Synthesis and immunological evaluation of a low molecular weight saccharide with TLR-4 agonist activity

Article abstract of DOI:10.1016/j.bmc.2016.11.044

The paucity of FDA approved adjuvants renders the synthesis, characterization, and use of new compounds as vaccine adjuvants, a necessity. For this purpose, a novel saccharide analog has been synthesized from glucosamine, pyruvylated galactose and 1,4-cyclohexanediol and its biological efficacy was determined in innate immune cells. More specifically, we assessed the production of pro-inflammatory cytokines from the murine monocyte cell line, Raw 264.7 and from C57 BL/6 mouse peritoneal macrophages following exposure to the saccharide analog. Our data conclude that the novel saccharide has immunostimulatory activity on mouse macrophages as indicated by the elevated levels of IL-6 and TNF-α in culture supernatants. This effect was TLR-4-dependent but TLR-2-independent. Our data, suggest TLR-4 agonism; a key feature of vaccine adjuvants.

Full text of DOI:10.1016/j.bmc.2016.11.044

Accepted Manuscript
Synthesis and immunological evaluation of a low molecular weight saccharide
with TLR-4 agonist activity
Vikram Basava, Heather Romlein, Constantine Bitsaktsis, Cecilia H. Marzabadi
PII:
S0968-0896(16)31266-4
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.11.044
BMC 13411
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
8 September 2016
21 November 2016
23 November 2016
Please cite this article as: Basava, V., Romlein, H., Bitsaktsis, C., Marzabadi, C.H., Synthesis and immunological
evaluation of a low molecular weight saccharide with TLR-4 agonist activity, Bioorganic & Medicinal Chemistry
(
2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.11.044
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Vikram Basava, Heather Romlein, Constantine Bitsaktsis and Cecilia H. Marzabadi
Seton Hall University

Bioorganic & Medicinal Chemistry
Synthesis and immunological evaluation of a low molecular weight saccharide with
TLR-4 agonist activity
a
b
b
a
Vikram Basava, Heather Romlein, Constantine Bitsaktsis,  and Cecilia H. Marzabadi *
a
Department of Chemistry & Biochemistry, Seton Hall University, 400 South Orange Ave., South Orange, NJ 07079, USA
Department of Biological Science, Seton Hall University, 400 South Orange Ave., South Orange, NJ 07079, USA
b
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
The paucity of FDA approved adjuvants renders the synthesis, characterization, and use of new
compounds as vaccine adjuvants, a necessity. For this purpose, a novel saccharide analog has
been synthesized from glucosamine, pyruvylated galactose and 1,4-cyclohexanediol and its
biological efficacy was determined in innate immune cells. More specifically, we assessed the
production of pro-inflammatory cytokines from the murine monocyte cell line, Raw 264.7 and
from C57 BL/6 mouse peritoneal macrophages following exposure to the saccharide analog. Our
data conclude that the novel saccharide has immunostimulatory activity on mouse macrophages
as indicated by the elevated levels of IL-6 and TNF- in culture supernatants. This effect was
TLR-4-dependent but TLR-2-independent. Our data, suggest TLR-4 agonism; a key feature of
vaccine adjuvants.
Available online
Keywords:
agonist
LPS
Saccharide synthesis
TLR-4
Innate immunity
2009 Elsevier Ltd. All rights reserved.
—
——

Corresponding author. Tel.: +0-000-000-0000; fax: +0-000-000-0000; e-mail: author@university.edu

.
TLR-4 agonists are of particular interest to us due to the range
of pathophysiological responses to TLR-4 binding, the diversity
of known TLR-4 ligands (Figure 1), and the different types of
1
. Introduction
11
molecular signals elicited by different TLR-4 agonists. Also,
Vaccination is the only artificial way to prevent disease by
the mechanism of TLR-4 binding has been extensively studied
eliciting robust, long-lived, and specific immune responses.
Pathogenic proteins are important vaccine candidates due to their
ability to stimulate both antibody and cell-mediated immunity.
However, their frequent low immunogenicity can impact their
efficacy as vaccine candidates. Also the use of “killed” pathogen
12
and x-ray structures of bound ligands have been determined.
particles results in an attenuated response versus vaccination with
1
“
live attenuated” particles.
The use of adjuvants is a promising approach to circumvent
these problems but the restricted number of FDA-approved
compounds renders the discovery of novel molecules very
important. Adjuvants are compounds that enhance the specific
immune response against co-inoculated antigens, ideally with
minimal toxicity. Adjuvants have been used with vaccines since
1
the 1920s. The most common vaccine adjuvant that is still in use
today is alum (aluminum phosphate or hydroxide). Both long-
term and short term side effects associated with alum and other
2
adjuvants has lead researchers to look for alternative molecules.
In recent years, the adjuvanticity of many organic molecules,
including polysaccharides and smaller carbohydrates, has been
tested with variable outcomes. The notion of using carbohydrate
Figure 1. Diversity of TLR-4 ligands.
3
recognition to foster an immune response comes from the
important roles they play in nearly every aspect of the innate
immune response. For example, surface carbohydrates of
circulating foreign particles interact with a variety of immune
receptors including pattern recognition receptors (PRRs) such as
toll-like receptors (TLRs), and NOD-like receptors (nucleotide-
The activation of cells via LPS and its analogues has been
12, 36
shown to occur via a series of steps.
First, the Lipid A portion
of LPS binds to the LPS-binding protein (LBP). LBP is believed
to help disaggregate LPS. Then, an LPS-LBP complex transfers
monomeric LPS to CD14 (Cluster of Differentiation 14). CD14
delivers LPS to TLR-4. Binding of LPS to TLR-4 is assisted by
the formation of an MD-2 (lymphocyte antigen 96)-TLR-4
complex. Two of the TLR-4-MD-2 complexes dimerize resulting
in the activation of the toll-like receptor.
Binding of the two complexes leads to one of two paths of
signaling; MyD88 (Myeloid differentiation primary response
gene 88)-dependent or MyD88-independent pathways. When
signaling occurs through the adaptor protein MyD88, a series of
4
binding oligomerization domain-like). PRRs recognize highly
conserved structures called “pathogen associated molecular
patterns” (PAMPs) that are on the surface of microorganisms
5
such as bacteria and viruses. Examples of microbial PAMPs are
lipopolysaccharide (LPS) from Gram-negative bacteria and
peptidoglycan (PGN) from Gram positive organisms. PRRs help
to destroy invading microorganisms mainly by triggering
inflammatory responses upon activation by a PAMP. This
response can lead to the release of cytokines and to the activation
and maturation of T- and B-cells.
Whereas TLRs are located both inside and outside the cell
membrane, NLRs are limited to the cytosol. The major cell
surface TLRs that bind microbial components are TLR-4 (LPS)
and TLR-2 (PGN) (either alone or in combination with TLR-1 or
TLR-6). Collectively, TLRs are transmembrane proteins with a
conserved intracellular Toll-interleulin1 receptor (TIR) domain—
responsible for the induction of signal transduction following
binding and an extracellular leucine-rich repeat (LRR)
6
responsible for ligand binding. Signal transduction occurs
following activation of TLRs through various effector proteins.
TLR signaling induces the activation and maturation of APCs
kinases are activated including mitogen-activated protein kinases
(
antigen presenting cells) and the release of inflammatory
(
MAPKs) and nuclear factor-NF). This ultimately leads
7
19, 20
cytokines such as TNF- and IL-6.
to the production of pro-inflammatory cytokines.
The latter
A variety of molecules have been shown to bind to and
activate TLRs on cells. These include analogs of naturally-
occurring bacterial ligands and a range of other structurally
pathway is based upon the TRIF and TRAM adaptor molecules
that also leads to activation of IRF3 (Interferon Regulatory
Factor 3) and to the production of Type I interferons. This
stimulates dendritic cells (DCs) to express the co-stimulatory
molecules CD40, CD80 and CD86 and this leads to T-cell
8
diverse compounds (Figure 1). Some of these TLR ligands have
9
been utilized as successful vaccine adjuvants. As adjuvants, they
2
5
maturation and differentiation.
target APCs, intermediates to both innate and adaptive immunity.
Internalization of PAMPs by APCs and subsequent
representation to naïve T cells leads to the differentiation to T
cells with specific functions (e.g. Th1, Th2, Th17, Treg) (Th =
helper T cells, Treg = regulatory T cells). Signaling through
TLR-4 leads to Th1 differentiation, whereas signaling through
The modified Lipid A product from the LPS of Salmonella
Minnesota R595, Monophosphoryl Lipid A (MPLA) (Figure 1),
was the first TLR-4 agonist approved for human use as a vaccine
13
adjuvant. It is currently utilized for both HPV and Hepatitis B
14,15
vaccines in the United States.
The reduced toxicity of MPLA
10
has been attributed to the selective induction of the TLR-4-TRIF
TLR-2/TLR-1 or TLR-2/TLR-6 favors Th2 responses.
16
signaling pathway over the TLR-4-MyD88 pathway. Another

smaller analog of Lipid A and MPLA, GLA (Glucopyranosyl
Lipid A), is also being used as a vaccine adjuvant.
4- and 6-hydroxyl groups as a lipophilic pyruvate ester ketal. All
pieces were coupled via thioglycoside donors using standard
17,18
26
Substantial efforts have been made to make other modified
Lipid A analogues with an emphasis on both increasing the
potency and reducing the toxicity of the molecules. The
importance of the number and the type of alcohol and amine
protecting groups has been well-established for the sugar-based
TLR-4 ligands. Lipid A analogues that are not fully acylated
have been shown to act as antagonists, rather than as agonists, for
glycosylation conditions.
For the synthesis of sugar subunit 2, the hydrochloride salt of
27
glucosamine 5 was treated with sodium methoxide to give the
free amino sugar 6 which was then treated with phthalic
anhydride and acetic anhydride, in separate steps, to afford the
peracetylated N-phthalimido protected glucosamine 8 in 50%
28
overall yield (40% of the desired -anomer) (Scheme 1).
2
1
TLR-4. This antagonism is believed to be due to the inability of
TLR-4 to form a dimer with MD-2. This lack of dimer formation
renders TLR-4 unresponsive.
Treatment of the anomeric acetate 8 with ethanethiol and boron
trifluoride – diethyletherate gave 79% of the -thioethyl glycosyl
26
donor 2.
Besides underacylated analogues, ether protection of Lipid A
22
derivatives has also led to reduced TLR-4 signaling. To our
knowledge, the effect of protection of Lipid A analogues as
pyruvate ketals has not yet been described. This interesting
residue has been found in the terminal chain in a variety of
Scheme 1. Synthesis of donor 2.

Thiophenyl glycoside 10 was synthesized from penta-O-
acetyl-D-galactopyranose 9 by treatment with thiophenol and
26, 29
boron trifluoride – diethyletherate (84% yield) (Scheme 2).
Deacetylation of 10 with sodium methoxide and methanol
produced 11 in quantitative yield. The 4,6-benzylidene 12 was
then installed with benzaldehyde/zinc chloride, followed by the
23
capsular polysaccharides including Streptococcus pneumonia,
24
25
Klebsiella sp. and Bacteroides fragilis. In S. pneumoniae, the
presence of a trans-3,4-pyruvate ketal in a galactopyranose
residue is believed to impart resistance of the bacteria to reaction
2
7
23
30
with antibodies to the capsular cell wall. In Klebsiella sp. and
in B. fragilis the immunogenic pyruvate ketal moiety is located
selective benzoylation of the C3 hydroxyl and the selective
2
4
between C4 and C6 of the galactopyranose.
Because of the
acetylation of the C4 –OH group to give 14 (13% over three
31
occurrence of this residue across a spectrum of bacterial types,
we wanted to investigate its ability to elicit and/or augment TLR-
steps). Treatment with trifluoroacetic acid, then methyl
pyruvate, led to replacement of the 4,6-benzylidene functionality
with the pyruvate ester ketal (36% over 2 steps) and to the
4
agonism.
3
2
formation of 3.
In this study, we prepared a small lipophilic molecule with
properties that could potentially mimic those of known TLR-4
agonists such as Lipid A and MPLA. A cyclohexane diol was
substituted for a central carbohydrate residue. Besides acyl
protection, we incorporated a pyruvate ketal on a galactopyranose
residue. The effects of our newly synthesized molecule and its
precursors in models of innate and adaptive immunity were
studied. Preliminary investigations into the mechanism of TLR
activation were also conducted.
2
2
. Results and Discussion
.1 Organic Synthesis of TLR ligand
In designing a potential TLR-4 agonist, we believed it was
important to retain the glucosamine moiety found in the
molecules Lipid A and MPLA. However, instead of protecting
the amino and alcohol functionalities as complex fatty acid
derivatives, as in Lipid A, we opted for simple acetyl protection.
Our analog 1 was prepared in multiple steps from three smaller
units; two saccharide derivatives, 2 and 3, and a protected 1,4-
cyclohexane diol 4 (Figure 2). The diol spacer was utilized to
connect glucosamine-derived sugar fragment 2 to galactose sugar
fragment 3.
Scheme 2. Synthesis of donor 3
Reaction of cis,trans- 1,4-cyclohexanediol 15 with tert-
butyldimethylsilyl chloride (1.25 equiv.) and imidazole (2.5
3
3
equiv.) afforded a mixture of cis, trans- monosilylated diols.
This mixture was chromatographically separated to give cis-3 as
a single isomer (50% yield) (Scheme 3).Treatment of sugar 1
with N-iodosuccinimide (NIS) and trimethylsilyl triflate in the
26
presence of 3 afforded the cyclohexyl glycoside 17, as well as
some of the silylated cyclohexyl glycoside 16. The latter could be
converted to the free alcohol 17 by treatment with tetra-n-
Figure 2. Retrosynthetic analysis for the preparation of analog 1
3
3
butylammonium fluoride (TBAF) in THF. Overall conversion
to 17 (either directly or via 16) was 65%. Thiophenyl glycoside
26
donor 3 was then coupled to the free hydroxyl of 17 to give
compound 1 in 58% yield. Both couplings afforded -anomers in
high yields and selectivities due to the presence of a participatory
It was used as a “sugar surrogate” that would not require the
multiple protection-deprotection steps, as would a central sugar
moiety. Besides simple esterification, 3 was also protected at the
34
C2 ester group.

cells.
Figure 3. Synthetic oligosaccharide analog 1 stimulates IL-6 and TNF-
production in vitro by mouse macrophages.
The murine macrophage cell line, RAW 264.7, and mouse peritoneal
macrophages (PECs) from C56BL/6 wildtype mice were cultured with 1
and g/ml of our synthetic oligosaccharide analog or LPS. At the time
points indicated, supernatants were collected and the levels of IL-6 (A)
and TNF- (B) were measured by ELISA. Cytokine levels were
compared to the negative control (media alone) and significance was
calculated by a one-tailed student t-test (*: p<0.1, **: p<0.05, ***:
p<0.01).
2.3. Pro-inflammatory properties of the saccharide analog
are TLR-4 dependent
The adjuvant effect of many compounds is mediated via TLR
35
Scheme 3. Preparation of analog 1.
signaling. Therefore, following our observation of the pro-
inflammatory properties of our saccharide 1 on murine
To summarize, compound 1 was synthesized in a
macrophages, we sought to identify whether the effect was TLR-
dependent. For this purpose, PECs (C57BL/6) from WT, TLR-2
deficient, and TLR-4 deficient mice were obtained and cultured
at various concentrations of the saccharide molecule 1 and the
pro-inflammatory cytokines IL-6, and TNF-, were measured as
previously described (Figure 4). The adjuvant effect of the
saccharide analogue 1 that was previously observed was
ameliorated in the absence of TLR-4 but was unaffected in the
absence of TLR-2 suggesting that the pro-inflammatory effect
observed is TLR-4 dependent (Figure 4). To verify that in order
to exert its adjuvanticity, the saccharide compound needs to bind
directly on TLR-4 expressed on innate immune cells, we carried
out competitive binding studies between the compound and a PE-
conjugated anti-TLR-4 monoclonal antibody and assessed
binding using flow cytometry (Figure 5). By pre-incubating
mouse PECs with increasing amounts of the saccharide
compound, binding of anti-TLR-4 mAb significantly decreased
indicating not just specificity to this TLR but also that the
saccharide and the antibody share the same binding site on the
receptor. Furthermore, pre-incubation of mouse PECs with the
highest concentration of the saccharide compound did not affect
binding of a PE-conjugated anti-TLR-2 mAb, further confirming
its specificity for TLR-4 (Figure 5).
straightforward manner from easily accessible starting materials.
The sequence carried out (Schemes 1-3) uses known synthetic
procedures to prepare and couple the different components. The
yields ranged from modest to good for all steps in the synthesis.
All compounds were characterized using NMR spectroscopy (1D
and 2D proton and carbon) and by mass spectrometry (ESI).
2
. The novel, saccharide analog has pro-inflammatory
properties on innate immune cells.
Following the synthesis of our saccharide compound 1, we
addressed its potential adjuvanticity by analyzing its pro-
inflammatory properties on the murine monocyte/macrophage
cell line, RAW 264.7, and on mouse (C57 BL/6) peritoneal
exudate macrophages (PECs). For this purpose, both cell types
were cultured with varying concentrations of the saccharide
compound and at different time points supernatants were
harvested. The IL-6 and TNF- cytokine concentrations in the
supernatants were assessed by ELISA (Figure 3). The saccharide
compound had an inflammatory effect on both RAW 264.7 cells
and the PECs in terms of IL-6 and TNF- production. More
specifically, IL-6 production was significantly increased at
different saccharide concentrations after 48 hours of culture
while a significant increase in TNF- concentration was
observed within 2 hours of culture (Figures 3A and 3B,
respectively).
Monosaccharides 2 and 3 were also evaluated separately for
their ability to elicit pro-inflammatory cytokine production in
mouse peritoneal macrophages. Both monosaccharides led to
only modest increases in IL-6 production (~ 50% of 1) at 24 hrs.
When TNF-was measured at the same time point, only 1
produced any significant cytokine response (~ 50 % of 3) (data
not shown).
2.4. Discussion
In this study, we have synthesized a new, small saccharide analog
in its protected form and through various immunoassays we
assessed its biological activity on innate and adaptive immunity
in mouse cells. Our biological studies showed that our saccharide
analog triggered the production of pro-inflammatory cytokines
by murine macrophages via a TLR-4 dependent mechanism.
Also, the molecule failed to activate T- and B- lymphocytes,
indicating that 1 has probably no direct effects on the murine
adaptive immune system but rather, exerts its adjuvanticity.
exclusively via its inflammatory properties on innate immune
Further studies with the trisaccharide showed that when 1 was
cultured with mouse splenocytes, no polyclonal activation of T-
or B-lymphocytes was observed (data not shown) indicating that
this molecule has probably no direct effects on the murine
adaptive immune system but rather, exerts its adjuvanticity
exclusively via its inflammatory properties on innate immune

cells.
by a Lipid A derivative. Binding of our small saccharide analog
to TLR-4 was confirmed by competitive assays using flow
cytometry, although in the absence of additional studies it is
difficult to say what the exact mechanism of binding of our
molecule to TLR-4 or to the TLR-4 complex really is.
Figure 4. Pro-inflammatory properties of trisaccharide analog 1 are `
TLR-4-dependent.
Mouse PECs from C56BL/6 wildtype mice, TLR-2 and TLR-4 deficient mice
were cultured with 1 and 5g/ml of the synthetic trisaccharide analog or LPS.
At the time points indicated, supernatants were collected and the levels of IL-
Several factors may contribute to the activity of our
trisaccharide analog 1. First, the large number of lipophilic acetyl
groups present in the molecule may contribute to its binding to
TLR-4 and its co-receptors. The acyl groups in Lipid A have
6
(A) and TNF-(B) were measured by ELISA. Cytokine levels were
x
compared to the negative control (media alone) and significance was
calculated by a one-tailed student t-test (**: p<0.05, ***: p<0.01).
been shown to associate with CD-14, as well as, with a
y
hydrophobic pocket in MD-2. Their interaction with MD-2 has
been shown to be essential for dimerization with TLR-4 and
subsequent activation of the complex.
3
6
Lipopolysaccharide (LPS), the natural ligand for TLR-4, is
the major structural component of the outer membrane of Gram-
negative bacteria. Due to its potent immunostimulatory ability,
LPS acts as a potent adjuvant for vaccines by initiating a strong
Th1 response. LPS activates TLR-4 signaling pathways mediated
Figure 5. Oligosaccharide analog 1 binds on TLR-4 expressed on mouse
PECs.
Mouse PECs from C56BL/6 wildtype mice were cultured for 4 hr. in the
presence or absence of the synthetic oligosaccharide analog at 1 , 5 and 10
g/ml. Cells were harvested and surface stained with either anti-TLR-4 PE or
anti-TLR-2 PE mAb; (A) Blocking of antibody binding to the relevant TLRs
by the oligosaccharide was assessed by flow cytometry at various
concentrations of 1; (B) Levels of TLR-4 binding were graphically presented
and statistically analyzed by a one-paired student t-test (N.S.: not significant,
3
7
by both MyD88 and TRIF. Though LPS is clearly a potent
adjuvant, its clinical use is also precluded due to its toxicity as
well as the association of TLR-4 with a variety of diseases
38
39
40
including: atherosclerosis, rheumatoid arthritis, allergy, and
41`
alcohol-induced neuroinflammation. Thus, due to the
importance of TLR-4 signaling in the regulation of inflammatory
processes and its connection between innate and adaptive
immunity, the evaluation of new synthetic TLR ligands, capable
of agonizing or antagonizing this signaling pathway, is very
important.
In our study, culturing of murine macrophages with the
saccharide analog 1 had a pro-inflammatory effect based on the
production of IL-6 and TNF-. The absence of TLR-4 expression
by the mouse macrophages ameliorated production of the pro-
inflammatory cytokines, indicating that the effect was TLR-4
dependent, rather than TLR-2 dependent, as it would be expected
*: p<0.1, **: p<0.05).
Also, the nature of the other protecting groups may lead to the
observed immunogenicity of 1. Cytokine production with the
monosaccharide precursors for 1 showed diminished levels of IL-
6
and TNF- relative to the trisaccharide analogue. Levels were
slightly higher with the galactose-based pyruvate ketal 3. The
immunogenicity of the pyruvate ketal moiety has also been
observed when it is present in the capsular saccharides from a
23-25
range of Gram positive and Gram negative bacteria.
Finally,
because reduced cytokine levels were observed with the
truncated sugars, it may also be possible that the combination of
the residues and/or a specific chain length may be necessary for
optimized TLR-4 agonism. Further structure-activity studies will
be carried out to more fully address these questions.
A TLR-4 control
TLR-4 + 1g block
Competition studies with 1 and mouse anti-TLR-4 mAb
suggest that the saccharide directly binds with TLR-4 and more
specifically at the same site at which the antibody binds. Mouse
PECs were pre-treated with varying concentrations of 1, followed
by the addition of PE conjugated TLR-4 mAb (10 g/mL). In the
absence of 1, more than 50 % of the cells showed binding to PE-
labeled mouse anti-TLR-4 mAb. Binding decreased to ~ 45%
when the cells were pre-treated with 1 at a concentration of 1
TLR-4 + 5g block
TLR-4 + 10g block
TLR-2 + 10g block

g/mL. At 10 g/mL in 1, binding to anti-TLR-4 mAb had
decreased to only 24.4%; more than half the binding observed in
the control cells. So at equivalent concentrations of mAb and if
saccharide, 1 appears to compete more effectively for binding at
the same site on TLR-4. No change was observed for the binding
of anti-TLR-2 mAb to the TLR-2 receptor even at the high
concentration of 10 g/mL of 1. This supports the data observed
for PECs from TLR-2 knockout mice.
TLR-2 control
The exact nature of binding to TLR-4 is not known, nor is it
known if additional co-receptors (e.g. MD-2 or CD-14) are
needed to interact with 1 in order to initiate TLR-4 signaling.
Additional blocking or co-blocking studies with other antibodies
may help clarify these questions.
3. Conclusions
We believe that our study is an important step towards the
discovery of new vaccine adjuvants through the chemical
synthesis of a novel, low molecular weight saccharide. Using
well established protocols, sugar precursors were prepared in
good yields and with good anomeric selectivities. Thioalkyl
glycosides were used to couple sugar fragments. A cyclohexane
PECs
8
6
4
2
0
0
0
0
0
**
N.S.
B
*
g/ml
g/ml
g/ml



control
1
5
10

diol was used as a central “sugar surrogate”. The fully-protected
trisaccharide analog 1 and its precursor sugars 3 and 8 were
tested in cells and their supernatents were analyzed for the
production of cytokines. The component sugar fragments
produced lower levels of IL-6 and TNF- than the fully
assembled trisaccharide.
Using TLR-2 and TLR-4 knockout mice, the mode of
signaling by the innate immune system was elucidated to be
dependent on TLR-4 expression. Compound 1 did not directly
activate adaptive immunity. When anti-TLR-4 mAbs were added
to cells that had been pretreated with 1, competitive displacement
of the saccharide was observed in a dosage-dependent fashion.
This suggests that 1 and the mouse anti-TLR-4 mAb utilize the
same TLR-4 binding site, though the involvement of other co-
receptors in binding and subsequent activation of the TLR are
unclear from these studies.
Further cell-based experiments need to be conducted to fully
elucidate the structural effects of the chain length and
presence/absence of key sugar protecting groups on the agonism
of TLR-4. Also, the effects of this molecule in vivo, in the
context of an orally-administrable drug, need to be studied.
Finally, downstream signaling of the activated TLR needs to be
examined for dependency on MyD88 and TRIF/TRAM effector
proteins.
4.2.1 Thioethyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-
glucopyranose (2): Starting from commercially-available
glucosamine hydrochloride, compound 2 was prepared in four
26-28
steps using known literature procedures.
First, neutralization
of the hydrochloride salt, then installation of the phthalamido
protecting group on nitrogen, followed by peracetylation of the
hydroxyl groups gave 8. Then, treatment with ethanethiol and
boron trifluoride etherate gave the thioethyl glycosyl donor (79
%) 2. Yields, melting points and compound spectroscopic data
for each product were in good agreement with prior literature
26-28
reports.
4.3 Synthesis of Fragment 3
4.3.1. Known Precursors: Compounds 10, 11, 12 and 13
were synthesized sequiv.uentially, starting with 1,2,3,4,6- penta-
O-acetyl- D-galactopyranose 9 using procedures described in
the literature. Their yields and spectroscopic properties were in
26, 27, 29, 30
good agreement with the literature.
4
.3.2.
Thiophenyl-2-O-acetyl-3-O-benzoyl-4,6-O-
31
benzylidene-β-D-galactopyranose (14):
To a solution of thiophenyl-3-O-benzoyl-4,6-O-benzylidene-
β-D-galactopyranose 13 (2.0 g, 4.31 mmol) in 10 mL of N, N, N-
triethylamine was added N, N-dimethylaminopyridine (39 mg,
0
0
.08 equiv.) and the mixture was stirred at 0 C for 30 min. To
4
4
4
. Experimental Section
.1 Chemistry
the ice-cold solution was added 508 mg (1.5 equiv.) of acetyl
chloride dropwise and the reaction was vigorously stirred at 0 C
initially, then at r.t. for 2 days. The reaction was terminated by
2 2
diluting with CH Cl (50 mL) and, by washing with 2 N HCl (2
0
.1.1 General methods.
1
Proton nuclear magnetic resonance ( H NMR) spectra were
recorded on a Varian Inova (500 MHz) spectrometer. NMR
samples were dissolved in d
DMSO and chemical shifts were reported in ppm relative to the
residual non-deuterated solvent or to the methyl protons on
x 25 mL) and saturated NaHCO solution (2 x 25 mL). The
3
organic layer was dried over anhydrous MgSO and evaporate in
4
6
-acetone, d
1
-chloroform or d
6
-
vacuo to give 2.44 g of the crude product as an orange fluffy
solid. Purification was carried out by flash column
chromatography using 30% EtOAc in hexanes as the eluent. 1.3 g
(60%) of the desired product was obtained as a yellow oily
1
tetramethylsilane (TMS, 0.00 ppm). H NMR data are reported as
follows: chemical shift, integration, multiplicity (s = singlet; d =
doublet; dd = doublet of doublet; ddd = doublet of doublet of
liquid. R (30% EtOAc/hexanes): 0.44.
f
1
13
H NMR (500 MHz, CDCl ) δ 7.99-7.97 (m, 2H, Ar), 7.63-7.64
3
doublets; m = multiplet), coupling constant, and assignment. 1
3
C
C
(m, 2H, Ar), 7.52-7.55 (m, 1H, Ar), 7.31-7.41 (m, 8H, Ar), 5.56
NMR were recorded on the same instrument (125 MHz).The
(d, 1H, J2,3 = 9.9 Hz, H-2), 5.48 (s, 1H, Bn-Ar), 5.20 (dd, 1H, J2,1
chemical shift were reported relative to the references at 29.84,
7.23 and 39.51 ppm, respectively. All the NMR experiments
=
9.9 Hz, J2,3 = 9.8 Hz, H-3), 4.79 (d, 1H, J1,2 = 9.8 Hz, H-1), 4.54
7
0
(s, 1H, H-4), 4.42 (d, 1H, J6,6’ = 12.4 Hz, H-6), 4.06 (d, 1H, J
=
C
6’,6
were run at room temperature (25 C) and assignments were done
by using gCOSY and gHMQC. The mass spectra were obtained
at the University of Illinois (Champaign-Urbana). Both the
higher resolution and the low resolution mass spectra were
obtained by ESI (electron spray ionization).
1
3
1
2.4 Hz, H-6’), 3.69 (s, 1H, H-5), 2.01 (s, 3H, -CO-CH
NMR (125 MHz, CDCl ) δ 170.1 (g-CO-CH
37.3, 133.9, 133.0, 130.0, 129.3, 128.6, 127.8, 125 (CH, Ar),
08.9 (Bn-Ar), 89.8 (C-1), 79.6 (C-4), 78.9 (C-5), 73.8 (C-3),
0.4 (C-2), 67.8 (C-6), 21.0 (CH ); LRMS (ESI): 524.2
3
);
3
3
), 165.9 (-CO-Ar),
1
1
7
3
Melting points (mp) were measured on a Mel-Temp melting
point apparatus (Laboratory Devices, Inc., USA).The optical
rotations were recorded on automatic polarimeter (Rudolph
Instruments, New Jersey USA). Reactions were monitored by
thin-layer chromatography (TLC) on 0.25 mm silica gel coated
aluminum sheets 60 F254 (Analtech). Silica gel (particle size 40–
+
(M+NH
4
) :.
4
.3.3. Thiophenyl-2-O-acetyl-3-O-benzoyl-4,6-O-pyruvate-
32
β-D-galactopyranose (3):
To a solution of thiophenyl-2-O-acetyl-3-O-benzoyl-4,6-O-
benzylidene-β-D-galactopyranose 14 in anhydrous
63 Å, 230–400 mesh) (Silicycle) was used for flash column
0
dichloromethane (20 mL) at -15 C was added dropwise a
chromatography. Preparative, thin-layer, chromatographic
separations were carried out on 2000 m silica gel 60 coated
glass plates.
All the reactions were carried out in oven-dried glassware
under nitrogen. Sugar precursors were purchased either from
Aldrich Chemical Co. or from Carbosynth. Other reagents were
purchased from either Aldrich Chemical Company or Alfa Aesar
and were used as supplied.
mixture of 20% v/v trifluoroacetic acid (880 mg, 6 equiv.uiv.) in
CH
an atmosphere of N
of excess CH Cl (30 mL), and washed with saturated NaHCO
solution (2 x 25 mL) and water (2 x 25 mL). The organic layer
was separated and dried over anhydrous Na SO , and evaporated
2 2
Cl . The mixture was brought to r.t. and stirred overnight in
2
. The reaction was terminated by the addition
2
2
3
2
4
in vacuo to yield 820 mg of the crude 4,6-diol sugar. To a
suspension of the intermediate (10 mg, 0.024 mmol) in methyl
pyruvate (58 mg, 23.5 equiv.uiv.) was added BF
dropwise. The mixture was allowed to stir at r.t. overnight in an
atmosphere of N . The reaction was terminated by the addition of
mL of triethylamine to neutralize the acid. The mixture was
3
-etherate
4.2. Synthesis of Fragment 2
2
1

poured into 25 mL of saturated NaHCO
extracted with CH Cl (2 x 50 mL). The organic layer was dried
over anhydrous Na SO and concentrated in vacuo to give the
3
solution and was
50%, 60%, 70%, 80%, 90% EtOAc in hexanes) which yielded
503 mg (65%) of the product gas a pale, yellow, oily syrup. R
f
(60% EtOAc/hexanes): 0.83.
2
2
2
4
desired pyruvate adduct in its crude form. The crude product was
purified by preparatory TLC using 50% EtOAc in hexanes
mixture as the eluent (with trace amounts of triethylamine) to
1
3
H NMR (500 MHz, CDCl ) δ 7.84-7.87 (m, 2H, Ar), 7.71-7.75
(
J
m, 2H, Ar), 5.76-5.83 (m, 1H, H-3), 5.44 (dd, 1H, J1,2 = 8.75 Hz,
1,3 = 2.4 Hz, H-1), 5.13-5.18 (m, 1H, H-4), 4.28-4.35 (m, 2H, H-
2, 6), 4.16 (d, 1H, J6,6’ = 12.2 Hz, H-6’) 3.84-3.87 (m, 1H, H-5),
.59-3.68 (m, 1H, Cyclohexyl-H-1), 3.49-3.53 (m, 1H,
Cyclohexyl-H-4), 2.11 (s, 3H, -CO-CH ), 2.03 (s, 3H, -CO-CH ),
give 10 mg (80%) of the pyruvate adduct.
R
f
(50%
) δ 7.97-8.01
m, 3H, Ar), 7.92-7.95 (m, 2H, Ar), 7.5-7.54 (m, 2H, Ar), 7.44-
1
EtOAc/hexanes): 0.77; H NMR (500 MHz, CDCl
3
3
(
3
3
7
3
.49 (m, 3H, Ar), 5.45-5.47 (m, 1H, H-2), 5.29-5.34 (m, 1H, H-
), 5.08 (dd, 1H, J1,2 = 10 Hz, J1,3 = 2.8 Hz, H-1), 4.63 (dd, 1H,
e
e
1
1
.86 (s, 3H, -CO-CH
.24-1.40 (m, 4H, Cyclohexyl-H-3 , 2 , 5 , 6 ), 1.19-1.40 (m, 1H,
3
), 1.68-1.8 (m, 2H, Cyclohexyl-H-2 , 6 ),
e a e a
J
4,3 = 7.8 Hz, J4,5 = 3.4, H-4), 4.09-4.1 (m, 1H, H-6), 4.06-4.07
m, 1H, H-6’), 3.89-3.9 (m, 1H, H-5), 3.27 (s, 3H, -OCH ), 2.01
); C NMR (125 MHz,
), 168.9 (-CO-OCH ), 165.9 (-CO-Ar)
33.9, 133, 130.1, 129.3, 128.6, 125.1 (Ar), 114.9 (Pyr), 89.8 (C-
), 79.1 (C-4), 78.9 (C-5), 73.8 (C-3), 70.4 (C-2), 65.1 (C-6),
2.5 (-CO-OCH ), 25.5 (CH ), 21.0 b(-CO-CH ); HRMS (ESI):
NaS for (M+Na) : 525.1195. Found: 525.1205.
a
Cyclohexyl-H-3 ), 0.83 (s, 5H, H-t-butyl), 0.74 (s, 4H, H-t-
butyl), -0.04 (s, 3H, CH ), -0.08 (s, 3H, CH ), -0.12 (s, 3H, CH );
C NMR (125 MHz, CDCl ) δ 172.96, 171.06 (-CO-CH ),
(
(
3
13
3
3
3
s, 3H, -CO-CH
3
), 1.63 (s, 3H, CH
3
13
3
3
CDCl
3
) δ 170.2 (-CO-CH
3
3
169.84 (-CO-N-), 134.57, 123.92 (Ar), 97.05 (C-1), 72.11 (C-5),
1
1
5
71.21 (C-3), 69.59 (C-4), 62.60 (C-6), 49.66 (C-2), 31.13 (-
CCH
(-CO-CH
3
, t-butyl), 26.15, 26.02 (-CCH
3
, t-butyl), 21.13, 21.0, 20.82
+
3
3
3
+
3
), -4.43 (CH ); LRMS (ESI): (M+H) : 648.3.
3
26 9
Calcd C25H O
4
.5.2.
Cyclohexyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-
26
4
4
.4. Synthesis of Central Cyclohexane Core
glucopyranose (17):
33
.4.1. 4-O-tert-butyldimethylsilylcyclohexanol (4):
Into a clean oven dried 10 mL rb flask was taken 25 mg
To a solution of cis/trans mixture of 1, 4-cyclohexanediol 15
200 mg, 1.72 mmol) in N, N-dimethylformamide (5 mL) was
added 2.5 equiv.uiv. of imidazole (293 mg) and the mixture was
stirred at r.t. for 30 min. To the solution was added tert-
butyldimethylsilyl chloride (1.25 equiv.uiv., 293 mg) and the
(0.052 mmol) of thioethyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-
(
glucopyranose, 2. To it was added 5 mL anhydrous CH Cl and
2 2
0
the mixture was cooled to -78 C upon stirring in an inert
atmosphere. To the mixture was added N-iodosuccinimide (2.5
0
equiv., 30 mg) and it was stirred at -78 C for 30 min. Keeping
mixture was further stirred for 3 days in an atmosphere of N
the end of the third day, the mixture was diluted with excess
CH Cl (30 mL), washed with water (5 x 25 mL) and saturated
NaCl (aq) solution (1 x 25 mL). The organic layer was dried over
anhydrous Na SO and then evaporated in vacuo to give 276 mg
2
. At
the temperature constant, 0.46 µL (0.05 equiv.) of trimethylsilyl
trifluoromethanesulfonate (TMSOTf) was added dropwise to the
mixture upon vigorous stirring. To the resulting mixture was
2
2
added dropwise
a
solution of 4-O-tert-butyldimethylsilyl
Cl (1 mL) and the
reaction was stirred at -78 C for 7 h. The reaction was
terminated and the mixture was diluted with CH Cl (25 mL) and
washed with saturated NaHCO solution (1 x 25 mL). The
organic layer was dried over anhydrous MgSO , filtered and
2
4
cyclohexanol 4 (1.2 equiv., 14.35 mg) in CH
2
2
0
of the crude product. Purification was done by flash column
chromatography using 50% EtOAc in hexanes as the eluent
2
2
which gave 196 mg (50%) of the product 4. R
f
(50%
3
EtOAc/hexanes): 0.87.
4
1
concentrated to dryness in vacuo to yield the crude product as an
orange-brown, oily syrup. Purification was done by preparatory
TLC using 80% EtOAc in hexanes as the solvent which yielded
H NMR (500 MHz, CDCl ) δ 3.79-3.83 (m, 1H, H-1), 3.65-3.70
3
a e e e a e
(
(
(
m, 1H, H-4), 1.6-1.77 (m, 6H, H-2 , 2 , 3 , 5 , 6 , 6 ), 1.46-1.5
m, 1H, H-3 ), 1.28-1.32 (m, 1H, H-5 ), 0.89 (s, 9H, t-butyl), 0.04
s, 6H, CH
), 32.1 (C-2, 6), 1.3 (C-3, 5), 30.9 (-CCH
CCH , t-butyl), -2.0 (CH ).
a
a
1
3
32 mg (95%) of the product 17 as a yellowish oily syrup. R
f
3
); C NMR (125 MHz, CDCl
3
) δ 78.1 (C-4), 69.7 (C-
(60% EtOAc/hexanes): 0.157.
1
3
, t-butyl), 25.9 (-
1
3
3
H NMR (500 MHz, CDCl
3
) δ 7.84-7.86 (m, 2H, Ar), 7.72-7.76
(
J
m, 2H, Ar), 5.75-5.82 (m, 1H, H-3), 5.44 (dd, 1H, J1,2 = 8.4 Hz,
1,3 = 6.4 Hz, H-1), 5.15-5.19 (m, 1H, H-4), 4.28-4.36
4.5. Formation of Trisaccharide Analog
4
.5.1.4-tert-butyldimethylsilyl-cyclohexyl)-2-phthalimido-
(overlapping m, 2H, H-2, H-6), 4.16 (m, 1H, J6’,6 = 10.8 Hz, H-
6’), 3.85-3.88 (m, 1H, H-5), 3.73-3.77 (m, 1H, Cyclohexyl-H-1),
26
3
,4,6-tri-O-acetyl-β-D-glucopyranose (16):
3
.56-3.65 (m, 1H, Cyclohexyl-H-4), 2.11 (s, 3H, -CO-CH
3
), 2.03
Into a clean oven dried 25 mL rb flask was taken 575 mg (1.2
mmol) of thioethyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-
glucopyranose 2. To it was added 15 mL anhydrous CH Cl and
the mixture was cooled to -78 C upon stirring in an inert
atmosphere. To the mixture was added N-iodosuccinimide (2.5
e
(s, 3H, -CO-CH ), 1.95-1.98 (m, 1H, Cyclohexyl-H-2 ), 1.86 (s,
3
e
3H, -CO-CH ), 1.69-1.76 (m, 1H, Cyclohexyl-H-6 ), 1.33-1.43
3
e e
2
2
(m, 2H, Cyclohexyl-H-3 , 5 ), 1.17-1.31 (m, 2H, Cyclohexyl-H-
0
a
a
13
3
, 5 ); C NMR (125 MHz, CDCl
3
) δ 170.73, 170.22 (-CO-
3
CH ), 169.51, 165.52 (-CO-N-), 134.35, 131.38, 123.6 (Ar),
0
equiv., 675 mg) and it was stirred at -78 C for an additional 30
9
4
3
6.85 (C-1), 76.86 (Cyclohexyl-C-1), 71.78 (C-3, 5), 70.88 (C-
), 69.19 (Cyclohexyl-C-4), 62.23 (C-6), 54.86 (C-2), 30.47,
min. Keeping the temperature constant, 10 µL (0.05 equiv.) of
trimethylsilyl trifluoromethanesulfonate (TMSOTf) was added
dropwise to the mixture upon vigorous stirring. To the mixture
was added dropwise a solution of 4-O-tert-butyldimethylsilyl-
3
0.06 (Cyclohexyl-C-2, 3, 5, 6), 20.78, 20.65, 20.46 (-CO-CH ).
+
LRMS (ESI): 534.2 (M+H) .
cyclohexanol 4 (1.2 equiv., 331 mg) in CH
2
Cl
reaction was stirred at -78 C for 90 min. The reaction was
terminated and the mixture was diluted with excess CH Cl (100
mL) and washed with saturated NaHCO solution (1 x 50 mL).
The organic layer was dried over anhydrous MgSO , filtered and
2
(2 mL) and the
4.5.3. (Cyclohexyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-
glucopyranosyl)-2-O-acetyl-3-O-benzoyl-4,6-O-pyruvate-β-D-
galactopyranose (1):
0
2
2
3
Thiophenyl-2-O-acetyl-3-O-benzoyl-4,6-O-pyruvate-β-D-
galactopyranose 2 (62 mg, 1.3 equiv.) and 5 mL of anhydrous
4
concentrated to dryness in vacuo to yield 1.0 g of the crude
product as a dark brown oily syrup. Purification was done by
flash column chromatography using gradient elution (30%, 40%,
CH
mixture was stirred at -42 C (CH
atmosphere for 15 min. N-iodosuccinimide (69 mg, 2.5 equiv.)
2 2
Cl were taken in a clean oven tried 10 mL flask and the
0
3
CN/dry ice) in an inert

was added to the mixture and it was stirred for an additional 30
4.6.1 Mice
0
26
min at -42 C. Keeping the temperature constant, 1.3 µL of
TMSOTf (0.05 equiv.) was added to the mixture dropwise with
vigorous stirring. To the mixture was added a solution of
cyclohexyl-2-phthalimido-3,4,6-tri-O-acetyl-β-D-glucopyranose
C57BL/6, TLR-2, and TLR-4 genetically deficient mice were
purchased from Jackson Laboratories (Bar Harbor, Maine). All
mice were housed at the Animal Research Facility at Seton Hall
University. The mice were used at 6–10 weeks of age. All
protocols were reviewed and approved by the Seton Hall
University Ethics Committee utilizing NIH standards.
1
7 (50 mg, 0.094 mmol) in anhydrous CH
2
Cl
reaction was run at -42 C for 3 h with continuous stirring. The
reaction was terminated by adding excess CH Cl (40 mL), it was
washed with saturated NaHCO solution (1 x 50 mL) and 10%
w/v Na solution (1 x 50 mL). The organic layer was
separated, dried over anhydrous MgSO filtered and
2
(1 mL) and the
0
2
2
3
4.6.2. Peritoneal exudate cell (PEC) isolation
2 2 3
S O
PECs were harvested from wildtype, TLR-2 deficient, or
TLR-4 deficient mice and centrifuged in a refrigerated centrifuge
at 4,000g for 10 minutes. Following centrifugation, the interface
containing PECs was resuspended in RPMI with 10% FBS, prior
to enumeration. Identification and enumeration of PECs was
based on the expression of surface antigens F4/80 (data not
shown).
4
,
concentrated to dryness in vacuo to yield 135 mg of the crude
product as an orange oily syrup. Purification was done by
preparatory TLC technique using 60 % EtOAc in hexanes as the
solvent, to afford 50 mg (57.6%) of the product 1 as an off-white
solid.
1
H NMR (500 MHz, CDCl
3
) δ 7.99-8.04 (m, 2H, Ar), 7.91-7.96
4.6.3. Cytokine assays
(m, 1H, Ar), 7.83-7.88 (m, 1H, Ar), 7.73-7.77 (m, 1H, Ar), 7.49-
7
5
.54 (m, 1H, Ar), 7.36-7.41 (m, 1H, Ar), 7.35-7.41 (m, 2H, Ar),
.73-5.80 (m, 1H, Hglu-1), 5.62-5.66 (m, 1H, Hgal-1), 5.36-5.46
Mouse PECs or the murine monocyte cell line, RAW 267.4
cells, were cultured in the presence of our novel saccharide
compound 1, diluted in PBS / 2% DMSO, at a concentration of
either 1 or 5g/ml. As a positive control, cells were cultured
with 10g/ml of E.coli LPS (Sigma). Supernatants were collected
at designated time points and the levels of IL-6 and TNF-α
cytokines were measured using ELISA kits and by following
vendor instructions (Biolegend).
(m, 2H, Hglu-3, Hgal-2), 5.13-5.19 (m, 1H, Hgal-3), 4.48-4.60 (m,
1
H
H
H, Hglu-4), 4.26-4.34 (m, 2H, Hgal-4, Hglu-2), 4.10-4.27 (m, 2H,
glu-5, 6), 3.93-4.08 (m, 2H, Hglu-6’, Hgal-6), 3.80-3.90 (m, 2H,
gal-6’, Cyclohexyl-H-1), 3.73-3.78 (m, 1H, Hgal-5), 3.61-3.7 (m,
3
H, -5OCH
CO-CH ), 2.10 (s, 1H, -CO-CH
H, -CO-CH ), 1.86 (s, 1H, -CO-CH
.65-1.83 (m, 4H, Cyclohexyl-H-2 , 2 , 6 , 6 ), 1.57 (s, 3H, -CO-
3
), 3.41-3.53 (m, 1H, Cyclohexyl-H-4), 2.11 (s, 1H, -
), 2.06 (s, 1H, -CO-CH ), 1.87 (s,
), 1.85 (s, 1H, -CO-CH ),
3
3
3
1
1
3
3
3
a
e
a
e
4.6.4. Flow cytometry blocking experiments
e
CH
3
), 1.37-1.42 (m, 1H, Cyclohexyl-H-3 ),1.27-1.31 (m, 1H,
PECs from wildtype, C57BL/6 mice were harvested and
e
0
Cyclohexyl-H-5 ) 1.25 (s, 3H, CH
3
), 0.86-0.92 (m, 1H,
Cyclohexyl-H-3 ); C NMR (125 MHz, CDCl ) δ 170.70, 170.20
-CO-CH ), 169.49 (-CO-OCH ), 165.89 (-CO-N-), 134.37,
33.16, 131.31, 129.88, 129.77, 129.67, 128.42, 128.36, 127.47,
23.61 (Ar), 100.36 (pyr), 98.69 (Cgal-1), 96.65 (Cglu-1), 96.12
cultured for 20 min at 37 C with varying concentrations of the
a
13
3
saccharide compound. Following incubation, PECs were washed
and stained with anti-TLR-4-PE or anti-TLR-2-PE mAbs (BD
Pharmingen). The level of TLR blocking by the saccharide
compound was determined by flow cytometry (MACSQuant
Analyzer, Miltenyi Biotech).
(
1
1
3
3
(
Cyclohexyl-C-1), 95.91 (Cyclohexyl-C-4), 72.04 (Cgal-4), 71.93
gal-5), 71.79 (Cglu-5), 70.82 (Cgal-3), 69.22 (Cglu-3), 69.13 (Cgal
), 66.7 (Cgal-2), 65.34 (Cgal-6), 64.01 (Cglu-6), 62.17 (Cglu-2),
4.8 (CO-OCH ), 31.93, 29.70, 29.36, 28.98 (Cyclohexyl-C-2, 3,
, 6), 25.72 (CH ), 20.78, 20.65, 20.47 (-CO-CH ); HRMS (ESI):
51NO20Na for (M+Na) : 948.2902. Found: 948.2899.
(C
-
4.7. Statistical Analysis
4
5
5
3
Statistical differences among the groups were analyzed using
a one-way analysis of variances (ANOVA) or the unpaired, one-
tailed student t-test. GraphPad Prism 4 provided the software for
3
3
+
Calcd C45
H
the
statistical
analysis
(San
Diego,
CA.).
4.6. Biological Evaluation
4
5
.
.
a) Cobb, B. “Glycobiology of Immune Response”, Annals
NYAcad. Sci. 2012, 1253, pp. 1-15. b) Marzabadi, C. H.; Franck,
R. W. Chem. Eur. J. 2016, 22, 1-16, doi:
Acknowledgments
1
0.1002/chem.201601539
This research was supported in part by the National Science
Foundation (MRI 1337426) and the Seton Hall University
Department of Biological Sciences and Department of Chemistry
and Biochemistry research funds.
The authors would like to thank Emi Hanawa-Romero and
Kari Wiedinger for their assistance with experiments performed
in this manuscript.
Janeway, C. A. Cold Spring Harb. Symp. Quant. Biol. 1989, 54(Pt
1):1–13.
a) Iwasaki, A.; Medzhitov, R. Science 2010, 327, 291-295; b)
Iwasaki, A.; Medzhitov, R. Nature Immunol. 2004, 5(10), 987-
6.
9
95.
7
.
Liu, J. J.; Buisman-Pijlman, F.; Hutchinson, M. R. Front.
Neurosci. 2014, doc:10.3389/fnins 2014.00309.
Peri, F.; Calabrese, V. J. Med. Chem. 2014, 57, 3612-3622.
Bhardwa, N.; Gnjatic, S.; Sawhney, N. B. Cancer J. 2010, 16(4),
8.
9
.
3
82–391. doi:10.1097/PPO.0b013e3181eaca65
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