Synthetic Lipomannan Glycan Microarray Reveals the Importance of α(1,2) Mannose Branching in DC-SIGN Binding

Article abstract of DOI:10.1021/acs.joc.8b02944

Lipomannan (LM), a glycophospholipid found on the cell surface of mycobacteria, involves the virulence and survival in host cells. However, there is little to no information on how exactly mannan alignment, including the number of mannose units and the br

Full text of DOI:10.1021/acs.joc.8b02944

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Article
Synthetic Lipomannan Glycan Microarray Reveals the
Importance of #(1,2) Mannose Branching in DC-SIGN Binding
Nithinan Sawettanai, Harin Leelayuwapan, Nitsara Karoonuthaisiri,
Somsak Ruchirawat, and Siwarutt Boonyarattanakalin
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.8b02944 • Publication Date (Web): 17 May 2019
Downloaded from http://pubs.acs.org on May 19, 2019
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Synthetic Lipomannan Glycan Microarray Reveals the Importance of α(1,2)
Mannose Branching in DC-SIGN Binding
†
†
‡
Nithinan Sawettanai , Harin Leelayuwapan , Nitsara Karoonuthaisiri , Somsak
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†
§
∥
Ruchirawat , and Siwarutt Boonyarattanakalin*
†Program in Chemical Biology, Chulabhorn Graduate Institute, Chulabhorn Royal Academy, Bangkok 10210,
Thailand
‡Microarray Laboratory, National Center for Genetic Engineering and Biotechnology (BIOTEC), National
Science and Technology Development Agency (NSTDA), Pathum Thani 12120, Thailand
∥
Laboratory of Medicinal Chemistry, Chulabhorn Research Institute, and Centre of Excellence on Environmental
Health and Toxicology Bangkok 10210, Thailand
§School of Bio-Chemical Engineering and Technology, Sirindhorn International Institute of Technology,
Thammasat University, Pathum Thani 12121, Thailand
*Corresponding Author, E-mail: siwarutt@siit.tu.ac.th, siwarutt.siit@gmail.com
ABSTRACT
Lipomannan (LM), a glycophospholipid found on the cell surface of mycobacteria,
involves the virulence and survival in host cells. However, there is little to no information on
how exactly mannan alignment, including the number of mannose units and the branched motif
of LM, affects protein engagement during host pathogen interactions. In this study, we
synthesized the exact substructures of the LM glycan which consist of an α(1,6) mannan core,
with and without the complete α(1,2) mannose branching, and comparatively studied their
protein-carbohydrate interactions. The synthetic LM glycans were equipped with a thiol linker
for immobilizations on the surfaces of microarrays. As per our findings, the presence of the
branching α(1,2) mannose on the LM glycans increases their binding toward DC-SIGN
receptor. An increase in the number of mannose units on the glycans also increases the binding
with the mannose receptor. Thus, the set of synthetic glycans can serve as a useful tool to study
the biological activities of LM and can provide better understanding of host-pathogen
interactions.
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INTRODUCTION
Tuberculosis (TB) is a severe airborne infectious disease that is caused by a single
infectious agent—Mycobacterium tuberculosis (Mtb). In 2017 alone, approximately 10.0
1
million people developed TB. There were 1.3 million deaths from TB among HIV-negative
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people and an additional 300,000 deaths among HIV-positive people. Mtb has unique cell
wall characteristics, including a thick layer of lipid conjugated with a complex array of glycans.
During an infection, lipomannan (LM) is one of the key cell surface glycophospholipids
implicated in the virulence and survival of the mycobacteria in the host’s mammalian cells.2,3
The structural features of LM from the Mtb surface are illustrated in Figure 1. The LM
structure consists of a phospholipid, a lipidated mannose, and a complex polymannan attached
to the myo-inositol at 1-O, 2-O, and 6-O positions, respectively. The polymannan residue is
composed of an α(1,6) linked backbone branched with additional mannose units at the 2-O
position.4,5 The numbers and positions of the acyl chains vary for different strains of
mycobacteria. The acyl chains can be attached to the glycerol unit and the mannose on the 2-
O position of the inositol.6
Figure 1. Structural features of the lipomannan (LM) of Mycobacterium tuberculosis (Mtb).7
Glycan is composed of an α(1,6) mannosyl backbone branched with additional mannose units
1
2
3
at the 2-O position (a and b are varied; R is tuberculostearic acid, R and R are various fatty
acids).
LM is a highly active pro-inflammatory activator when compared to other mycobacteria
surface glycolipids. Lipoarabinomannan (LAM) was unable to stimulate a significant cytokine
secretion. The cytokine expression increased after the chemical degradation of the arabinan
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chain of LAM to reveal the core LM and the mutation of mycobacterial LAM with a truncated
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arabinan domain. The results indicate that the arabinan portion of LAM may cover the LM
structure and thus blocks the immunostimulatory effects.
Despite LM being isolated from different species of mycobacteria, LM were potent
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inducer of interleukin-12 (IL-12) and apoptosis , and it causes the secretion of interleukin-8
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(IL-8) and tumor necrosis factor alpha (TNF-α). The precursor of LM, phosphatidyl-myo-
inositol dimannoside (PIM
), has been proven to show no activity.8,10 Thus, it can be inferred
2
that the activity of LM must arise from the mannan core of LM which consists of the α(1,6)
mannan backbone and multiple monosaccharide branching.
The role of α(1,2) mannose branching was studied in the Mycobacterium marinum
zebrafish infection model. A disruption of M. marinum mptC (α(1,2) mannosyltransferase)
caused the mutant to lack the mannose caps on LAM and α(1,2) mannose branching on the
mannan core of LM. The M. marinum mptC mutant was strongly attenuated in the zebrafish.
To clearly observe the effect of mannose core branching, the M. smegmatis mptC gene was
introduced into the mutant to restore the branching. Restoring the mannan core branching but
not the mannose cap elongation could fully complement the virulence defect in embryonic
zebrafish. These results show that α(1,2) mannose branching, and not the mannose cap, play
an important role in mycobacterial pathogenesis in the context of innate immunity.11
An alteration of the lipomannan structure by hypermannosylation could enhance innate
immune responses. The deletion of the glycosyltransferase, NCgl2096, from Corynebacterium
glutamicum that encodes for an α(1,2) arabinofuranosyltransferase, resulted in the absence of
arabinose from LAM and the additional mannosylation of the branching sites. Such
hypermannosylated LM was a stronger inducer of cytokine responses than LAM in both in
vitro and in vivo.12
With its strong pro-inflammatory activity and uniqueness of structure that is found to
be common in every species of mycobacteria, LM becomes an interesting molecule to study as
a potential antigen and/or adjuvant for vaccine development. However, the extraction of LM
from a mycobacterial cell wall is a tedious process and even the most efficient methods usually
13
only yield small amounts of the product with contaminations. Moreover, the uncertainty of
the exact structure limits the use of isolated LM in structure-activity relationship (SAR) studies
and immunological studies. SAR studies that employ computational chemistry can only be
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done precisely if they use a well-characterized target structure.14 The chemical synthesis
approach offers a defined structure of LM with high purity and yield. An efficient chemical
synthesis of LM compound from n-pentenyl orthoesters facilitated by the use of ytterbium
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triflate/N-iodosuccinimide has been reported. The exact structure of the synthetic LM can be
used as a biochemical tool to study the interactions between the Mtb surface components and
the human immune system. A better understanding of such interactions is essential for the
development of preventive measures and treatments for TB.
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The chemically defined exact structures of the LM glycan, which is an α(1,6) mannan
core with and without the complete α(1,2) mannose branching, have never been synthesized
and comparatively investigated for its immunological properties before this study. To observe
the effect of LM α(1,2) mannose branching and the size of the α(1,6) mannoside core, we
performed systematic investigations on the syntheses and immune receptor bindings of the
different patterns of the LM glycans. The comparisons were done among the linear α(1,6)
mannosides at different chain lengths and the corresponding α(1,2) mannose branching on the
linear mannoside core (Figure 2). There are four different patterns of synthetic glycans. The
linear α(1,6) mannosides, consisting of 2 and 5 units, are a feature of the targets—Man
Man . The complete mannosylation at the 2-O-positions of the linear di- and pentamannosides
backbone gives rise to the branched targets—BMan and BMan10, respectively.
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and
5
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Figure 2. Structures of synthetic LM glycans, with and without branched mannose at C-2.
The synthetic glycans were equipped with a thiol linker to facilitate the process of
glycan immobilization on the solid surfaces and on the proteins. The bindings of the different
synthetic glycans with dendritic cell-specific intercellular adhesion molecule-3 grabbing
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nonintegrin (DC-SIGN) and mannose receptors, which are the key immune receptors on
dendritic cells, were studied side by side by microarrays. The experimental results would
determine whether, and to what extent, the α(1,2) mannose branching pattern is implicated
during immune receptor binding. The synthetic glycans are useful for various biological and
immunological studies of lipomannan. This study provides a better understanding of the
interactions between the pathogen and the host’s immune system.
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RESULTS AND DISCUSSION
Retrosynthetic Analysis
The overall structure of the synthetic targets was obtained by the convergent assembly
of the mannosyl residue and a thiol linker (Scheme 1). The key steps in these syntheses are the
glycosylation of the mannosyl acceptors and donors to construct the linear and branched
mannoside patterns. The linear pentamannoside is derived from the coupling of trimannoside
3 and dimannosyl imidate 4, which can be prepared from three mannosyl building blocks
(compounds 1, 5, and 6). The coupling of the linear mannosyl residue with the thiol linker 2
by a glycosidic bond affords the masked linear di- and pentamannosides—Man and Man ,
2
5
respectively. Further complete glycosylation at the 2-O-position of the linear backbone
mannose with mannosyl phosphate 1 gives rise to the masked branched di- and branched
pentamannosides—BMan and BMan10, respectively. The protecting groups were globally
4
removed by Birch reduction to yield the unmasked target compounds. The synthetic approach
in this project is highly convergent, relying on only four types of building blocks, all of which
can be synthesized in parallel without relying on one another.
Scheme 1. Retrosynthetic analysis for the assembly of synthetic targets
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The glycosylation of the linear mannosyl residue and the thiol linker needed to be done
prior to the installation of the branching α(1,2) mannose in order to form an alpha glycosidic
bond. The stereoselectivity of every glycosidic bond formation is ensured by neighboring C-2
ester protecting groups which are either O-benzoyl or O-acetyl groups. Consequently, the
nucleophile (thiol linker or mannosyl acceptor) necessarily approach the activated mannoside
from the bottom face because the top face is not accessible. This leads to alpha glycosidic bond
formations.
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Assembly of Oligomannoside Fragments
Only three types of mannosyl building blocks were needed to prepare mannosides in
the series in this study. The preparation of trimannoside 3 required two cycles of glycosylation
in the presence of TMSOTf (Scheme 2). To allow the formation of two glycosidic bonds in the
α(1,6) direction, mannosyl phosphate 5 was used to synthesize trimannoside 3. Mannosyl
building blocks 5 and 6 were first assembled to give disaccharide 7 at 75% yield. Due to
TMSOTf, the TIPS group was also removed. The glycosylation of dimannoside 7 with
mannosyl phosphate 5 provided trimannoside 3 at 66% yield. A formation of oligomers might
occur but did not appear as a major side product. It is possible that the TIPS removal occurred
after the glycosylation had almost completed. The glycosylation reaction was also controlled
to limit the formation of oligomers by conducting the reaction at low temperature, maintaining
the temperature uniformly throughout the reaction time, and holding the reaction time for not
too long.
Not all of mannose 6 was coupled with mannosyl phosphate 5 during the first
glycosylation. The remaining mannose 6 had to be completely removed before another
glycosylation; otherwise it would form dimannoside 7 which was co-eluted with trimannoside
3 during the purifications by silica gel column chromatography and size exclusion
chromatography. Using the newly prepared mannosyl phosphate 5 also facilitated
glycosylation by increasing the reaction yield.
Scheme 2. Synthesis of trimannoside 3a
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a
Reagents and conditions: (a) 5 (1.3 equiv.), TMSOTf, CH
2
Cl , 0 °C, 75%; (b) 5 (1.2 equiv.),
2
TMSOTf, CH Cl , 0 °C, 66%.
2
2
Dimannoside 8 was obtained by glycosylation between mannosyl building blocks 1 and
by TMSOTf activation (Scheme 3). Mannosyl phosphate 1 was suitable to be the terminal
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unit of the non-reducing end of mannose because 1 has a permanent protecting group (O-benzyl
group) at the C-6 position and a temporally O-acetyl group at the C-2 position for further
branching mannosylation. Removal of the allyl group of compound 8 was efficiently conducted
using palladium chloride. The N-phenyltrifluoroacetimidoyl group was then installed at the C-
1
position of compound 9 to provide the leaving group for dimannoside 4. The union of
dimannosyl imidate 4 and trimannoside 3, using standard TMSOTf-catalyzed glycosylation,
furnished pentamannoside 10 (Scheme 3). The allyl group of compound 10 was cleaved and
replaced with the N-phenyltrifluoroacetimidoyl group to provide the site for the attachment
with the thiol linker moiety.
Scheme 3. Assembly of oligosaccharide donors 4 and 12a
a
Reagents and conditions: (a) TMSOTf, CH
2
Cl
2
, 0 °C (Molar ratio 1:6 = 3:2), 99%; (b) PdCl
(Cl)CNPh, Cs CO , Acetone, 0 °C, 85%
, 0 °C (Molar ratio 4:3 = 3:2), 88%.
2
,
NaOAc, AcOH, rt, 87% for 9 and 85% for 11; (c) CF
3
2
3
for 4 and 65% for 12; (d) 3, TMSOTf, CH Cl
2
2
The removal of the allyl protecting group on the reducing end of compound 8 with
catalytic Pd(OAc) , PPh , and Et NH yielded 60% of hemiacetal 9 (Scheme 3). Pd(OAc) (2
equiv.) in MeOH (1 mL) was allowed to form a complex with PPh (6 equiv.) for 6 hours prior
2
3
2
2
3
to the addition of Et
2
NH (6 equiv.) and the starting material (~100 mg) at room temperature
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(
rt). The reaction solution was further stirred for 12 hours to convert compound 8 to compound
9. Palladium residues remained with the compound even after filtration through a pad of Celite
and purification by a silica gel column chromatography. Using PdCl under the buffered
2
conditions of AcOH and NaOAc (pH 2 after mixing) was a better way to remove the allyl
group. Palladium residue was more easily removed by filtration through a pad of Celite. The
reaction could be finished in one step (12 h), and the reaction yield increased to 87% for
compound 9 (Scheme 3). A small amount of the side product from the Wacker-type oxidative
side reaction also formed during the Pd-mediated anomeric deallylation. The corresponding
ketone product could be observed by mass spectrometry and as a minor spot on Thin Layer
Chromatography (TLC).
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The syntheses of mannosyl imidates 4 and 12 were achieved by the treatment of
hemiacetals 9 and 11 with N-phenyltrifluoroacetimidoyl chloride under basic conditions
(Scheme 3). Cs
2% and 51%, respectively. Due to the acidic conditions of the deallylation step, the crudes of
hemiacetals 9 and 11 were neutralized prior to the installation of the imidates. Adjusting the
reaction pH to 7 by Na CO improved the yields to 85% and 65%, respectively.
2
CO
3
proved to work better than K
2
CO
3
and provided compounds 4 and 12 at
7
2
3
Assembly of Mannosides with a Thiol Linker and Branching
The thiol terminated linker 2 was prepared from 6-mercapto-1-hexanol by benzylating
16
the thiol functional group with benzyl bromide. The assembly of mannosyl residue and the
thiol linker was done by glycosylation (Scheme 4). N-phenyltrifluoroacetimidoyl mannosides
(compounds 4 and 12) were treated with thiol linker 2 in the presence of TMSOTf to afford
compounds 13 and 16 in good yield. Mannosyl imidates 4 and 12 were treated with an excess
amount of the thiol linker (5 equiv.) to increase the linker glycosylated product yields.
However, there was some difficulty in separating the excess linker from mannosides 13 and
1
6. The thiol linker was co-eluted with products during silica gel column chromatography
despite the moderate difference in R values. The excess thiol linker is needed to be separated
f
from oligomannosides using size exclusion column chromatography. Silica gel column
chromatography was then used to separate the products 13 and 16 from the corresponding
hemiacetal byproducts.
Removing all of the protecting groups by Birch reduction gave rise to the final products:
linear di- and pentamannosides, equipped with a thiol linker (Man
2
and Man
5
, Figure 2). In
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parallel, hydrolyzing the O-acetyl and O-benzoyl groups of compounds 13 and 16 by refluxing
with NaOMe at 50 °C gave mannan intermediates 14 and 17. The free C-2 hydroxyl groups on
mannan intermediates 14 and 17 were able to accept mannoside 1 to form the branched targets
(Scheme 4). The α(1,2) branched mannosylation by mannosyl phosphate 1 with TESOTf as a
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0
catalyst yielded compounds 15 and 18. The global protecting group removals provided the final
products, which are branched di- and branched pentamannosides, equipped with a thiol linker
(
BMan and BMan10, Figure 2).
4
Scheme 4. Conjugation of mannosides with a thiol linker and the removal of protecting groups
using Birch reductiona
a
Reagents and conditions: (a) 2 (5 equiv. for 4 and 4 equiv. for 12), TMSOTf, CH
7% for 13 and 76% for 16; (b) Na, NH (l), THF, -78 °C, 25% for Man , 27% for Man
for BMan and 31% for BMan10; (c) NaOMe, CH Cl , MeOH, 50 °C, 93% for 14 and 94% for
17; (d) 1 (5 equiv.), TESOTf, Et O, 0 °C, 88% for 15 and 60% for 18.
2
Cl
2
, -10 °C,
7
3
2
5
, 33%
4
2
2
2
Branching α(1,2) mannose was installed completely onto all the C-2 hydroxyl groups
1
on intermediates 14 and 17, as confirmed by NMR spectroscopy. The H NMR of compound
1
3
1
5 displays four anomeric proton signals at 5.08, 5.07, 4.92, and 4.76 ppm; the C NMR shows
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2
2
2
2
2
2
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2
2
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four anomeric carbon signals at 99.59 (two overlapping), 98.51, and 98.42 ppm. Similarly, the
1H NMR of compound 18 shows ten anomeric proton signals between 5.12 and 4.88 ppm. Ten
1
3
anomeric carbon signals between 99.90 and 98.70 ppm are found in the C NMR spectrum.
The molecular weights of 15 and 18 were also confirmed by high resolution mass spectrometry
+
2+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(
[M+Na] calculated for 15:1969.8621, found 1969.8616; and [M+2Na] calculated for 18:
2220.9524, found 2220.9544).
For the global protecting group removal by Birch reduction, both linear mannan
intermediates (13 and 16) and branched mannan intermediates (15 and 18) were treated with
sodium in liquid ammonia to yield the final products at around 30% yields. Some of the starting
materials and the partially unmasked products remained in the organic layer after extracting
the aqueous layer of the crude product with CH
2
Cl . The side products were subjected to Birch
2
reduction again to convert them into the desired products. The unmasked final products were
achieved as a mixture of a single molecule and the corresponding disulfide dimer. The
molecular peak of each product was found by MALDI-TOF mass spectroscopy as reported in
the Experimental section. Moreover, the mass peak of each corresponding disulfide dimer was
+
also found as the followings: [2M+Na] calculated for dimer for Man
2
: 937.3385, found
+
: 1887.6735, found 1887.6759; [2M+Na]+
937.3356; [2M+H] calculated for dimer of Man
5
2
+
calculated for dimer of BMan : 1585.5498, found 1585.5461; and [2M+2Na] calculated for
4
dimer of BMan10: 1776.5867, found 1776.5878.
The final products in this study were synthesized in a stepwise approach which resulted
1
in chemically well-defined structures. According to the H NMR spectroscopic data, all of the
protecting groups were removed completely as the aromatic and acetate peaks disappeared.
The integrated areas of the anomeric protons correspond to the number of mannosyl units in
1
each compound of the final products. The H NMR anomeric signals are displayed as two
discrete peaks at 4.89 and 4.84 ppm with the integrated area of 2 for Man and are displayed
2
as two overlapping peaks at 4.87 and 4.83 ppm with the integrated area of 5 for Man . For the
5
1
branched mannoside products, the anomeric peaks shift to lower field. The H NMR chemical
shift of the anomeric protons are at 5.12, 5.06, and 5.01ppm for BMan and at 5.11, 5.07, 5.03,
4
and 4.90 ppm for BMan10, of which the areas under peaks correspond to the 4 and 10 mannosyl
residues, respectively. High resolution mass spectrometry of the final products confirms their
+
expected exact masses to be the following: [M+Na] calculated for Man
2
: 481.1720, found
+
+
4
81.1739; [M+Na] calculated for Man
5
: 967.3304, found 967.3343; [M+Na] calculated for
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+
BMan
4
: 805.2776, found 805.2798; and [M+Na] calculated for BMan10: 1777.5945, found
1777.5980. MALDI-TOF mass spectra of the final products were obtained in a positive mode
using 2,5-dihydroxybenzoic acid (DHB) as the matrix.
All of the glycosidic bonds were formed in alpha configuration as a result of the
neighboring acyl group participation. The stereoselectivity was evidenced by the measurement
of the coupling constants between the anomeric carbon and the corresponding anomeric proton
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
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3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
0
1
2
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8
9
0
1
2
3
4
5
6
7
8
9
0
(JC1,H1). The typical JC1,H1 value for alpha mannoside is 171 Hz and for beta mannoside is 159
Hz.17,18 The JC1,H1 in all mannosides, with and without protecting groups, are between 171-173
Hz, which corresponds to the typical values of alpha mannoside.
Mannan Microarrays to Determine Bindings to DC-SIGN and MR
Interactions between the mycobacterium cell membrane and immune receptors
determine the immunomodulatory outcomes of Mtb. These protein-carbohydrate interactions
play a key role in the infectious, virulent, and survival events of Mtb in mammalian host cells.19-
2
1
Subtle variations in the LM structure, with regard to the mannan chain lengths, along with
the pattern of the glycan alignment present in LM, dramatically impact LM’s unique roles in
immunoregulation.8,10,22-24 Several studies have addressed the significance of the mannan motif
present in LM toward pro-inflammatory activities;8,10,22,25 however, insights into how exactly
the mannan alignment affects protein engagement have never been revealed. Thus, it was vital
to determine how different LM substructures affect biological interactions. To gain a better
understanding of the protein-carbohydrate interactions, a carbohydrate microarray was
assembled as a biochemical tool to study the influence of the linear α(1,6) mannan, along with
branching α(1,2) mannan present in LM, on their binding affinities with the relevant immune
receptors, DC-SIGN and MR.
DC-SIGN and MR are important pattern-recognition receptors of antigen presenting
cells. DC-SIGN and MR contribute to the initiation of pro-inflammatory responses, and antigen
internalization and later presentation to lymphocytes, which trigger an adaptive immune
response. DC-SIGN and MR targeting is an efficient strategy of pathogens like Mtb and HIV
to evade the immune system.2,26-28 Mannosylated moieties on the mycobacterial cell wall have
previously been shown to exploit these receptors to enter antigen presenting cells.2,26,28-30
However, the interactions between the chemically defined LM glycan, with and without α(1,2)
branched mannose, with DC-SIGN and MR have never been comparatively investigated before.
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The synthetic LM glycans were already equipped with a thiol linker for immobilization on the
maleimide-functionalized microarray surfaces. The glass slides printed with the synthetic
glycans were incubated with the target receptors in a buffer solution at rt to allow the receptors
to bind to the immobilized LM glycans. Excess receptors were washed off and the remaining
bound receptors were detected through incubation with the fluorescein-conjugated antibody.
The α(1,6) mannan chain with 13 repeating units (Man13)18 was also investigated in parallel
as a positive control for comparison, in order to assess the effects of the differing numbers of
mannose units and the α(1,2) branched mannosyl pattern. The synthetic LM glycans bound to
both DC-SIGN and MR in a specific manner (Figure 3), which was statistically significant
when compared to the fluorescence levels of the thiol linker and the buffer (negative controls).
As expected, the linear α(1,6) mannan chain with 13 repeating units bound significantly to both
receptors. The binding affinity of MR increased with the number of mannose units on the
glycan. The increase in the observed fluorescence intensities is in line with the increase in the
0
1
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9
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numbers of mannose units of the polysaccharides (Man13 > BMan10 > Man
Man ).
5
> BMan >
4
2
Previous studies of lectin binding interactions with Mtb have suggested that α(1,2)
terminal mannose residues on LAM are responsible for the engagement.2,28-30 From our
microarray results, MR recognized the mannan well and the binding was enhanced when the
units of mannose were increased, but the enhancement was not necessarily specific to α(1,2)
or α(1,6) linkage. Multiple C-type carbohydrate recognition domains (CRDs) of MR are
required to achieve tight binding to multivalent ligands unit.31,32 Multiple CRDs are arranged
spatially to accommodate the geometric configurations of natural oligosaccharides.31,32 In this
case, the increasing in terminal mannose units in α(1,2) branched structure might not create the
conformation that favors MR-carbohydrate interactions. It was also shown by Geijtenbeek et
al. that ManLAM (with α(1,2) terminal mannose units) bound to dendritic cells preferably
2
through DC-SIGN, not MR. The presence of the α(1,2) mannose branching pattern on the LM
glycan significantly increased the binding ability of the LM glycan with DC-SIGN (Figure 3).
When comparing mannans with the same backbone lengths (Man
BMan ), the mannans with the α(1,2) mannose branching motif strikingly bound with DC-
SIGN with higher affinity (BMan10 > Man and BMan > Man ). This implies that the
5
Vs. BMan10 and Man Vs.
2
4
5
4
2
increase in the observed fluorescence intensities profoundly depends on the increase in the
numbers of α(1,2) mannose branching units of the polysaccharides rather than the total number
of mannose units of the polysaccharides. For example, BMan10 binds with DC-SIGN at a
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higher affinity than Man13, although BMan10 contains fewer mannan repeating units than
Man13 does. The same thing is also true for a higher DC-SIGN affinity of BMan
4
, as compared
to that of Man . This suggests that the α(1,2) mannose branching motif and α(1,6) mannan
5
chain could be a key mycobacteria glycan epitope that can be recognized and detected by the
human immune receptors DC-SIGN and MR, respectively.
1
1
1
1
1
1
1
1
1
1
2
2
2
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0
Several publications suggested that high mannose oligosaccharides interact with DC-
SIGN in multiple orientations resulting in statistical affinity enhancements.33-36 The different
binding orientations feature contacts between mannan and different regions of the protein at
the same time. The multiple interactions occur more likely due to the more available mannose
units on larger mannan and/or the branched mannan. The observed microarray fluorescence
intensities suggested that other contacts between DC-SIGN’s carbohydrate recognition
domains and mannan might occur and provide substantial affinity enhancements.
Blattes et al. studied different types of mannodendrimers binding affinity toward DC-
37
SIGN. Spatial geometry, and stereo- and regiochemistry of glycan have strong influences on
lectin binding. Subsequently, density also play a role in determining the spatial geometry of
glycan. The optimal glycan density would create a perfect environment for lectin binding.32,38,39
The fluorescent intensities that showed dose independent are probably because at the lower
printing concentrations, glycan molecules might already saturate the array surface. Thus,
glycan-protein interactions reached their saturation point. Consequently, there was no
significant difference on the protein binding affinity among different concentrations of the
same glycan structure.
The activation of macrophages and dendritic cells depends on the net balance between
positive and negative signals. The results from this study underscore the significance of the
linear α(1,6) mannan and the α(1,2) branched mannosyl pattern presented in the LM structures
and highlight their potential use as an immune modulator that may exert desired immunological
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properties such as adjuvant activities. Studies of these interactions may lead to the development
of novel, effective, and highly selective therapeutic practices.
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9
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Figure 3. Analysis of MR and DC-SIGN bindings with synthetic Mtb glycan substructures at
various printing concentrations. Fluorescence intensity was normalized with buffer spots. Data
are presented as mean±SD for each triplicate.
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CONCLUSION
In this study, we have developed an efficient synthesis of the α(1,6) mannan cores and
the chemically exact α(1,2) mannosyl branch on the α(1,6) mannan cores. The synthetic
glycans serve as biochemical tools to determine the effects of mannosyl unit lengths and the
branched motif of the Mtb LM on the binding with DC-SIGN and mannose receptors. The
stepwise synthetic strategy is highly convergent, relying on only three types of mannosyl
acceptors and donors which can be prepared from a mannosyl tricyclic orthoester. Each
synthetic LM glycan was equipped with a thiol linker at the reducing end of the mannan to be
immobilized on its surface for a carbohydrate microarray. The results show that the presence
of the branching α(1,2) mannose motif on the LM glycan, which is more important than the
total number of mannose units, enhances the binding affinity toward the relevant immune DC-
SIGN receptor. Additionally, the more mannose units on the glycans, though not necessarily
specific to α(1,2) or α(1,6) linkage, result in the greater the binding with the mannose receptor.
Thus, synthetic LM glycans could serve as a biochemical tool for further studies. This
experiment provides a much better understanding of the protein-carbohydrate interactions
between the pathogens and the host’s immune system.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
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EXPERIMENTAL SECTION
General information for Chemical Synthesis
All the chemicals used were reagent grade and used as supplied, except where noted.
All the reactions were performed in oven-dried glassware under an inert atmosphere, unless
noted otherwise. Dry dichloromethane (CH
2
Cl ) was obtained from a solvent purification
2
system (PureSolv MD 5, Innovative Technology). Benzyl bromide was treated with aluminum
oxide prior to use. Analytical thin layer chromatography (TLC) was performed on Merck silica
gel 60 F254 plates (0.25 mm). Compounds were visualized by staining with cerium ammonium
molybdate (Hanessian’s Stain) solution. Flash column chromatography was carried out using
a forced flow of the indicated solvent on Fluka Kieselgel 60 (230-400 mesh). Gel filtration
chromatography was carried out using Sephadex LH-20 from Biosciences.
1
13
All the new compounds were characterized by NMR spectroscopy ( H, C), ESI-MS,
MALDI-TOF, optical rotation, and melting point (for a solid). NMR spectra were recorded on
Bruker AVANCE III (300 MHz), Bruker Fourier 300 (300 MHz), and Bruker AVANCE 400
(400 MHz) spectrometer. NMR chemical shifts (δ) are reported in ppm relative to internal
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1
13
1
standards (CDCl
3
, δ 7.26 ppm for H, and δ 77.00 ppm for C; D O, δ 4.79 ppm for H) and
2
coupling constants (J) are reported in Hz. Splitting patterns are indicated as s, singlet; d,
1
doublet; t, triplet; q, quartet; m, multiplet; brs, broad singlet for H NMR data. High resolution
ESI mass spectra were obtained by Bruker microTOF mass spectrometer and Thermo Scientific
Q Exactive Focus Orbitrap mass spectrometer. MALDI-TOF was carried out using JEOL JMS-
S3000 MALDI-TOF mass spectrometer and Shimadzu AXIMA Performance MALDI TOF-
TOF mass spectrometer. Optical rotations were measured using JASCO P-1020 polarimeter.
Melting points were measured in capillaries using Staurt SMP30 apparatus.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
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8
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3
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2
3
4
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9
0
General Procedures for Glycosylations
The mannosyl donor and acceptor were co-evaporated with anhydrous toluene three
times in vacuo and placed under high vacuum for at least 4 h. Glycosylations were performed
without molecular sieves. Under argon atmosphere, a solvent was added to the mixture of
starting materials at rt and the solution was cooled to the desired temperature (0 °C by ice-
water bath and -10 °C by ice-acetone bath). A promoter (TMSOTf or TESOTf) was introduced
to the reaction solution in one portion via syringe. After the reaction had finished, excess
triethylamine (NEt ) was added to quench the reaction at the particular reaction temperature.
3
The reaction mixture was concentrated in vacuo and purified by flash silica gel column
chromatography.
Allyl O-(2-O-Benzoyl-3,4-di-O-benzyl-α-D-mannopyranosyl)-(1→6)-2-O-benzoyl-3,4-di-
O-benzyl-α-D-mannopyranoside (7):
Following the general procedures for glycosylations, a glycosylation of mannosyl
7
7
phosphate 5 (339.4 mg, 0.417 mmol) and mannose 6 (162.0 mg, 0.321 mmol) promoted by
TMSOTf (82.1 µL, 0.417 mmol) was carried out in CH Cl (0.6 mL) at 0 °C for 1.5 h. After
being quenched by NEt (100 µL), the reaction mixture was concentrated in vacuo and
subjected to the silica gel column chromatography (Hexane/EtOAc = 3:2) to obtain compound
2
2
3
7
(227.0 mg, 75%) as a colorless syrup. The spectral data were in agreement with those in the
literature.40 HRMS-ESI (m/z): [M+Na] calculated for C57
+
H
58NaO13, 973.3775; Found:
9
73.3778.
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Allyl O-(2-O-Benzoyl-3,4-di-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-benzoyl-3,4-
di-O-benzyl-α-D-mannopyranosyl)-(1→6)-2-O-benzoyl-3,4-di-O-benzyl-α-D-
annopyranoside (3):
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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3
3
3
3
3
3
4
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6
0
1
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9
0
Following the general procedures for glycosylations, a glycosylation of mannosyl
phosphate 5 (209.2 mg, 0.257 mmol) and dimannoside 7 (203.9 mg, 0.214 mmol) promoted by
TMSOTf (50.6 µL, 0.257 mmol) was carried out in CH
2
Cl
2
(0.6 mL) at 0 °C for 45 min. After
being quenched by NEt (100 µL), the reaction mixture was concentrated in vacuo and
3
subjected to flash silica gel column chromatography (Hexane/EtOAc = 3:2) to obtain
r.t.
compound 3 (197.0 mg, 66%) as a colorless syrup. R
f
0.30 (Hexane/EtOAc = 3:1); [α]
D
=
1
+
22.40 (c = 1.0, CH
2
Cl
2
); H NMR (400 MHz, CDCl
3
)  8.15-8.07 (m, 6H, Ar-H), 7.60-7.44
CH=CH ), 5.82 (dd, J = 1.9,
.1 Hz, 1H, H2), 5.75 (dd, J = 1.9, 3.1 Hz, 1H, H2), 5.69 (dd, J = 1.9, 3.1 Hz, 1H, H2), 5.29
(dd, J = 1.5, 17.2 Hz, 1H, CH CH=CH ), 5.20 (dd, J = 1.4, 10.4 Hz, 1H, CH CH=CH ), 5.10
d, J = 1.5 Hz, 1H, H1), 5.04 (d, J = 1.5 Hz, 1H, H1), 4.98 (d, J = 1.6 Hz, 1H, H1), 4.92-4.39
(
m, 9H, Ar-H), 7.30-7.08 (m, 30 H, Ar-H), 5.94-5.84 (m, 1H, CH
2
2
3
2
2
2
2
(
(
13
1
m, 12H), 4.20-3.60 (m, 17H); C{ H} NMR (100 MHz, CDCl ) δ 165.6 (COPh), 165.5
3
(COPh), 165.3 (COPh), 138.3, 138.3, 138.2, 137.8, 137.5, 133.2, 129.8, 128.5, 128.4, 128.2,
1
7
28.1, 127.9, 127.6, 127.4, 127.3, 118.0, 98.1 (C1), 97.9 (C1), 96.9 (C1), 78.5, 78.0, 77.6, 75.0,
4.0, 73.9, 73.6, 72.0, 71.5, 71.3, 71.1, 70.7, 68.9, 68.6, 68.4, 68.1, 66.0, 65.7, 61.7; HRMS-
+
ESI (m/z): [M+Na] calculated for C84
H84NaO19, 1419.5505; Found: 1419.5500.
Allyl O-(2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-2-O-benzoyl-3,4-
di-O-benzyl-α-D-mannopyranoside (8):
Following the general procedures for glycosylations, a glycosylation of mannosyl
phosphate 141,42 (300.5 mg, 0.439 mmol) and mannose 6 (147.6 mg, 0.293 mmol) promoted by
TMSOTf (86 µL, 0.439 mmol) was carried out in CH
2
Cl (0.6 mL) at 0 °C for 20 min. After
2
being quenched by NEt (100 µL), the reaction mixture was concentrated in vacuo and purified
3
by flash silica gel column chromatography (Hexane/EtOAc = 4:1) to obtain compound 8 (285.6
mg, 99%) as a colorless syrup. The spectral data were in agreement with those in the
literature.43 HRMS-ESI (m/z): [M+Na] calculated for C59
+
H62NaO13, 1001.4088; Found:
1
001.4080.
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(
2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-2-O-benzoyl-3,4-di-O-
benzyl-α-D-mannopyranose (9):
To a solution of compound 8 (266.4 mg, 0.272 mmol) in a mixture of acetic acid (6.0
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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6
7
8
9
0
1
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7
8
9
0
1
2
3
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5
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7
8
9
0
mL) and water (0.3 mL), NaOAc (223.2 mg, 2.721 mmol) and PdCl (241.3 mg, 1.361 mmol)
2
were added. The reaction mixture was stirred at rt for 12 h. After the reaction had finished, the
reaction was filtered through a pad of Celite and evaporated to dryness. The crude was extracted
with EtOAc and saturated NaHCO
3
(3×100.0 mL). The solid Na
2
CO was added to carefully
3
adjust the pH of the reaction mixture to 7. The combined organic layer was washed with
saturated brine, dried over Na
2
SO
4
and concentrated in vacuo. Purification of the crude by flash
7
silica gel column chromatography (Hexane/EtOAc = 13:7) gave compound 9 (221.6 mg, 87%)
r.t.
1
as a colorless syrup. R
f
0.33 (Hexane/EtOAc = 3:2); [α]
D
= + 0.18 (c = 1.0, CH
2
Cl ); H NMR
2
(300 MHz, CDCl ) δ 8.09-8.07 (m, 2H, Ar-H), 7.52-7.42 (m, 3H, Ar-H), 7.29-7.10 (m, 25H,
3
Ar-H), 5.63 (dd, J = 1.9, 3.0 Hz, 1H, H2), 5.45 (dd, J = 1.9, 3.0 Hz, 1H, H2), 5.25 (brs, 1H,
H1), 4.96 (d, J = 1.4 Hz, 1H, H1), 4.89-4.41 (m, 10H), 4.17-3.60 (m, 11H), 2.13 (s, 3H,
13
1
COCH
3
); C{ H} NMR (75 MHz, CDCl
3
) δ 170.6 (COCH
3
), 165.8 (COPh), 138.3, 138.0,
137.8, 133.3, 129.9, 128.5, 128.5, 128.4, 128.3, 128.2, 128.0, 127.8, 127.7, 98.2 (C1), 92.5
(C1), 78.2, 77.8, 75.2, 75.1, 74.4, 73.4, 71.6, 71.5, 70.8, 69.4, 69.1, 68.6, 67.7, 21.2 (CH );
3
+
HRMS-ESI (m/z): [M+Na] calculated for C56
H58NaO13, 961.3775; Found: 961.3769.
N-Phenyltrifluoroacetimidoyl O-(2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1
→
6)-2-O-benzoyl-3,4-di-O-benzyl-α-D-mannopyranoside (4):
N-phenyltrifluoroacetimidoyl chloride (103.7 µL, 7.645 mmol) was added to a solution
of disaccharide 9 (151.3 mg, 0.161 mmol) in acetone (3.0 mL) at 0 °C, followed by cesium
carbonate (105.0 mg, 0.322 mmol). The reaction was stirred overnight, allowing it to warm to
rt. After the reaction had finished, it was filtered through a pad of Celite and evaporated to
dryness. The crude was purified by flash silica gel column chromatography (Hexane/EtOAc =
4
:1) to obtain compound 4 (152.3 mg, 85%) as a yellowish syrup. R
f
0.50 (Hexane/EtOAc =
r.t.
1
7:3); [α]
D
= +37.16 (c = 1.0, CH
2
Cl
2
); H NMR (300 MHz, CDCl
3
) δ 8.11-8.09 (m, 2H, Ar-
H), 7.51-7.45 (m, 3H, Ar-H), 7.34-7.04 (m, 28H, Ar-H), 6.84 (d, J = 7.8 Hz, 2H, Ar-H), 5.78
(brs, 1H, H2), 5.53 (brs, 1H, H2), 5.00 (brs, 2H, H1), 4.88-4.40 (m, 10H), 4.15-3.57 (m, 10H),
13
1
2.15 (s, 3H, COCH
3
); C{ H} NMR (75 MHz, CDCl
3
) δ 170.4 (COCH ), 165.5 (COPh),
3
1
43.2, 138.6, 138.3, 138.1, 137.8, 137.6, 133.6, 130.0, 129.6, 128.9, 128.8, 128.5, 128.4, 128.3,
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28.2, 128.0, 127.9, 127.7, 127.6, 124.7, 119.6, 98.3, (C1), 94.6 (C1), 77.9, 75.5, 75.2, 74.2,
+
73.5, 72.2, 71.9, 71.6, 68.8, 68.4, 67.7, 66.0, 21.2 (CH
3
); HRMS-ESI (m/z): [M+Na] calculated
for C64
H F
62 3
NNaO13, 1132.4071; Found: 1132.4025.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
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3
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5
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0
1
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8
9
0
1
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6
7
8
9
0
1
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6
7
8
9
0
1
2
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4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Allyl O-(2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-benzoyl-3,4-
di-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-benzoyl-3,4-di-O-benzyl-α-D-
mannopyranosyl)-(1→6)-O-(2-O-benzoyl-3,4-di-O-benzyl-α-D-mannopyranosyl)-(1→6)-
2
-O-benzoyl-3,4-di-O-benzyl-α-D-mannopyranoside (10):
Following the general procedures for glycosylations, a glycosylation of dimannosyl
imidate 4 (370.0 mg, 0.333 mmol) and trimannoside 3 (310.5 mg, 0.222 mmol) promoted by
TMSOTf (65.6 µL, 0.333 mmol) was carried out in CH Cl (2.7 mL) at 0 °C for 30 min. After
being quenched by NEt (100 µL), the reaction mixture was concentrated in vacuo and purified
2
2
3
by flash silica gel column chromatography (Hexane/EtOAc = 7:3) to obtain compound 10
r.t.
(453.8 mg, 88%) as a colorless syrup. R
f
0.45 (Hexane/EtOAc = 7:3); [α]
D
= + 1.17 (c = 0.1,
1
CH
2
Cl
2
); H NMR (300 MHz, CDCl
3
)  8.19-8.13 (m, 8H, Ar-H), 7.51-7.46 (m, 12H, Ar-H),
CH=CH ), 5.84 (brs, 3H, H2), 5.70 (dd, J
= 1.7, 2.8 Hz, 1H, H2), 5.58 (dd, J = 1.7, 2.8 Hz, 1H, H2), 5.29 (dd, J = 1.5, 17.2 Hz, 1H,
CH CH=CH ), 5.19 (dd, J = 1.1, 10.4 Hz, 1H, CH CH=CH ), 5.14 (d, J = 1.0 Hz, 1H, H1),
.09 (d, J = 0.9 Hz, 1H, H1), 5.06 (d, J = 1.0 Hz, 1H, H1), 5.00 (d, J = 1.2 Hz, 1H, H1), 4.99
7
.34-7.07 (m, 55 H, Ar-H), 5.95-5.87 (m, 1H, CH
2
2
2
2
2
2
5
13
1
(d, J = 1.4 Hz, 1H, H1), 4.95-4.34 (m, 22H), 4.20-3.49 (m, 27H), 2.14 (s, 3H, COCH ); C{ H}
3
NMR (75 MHz, CDCl
3
) δ 170.4 (COCH
3
), 165.9 (COPh), 165.7 (COPh), 165.6 (COPh), 165.6
(COPh), 138.6, 138.3, 137.7, 133.4, 129.9, 128.7, 128.4, 128.3, 127.9, 127.7, 127.4, 118.2,
9
8.5 (C1), 98.3 (3×C1), 97.1, (C1), 78.7, 78.4, 77.9, 75.2, 74.3, 74.1, 74.1, 73.5, 71.8, 71.5,
+
71.1, 69.1, 68.7, 68.3, 66.2, 65.9, 65.8, 21.3 (CH
3
); HRMS-ESI (m/z): [M+Na] calculated for
C
140
H140NaO31, 2339.9276; Found: 2339.9285.
(2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-benzoyl-3,4-di-O-
benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-benzoyl-3,4-di-O-benzyl-α-D-
mannopyranosyl)-(1→6)-O-(2-O-benzoyl-3,4-di-O-benzyl-α-D-mannopyranosyl)-(1→6)-
2
-O-benzoyl-3,4-di-O-benzyl-α-D-mannopyranose (11):
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6
To a solution of compound 10 (261.7 mg, 0.113 mmol) in a mixture of acetic acid (7.5
mL), EtOAc (0.4 mL) and water (0.4 mL), NaOAc (92.6 mg, 1.129 mmol) and PdCl (100.0
2
mg, 0.5650 mmol) were added. The reaction mixture was stirred at rt for 12 h. After the reaction
had finished, the reaction was filtered through a pad of Celite and evaporated to dryness. The
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
crude was extracted with EtOAc and saturated NaHCO
added to carefully adjust the pH of the reaction mixture to 7. The combined organic layer was
washed with saturated brine, dried over Na SO , and concentrated in vacuo. Purifying the crude
3
(3×100.0 mL). Solid Na
2
CO was
3
2
4
by flash silica gel column chromatography (Hexane/EtOAc = 3:2) gave the desired compound
r.t.
1
1 (217.0 mg, 85%) as a colorless syrup. R
f
0.50 (Hexane/EtOAc = 3:2); [α]
D
= +36.33 (c =
1
0
.1, CH
2
Cl
2
); H NMR (300 MHz, CDCl
3
) δ 8.16-8.07 (m, 8H, Ar-H), 7.49-7.47 (m, 12H, Ar-
H), 7.31-7.09 (m, 55H, Ar-H), 5.80 (brs, 3H, H2), 5.64 (brs, 1H, H2), 5.56 (brs, 1H, H2), 5.21-
13
1
4
.98 (m, 5H, H1), 4.92-4.31 (m, 22H), 4.17-3.46 (m, 25H), 2.15 (s, 3H, COCH
NMR (75 MHz, CDCl ) δ 170.4 (COCH ), 165.8 (COPh), 165.8 (COPh), 165.7 (COPh), 165.6
COPh), 138.6, 138.4, 138.2, 137.7, 133.4, 129.9, 128.7, 128.5, 127.8, 127.7, 127.4, 98.5 (C1),
3
); C{ H}
3
3
(
9
7
8.3 (C1), 98.2 (C1), 97.9 (C1), 92.7 (C1), 78.3, 78.2, 77.9, 75.2, 74.5, 74.1, 73.8, 73.5, 72.1,
1.7, 71.5, 71.0, 69.4, 68.6, 68.3, 66.4, 65.8, 26.5, 21.2 (CH
); HRMS-ESI (m/z): [M+Na]+
136NaO31, 2299.8963; Found: 2299.8955.
3
calculated for C137
H
N-Phenyltrifluoroacetimidoyl O-(2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-
1→6)-O-(2-O-benzoyl-3,4-di-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-benzoyl-
(
3
,4-di-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-benzoyl-3,4-di-O-benzyl-α-D-
mannopyranosyl)-(1→6)-2-O-benzoyl-3,4-di-O-benzyl-α-D-mannopyranoside (12):
N-phenyltrifluoroacetimidoyl chloride (125.3 µL, 0.779 mmol) was added to a solution
of pentasaccharide 11 (354.8 mg, 0.156 mmol) in acetone (7.7 mL) at 0 °C, followed by cesium
carbonate (101.5 mg, 0.311 mmol). The reaction was stirred overnight, allowing it to warm to
rt. After the reaction had finished, the reaction was filtered through a pad of Celite and
evaporated to dryness. The crude was purified by flash silica gel column chromatography
(
Hexane/EtOAc = 7:3) to obtain compound 12 (248.4 mg, 65%) as a colorless syrup. R
f
0.45
) δ 8.16-
.12 (m, 8H, Ar-H), 7.51-7.46 (m, 12H, Ar-H), 7.30-7.07 (m, 58H, Ar-H), 6.83 (d, J = 7.9 Hz,
2H, Ar-H), 5.81 (brs, 4H, H2), 5.56 (brs, 1H, H2), 5.09-4.91 (m, 5H, H1), 4.89-4.31 (m, 22H),
r.t.
1
(
Hexane/EtOAc = 7:3); [α]
D
= +57.93 (c = 0.1, CH
2
Cl
2
); H NMR (400 MHz, CDCl
3
8
1
3
1
4
.17-3.45 (m, 25H), 2.15 (s, 3H, COCH
3
); C{ H} NMR (100 MHz, CDCl
3
) δ 170.2 (COCH ),
3
2
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65.6 (COPh), 165.5 (COPh), 138.5, 138.0, 137.6, 133.3, 129.8, 128.6, 128.3, 128.1, 127.7,
127.2, 119.4, 98.5 (C1), 98.4, (3×C1) 98.2 (C1), 78.2, 77.7, 75.0, 73.9, 73.3, 72.1, 71.6, 71.4,
+
7
0.9, 68.4, 67.5, 65.9, 65.6, 21.1 (CH
3
); HRMS-ESI (m/z): [M+Na] calculated for
C
145
H F
140 3
NNaO31, 2470.9259; Found: 2470.9229.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6
-(S-Benzyl)thiohexyl O-(2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-2-
O-benzoyl-3,4-di-O-benzyl-α-D-mannopyranoside (13):
Following the general procedures for glycosylations, a glycosylation of dimannosyl
imidate 4 (226.8 mg, 0.204 mmol) and 6-(benzylthio)-1-hexanol 2 (229.0 mg, 1.022 mmol),
promoted by TMSOTf (60.0 µL, 0.307 mmol), was carried out in CH
for 1 h. After being quenched by NEt (60.0 µL), the reaction mixture was concentrated in
vacuo and purified by size exclusion column chromatography (Sephadex LH-20,
CH Cl /MeOH = 7:3) and by flash silica gel column chromatography (Hexane/EtOAc = 9:1)
to obtain compound 13 (180.0 mg, 77%) as a yellowish syrup. R 0.55 (Hexane/EtOAc = 7:3);
) δ 8.11-8.08 (m, 2H, Ar-H),
.49-7.43 (m, 3H, Ar-H), 7.31-7.09 (m, 30H, Ar-H), 5.61 (dd, J = 1.8, 2.9 Hz, 1H, H2), 5.52
2
Cl
2
(0.7 mL) at -10 °C
3
2
2
f
r.t.
1
[
α]
D
= +20.46 (c = 1.0, CH
2
Cl
2
); H NMR (300 MHz, CDCl
3
7
(dd, J = 1.8, 2.6 Hz, 1H, H2), 5.01 (d, J = 1.2 Hz, 1H, H1), 4.88 (brs, 1H, H1), 4.86-4.40 (m,
1
0H), 4.10-3.35 (m, 12H), 3.69 (brs, 2H), 2.40 (t, J = 7.5 Hz, 2H, CH
2
SBn), 2.15 (s, 3H,
), 1.59-1.50 (m, 4H), 1.38-1.28 (m, 4H); C{ H} NMR (75 MHz, CDCl ) δ 170.2
), 165.7 (COPh), 138.5, 138.4, 138.3, 138.1, 137.9, 137.6, 133.2, 129.9, 129.8, 128.8,
128.5, 128.4, 128.3, 128.2, 128.1, 128.1, 128.0, 127.7, 127.6, 127.5, 127.4, 126.8, 97.9 (C1),
13
1
COCH
3
3
(COCH
3
9
3
7.7 (C1), 78.5, 77.9, 75.1, 75.0, 74.1, 74.0, 73.3, 71.5, 71.4, 70.6, 69.0, 68.6, 68.3, 67.8, 66.1,
+
6.2, 31.2, 29.2, 29.0, 28.6, 25.7, 21.1 (CH
3
); HRMS-ESI (m/z): [M+Na] calculated for
C H
69
76NaO13S, 1167.4904; Found: 1167.4900.
6
-(S-Benzyl)thiohexyl O-(3,4,6-Tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-3,4-di-O-
benzyl-α-D-mannopyranoside (14):
To a solution of compound 13 (65.4 mg, 0.057 mmol) in a mixture of CH
2
Cl and
2
MeOH (2.0 mL each), NaOMe (12.4 mg, 0.228 mmol) was added. The reaction mixture was
refluxed at 50 °C. After 12 h, the reaction was evaporated to dryness. The crude was purified
by flash silica gel column chromatography (Hexane/EtOAc = 3:2) to obtain compound 14 (53.0
r.t.
mg, 93%) as a colorless syrup. R
f
0.20 (Hexane/EtOAc = 3:2); [α]
D
= +46.37 (c = 1.0,
2
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1
1
2
2
2
2
2
2
2
2
2
2
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
CH
2
Cl
2
); H NMR (300 MHz, CDCl ) δ 7.34-7.13 (m, 30H, Ar-H), 5.03 (brs, 1H, H1), 4.88-
3
4.41 (m, 10H), 4.81 (brs, 1H, H1) 4.12-4.00 (brs, 2H, H2), 3.94-3.29 (m, 12H), 3.67 (brs, 2H),
1
3
1
2
.38 (t, J = 7.3 Hz, 2H, CH
MHz, CDCl ) δ 138.4, 138.2, 138.1, 137.9, 128.7, 128.4, 128.4, 128.3, 128.3, 128.2, 128.1,
27.8, 127.8, 127.7, 127.6, 127.4, 126.8, 99.7 (C1), 98.9 (C1), 80.3, 79.6, 75.0, 74.9, 74.1,
2
SBn), 1.58-1.45 (m, 4H) 1.38-1.22 (m, 4H); C{ H} NMR (75
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
74.0, 73.3, 71.8, 71.6, 71.1, 70.5, 68.7, 68.2, 68.0, 67.5, 66.0, 36.2, 31.2, 29.1, 29.0, 28.5, 25.7;
+
HRMS-ESI (m/z): [M+Na] calculated for C60
H70NaO11S, 1021.4537; Found: 1021.4557.
6
3
-(S-Benzyl)thiohexyl O-(2-O-{2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl}-
,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-2-O-{2-O-acetyl-3,4,6-tri-O-benzyl-α-D-
mannopyranosyl}-3,4-di-O-benzyl-α-D-mannopyranoside (15):
Following the general procedures for glycosylations, a glycosylation of dimannoside
1
4 (44.4 mg, 0.044 mmol) and mannosyl phosphate 1 (152.0 mg, 0.222 mmol) promoted by
TESOTf (51.0 µL, 0.222 mmol) was carried out in Et O (3.0 mL) at 0 °C for 1 h. After being
quenched by NEt (60 µL), the reaction mixture was concentrated in vacuo and purified by
flash silica gel column chromatography (Hexane/EtOAc = 7:3) and by size exclusion column
2
3
chromatography (Sephadex LH-20, CH
8%) as a colorless syrup. R
1H NMR (400 MHz, CDCl
.53 (dd, J = 1.9, 3.0 Hz, 1H, H2), 5.08 (d, J = 1.9 Hz, 1H, H1), 5.07 (d, J = 1.8 Hz, 1H, H1),
4.92 (d, J = 1.5 Hz, 1H, H1), 4.76 (d, J = 1.2 Hz, 1H, H1), 4.86-4.32 (m, 22H), 4.05-3.17 (m,
4H), 3.66 (brs, 2H), 2.35 (t, J = 7.5 Hz, 2H, CH SBn), 2.10-2.09 (s, 6H, COCH ), 1.53-1.40
m, 4H) 1.32-1.16 (m, 4H); C{ H} NMR (100 MHz, CDCl ) δ 170.1 (COCH ), 138.6, 138.6,
38.5, 138.2, 138.2, 138.2, 138.1, 138.0, 137.9, 128.8, 128.4, 128.4, 128.3, 128.3, 128.3, 128.2,
128.2, 127.9, 127.8, 127.8, 127.8, 127.7, 127.6, 127.5, 127.4, 127.4, 126.8, 99.6 (2×C1), 98.5
C1), 98.4 (C1), 80.0, 79.8, 78.2, 78.1, 77.2, 75.2, 75.0, 75.0, 74.9, 74.8, 74.7, 74.6, 74.3, 74.2,
2
Cl
2
/MeOH = 7:3) to obtain compound 15 (76.4 mg,
r.t.
8
f
0.50 (Hexane/EtOAc = 7:3); [α]
D
= +229.57 (c = 1.0, CH
2
Cl );
2
3
) δ 7.33-7.10 (m, 60H, Ar-H), 5.54 (dd, J = 1.7, 3.1 Hz, 1H, H2),
5
2
2
3
13
1
(
3
3
1
(
7
4.1, 73.4, 73.3, 73.1, 72.0, 71.9, 71.9, 71.8, 71.7, 71.6, 70.8, 68.9, 68.8, 68.7, 68.6, 67.4, 67.1,
+
36.2, 31.3, 29.2, 29.1, 28.7, 25.9, 21.1 (CH
3
); HRMS-ESI (m/z): [M+Na] calculated for
C
118
H
130NaO23S, 1969.8621; Found: 1969.8616.
6-(S-Benzyl)thiohexyl O-(2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-
(
2-O-benzoyl-3,4-di-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-benzoyl-3,4-di-O-
2
2
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benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-benzoyl-3,4-di-O-benzyl-α-D-
mannopyranosyl)-(1→6)-2-O-benzoyl-3,4-di-O-benzyl-α-D-mannopyranoside (16):
Following the general procedures for glycosylations, a glycosylation of pentamannosyl
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
imidate 12 (57.9 mg, 0.024 mmol) and 6-(benzylthio)-1-hexanol 2 (21.2 mg, 0.095 mmol)
promoted by TMSOTf (8.4 µL, 0.043 mmol) was carried out in CH
h. After being quenched by NEt (10.0 µL), the reaction mixture was concentrated in vacuo
and purified by size exclusion column chromatography (Sephadex LH-20, CH Cl /MeOH =
:3) and by flash silica gel column chromatography (Hexane/EtOAc = 4:1) to obtain compound
2
Cl (0.5 mL) at -10 °C for
2
1
3
2
2
7
r.t.
16 (44.5 mg, 76%) as a colorless syrup. R
f
0.50 (Hexane/EtOAc = 7:3); [α]
D
= +39.20 (c =
1
1
.0, CH
2
Cl
2
); H NMR (400 MHz, CDCl
3
) δ 8.19-8.14 (m, 8H, Ar-H), 7.51-7.46 (m, 12H, Ar-
H), 7.34-7.10 (m, 60H, Ar-H), 5.84-5.67 (brs, 4H, H2), 5.58 (brs, 1H, H2), 5.14-4.99 (m, 5H,
H1), 4.93-4.32 (m, 22H), 4.13-3.38 (m, 27H), 3.67 (brs, 2H), 2.39 (t, J = 7.6 Hz, 2H, CH SBn),
), 1.70-1.53 (m, 4H), 1.36-1.31 (m, 4H); C{ H} NMR (100 MHz, CDCl
), 166.0 (COPh), 165.7 (COPh), 165.7 (COPh), 165.6 (COPh), 138.8, 138.6,
38.5, 138.3, 138.1, 137.8, 137.8, 137.7, 137.7, 133.4, 130.1, 130.0, 128.9, 128.8, 128.7, 128.6,
128.5, 128.5, 128.4, 128.4, 128.4, 128.3, 128.3, 128.2, 127.9, 127.9, 127.8, 127.7, 127.7, 127.4,
2
1
3
1
2
.15 (s, 3H, COCH
3
3
)
δ 170.4 (COCH
3
1
1
7
6
27.4, 127.3, 127.0, 98.5 (2×C1), 98.4 (C1), 98.3 (C1), 98.0 (C1), 78.8, 78.5, 78.4, 78.2, 77.9,
5.3, 75.2, 74.4, 74.2, 74.1, 74.0, 73.9, 73.5, 71.8, 71.6, 71.5, 71.4, 71.1, 71.0, 70.9, 69.2, 68.7,
8.6, 68.5, 68.4, 68.0, 66.2, 65.9, 65.8, 65.7, 36.4, 31.4, 29.4, 29.2, 28.8, 26.0, 21.3 (CH );
3
+
HRMS-ESI (m/z): [M+Na] calculated for C150
H154NaO31S, 2506.0092; Found: 2506.0058.
6-(S-Benzyl)thiohexyl O-(3,4,6-Tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-(3,4-di-O-
benzyl-α-D-mannopyranosyl)-(1→6)-O-(3,4-di-O-benzyl-α-D-mannopyranosyl)-(1→6)-
O-(3,4-di-O-benzyl-α-D-mannopyranosyl)-(1→6)-3,4-di-O-benzyl-α-D-mannopyranoside
17):
(
To a solution of compound 16 (142.8 mg, 0.058 mmol) in a mixture of CH
2
Cl and
2
MeOH (3.0 mL each), NaOMe (31.0 mg, 0.575 mmol) was added. The reaction mixture was
refluxed at 50 °C. After 12 h, the reaction was evaporated to dryness. The crude was purified
by size exclusion column chromatography (Sephadex LH-20, CH
2
Cl
2
/MeOH = 7:3) to obtain
r.t.
compound 17 (109.1 mg, 94%) as a colorless syrup. R 0.38 (CH Cl
f
2
2
/MeOH = 19:1); [α]
D
=
2
3
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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5
5
5
5
5
5
5
5
5
5
6
1
Pd(+65.07 (c = 1.0, CH
2
Cl
2
); H NMR (300 MHz, CDCl ) δ 7.32-7.13 (m, 60H, Ar-H), 5.01-
3
4.94 (m, 5H, H1), 4.88-4.41 (m, 22H), 4.11-3.99 (brs, 5H, H2), 3.88-3.28 (m, 27H), 3.66 (brs,
1
3
1
2
H), 2.36 (t, J = 7.4 Hz, 2H, CH
75 MHz, CDCl ) δ 138.5, 138.4, 138.3, 138.3, 138.2, 138.0, 137.9, 137.8, 137.8, 137.6, 128.7,
28.4, 128.4, 128.4, 128.3, 128.3, 128.2, 128.2, 128.1, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4,
2
SBn), 1.88-1.41 (m, 4H), 1.36-1.18 (m, 4H); C{ H} NMR
(
3
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
127.3, 126.8, 99.7 (C1), 99.0 (2×C1), 98.8 (C1), 98.7 (C1), 80.2, 79.8, 79.8, 79.5, 75.0, 74.9,
7
6
4.8, 74.7, 74.2, 74.1, 73.8, 73.3, 71.8, 71.6, 71.5, 71.4, 71.1, 70.7, 70.6, 70.1, 70.0, 68.8, 68.2,
+
7.4, 67.9, 65.9, 36.2, 31.2, 29.1, 29.0, 28.5, 25.8; HRMS-ESI (m/z): [M+Na] calculated for
C
120
H136NaO26S, 2047.8938; Found: 2047.8972.
6-(S-Benzyl)thiohexyl O-(2-O-{2-O-Acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl}-
3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-{2-O-acetyl-3,4,6-tri-O-benzyl-
α-D-mannopyranosyl}-3,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-O-(2-O-{2-O-
acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl}-3,4,6-tri-O-benzyl-α-D-
mannopyranosyl)-(1→6)-O-(2-O-{2-O-acetyl-3,4,6-tri-O-benzyl-α-D-mannopyranosyl}-
3
,4,6-tri-O-benzyl-α-D-mannopyranosyl)-(1→6)-2-O-{2-O-acetyl-3,4,6-tri-O-benzyl-α-D-
mannopyranosyl}-3,4-di-O-benzyl-α-D-mannopyranoside (18):
Following the general procedures for glycosylations, a glycosylation of
pentamannoside 17 (101.2 mg, 0.050 mmol) and mannosyl phosphate 1 (342.0 mg, 0.499
mmol), promoted by TESOTf (28.7 µL, 0.125 mmol), was carried out in Et
for 25 min. After being quenched by NEt (100 µL), the reaction mixture was concentrated in
vacuo and purified by flash silica gel column chromatography (Hexane/EtOAc = 7:3) and by
2
O (5.0 mL) at 0 °C
3
size exclusion column chromatography (Sephadex LH-20, CH
2
Cl
2
/MeOH = 7:3) to obtain
r.t.
compound 18 (132.2 mg, 60%) as a colorless syrup. R
f
0.73 (Hexane/EtOAc = 3:2); [α]
D
=
1
+
35.45 (c = 1.0, CH
2
Cl
2
); H NMR (400 MHz, CDCl
3
) δ 7.27-7.07 (m, 135H, Ar-H), 5.56-5.50
(
brs, 5H, H2), 5.12-4.88 (m, 10H, H1), 4.84-4.23 (m, 52H), 4.14-3.05 (m, 57H), 3.64 (brs, 2H),
2.34 (t, J = 7.1 Hz, 2H, CH
2
SBn), 2.09-1.99 (s, 15H, COCH
) δ 170.0 (COCH
), 138.6, 138.6, 138.5, 138.4, 138.3, 138.2, 138.2,
38.1, 138.0, 137.8, 137.7, 128.7, 128.4, 128.2, 128.0, 127.9, 127.8, 127.8, 127.7, 127.6, 127.5,
27.4, 127.4, 127.3, 126.8, 99.9 (C1), 99.8 (C1), 99.7 (C1), 99.5 (C1), 99.3 (C1), 99.2 (C1),
3
), 1.66-1.43 (m, 4H), 1.28-1.21
1
(
(
m, 4H); 13C{ H} NMR (100 MHz, CDCl
3
3
), 170.0 (COCH ), 169.9
3
COCH
3
), 169.8 (COCH
3
), 169.7 (COCH
3
1
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2
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9.0 (C1), 98.9 (C1), 98.8 (C1), 98.7 (C1), 80.0, 79.7, 79.6, 79.6, 79.2, 79.1, 78.5, 78.4, 78.3,
78.2, 75.0, 74.9, 74.8, 74.6, 74.2, 74.0, 73.3, 73.2, 73.1, 72.0, 71.8, 71.6, 69.0, 68.7, 68.6, 68.5,
2
+
6
7.4, 36.2, 31.2, 29.2, 29.0, 28.6, 25.8, 21.0, 20.9; HRMS-ESI (m/z): [M+2Na] calculated for
56S, 2220.9524; Found: 2220.9544.
C
265
H
286Na O
2
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
General Procedures for Birch Reductions
Each sample of the conjugated oligomannoside with a thiol linker (13, 15, 16 and 18,
~
100 mg) was dissolved in dried THF (5.0 mL) in a three-neck round bottom flask. At -78 °C
(dry ice/acetone bath), ammonia was condensed into the flask. Small pieces of sodium metal
were added to generate a stable dark blue solution. The dark blue solution was stirred at -78 °C
for 6 hours. The reaction was allowed to slowly warm up to rt. The remaining ammonia in the
reaction solution was blown off by argon stream. The reaction solution was concentrated in
vacuo and redissolved in DI water (10.0 mL). The solution was neutralized by adding a small
amount of formic acid until it reached pH 7. The aqueous solution was extracted with CH
3×10.0 mL) to remove the less polar partially debenzylated side products and the remaining
starting material. In case of Man and BMan , the remaining materials in the organic layer
2
Cl
2
(
2
4
were subjected to Birch Reduction as described above. The aqueous layer containing the
debenzylated products was dialyzed in DI water at 4 °C for 48 h to give the final products.
6-Thiohexyl O-(α-D-Mannopyranosyl)-(1→6)-α-D-mannopyranoside (Man ):
2
r.t.
Colorless solid (13.7 mg, 25%). [α]
D
= +15.47 (c = 0.1, Milli-Q water); mp = 179.1-
1
183.2 °C; H NMR (400 MHz, D
2
O) δ 4.89-4.84 (brs, 2H, H1), 3.96-3.53 (m, 14H), 2.90 (t, J
=
8.1 Hz, 0.58H), 2.76 (t, J = 7.0 Hz, 0.42H), 2.36 (t, J = 7.9 Hz, 1H), 1.77-1.54 (m, 4H), 1.47-
13
1
1
6
.37 (m, 4H); C{ H} NMR (100 MHz, D O) δ 99.9 (C1), 99.5 (C1), 72.7, 70.8, 70.6, 70.1,
2
8.0, 66.8, 65.8, 61.0, 60.8, 51.0, 28.3, 27.9, 27.4, 25.2, 23.9, 21.7; MS-MALDI (m/z):
+
[M+Na] calculated for C18
H34NaO11S, 481.1720; Found: 481.1739.
6-Thiohexyl O-(α-D-Mannopyranosyl)-(1→6)-O-(α-D-mannopyranosyl)-(1→6)-O-(α-D-
mannopyranosyl)-(1→6)-O-(α-D-mannopyranosyl)-(1→6)-α-D-mannopyranoside
(Man ):
5
r.t.
Colorless solid (12.3 mg, 27%). [α]
D
= +77.53 (c = 0.1, Milli-Q water); mp = 187.2-
1
1
92.0 °C; H NMR (400 MHz, D
2
O) δ 4.87-4.83 (brs, 5H, H1), 3.97-3.52 (m, 32H), 2.89 (t, J
2
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2
2
2
2
2
2
2
3
3
3
3
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3
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3
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4
4
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5
5
5
5
5
5
5
5
5
6
=
8.6 Hz, 0.58H), 2.75 (t, J = 7.0 Hz, 0.42H), 2.36 (t, J = 7.7 Hz, 1H), 1.76-1.53 (m, 4H), 1.46-
1
3
1
1.35 (m, 4H); C{ H} NMR (100 MHz, D
2
O) δ 99.9 (C1), 99.5 (C1), 99.3 (3×C1), 72.7, 70.9,
7
3
0.8, 70.7, 70.6, 70.5, 70.1, 70.0, 69.9, 68.0, 66.8, 66.7, 66.6, 65.7, 65.6, 65.5, 61.0, 60.7, 51.0,
8.2, 28.4, 28.3, 28.2, 27.9, 27.4, 27.1, 25.2, 25.0, 24.9, 23.9, 21.7; MS-MALDI (m/z):
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
[
M+Na] calculated for C36
H64NaO26S, 967.3304; Found: 967.3343.
6
-Thiohexyl O-(2-O-{α-D-Mannopyranosyl}-α-D-mannopyranosyl)-(1→6)-2-O-{α-D-
mannopyranosyl}-α-D-mannopyranoside (BMan ):
4
r.t.
Colorless solid (11.7 mg, 33%). [α]
D
= +25.80 (c = 0.1, Milli-Q water); mp = 183.5-
1
188.9 °C; H NMR (400 MHz, D
2
O) δ 5.12-5.01 (brs, 4H, H1), 4.06-3.52 (m, 26H), 2.91 (t, J
=
8.0 Hz, 0.93H), 2.77 (t, J = 6.8 Hz, 0.53H), 2.36 (t, J = 7.8 Hz, 0.75H), 1.76-1.53 (m, 4H)
13
1
1
7
.47-1.36 (m, 4H); C{ H} NMR (100 MHz, D O) δ 102.3 (2×C1), 98.3 (C1), 98.1 (C1), 78.7,
2
3.4, 73.2, 72.8, 71.2, 70.5, 70.4, 70.0, 69.9, 68.1, 66.9, 66.8, 66.7, 65.8, 61.1, 60.9, 51.0, 28.3,
+
28.2, 28.0, 27.4, 25.2, 25.0, 24.9, 23.9, 21.7; MS-MALDI (m/z): [M+Na] calculated for
C H54NaO21S, 805.2776; Found: 805.2798.
30
6
-Thiohexyl O-(2-O-{α-D-Mannopyranosyl}-α-D-mannopyranosyl)-(1→6)-O-(2-O-{α-D-
mannopyranosyl}-α-D-mannopyranosyl)-(1→6)-O-(2-O-{α-D-mannopyranosyl}-α-D-
mannopyranosyl)-(1→6)-O-(2-O-{α-D-mannopyranosyl}-α-D-mannopyranosyl)-(1→6)-2-
O-{α-D-mannopyranosyl}-α-D-mannopyranoside (BMan10):
r.t.
Colorless solid (15.4 mg, 31%). [α]
D
= +91.07 (c = 0.1, Milli-Q water); mp = 208.4-
1
2
13.9 °C; H NMR (400 MHz, D
2
O) δ 5.11-4.90 (brs, 10H, H1), 4.07-3.56 (m, 62H), 2.91, (t,
J = 8.0 Hz, 0.71H), 2.77 (t, J = 6.8 Hz, 0.72H), 2.39 (t, J = 7.2 Hz, 0.57H), 1.77-1.56 (m, 4H),
1
3
1
1.48-1.36 (m, 4H); C{ H} NMR (100 MHz, D
2
O) δ 102.2 (5×C1), 98.3 (C1), 98.2 (4×C1),
7
3
8.8, 78.6, 73.3, 72.8, 71.2, 70.5, 70.3, 70.1, 68.1, 66.9, 66.7, 66.5, 65.8, 65.6, 61.1, 60.9, 51.0,
+
8.2, 28.3, 28.0, 27.5, 27.1, 25.2, 25.0, 23.9, 21.6; MS-MALDI (m/z): [M+Na] calculated for
114NaO51S, 1777.5945; Found: 1777.5980.
C H
66
Mannan Microarrays to Determine Binding to DC-SIGN and MR
The synthetic glycans were immobilized on maleimide activated glass slides via their
thiol handle following established protocols.7,44 Amine-coated GAPS slides (purchased from
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Corning) were incubated overnight at rt in a 35 mL DMF solution containing succinimidyl 4-
(N-maleimidomethyl)cyclohexane-1-carboxylate
(SMCC,
0.7
mM)
and
N,N-
diisopropylethylamine (DIPEA, 100 mM). Then, the slides were washed four times with
methyl alcohol and dried under a stream of argon. After preparing the maleimide functionalized
surfaces, the synthetic glycans were immobilized on the slides using the following protocol:
the synthetic glycans were treated with tris(2-carboxyethyl)phosphine (TCEP, 1 equiv.) in a
PBS buffer (10 mM; pH, 7.4) for 1 h at rt. An array printer (NanoPrint 210 equipped with a
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46MP10 stealth pin, Arrayit Corporation) was used to deliver as little as 3.9 nL/spot of the
solutions containing carbohydrates that ranged in concentrations from 40 µM to 2,000 µM.
Each concentration of a compound was spotted three times for technical replicates. Thereafter,
the slides were stored in a humid chamber for 12 h at rt, then incubated for 1 h in a solution of
3
-mercaptopropionic acid (1 mM) in 35 mL of the PBS buffer to quench all the remaining
maleimide groups. The slides were washed three times with distilled water, and then twice with
ethanol (95%). The slides were incubated for 1 h in a solution of 2.5% w/v BSA, then washed
three times with PBST. The glass slides printed with the immobilized glycans were incubated
with 30 µL solution of target protein (2 µg/100 µL, recombinant human DC-SIGN/CD209 Fc
Chimera or recombinant human MR, purchased from R&D Systems) in a HEPES buffer (50
mM, pH 7.5) solution containing 1% BSA, 20 mM CaCl , and 0.5% Tween-20 at rt for 2 h to
2
allow the protein to bind to the immobilized glycans. The excess protein was washed off
thoroughly. The bound protein was incubated with 30 µL solution of fluorescein conjugated
antibody (1 µg/100 µL, anti-human IgG Fc specific Cy3 purchased from Sigma-Aldrich or
anti-human MR Alexa Fluor 647 purchased from Biolegend) in a HEPES buffer containing 1%
BSA and 0.5% Tween-20 at rt for 1 h. The differences in protein binding affinity to synthetic
glycans were assessed semi-quantitatively by monitoring fluorescence intensity via a
fluorescence scanner (Genepix 4000B, Molecular Devices).
ACKNOWLEDGMENTS
This work was supported by the Thailand Research Fund (RSA6280106) and the
National Vaccine Institute, Thailand. We thank Chulabhorn Research Institute for the required
chemicals and equipment.
Supporting Information: Complete NMR spectral copies of all compounds and MALDI-TOF
spectra.
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