A simple glycolipid mimic of the phosphatidylinositol mannoside core from mycobacterium tuberculosis inhibits macrophage cytokine production

Article abstract of DOI:10.1002/cbic.201800150

Glycolipids from Mycobacterium tuberculosis have a profound impact on the innate immune response of the host. Macrophage-inducible C-type lectin (Mincle) is a pattern-recognition receptor that has been shown to bind trehalose dimycolate (TDM) from the mycobacterium and instigate intracellular signalling in the immune cell. There are structural similarities between the structures of TDM and phosphatidyl inositol mannoside (PIM). We thus hypothesized that these latter structures might also modulate an immune response in a similar manner. To test this, we synthesized a series of new mannose derivatives modified with fatty esters at the 6-position and assessed the release of inflammatory cytokines in human U937 macrophages under the induction of lipopolysaccharides (LPS) after glycolipid treatment. The results showed that the amount of two major cytokines—tumour necrosis factor (TNF)-α and interleukin (IL)-6—released from LPS-stimulated U937 cells decreased significantly when compared to a control upon treatment with the prepared glycolipids, thus indicating a reduction in cytokine production by the macrophages.

Full text of DOI:10.1002/cbic.201800150

Accepted Article
Title: Simple Glycolipid Mimic of Phosphatidylinositol Mannoside Core
from Mycobacterium tuberculosis Inhibits Macrophage Cytokine
Production
Authors: Tamim Mosaiab, Sandra Boiteux, Abu Zulfiker, Ming Wei,
Milton J. Kiefel, and Todd A. Houston
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemBioChem 10.1002/cbic.201800150
Link to VoR: http://dx.doi.org/10.1002/cbic.201800150
A Journal of
www.chembiochem.org

10.1002/cbic.201800150
ChemBioChem
COMMUNICATION
Simple Glycolipid Mimic of Phosphatidylinositol Mannoside Core
from Mycobacterium tuberculosis Inhibits Macrophage Cytokine
Production
Tamim Mosaiab,[a] Sandra Boiteux, [a] Abu Hasanat Md Zulfiker, [b,c] Ming Wei, [b] Milton J.
Kiefel,*[a] and Todd A. Houston*[a]
Abstract: Glycolipids from Mycobacterium tuberculosis have a
profound impact on the innate immune response of the host.
Macrophage inducible C-type Lectin (Mincle) is a pattern recognition
receptor that has been shown to bind trehalose dimycolate (TDM)
from the mycobacteria and instigate intracellular signaling in the
immune cell. There are structural similarities between TDM and
phosphatidyl inositol mannoside (PIM) structures. We thus
hypothesized that these latter structures might also modulate an
immune response in a similar manner. To test this, we have
synthesized a series of novel mannose derivatives modified with
fatty esters at the 6-position and assessed the release of
inflammatory cytokines in human U937 macrophages under
induction of lipopolysaccharides (LPS) after glycolipid treatment. The
results showed that the amount of two major cytokines such as
tumor necrosis factor (TNF)-α and interleukin (IL)-6 released from
LPS stimulated U937 cells decreased significantly when compared
to control upon treatment with the prepared glycolipids indicating the
reduction of cytokines production by macrophages.
additional lipid modifications at various positions including the 6-
position of mannose attached directly to the PI anchor.
Scheme 1. Structural similarity between core structures of trehalose
dimycolate (TDM) and phosphatidylinositol mannoside (PIM) from
Mycobacteria and phospholipomannan (PLM) from Candida [Note: PLM
contains a phosphodiester in place of the ester at C-6 of mannose in PIM].
These two families possess different immunomodulatory activity;
whereas PIM facilitates early endosome-phagosome fusion, LAM
inhibits the phagosome’s maturation and late endosome-lysosome
fusion.[3] This is in one sense a holding pattern that may allow
access to nutrients during MTB latency while avoiding lysosome
destruction. LAM from MTB also plays a role in intracellular survival
by promoting phosphorylation of the proapoptotic protein Bad from
the Bcl-2 family.[4] Based on the diverse structural features and
varied immune modulation profile of these glycolipids, it is certain
that they interact with multiple targets. For example, the mannose
trisaccharide caps found on LAM from pathogenic mycobacteria bind
the lectin DC-SIGN and dampen IL-12 production in macrophages
through a TLR2-dependent mechanism.[5] To investigate the role of
the PIM core on macrophage cytokine production, we have created
a simple monosaccharide mimic of this structure, shown in Scheme
2.
1. Introduction
Glycolipids from Mycobacterium tuberculosis (MTB) have a profound
impact on the immune response of a human host allowing the
microorganism to survive intracellularly upon capture by
macrophages. An important core structure common to several
complex glycolipids is the phosphatidylinositol mannoside (PIM)
(Scheme 1). Importantly, it contains a mannose at the 2-position of
inositol,
a
position
that
is
unsubstituted
in
human
phosphatidylinositol (PI) species. It is likely that such substitution
would identify it as “non-self” to pattern recognition receptors (PPRs)
of the innate immune system. A second mannose is added to the 6-
position of inositol and this PIM2 core can be further decorated with
additional mannose (PIM3-PIM6).[1] This core is also the starting
point for biosynthesis of the large polysaccharide derivatives, the
lipoarabinomannans (LAMs).[2] Both PIMs and LAMs contain
[a]
Tamim Mosaiab, Sandra Boiteux, Dr. Milton J. Kiefel, Dr. Todd A.
Houston
Institute for Glycomics, Griffith University (Gold Coast Campus)
Parklands Drive, Southport, Queensland, Australia 4215
E-mail: t.houston@griffith.edu.au
m.kiefel@griffith.edu.au
[b]
[c]
Dr Abu Hasanat Md Zulfiker and Dr Ming Wei
Menzies Health Institute Queensland and School of Medical Science
Griffith University (Gold Coast Campus)
Parklands Drive, Southport, Queensland, Australia 4215
Dr Abu Hasanat Md Zulfiker
Department of Biomedical Sciences, John C. Edwards School of
Medicine, Marshall University, 1 John Marshall Dr., Huntington WV
25701 USA
Supporting information for this article is given via a link at the end of
the document
Scheme 2. Fatty acylated mannose derivatives as mimics of the PIM structure.
One PRR that may bind the PIM core is macrophage inducible C-
type lectin (Mincle), an important element of the innate immune
response to both mycobacterial and fungal infections.[6] It was
initially discovered to play a role in identifying damaged or dead cells
by responding to spliceosome-associated protein 130.[7] Several
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800150
ChemBioChem
COMMUNICATION
carbohydrate ligands have been identified—trehalose dimycolate
(TDM) from mycobacteria [8] and glycolipids from Malessezia
pachydermatis [9] —and undoubtedly many more will be discovered
for this critical component of immune surveillance. We have noted
the close structural similarity between TDM and both
phosphatidylinositol mannosides (PIMs) [10] from mycobacteria and
phospholipomannan (PLM) [11] from Candida albicans and believe
that this motif (2-mannosylinositol) is likely to be recognized by
Mincle. Since inositol modified at the 2-position has not been
identified in mammalian cell-surface components, it is reasonable to
assume that Mincle, as a pattern-recognition receptor scouting for
pathogen-associated molecular patterns, might have evolved to
identify this motif in macrophage-resident microorganisms such as M.
tuberculosis and C. albicans. In fact, Lee and colleagues have
shown that Mincle has a higher affinity for simple mannosides than
the corresponding glucosides. [12] Thus, it is possible that TDM may
not be the only cell-surface glycoconjugate on mycobacteria
recognized by this lectin as M. tuberculosis is replete with mannose
conjugates on its exterior. The structures of trehalose and 2-
mannosylated inositol are quite similar as shown in Figure 1,
particularly along the sides of both rings opposite the lipid anchors of
TDM and PIM/PLM. It is important to note that these structures have
identical stereochemistry in eight out of nine contiguous
stereocentres as highlighted by the red oxygen atoms in Figure 1.
The difference between PIM and PLM occurs at C-6 of mannose
where some PIM derivatives contain fatty esters and PLM extends a
large oligosaccharide (mannan) at this site.
activation to facilitate similar transformations. [16] However, alkanoic
acids tend to be less reactive partners relative to aryl carboxylic
acids in these reactions. Yamamoto and co-workers have shown
the C-6 primary alcohol of hexopyranosides can be selectively
esterified using sym-collidine activation of acid chlorides. [17] 2,6-
lutidine also contains methyl substituents on the carbons adjacent to
the pyridine nitrogen. The pyridinium intermediate shown in Figure
3 is more reactive toward primary over secondary alcohols due to
steric shielding of the carbonyl carbon by the ortho-type methyl
groups on collidine. We chose to compare sym-collidine and 2,6-
lutidine for selective esterification of benzyl α-D-mannoside (1).
Scheme 3. Synthesis of C-6 esterified benzyl α-D-mannosides. Reagents and
conditions: 1, corresponding fatty acid chloride, 2,6-lutidine, 0oC, 3 h, rt, 1h.
Progenitor benzyl α-D-mannoside 1 was synthesized using the
method of Zunk, et al., in 98% yield.[18] Selective esterification of
this monosaccharide was attempted using a range of fatty acid
chlorides and either sym-collidine or 2,6-lutidine as promoter. The
2,6-lutidine required higher temperatures than sym-collidine (0-20˚C
vs. -40˚C) and did lead to small amounts of diesters being formed.
However, these undesired compounds were easily removed by flash
column chromatography. The higher reaction temperature used with
2,6-lutidine allowed conversion to the desired monoesters in a
shorter time frame and thus was the preferred method. Four novel
compounds namely benzyl 6-lauroyl-α-D-mannoside (LBM, 2),
benzyl 6-myristoyl-α-D-mannoside (MBM, 3), benzyl 6-palmitoyl-α-D-
mannoside (PBM, 4), benzyl 6-stearoyl-α-D-mannoside (SBM, 5),
were synthesized by this protocol in reasonable yields (65-75%). Of
particular note is compound 4 derived from palmitoyl chloride as its
lipid matches the chain length common in some PIM structures. [10]
We are interested in novel methods of drug delivery to treat
macrophage infections [13] and are seeking to identify targeting
ligands for this purpose. As Mincle appears to be up-regulated in
response to these infections, it could be a target for selective drug
delivery to infected cells. Ideally, we require a Mincle ligand that
binds the lectin with some avidity but does not activate an
inflammatory response in uninfected macrophages. It is important to
note that the dimeric structure of TDM is important to both Mincle
affinity and its subsequent activation and glucose monoesters do not
activate the Mincle signalling cascade.[8] Here, we report the direct
synthesis of 6-acyl and -amido mannosides with varying lipid lengths
to determine the optimal chain length to balance maximal affinity and
minimal activation. Ainge, et al., have reported evidence the inositol
is responsible for TLR agonist activity, [10b] whereas more complex,
natural PIM6 structures negatively regulate TLR-4.[10a] Thus, we
have targeted benzyl mannoside derivatives as the aromatic ring is
expected to mimic the hydrophobic face of inositol that contains
three axial hydrogens (Scheme 2). In addition, inositol groups often
bind aromatic groups on proteins or synthetic receptors [14] and the
benzyl group may also interact with cationic side chains via π-cation
interaction. Moreover, mannose and its derivatives are widely used
to target the macrophage mannose receptor (MMR), a pattern
recognition receptor involved in host defense, innate immunity,
triggering cytokine production, and modulating cell surface receptors.
Scheme 4. Synthesis of benzyl palmitoylamido-α-D-mannoside from 1.
Reagents and conditions: (i) p-TsCl, pyridine, 0oC, 12 h; (ii) NaN3, DMF, 100oC,
24 h; (iii) Zn/NH4Cl, Methanol/Water (9:1), rt, 1 h; (iv) Palmitoyl chloride,
pyridine, 0oC, 12 h; (v) Na, Methanol, rt, 24 h.
2. Results and Discussion
Since these esters may be subject to cleavage by lipases, we
synthesized an amide derivative as a control. To synthesize benzyl
palmitoylamido-α-D-mannoside (PABM), benzyl 6-azido-6-deoxy-α-D-
mannoside (6) was obtained following published protocol of Wang
and co-workers, omitting the acetylation/deacetylation sequence
Several methods are available for the selective introduction of an
ester linkage at the C-6 primary hydroxyl group of a hexopyranoside.
[15] Previously, we have investigated esterification under Mitsunobu
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800150
ChemBioChem
COMMUNICATION
(Scheme 4).[19] The azido group was reduced to the benzyl 6-
amino-6-deoxy-α-D-mannoside (7) with zinc and ammonium chloride
under mild conditions.[20] The resulting primary amine was then
reacted with palmitoyl chloride to give benzyl 6-palmitoylamido-α-D-
mannoside with small amounts of acylation at C3 and C4 positions
(confirmed via NMR, data not shown). Deacylation of the esters
using sodium in methanol provide benzyl 6-palmitoylamido-α-D-
mannoside (PABM, 8).
variation of cytokine production by the amide-linked chain at 6-OH
position of mannose, 6-palmitoylamido-benzyl-α-D-mannoside
(PABM) was tested in a similar manner. Upon treatment with PABM,
both IL-6 and TNF-α secretions decreased in the LPS treated cells
(Fig. 2 e and f). However, the amount of IL-6 was enhanced at 40
μM of PABM concentration (Fig. 2 e), opposite to that observed with
the related ester PBM.
Having successfully prepared the acyl and amido benzyl α-D-
mannosides, the toxicity of these compounds was investigated
against human U937 macrophages at different concentrations by
MTT assay, to allow exclusion of a nonspecific cellular toxicity as a
credible explanation for any altered cytokine output. As shown in
Figure 1, the glycolipids displayed little cytotoxicity on the
differentiated U937 cells up to 40 µM. The results show that these
mannosides are relatively non-toxic even at fairly high
concentrations and are suitable for interrogating macrophage
cytokine output.
D M S O
 M
120
5
100
80
60
40
20
0
1 0  M
2 0  M
4 0  M
8 0  M
Figure 2. Production of proinflammatory (IL-6 and TNF-α) cytokines upon 24 h
treatment with PBM (2a and 2b), SBM (2c and 2d) and PABM (2e and 2f)
against LPS treated (10 nm) and untreated U937 cells. The supernatants were
analysed for IL-6 and TNF-α production at 24 h by ELISA. For all graphs, the
means and S.D. of triplicate samples from representative experiment
performed in duplicate are shown. * p <0.05, ** p <0.01, *** p <0.001 vs. blank
control group.
M B M
P B M
S B M
P AB M
Figure 1. Effects of glycolipids on U937 cell viability. A MTT assay was
performed to detect In vitro cytotoxic effects of MBM, PBM, SBM, and PABM
against U937 cells after 24 h incubation. Cells were treated with 0-80 µM of
corresponding glycolipids. Results were expressed as mean ± S.D (n = 6) of
three independent experiments.
Macrophage phagocytosis of virulent MTB involves the MMR, and
our results are consistent with previously published results that
simple mannose can inhibit the secretion of proinflammatory
cytokines by upregulated expression of MMR.[22] The MMR
inhibitor mannan (1 mg/ml) was used to block mannose receptors by
treating the differentiated U937 macrophages for an hour. The cells
were then treated with the synthesized glycolipids to determine the
cytokine production after 24 h at a fixed concentration of 20 µM. We
still observed significant inhibition of IL-6 and TNF-α secretion in
LPS treated cells (Fig. 3a and b); however, there was no
considerable amount of cytokines secreted from the LPS untreated
cells. This indicates these glycolipids exert their anti-inflammatory
potential through receptors other that the MMR.
Macrophages play a critical role in the initiation, maintenance, and
resolution of inflammation. Stimulated by bacterial endotoxins, e.g.,
lipopolysaccharide (LPS), macrophages can secrete large amounts
of inflammatory cytokines, such as TNF- α and IL-6, and these
inflammatory cytokines further activate macrophages, creating a
vicious feedback cycle increasing the inflammatory response.[21] To
explore the abilities of these glycolipids to interact with macrophages,
we set out to determine the proinflammatory cytokine (TNF-α and IL-
6) secretion by ELISA after incubation with the synthesized
glycolipids. Interestingly, all the glycolipids significantly inhibited
cytokine production compared to control in a dose dependent
manner in LPS primed macrophages, and this inhibition was greater
for compounds with longer lipid chains (C16/C18). For instance, the
secretions of IL-6 and TNF-α were significantly lower at higher
concentration (20 μM) compared to untreated macrophages (PBM,
Fig. 2 a and b). Similar results were observed with extended steroyl
group on the 6-OH position of the mannose (SBM, Fig. 2 c and d).
The levels of TNF-α were not significantly altered upon treatment
with PBM and SBM in the LPS untreated cells. To explore the
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800150
ChemBioChem
COMMUNICATION
Figure 3. In Figure 3a and 3b, LPS treated (10 nm) and untreated cells were
incubated 24 h with 20 µM of corresponding glycolipids following treatment
with Mannan (1mg/ml) for 1 hour. The supernatants were analysed for IL-6
and TNF-α production at 24 h by ELISA. For all graphs, the means and S.D of
triplicate samples from representative experiment performed in duplicate are
shown. * p <0.05, ** p <0.01, *** p <0.001 vs. blank control group.
Benzyl 6-myristoyl-α-D-mannopyranoside (MBM, 3):
6-myristoyl-benzyl-α-D-mannopyranoside (3) was obtain from benzyl-α-D-
mannopyranoside in 72% yield.
1H-NMR (400 MHz, CDCl3) δ: 0.9 (t, 3 H, J = 6.8 Hz, RCH3), 1.27-1.31 (m,
20 H, RCH2R), 1.67 (dt, 2 H, J = 14.8, 7.4 Hz, RCH2R), 2.34-2.46 (m, 2 H,
-COCH2R), 3.61 (t, 1 H, J = 9.6 Hz, H-4), 3.75-3.79 (m, 1 H, H-5), 3.92
(dd, 1 H, J = 9.3, 3.4 Hz, H-3), 4.02 (dd, 1 H, J = 3.3, 1.4 Hz, H-2), 4.17
(dd, 1 H, J = 12.3, 2.2 Hz, H-6a), 4.55 (d, 1 H, J = 11.9 Hz, -OCH2Ph),
4.62 (dd, 1 H, J = 12.3, 4.0 Hz, H-6b), 4.73 (d, 1 H, J = 11.9 Hz, -
OCH2Ph), 4.97 (s, 1 H, H-1), 7.31-7.40 (m, 5 H, Ar-H); 13C NMR (CDCl3)
δ: 14.13 (RCH3), 22.70 (RCH2R), 24.77 (RCH2R), 24.96 (RCH2R), 29.13
(RCH2R), 29.20 (RCH2R), 29.33 (RCH2R), 29.37 (RCH2R), 29.51
(RCH2R), 29.67 (RCH2R), 29.68 (RCH2R), 29.71 (RCH2R), 31.94
(RCH2R), 34.25 (-COCH2R), 63.69 (C-6), 67.75 (C-4), 69.2 (OCH2Ph),
70.58 (C-2), 70.72 (C-5), 71.50 (C-3), 98.99 (C-1), 128.01, 2 x 128.06, 2
x 128.52 (Ar-H), 136.93 (ipso-Ar), 174.87 (-COCH2R) ; ES-MS: m/z =
503.3 [M + Na]+, HRMS (ESI): m/z calcd for [C27H44O7+Na]+: 503.2979;
found: 503.2985.
3. Conclusions
We have successfully synthesized novel mannose derivatives and
further tested against human macrophages for pro-inflammatory
cytokines measurement. The results showed significant reduction of
cytokine production in the LPS-stimulated macrophages. These
novel mannose derivatives follow the same trend of activity as
monoacylated trehalose derivatives where decreasing affinity to
Mincle based on fatty ester lipid chain length has been observed.
[23] Further work is necessary to verify the immune cell target(s) of
the compounds presented here. These derivatives may be used in
liposome preparations or as components in nanoparticle drug
delivery systems for the treatment of macrophage-resident infections.
In addition, a recent report suggests that compounds of this type (e.g.
the methyl mannoside analogous to 2) may have antimicrobial
activity as well. [24]
Benzyl 6-palmitoyl-benzyl-α-D-mannopyranoside (PBM, 4):
Benzyl 6-palmitoyl-α-D-mannopyranoside (4) was obtain from α-D-
benzylmannopyranoside in 68% yield.
1H-NMR (400 MHz, CDCl3) δ: 0.9 (t, 3 H, J = 6.9 Hz, RCH3), 1.27-1.31 (m,
20 H, RCH2R), 1.67 (dt, 2 H, J = 12.3, 2.3 Hz, RCH2R), 2.37-2.49 (m, 2 H,
-COCH2R), 3.60 (t, 1 H, J = 9.6 Hz, H-4), 3.77 (ddd, 1 H, J = 9.8, 3.7, 2.3
Hz, H-3), 3.92 (dd, 1 H, J = 9.2, 3.1 Hz, H-2), 4.02 (dd, 1 H, J = 3.4, 1.5
Hz, H-5), 4.16 (dd, 1 H, J = 12.3, 2.3 Hz, H-6), 4.56 (d, 1 H, J = 11.9 Hz, -
OCH2Ph), 4.64 (dd, 1 H, J = 12.3, 3.9 Hz, H-6a), 4.73 (d, 1 H, J = 11.9
Hz, -OCH2Ph), 4.97 (d, 1 H, J = 1.1 Hz, H-1), 7.31-7.40 (m, 5 H, Ar-H);
13C NMR (CDCl3) δ: 14.14 (RCH3), 22.71 (RCH2R), 24.97 (RCH2R),
29.19 (RCH2R), 29.33 (RCH2R), 29.38 (RCH2R), 29.51 (RCH2R), 29.66
(RCH2R), 29.68 (RCH2R), 29.72 (RCH2R), 31.94 (RCH2R), 34.25 (-
COCH2R), 63.62 (C-6), 67.74 (C-4), 69.24 (OCH2Ph), 70.55 (C-2), 70.73
(C-5), 71.46 (C-3), 99.05 (C-1), 128.02, 2 x 128.06, 2 x 128.52 (Ar-H),
136.93 (ipso-Ar), 174.93 (-COCH2R) ; ES-MS: m/z = 531.1 [M + Na]+,
HRMS (ESI): m/z calcd for [C29H48O7+Na]+: 531.3292; found: 531.3302.
4. Experimental Section
4.1 Preparation of acylated benzyl α-D-mannopyranosides:
General Procedure
A solution of benzyl α-D-mannoside (0.2g, 0.75 mmol) in 2,6-lutidine (5 ml,
43.2 mmol) at 0˚C under nitrogen was prepared and a solution of desired
fatty acid chloride (~1.0 mmol) in 3 ml dichloromethane was added
dropwise. The resulting mixture was stirred vigorously at that
temperature for 3 hours, and at room temperature (20˚C) for 1 h. The
reaction was monitored by TLC and visualization of TLC was performed
by UV fluorescence and by charring plates with a mixture of 5% H2SO4 in
ethanol. Following reaction time, 2 mL of methanol was poured into the
suspension, and the mixture was concentrated under reduced pressure.
The solid residue was purified by column chromatography on silica gel
(CH2Cl2/MeOH, 10:1) to yield desired fatty acylated benzyl α-D-
mannosides (~ 65-70%) as colorless oils.
Benzyl 6-stearoyl-α-D-mannopyranoside (SBM, 5):
Benzyl 6-stearoyl-α-D-mannopyranoside (5) was obtain from benzyl α-D-
mannopyranoside in 65% yield.
1H-NMR (400 MHz, CDCl3) δ: 0.81 (t, 3 H, J = 6.6 Hz, RCH3), 1.17-1.18
(m, 28 H, RCH2R), 1.53 (dd, 2 H, J = 14.0, 6.9 Hz, RCH2R), 2.27-2.31 (m,
2 H, -COCH2R), 3.56 (t, 1 H, J = 9.4 Hz, H-4), 3.69-3.72 (m, 1 H, H-5),
3.79 (d, 1 H, J = 7.1 Hz, H-3), 3.89 (s, 1 H, H-2), 4.19 (d, 1 H, J = 11.6 Hz,
H-6a), 4.36 (d, 1 H, J = 5.5 Hz, -OCH2Ph), 4.41 (d, 1 H, J = 11.7 Hz, H-
6b), 4.62 (d, 1 H, J = 11.8 Hz, -OCH2Ph), 4.82 (s, 1 H, H-1), 7.21-7.28 (m,
5 H, Ar-H); 13C NMR (CDCl3) δ: 14.13 (RCH3), 22.71 (RCH2R), 24.97
(RCH2R), 29.20 (RCH2R), 29.33 (RCH2R), 29.38 (RCH2R), 29.52
(RCH2R), 29.68 (RCH2R), 29.69 (RCH2R), 29.73 (RCH2R), 31.94
(RCH2R), 34.25 (-COCH2R), 63.63 (C-6), 67.74 (C-4), 69.23 (OCH2Ph),
70.56 (C-2), 70.72 (C-5), 71.47 (C-3), 99.0 (C-1), 128.01, 2 x 128.06, 2 x
128.52 (Ar-H), 136.93 (ipso-Ar), 174.91 (-COCH2R) ; ES-MS: m/z =
559.4 [M + Na]+, HRMS (ESI): m/z calcd for [C31H52O7+Na]+: 559.3605;
found: 559.3618.
Benzyl 6-lauroyl-α-D-mannopyranoside (LBM, 2):
Benzyl 6-lauroyl-α-D-mannopyranoside (2) was obtain from benzyl α-D-
mannopyranoside in 70% yield.
1H-NMR (400 MHz, CDCl3) δ: 0.81 (t, 3 H, J = 6.9 Hz, RCH3), 1.13-1.24
(m, 16 H, RCH2R), 1.54-1.61 (m, 2 H, RCH2R), 2.31-2.35 (m, 2 H, -
COCH2R), 3.51 (t, 1 H, J = 9.6 Hz, H-4), 3.66-3.70 (m, 1 H, H-5), 3.83
(dd, 1 H, J = 9.3, 3.4 Hz, H-3), 3.93 (d, 1 H, J = 1.9 Hz, H-2), 4.07 (dd, 1
H, J = 12.3, 2.2 Hz, H-6a), 4.46 (d, 1 H, J = 11.9 Hz, -OCH2Ph), 4.54 (dd,
1 H, J = 12.3, 4.0 Hz, H-6b), 4.64 (d, 1 H, J = 11.9 Hz, -OCH2Ph), 4.88 (d,
1 H, J = 1.1 Hz, H-1), 7.24-7.29 (m, 5 H, Ar-H); 13C NMR (CDCl3) δ:
14.13 (RCH3), 22.69 (RCH2R), 24.77 (RCH2R), 24.96 (RCH2R), 29.18
(RCH2R), 29.31 (RCH2R), 29.35 (RCH2R), 29.49 (RCH2R), 29.62
(RCH2R), 29.63 (RCH2R), 31.92 (RCH2R), 34.24 (-COCH2R), 63.53 (C-6),
67.72 (C-4), 69.9 (OCH2Ph), 70.54 (C-2), 70.72 (C-5), 71.44 (C-3), 99.01
(C-1), 128.02, 2 x 128.05, 2 x 128.52 (Ar-H), 136.93 (ipso-Ar), 175.0 (-
COCH2R) ; ES-MS: m/z = 475.3 [M + Na]+, HRMS (ESI): m/z calcd for
[C25H40O7+Na]+: 475.2666; found: 475.2676.
4.2 Synthesis of benzyl palmitoylamido-α-D-mannopyranoside:
General Procedure
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800150
ChemBioChem
COMMUNICATION
Benzyl 6-azido-6-deoxy-α-D-mannopyranoside (6) was synthesized
according to the procedure by Wang et al. [19] Reduction of 6 using zinc
and ammonium chloride in methanol/water (9:1) produced the desired
product benzyl 6-amino-6-deoxy-α-D-mannopyranoside (7, 92.5% yield).
To a solution of 7 (1g, 3.7 mmol) in 20 mL of dry pyridine at 0˚C under
nitrogen environment palmitoyl chloride (4.5 mM) was added dropwise.
The reaction was monitored via TLC. and the starting material was
consumed completely by 12h. After quenching with methanol, the solvent
was evaporated under vacuum. The crude compound was then dissolved
in CH2Cl2 (50 mL) and washed with 1 M HCl (3x 50 mL), NaHCO3 (50
mL), and brine (50 mL), dried over Na2SO4, filtered and concentrated to
yield yellowish solid compound. The residues were purified by flash
chromatography using hexane/EtOAc 5:1 as eluent and obtain a mixture
of acylated benzyl palmitoylamido-α-D-mannopyranosides as white solid
(confirmed by 1H-NMR, 13C-NMR and mass spectroscopy, data not
shown).
4.4 Cell Viability: MTT Assays
Cell proliferation was evaluated using the MTT assay. U937 cells with 50
ng/mL of PMA in 100 µL RPMI 1640 were seeded into 96-well plate (5 x
103 cells/well) for 48 h, washed, and treated with 10, 20, 40 and 80 µM of
different glycolipids for 24 h at 370C. After 24 h treatment, cells were
washed with PBS and MTT stock solution (5mg/mL) was then added to
each well and incubated for an additional 4 h. At the end of the
experiment, the medium was replaced with 100 μL of DMSO to dissolve
the formazan crystals, and the absorbance was measured at 540 nm
using Polarstar Omega 96-well microplate reader (BMG Labtech GmBH,
Germany). The cell viability was calculated using the following formula:
Cell viability (%) = (ODexp − ODblank)/(ODcontrol − ODblank) × 100%.
4.5 ELISA: Determination of the TNF-α and IL-6 Release
Both cytokines were quantified by enzyme-linked immunosorbent assay
(ELISA) obtained from elisakit.com.au, Australia and the ELISA kit was
performed according to producer’s instructions. Briefly, diluted
supernatants (1:5, in RPMI 1640 for U937) were incubated in antibody-
coated 96-well plates to facilitate binding of released cytokines. The
incubation with a biotinylated primary antibody was performed. The next
step was incubation with streptavidin-horseradish peroxidase. All
incubation steps were followed by washing, which guarantees the
removal of unbound molecules. Horseradish peroxidase is capable of
oxidizing its substrate tetramethylbenzidine (TMB), which develops a
stable staining complex with sulfuric acid detectable at 450 nm by means
of a plate reader. The limit of sensitivity for detection of TNF-α and IL-6
was <5 and <1 pg/mL, respectively, with both intra-assay and inter-assay
variability < 10%.
For the deacylation of the acylated compound 0.1 mmol was dissolved in
dry methanol (50 mL) and sodium metal (0.023g, 1.0 mmol) was added
to it slowly at 0oC. The ice bath was removed when the sodium was
dissolved completely and the solution was stirred for 24 h at room
temperature. After reaction time, the solution was neutralized with ion
exchange resin (Dowex 50 WX 8-400 ion exchange resin, Sigma-Aldrich),
filtered and concentrated under reduced pressure to give a yellowish
solid. The residue was purified by flash chromatography using
EtOAc/Hexane 2:3 to 3:2 to obtain the desired benzyl palmitoylamido-α-
D-mannopyranoside as a white solid with yields between 30 and 55%.
Benzyl 6-palmitoylamido-α-D-mannopyranoside (PABM, 9):
Benzyl 6-palmitoylamido-α-D-mannopyranoside (9) was obtain from
benzyl 6-amino-6-deoxy-α-D-mannopyranoside (7) in 40% yield.
4.6 Statistical Analysis
1H-NMR (400 MHz, CDCl3/Methanol; 9:1) δ: 0.81 (t, J = 6.8 Hz, 3H,
RCH3), 1.09-1.25 (m, 24 H, RCH2R), 1.51-1.59 (m, 2 H, RCH2R), 2.14-
2.21 (m, 2 H, -COCH2R), 2.95 (d, 1 H, J = 14.8 Hz, H-4), 3.40 (dd, 1 H, J
= 11.5, 7.7 Hz, H-3), 3.54 (d, 1 H, J = 9.7 Hz, H-2), 3.92 (s, 1 H, H-5),
4.44 (d, 1 H, J = 11.9, H-6), 4.60 (d, 1 H, J = 11.9 Hz, -OCH2Ph), 4.81 (d,
1 H, J = 1.1 Hz, H-1), 7.27 (dt, 5 H, J = 13.5, 4.6 Hz, Ar-H); 13C NMR
(CDCl3) δ: 13.96 (RCH3), 22.59 (RCH2R), 25.78 (RCH2R), 29.27
(RCH2R), 29.42 (RCH2R), 29.56 (RCH2R), 29.59 (RCH2R), 29.66
(RCH2R), 29.68 (RCH2R), 31.83 (RCH2R), 36.23 (RCH2R), 39.65 (-
COCH2R), 67.42 (C-6), 69.25 (C-4), 70.37 (OCH2Ph), 71.21 (C-2), 76.78
(C-5), 77.30 (C-3), 99.26 (C-1), 127.80, 2 x 127.87, 2 x 128.39 (Ar-H),
137.01 (ipso-Ar), 175.95 (-COCH2R) ; ES-MS: m/z = 530.4 [M + Na]+.
Data from all samples within each group were combined with means ±
standard error of mean (S.E.M.). Data were averaged from three
independent experiments each containing at least 4 replicates. Statistical
significant was determined by one-way analysis of variance (one-way
ANOVA) and a Dunnett’s multiple comparison was used to compare the
effect among groups using GraphPad Prism software (version 6.01, La
Jolla, CA). Values with P<0.05 were considered significant.
Acknowledgements
The authors wish to thank the Institute for Glycomics and Griffith
University for financial support through a PhD scholarship to T.M.
and infrastructure funding within the Institute.
4.3 Cell culture, differentiation and sample collection for ELISA
Human leukemic monocyte lymphoma cells U937 were obtained from
American Type Culture Collection (ATCC, Manassas, VA, USA) and
cultured according to the ATCC procedure. Briefly, cells were cultured in
RPMI 1640 (Sigma-Aldrich) medium supplemented with 10% heat-
inactivated foetal bovine serum (FBS), 100U/mL of penicillin, and 100
µg/mL of Streptomycin (Sigma-Aldrich) in a humidified atmosphere of 5%
CO2 at 37°C and used for experiments between passages 12 and 16. To
differentiate the cell line into macrophage-like cells, 50 ng/mL phorbol 12-
myristate 13-acetate (PMA, Sigma-Aldrich) was added to 5 x 105 cells in
1 mL of RPMI 1640 medium for 2 days in 12 wells plate. After
differentiation, the adherent cells were washed with PBS to remove PMA
and non-adherent cells. The adherent cells, referred to as U937
macrophages, were treated with various concentration of fatty acylated
and amido mannose derivatives with or without further stimulation by the
lipopolysaccharide (LPS, 100 ng/mL, Sigma-Aldrich, USA) in fresh
culture medium for 6, 12 and 24 h. Then, 500 µL aliquots were taken
from the supernatant and stored at -80°C until they were used for
measurements by sandwich ELISA assays.
Keywords: mannose • macrophage • lectin • inflammation •
Mincle
[1]
V. Briken, S. A. Porcelli, G. S. Besra, L. Kremer, Mol. Microbiol. 2004,
53, 391-403.
[2]
[3]
[4]
[5]
J. Nigou, M. Gilleron, G. Puzo, Biochimie 2003, 85, 153-166.
R. A. Fratti, J. Chua, I. Vergne, V. Deretic, PNAS 2003, 100, 5437-5442.
D. Maiti, A. Bhattacharyya, J. Basu, J. Biol. Chem. 2001, 276, 329-333.
E. H. Song, A. O. Osanya, C. A. Petersen, N. L. Pohl, J. Am. Chem.
Soc. 2010, 132, 11428-11430. Mannose caps may not dominate LAM
interactions with the host: see, B. J. Appelmelk, et al. Cellular Microbiol.
2008, 10, 930-944.
[6]
[7]
M. B. Richardson, S. J. Williams, Frontiers Immunol. 2014, 5, Article
288, 1-9.
S. Yamasaki, E. Ishikawa, M. Sakuma, H. Hara, K. Ogata, T. Saito,
Nature Immunol. 2008, 9, 1179-1188.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800150
ChemBioChem
COMMUNICATION
[8]
[9]
E. Ishikawa, T. Ishikawa, Y. S. Morita, K. Toyonaga, H. Yamada, O.
Takeuchi, T. Kinoshita, S. Akira, Y. Yoshikai, S. Yamasaki, J. Exp. Med.
2009, 206, 2879-2888.
T. Ishikawa, F. Itoh, S. Yoshida, S. Saijo, T. Matsuzawa, T. Gonoi, T.
Saito, Y. Okawa, N. Shibata, T. Miyamoto, S. Yamasaki, Cell Host
Microbe 2013, 13, 477-488.
[10] a) E. Doz, S. Rose, et al. J. Biol. Chem. 2009, 284, 23187-23196. b) G.
D. Ainge, W. J. Martin, , B. J. Compton, C. M. Hayman, D. S. Larsen, G.
F. Painter, S. Yoon, I. A. Wilson, J. L. Harper, J. Med. Chem. 2011, 54,
7268-7279; c) G. M. Rankin, B. J. Compton, K. A. Johnston, C. M.
Hayman, G. F. Painter, D. S. Larsen, J. Org. Chem. 2012, 77, 6743-
6759;
[11] P. A. Trinel, E. Maes, J. P. Zanetta, F. Delplace, B. Coddeville, T.
Jouault, G. Strecker, D. Poulain, J. Biol. Chem. 2002, 277, 37260-71.
[12] R. T. Lee, T. L. Hsu, S. K. Huang, S. L. Hsieh, C. H. Wong, Y. C. Lee,
Glycobiology 2011, 21, 512-520.
[13] a) C. W. Gray Jr, B. T. Walker, R. A. Foley, T. A. Houston, Tetrahedron
Lett. 2003, 44, 3309-3312; b) K. L. Kramp, K. DeWitt, J. W. Flora, D. C.
Muddiman, K. M. Slunt, T. A. Houston, Tetrahedron Lett. 2005, 46, 695-
698; c) T. A. Houston, Curr. Drug Deliv. 2007, 4, 264-268; d) B. T.
Walker, T. A. Houston, Tuberculosis 2013, 93, 102-103.
[14] a) D. W. Heinz, M. Ryan, T. L. Bullock, O. H. Griffith, EMBO 1995, 14,
3855-60; b) K. Niikura, A. Metzger, E. V. Anslyn, J. Am. Chem. Soc.
1998, 120, 8533-34; c) C. W. Gray, K. Barry, E. J. Lindberg, T. A.
Houston, Tetrahedron Lett. 2007, 48, 2683-2686.
[15] M. Therisod, A. M. Klibanov, J. Am. Chem. Soc. 1986, 108, 5638-5640.
[16] a) S. Quader, S. E. Boyd, I. D. Jenkins, T. A. Houston, Org. Biomol.
Chem. 2006, 4, 36-37; b) S. Quader, S. E. Boyd, I. D. Jenkins, T. A.
Houston, J. Org. Chem. 2007, 72, 1962-1979; c) S. Quader, S. E. Boyd,
I. D. Jenkins, T. A. Houston, Carbohydr. Res. 2015, 413, 16-21.
[17] K. Ishihara, H. Kurihara, H. Yamamoto, J. Org. Chem. 1993, 58, 3791-
3793.
[18] M. Zunk, M. J. Kiefel, Tetrahedron Lett. 2011, 52, 1296-1299.
[19] P. Wang, G. J. Shen, Y. F. Wang, Y. Ichikawa, C. H. Wong, J. Org.
Chem. 1993, 58, 3985-3990.
[20] W. Lin, X. Zhang, Z. He, Y. Jin, L. Gong, A. Mi, Synthetic Commun.
2002, 32, 3279-3284.
[21] X. He, J. Shu, L. Xu, C. Lu, A. Lu, Molecules 2012, 17, 3155-3164.
[22] a) L. S. Schlesinger, J. Immunol. 1993, 150, 2920-2930; b) X.-L. Xu, P.
Zhang, Y.-H. Shen, H.-Q. Li, Y.-H. Wang, G.-H. Lu, J.-Y. Zhou, Intl. J.
Clin. Exp. Pathology 2015, 8, 6214-6224.
[23] B. L. Stocker, A. A. Khan, S. H. Chee, F. Kamena, M. S. M. Timmer,
ChemBiochem 2014, 15, 382-388. See also: A. A. Khan, et al.
ChemBiochem 2011, 12, 2572.
[24] A. Smith, P. Nobmann, G. Henehan, P. Bourke, J. Dunne, Carbohydr.
Res. 2008, 343, 2557-2566.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.

10.1002/cbic.201800150
ChemBioChem
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Tamim Mosaiab, Sandra Boiteux, Abu
Hasanat Md Zulfiker, Ming Wei, Milton J.
Kiefel,* and Todd A. Houston*
Page No. – Page No.
Simple Glycolipid Mimic of
Phosphatidylinositol Mannoside Core
from Mycobacterium tuberculosis
Inhibits Macrophage Cytokine
Production
Novel mannose derivatives modified with fatty esters at the C6-position reduce the
release of inflammatory cytokines in human U937 macrophages under induction of
lipopolysaccharides (LPS) after synthetic glycolipid treatment.
For internal use, please do not delete. Submitted_Manuscript
This article is protected by copyright. All rights reserved.