Synthesis of new sulfated disaccharides for the modulation of TLR4-dependent inflammation

Article abstract of DOI:10.1039/d1ob00692d

Natural sulfated glycans are key players in inflammation through TLR4 activation; therefore synthetic exogenous sulfated saccharides can be used to downregulate inflammation processes. We have designed and synthesized new sulfated compounds based on small and biocompatible carbohydrates that are able to cross the BBB. A suitable protected donor and acceptor, obtained from a unique precursor, have been stereoselectively glycosylated to give an orthogonally protected cellobiose disaccharide. Selective deprotection and sulfation allowed the syntheses of four differentially sulfated disaccharides, which have been characterized by NMR, HRMS and MS/MS. Together with their partially protected precursors, the new compounds were tested on HEK-TLR4 cells. Our results show the potential of small oligosaccharides to modulate TLR4 activity, confirming the need for sulfation and the key role of the 6-sulfate groups to trigger TLR4 signalization.

Full text of DOI:10.1039/d1ob00692d

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Synthesis of new sulfated disaccharides for the
modulation of TLR4-dependent inflammation†
Cite this: DOI: 10.1039/d1ob00692d
Rachid Naïtaleb,a Agnès Denys,b Fabrice Allain,b Jérôme Ausseil,c
a
Sylvestre Toumieux *a and José Kovensky
*
Natural sulfated glycans are key players in inflammation through TLR4 activation; therefore synthetic
exogenous sulfated saccharides can be used to downregulate inflammation processes. We have designed
and synthesized new sulfated compounds based on small and biocompatible carbohydrates that are able
to cross the BBB. A suitable protected donor and acceptor, obtained from a unique precursor, have been
stereoselectively glycosylated to give an orthogonally protected cellobiose disaccharide. Selective de-
protection and sulfation allowed the syntheses of four differentially sulfated disaccharides, which have
been characterized by NMR, HRMS and MS/MS. Together with their partially protected precursors, the
new compounds were tested on HEK-TLR4 cells. Our results show the potential of small oligosaccharides
to modulate TLR4 activity, confirming the need for sulfation and the key role of the 6-sulfate groups to
trigger TLR4 signalization.
Received 8th April 2021,
Accepted 20th April 2021
DOI: 10.1039/d1ob00692d
rsc.li/obc
the NF-κB pathway.11 It can be found on the surface of micro-
glial cells in the central nervous system (CNS), brain or spinal
Introduction
Toll-like receptors (TLRs) are a type of pattern recognition recep- cord. Interestingly, neuroinflammation is known to play a deci-
tor (PRR) that identify molecules shared by pathogens but dis- sive role in neurological diseases such as neurodegeneration.10
tinguishable from host molecules collectively referred to as Thus, targeting brain inflammation represents a potential
pathogen-associated molecular patterns (PAMPs). TLRs play a clinical intervention strategy for such pathologies but the
critical role by triggering the molecular activation cascade that blood–brain barrier (BBB) is one of the main hurdles to access
regulates the innate immune response/inflammatory process.1–3 the CNS. Some molecules that are able to modulate the TLR4
The upregulation of TLRs with the development of TLR agonists response have been tested previously and are often too big
could be useful for the treatment of tumors,4 allergies,5 and and/or not specific enough. Nevertheless, some small mole-
infectious diseases (HBV, malaria).6 The downregulation with cule modulators that are able to cross the BBB have been
antagonists is implicated in pathologies such as sepsis,7 type 1 reported to have antagonist activities.12–16
and 2 diabetes,8,9 or neuroinflammation.10
Sulfated glycans such as glycosaminoglycans on proteogly-
Among TLRs, TLR4 is a key cell surface receptor involved in cans are key players in both molecular and cellular events of
innate and adaptive immune responses. This receptor is acti- inflammation through TLR4 activation. For example, it has
vated through exposure to lipopolysaccharides (LPS), lipid A or been shown that heparan sulfate is degraded during inflam-
lipooligosaccharides (LOS) and initiates the production of a mation to become a potent TLR4 ligand and that TLR4 can be
number of inflammatory mediators, including IL-1β, TNF-α, activated by small soluble fragments of heparan sulfate.
and macrophage inflammatory protein 1 alpha (MIP1α) via Therefore, exogenous sulfated glycans of various structures and
TLR4-dependent activation through the MyD88 adaptor and origins can be used to interventionally downregulate inflam-
mation processes.17–19 For example, disaccharides like lipid A
and its analogs have been proven to establish interaction with
TLR4 and could be potent interesting modulators.20,21
aLaboratoire de Glycochimie, des Antimicrobiens et des Agroressources, LG2A CNRS
Herein, we propose to modulate TLR4 activity to reduce
UMR 7378, Université de Picardie Jules Verne, 33 rue Saint Leu, 80039 Amiens,
neurodegeneration by the modulation of neuroinflammation.
We have designed and synthesized new sulfated saccharides
acting as inflammation regulators in order to restrain the neu-
roinflammation. Those modulators are based on small and
biocompatible carbohydrates and therefore, would be able to
pass the BBB.
France. E-mail: jose.kovensky@u-picardie.fr, sylvestre.toumieux@u-picardie.fr
bUnité de Glycobiologie Structurale et Fonctionnelle, Université de Lille, UGSF CNRS
UMR 8576, F-59000 Lille, France. E-mail: fabrice.allain@univ-lille.fr
cInstitut Toulousain des Maladies Infectieuses et Inflammatoires, CHU Purpan, BP
3028, 31024 Toulouse, France. E-mail: ausseil.j@chu-toulouse.fr
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d1ob00692d
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benzyl groups, leading to triol 10. Sulfation using the SO3–tri-
methylamine complex (5 equiv./OH) afforded 11, which by dea-
Results and discussion
The synthetic route is shown in Scheme 1. Regioselective cetylation gave compound 12. On the other hand, deacetyla-
reductive opening of the benzylidene acetal of the known tion of 9 afforded compound 13 which has five free hydroxyls.
phenyl 2,3-di-O-acetyl-4,6-O-benzylidene-1-thio-β-D-glucopyra- Sulfation of 13 as above followed by hydrogenolysis led to com-
noside (1)22 was performed using trifluoroacetic acid (TFA) pound 14. For comparison, the known methyl cellobioside 15
and triethylsilane (TES) to give 6-O-benzyl derivative 2 in 94% was also sulfated to give compound 16.
yield. The 4-hydroxyl was temporarily protected as a 2,2,2-tri-
All the sulfation reactions proceeded well, but extensive
chloroethoxycarbonyl (Troc), followed by the selective removal purifications had to be performed to completely eliminate the
of the thiophenyl group of compound 3 in the presence of tri- excess sulfating agent on Sephadex LH-20, and the isolated
chloroisocyanuric acid (TCCA),23 affording 4 in 80% yield. This yields were poor (24%). Nevertheless, the sequence allowed us
method is cleaner than the traditional method using NBS to to obtain a family of differently sulfated disaccharides useful
cleave the C–S linkage. Adding dichloromethane at the end of to analyze the structure–activity relationships.
the reaction leads to precipitation of the isocyanuric acid and
makes the workup very simple.
We expected some regioselectivity of the sulfation reaction.
However, the analysis of sulfated disaccharides was not
Reaction with trichloroacetonitrile in the presence of DBU straightforward, and a combination of NMR and MS analyses
1
led to trichloroacetimidate donor 5 (75% yield). The H NMR was necessary to determine the structure of compounds 11,
spectrum revealed its α configuration (δ 6.50, d, J = 3.6 Hz, 1H, 12, 14 and 16.
H-1). The glycosyl acceptor was prepared from compound 5 in
The simplest case was the sequence 10 → 11 → 12. HRMS
two steps. First, glycosylation with methanol was achieved (negative mode) of compound 11 showed unambiguously the
using TMSOTf as the promotor to give methyl glucopyranoside presence of three sulfates, m/z 807.0028 [M − 3H + 2Na]−. Thus,
6 with total β stereoselectivity shown by the H-1 doublet at δ under our conditions, the sulfation of the primary positions was
1
4.39 (J = 7.9 Hz) in the H NMR spectrum, as expected in the accompanied by the sulfation of the 4-position. In the 13C NMR of
presence of an acetate participating group at C-2. Second, the compound 11, the resonances of the sulfated primary positions
Troc protecting group was selectively removed by activated Zn/ C-6 and C-6′ appeared at δ 65.5 and 66.5, a deshielding of about
AcOH in THF. Acceptor 7 was obtained in 90% yield.24
7 ppm when compared to those of compound 10. The corres-
Glycosylation between donor 5 and acceptor 7 promoted by ponding signal of C-4′ shifted from δ 69.2 to 73.9 upon sulfation.
TMSOTf proceeded smoothly at room temperature, affording The deacetylation step led to compound 12, and the pres-
disaccharide 8 in 80% yield. In the 1H NMR spectrum, the ence of the three sulfate groups was confirmed on positions 6,
anomeric protons appeared at 4.42 ppm (d, J = 8.0 Hz, H-1) 4′ and 6′ by NMR. The HRMS at m/z 638.9576 fitted well with
and 4.33 ppm (d, J = 7.9 Hz, 1H, H-1), whereas the 13C NMR the trisulfated disaccharide structure. MS/MS of this ion
spectrum showed the signals of C-1′ and C-1 at δ 101.0 and showed the main fragments at m/z 519.00 (corresponding to
100.0, respectively. Treatment of compound 8 under the same the loss of NaSO3 and water) and the peaks arising from clea-
conditions used above to remove the Troc group gave the key vages at the interglycosidic linkage (Scheme 3). The peak at
disaccharide 9 in 80% yield.
m/z 342.94 (C cleavage, loss of water) is consistent with a
Disaccharide 9 can be orthogonally deprotected in order to monosaccharide fragment carrying two sulfate groups,
obtain regioselective sulfated products (Scheme 2). Catalytic whereas the other sulfate is found in the reducing end frag-
hydrogenation over 10% Pd/C allowed the cleavage of the ment (Z cleavage, m/z 273.03).
Scheme 1 Synthesis of orthogonally protected disaccharide 9.
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Scheme 2 Synthesis of diversely sulfated products.
the aim is to determine the exact location of the sulfate group
in compound 14.
MS/MS of the molecular ion gave limited information. The
main fragments observed were at m/z 273.00 (Y interglycosidic
cleavage), m/z 254.99 and m/z 240.98 (loss of water and metha-
nol, respectively, from the precedent ion), consistent with the
location of the sulfate group at the reducing end.
The NMR spectra of compound 14 were assigned by com-
parison with those of methyl cellobioside 15. The HSQC NMR
spectrum of compound 14 showed a downfield shifted triplet
at δ 4.35 (that can be assigned to H-3) that correlates with a
peak at δ 84.0 (thus assigned to C-3). In addition, the little
difference in the chemical shifts of C-1 between the non-sul-
fated and the sulfated product indicated that the sulfate group
cannot be at C-2. Therefore, the structure of compound 14
corresponds to a 3-sulfate methyl cellobioside.
This regioselectivity for O-3 can be explained by assuming a
conformation of cellobiose similar to those found in cellulose
(Scheme 4). In methyl cellobioside 15, the reactivity would be
enhanced for both O-3 (linked through a hydrogen bond to the
sugar O-5) and O-2′ (linked through a hydrogen bond to O-6).
Scheme 3 MS/MS analysis of the molecular ion of compound 12.
Sulfation of compound 13 was performed under the same However, in compound 13, the 6-oxygen is blocked with a
conditions used above. Unfortunately, different purifications benzyl group, therefore O-2′ is no longer activated for substi-
by flash chromatography (reverse phase) of the sulfated tution and sulfation takes place at position 3.
product did not allow the obtaining of this intermediate in the
Finally, methyl cellobioside 15 was directly sulfated and the
pure form, and hydrogenolysis of the benzyl groups was major compound 16 was purified and analyzed. HRMS showed
accomplished and the deprotected sulfated molecule 14 was the molecular ion at m/z 638.9604, indicating a trisulfated dis-
finally purified using Sephadex LH20. HRMS of compound 14 accharide. The preferential substitution is expected to occur at
showed the presence of one sulfate group, as the molecular the 6- and 6′-positions. In the 13C NMR spectrum of 16, a shift
ion [M − H]− appeared at m/z 435.0860. As the secondary was observed for the resonances corresponding to C-6 and C-6′
hydroxyls are less reactive than the primary positions (blocked when compared to those in the spectrum of 15, from δ 61.4
as benzyl ethers in compound 13), the sulfation did not lead and 61.9 to δ 68.9 and 69.3, respectively, confirming the
to an oversulfated product. From the five available positions, assignment. The NMR spectra also showed that this trisulfated
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Scheme 4 Conformation of cellulose and methyl cellobiosides and regioselectivity of the sulfation of compound 13.
disaccharide 16 is different from compound 12, which is also HEK-Null cells, is informative in terms of their capacity to
sulfated at C-6 and C-6′ but possesses an additional sulfate at trigger signalization through TLR4.26
C-4′. MS/MS of the molecular ion showed a diagnostic fragment
First, we checked that compounds 9 to 16 did not induce
at m/z 374.97, indicating the presence of the remaining sulfate any activation of HEK cells independent of TLR4 expression.
on the reducing unit (Scheme 5). From the two possible To this end, HEK-Null cells were exposed to compounds 9 to
hydroxyls, the NMR spectra of compound 16 are consistent with 16 (each at 10, 30 and 100 µM) for 16 h, after which time the
the presence of the additional sulfate group at position C-3.
activity of SEAP was measured. As expected, we found that
The sulfated molecules synthesized, together with some these concentrations did not interfere with the production of
precursors or partially protected molecules, have been then SEAP from HEK cells that are devoid of TLR4.
tested for TLR4 interaction.
Then, we tested the ability of HEK-TLR4 cells to detect com-
To test the capacity of compounds 9 to 16 to trigger signali- pounds 9 to 16 at the same final concentrations (Fig. 1). In our
zation through TLR4, we decided to use a TLR4 transfected
cell line. A number of enzymatic bio-assays have been indeed
developed, based on the use of TLR stably transfected cell
lines, and designed to provide a sensitive method for the
detection of TLR agonists.25 Among them, HEK-Blue™/hTLR4
(HEK-TLR4) is a commercially available cell line, which is
stably co-transfected to express the human TLR4 gene and a
TLR-inducible reporter gene encoding a secreted embryonic
alkaline phosphatase (SEAP). Parental HEK-293 cells do not
express TLRs on their plasma membrane. Accordingly, TLR4
stimulation can be conveniently monitored through the
release of SEAP from HEK-TLR4 cells by using a phosphatase
detection assay. In addition, we used the HEK-Blue™/Null1
(HEK-Null) cell line as a negative control. Indeed, this cell line
is only transfected with the reporter gene encoding SEAP but
is devoid of TLR4. Thus, the way by which compounds 9 to 16
induced the production of SEAP in HEK-TLR4 cells, but not in
Fig. 1 Stimulation of TLR4-expressing HEK cells by compounds 9 to 16.
HEK-TLR4 cells were stimulated by the addition of compounds 9 to 16,
each at the final concentration of 10, 30 and 100 µM. After 16 h of incu-
bation, the production of SEAP related to the reporter gene activation
was quantified by measuring the phosphatase activity released in cell-
free supernatants with a chromogenic substrate (620 nm). Data of SEAP
activity are mean values
SEM from three separate experiments and
expressed as percentages of the maximal cellular response induced by
LPS (10 ng mL−1).
Scheme 5 MS/MS analysis of the molecular ion of compound 16.
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hands, non-sulfated molecules 9, 13 and 15 did not show any
interaction with TLR4. Only the partially acetylated 10 showed
a slight response at a high concentration (100 μM). Compound
11, bearing both acetate and sulfate esters, induced a TLR4
response at 30 μM. Unexpectedly, the trisulfated (and non-
acetylated) disaccharide 12 (obtained from 11) seemed inac-
tive. Monosulfated compound 14 showed a very low response,
and no significant difference was observed when compared
with the non-substituted analog 15. The highest response was
shown by compound 16, the 3,6,6′-trisulfated disaccharide.
Clearly, these findings indicate that sulfate groups on the
primary positions favored the interaction with TLR4, thus trig-
gering inflammatory signalization. It is difficult however to
explain the absence of activity of the trisulfated molecule 12
because the 6,6′-disulfation pattern of 16 is also present.
Further investigations and other synthetic derivatives may help
explain this result and the role of the specific position of
sulfate groups for TLR4 binding. Anyway, our results on TLR4
binding make compound 16 a promising starting point for the
regulation of inflammatory response.
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groups to trigger TLR4 signalization.
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Author contributions
S. T. and J. K. designed and supervised the synthetic
work. R. N. performed the synthesis and the characterization
of the compounds. F. A. designed the biological test performed 12 F. Peri and V. Calabrese, Toll-like Receptor 4 (TLR4)
by A. D. S. T., J. K. and F. A. wrote the paper and together with
J. A. revised its final version.
Modulation by Synthetic and Natural Compounds: An
Update, J. Med. Chem., 2014, 57, 3612.
13 J. Li, A. Csakai, J. Jin, F. Zhang and H. Yin, Therapeutic
Developments Targeting Toll-like Receptor-4-Mediated
Neuroinflammation, ChemMedChem, 2016, 11, 154.
14 M. Molteni, A. Bosi and C. Rossetti, Natural Products with
Conflicts of interest
The authors declare no conflicts of interest.
Toll-Like
Receptor
4
Antagonist
Activity,
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15 F. A. Facchini, L. Zaffaroni, A. Minotti, S. Rapisarda,
V. Calabrese, M. Forcella, P. Fusi, C. Airoldi, C. Ciaramelli,
J.-M. Billod, A. B. Schromm, H. Braun, C. Palmer,
R. Beyaert, F. Lapenta, R. Jerala, G. Pirianov, S. Martin-
Santamaria and F. Peri, Structure–Activity Relationship in
Acknowledgements
The authors thank the Conseil Régional de Hauts-de-France
(grant SOLIDE) for financial support and a PhD fellowship for
R. N.
Monosaccharide-Based Toll-Like Receptor
4
(TLR4)
Antagonists, J. Med. Chem., 2018, 61, 2895.
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M. R. Hutchinson, H. Yin and X. Wang, Small-Molecule
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