Synthesis of 12- O-Mono- and Diglycosyl-oxystearates, a New Class of Agonists for the C-type Lectin Receptor Mincle

Article abstract of DOI:10.1021/acsmedchemlett.8b00413

Fifteen glycosyl-oxystearates were synthesized by Crich's 4,6-benzylidene and K?ening-Knorr strategies. Assessment of structure-activity relationships using macrophage-inducible C-type lectin (Mincle) receptor cells expressing nuclear factor of activated

Full text of DOI:10.1021/acsmedchemlett.8b00413

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Letter
Synthesis of 12-O-Mono- and Di-glycosyl-oxystearates, a
New Class of Agonists for the C-type Lectin Receptor Mincle
Le Van Huy, Chiaki Tanaka, Takashi Imai, Sho Yamasaki, and Tomofumi Miyamoto
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.8b00413 • Publication Date (Web): 13 Dec 2018
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ACS Medicinal Chemistry Letters
Synthesis of 12-O-Mono- and Di-glycosyl-oxystearates, a New Class
of Agonists for the C-type Lectin Receptor Mincle
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Le Van Huy, † Chiaki Tanaka, † Takashi Imai, ‡ Sho Yamasaki,‡ and Tomofumi Miyamoto*, †
† Department of Natural Products Chemistry, Graduate School of Pharmaceutical Sciences, Kyushu
University, Fukuoka 812-8582, Japan
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‡ Department of Molecular Immunology, Immunology Frontier Research Center, Osaka University,
Osaka 565-0871, Japan
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KEYWORDS: glycosyl-oxystearate, Mincle, -mannosylation, TDM.
ABSTRACT: Fifteen glycosyl-oxystearates were synthesized by Crich’s 4,6-benzylidene and Köening-Knorr strategies.
Assesment of structure-activity relationships using macrophage-inducible C-type lectin (Mincle) receptor cells expressing
nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) revealed that four di-mannopyranosyl-
oxystearate analogues were Mincle agonists and that 12-O-(2-O--D-mannopyranosyl)--D-mannopyranosyl-oxystearate
was as an activator of both mouse and human Mincle.
The finding, that macrophage-inducible C-type lectin (Mincle) was
a trehalose dimycolate (TDM) receptor, suggested a strategy for
developing a potent adjuvant from ligands of Mincle receptors.1-3
Mincle binding of glycolipids from pathogenic bacteria and fungi,
such as TDM, can initiate a signaling cascade through the
immunoreceptor tyrosine-base activation motif (ITAM)-containing
adaptor molecule Fc receptor -chain (FcR). Spleen tyrosine
kinase (Syk) recruitment by FcR leads to the activation of nuclear
factor kappa-light-chain enhancer of activated B cells (NF-B)
through Card9-Bcl10-Malt1 signalosomes, induces the
transcription of genes encoding pro-inflammatory chemokines,
growth factors and cytokines, and consequently elicits responses by
Th1/Th17 immune cells. The mechanism by which these effects are
induced by interactions between Mincle and its ligand remain
unclear.4-9 Mincle contains three ligand binding sites, including
carbohydrate recognition domain (CRD), cholesterol and
SAP130.10,11 CRD have four sub-site to recognize glycolipid which
consist of two sugar binding sites, and two lipid binding sites. The
binding of glycol/glycerolipid ligands to at least three of the four
binding subsites of the Mincle CRD is regarded as essential to
activate the receptor.4,12 Research involving TDM, trehalose-6,6-
dibehenate (TDB), and other glycolipid ligands has provided clues
about the pure and structurally well-defined potential candidate
adjuvants.13-16 The effectiveness of this research strategy was the
identification of the CRD of Mincle complexed to trehalose. Indeed,
the potential of Mincle ligands as vaccine adjuvants can be
illustrated by the use of TDB in combination with a cationic
synthesized a series of fifteen 12-O-mono- and di-glycosyl
analogues of 44-2-1 and 44-2-2, respectively.
OH
-D-Man
HO
HO
HO
O
OH
O
O
-D-Man
HO
HO
HO
18
O
O
10
-D-Man
HO
HO
HO
O
O
3
OH
O
-D-Man
OH
HO
OH HO
HO
O
O
4
18
O
O
1
18
OH
L-manitol
O
10
O
44-2
10
OH
O
-D-Man
HO
HO
HO
NaOMe
O
OH
O
-D-Man
-D-Man
HO
HO
HO
HO
O
HO
O
O
HO
18
18
OMe
O
M
10
10
e
O
O
44-2-2
44-2-1
Figure 1. Structures of a novel Mincle ligand 44-2 found from
pathogenic fungus M. pachydermatis and its alkaline methanolysis
products 44-2-1 and 44-2-2.
Synthesis of the 12-O-mono- and di-glycosyl analogues of 44-2-1
and 44-2-2 required the synthesis of a -mannosidic linkage, a
synthesis that may be inhibited due to steric hinderance by
secondary glycosyl acceptors.19 Use of Crich’s 4,6-benzylidene,20-
24 however, enabled the construction of a -mannosidic linkage, as
well as the successful construction of a -mannosidic linkage with
less active secondary glycosyl acceptors.25,26 This method was
therefore selected to construct the -mannosidic linkage of 12-O-
mono- (1) and di-(2) -mannosyl-oxystearates.
quaternary ammonium in
a phase 1 clinical trial against
Briefly, the hydroxyl groups of D-mannose were protected with
benzoyl groups, followed by conversion to the thioethyl mannoside.
Debenzoylation, and then benzylidenation afforded S-ethyl 4,6-O-
benzylidene--mannoside14. 21 The hydroxyl groups at C-2 and C-
3 were protected with benzyl groups to yield the dibenzyl ether15.
The dibenzyl ether 15 was oxidized to a mannosyl sulfoxide 16.
Using Crich’s 4,6-benzylidene, the glycosyl donor 16 (1
equivalent) was treated with a glycosyl acceptor (2 equivalents of
methyl 12-hydroxystearate) to form the -mannosylated derivative
17, in a yield of 44%. The -mannosidic linkage of intermediate 17
was characterized by a consistent up-field shift at H-5 [H 3.33 (m,
1H)].25 Finally, the benzylidene and benzoyl groups of 17 were
deprotected, generating the 12-O--mannosyl oxystearate analogue
1 in a yield of 91%. The up-field chemical shifts of H-1 and H-5
[H 4.92 (s, 1H), 3.83 (m, 1H) in py-d5], and the 1JCH (155.4 Hz) of
tuberculosis.17
In 2013, we isolated the novel potent Mincle glycolipid ligand 44-
2 from the pathogenic fungus Malassezia pachydermatis.18 The
structure of 44-2 was unusual, being composed of -linked D-
mannopyranosyl residues attached to 10-hydroxystearic acid, with
the latter esterified onto a L-mannitol core. Testing with mouse
Mincle (mMincle) showed that this compound was active as TDM.
Moreover, the -mannosyl oxystearates 44-2-1 and 44-2-2 (Fig. 1),
generated by the alkaline methanolysis of 44-2, showed weak
Mincle ligand activity. To distinguish between the -anomeric
structures of the active fragments 44-2-1 and 44-2-2, and to gain
insight into the relationship between 44-2 structure and activity, we
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1,27 were not only similar with those of typical -mannoside but
differed from those of its -mannoside 6 (Table S1 and Scheme 3).
The 1H NMR spectra of synthetic analogue 1 was similar to that of
the natural ligand 44-2-1 (Fig. 2).
prototypical glycosyl donor 16 was modified to yield compound 20
(Scheme 1). Briefly, the hydroxyl group at C-3 of compound 14
was selectively benzylated to yield the glycosyl acceptor 18, which
was treated with the glycosyl donor 16 to form S-ethyl -di-
mannoside 19, in a yield of 51%. Compound 19 was converted to
the glycosyl donor 20. The reaction between the glycosyl donor 20
and the glycosyl acceptor methyl 12-hydroxystearate resulted in the
-di-mannosylated intermediate 21, in a yield of 38%. Finally,
deprotection of compound 21 resulted in 12-O-(2-O--D-
mannopyranosyl)--D-mannopyranosyl-oxystearate (analogue 2),
in a yield of 80%. The 1JCH values of -di-mannosyl oxystearate 2
were estimated to be 158.8 Hz (C1-H1) and 156.3 Hz (C1’-H1’),
and the 1H NMR spectrum was almost identical to that of the natural
ligand 44-2-2 (Fig. 3).
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Figure 2. 1H NMR spectra of the natural ligand 44-2-1, the synthetic
analogue 1 and -mannosyl-oxystearate 6 (600MHz, py-d5, 303K).
Crich’s method was also used to synthesize the 12-O--di-
mannosyl oxystearate analogue 2. Using this method, the
1
Figure 3. H NMR spectra of the natural ligand 44-2-2 and the
synthetic analogue 2 (600MHz, py-d5, 303K).
Scheme 1. Synthesis of 12-O-mono- and di--mannosyl-oxystearate analogues 1 and 2
OBn
OH
Ph
Ph
OBn
O
Ph
OR
O
Ph
O
HO
O
O
O
O
O
HO
12OH-MeST
d
O
c
O
O
a
O
BnO
H3CO
17
HO
D-mannose
RO
BnO
e
SEt
S+
H3CO
16
14 R = H
b
12
12
-O
Et
4
9
4
9
1
O
O
15 R = Bn
OBn
O
OH
O
O
HO
HO
HO
O
O
O
OBn
O
Ph
c
BnO
OBn
O
Ph
O
O
O
Ph
O
O
O
O
O
HO
HO
HO
BnO
Ph
O
O
BnO
Ph
Ph
OH
O
O
16
12OH-MeST
e
O
O
f
O
BnO
O
O
14
O
O
BnO
O
O
d
H3CO
H3CO
d
BnO
BnO
18
SEt
12
12
4
S+
9
4
9
19
21
SEt
2
-O
Et
OH
O
20
O
H3COOC
12OH-MeST
(a): i) BzCl, Py, ii) EtSH, BF3, Et2O, 1,2-dichloroethane (DCE), iii) NaOMe, MeOH (81% over 3 steps), (iv) PhCHO/ ZnCl2 53%; (b):
NaH, BnBr, DMF 91%; (c): meta-chloroperoxybenzoic acid (m-CPBA), CH2Cl2, -78 ℃ 83% for 16, 78% for 20; (d): 2,4,6-tri-tert-
butylpyrimidine (TTBP), Tf2O, molecular sieve 4A (MS4A), CH2Cl2, -78 ℃, 44% for 17, 51% for 19, 38% for 21; (e): EtOAc/ n-propanol/
THF/ H2O = 2:1:1:1, 1 atm H2, Pd-C, 1% for 1, 80% for 2; (f) n-Bu2SnO, PhMe, reflux, CsF, BnBr, DMF, 97%.
were synthesized using 12-O--D-mannopyranosyl-oxystearate 6 as a
1
starting material. The JCH value of each mannosidic linkage was
estimated by the non-decoupled HSQC method, resulting in 1JCH value
for -anomers of 168.2-177.5 Hz and for -anomers of 154.0-158.7 Hz
(Fig. 4).
To investigate the structure-activity relationship of Mincle ligands, we
synthesized three di-mannosyl isomers 3-5 of 2 as shown in Scheme 2,
together with ten 12-O-mono or di-glycosyl oxystearate 6-13 as shown
in Scheme 3. The di-mannosyl isomers 3-5 were synthesized using a
combination of Köenings-Knorr and Crich’s 4,6-benzylidene method.
The other 12-O-mono- and di-glycosyl-oxystearates 6-13 were
synthesized using the Köenings-Knorr method (Scheme 3).
The
mannopyranosyl-oxystearate
methoxybenzyl-S-ethyl sulfoxide 22 as a glycosyl donor, whereas 12-O-
compound
12-O-(2-O--D-mannopyranosyl)--D-
3
was synthesized using 2-O-p-
(2-O--D-mannopyranosyl)--D-mannopyranosyl-oxystearate
4
and
12-O-(2-O--D-mannopyranosyl)--D-mannopyranosyl-oxystearate
5
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Scheme 2. Synthesis of 12-O-di-mannosyl-oxystearate isomers 3, 4, and 5
OBz
BzO
OH
O
HO
HO
HO
OBz
O
O
BzO
BzO
BzO
BzO
BzO
OH
O
O
O
O
Ph
Ph
OH
OPMB
O
Ph
Ph
d
4
a
O
O
HO
HO
HO
Br
O
O
12OH-MeST
b
O
O
O
O
O
O
O
O
O
BnO
H3CO
23
BnO
H3CO
24
BnO
BnO
c
S+
18
22
SEt
H3CO
-O
Et
12
12
12
4
4
9
9
9
3
O
O
O
9
OH
O
OBz
O
10
11
12
13
14
15
16
17
18
HO
HO
BzO
BzO
OBz
O
BzO
BzO
BzO
HO
BzO
O
O
O
Ph
HO
O
h
Br
O
HO
HO
O
BnO
g
O
O
H3CO
H3CO
12
12
OH
O
OH
O
OH
4
Ph
Ph
f
4
9
9
4
HO
HO
HO
O
O
27
O
O
O
O
O
e
HO
BnO
Ph
OH
O
O
O
O
OBn
O
HO
HO
HO
O
O
O
O
H3CO
H3CO
H3CO
BnO
Ph
12
12
12
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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44
45
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4
4
4
9
9
9
6
25
26
HO
O
j
O
O
O
O
O
HO
O
16
HO
BnO
O
O
i
H3CO
H3CO
12
12
4
4
9
9
5
28
O
O
(a): i) Bu4NI, NaH, para-methoxybenzyl chloride (PMBCl), DMF, 81.0%, ii) m-CPBA, CH2Cl2, -78oC, -20oC, 90% ; (b): i) TTBP, Tf2O,
MS4A, CH2Cl2, -78oC, 25%, ii) 2,3-dichloro-5,6-dicyano-para-benzoquinone (DDQ), CH2Cl2/ H2O, 80% ; (c): AgClO4, Ag2CO3, MS4A
CH2Cl2, rt., 5h, 89% (d): i) NaOMe/ MeOH, 90%, ii) EtOAc/ n-propanol/ THF/ H2O = 2:1:1:1, 1 atm H2, Pd-C, 84%; (e) PhCHO/ ZnCl2,
31%; (f) n-Bu2SnO, PhMe, reflux, CsF, BnBr, DMF, 84%; (g) AgClO4, Ag2CO3, MS4A, CH2Cl2, rt., 5h, 99%; (h) i) NaOMe/ MeOH, 61%,
ii) EtOAc/ n-propanol/ THF/ H2O = 2:1:1:1, 1 atm H2, Pd-C, 72%.; (i) TTBP, Tf2O, MS4A, CH2Cl2, 73%, -78oC; (j) EtOAc/ n-propanol/
THF/ H2O = 2:1:1:1, 1 atm H2, Pd-C, 75%.
weak ability to signal through mMincle. The other mono-glycosyl-
oxystearates, 7 and 8, did not signal through Mincle at
concentrations of 3 nmol/well (data not shown). Surprisingly, the
- and -di-mannosyl isomers 3-5 showed a greater ability to
signal through hMincle than -mannosyl-oxystearate 2. In
contrast, gentiobiosyl-oxystearate 10 signaled only through
mMincle, whereas the other biosyl-oxystearates did not through
Mincle. These results indicated that these di-mannosyl-
oxystaerates 2-5 acted as the agonist of Mincle ligands.
Scheme 3. Synthesis of 12-O-mono- and di-glycosyl-oxystearates
6-13
Figure 4. 1H NMR spectra of the synthetic isomers 3 and 4
(CD3OD), 5 [CD3OD/CDCl3 (9/1)] (600MHz, 298K).
O
O
O
b
a
c
Saccharides
BzO
Glycosyl bromide
BzO
HO
Br
OBz
Perbenzoylation
OR
Synthetic analogues 6-13
To assess the structure-activity relationships of these synthesized
compounds, we evaluated their ability to activate a nuclear factor
of activated T cells (NFAT)-green fluorescent protein (GFP)
through Mincle.28 NFAT-GFP reporter cells expressing human
Mincle (hMincle)/FcR, mouse Mincle (mMincle)/ FcR or FcR
alone were cultured for 16 hr on plates that were pre-coated with
the indicated amounts of synthetic mono- or di-glycosyl
oxystearates or TDB as a positive control. The NFAT-dependent
activation of cells was monitored by the detection of GFP-positive
cells by flow cytometry.
OH
OH
O
OH
OH
OH
HO
HO
HO
HO
HO
O
O
O
O
HO
HO
HO
HO
HO
OR
OR
OR
HO
OH
OH
OH
OH
OR
OR
HO
OR
8
6

7
8

7

OH
OH
HO
HO
O
OH
O
OH
O
O
O
HO
O
HO
HO
O
HO
9
OH
OR
OH O
HO
HO
HO
OR
O
OH
OH
10
OH
11
HO
OH
OH
OH
O
O
HO
HO
HO
HO
OH
H3CO
O
OH
R=
O
O
OH
12
OH
4
9
O
HO
OR
OR
O
12
13
HO
HO
OH
The compound ,-mannosyl-oxystearate 2, an analogue of the
natural ligand 44-2-2, showed the ability to signal through both
hMincle and mMincle, whereas the compound mono--mannosyl-
oxystearate 1, an analogue of the natural ligand 44-2-1, was unable
to signal through either Mincle at a concentration of 3 nmol/well
(Fig. 5). Interestingly, the -mannosyl-oxystearate 6 showed a
(a): BzCl, pydine; (b): 30%HBr/ AcOH, CH2Cl2; (c): (i) AgClO4,
Ag2CO3, MS4A, CH2Cl2, (ii) α/β separation by silica gel column
chromatography, (iii) NaOMe/ MeOH.
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Figure 5. NFAT-GFP reporter cells expressing human Mincle (hMincle)/FcR, mouse Mincle (mMincle)/ FcR or FcR alone were tested
for their reactivity to trehalose dibehenate (TDB; 0.8nmol/well) and 12-O-mono- and di-glycosyl-oxystearates 1-6 and 9-13.
decipher the interaction of the CRD of human and mouse Mincle
Pathogen-derived Mincle ligands, including trehalose, glucose,
glycerol mycolates, corynomycolates and various -gentiobiosyl
diacylglycerides, have the ability to signal through Mincle.
Gentiobiosyl diacylglycerides were isolated from Mycobacterium
tuberculosis and M. pachydermatis, together with the novel potent
ligand 44-2. Recently, -glucosyl diacylglycerides were identified
from the pathogenic bacterium Streptococcus pneumoniae and the
other monoglucosyldiacylglycerol (MGDG) produced from Group
A Streptococcus, induced proinflammatory cytokines in a Mincle-
dependent manner. 9,29
with dimannosyl-oxystearates.
In conclusion the results of this study indicated that di-mannosyl-
oxystearates can act as novel agonist of Mincle ligands and the
compound which consists of di-mannosyl oxystearates esterified to
mannitol may be act as Mincle ligand. These results show that 44-
2-2, and analogues in which the position of sugar modification on
the lipid is varied, as well as the nature of the sugar group itself is
changed, may have potential as adjuvants.
Figure 5. NFAT-GFP reporter cells expressing human Mincle
(hMincle)/FcR, mouse Mincle (mMincle)/ FcR or FcR alone
were tested for their reactivity to trehalose dibehenate (TDB;
0.8nmol/well) and 12-O-mono- and di-glycosyl-oxystearates 1-6
and 9-13.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the ACS
Publications website.
A study of the structure-activity relationships of gentiobiosyl diacyl
glycerides with various alkyl chains showed that signaling through
both human and mouse Mincle was dependent on acyl chain length,
as had been previously determined for the activation of mMincle
by trehalose glycolipids of different length.16 Although the
gentiobiosyl-oxystearate 10 showed weak signaling through
mMincle, the di-mannosyl-oxystearates 2-5 signaled through both
human and mouse Mincle. The amino acid structures of hMincle
and mMincle are 85% identical,30 with both sharing the
conservative binding motif responsible for the interaction between
mMincle and the sugar head group of TDM.31-33 The carbohydrate
moieties of gentiobiosyl diacylglycerides have been reported to
bind to the same region of Mincle as trehalose, in the trehalose-
Mincle-carbohydrate recognition domain (CRD) complex. Both
human and mouse Mincle can bind to mannose
General experimental procedures, characterization data, and 1H and
13C NMR spectra, and ESI-MS of new compounds (PDF).
AUTHOR INFORMATION
Corresponding Author
* Tel: +81-92-642-6636. E-mail: miyamoto@phar.kyushu-u.ac.jp.
ORCID
Tomofumi Miyamto: 0000-0003-1589-5807
Notes
The authors declare no competing financial interest.
monocorynomycolate
(ManMCM),
and
mannose
2-
ACKNOWLEDGMENT
Dr. Le Van Huy would like to thank the Ministry of Education,
Culture, Sports, Science and Technology (MEXT) of Japan for
tetradecyloctadecanoate (ManC14C18), as well as to their
corresponding glucose derivatives.4 However, the monomannosyl-
oxystearates 1 and 6 and the monoglycosyl-oxystearates 7 and 8
did not signal through Mincle. Additional research is required to
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(16) Richardson, M. B.; Torigoe, S.; Yamasaki, S.; Williams, S. J.
financial support (scholarship). This research was supported by a
Mycobacterium Tuberculosis -gentiobiosyl Diacylglycerides
Signal Through the Pattern Recognition Receptor Mincle: Total
Synthesis and Structure Activity Relationships. Chem. Commun.,
2015, 51, 15027-15030.
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Grant-in-Aid for Research on Innovative Areas (No. 26100).
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28465.
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SYNOPSIS TOC.
OH
HO
4
5
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8
9
O
HO
HO
-D-Man
OH
HO
O
O
-D-Man
HO
HO
HO
O
HO
HO
O
-D-Man
optimization
HO
HO
HO
O
-D-Man
O
O
18
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18
O
O
R
12
a synthetic agonist for Mincle
R
10
O
O
a natural ligand for Mincle
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Lay Summary
The structure of -linked mannose residues of the natural ligand 44-2-2 obtained from the pathogenic
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fungus Malassezia pachydermatis has been optimized to -linked 12-O-di-mannosyl-oxystearate, which
can act as novel agonist of Mincle ligands and the compound which consists of di-mannosyl
oxystearates esterified to mannitol may be act as Mincle ligand to develop a potent adjuvant.
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Synthesis of 12-O-Mono- and Di-glycosyl-oxystearates, a New Class of Agonists for the
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C-type Lectin Receptor Mincle
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Le Van Huy, † Chiaki Tanaka,† Takashi Imai, ‡ Sho Yamasaki,‡ and Tomofumi
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Miyamoto*,
†
† Department of Natural Products Chemistry, Graduate School of Pharmaceutical
Sciences, Kyushu University, Fukuoka 812-8582, Japan
‡ Department of Molecular Immunology, Immunology Frontier Research Center, Osaka
University, Osaka 565-0871, Japan
Table of Contents Graphic
Figure 1. Structures of a novel Mincle ligand 44-2 found from pathogenic fungus M.
pachydermatis and its alkaline methanolysis products 44-2-1 and 44-2-2.
Figure 2. 1H NMR spectra of the natural ligand 44-2-1, the synthetic analogue 1 and α-
mannosyl-oxystearate 6 (600MHz, py-d5, 303K).
Figure 3. 1H NMR spectra of the natural ligand 44-2-2 and the synthetic analogue 2
(600MHz, py-d5, 303K).
Figure 4. 1H NMR spectra of the synthetic isomers 3 and 4 (CD3OD), 5 [CD3OD/CDCl3
(9/1)] (600MHz, 298K).
Figure 5. NFAT-GFP reporter cells expressing human Mincle (hMincle)/FcRγ, mouse
Mincle (mMincle)/ FcRγ or FcRγ alone were tested for their reactivity to trehalose
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dibehenate (TDB; 0.8nmol/well) and 12-O-mono- and di-glycosyl-oxystearates 1-6 and
9-13.
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Scheme 1. Synthesis of 12-O-mono- and di-β-mannosyl-oxystearate analogues 1 and 2
Scheme 2. Synthesis of 12-O-di-mannosyl-oxystearate isomers 3, 4, and 5
Scheme 3. Synthesis of 12-O-mono- and di-glycosyl-oxystearates 6-13
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Figure 1.
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OH
O
-D-Man
β
HO
HO
HO
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OH
O
HO
HO
-D-Man
β
O
18
HO
O
O
10
-D-Man
β
HO
HO
HO
O
O
3
OH
O
-D-Man
β
OH
O
HO
OH HO
HO
O
4
18
O
O
1
18
OH
L-manitol
O
10
44-2
O
10
OH
O
HO
HO
HO
-D-Man
β
O
NaOMe/MeOH
OH
O
-D-Man
β
-D-Man
10
β
HO
HO
HO
HO
O
HO
O
O
HO
18
18
OMe
OMe
10
O
O
44-2-2
44-2-1
OH
-D-Man
β
HO
O
HO
OH
O
O
-D-Man
HO
HO
HO
HO
β
18
O
O
10
-D-Man
β
HO
HO
HO
O
O
3
OH
O
-D-Man
β
OH
O
HO
HO
HO
O
4
OH
18
O
1
O
18
OH
L-manitol
O
10
O
44-2
10
OH
O
-D-Man
HO
HO
HO
β
O
NaOMe/MeOH
OH
O
-D-Man
β
-D-Man
10
β
HO
HO
HO
HO
HO
HO
O
O
O
18
18
OMe
OMe
10
O
O
44-2-2
44-2-1
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Figure 2.
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Figure 3
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Figure 4
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Figure 5
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Scheme 1.
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OBn
O
OH
Ph
Ph
OBn
O
HO
HO
HO
Ph
OR
Ph
O
O
O
O
O
O
c
12OH-MeST
d
a
O
O
O
O
BnO
3CO
17
D-mannose
RO
BnO
e
S+
H
3CO
H
SEt
16
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-O
Et
14 R = H
b
12
9
12
4
4
9
1
O
O
15 R = Bn
OBn
O
OH
O
HO
HO
HO
O
O
O
O
Ph
c
OBn
BnO
Ph
OBn
O
O
O
O
O
Ph
O
O
HO
O
O
O
BnO
Ph
O
O
BnO
Ph
Ph
OH
O
O
HO
O
e
16
12OH-MeST
O
f
O
BnO
HO
3CO
O
O
14
O
O
BnO
O
O
d
H
3CO
H
d
BnO
BnO
18
SEt
12
12
S+
4
9
4
9
-O
Et
19
21
SEt
2
OH
20
O
O
H
3COOC
12OH-MeST
(a): i) BzCl, Py, ii) EtSH, BF3, Et2O, DCE, iii) NaOMe, MeOH (81% over 3 steps), (iv) PhCHO/ ZnCl2 53%; (b): NaH, BnBr,
DMF 91%; (c): meta-chloroperoxybenzoic acid (m-CPBA), CH2Cl2, -78 ℃ 83% for 16, 78% for 20; (d): 2,4,6-tri-tert-
butylpyrimidine (TTBP), Tf2O, molecular sieve 4A (MS4A), CH2Cl2, -78 ℃, 44% for 17, 51% for 19, 38% for 21; (e): EtOAc/
n-propanol/ THF/ H2O = 2:1:1:1, 1 atm H2, Pd-C, 1% for 1, 80% for 2; (f ) n-Bu2SnO, PhMe, reflux, CsF, BnBr, DMF, 97%.
OBn
O
OH
O
Ph
OBn
O
Ph
OR
O
Ph
O
HO
O
O
HO
O
12OH-MeST
d
c
a
O
O
O
O
BnO
H3CO
17
HO
D-mannose
RO
BnO
e
S+
H3CO
SEt
16
-O
Et
14 R = H
b
12
12
4
4
9
9
1
O
O
15 R = Bn
Ph
OBn
O
OH
O
O
HO
HO
HO
O
O
O
Ph
c
OBn
O
BnO
Ph
OBn
O
O
O
O
Ph
O
O
O
HO
HO
HO
O
O
BnO
Ph
O
O
BnO
Ph
Ph
OH
O
e
O
16
12OH-MeST
O
O
f
O
BnO
O
O
14
O
O
BnO
O
O
d
H3CO
H3CO
d
BnO
BnO
18
SEt
12
12
S+
4
9
4
9
-O
Et
19
21
SEt
2
OH
O
20
O
H3COOC
12OH-MeST
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Page 17 of 18
ACS Medicinal Chemistry Letters
1
2
3
4
5
6
Scheme 2
7
8
9
OBz
O
OH
HO
BzO
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
OBz
O
O
BzO
BzO
HO
HO
BzO
BzO
Ph
BzO
Ph
OH
O
O
O
Ph
OH
a
Ph
OPMB
O
d
4
O
Br
O
O
HO
HO
HO
O
O
O
12OH-MeST
b
O
O
O
O
O
O
O
O
BnO
3CO
23
BnO
3CO
24
BnO
BnO
c
S+
18
22
SEt
H
H
H
3CO
-O
Et
12
12
12
9
4
4
9
9
3
O
O
O
OBz
O
OH
O
BzO
BzO
BzO
HO
OBz
O
BzO
HO
BzO
BzO
HO
O
O
O
Ph
HO
HO
HO
O
Br
h
O
O
BnO
g
O
O
H
3CO
H
3CO
12
12
9
OH
O
OH
O
OH
4
Ph
Ph
f
4
9
4
HO
O
O
O
27
O
O
O
HO
O
e
HO
HO
BnO
Ph
OH
O
O
O
O
OBn
O
HO
HO
HO
O
O
H
3CO
H
3CO
H
3CO
O
O
BnO
Ph
12
12
12
9
4
4
4
9
9
6
25
26
HO
O
j
O
O
O
O
O
HO
O
16
BnO
HO
O
O
i
H
3CO
H
3CO
12
12
4
9
4
9
28
5
O
O
(a): i) Bu4NI, NaH, para-methoxybenzyl chloride (PMBCl), DMF, 81.0%, ii) m-CPBA, CH2Cl2, -78oC, -20oC, 90% ; (b): i)
TTBP, Tf2O, MS4A, CH2Cl2, -78oC, 25%, ii) 2,3-dichloro-5,6-dicyano-para-benzoquinone (DDQ), CH2Cl2/ H2O, 80% ; (c):
AgClO4, Ag2CO3, MS4A CH2Cl2, rt., 5h, 89% (d): i) NaOMe/ MeOH, 90%, ii) EtOAc/ n-propanol/ THF/ H2O = 2:1:1:1, 1
atm H2, Pd-C, 84%; (e) PhCHO/ ZnCl2, 31%; (f) n-Bu2SnO, PhMe, reflux, CsF, BnBr, DMF, 84%; (g) AgClO4, Ag2CO3, MS4A,
CH2Cl2, rt., 5h, 99%; (h) i) NaOMe/ MeOH, 61%, ii) EtOAc/ n-propanol/ THF/ H2O = 2:1:1:1, 1 atm H2, Pd-C, 72%.; (i)
TTBP, Tf2O, MS4A, CH2Cl2, 73%, -78oC; (j) EtOAc/ n-propanol/ THF/ H2O = 2:1:1:1, 1 atm H2, Pd-C, 75%.
OBz
OH
O
BzO
HO
HO
HO
OBz
O
O
BzO
BzO
BzO
BzO
Ph
BzO
OH
O
O
O
Ph
Ph
OH
a
Ph
OPMB
O
d
4
HO
HO
HO
O
Br
O
O
O
12OH-MeST
b
O
O
O
O
O
O
O
O
O
O
BnO
BnO
c
BnO
BnO
SEt
S+
18
22
H
H
H
3CO
3CO
23
3CO
24
-O
Et
12
12
12
4
4
9
9
9
3
O
O
O
OH
O
OBz
O
HO
BzO
BzO
BzO
OBz
O
BzO
HO
BzO
BzO
HO
O
O
O
Ph
HO
O
h
Br
O
HO
O
HO
BnO
g
O
O
H
3CO
H
3CO
12
12
OH
O
OH
O
OH
4
Ph
Ph
f
4
9
9
4
HO
27
O
O
O
O
O
HO
O
O
e
HO
HO
BnO
Ph
OH
O
O
O
O
OBn
O
HO
HO
HO
O
O
O
H
H
H
O
3CO
3CO
3CO
BnO
Ph
12
12
12
4
4
4
9
9
9
6
25
26
HO
O
j
O
O
O
O
O
HO
O
16
HO
BnO
O
O
i
H
3CO
H
3CO
12
12
4
4
9
9
5
28
O
O
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ACS Medicinal Chemistry Letters
Page 18 of 18
1
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6
Scheme 3.
7
8
9
a
O
O
c
O
b
Saccharides
BzO
BzO
HO
Br
OBz
OR
-
Perbenzoylation
Glycosyl bromide
6 13
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Synthetic analogues
OH
OH
O
OH
OH
OH
HO
HO
HO
HO
HO
O
O
O
O
HO
HO
HO
HO
HO
OR
OR
OR
HO
OH
OH
OH
OH
OR
OR
HO
OR
α
α
8
β
6
7
β
8
7
OH
OH
HO
HO
O
OH
O
OH
O
O
O
HO
O
HO
HO
O
HO
9
OH
OR
O
HO
HO
OH
HO
HO
OR
O
OH
OH
10
OH
11
OH
OH
OH
O
O
HO
HO
HO
HO
OH
H3CO
O
OH
R=
O
O
OH
12
OH
4
9
O
HO
OR
OR
O
12
13
HO
HO
OH
(a): BzCl, pydine; (b): 30%HBr/ AcOH, CH2Cl2; (c): (i) AgClO4, Ag2CO3, MS4A, CH2Cl2, (ii) α/β separation by silica
gel column chromatography, (iii) NaOMe/ MeOH.
a
O
O
c
O
b
Saccharides
OH
BzO
BzO
HO
Br
OBz
OR
-
Perbenzoylation
Glycosyl bromide
6 13
Synthetic analogues
OH
O
OH
OH
OH
HO
HO
HO
HO
HO
O
O
O
O
HO
HO
HO
HO
HO
HO
OR
OR
OR
OH
OH
OH
OH
OR
OR
HO
OR
α
α
8
β
6
7
8
7
β
OH
OH
HO
HO
O
OH
O
OH
O
O
O
HO
O
HO
HO
O
HO
9
OH
O
HO
HO
OR
OH
HO
HO
OR
O
OH
OH
10
OH
11
OH
OH
OH
O
O
HO
HO
HO
HO
OH
H
3CO
O
OH
R=
O
O
OH
12
OH
4
9
O
HO
OR
OR
O
13
12
HO
HO
OH
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