Inhibition of cytokine release by mycobacterium tuberculosis phenolic glycolipid analogues

Article abstract of DOI:10.1002/cbic.201402001

Infection by Mycobacterium tuberculosis causes tuberculosis, a disease characterized by alteration of host innate and adaptive immunity. These processes are mediated by a series of bacterial biomolecules, among which phenolic glycolipids (PGLs) and the related p-hydroxybenzoic acid derivatives have been suggested to play important roles. To probe the importance of structural features of these glycans on cytokine modulation, we synthesized three M. tuberculosis PGL analogues (1-3), which differ from the native glycoconjugates by possessing a simplified lipid algycone. The ability of 1-3 to modulate the release of proinflammatory cytokines (TNF-α, IL-1β, IL-6, MCP-1) and nitric oxide (NO) was evaluated. None of the compounds stimulated the secretion of these signalling molecules. However, all showed a Toll-like Receptor 2-mediated, concentration-dependent inhibition profile that was related to the methylation pattern on the glycan. Inflammatory contribution: Three simplified analogues of phenolic glycolipids from Mycobacterium tuberculosis were synthesized, and their ability to modulate the release of proinflammatory cytokines (TNF-α, IL-1β, IL-6, and MCP-1) and nitric oxide was evaluated. All demonstrated a Toll-like Receptor 2-mediated, concentration-dependent inhibition profile related to the methylation pattern on the glycan.

Full text of DOI:10.1002/cbic.201402001

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DOI: 10.1002/cbic.201402001
Inhibition of Cytokine Release by Mycobacterium
tuberculosis Phenolic Glycolipid Analogues
Hassan R. H. Elsaidi and Todd L. Lowary*[a]
Infection by Mycobacterium tuberculosis causes tuberculosis, a
disease characterized by alteration of host innate and adaptive
immunity. These processes are mediated by a series of bacteri-
al biomolecules, among which phenolic glycolipids (PGLs) and
the related p-hydroxybenzoic acid derivatives have been sug-
gested to play important roles. To probe the importance of
structural features of these glycans on cytokine modulation,
we synthesized three M. tuberculosis PGL analogues (1–3),
which differ from the native glycoconjugates by possessing
a simplified lipid algycone. The ability of 1–3 to modulate the
release of proinflammatory cytokines (TNF-a, IL-1b, IL-6, MCP-
1) and nitric oxide (NO) was evaluated. None of the com-
pounds stimulated the secretion of these signalling molecules.
However, all showed a Toll-like Receptor 2-mediated, concen-
tration-dependent inhibition profile that was related to the
methylation pattern on the glycan.
Introduction
Tuberculosis, caused by infection with Mycobacterium tubercu-
losis, remains a serious disease that leads to more than a million
deaths annually.[1] Moreover, the emergence of multidrug re-
sistant (MDR) and extensively drug resistant (XDR) M. tuberculo-
sis strains, as well as coinfection with HIV, poses a worldwide
threat.[2] There has therefore been increased interest in under-
standing how the organism manipulates the host immune
response, a process that is essential to disease progression.[3–6]
One characteristic of M. tuberculosis is its ability to invade and
neutralize the antimicrobial arsenal of alveolar macrophag-
es.[3–6] This ability is believed to be attributable, in part, to
unique cell wall components that help the organism invade
macrophages and control phagosomal pH, thus rendering
these cells a protective niche for the bacilli.[6] In addition, cell-
wall components help the organism suppress cytokine-induced
apoptosis of macrophages, as well as the action of other mac-
rophage-mediated antimicrobial mechanisms.[5a]
Phenolic glycolipids (PGLs, Scheme 1) are important immu-
nomodulatory components of the mycobacterial cell wall.[7] It
has been shown that most of the M. tuberculosis strains that
produce PGLs belong to Beijing genotype strains,[8] which are
responsible for major disease outbreaks in different areas of
the world.[9] Beijing genotype strains are also associated with
multidrug resistance, especially in HIV-positive individuals.[10]
Another study has demonstrated that the Beijing genotype
strains are hypervirulent in a murine disease model.[11] Unlike
PGLs, p-hydroxybenzoic acid derivatives (p-HBADs, Scheme 1)
are produced by all strains of M. tuberculosis.[12] p-HBADs share
Scheme 1. Structures of M. tuberculosis PGLs and p-HBADs.
a close structural similarity with PGLs and are produced by re-
lated pathways.[13]
The impact of tuberculosis infection on proinflammatory cy-
tokine production by the host immune defense system is influ-
enced by the presence of PGLs.[7,14] For example, it was dem-
onstrated that infection with a Beijing genotype strain (or ex-
posure to its lipid extract) elicited a TH2-dependent immune re-
sponse and inhibition of TNF-a, IL-10, and IL-12.[15] In contrast,
non-Beijing strains induced a TH1-dependent response. In an-
other study,[14] infection of mice with a Beijing HN 878 knock-
out strain (PGL biosynthesis disrupted) resulted in longer sur-
vival than for the wild-type strain. In the same study, it was
found that the bone-marrow-derived macrophages infected
with the wild-type strains showed decreased secretion of the
proinflammatory cytokines, TNF-a, IL-6, and IL-12, when com-
[a] Dr. H. R. H. Elsaidi, Dr. T. L. Lowary
Alberta Glycomics Centre and Department of Chemistry
University of Alberta, Gunning-Lemieux Chemistry Centre
11225 Saskatchewan Drive, Edmonton, AB T6G 2G2 (Canada)
E-mail: tlowary@ualberta.ca
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cbic.201402001.
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pared to those infected with the mutant strain.[14]
The results of this study implicate PGLs as virulence
factors that modulate the host innate immunity, at
least in the Beijing family. Finally, the protective role
of TNF-a against tuberculosis infection was found to
be influenced by the presence of PGLs.[16]
Previously, we used synthetic analogues of PGLs
from Mycobacterium leprae and Mycobacterium bovis;
the complex lipid core was truncated to a p-methox-
yphenol aglycone to probe the effect of the methyla-
tion pattern on cytokine-modulating activity.[17] The
use of these synthetic analogues allowed us to over-
come some of the difficulties associated with the use
of the natural PGLs in studies of this type. These diffi-
culties include their extremely poor aqueous solubili-
ty and the possible presence of small quantities of
other immunoactive impurities in material isolated
from mycobacteria. We report here the synthesis of
a similar panel of analogues of known PGLs from
M. tuberculosis (1–3, Scheme 2),[7,18] and an evaluation
of their ability to modulate the production of various
cytokines and nitric oxide (NO). This work comple-
ments a very recent study by Scanlan and co-workers
on p-HBAD-I and p-HBAD-II obtained by chemical
synthesis.[19]
Scheme 2. Structures and retrosynthetic analysis of 1–3; Tol=p-toluyl.
Results and Discussion
Synthesis of 1–3
In previous related synthetic work, van Boom and co-
workers prepared the aminopropyl trisaccharide de-
rivative of PGL-Tb-1 and two disaccharide frag-
ments.[20a] Subsequently, Gurjar and Viswanadham
synthesized the methyl glycoside derivative of the
PGL-Tb-1 trisaccharide and a disaccharide derivative
lacking the tri-O-methylated fucose residue.[20b] Re-
cently, the total synthesis of the entire PGL-Tb-1 mole-
cule, including the complex lipid, was reported by
Minnaard and co-workers.[21]
Similarly to the routes developed by Gurjar and
Viswanadham[20b] and by Minnard,[21] we chose to
build up the trisaccharides by sequential addition of
monosaccharide residues from the reducing to the
nonreducing end of the molecule. Thus, the synthesis
of the target trisaccharides 1–3 was achieved from
building blocks 4–7 (Scheme 2). The routes used for
the synthesis of 4–7 are shown in the Supporting
Information, and the assembly of 1–3 is depicted in
Scheme 3.
Scheme 3. Assembly of 1–3. A) a) NIS, AgOTf, CH2Cl2, ꢀ208C, 30 min, 87%;
b) NH2NH2·HOAc, CH2Cl2, 4 h, 77%; c) 6, NIS, AgOTf, CH2Cl2, ꢀ408C 30 min; d) NaOCH3,
CH3OH/CH2Cl2 (1:1), 5 h, 70% over two steps; e) CH3I, NaH, DMF, 1 h; f) Pd/C, H2, CH3OH/
CH2Cl2 (1:1), three days, 71% over two steps; g) Pd/C, H2, CH3OH/CH2Cl2 (1:1), overnight,
79%. B) Assembly of 3: h) NIS, AgOTf, CH2Cl2, ꢀ408C, 30 min; i) NaOCH3, CH3OH/CH2Cl2
(1:1), 5 h, 79% over two steps; j) CH3I, NaH, DMF, 1 h; k) Pd/C, H2, CH3OH/CH2Cl2 (1:1),
two days, 77% over two steps.
Conversion of 9 into a trisaccharide was explored by using
The synthesis was initiated by the construction of disacchar-
NIS-AgOTf-mediated coupling with thioglycoside 6.[22] Unex-
pectedly, this reaction failed to give the desired a-glycoside as
a single product; instead, a 1:1 a/b mixture was obtained, and
this was not separable by chromatography. The use of an “in-
verse” glycosylation procedure[23] circumvented this problem:
a solution of 6 was added slowly to a mixture of 9, NIS, and
ide 8 (87% yield) through the coupling of thioglycoside 5 and
acceptor
4 by using NIS-AgOTf-promoted glycosylation
(Scheme 3A). Subsequent treatment of 8 with hydrazine ace-
tate resulted in selective removal of the levulinoyl group to
afford 9 in 77% yield.
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AgOTf over 10 min at ꢀ408C. This addition sequence provided
exclusively the a-anomer. Subsequent Zemplꢁn deacylation of
the product trisaccharide gave 10 in 70% yield over two steps.
The resulting hydroxyl group was converted to a methyl ether
by using methyl iodide and sodium hydride. Finally, debenzyla-
tion over palladium on charcoal under a hydrogen atmosphere
afforded 1 in 71% yield over two steps. The synthesis of 2 was
also achieved from trisaccharide 10 (79% yield) through hydro-
genolytic cleavage of the benzyl ethers.
on cytokine inhibition. Among the tested trisaccharides, 1
showed the highest inhibition activity; 2 (lacking the O-2
methyl group) showed 12–14% lower activity; and 3 (lacking
the O-3’’ methyl group) showed a more substantial loss of in-
hibitory activity (29–44% relative to 1). Thus, the methylation
pattern is important for immunomodulatory activity, with re-
moval of the methyl group at O-3’’ having the most deleteri-
ous effect.
The synthesis of 3 (Scheme 3B) started by glycosylation of
acceptor 9 with donor 7 by using NIS-AgOTf-mediated inverse
glycosylation, as described for the preparation of 10. Subse-
quent Zemplꢁn deacetylation of the trisaccharide product fur-
nished 11 in 79% yield over two steps. The synthesis was com-
pleted by methylation of the hydroxyl group and then hydro-
genolysis over palladium on charcoal to afford 3 over two
steps in 77% yield. In all newly formed glycosidic bonds, the
Conclusions
We synthesized three M. tuberculosis PGL analogues (1–3) and
evaluated their ability to modulate cytokine and NO release.
None induced release of NO or of the evaluated cytokines
(TNF-a, IL-1b, IL-6, MCP-1); they all showed a concentration-de-
pendent inhibitory effect on the release of these signaling mol-
ecules. The effect was shown to be dependent on the methyla-
tion pattern of the molecule and mediated through TLR2.
These results are in agreement with our earlier work on syn-
thetic analogues from M. leprae and M. bovis[17] and, taken to-
gether, demonstrate that the glycan portion of PGLs alone can
inhibit release of the evaluated cytokines and NO. This conclu-
sion is further supported by a recent investigation on p-HBAD-I
and p-HBAD-II.[19] In this study, Scanlan and co-workers showed
that p-HBAD-I and an analogue lacking the methyl group on
the rhamnose moiety exhibit an inhibitory effect on the release
of some cytokines, in particular TNF-a and IL-12p40. However,
no effect was observed with the more complex p-HBAD-II, al-
though this glycan did suppress the release of IL-6. Trisacchar-
ide 1 and p-HBAD-II differ only in the nature of the aglycone
(OCH3 vs. CO2CH3); this, similarly to the data obtained on 1–3,
underscores the importance of subtle structural differences on
immunomodulatory activity.
1
required a-linkage was clearly evident from the value of JC-1’’,H-
of the newly introduced monosaccharide residue (169.8–
1’’
170.0 Hz).[24]
Immunomodulatory activity of 1–3
The ability of 1–3 to modulate cytokine and NO release was
evaluated with activated THP-1 (A-THP-1) cells (Figure 1). THP-
1 (human acute leukemic monocyte/macrophage) cells were
chosen because they provide a close model of alveolar macro-
phages, the first point of contact between the immune system
and mycobacteria, as well as reproducibility in both growth
and macrophage functions.[25] The proinflammatory cytokines
TNF-a, IL-1b, IL-6, chemokine monocyte chemotactic protein-
1 (MCP-1), and nitric oxide (NO) were selected for evaluation;
all have been implicated in mycobacterial infection.[26]
Compounds 1–3 were first tested for their ability to stimu-
late cytokine and NO release. In this assay, A-THP-1 cells were
challenged with each compound, and then cytokine concen-
tration in the cell supernatant was determined by ELISA. As a
positive control, A-THP-1 cells were treated with Pam3CSK4 (a
synthetic Toll-like receptor 2 (TLR2) agonist).[27] In all cases, the
cytokine concentration was comparable to that of the negative
control (unstimulated A-THP-1 cells), thus demonstrating that
none of the synthetic analogues induces production of cyto-
kines under these conditions (Figure 1A).
Experimental Section
General methods for chemical synthesis: Solvents used in reac-
tions were purified by successive passage through columns of alu-
mina and copper under an argon atmosphere. All reagents were
purchased from commercial sources and were used without further
purification unless noted otherwise. All reactions were carried out
under a positive pressure of argon atmosphere and monitored by
TLC on Silica Gel G-25 UV254 (0.25 mm) plates unless stated other-
wise. Spots were detected under UV light and/or by charring with
a solution of anisaldehyde in ethanol, acetic acid, and H2SO4.
Column chromatography was performed on Silica Gel 60 (40–
60 mm; silica gel/residue, 100:1–20:1, w/w). Organic solutions were
We next evaluated the ability of the synthetic analogues to
inhibit stimulus-induced cytokine release. Pam3CSK4-stimulat-
ed A-THP-1 cells were challenged with 1–3, and cytokine con-
centrations were determined. All synthetic analogues demon-
strated concentration-dependent inhibition of the release of all
tested cytokines and of NO (Figure 1B–F). Further insight into
the receptor that mediates this inhibitory activity was obtained
by a series of experiments with ultra-pure E. coli LPS (a TLR4
agonist)[28] as the stimulus. These showed no inhibition of the
release of the tested cytokines (see the Supporting Informa-
tion), thus demonstrating that the inhibitory activity of 1–3 is
mediated through TLR2, and not TLR4.
1
concentrated under vacuum below 508C. H NMR spectra were re-
corded at 400 or 500 MHz, and H NMR chemical shifts are refer-
1
enced to TMS (0.0, CDCl3). 13C NMR spectra were recorded at
125.7 MHz, and chemical shifts are referenced to CDCl3 (77.23,
CDCl3). 1H NMR data are reported as though they are first order,
and the peak assignments were made on the basis of 2D NMR
(1H,1H COSY and HMQC) experiments. In reporting 13C chemical
shifts, when two signals were so closely spaced that rounding to
one decimal place would have led to identical values, the second
decimal place is included in parenthesis (e.g., “78.9(1) and 78.8(8)”).
Optical rotations were measured at (21ꢁ2)8C at the sodium D line
Testing compounds with different methylation patterns ena-
bled us to probe the effect of these hydrophobic substituents
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Figure 1. A) Cytokine stimulation assay. THP-1 cells were differentiated (activated) into mature macrophages (A-THP-1) by treatment with 5 ngmLꢀ1 PMA for
18 h, and then subjected to synthetic compounds at 50 mgmLꢀ1 (dissolved in 0.1% DMSO in RPMI) for 24 h. Culture supernatants were tested for cytokine
levels by ELISA. Pam3CSK4-stimulated A-THP-1 was used as positive control; unstimulated A-THP-1 was the negative control. B)–F) Cytokine inhibition assays
with Pam3CSK4 (a TLR2 agonist; 25 ngmLꢀ1) as the stimulant and compounds (10 or 50 mgmLꢀ1). After 24 h, supernatants were analyzed for TNF-a, MCP-1,
IL-6, IL-1b, and NO by ELISA. Each experiment was repeated twice; concentrations from each experiment were determined in triplicate; data are averages of
six readings (ꢁSD). Solv: solvent, 1% DMSO in RPMI.
(589 nm), and are in degmLdmꢀ1 gꢀ1. ESI-MS spectra were carried
out on samples suspended in THF or CH3OH and added NaCl.
15:1) to give 1 (6 mg, 71%) as an amorphous solid: Rf =0.55
(CH2Cl2/CH3OH, 15:1); ½aꢂD =ꢀ14.8 (c=0.6, CHCl3); 1H NMR
(500 MHz, CDCl3): d=7.02–6.99 (m, 2H; Ar-2,6), 6.84–6.81 (m, 2H;
Ar-3,5), 5.40 (d, J1,2 =1.8 Hz, 1H; H-1), 5.14–5.13 (m, 2H; H-1’, H-1’’),
4.11 (dd, J1’,2’ =1.8 Hz, J2’,3’ =3.4 Hz, 1H; H-2’), 4.05 (dq, J4,5 =9.5 Hz,
p-Methoxyphenyl 2,3,4-tri-O-methyl-a-l-fucopyranosyl-(1!3)-a-
l-rhamnopyranosyl-(1!3)-2-O-methyl-a-l-rhamnopyranoside
(1): NaH (60% in mineral oil, 4 mg) was added to a solution of 10
(12 mg, 0.014 mmol) and CH3I (20 mL) in DMF (2 mL) at 08C. The re-
action mixture was stirred for 1 h, then water (5 mL) was added.
The solution was diluted with CH2Cl2 (10 mL) and washed with
water (2ꢂ10 mL). The organic layer was dried (Na2SO4), filtered,
and concentrated, and the resulting oil was dissolved in CH2Cl2/
CH3OH (1:1, 20 mL), and Pd/C (2.5 mg, 20%, w/w) was added. The
solution was stirred for two days under hydrogen, then filtered,
concentrated, and purified by chromatography (CH2Cl2/CH3OH,
J
5,6 =6.0 Hz, 1H; H-5), 4.03 (dd, J2,3 =3.3 Hz, J3,4 =9.5 Hz, 1H; H-3),
3.93 (dq, J4’,5’ =9.5 Hz, J5’,6’ =6.2 Hz, 1H; H-5’), 3.79 (dd, J2’,3’ =3.4 Hz,
3’,4’ =9.5 Hz, 1H; H-3’), 3.78–3.76 (m, 1H; H-5’’), 3.77 (s, 3H;
J
ArOCH3), 3.74 (dd, J1,2 =1.8 Hz, J2,3 =3.3 Hz, 1H; H-2), 3.69–3.61 (m,
4H; H-2’’, H-4, H-4’, H-4’’), 3.58 (s, 3H; OCH3), 3.57 (s, 3H; OCH3),
3.51 (s, 3H; OCH3), 3.49 (s, 3H; OCH3), 3.47–3.46 (m, 1H; H-3’’), 1.36
(d, J5’’,6’’ =6.0 Hz, 3H; H-6’’), 1.29 (d, J5,6 =6.0 Hz, 3H; H-6), 1.26 ppm
(d, J5’,6’ =6.2 Hz, 3H; H-6’); 13C NMR (125.7 MHz, CDCl3): d=155.0
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(Ar), 150.5 (Ar), 117.5 (2Ar), 114.6 (2Ar), 102.1 (C-1’’), 100.8 (C-1’),
95.6 (C-1), 83.1 (C-3’), 81.0 (C-4’’), 80.0 (C-2), 79.8 (C-3), 79.0 (C-3’’),
78.8 (C-2’’), 71.8 (C-4), 71.6 (C-4’), 71.2 (C-2’), 69.0 (C-5’’), 68.7 (C-5’),
67.5 (C-5), 61.8 (OCH3), 60.2 (OCH3), 58.6 (OCH3), 57.7 (OCH3), 55.7
(OCH3), 17.9 (C-6), 17.8 (C-6’), 16.7 ppm (C-6’’); ESI: calcd for
C29H46O14Na: 641.2785 [M+Na]+; found: 641.2784.
p-Methoxyphenyl 2,4-di-O-benzyl-3-O-levulinyl-a-l-rhamnopyra-
nosyl-(1!3)-4-O-benzyl-2-O-benzoyl-a-l-rhamnopyranoside (8):
Crushed 4 ꢃ molecular sieves (300 mg) was added to a solution of
4 (0.5 g, 1.07 mmol) and 5 (0.65 g, 1.2 mmol) in CH2Cl2 (30 mL). The
reaction mixture was stirred at RT for 30 min, cooled to ꢀ208C,
and NIS (292 mg, 1.3 mmol) and AgOTf (62 mg, 0.24 mmol) were
added. The solution was stirred for another 30 min. The reaction
mixture was then neutralized with Et3N (1 mL), and the solution
was concentrated to a crude residue that was purified by chroma-
tography (hexane/EtOAc, 2:1) to give 8 (0.85 g, 87%) as a syrup:
p-Methoxyphenyl 2,3,4-tri-O-methyl-a-l-fucopyranosyl-(1!3)-a-
l-rhamnopyranosyl-(1!3)-a-l-rhamnopyranoside
(2):
Pd/C
(3 mg, 20% (w/w)) was added to a solution of 10 (15 mg,
0.016 mmol) in CH2Cl2/CH3OH (1:1, 20 mL), and the reaction mix-
ture was stirred for three days under hydrogen. The solution was
then filtered and concentrated, and the resulting residue was puri-
fied by chromatography (CH2Cl2/CH3OH, 15:1) to give 2 (8.2 mg,
79%) as an amorphous solid: Rf =0.45 (CH2Cl2/CH3OH, 15:1); ½aꢂD =
ꢀ12.3 (c=0.8, CHCl3); 1H NMR (500 MHz, CDCl3): d=7.00 (d, J=
9.1 Hz, 2H; Ar-2,6), 6.84 (d, J=9.1 Hz, 2H; Ar-3,5), 5.37 (brs, 1H; H-
1), 5.19 (brs, 1H; H-1’), 5.16 (d, J1’’,2’’ =3.4 Hz, 1H; H-1’’), 4.17 (dd,
1
Rf =0.29 (hexane/EtOAc, 2:1); ½aꢂD =ꢀ19.2 (c=0.5, CHCl3); H NMR
(500 MHz, CDCl3): d=8.10–8.08 (m, 2H; Ar), 7.61–7.58 (m, 1H; Ar),
7.50–7.47 (m, 2H; Ar), 7.46–7.22 (m, 13H; Ar), 7.22–7.11 (m, 2H;
Ar), 6.99 (d, J=9.1 Hz, 2H; Ar-2,6), 6.83 (d, J=9.1 Hz, 2H; Ar-3,5),
5.55 (dd, J1,2 =2.0 Hz, J2,3 =3.4 Hz, 1H; H-2), 5.51 (d, J1,2 =2.0 Hz,
1H; H-1), 5.19 (dd, J2’,3’ =3.3 Hz, J3’,4’ =9.4 Hz, 1H; H-3’), 5.10 (d,
J
1’,2’ =2.2 Hz, 1H; H-1’), 4.92, 4.72 (ABq, J=11.2 Hz, 2H; ArCH2),
4.58, 4.51 (ABq, J=11.7 Hz, 2H; ArCH2), 4.48, 4.42 (ABq, J=12.0 Hz,
2H; ArCH2), 4.39 (dd, J2,3 =3.4 Hz, J3,4 =9.5 Hz, 1H; H-3), 3.97 (dq,
J
1,2 =1.8 Hz, J2,3 =3.1 Hz, 1H; H-2), 4.12 (dd, J1’,2’ =1.5 Hz, J2’,3’ =
J
4,5 =9.5 Hz, J5,6 =6.3 Hz, 1H; H-5), 3.91 (dd, J1’,2’ =2.2 Hz, J2’,3’ =
3.1 Hz, 1H; H-2’), 4.08 (dq, J4’’,5’’ =9.5 Hz, J5’’,6’’ =6.2 Hz, 1H; H-5’’),
4.03 (dd, J2,3 =3.1 Hz, J3,4 =9.5 Hz, 1H; H-3), 3.87 (dq, J4,5 =9.4 Hz,
3.3 Hz, 1H; H-2’), 3.81 (dq, J4’,5’ =9.4 Hz, J5’,6’ =6.3 Hz, 1H; H-5’), 3.66
(appt, J3,4 =J4,5 =9.5 Hz, 1H; H-4), 3.58 (appt, J3’,4’ =J4’,5’ =9.4 Hz,
1H; H-4’), 2.65–2.35 (m, 4H; CH2CH2), 2.11 (s, 3H; CH2-CH3), 1.32 (d,
J
5,6 =6.2 Hz, 1H; H-5), 3.83–3.78 (m, 2H; H-3’, H-5’), 3.77 (s, 3H;
ArOCH3), 3.69–3.62 (m, 4H; H-2’’, H-3’’, H-4, H-4’), 3.59 (s, 3H;
OCH3), 3.57 (s, 3H; OCH3), 3.51 (s, 3H; OCH3), 3.47–3.46 (m, 1H; H-
4’’), 1.35 (d, J5’’,6’’ =6.2 Hz, 3H; H-6’’), 1.28 (d, J5,6 =6.2 Hz, 3H; H-6),
1.26 ppm (d, J5’,6’ =6.2 Hz, 3H; H-6’); 13C NMR (125.7 MHz, CDCl3):
d=155.0 (Ar), 150.2 (Ar), 117.6 (2Ar), 114.6 (2Ar), 101.8 (C-1’), 100.7
(C-1’’), 98.4 (C-1), 82.8 (C-3’), 80.8 (C-3’’), 78.9(1) (C-3), 78.9(0) (C-2’’),
78.7 (C-4’’), 72.1 (C-4’), 71.5 (C-4), 71.0 (C-2’), 70.8 (C-2), 69.0 (C-5’),
68.8 (C-5), 67.5 (C-5’’), 61.9 (OCH3), 60.2 (OCH3), 57.7 (OCH3), 55.7
(OCH3), 17.7 (C-6), 17.6 (C-6’), 16.7 ppm (C-6’’); ESI: calcd for
C28H44O14Na: 627.2629 [M+Na]+; found: 627.2628.
J
5,6 =6.3 Hz, 3H; H-6), 1.16 ppm (d, J5’,6’ =6.3 Hz, 3H; H-6’); 13C NMR
(125.7 MHz, CDCl3): d=206.2 (C=O), 172.0 (C=O), 166.0 (Ar), 155.1
(Ar), 150.2 (Ar), 138.4 (Ar), 138.3 (Ar), 138.0 (Ar), 133.3 (Ar), 129.9
(2Ar), 129.7 (Ar), 128.5 (2Ar), 128.4 (2Ar), 128.3 (2Ar), 128.2 (2Ar),
127.7 (2Ar), 127.6(7) (2Ar), 127.6(5) (Ar), 127.4 (Ar), 127.3 (2Ar),
117.8 (2Ar), 114.6 (2Ar), 100.4 (C-1), 96.1 (C-1’), 80.0 (C-5’), 78.9 (C-
5), 78.6 (C-4’), 76.7 (C-4), 75.4 (ArCH2), 73.8 (ArCH2), 73.5 (C-3’), 73.0
(ArCH2), 72.8 (C-2), 68.6 (C-2’), 68.4 (C-3), 55.7 (ArOCH3), 37.8
(CH2CH2), 29.8 (CH2CH2), 28.0 (CH3CO), 18.2 (C-6’), 17.8 ppm (C-6);
ESI: calcd for C52H56O13Na: 911.3613 [M+Na]+; found: 911.3613.
p-Methoxyphenyl 2,4-di-O-methyl-a-l-fucopyranosyl-(1!3)-a-l-
rhamnopyranosyl-(1!3)-2-O-methyl-a-l-rhamnopyranoside (3):
NaH (60% in mineral oil, 5 mg) was added to a solution of 11
(15 mg, 0.016 mmol) and CH3I (20 mL, 0.27 mmol) in DMF (2 mL) at
08C. The solution was stirred for 1 h, then water (5 mL) was added.
The mixture was diluted with CH2Cl2 (10 mL) and washed with
water (2ꢂ10 mL). The organic layer was dried (Na2SO4), filtered,
and concentrated, and the resulting oil was dissolved in CH2Cl2/
CH3OH (1:1, 20 mL), and Pd/C (3 mg) was added. The solution was
stirred for two days under hydrogen then filtered. The filtrate was
concentrated, and the resulting residue was purified by chroma-
tography (CH2Cl2/CH3OH, 15:1) to give 3 (7.7 mg, 77%) as an amor-
phous solid: Rf =0.39 (CH2Cl2/CH3OH, 15:1); ½aꢂD =ꢀ18.4 (c=0.8,
CHCl3); 1H NMR (500 MHz, CDCl3): d=7.00–6.97 (m, 2H; Ar-2,6),
6.84–6.81 (m, 2H; Ar-3,5), 5.41 (d, J1,2 =1.8 Hz, 1H; H-1), 5.26 (d,
p-Methoxyphenyl 2,4-di-O-benzyl-a-l-rhamnopyranosyl-(1!3)-
4-O-benzyl-2-O-benzoyl-a-l-rhamnopyranoside (9): NH2NH2·HOAc
(77 mg, 0.84 mmol) was added to a solution of 8 (0.5 g, 0.56 mmol)
in CH2Cl2 (30 mL). The reaction mixture was stirred for 4 h, then fil-
tered. The filtrate was concentrated, and the resulting residue was
purified by chromatography (hexane/EtOAc, 1:1) to give 9 (350 mg,
77%) as a colorless oil: Rf =0.41 (hexane/EtOAc, 1:1); ½aꢂD =ꢀ20.6
(c=0.4, CHCl3); 1H NMR (500 MHz, CDCl3): d=8.10–8.08 (m, 2H;
Ar), 7.63–7.60 (m, 1H; Ar), 7.51–7.48 (m, 2H; Ar), 7.36–7.26 (m,
12H; Ar), 7.23–7.21 (m, 3H; Ar), 7.02 (d, J=9.1 Hz, 2H; Ar-2,6), 6.84
(d, J=9.1 Hz, 2H; Ar-3,5), 5.56 (dd, J1,2 =2.0 Hz, J2,3 =3.3 Hz, 1H; H-
2), 5.52 (d, J1,2 =2.0 Hz, 1H; H-1), 5.21 (d, J1’,2’ =1.6 Hz, 1H; H-1’),
4.85, 4.75 (ABq, J=11.5 Hz, 2H; ArCH2), 4.73, 4.57 (ABq, J=11.5 Hz,
J
J
1’’,2’’ =3.8 Hz, 1H; H-1’’), 5.02 (d, J1’,2’ =1.7 Hz, 1H; H-1’), 4.15 (dq,
2H; ArCH2), 4.50, 4.30 (ABq, J=11.5 Hz, 2H; ArCH2), 4.44 (dd, J2,3 =
4,5 =9.5 Hz, J5,6 =6.2 Hz, 1H; H-5’’), 4.05 (dd, J1’,2’ =1.7 Hz, J2’,3’
=
3.3 Hz, J3,4 =9.5 Hz, 1H; H-3), 4.00 (dq, J4,5 =9.5 Hz, J5,6 =6.2 Hz, 1H;
H-5), 3.88 (ddd, J2’,3’ =3.6 Hz, J3’,4’ =9.5 Hz, J3’,OH-3’ =8.9 Hz, 1H; H-3’),
3.79–3.76 (m, 1H; H-5’), 3.78 (s, 3H; ArOCH3), 3.71 (dd, J1’,2’ =1.6 Hz,
3.2 Hz, 1H; H-2’), 3.98–3.96 (m, 2H; H-3, H-3’’), 3.81 (dq, J4’,5’
=
9.4 Hz, J5’,6’ =6.2 Hz, 1H; H-5’), 3.76 (dd, J2’,3’ =3.2 Hz, J3’,4’ =9.4 Hz,
1H; H-3’), 3.69 (dd, J1,2 =1.8 Hz, J2,3 =3.3 Hz, 1H; H-2), 3.72 (s, 3H;
ArOCH3), 3.68–3.63 (m, 1H; H-5), 3.61–3.56 (m, 1H; H-4’), 3.55 (s,
3H; OCH3), 3.52–3.48 (m, 1H; H-4), 3.51 (s, 3H; OCH3), 3.50 (s, 3H;
OCH3), 3.45–3.42 (m, 1H; H-2’’), 3.33–3.32 (m, 1H; H-4’’), 1.29 (d,
J
2’,3’ =3.6 Hz, 1H; H-2’), 3.67 (appt, J3,4 =J4,5 =9.5 Hz, 1H; H-4), 3.31
(appt, J3’,4’ =J4’,5’ =9.5 Hz, 1H; H-4’), 2.20 (d, J3’,OH-3’ =8.9 Hz, 1H; OH-
3’), 1.33 (d, J5,6 =6.2 Hz, 3H; H-6), 1.24 ppm (d, J5’,6’ =6.5 Hz, 3H; H-
6’); 13C NMR (125.7 MHz, CDCl3): d=171.4 (C=O), 165.8 (Ar), 155.1
(Ar), 150.2 (Ar), 138.6 (Ar), 138.2 (Ar), 137.7 (Ar), 133.4 (Ar), 129.8
(2Ar), 129.7 (Ar), 128.6 (2Ar), 128.5(2) (Ar), 128.4(8) (Ar), 128.4(5)
(Ar), 128.3 (2Ar), 127.9 (Ar), 127.8 (Ar), 127.7 (2Ar), 127.6 (2Ar),
127.5 (Ar), 127.3 (2Ar), 117.8 (2Ar), 114.6 (2Ar), 99.5 (C-1’), 96.2 (C-
1), 81.7 (C-4’), 80.5 (C-4), 79.8 (C-2’), 77.7 (C-3), 75.2 (ArCH2), 73.9
(ArCH2), 72.8 (C-2), 72.8 (ArCH2), 71.0 (C-3’), 68.5 (C-5), 68.1 (C-5’),
55.7 (ArCH3), 18.2 (C-6’), 17.9 ppm (C-6); ESI: calcd for C47H50O11Na:
813.3245 [M+Na]+; found: 813.3246.
J
5’,6’ =6.2 Hz, 3H; H-6’), 1.20 (d, J5’’,6’’ =6.2 Hz, 3H; H-6’’), 1.19 ppm
(d, J5,6 =6.2 Hz, 3H; H-6); 13C NMR (125.7 MHz, CDCl3): d=156.6
(Ar), 151.7 (Ar), 119.0 (2Ar), 115.7 (2Ar), 104.0 (C-1’), 100.0 (C-1’’),
97.5 (C-1), 84.5 (C-4’’), 81.6 (C-3), 80.6 (C-2), 80.2 (C-2’’), 79.3 (C-3),
73.5 (C-4’), 73.1 (C-4), 72.1 (C-2’), 71.1 (C-3’’), 70.9 (C-5), 70.5 (C-5’),
68.0 (C-5’’), 62.7 (OCH3), 59.5 (OCH3), 58.7 (OCH3), 56.2 (OCH3), 18.2
(C-6), 18.0 (C-6’’), 16.8 ppm (C-6’); ESI: calcd for C28H44O14Na:
627.2623 [M+Na]+; found: 627.2629.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 8
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p-Methoxyphenyl
2,3,4-tri-O-methyl-a-l-fucopyranosyl-(1!3)-
2H; Ar-2,6), 6.84 (d, J=9.1 Hz, 2H; Ar-3,5), 5.40 (brs, 1H; H-1), 5.26
(brs, 1H; H-1’), 5.24 (d, J1’’,2’’ =3.7 Hz, 1H; H-1’’), 5.19, 4.61 (ABq, J=
11.1 Hz, 2H; ArCH2), 4.83, 4.72 (ABq, J=11.8 Hz, 2H; ArCH2), 4.80,
4.69 (ABq, J=11.2 Hz, 2H; ArCH2), 4.54, 4.29 (ABq, J=11.2 Hz, 2H;
2,4-di-O-benzyl-a-l-rhamnopyranosyl-(1!3)-4-O-benzyl-a-l-
rhamnopyranoside (10): Two solutions were prepared. Solution A
was prepared by dissolving acceptor 9 (0.11 g, 0.14 mmol) in
CH2Cl2 (15 mL) containing crushed 4 ꢃ molecular sieves (100 mg).
Solution B was prepared by dissolving donor 6 (53 mg, 0.17 mmol)
in CH2Cl2 (10 mL) containing crushed 4 ꢃ molecular sieves
(100 mg). Both solutions were stirred for 30 min at RT; then solu-
tion A was cooled to ꢀ408C, and NIS (36 mg, 0.17 mmol) and
AgOTf (10 mg, 0.04 mmol) were added. Solution B was then added
dropwise to solution A over 10 min while stirring, and the reaction
mixture was stirred for additional 30 min at ꢀ408C before it was
neutralized with Et3N (2 mL). The solution was then filtered, the fil-
trate concentrated, and the resulting residue was dissolved in
CH2Cl2/CH3OH (1:1, 10 mL) and 1m NaOCH3 in CH3OH (0.5 mL) was
added. After stirring for 5 h at RT, the reaction mixture was neutral-
ized with Amberlite IR-120 H+ resin, filtered, and concentrated.
The resulting oil was purified by chromatography (hexane/EtOAc,
1:1) to give 10 (67 mg, 70%) as a colorless liquid: Rf =0.55
(hexane/EtOAc, 1:1); [a]D =ꢀ106.8 (c=1.3, CHCl3); 1H NMR
(500 MHz, CDCl3): d=7.38–7.35 (m, 15H; Ar), 7.00–6.97 (m, 2H; Ar-
2,6), 6.84–6.81 (m, 2H; Ar-3,5), 5.40 (d, J1,2 =1.9 Hz, 1H; H-1), 5.27
(d, J1’,2’ =1.8 Hz, 1H; H-1’), 5.20 (d, J1’’,2’’ =3.3 Hz, 1H; H-1’’), 5.17,
4.83 (ABq, J=11.3 Hz, 2H; ArCH2), 4.69, 4.60 (ABq, J=11.2 Hz, 2H;
ArCH2), 4.26–4.21 (m, 2H; H-2, H-3), 4.08 (dd, J2’,3’ =3.1 Hz, J3’,4’
=
9.4 Hz, 1H; H-3’), 3.93–3.87 (m, 2H; H-5, H-5’), 3.84–3.82 (m, 2H; H-
2’, H-3’’), 3.78 (s, 3H; ArOCH3), 3.73–3.63 (m, 3H; H-2’’, H-4’, H-5’’),
3.59 (s, 3H; OCH3), 3.58–3.54 (m, 1H; H-4), 3.41 (s, 3H; OCH3), 3.56–
3.54 (m, 1H; H-4’’), 2.44 (br s, 1H; OH-2), 1.37 (d, J5’,6’ =6.2 Hz, 3H;
H-6’), 1.27 (d, J5,6 =6.2 Hz, 3H; H-6), 1.01 ppm (d, J5’’,6’’ =6.3 Hz, 3H;
H-6’’); 13C NMR (125.7 MHz, CDCl3): d=154.9 (Ar), 150.2 (Ar), 138.9
(2Ar), 138.3 (2Ar), 128.5(1) (Ar), 128.4(6) (2Ar), 128.7 (2Ar), 128.4
(2Ar), 128.3(1) (2Ar), 128.2(5) (2Ar), 127.7 (Ar), 127.5 (Ar), 127.5 (Ar),
127.4 (2Ar), 127.2 (2Ar), 127.1 (2Ar), 117.6 (2Ar), 114.6 (2Ar), 99.7
(C-1’), 99.3 (C-1’’), 98.0 (C-1), 80.5 (C-4), 80.2 (C-4’), 79.9 (C-2), 79.7
(C-3), 79.1 (C-3’’), 78.6 (C-3’), 78.5 (C-2’), 78.4 (C-2’’), 75.0 (ArCH2),
74.8 (ArCH2), 72.6 (ArCH2), 71.6 (ArCH2), 71.1 (C-4’’), 69.2 (C-5’), 68.3
(C-5), 66.6 (C-5’’), 61.8 (OCH3), 59.6 (OCH3), 55.7 (OCH3), 18.1 (C-6),
17.9 (C-6’’), 16.5 ppm (C-6’); ESI: calcd for C55H66O14Na: 973.4342
[M+Na]+; found: 973.4345.
General methods for immunoassays
Chemicals, reagents and culture medium: Ultrapure LPS from E. coli
0111:B4 (a TLR4 agonist) and the synthetic bacterial lipoprotein
Pam3CSK4 (a TLR2/TLR1 agonist) were purchased from Invivogen
(San Diego, CA). BD OptEIA human IL-1b, IL-6, MCP-1, TNF-a, and
IL-10 assay kits were purchased from BD Biosciences (Mississauga,
Canada). The FLICA polycaspases kit for measuring apoptosis was
purchased from ImmunoChemistry Technologies, LLC (Blooming-
ton, MN). The Griess Reagent System was purchased from Promega
(Madison, WI). The human myeloid THP-1 monocyte/macrophage
human cell line (a generous donation from Prof. Daniel Barreda)
was maintained in RPMI 1640 with l-glutamine (10 mm, Gibco/Life
Technologies) supplemented with fetal bovine serum (FBS (10%);
ArCH2), 4.56, 4.26 (ABq, J=12.3 Hz, 2H; ArCH2), 4.22 (dd, J1,2
2.0 Hz, J2,3 =3.3 Hz, 1H; H-2), 4.05 (dd, J2,3 =3.3 Hz, J3,4 =9.1 Hz, 1H;
=
H-3) 3.92–3.84 (m, 2H; H-5, H-3’), 3.81 (dd, J1’,2’ =1.5 Hz, J2’,3’
3.1 Hz, 1H; H-2’), 3.77 (s, 3H; ArOCH3), 3.69 (dq, J4’,5’ =9.3 Hz, J5’,6’
6.5 Hz, 1H; H-5’), 3.65–3.59 (m, 2H; H-2’’, H-3’’), 3.57–3.53 (m, 3H;
H-4, H-4’, H-5’’), 3.55 (s, 3H; OCH3), 3.52 (s, 3H; OCH3), 3.37 (s, 3H;
OCH3), 3.22 (dd, J3’’,4’’ =2.9 Hz, J4’’,5’’ =1.9 Hz, 1H; H-4’’), 1.35 (d,
=
=
J
J
5’,6’ =6.5 Hz, 3H; H-6’), 1.25 (d, J5,6 =6.5 Hz, 3H; H-6), 1.01 ppm (d,
5’’,6’’ =6.3 Hz, 3H; H-6’’); 13C NMR (125.7 MHz, CDCl3): d=154.9 (Ar),
150.2 (Ar), 138.8(3) (Ar), 138.8(1) (Ar), 138.3 (Ar), 128.5 (2Ar),
128.2(8) (2Ar), 128.2(7) (2Ar), 127.6 (2Ar), 127.4(9) (Ar), 127.4(5)
(Ar), 127.4(4) (2Ar), 127.2 (2Ar), 127.1 (2Ar), 117.6 (2Ar), 114.6 (Ar),
99.6 (C-1’’), 99.3 (C-1’), 98.0 (C-1), 80.4 (C-4), 80.1 (C-2), 79.9 (C-3),
79.7(1) (C-3’), 79.6(5) (C-3’’), 79.2 (C-2’), 78.7 (C-2’’), 77.8 (C-4’), 75.0
(ArCH2), 74.8 (ArCH2), 71.6 (ArCH2), 71.1 (C-4’’), 69.2 (C-5’), 68.3 (C-5),
66.4 (C-5’’), 61.7 (OCH3), 59.2 (OCH3), 58.0 (OCH3), 55.7 (OCH3), 18.2
(C-6’), 17.9 (C-6), 16.6 ppm (C-6’’); ESI: calcd for C49H62O14Na:
897.4032 [M+Na]+; found: 897.4034.
) ;
Gibco), penicillin (100 UmLꢀ1 and streptomycin (100 mgmLꢀ1
Gibco) in an atmosphere of 5% CO2 at 378C.
Cell inhibition assays: Cells were first differentiated into mature
macrophages by treatment with PMA (5 ngmLꢀ1; Sigma–Aldrich)
for 24 h, and then washed three times with phosphate buffered
saline (PBS). Cells were then rested for two days, treated with tryp-
sin, then washed with and resuspended in serum-free RMPI (5.4ꢂ
105 cellsmLꢀ1). After 2 h, 1 mL was transferred to a 24-well plate
(BD Falcon), then stimulator (Pam3CSK4, Ultra Pure LPS;
25 ngmLꢀ1) and test compound (10 or 50 mgmLꢀ1) were added fol-
lowed by incubation for 24 h in an atmosphere of 5% CO2 at 378C
(triplicate experiments). Unstimulated cells and the solvent used to
dissolve the test compounds were used as negative controls;
stimulated cells without test compound served as the positive con-
trol. Culture supernatants were harvested after 24 h and stored at
ꢀ808C for cytokine measurement by ELISA; the remaining cells
were washed twice with PBS and then tested for apoptosis.
p-Methoxyphenyl 3-O-benzyl-2,4-di-O-methyl-a-l-fucopyranosyl-
(1!3)-2,4-di-O-benzyl-a-l-rhamnopyranosyl-(1!3)-4-O-benzyl-
a-l-rhamnopyranoside (11): Two solutions were prepared. Solu-
tion A was prepared by dissolving 10 (0.11 g, 0.13 mmol) in CH2Cl2
(10 mL) containing crushed 4 ꢃ molecular sieves (100 mg). Solution
B was prepared by dissolving 7 (60 mg, 0.15 mmol) in CH2Cl2
(10 mL) containing crushed 4 ꢃ molecular sieves (100 mg). Both
solutions were stirred for 30 min at RT. Solution A was then cooled
to ꢀ408C, and NIS (43.3 mg, 0.16 mmol) and AgOTf (10 mg,
0.04 mmol) were added. Solution B was added dropwise to solu-
tion A over 10 min while stirring, and then the reaction mixture
was stirred for additional 30 min at ꢀ408C before it was neutral-
ized with Et3N (2 mL). The reaction mixture was filtered and con-
centrated, and the resulting residue was dissolved in CH3OH/CH2Cl2
(1:1, 30 mL), and a catalytic amount of NaOCH3 (1m) in CH3OH
(0.25 mL) was added. After stirring for 5 h at RT, the reaction mix-
ture was neutralized with Amberlite IR-120 H+ resin, filtered, and
concentrated. The resulting oil was purified by chromatography
(hexane/EtOAc, 2:1) to give 11 (105.1 mg, 79%) as a colorless oil:
Cell stimulation assay: The assay was carried out as described for
the cell inhibition assay, but without stimulators.
ELISA cytokine production assay: Cytokine concentrations in the cul-
ture supernatants were determined by ELISA using BD OptEIA
human IL-1b, IL-6, MCP-1, TNF-a and IL-10 assay kits purchased
from BD Biosciences (Mississauga, Canada) according to the manu-
facturer’s procedure. First, microwells of a 96-well plate (BD) were
coated with capture antibody (100 mL per well) diluted in coating
buffer (Na2CO3 (0.1m, pH 9.5)). After coating, the solution in the
wells was aspirated, and the wells were washed (ꢃ300 mL, ꢂ3)
with washing buffer (PBS with Tween-20 (0.05%)). Next, the wells
1
Rf =0.45 (hexane/EtOAc, 2:1); [aD]=ꢀ88.1 (c=0.6, CHCl3); H NMR
(500 MHz, CDCl3): d=7.41–7.21 (m, 20H; Ar), 7.01 (d, J=9.1 Hz,
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 8
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CHEMBIOCHEM
FULL PAPERS
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were blocked with assay diluent (PBS (pH 7.0) with FBS (10%);
ꢃ200 mL per well) and then incubated for 1 h at RT. After aspira-
tion and washing (ꢂ3) as described for the coating step, samples
and standard dilutions were prepared in the assay diluent. The
samples (100 mL), standard, and control were then added to the
appropriate wells (in triplicate), and the plate was sealed and incu-
bated for 2 h at RT. After samples were aspirated, wells were
washed five times (as above). Then, detector solution (100 mL; de-
tecting antibody conjugated to streptavidin–horseradish perox-
idase reagent) was added to each well, and the plate was sealed
and incubated for 1 h at RT. All wells were then aspirated and
washed (ꢂ7, as above). Substrate solution (100 mL, tetramethylben-
zidine (TMB) and hydrogen peroxide) was then added to each well,
and the plate was sealed and incubated for 30 min at RT in the
dark. To stop the reaction after 30 min, stop solution (50 mL; H3PO4
(1m) was added to each well; the intensity of the resulting yellow
color is dependent on the concentration. The absorbance was
measured at 450 nm on a SPECTRAmax 340PC plate reader from
Molecular Devices. To determine the concentrations of the sam-
ples, a standard curve was generated by using data from the
standards, and then the concentration of each sample was deter-
mined from the curve from the measured absorbance.
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Nitric oxide (NO) assay: Nitric oxide concentration was determined
in each sample by measuring the nitrite concentration in each
sample by using the Promega Griess reagent system according to
the manufacturer’s procedure. Samples (50 mL) and nitrite standard
solution (100, 50, 25, 12.5, 6.25, 3.13, and 1.56 mm) were added to
microwells of a 96-well plate (BD). Sulfanilamide solution (50 mL)
was added to each sample and to the standards wells, and the
plate was incubated for 10 min at RT in the dark. After 10 min, N-1-
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Acknowledgements
We acknowledge NSERC, Alberta Innovates Technology Futures,
and the Alberta Glycomics Centre for financial support for this
project.
Keywords: cytokines
· glycolipids · immunomodulators ·
mycobacteria · Toll-like receptors · tuberculosis
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Received: February 2, 2014
Published online on && &&, 0000
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 8
&
7
&
ÞÞ
These are not the final page numbers!

FULL PAPERS
H. R. H. Elsaidi, T. L. Lowary*
Inflammatory contribution: Three sim-
plified analogues of phenolic glycolipids
from Mycobacterium tuberculosis were
synthesized, and their ability to modu-
late the release of proinflammatory cy-
tokines (TNF-a, IL-1b, IL-6, and MCP-1)
and nitric oxide was evaluated. All dem-
onstrated a Toll-like Receptor 2-mediat-
ed, concentration-dependent inhibition
profile related to the methylation pat-
tern on the glycan.
&& – &&
Inhibition of Cytokine Release by
Mycobacterium tuberculosis Phenolic
Glycolipid Analogues
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemBioChem 0000, 00, 1 – 8
&
8
&
ÞÞ
These are not the final page numbers!