basis of procedures reported in the literature [29], we then
prepared compound 6, a protected derivative of 3-aminopropyl N-
acetyl-β-D-mannosamine, benzylated at the sugar hydroxyls. Mono-
saccharide 6 was deprotected from the Cbz group by hydrogenolysis
producing the amino derivative 7 that was subsequently reacted with
cone and 1,3-alternate tetraisothiocyanate calix[4]arenes 4b and 4c,
obtaining in good yields the corresponding tetraglycosylated macro-
cycles (see SI). Nevertheless, unexpectedly, their deprotection from
benzyl groups to give the two planned glycoclusters failed, despite the
use of different methods, discouraging the use of this pathway for the
preparation of the target calixarenes 1a-d.
Therefore, we went back to monosaccharide 6 to replace benzyl
protecting groups with acetyls being proved that the latter one can
efficiently be removed from saccharide units attached to a calixarene
scaffold [13,14,30,31]. Therefore, a sequence of protection and de-
protection reactions starting from 6 was carried out to finally obtain
compound 11 with an overall yield of 50% (Scheme 2). More in detail,
after the selective removal of N-Cbz from 6 by hydrogenolysis, com-
pound 7 was protected with a Boc group to give 8. The subsequent
catalytic hydrogen transfer under MW irradiation yielded 9, fully de-
protected on the OH groups, without the need of purification. The
Fig. 1. Streptococcus pneumoniae 19F capsular polysaccharide repeating unit.
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M. Giuliani, et al.
BioorganicChemistry93(2019)103305
Fig. 2. Library of multivalent glycocalixarenes decorated with saccharide units of SP 19F CPS.
acetylation step, performed with acetic anhydride and pyridine, af-
forded in high yield sugar 10, which was treated with trifluoroacetic
anhydride to remove the Boc group providing 11 in quantitative yield.
The mannosamine derivative 11 was then reacted with the iso-
thiocyanate calixarenes 4a-d. The click chemistry reaction between
isothiocyanate and amine, used for the conjugation reaction, is one of
the most commonly employed methodology for the design and synth-
esis of multivalent glycoconjugates [32,33]. This methodology does not
involve the anomeric position of the glycoside and the newly formed
thioureido unit presents an enhanced potential for hydrogen bonding
that can increase both the solubility in water of the ligand and the
number of interactions with the receptor. Glycocalixarenes 5a [34], 5b,
5c [34] and 5d were obtained in moderate yields (from 25% to 71%)
after column chromatography performed to remove the sugar excess
and traces of partially functionalized derivatives. Finally, compounds
5a-d were deacetylated under Zemplen conditions at 0 °C to give the
target calixarenes 1a-d. For the two conformationally mobile deriva-
tives 1a and 1d, we proceeded in the investigation by 1H NMR spec-
troscopy of their behavior in solution. The 1H NMR spectrum in MeOD
of the former one did not give any clear information about the con-
formation adopted by the calixarene in solution. A spectrum in D2O was
then recorded, knowing that conformationally mobile calix[4]arene
amphiphiles usually tend to adopt in water a 1,3-alternate geometry
which minimizes the lipophilic surfaces exposed to the solvent [35]. At
room temperature the spectrum still presented broad signals, difficult to
assign, while by raising the temperature up to 80 °C a rather well-de-
fined spectrum was obtained (see SI at page SI22). It was possible to
identify the signals of the H1 and H2 protons of the sugar moiety, at 4.69
and 4.43 ppm respectively, and, at the same time, the single signal at
around 7 ppm for all the aromatic protons together with the absence of
the typical doublets for the methylene bridge of cone and partial cone
conformers demonstrated the adoption of a 1,3-alternate geometry. This
should plausibly be the geometry adopted by glycocalixarene 1a also in
the aqueous environment of the biological tests.
As observed for 1a, the 1H NMR spectrum in D2O at room tem-
perature of calix[6]arene 1d was characterized by broad signals that
became sharp by increasing the temperature at 80 °C (see SI at page
SI24). This behaviour could be explained with the low mobility of the
aromatic units through the macrocyclic annulus, slowed down by the
steric hindrance of the bulky substituents at the upper rim despite the
bigger size of the macrocycle. On the other hand, interestingly, in the
past it was observed, rather unexpectedly, that methoxyglucocalix[6]-
and [8]-arenes tend to self-assemble in aqueous solution [36], despite
the absence of a well-defined amphiphilicity, as indeed for 1a and 1d.
For glycocalixarenes 1a and 1d self-aggregation cannot then be ex-
cluded, determining or at least contributing to the broadening of the
signals in the 1H NMR spectrum. In both cases, the sharpening of the
signals by increasing the temperature can be read as a consequence of
the assembly disaggregation and/or of the increased mobility of these
derivatives in solution.
Following a similar synthetic strategy, monomeric derivative 1-
MON was prepared (Scheme 3) starting from 4-propoxyaniline 12 that
was transformed into the corresponding isothiocyanate 13. Compound
13 was then reacted with sugar unit 11 to give compound 14 which was
subsequently deprotected to the final 1-MON in 78% yield.
On the basis of the results obtained from biological tests on com-
pounds 1a-d (see below), the conformationally mobile calix[6]arene
was selected as the best scaffold for obtaining the glycocalixarenes
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M. Giuliani, et al.
BioorganicChemistry93(2019)103305
Scheme 1. Synthesis of glycocalix[n]arenes 1a–d.
displaying the trisaccharide repeating unit of SP19F. To this aim, ex-
ploiting our expertise in the synthesis of synthetic fragments of
Streptococcus pneumoniae [37–39], we planned the preparation of
compound 15, the peracetylated derivative of the SP19F trisaccharide
repeating unit, functionalized at the downstream residue with an ami-
nopropyl linker. Compound 15 was synthesized starting from the tri-
saccharide trichloroacetimidate donor 16 [37], which was glycosylated
with Z-aminopropanol (Scheme 4) [38]. The reaction was promoted
with trimethylsilyl triflate and trisaccharide 17 was obtained in high
yields (96%) as an alpha/beta mixture of the two anomers 17α and
17β, which were separated by flash chromatography. Each anomer was
separately subjected to initial exchange of the CBZ amino protecting
group of the linker for the BOC one, followed by hydrogenolysis of the
benzyl ethers, and final acetylation of the hydroxyl groups with acetic
anhydride. This protecting group manipulation sequence was very ef-
ficient and allowed to recover in both cases the final trisaccharides 15α
and 15β in 88% yield.
Hexaisothiocyanate 4d was separately reacted in good yields with
both the α and the β anomer of compound 15, to evaluate if a different
stereochemistry of the anomeric position on the rhamnose residue may
have an influence on the biological activity of the glycocalixarene. If
not, in perspective, the calixarene could be reacted with the mixture of
Scheme 2. Sequence of protection and deprotection steps to obtain compound 11.
4

M. Giuliani, et al.
BioorganicChemistry93(2019)103305
Scheme 3. Synthesis of monomer 1-MON.
antibodies was evaluated in a classical competitive ELISA. Fig. 3 shows
the inhibition curves obtained with the compounds under evaluation.
The relative efficacy of each compound was assessed by determining
its maximum effect of inhibition at 1 mg/mL, while the concentration
that produces the 50% of the possible maximum effect (IC50) was cal-
culated when the curve reaches a plateau and taken as indirect index of
the relative potency (Table 1). The activities of the new compounds
were compared with those of the natural 19F polysaccharide and the
19F trisaccharide repeating unit (SP19F-RU, see structure in SI)
[38,40] as the reference compound. The maximum inhibition observed
for 19F CPS was fixed as the 100%.
Our results demonstrate that the spatial preorganization of the
mannosamide units on the calixarene effectively increases their efficacy
in a way that depends on the valency and on the tridimensional ar-
chitecture of the macrocyclic scaffold. In fact, comparing the maximum
% of inhibition (Table 1) found for the monomeric model 1-MON (16%)
with that of calixarene 1a (34%), also considering that the concentra-
tion of the ManAc epitope is substantially the same for the two com-
pounds (2.1 and 2.2 mM for 1-MON and 1a, respectively), the single
mannosamide unit evidences an efficacy almost 2 fold higher when it is
displaced on the calix[4]arene scaffold, which raises to 3 fold with the
glycocalix[6]arene 1d. The efficacy of the tetravalent calixarenes 1a-c
appears to be related to their conformational properties and the con-
sequent geometry of presentation of the mannosamine units. In fact, the
percentage of inhibition increases from the cone isomer to the 1,3-al-
ternate and to the conformationally mobile (1b 10
2%, 1c
21 3%, 1a 34 7). This suggests that, despite the same number of
saccharide units in these three ligands, the upper rim of ligand 1b is too
crowded for an effective interaction with the antibodies resulting even
worse than the monovalent model 1-MON. The better behavior of 1c
supports this hypothesis since it exposes only two sugar units per part of
the space with a reduced steric hindrance and perhaps an overall more
convenient arrangement with respect to 1b. However, also this blocked
display of 1c seems not to be the optimal one, as reflected in its lower
level of efficacy with respect to the mobile isomer 1a that evidently can
adapt itself for a better interaction with the antibodies as allows the
epitope units to better adapt to the binding sites. Flexibility appears
then to play an important role in determining the efficacy. The inability
of compound calix[4]-Gal, a tetra thioureidoglycocalixarene based on
the same calix[4]arene scaffold as 1a but bearing a non-related sugar
(galactose), in inhibiting the binding between 19F CPS and anti-19F
antibodies confirms the specificity of the sugar recognition and ex-
cludes an unspecific cross-reaction with the calixarene scaffold. On the
whole, the conformational properties, the related geometry of exposi-
tion of the mannosamine units, the flexibility together with the higher
Scheme 4. Synthesis of trisaccharides 15α and 15β.
the two anomers that is produced in the final step of the synthesis of the
trisaccharide when the aminopropyl chain is introduced at the
anomeric position of rhamnose. The two glycocalixarenes 18a and 18b
obtained by this condensation were deprotected under Zemplen con-
ditions at 0 °C to give the final glycoclusters 2a and 2b in 81% and 82%
yield respectively (Scheme 5).
2.2. Binding affinity measurements
The ability of increasing concentrations (from 10−7 mg/mL to
1 mg/mL) of each new compound to inhibit the binding between the
native 19F CPS, coated onto plates, and the mouse anti-19F polyclonal
5

M. Giuliani, et al.
BioorganicChemistry93(2019)103305
Scheme 5. Synthesis of 2a and 2b.
Fig. 3. Results of the Elisa experiments. Concentration/response curves of tested compounds on the inhibition of the binding between SP19F native polysaccharide,
coated onto the plates, and the anti-19F antibodies, evaluated by a competitive ELISA method. Values are means of at least four experiments run in triplicate.
efficacy of antibody binding. In this framework, at the maximum of
inhibition, the trisaccharide attached to the calix[6]arene platform is at
a very similar concentration (1.31 mM) as when used alone (1.47 mM),
but induces an efficacy 1.5 fold higher than SP19F-RU, determining an
inhibition of the binding over 70% significantly higher than the 52% of
the SP19F-RU. These data strongly suggest that the glycocalix[6]arene
thanks to its peculiar conformational mobility enables a proper pre-
sentation of the trisaccharide repeating units that improves the strength
of the ligand-receptor interactions well mimicking the conformational
organization of the epitopes generated by the natural 19F poly-
saccharide and recognized by the specific antibodies. This result is even
more significant if we consider that the chemical structure of calixar-
enes 2a and 2b is much simpler than the one of the natural poly-
saccharide and yet able to provide a significant inhibitory effect. As a
consequence, the maximum of inhibition compared with that of 19F
CPS is relevant, 75% of inhibition versus the 100%. Furthermore, the
structural difference due to the α and β anomeric connection of the
trisaccharide to the spacer has no significant influence on the efficacy of
the two ligands 2a and 2b, although resulted in slightly different po-
tencies. This means that, in perspective, the separation of the two
anomers 15α and 15β could even be avoided, proceeding in the cou-
pling as a mixture of both with the calixarene isothiocyanate. The
statistic products containing randomly both anomers should sub-
stantially show the same biological activity as 2a and 2b with a not
negligible save in the procedure of preparation.
Table 1
Results of the competitive Elisa assay.
Compound
IC50(mg/mL)
Max inhibition (%)a
19F
1.8 × 10−4
100
34
10
21
48
16
7
2
1a
7
2
3
8
7
1b
1c
1.2 × 10−1
9.5 × 10−2
1d
1-MON
calix[4]-Gal
SP19F-RU
2a
2
2.1 × 10−2
1.9 × 10−3
8.6 × 10−4
52
78
75
4
8
7
2b
a
The maximum inhibition elicited by each compound at 1 mg/mL.
valency could explain the highest efficacy (maximal inhibition
48
8%) and potency (IC50 = 9.5 × 10−2 mg/mL) showed by glyco-
calix[6]arene 1d. Calix[6]arene was then selected as the more pro-
mising scaffold for the functionalization with the SP19F trisaccharide
repeating unit analogs 2a and 2b.
As shown in Fig. 3 and reported in Table 1, glycocalix[6]arenes 2a
and 2b, decorated with six trisaccharide SP19F repeating units, sig-
nificantly improve efficacy (75–78% of inhibition) and affinity
(IC50 = 1.9 × 10−3 and 8.6 × 10−4 mg/mL, respectively) towards the
antibodies with respect to the simpler cluster 1d (48% of inhibition,
IC50 = 9.5 × 10−2 mg/mL). This result can be explained considering
the nature of the saccharide ligands on the two clusters: the tri-
saccharide repeating unit is a more specific epitope for the anti-19F
antibodies, whereas the lower, but still non-negligible, activity of
compound 1d could be ascribed to a predominant role of the manno-
samine unit into the trisaccharide. Moreover, and more importantly,
both glycocalixarenes 2a and 2b are more active than the single tri-
saccharide unit (52% of inhibition and IC50 = 2.1 × 10−2 mg/mL)
showing once more and more significantly that the presentation of the
epitope units on this type of scaffold increases the strength and the
3. Conclusions
Fully synthetic carbohydrate-based vaccines offer the advantage to
allow site-selective conjugation of saccharide epitopes [37,41,42] and
to incorporate into the nanosystems active mediators to increase vac-
cine efficacy. The possibility to accurately control the number and type
of vaccine active species ensures homogeneous composition, which is
important to induce highly reproducible biological properties with a
better safety profile. In this work, the calix[6]arene scaffold represents
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M. Giuliani, et al.
BioorganicChemistry93(2019)103305
a valuable platform for the simultaneous presentation of multiple co-
pies of minimized portions of the 19F CPS. In fact, this system, bearing
six trisaccharide repeating units linked through a thiourea group to the
macrocycle via an aminopropyl spacer, has been able to efficiently bind
to anti-19F antibodies with a significant improvement of the inhibition
activity compared to the one of the single repeating unit. The overall
efficiency is very high, especially considering the low number of ex-
posed saccharide antigens compared to the large number of repeating
units that are present in the natural polymer. Evidently, the structural
properties of the calixarene can properly present the exposed sac-
charide units in a tridimensional arrangement that is significantly si-
milar to that of the natural epitopes in CPS. Our data clearly suggest
that, in perspective, glycocalixarenes 2a and 2b, or even closely related
systems with slightly longer saccharide fragments, could be functiona-
lized with immunogenic peptides in order to elicit an antibacterial
specific immune response. A structurally well-defined and easily re-
producible multivalent glycocalixarene should avoid the use of complex
oligosaccharide species, which are generally required for im-
munogenicity. Calixarenes seem then to have great potential as carriers
for the development of fully synthetic carbohydrate-based vaccines, and
this work contributes towards this direction.
4.1.1. 5,11,17,23-Tetraisothiocyanate-25,26,27,28-tetramethoxycalix[4]
arene (4a)
In
a
two-neck round-bottom flask 5,11,17,23-tetraamino-
25,26,27,28-tetramethoxycalix[4]arene 3a (0.82 g, 1.22 mmol) was
dissolved in 65 mL of dry toluene under N2 atmosphere. Then thio-
phosgene (1.11 mL, 14.64 mmol, caution) and Et3N (4.07 mL,
29.28 mmol) were added and the mixture was allowed to react at room
temperature for 48 h. The solvent was removed under reduced pressure
and the crude was redissolved in dichloromethane. The organic phase
was washed with water (2 × 70 mL), 1 N HCl (70 mL) and then with a
saturated solution of NaCl (2 × 80 mL). The solvent was removed under
reduced pressure and the residue purified by column chromatography
(hexane/DCM 7/3, v/v) to afford a yellowish solid (0.19 g, 0.28 mmol,
23% yield). Mp: dec > 180 °C. 1H NMR (400 MHz, CDCl3) δ (ppm):
7.14 (s, 1.6H, ArH, partial cone), 7.02 (s, 1.6H, ArH, partial cone), 6.81
(s, 1.6H, ArH, partial cone), 6.65 (s, 1.6H, ArH, cone), 6.29 (s, 1.6H,
ArH, partial cone), 4.26 (d, J = 13.5 Hz, 0.8H, ArCHHaxAr, cone), 3.97
(d, J = 14.0 Hz, 1.6H, ArCHHaxAr, partial cone), 3.78 (s, 2.4H, OCH3,
cone), 3.74 (s, 2.4H, OCH3, partial cone), 3.68 (s, 4.8H, OCH3, partial
cone), 3.56 (s, 3.2H, ArCHHeqAr, partial cone), 3.13 (d, J = 13.5 Hz,
0.8H, ArCHHeqAr, cone), 3.06 (d, J = 14.0 Hz, 1.6H, ArCHHeqAr, par-
tial cone), 3.04 (s, 2.4H, OCH3, partial cone). 13C NMR (100 MHz,
CDCl3) δ (ppm): 156.9, 156.4 (CAr ipso), 135.6, 134.3, 132.5, 127.7,
126.5, 126.2, 125.7, 125.6 (CAr, NCS), 61.9, 61.4, 60.2, 59.8 (OCH3),
35.0 (ArCH2Ar, partial cone), 30.3, 30.1 (ArCH2Ar, cone and partial
cone). HRMS (ESI-TOF) m/z: calcd for C36H28N4O4S4Na [M + Na]+
731.0886, found 731.0861.
4. Experimental section
4.1. Chemical procedures
General information. All moisture sensitive reactions were carried
out under a nitrogen or argon atmosphere, using previously oven-dried
glassware. All dry solvents were prepared according to standard pro-
cedures, distilled before use and stored over 3 or 4 Å molecular sieves.
All other reagents were commercial samples and used without further
purification. Analytical TLC were performed using prepared plates of
silica gel (Merck 60F-254 on aluminum) and then, according to the
functional groups present on the molecules, revealed with UV light or
using staining reagents: FeCl3 (1% in H2O/MeOH 1:1), H2SO4 (5% in
EtOH), ninhydrin (5% in EtOH), basic solution of KMnO4 (0.75% in
H2O), molybdic acid solution (molybdatophosphorus acid and Ce(IV)
sulphate in 4% sulphuric acid). Reverse phase TLC were performed
using silica gel 60 RP-18F-254 on aluminium sheets. Merck silica gel 60
was used for flash chromatography (40–63 μm) and for preparative TLC
plates (10–12 μm). Sigma Aldrich C18 reverse phase silica gel was used
for flash chromatography. 1H NMR and 13C NMR spectra were recorded
on Bruker AV300 and Bruker AV400 spectrometers (observation of 1H
nucleus at 300 MHz and 400 MHz, respectively, and of 13C nucleus at
75 MHz and 100 MHz, respectively) and partially deuterated solvents
were used as internal standards to calculate the chemical shifts (δ va-
lues in ppm). All 13C NMR spectra were performed with proton de-
coupling. For 1H NMR spectra recorded in D2O at temperatures higher
that 25 °C the correction of chemical shifts was performed using the
expression δ = 5.060–0.0122 × T(°C) + (2.11 × 10−5) × T2(°C) [43]
to determine the resonance frequency of water protons. Electrospray
ionization (ESI) mass analyses were performed with a Waters single-
quadrupole spectrometer or with a LTQ Orbitrap XL spectrometer.
Melting points were determined on an Electrothermal apparatus in
capillaries sealed under N2 atmosphere. Microwave reactions were
performed using a CEM Discovery System reactor.
4.1.2. 5,11,17,23-Tetraisothiocyanate-25,26,27,28-tetrapropoxycalix[4]
arene 1,3-alternate (4c)
In
a
two-neck round-bottom flask 5,11,17,23-tetraamino-
25,26,27,28-tetrapropoxycalix[4]arene 1,3-alternate 3c (1.23 g,
1.96 mmol) was dissolved in 25 mL of dry toluene under N2 atmo-
sphere. Then thiophosgene (1.8 mL, 23.47 mmol, caution) and Et3N
(6.53 mL, 46.94 mmol) were added and the mixture was allowed to
react at room temperature for 48 h. The solvent was removed under
reduced pressure and the crude was redissolved in dichloromethane.
The organic phase was washed twice with distilled H2O (30 mL), once
with 1 N HCl (30 mL) and then twice with a saturated solution of NaCl
(40 mL). The solvent was removed under reduced pressure and the
desired compound was obtained in 33% yield as an off-white solid after
purification by column chromatography (cyclohexane/DCM 4/1 v/v)
(0.53 g, 0.65 mmol). Mp: 220–221 °C. 1H NMR (400 MHz, CDCl3) δ
(ppm): 6.90 (s, 8H, ArH), 3.65 (t, J = 7.2 Hz, 8H, OCH2), 3.47 (s, 8H,
ArCH2Ar), 1.87–1.78 (m, 8H, OCH2CH2CH3), 1.09 (t, J = 7.2 Hz, 12H,
CH2CH3). 13C NMR (100 MHz, CDCl3) δ (ppm): 155.3 (CAr ipso), 134.0
(NCS), 127.1 (CAr ortho), 125.0 (CAr para), 74.7 (OCH2), 35.1
(ArCH2Ar), 23.9 (OCH2CH2CH3), 10.7 (CH2CH3). HRMS (ESI-TOF) m/z:
calcd for C44H44N4O4S4Na [M + Na]+ 843.2138, found 843.2156.
4.1.3. 5,11,17,23,29,35-Hexaisothiocyanate-37,38,39,40,41,42-
hexamethoxycalix[6]arene (4d)
In a round-bottom flask 3d (0.15 g, 0.185 mmol) was dissolved in
DCM (5 mL) under N2 atmosphere. Then thiophosgene (254 µL,
3.33 mmol, caution), BaCO3 (0.657 g, 3.33 mmol) and H2O (3 mL) were
added with the remaining amount of DCM (5 mL). The mixture was was
allowes to react at rt for 48 h and then diluted with DCM/H2O. The
organic phase was separated from the aqueous layer and evaporated
under reduced pressure. The crude thus obtained was purified by flash
chromatography (hexane/EtOAc 8/2, v/v) and crystallized with CH3CN
to yield the pure product as a white solid (39 mg, 0.037 mmol, 20%).
Mp: dec > 200 °C. 1H NMR (300 MHz, CDCl3) δ (ppm): 6.74 (s, 12H,
ArH), 3.89 (s, 12H, ArCH2Ar), 3.54 (s, 18H, OCH3). 13C NMR (75 MHz,
CDCl3) δ(ppm): 155.3 (CAr ipso), 135.2 (CAr ortho), 133.9 (NCS), 126.6
(CAr para), 126.2 (CAr meta), 61.0 (OCH3), 30.2 (ArCH2Ar). HRMS (ESI-
TOF) m/z: calcd for C54H42N6O6S6Na [(4d + Na)+] 1085.1388, found
5,11,17,23-Tetraamino-25,26,27,28-tetramethoxycalix[4]arene
[25],
5,11,17,23-tetraamino-25,26,27,28-tetrapropoxycalix[4]arene
cone [27], 5,11,17,23-tetraamino-25,26,27,28-tetrapropoxycalix[4]
arene 1,3-alternate [25], 5,11,17,23,29,35-hexaamino-37,38,39,40,
41,42-hexamethoxycalix[6]arene
[26],
4-propoxyaniline
[44],
5,11,17,23-tetraisohiocyanate-25,26,27,28-tetrapropoxycalix[4]arene
cone [28], N-(benzyloxycarbonyl)aminopropyl 2-acetamido-3,4,6-tri-O-
benzyl-2-deoxy-β-D-mannopyranoside [29] were prepared according to
literature procedures.
7

M. Giuliani, et al.
BioorganicChemistry93(2019)103305
4.1.7. N-(Boc)aminopropyl 2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
1085.1414 (80%); calcd for C54H42N6O6S6K [M + K]+ 1101.1127,
found 1101.1151 (100%).
mannopyranoside (10)
N-(Boc)aminopropyl
2-acetamido-2-deoxy-β-D-mannopyranoside
4.1.4. Aminopropyl
mannopyranoside (7)
2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-
(9) (0.18 g, 0.47 mmol) was dissolved in pyridine (6 mL) and then
acetic anhydride was added (740 μL, 7.91 mmol). The reaction mixture
was stirred at room temperature for 1 h, then the solvent removed
under reduced pressure. The pure compound was obtained after pur-
ification via flash column chromatography (EtOAc/hexane 4/1, v/v) as
a white foam in quantitative yield (0.23 g, 0.46 mmol). 1H NMR
(300 MHz, CDCl3) δ (ppm): 5.94 (br, 1H, NHAc), 5.06 (t, J = 9.2 Hz,
1H, H4), 4.97 (dd, J3,2 = 4.0, J3,4 = 9.2 Hz, 1H, H3), 4.67–4.60 (m, 3H,
H1, H2, NHBoc), 4.27 (dd, J6a,5 = 6.0, J6a,6b = 15.0 Hz, 1H, H6a), 4.12
To a solution of N-(benzyloxycarbonyl)aminopropyl 2-acetamido-
3,4,6-tri-O-benzyl-2-deoxy-β-D-mannopyranoside
(6)
(0.54 g,
0.79 mmol) in a 4:1 mixture of AcOEt/EtOH (10 mL), Pd/C (10%) was
added and the suspension was shaken in a Parr hydrogenator for 90 min
under 1.5 bar of H2 at room temperature, after which the catalyst was
filtered off and the filtrate evaporated under vacuum, to give the de-
sired product as an orange oil (0.373 g, 0.68 mmol, 86%). 1H NMR
(300 MHz, CDCl3)
δ
(ppm): 7.38–7.13 (m, 15H, ArH), 6.20 (d,
(dd, J6b,5 = 6.0, J6b,6a = 15.0 Hz, 1H, H6b), 3.90–3.83 (m, 1H,
J = 9.9 Hz, 1H, NHAc), 4.90–4.79 (m, 3H, H2, 2 × CHHPh), 4.57 (d,
J = 11.9 Hz, 1H, CHHPh), 4.52–4.42 (m, 4H, H1, 3 × CHHPh),
3.93–3.82 (m, 1H, OCHHCH2), 3.73 (br, 2H, H6a,b), 3.68–3.62 (m, 2H,
H4, H5), 3.61–3.51 (m, 1H, OCHHCH2), 3.46–3.38 (m, 1H, H3),
2.83–2.74 (m, 2H, CH2CH2NH2), 2.03 (s, 3H, CH3CO), 1.77–1.69 (m,
2H, CH2CH2CH2). 13C NMR (75 MHz, CDCl3) δ (ppm): 171.9 (COCH3),
138.2, 137.9, 137.7, 128.4, 128.33, 128.31, 127.9, 127.85, 127.8,
127.7 (CAr), 99.5 (C1), 79.8 (C5), 75.0 (C3), 74.8 (CH2Ph), 74.1 (C4),
73.3 (CH2Ph), 71.1 (CH2Ph), 68.9 (C6), 67.6 (OCH2), 49.2 (C2), 38.5
(CH2NH2), 27.9 (CH2CH2CH2), 23.6 (COCH3). ESI-MS m/z: calcd for
OCHHCH2), 3.68–3.58 (m, 1H, H5), 3.55 (br, 1H, OCHHCH2),
3.22–3.15 (m, 2H, CH2CH2NHBoc), 2.09 (s, 3H, CH3CO), 2.06 (s, 3H,
CH3CO), 2.04 (s, 3H, CH3CO), 2.00 (s, 3H, NHCOCH3), 1.77–1.68 (m,
2H, CH2CH2CH2), 1.42 (s, 9H, C(CH3)3). 13C NMR (75 MHz, CDCl3) δ
(ppm): 171.4, 170.5, 170.2, 169.7 (COCH3), 156.1 (CO(CH3)3), 98.7
(C1), 78.9 (C(CH3), 72.2 (C5), 71.4 (C3), 67.0 (OCH2), 66.2 (C4), 62.6
(C6), 49.9 (C2), 37.3 (CH2NH2), 29.5 (CH2CH2CH2), 28.3 (C(CH3)3),
22.9, 20.6, 20.5 (COCH3). ESI-MS m/z: calcd for C22H36N2O11Na
[M + Na]+ 527.2, found 527.4.
C
32H41N2O6 [M + H]+ 549.3, found 549.0.
4.1.8. Aminopropyl
mannopyranoside (11):
N-(Boc)aminopropyl
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
4.1.5. N-(Boc)aminopropyl 2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-
mannopyranoside (8)
2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
mannopyranoside (10) (0.24 g, 0.47 mmol) was dissolved in dry DCM
(15 mL), then trifluoroacetic acid (1.27 mL, 16.45 mmol) was added
dropwise. The reaction was allowed to stir at room temperature for 1 h,
then it was quenched by the addition of Et3N. The solvent was evapo-
rated under reduced pressure and the desired compound was obtained
as a yellow-orange oil in quantitative yield (0.19 g, 0.46 mmol). 1H
NMR (300 MHz, CDCl3) δ (ppm): 6.85 (d, J = 9.0 Hz, 1H, NHAc), 5.08
(t, J = 9.6 Hz, 1H, H4), 4.99 (dd, J3,2 = 4.6, J3,4 = 9.6 Hz, 1H, H3),
4.78–4.70 (m, 2H, H1, H2), 4.24 (dd, J6a,5 = 6.0, J6a,6b = 12.4 Hz, 1H,
Aminopropyl 2-acetamido-3,4,6-tri-O-benzyl-2-deoxy-β-D-manno-
pyranoside (7) (0.34 g, 0.63 mmol) was dissolved in MeOH (8 mL), then
Et3N (348 μL, 2.5 mmol) and Boc2O (0.68 g, 3.13 mmol) were subse-
quently added. The reaction was allowed to react for 2 h, then the
solvent was removed under vacuum. The pure product was obtained as
a pale oil in quantitative yield (0.40 g, 0.61 mmol). 1H NMR (300 MHz,
CDCl3) δ (ppm): 7.26–7.03 (m, 15H, ArH), 6.18 (d, J = 9.4 Hz, 1H,
NHAc), 5.26 (br, 1H, NHBoc), 4.78–4.68 (m, 3H, H2, 2 × CHHPh), 4.44
(d, J = 12.0 Hz, 1H, CHHPh), 4.40–4.31 (m, 4H, 3 × CHHPh, H1),
3.75–3.66 (m, 1H, OCHHCH2), 3.64–3.58 (m, 2H, H6a,b), 3.56–3.51 (m,
2H, H4, H5), 3.50–3.39 (m, 1H, OCHHCH2), 3.37–3.29 (m, 1H, H3),
3.11–3.05 (m, 2H, CH2CH2NHBoc), 1.89 (s, 3H, COCH3), 1.66–1.54 (m,
2H, CH2CH2CH2), 1.31 (s, 9H, C(CH3)3). 13C NMR (100 MHz, CDCl3) δ
(ppm): 170.8 (COCH3), 156.1 (CO(CH3)3), 146.7, 138.2, 137.9, 137.8,
128.4, 128.3, 128.2, 127.8, 127.78, 127.73, 127.6 (CAr), 99.5 (C1), 85.0
(C(CH3), 80.3 (C5), 74.9 (C3), 74.7 (CH2Ph), 74.0 (C4), 73.3 (CH2Ph),
71.0 (CH2Ph), 68.8 (C6), 67.1 (OCH2), 49.2 (C2), 37.6 (CH2NH2), 29.7
(CH2CH2CH2), 27.2 (C(CH3)3), 23.3 (COCH3). ESI-MS m/z: calcd for
H
6a), 4.13 (dd, J6b,5 = 2.4, J6b,6a = 12.0 Hz, 1H, H6b), 4.01–3.92 (m,
1H, OCHHCH2), 3.81–3.74 (m, 2H, H5, OCHHCH2), 3.71–3.64 (m, 2H,
CH2CH2NH2), 2.07 (s, 3H, CH3CO), 2.02 (s, 6H, CH3CO), 1.99 (s, 3H,
NHCOCH3), 2.00–1.96 (m, 2H, CH2CH2CH2). 13C NMR (100 MHz,
CDCl3) δ (ppm): 161.5, 161.2, 160.4 (COCH3), 98.2 (C1), 72.3 (C5),
71.3 (C3), 66.6 (OCH2), 66.0 (C4), 62.4 (C6), 49.5 (C2), 37.4 (CH2NH2),
26.3 (CH2CH2CH2), 22.4, 20.4 (COCH3). HRMS (ESI-TOF) m/z: calcd
for C17H28N2O9Na [M + Na]+ 427.1687, found 427.1658.
4.1.9. N-(tertbutoxycarbonyl)-3-amminopropyl (2-acetamido-3,4,6-tri-O-
acetyl-2-deoxy-β-D-mannopyranosyl)-(1 → 4)-(2,3,6-tri-O-acetyl-α-D-
glucopyranosyl)-(1 → 2)-3,4-di-O-acetyl-L-rhamnopyranoside (15)
C
37H48N2O8Na [M + Na]+ 671.3, found 671.1.
4.1.6. N-(Boc)aminopropyl 2-acetamido-2-deoxy-β-D-mannopyranoside
(9):
Compound 16 [37] (0.19 g, 0.147 mmol) and N-(benzylox-
ycarbonyl)-3-amminopropyl (0.12 g, 0.586 mmol), as previously de-
scribed [38], were dissolved in dry CH2Cl2 (3 mL) and activated powder
molecular sieves 4 Å (0.10 g) were added. The suspension was stirred
under Ar atmosphere at room temperature for 15 min, then it was
cooled to 0 °C and TMSOTf 0.1 M in dry CH2Cl2 (0.29 mL, 0.029 mmol)
was added. After 15 min, the reaction was quenched by the addition of
TEA, filtered over a Celite pad and the solvent evaporated under re-
duced pressure. Purification of the crude through flash chromatography
(Hexane/Ethyl Acetate 6:4) afforded 0.48 g of the less polar α-anomer,
0.80 g of a mixture of the two anomers, and 0.63 g of the β-anomer (17
overall yield: 96%). 17α and 17β were reacted separately in the next
step. To a solution under Argon of compound SP3 in MeOH (0.01 M),
Boc2O (3.5 eq.) and then Pd(OH)2/C (1/1, w/wsubstrate) were added.
The mixture was stirred under hydrogen atmosphere for 3 h, and
checked by TLC (hexane/AcOEt, 1/1) to confirm that the Z-amino
protecting group has been exchanged with BOC. Then, one drop of HCl
1 N was added, and the reaction was stirred again under hydrogen at-
mosphere overnight. TLC (DCM/MeOH, 75/25) control showed that the
In a MW sealed vessel N-(Boc)aminopropyl 2-acetamido-3,4,6-tri-O-
benzyl-2-deoxy-β-D-mannopyranoside (8) (0.60 g, 0.93 mmol) was dis-
solved in a MeOH/H2O mixture (5 mL, 1/1 v/v) and Pd/C (10%) in
catalytic amount and then NH4COOH (0.23 g, 3.7 mmol) were added.
The mixture was heated at 60 °C under microwave irradiation (150 W)
for 1.5 h. The catalyst was filtered off and the solvent removed under
reduced pressure, to give the desired product as white foam (0.30 g,
0.79 mmol, 85%) 1H NMR (400 MHz, MeOD) δ (ppm): 4.65 (s, 1H, H1),
4.47 (d, J = 3.2 Hz, 1H, H2), 3.87 (br, 3H, H6a,b, OCHHCH2), 3.68 (dd,
J3,2 = 4.0, J3,4 = 9.5 Hz, 1H, H3), 3.63–3.57 (m, 1H, OCHHCH2), 3.53
(t, J = 9.5 Hz, 1H, H4), 3.31–3.25 (m, 1H, H5), 3.13 (t, J = 6.4 Hz, 2H,
CH2CH2NHBoc), 2.05 (s, 3H, COCH3), 1.73 (t, J = 6.0 Hz, 2H,
CH2CH2CH2), 1.45 (s, 9H, C(CH3)3). 13C NMR (100 MHz, MeOD) δ
(ppm): 173.4 (COCH3), 157.1 (CO(CH3)3), 99.4 (C1), 78.5 (C(CH3)3),
76.9 (C5), 73.0 (C3), 66.9 (C4), 66.3 (OCH2), 60.5 (C6), 53.5 (C2), 36.9
(CH2NH2), 29.5 (CH2CH2CH2), 27.4 (C(CH3)3), 21.4 (COCH3). ESI-MS
m/z: calcd for C16H32N2O8Na [M + Na]+ 401.2, found 401.3.
8

M. Giuliani, et al.
BioorganicChemistry93(2019)103305
reaction was completed, then few drops of dry Py were added, and the
reaction was filtered over filter paper. After evaporation of the solvent,
the crude was dissolved in dry Py (0.03 M), and acetic anhydride was
added (Ac2O/Py, 1/2) together with a catalytic amount of DMAP. The
reaction was stirred at room temperature for 19 h, diluted with MeOH,
and then the solvent evaporated. Purification of the crude by flash
chromatography (hexane/EtOAc, 1:9) gave compound 15 as an amor-
phous white solid.
2.01 (s, 12H, CH3CO), 1.96–1.90 (m, 16H, OCH2CH2CH3, CH2CH2CH2),
0.94 (t, J = 7.5 Hz, 12H, CH2CH3). 13C NMR (75 MHz, CDCl3) δ (ppm):
180.4 (CS), 172.1 (NHCOCH3), 170.7 (COCH3), 170.5 (COCH3), 169.8
(COCH3), 154.5 (CAr ipso), 136.2 (CAr ortho), 132.1 (CAr para), 124.8
(CAr meta), 99.1 (C1), 77.3 (OCH2CH2CH3) 72.4 (C5), 71.4 (C4), 68.7
(OCH2CH2CH2), 66.0 (C3), 62.5 (C6), 50.3 (C2), 43.0 (CH2NHCS), 30.9
(ArCH2Ar), 29.9 (CH2CH2CH2), 28.9 (OCH2CH2CH3), 23.4 (COCH3),
23.2 (COCH3), 20.8 (COCH3), 20.7 (COCH3), 10.2 (CH2CH2CH3). HRMS
15α: 0.32 g of 15α were obtained starting from 0.48 g of 17α (88%
yield). [α]D20 = +22.3 (c = 1 in chloroform). 1H NMR (CDCl3):
(ESI-TOF) m/z: calcd for
C
112H156N12O40S4Na2 [M + 2Na]2+
1241.4590, found 1241.4586.
δ = 5.90
(d,
1H,
J
2′,NH = 7.2 Hz,
NH),
5.39
(t,
1H,
J2′,3′ = J3′,4′ = 9.4 Hz, H3′), 5.29–5.20 (m, 2H, H1′,H3), 5.15–5.03 (m,
2H, H4,H4′), 4.92 (dd, 1H, J2″,3″ = 3.8 Hz, J3″,4″ = 10.0 Hz, H3″),
4.1.11. 5,11,17,23,29,35-Hexakis-N-[3-(2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-β-D-mannopiranosyloxy)-propyl-thioureido]-37,38,39,40,41,42-
hexamethoxycalix[6]arene (5d)
4.76–4.56 (m, 5H, H1, H1″
, H2′, H2″ and NH), 4.36 (dd, 1H,
J
5″,6a″ = 5.3 Hz, J6a″,6b″ = 12.5 Hz, H6a″), 4.30–4.22 (m, 2H, 2H6′),
To a solution of compound 4d (0.0416 g, 0.0392 mmol) in DCM
(8 mL) sugar 11 (0.111 g, 0.274 mmol) and NEt3 (273 μL, 1.96 mmol)
were added and the mixture stirred at rt for 72 h. The solvent was re-
moved under reduced pressure and the crude purified via flash column
chromatography and preparative TLC (DCM/MeOH 94/6, v/v) to afford
5d as a yellowish solid (0.0317 mg, 0.0091 mmol, 23%). 1H NMR
(400 MHz, CDCl3) δ (ppm): 8.66 (br, 6H, NHCS), 6.92 (br, 12H, ArH),
6.57 (s, 12H, CSNHCH2, NHAc), 5.18 (br, 6H, H4), 5.03 (br, 6H, H3),
4.77 (br, 6H, H2), 4.71 (br, 6H, H1), 4.29 (br, 6H, H6a), 4.14 (br, 6H,
4.15–4.03 (m, 2H, H5′, H6a″), 4.02–3.98 (m, 1H, H2), 3.84–3.69 (m, 3H,
Ha, H4′, H5), 3.67–3.59 (m, 1H, H5″), 3.49–3.40 (m, 1H, Ha′), 3.28–3.17
(m, 2H, 2Hc), 2.21–1.98 (9 s, 27H, 9 CH3), 1.85–1.75 (m, 2H, 2Hb), 1.46
(s ,9H, (CH3)3CO), 1.20 (d, 3H, J5,6 = 6.0 Hz ,3H6). 13C NMR (CDCl3):
δ = 171.9–169.6 (9C, C]O), 155.9 (C]O), 98.2 (C1″), 96.4 (C1), 93.3
(C1′), 75.5 (C4′), 73.7 (C2), 72.7 (C5″), 71.9 (C3″). 71.3 (C4), 71.0 (C2′),
70.7 (C3′), 69.9 (C3), 68.2 (C5′), 66.6 (C5), 65.9 (Ca), 65.7 (C4″), 62.2
(C6″), 62.0 (C6′), 50.8 (C2″), 37.83 (Cc), 31.6 (Me3C), 29.8 (Cb), 28.4
(Me3C), 21.2–20.6 (9C, CH3CO), 17.7 (C6). MS (ESI) m/z (%): 1045.3
H
6b), 4.03–3.17 (overlapped, 60H, OCHHCH2CH2, CHHNHCS, H5,
(1 0 0) [M + Na]+
.
ArCH2Ar, OCH3), 2.10 (s, 18H, CH3CO), 2.07 (s, 36H, CH3CO), 2.03 (s,
18H, CH3CO), 1.81 (br, 12H, CH2CH2CH2). 13C NMR (100 MHz, CDCl3)
δ (ppm): 180.6 (CS), 172.1 (COCH3), 170.7 (COCH3), 170.3 (COCH3),
169.7 (COCH3), 154.2 (CAr ipso), 134.8 (br, CAr ortho, CAr para), 125.3
(CAr meta), 99.9 (C1), 72.5 (C5), 71.3 (C3), 68.6 (OCH2CH2CH2), 66.1
(C4), 62.6 (C6), 61.0 (OCH3), 50.4 (C2), 42.9 (CH2NHCS), 29.7
(ArCH2Ar), 29.0 (CH2CH2CH2), 23.4 (NHCOCH3), 20.9 (COCH3), 20.8
(COCH3), 20.7 (COCH3). HRMS (ESI-TOF) m/z: calcd for
C156H210N18O60S6Na2 [M + 2Na]2+ 1766.6028, found 1766.6047.
15β: 0.42 g of 15β were obtained starting from 0.63 g of 17β (85%
yield). [α]D20 = +55.4 (c = 1 in chloroform). 1H NMR (CDCl3):
δ = 5.94 (d, 1H, J2′,NH = 7.2 Hz, NH), 5.62 (br d, 1H, J1′,2′ = 3.9 Hz,
H1′), 5.41 (t, 1H, J2′,3′ = J3′,4′ = 9.5 Hz, H3′), 5.13–5.04 (m, 2H, H4,
H
4″), 4.98 (dd, 1H, J2,3 = 3.0 Hz, J3,4 = 10.0 Hz, H3), 4.92 (dd, 1H,
J
2″,3″ = 4.0 Hz, J3″,4″ = 10.0 Hz, H3″), 4.75 (dd, 1H, J1′,2′ = 3.9 Hz,
J2′,3′ = 10.2 Hz, H2′), 4.69 (br s, 1H, H1″), 4.63–4.58 (m, 1H, H2″), 4.47
(s, 1H, H1), 4.36 (dd, 1H, J5″,6a″ = 5.4 Hz, J6a″,6b″= 12.4 Hz, H6″), 4.25
(dd, 1H, J5′,6a′ = 5.0 Hz, J6a′,6b′ = 11.8 Hz, H6a′), 4.21–4.12 (m, 3H, H2,
H5′, H6b′), 4.06 (dd, 1H, J5″,6a″ = 2.2 Hz, J6a″,6b″ = 12.4 Hz, H6a″),
3.87–3.79 (m, 1H, Ha), 3.69 (t, 1H, J3′,4′ = J4′,5′ = 9.5 Hz, H4′),
3.65–3.61 (m, 1H, H5″), 3.50–3.41 (m, 2H, Ha, H5), 3.29–3.19 (m, 1H,
Hc), 3.11–3.02 (m, 1H, Hc), 2.17–2.00 (9 s, 27H, 9 CH3), 1.82–1.66 (m,
2H, 2Hb), 1.45 (s ,9H, (CH3)3CO), 1.26 (d, 3H, J5,6 = 6.0 Hz, 3H6). 13C
NMR (CDCl3): δ = 172.1–169.5 (9C, C]O), 156.1 (C]O), 100.9 (C1),
98.2 (C1″), 94.3 (C1′), 75.7 (C4′), 72.7 (C5″), 72.3 (C2), 71.9 (2C, C3, C3″).
71.1 (C4), 70.7 (C5), 70.5 (C3′), 70.3 (C2′), 67.8 (C5′), 67.6 (Ca), 65.8
(C4″), 62.2 (2C, C6, C6″), 50.8 (C2″), 37.3 (Cc), 31.6 (Me3C), 29.4 (Cb),
28.4 (Me3C), 23.1–20.6 (9C, CH3CO), 17.7 (C6). MS (ESI) m/z (%):
4.1.12. N-[3-(2-acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-
mannopiranosyloxy)-propyl-thioureido]-4-propoxybenzene (14)
To a solution of 13 (0.03 g, 0.155 mmol) in dry DCM (4 mL) under
N2 atmosphere, sugar 11 (0.0943 g, 0.233 mmol) and NEt3 (324 μL,
2.33 mmol) were added and the mixture stirred for 12 h at rt. The
solvent was removed under reduced pressure and the crude purified by
flash column chromatography (DCM/MeOH 20/1, v/v) yielding 14 as
an off-white solid (0.0490 g, 0.0821 mmol, 53%). Mp: 90–91 °C. 1H
NMR (400 MHz, CDCl3) δ (ppm): 8.25 (br, 1H, NHCS), 7.18 (d,
J = 8.0 Hz, 2H, ArH), 6.86 (d, J = 8.0 Hz, 2H, ArH), 6.23 (br, 1H,
CH2NHCS), 6.05 (d, J = 7.6 Hz, 1H, NHAc), 5.08 (t, J = 9.6 Hz, 1H,
H4), 4.99 (dd, J3,2 = 4.0 Hz, J3,4 = 10.0 Hz, 1H, H3), 4.72 (d,
J = 4.0 Hz, 1H, H2), 4.63 (s, 1H, H1), 4.26 (dd, J6a,5 = 5.2 Hz,
1045.3 (1 0 0) [M + Na]+
.
4.1.10. 5,11,17,23-Tetrakis-N-[3-(2-acetamido-3,4,6-tri-O-acetyl-2-
deoxy-β-D-mannopiranosyloxy)-propyl-thioureido]-25,26,27,28-
tetrapropoxycalix[4]arene cone (5b)
J
6a,6b = 12.4 Hz, 1H, H6a), 4.08 (d, J = 12.0 Hz, 1H, H6b), 3.89–3.86
(m, 3H, OCHHCH2CH2, OCH2CH2CH3), 3.76 (br, 1H, CHHNHCS),
3.67–3.56 (m, 2H, H5, OCHHCH2CH2), 3.53 (br, 1H, CHHNHCS), 2.06
(s, 3H, COCH3), 2.03 (s, 3H, COCH3), 2.02(s, 3H, COCH3), 1.97 (s, 3H,
COCH3), 1.75 (m, 4H, OCH2CH2CH3, CH2CH2CH2), 0.98 (t, J = 7.2 Hz,
3H, CH2CH3). 13C NMR (100 MHz, CDCl3) δ (ppm): 181.7 (CS), 171.7
(NHCOCH3), 170.6 (COCH3), 170.1 (COCH3), 169.7 (COCH3), 157.9
(CAr ipso), 129.8 (CAr ortho), 127.3 (CAr para), 127.3, 125.6 (CAr meta),
99.1 (C1), 72.5 (C5), 71.0 (C3), 69.8 (OCH2CH2CH3), 68.5
(OCH2CH2CH2), 66.0 (C4), 62.4 (C6), 50.3 (C2), 43.1 (CH2NHCS), 29.7
(CH2CH2CH2), 23.4 (COCH3), 20.8 (OCH2CH2CH3), 20.8, 20.7
(COCH3), 10.5 (OCH2CH2CH3). HRMS (ESI-TOF) m/z: calcd for
In
a
two-neck round-bottom flask calixarene 4b (0.05 g,
0.0609 mmol) was dissolved in dry DCM (6 mL) under N2 atmosphere,
then sugar 11 (0.123 g, 0.305 mmol) and Et3N (255 μL, 1.83 mmol)
were added. The mixture was stirred at rt for 24 h, after which half
equivalents of sugar and Et3N were added and the mixture stirred for
additional 16 h. The solvent was removed under reduced pressure and
the crude was purified via flash column chromatography (DCM/MeOH
20/1, v/v) yieldig cone derivative 5b in 37% yield as a white solid
(0.0547 g, 0.0225 mmol). Mp: dec > 130 °C. 1H NMR (300 MHz,
CDCl3) δ (ppm): 8.36 (br, 4H, NHCS), 6.65 (br, 8H, ArH), 6.38 (br, 8H,
CSNHCH2, NHAc), 5.15–5.09 (m, 8H, H3, H4), 4.78–4.74 (m, 8H, H2,
H1), 4.41 (d, J = 13.2 Hz, 4H, ArCHHaxAr), 4.27 (dd, J6a,5 = 5.6,
J6a,6b = 12.0 Hz, 4H, H6a), 4.13 (dd, J6b,5 = 2.4, J6b,6a = 12.0 Hz, 4H,
C
27H39N3O10SNa [M + Na]+ 620.2254, found 620.2261.
4.1.13. 5,11,17,23,29,35-Hexakis-N-[3-(2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-β-D-mannopyranosyl-(1 → 4)-2,3,6-tri-O-acetyl-α-D-
H
6b), 3.97–3.96 (m, 4H, OCHHCH2CH2), 3.84 (br, 12H, OCH2CH2CH3,
CHHNHCS), 3.70–3.69 (m, 4H, H5), 3.57 (br s, 4H, OCHHCH2CH2),
3.49–3.48 (m, 4H, CHHNHCS), 3.14 (d, J = 13.2 Hz, 4H, ArCHHeqAr),
2.07 (s, 12H, CH3CO), 2.05 (s, 12H, CH3CO), 2.03 (s, 12H, CH3CO),
glucopyranosyl-(1 → 2)-3,4-di-O-acetyl-α-L-rhamnopyranosyloxy)-propyl-
thioureido]-37,38,39,40,41,42-hexamethoxycalix[6]arene (18a)
15α (22 mg, 0.0215 mmol) was dissolved in dry DCM (4 mL) under
9

M. Giuliani, et al.
BioorganicChemistry93(2019)103305
Ar atmosphere. The temperature was decreased to 0 °C and 0.5 mL of
trifluoroacetic were added. The reaction proceeded at room tempera-
ture for 1 h, after which the TLC showed the complete consumption of
the reagent, thus the solvent was evaporated at reduced pressure. The
obtained deprotected product was dissolved in dry DCM (2 mL) and
5,11,17,23,29,35-hexaisothiocyanate-37,38,39,40,41,42-hexamethox-
ycalix[6]arene (4d) (2.5 mg, 0.00239 mmol) and NEt3 (17 μL,
0.120 mmol) were added under Ar atmopshere. The mixture was stirred
for 48 h at room temperature, after which the reaction was quenched by
removal of the solvent at reduced pressure. The desired product was
obtained after column chromatography (DCM/MeOH 96:4 v/v) as a
white solid in 86% yield (13.5 mg, 0.00205 mmol). 1H NMR (400 MHz,
CDCl3) δ (ppm): 7.01 (br, 12H, ArH), 5.91 (br, 6H, NHAc), 5.38 (t,
4.1.15.1. 5,11,17,23-Tetrakis-N-[3-(2-acetamido-2-deoxy-β-D-
mannopiranosyloxy)-propyl-thioureido]-25,26,27,28-tetramethoxycalix[4]
arene (1a). The crude was first purified by C18 reverse phase column
chromatography (MeOH/H2O 5.5/4.5, v/v), and then by size exclusion
chromatography on Sephadex G-25 as stationary phase (MeOH/H2O
1.5/8.5, v/v). The final purification step was performed via standard
flash chromatography (i-PrOH/H2O/Et3N 8/2/0.5, v/v/v) to afford the
mobile calix[4]arene 1a in 40% yield (0.037 g, 0.0191 mmol). Mp:
dec > 140 °C. 1H NMR (400 MHz, D2O, 80 °C) δ (ppm): 6.98 (br, 8H,
ArH), 4.69 (br, 4H, H1), 4.43 (br, 4H, H2), 3.88–3.22 (m, 48H, H3, H4,
H5, H6a, H6b, OCH2CH2, CH2NHCS, OCH3), 1.97 (s, 12H, NHCOCH3),
1.80–1.74 (m, 8H, CH2CH2CH2). 13C NMR (100 MHz, MeOD, 55 °C) δ
(ppm): 180.6 (CS), 173.6 (NHCOCH3), 155.7 (CAr ipso), 135.32 (CAr
ortho), 132.4 (CAr para), 124.4 (CAr), 99.5 (C1), 77.0 (C5), 73.0 (C3),
67.2 (C4, OCH2CH2CH2), 61.0 (C6, OCH3), 53.6 (C2), 42.1 (CH2NHCS,
ArCH2Ar), 29.2 (CH2CH2CH2), 22.2 (NHCOCH3). HRMS (ESI-TOF) m/z:
J
3′,4′ = J3′,2′ = 9.5 Hz, 6H, H3′), 5.24 (br, 12H, H3, H1′), 5.14–5.03 (m,
12H, H4, H4′), 4.93 (br, 6H, H3″), 4.77–4.67 (m, 12H, H2′, H1″), 4.62 (br,
12H, H2″, H1), 4.37 (dd, J6a,5 = 5.1, J6a,6b = 12.5 Hz, 6H, H6a″), 4.25
(br, 12H, H6′), 4.12–4.03 (m, 12H, H5′, H6″), 4.00 (br, 6H, H2),
calcd for
1843.6812.
C
80H116N12O28S4Na [M + Na]+ 1843.6803, found
3.84–3.70 (m, 18H, H5, H4′, OCHHCH2CH2), 3.67 (br, 12H, H5″
,
OCHHCH2CH2), 3.45 (br, 12H, CH2NHCS), 2.16 (s, 18H, CH3CO), 2.13
(s, 18H, CH3CO), 2.12 (s, 18H, CH3CO), 2.11 (s, 18H, CH3CO), 2.09 (s,
18H, CH3CO), 2.06 (s, 36H, CH3CO), 2.04 (s, 18H, CH3CO), 2.02 (s,
18H, CH3CO), 1.88 (br, 12H, CH2CH2CH2), 1.19 (d, J = 5.8 Hz, 18H,
H6). HRMS (ESI-TOF) m/z: calcd for C288H390N18O144S6Na4
[M + 4Na]4+ 1672.0388, found 1672.0396.
4.1.15.2. 5,11,17,23-Tetrakis-N-[3-(2-acetamido-2-deoxy-β-D-
mannopiranosyloxy)-propyl-thioureido]-25,26,27,28-tetrapropoxycalix[4]
arene cone (1b). The pure product 1b was obtained after purification by
C18 reverse phase column chromatography (MeOH/H2O 4/1, v/v) in
78% yield as a white solid (0.034 g, 0.0176 mmol). Mp: dec > 178 °C.
1H NMR (400 MHz, MeOD) δ (ppm): 6.71 (br, 8H ArH), 4.71 (s, 4H,
H1), 4.53 (s, 4H, H2), 4.47 (d, J = 13.2 Hz, 4H, ArCHHaxAr), 3.98–3.86
(m, 24H, H6a,b, OCHHCH2CH2, OCH2CH2CH3), 3.71–3.63 (m, 16H, H3,
OCHHCH2CH2, CHHNHCS), 3.58 (t, J = 9.6 Hz, 4H, H4), 3.29–3-27 (m,
4H, H5), 3.18 (d, J = 13.2 Hz, 4H, ArCHHeqAr), 2.08 (s, 12H,
NHCOCH3), 2.02–1.96 (m, 8H, OCH2CH2CH3), 1.86 (br, 8H,
CH2CH2CH2), 1.06 (t, J = 6.8 Hz, 12H, OCH2CH2CH3). 13C NMR
(100 MHz, MeOD) δ (ppm): 179 (CS), 173.6 (NHCOCH3), 154.0 (CAr
ipso), 135.32 (CAr ortho), 132.2 (CAr para), 123.9 (CAr), 99.5 (C1), 76.9
(C5), 76.8 (C6), 72.8 (C3), 67.3 (OCH2CH2CH2), 66.9 (C4), 60.7
(OCH2CH2CH3), 53.6 (C2), 47.7 (CH2NHCS), 42.2 (C2), 30.5
(ArCH2Ar), 28.7 (CH2CH2CH2), 23.1 (OCH2CH2CH3), 21.7
(NHCOCH3), 9.5 (OCH2CH2CH3). HRMS (ESI-TOF) m/z: calcd for
4.1.14. 5,11,17,23,29,35-Hexakis-N-[3-(2-acetamido-3,4,6-tri-O-acetyl-
2-deoxy-β-D-mannopyranosyl-(1 → 4)-2,3,6-tri-O-acetyl-α-D-
glucopyranosyl-(1 → 2)-3,4-di-O-acetyl-β-L-rhamnopyranosyloxy)-propyl-
thioureido]-37,38,39,40,41,42-hexamethoxycalix[6]arene (6b)
15β (29 mg, 0.0283 mmol) was dissolved in dry DCM (4 mL) under
Ar atmosphere. The temperature was decreased to 0 °C and 0.5 mL of
trifluoroacetic were added dropwise. The reaction proceeded at room
temperature for 1 h, after which the TLC showed the complete con-
sumption of the reagent, thus the solvent was evaporated at reduced
pressure. The crude was then dissolved in dry DCM (3 mL) and
5,11,17,23,29,35-hexaisothiocyanate-37,38,39,40,41,42-hexamethoxy-
calix[6]arene (4d) (3.3 mg, 0.00314 mmol) and NEt3 (21 μL,
0.157 mmol) were added under Ar atmopshere. The mixture was stirred
for 48 h at room temperature, after which the reaction was quenched by
removal of the solvent at reduced pressure. The desired product was
obtained after column chromatography (DCM/MeOH 95/5, v/v) as a
white solid in 82% yield (17 mg, 0.00258 mmol). 1H NMR (400 MHz,
CDCl3) δ (ppm): 7.11 (br, 12H, ArH), 5.93 (d, J = 7.3 Hz, 6H, NHAc),
C
88H132N12O28S4Na [M + Na]+ 1955.8055, found 1955.8069.
4.1.15.3. 5,11,17,23-Tetrakis-N-[3-(2-acetamido-2-deoxy-β-D-
mannopiranosyloxy)-propyl-thioureido]-25,26,27,28-tetrapropoxycalix[4]
arene 1,3-Alternate (1c). Compound 1c was obtained after purification
via C18 reverse phase column chromatography (MeOH/H2O 3/2, v/v)
as a white solid (0.023 g, 0.0119 mmol, 83%). Mp: dec > 176 °C. 1H
NMR (400 MHz, MeOD) δ (ppm): 7.09 (s, 8H, ArH), 4.71 (s, 4H, H1),
4.56 (d, J = 3.6 Hz, 4H, H2), 4.06–3.94 (m, 4H, CHHNHCS), 3.87 (d,
J = 3.2 Hz, 8H, H6ab), 3.83–3.64 (m, 24H, H3, OCH2CH2CH2,
CHHNHCS, OCH2CH2CH3), 3.63–3.50 (m, 12H, ArCH2Ar, H4),
3.31–3.25 (m, 4H, H5), 2.10 (s, 12H, NHCOCH3), 2.01–1.81 (m, 8H,
CH2CH2CH2), 1.96–1.80 (m, 8H, OCH2CH2CH3), 1.04 (t, J = 7.6 Hz,
12H, OCH2CH2CH3). 13C NMR (100 MHz, MeOD) δ (ppm): 180.7 (CS),
173.5 (NHCOCH3), 153.9 (CAr ipso), 133.6 (CAr ortho), 131.6 (CAr
para), 125.4 (CAr), 99.52 (C1), 76.90 (C5), 74.9 (OCH2CH2CH3), 72.8
(C3), 67.2 (CH2NCS), 66.9 (C4), 60.7 (C6), 53.6 (C2), 42.2
(OCH2CH2CH2), 35.0 (ArCH2Ar), 28.8 (CH2CH2CH2), 23.6
(OCH2CH2CH3), 21.6 (NHCOCH3), 9.7 (OCH2CH2CH3). HRMS (ESI-
TOF) m/z: calcd for C88H132N12O28S4Na [M + Na]+ 1955.8055, found
1955.8070.
5.60 (br, 6H, H1′), 5.41 (t, J = 9.1 Hz, 6H, H3′) 5.17–5.02 (m, 12H, H4″
H4), 4.96 (dd, J3,2 = 3.0, J3,4 = 10.1 Hz, 6H, H3), 4.93 (dd, J3″,2″ = 5.8,
3″,4″ = 9.9 Hz, 6H, H3″) 4.74 (br, 6H, H1″), 4.69 (br, 6H, H2′), 4.62 (br,
,
J
6H, H2″), 4.50 (br, 6H, H1), 4.39 (dd, 6H, H6a″) 4.28 (br, 6H, H6a′), 4.13
(br, 18H, H2, H5′, H6b′), 4.05 (d, J = 12.0 Hz, 6H, H6b″), 3.86 (br, 12H,
CH2NHCS), 3.75 (br, 6H, H4′), 3.66 (br, 6H, H5″), 3.45 (br, 18H, H5,
OCH2) 2.16 (s, 18H, CH3CO), 2.12 (s, 36H, CH3CO), 2.11 (s, 18H,
CH3CO), 2.061 (s, 18H, CH3CO), 2.056 (s, 18H, CH3CO), 2.05 (s, 18H,
CH3CO), 2.03 (s, 18H, CH3CO), 2.01 (s, 18H, CH3CO), 1.75 (br, 12H,
CH2CH2CH2), 1.23 (d, J = 5.8 Hz, 18H, H6). 13C NMR (100 MHz,
CDCl3) δ (ppm): 172.1, 170.6, 170.5, 170.4, 170.1, 169.7, 169.6, 135.2,
125.0, 100.8, 98.3, 94.0, 75.8, 72.6, 72.3, 71.9, 71.8, 71.0, 70.7, 70.4,
67.8, 65.6, 62.3, 62.1, 60.8, 50.8, 41.8, 32.2, 29.7, 26.4, 23.4, 23.2,
21.3, 20.9, 20.8, 20.76, 20.68, 17.7, 14.1. HRMS (ESI-TOF) m/z: calcd
for C288H390N18O144S6Na4 [M + 4Na]4+ 1672.0388, found 1672.0399.
4.1.15. General procedure for the deacetylation reaction of glycocalix[n]
arenes 5a-d and monomer 14
4.1.15.4. 5,11,17,23,29,35-Hexakis-N-[3-(2-acetamido-2-deoxy-β-D-
mannopiranosyloxy)-propyl-thioureido]-37,38,39,40,41,42-
To a solution of the peracetylated compound in MeOH at 0 °C, freshly
prepared MeONa was added till pH 9. The solution was stirred for 3 h, after
which Amberlite IR-120 (H+), and 1 mL of H2O in the case of the hex-
acalixarenes, were added and the mixture stirred at rt till neutral pH. The
resin was filtered off and the solvent removed under reduced pressure.
hexamethoxycalix[6]arene (1d). Purification by C18 reverse phase
column chromatography (MeOH/H2O 64/36, v/v) afforded 1d in
54% yield as
a off-white solid (0.015 g, 0.00549 mmol). Mp:
186–187 °C. 1H NMR (400 MHz, D2O, 80 °C) δ (ppm): 7.45 (s, 12H,
ArH), 5.20 (s, 6H, H1), 4.97 (d, J = 4.4 Hz, 6H, H2), 4.44 (br, 12H,
10

M. Giuliani, et al.
BioorganicChemistry93(2019)103305
ArCH2Ar), 4.40–4.23 (m, 24H, H3, H6a,b, OCHHCH2CH2), 4.18–4.11 (m,
6H, OCHHCH2CH2), 4.07–4.00 (m, 18H, CH2NHCS, H4), 3.84 (br, 6H,
H5), 3.77 (br, 18H, CH3), 2.52 (s, 18H, NHCOCH3), 2.31 (br, 12H,
CH2CH2CH2). 13C NMR (100 MHz, D2O, 80 °C) δ (ppm): 180.7 (CS),
175.4 (NHCOCH3), 154.8 (CAr ipso), 135.6 (CAr ortho), 134.1 (CAr
para), 126.8 (CAr), 99.9 (C1), 77.2 (C5), 75.0 (C3), 67.8 (C4), 67.7
(OCH2CH2CH2), 61.2 (OCH3), 59.9 (C6), 53.8 (C2), 42.5 (CH2NHCS),
30.7 (CH2CH2CH2), 29.2 (ArCH2Ar), 22.8 (NHCOCH3). HRMS (ESI-
TOF) m/z: calcd for C120H174N18O42S6K2 [M + 2 K]2+ 1404.6177,
found 1404.6204.
with a mixture of S. pneumoniae CPS 19F (1 mg/mL,Sanofì-Aventis,
France) and methylated human serum albumin (1 mg/mL). A solution
of foetal calf serum (5%) in phosphate-buffered saline supplemented
with Brij-35 (0.1%) and sodium azide (0.05%) was applied to the plates
for blocking of nonspecific binding sites. The plates were incubated
overnight at 4–8 °C with a solution (1:200) of rabbit polyclonal anti-
19F, used as reference serum (Statents serum Institute, Artillerivej,
Denmark). When compounds were tested, they were added to each well
immediately before the addition of the reference serum. The plates
were then incubated with alkaline phosphatase conjugate goat anti-
rabbit IgG (Sigma-Aldrich, Milan, Italy), stained with p-ni-
trophenylphosphate, and the absorbance was measured at 405 nm with
an Ultramark microplate reader (Bio-Rad Laboratories S.r.l., Milan,
Italy).
4.1.15.5. N-[3-(2-acetamido-2-deoxy-β-D-mannopiranosyloxy)-propyl-
thioureido]-4-propoxybenzene (1-MON). The pure product was obtained
after purification by C18 reverse phase column chromatography
(MeOH/H2O 1/1, v/v) as a off-white solid (0.0272 g, 0.0577 mmol,
66%). Mp: dec > 76 °C. 1H NMR (400 MHz, MeOD) δ (ppm): 7.24 (d,
J = 8.9 Hz, 2H, ArH), 6.93 (d, J = 8.9 Hz, 2H, ArH), 4.65 (d,
J = 1.5 Hz, 1H, H1), 4.48 (dd, J2,1 = 1.5, J2,3 = 4.5 Hz, 1H, H2), 3.95
(t, J = 6.5 Hz, 2H, OCH2CH2CH3), 3.93–3.89 (m, 1H, OCHHCH2CH2),
3.88–3.84 (m, 1H, H6a,b), 3.69 (br, 1H, CHHNHCS), 3.67 (dd,
Declaration of Competing interest
The authors declared that there is no conflict of interest.
Acknowledgements
J
3,2 = 4.4, J3,4 = 9.6 Hz, 1H, H3), 3.61 (m, 2H, OCHHCH2CH2,
CHHNHCS), 3.53 (t, J = 9.6 Hz, 1H, H4), 3.29–3.23 (m, 1H, H5), 2.04
(s, 3H, NHCOCH3), 1.89–1.76 (m, 4H, OCH2CH2CH3, CH2CH2CH2),
1.06 (t, J = 7.2 Hz, 3H, OCH2CH2CH3). 13C NMR (100 MHz, MeOD) δ
(ppm): 181.1 (CS), 173.5 (NHCOCH3), 157.7 (CAr ipso), 127.3 (CAr
para), 126.7, 114.5 (CAr meta), 99.4 (C1), 76.9 (C5), 76.8
(OCH2CH2CH3), 69.4 (C4, OCH2CH2CH2), 60.6 (C6), 53.5 (C2), 41.9
(CH2NHCS), 28.6 (CH2CH2CH2), 22.3 (OCH2CH2CH3), 21.4
(NHCOCH3), 9.4 (OCH2CH2CH3). HRMS (ESI-TOF) m/z: calcd for
The authors are grateful to Prof. Luigi Lay for kindly providing
SP19F-RU used as reference compound in ELISA tests. This work was
supported by the Italian Ministry of University and Research (PRIN
2015 grant, prot. 2015RNWJAM, Nanoplatforms for enhanced immune
response). FS acknowledges the Centro Interdipartimentale Misure “G.
Casnati” of Parma University for the use of NMR and Mass
Spectrometry facilities. This work has benefited from the equipment
and framework of the COMP-HUB Initiative, funded by the
‘Departments of Excellence’ program of the Italian Ministry for
Education, University and Research (MIUR, 2018–2022).
C
21H33N3O7SNa [M + Na]+ 494.1937, found 494.1955.
4.1.16. 5,11,17,23,29,35-Hexakis-N-[3-(2-acetamido-2-deoxy-β-D-
mannopyranosyl-(1 → 4)-α-D-glucopyranosyl-(1 → 2)-α-L-
Appendix A. Supplementary material
rhamnopyranosyloxy)-propyl-thioureido]-37,38,39,40,41,42-
hexamethoxycalix[6]arene (2a)
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103305.
The pure product was obtained after trituration in diethyl ether
(7.5 mg, 0.00164 mmol, 80%). 1H NMR (400 MHz, MeOD/D2O 75/25)
δ (ppm): 8.49 (s, NH), 7.01 (br, 12H, ArH), 5.04 (br, OH), 4.94 (br, 6H,
H1′), 4.87 (br, 6H, H1″), 4.85 (br, 6H, H1), 4.54 (br, 6H, H2″), 4.14–3.22
(ovelapped, 144H, H3″, H4″, H5″, H6a,b″, H2′, H3′, H4′, H5′, H6a,b′, H2, H3,
H4, H5, OCH2CH2CH2, CH2NHCS, OCH3, ArCH2Ar), 2.08 (s, 18H,
NHCOCH3), 1.86 (br, 12H, CH2CH2CH2), 1.29 (br, 18H, H6). 13C NMR
(100 MHz, MeOD/D2O 1:1) δ (ppm): 174.5, 99.4, 98.0, 97.7, 78.8, 76.9,
76.8, 72.5, 72.3, 71.6, 71.5, 70.5, 70.1, 68.9, 66.7, 65.2, 60.4, 59.9,
53.5, 39.9, 28.7, 21.8, 16.8. HRMS (ESI-TOF) m/z: calcd for
References
[1] K.A. Geno, G.L. Gilbert, J.Y. Song, I.C. Skovsted, K.P. Klugman, C. Jones,
H.B. Konradsen, M.H. Nahm, Pneumococcal capsules and their types: past, present,
and future, Clin. Microbiol. Rev. 28 (2015) 871–899.
[2] B.H.A. Rehm, Bacterial polymers: biosynthesis, modifications and applications, Nat.
Rev. Microbiol. 8 (2010) 578–592.
[3] O. Haji-Ghassemi, R.J. Blackler, N.M. Young, S.V. Evans, Antibody recognition of
carbohydrate epitopes, Glycobiology 25 (2015) 920–952.
[4] M. Cohen, A. Varki, Modulation of glycan recognition by clustered saccharide
patches, Int. Rev. Cell Mol. Biol. 308 (2014) 75–125.
C
192H294N18O96S6Na3 [M + 3Na]3+ 1550.2248, found 1550.2276.
[5] T.K. Dam, C.F. Brewer, Effects of clustered epitopes in multivalent ligand-receptor
interactions, Biochemistry 47 (2008) 8470–8476.
4.1.17. 5,11,17,23,29,35-Hexakis-N-[3-(2-acetamido-2-deoxy-β-D-
mannopyranosyl-(1 → 4)-α-D-glucopyranosyl-(1 → 2)-β-L-
[6] E. Osinaga, S. Bay, D. Tello, A. Babino, O. Pritsch, K. Assemat, D. Cantacuzene,
H. Nakada, P. Alzari, Analysis of the fine specificity of Tn-binding proteins using
synthetic glycopeptide epitopes and a biosensor based on surface plasmon re-
sonance spectroscopy, FEBS Lett. 469 (2000) 24–28.
rhamnopyranosyloxy)-propyl-thioureido]-37,38,39,40,41,42-hexamethoxy
calix[6]arene (2b)
[7] R. Lo-Man, S. Vichier-Guerre, S. Bay, E. Deriaud, D. Cantacuzene, C. Leclerc, Anti-
tumor immunity provided by a synthetic multiple antigenic glycopeptide displaying
a tri-Tn glycotope, J. Immunol. 166 (2001) 2849–2854.
The pure product was obtained after trituration in diethyl ether
(9.6 mg, 0.00209 mmol, 81%). 1H NMR (400 MHz, MeOD/D2O 9/1) δ
(ppm): 7.04 (br, 12H, ArH), 5.11 (br, 6H, H1′), 4.83 (s, 6H, H1″), 4.67
(br, 6H, H1), 4.63 (br, OH), 4.53 (br, 6H, H2″), 4.24–3.27 (overlapped,
144H, H3″, H4″, H5″, H6a,b″, H2′, H3′, H4′, H5′, H6a,b′, H2, H3, H4, H5,
OCH2CH2CH2, CH2NHCS, OCH3, ArCH2Ar), 2.07 (s, 18H, NHCOCH3),
1.90 (br, 12H, CH2CH2CH2), 1.34 (br, 18H, H6). 13C NMR (100 MHz,
MeOD/D2O 9:1) δ (ppm): 173.4, 100.7, 100.4, 99.5, 78.9, 78.4, 77.0,
72.9, 72.6, 72.4, 72.1, 70.4, 66.7, 60.5, 60.0, 53.5, 29.5, 21.6, 16.8.
HRMS (ESI-TOF) m/z: calcd for C192H294N18O96S6Na3 [M + 3Na]3+
1550.2248, found 1550.2273.
[8] S. Grigalevicius, S. Chierici, O. Renaudet, R. Lo-Man, E. Deriaud, C. Leclerc,
P. Dumy, Chemoselective assembly and immunological evaluation of multiepitopic
glycoconjugates bearing clustered Tn antigen as synthetic anticancer vaccines,
Bioconjug. Chem. 16 (2005) 1149–1159.
[9] R. Lo-Man, S. Vichier-Guerre, R. Perraut, E. Deriaud, V. Huteau, L. BenMohamed,
O.M. Diop, P.O. Livingston, S. Bay, C. Leclerc, A fully synthetic therapeutic vaccine
candidate targeting carcinoma-associated Tn carbohydrate antigen induces tumor-
specific antibodies in nonhuman primates, Cancer Res. 64 (2004) 4987–4994.
[10] S. Behren, U. Westerlind, Glycopeptides and -mimetics to detect, monitor and in-
hibit bacterial and viral infections: recent advances and perspectives, Molecules 24
(2019).
[11] F. Compostella, O. Pitirollo, A. Silvestri, L. Polito, Glyco-gold nanoparticles:
synthesis and applications, Beilstein J. Org. Chem. 13 (2017) 1008–1021.
[12] B. Lepenies, J. Lee, S. Sonkaria, Targeting C-type lectin receptors with multivalent
carbohydrate ligands, Adv. Drug. Deliver. Rev. 65 (2013) 1271–1281.
[13] S.C. Purcell, K. Godula, Synthetic glycoscapes: addressing the structural and func-
tional complexity of the glycocalyx, Interf. Focus 9 (2019).
4.2. Competitive ELISA
96-well flat-bottomed plates were incubated overnight at 4–8 °C
11

M. Giuliani, et al.
BioorganicChemistry93(2019)103305
[14] F. Sansone, A. Casnati, Multivalent glycocalixarenes for recognition of biological
macromolecules: glycocalyx mimics capable of multitasking, Chem. Soc. Rev. 42
(2013) 4623–4639.
[30] M. Torvinen, R. Neitola, F. Sansone, L. Baldini, R. Ungaro, A. Casnati, P. Vainiotalo,
E. Kalenius, Glucosylthioureidocalix[4]arenes: synthesis, conformations and gas
phase recognition of amino acids, Org. Biomol. Chem. 8 (2010) 906–915.
[31] G.M.L. Consoli, F. Cunsolo, C. Geraci, T. Mecca, P. Neri, Calix[8]arene-based gly-
coconjugates as multivalent carbohydrate-presenting systems, Tetrahedron Lett. 44
(2003) 7467–7470.
[15] C.D. Gutsche, Calixarenes: an introduction, 2nd edition, Monogr. Supramol. Chem
(2008) 1–276.
[16] L. Legnani, F. Compostella, F. Sansone, L. Toma, Cone calix[4]arenes with or-
ientable glycosylthioureido groups at the upper rim: an in-depth analysis of their
symmetry properties, J. Org. Chem. 80 (2015) 7412–7418.
[32] J.M.G. Fernandez, C.O. Mellet, Chemistry and developments of N-thiocarbonyl
carbohydrate derivatives: Sugar isothiocyanates, thioamides, thioureas, thiocarba-
mates, and their conjugates, Adv. Carbohyd. Chem. Bi 55 (2000) 35–135.
[33] C. Fessele, S. Wachtler, V. Chandrasekaran, C. Stiller, T.K. Lindhorst, A. Krueger,
Thiourea-bridged nanodiamond glycoconjugates as inhibitors of bacterial adhesion,
Eur. J. Org. Chem. (2015) 5519–5525.
[17] S. Andre, C. Grandjean, F.M. Gautier, S. Bernardi, F. Sansone, H.J. Gabius,
R. Ungaro, Combining carbohydrate substitutions at bioinspired positions with
multivalent presentation towards optimising lectin inhibitors: case study with ca-
lixarenes, Chem. Commun. 47 (2011) 6126–6128.
[18] S. Andre, F. Sansone, H. Kaltner, A. Casnati, J. Kopitz, H.J. Gabius, R. Ungaro, Calix
[n]arene-based glycoclusters: bioactivity of thiourea-linked galactose/lactose moi-
eties as inhibitors of binding of medically relevant lectins to a glycoprotein and cell-
surface glycoconjugates and selectivity among human adhesion/growth-regulatory
galectins, ChemBioChem 9 (2008) 1649–1661.
[34] L. Toma, L. Legnani, F. Compostella, M. Giuliani, F. Faroldi, A. Casnati, F. Sansone,
Molecular architecture and symmetry properties of 1,3-alternate calix[4]arenes
with orientable groups at the para position of the phenolic rings, J. Org. Chem. 81
(2016) 9718–9727.
[35] M. Giuliani, I. Morbioli, F. Sansone, A. Casnati, Moulding calixarenes for bioma-
cromolecule targeting, Chem. Commun. 51 (2015) 14140–14159.
[36] F. Sansone, L. Baldini, A. Casnati, R. Ungaro, Conformationally mobile gluco-
sylthioureidocalix[6]- and calix[8]arenes: synthesis, aggregation and lectin
binding, Supramol. Chem. 20 (2008) 161–168.
[19] S. Cecioni, R. Lalor, B. Blanchard, J.P. Praly, A. Imberty, S.E. Matthews, S. Vidal,
Achieving high affinity towards a bacterial lectin through multivalent topological
isomers of calix[4]arene glycoconjugates, Chem.-Eur. J. 15 (2009) 13232–13240.
[20] C. Geraci, G.M.L. Consoli, E. Galante, E. Bousquet, M. Pappalardo, A. Spadaro, Calix
[4]arene decorated with four tn antigen glycomimetic units and P3CS im-
munoadjuvant: Synthesis, characterization, and anticancer immunological evalua-
tion, Bioconjug. Chem. 19 (2008) 751–758.
[37] L. Legnani, S. Ronchi, S. Fallarini, G. Lombardi, F. Campo, L. Panza, L. Lay,
L. Poletti, L. Toma, F. Ronchetti, F. Compostella, Synthesis, molecular dynamics
simulations, and biology of a carba-analogue of the trisaccharide repeating unit of
Streptococcus pneumoniae 19F capsular polysaccharide, Org. Biomol. Chem. 7 (2009)
4428–4436.
[21] F. Berti, R. Adamo, Antimicrobial glycoconjugate vaccines: an overview of classic
and modern approaches for protein modification, Chem. Soc. Rev. 47 (2018)
9015–9025.
[38] M. Vetro, D. Safari, S. Fallarini, K. Salsabila, M. Lahmann, S. Penades, L. Lay,
M. Marradi, F. Compostella, Preparation and immunogenicity of gold glyco-nano-
particles as antipneumococcal vaccine model, Nanomedicine-UK 12 (2017) 13–23.
[39] L. Morelli, S. Fallarini, G. Lombardi, C. Colombo, L. Lay, F. Compostella, Synthesis
and biological evaluation of a trisaccharide repeating unit derivative of
Streptococcus pneumoniae 19A capsular polysaccharide, Bioorg. Med. Chem. 26
(2018) 5682–5690.
[22] J.N. Weiser, D.M. Ferreira, J.C. Paton, Streptococcus pneumoniae: transmission,
colonization and invasion, Nat. Rev. Microbiol. 16 (2018) 355–367.
[23] T. Welte, A. Torres, D. Nathwani, Clinical and economic burden of community-
acquired pneumonia among adults in Europe, Thorax 67 (2012) 71–79.
[24] A. Arbo, D. Lovera, C. Martinez-Cuellar, Mortality predictive scores for community-
acquired pneumonia in children, Curr. Infect. Disease Rep. 21 (2019) 10.
[25] F. Sansone, M. Dudic, G. Donofrio, C. Rivetti, L. Baldini, A. Casnati, S. Cellai,
R. Ungaro, DNA condensation and cell transfection properties of guanidinium ca-
lixarenes: dependence on macrocycle lipophilicity, size, and conformation, J. Am.
Chem. Soc. 128 (2006) 14528–14536.
[40] G. Soliveri, A. Bertolotti, L. Panza, L. Poletti, C. Jones, L. Lay, Synthesis of phos-
phorylated fragments of Streptococcus pneumoniae type 19F capsular polysaccharide,
J. Chem. Soc. Perk Trans. 1 (2002) 2174–2181.
[41] C. Colombo, O. Pitirollo, L. Lay, Recent advances in the synthesis of glycoconjugates
for vaccine development, Molecules 23 (2018).
[26] M. Dudic, A. Colombo, F. Sansone, A. Casnati, G. Donofrio, R. Ungaro, A general
synthesis of water soluble upper rim calix[n]arene guanidinium derivatives which
bind to plasmid DNA, Tetrahedron 60 (2004) 11613–11618.
[42] I. Calloni, L. Unione, G. Jimenez-Oses, F. Corzana, L. Del Bino, A. Corrado,
O. Pitirollo, C. Colombo, L. Lay, R. Adamo, J. Jimenez-Barbero, The Conformation
of the mannopyranosyl phosphate repeating unit of the capsular polysaccharide of
neisseria meningitidis serogroup a and its carba-mimetic, Eur. J. Org. Chem. (2018)
4548–4555.
[27] F. Sansone, E. Chierici, A. Casnati, R. Ungaro, Thiourea-linked upper rim calix[4]
arene neoglycoconjugates: synthesis, conformations and binding properties, Org.
Biomol. Chem. 1 (2003) 1802–1809.
[28] A.M.A. vanWageningen, E. Snip, W. Verboom, D.N. Reinhoudt, H. Boerrigter,
Synthesis and application of iso(thio)cyanate-functionalized calix[4]arenes, Liebigs
Ann.-Recl (1997) 2235–2245.
[43] H.E. Gottlieb, V. Kotlyar, A. Nudelman, NMR chemical shifts of common laboratory
solvents as trace impurities, J. Org. Chem. 62 (1997) 7512–7515.
[44] I.T. Ho, J.H. Chu, W.S. Chung, Calix[4]arene with lower-rim beta-amino alpha,
beta-unsaturated ketones containing bis-chelating sites as a highly selective fluor-
escence turn-on chemosensor for two copper(II) ions, Eur. J. Org. Chem. (2011)
1472–1481.
[29] M.I. Torres-Sanchez, C. Zaccaria, B. Buzzi, G. Miglio, G. Lombardi, L. Polito,
G. Russo, L. Lay, Synthesis and biological evaluation of phosphono analogues of
capsular polysaccharide fragments from Neisseria meningitidis A, Chem.-Eur. J. 13
(2007) 6623–6635.
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