Total Synthesis of Phospholipomannan of Candida albicans

Article abstract of DOI:10.1021/acs.joc.0c00402

First, total synthesis of the cell surface phospholipomannan anchor [β-Manp-(1 → 2)-β-Manp]n-(1 → 2)-β-Manp-(1 → 2)-α-Manp-1 → P-(O → 6)-α-Manp-(1 → 2)-Inositol-1-P-(O → 1)-phytoceramide of Candida albicans is reported. The target phospholipomannan (PLM) anchor poses synthetic challenges such as the unusual kinetically controlled (1 → 2)-β-oligomannan domain, anomeric phosphodiester, and unique phytoceramide lipid tail linked to the glycan through a phosphate group. The synthesis of PLM anchor was accomplished using a convergent block synthetic approach using three main appropriately protected building blocks: (1 → 2)-β-tetramannan repeats, pseudodisaccharide, and phytoceramide-1-H-phosphonate. The most challenging (1 → 2)-β-tetramannan domain was synthesized in one pot using the preactivation method. The phytoceramide-1-H-phosphonate was synthesized through an enantioselective A3 three-component coupling reaction. Finally, the phytoceramide-1-H-phosphonate moiety was coupled with pseudodisaccharide followed by deacetylation to produce the acceptor, which on subsequent coupling with tetramannosyl-H-phosphonate provided the fully protected PLM anchor. Final deprotection was successfully achieved by Pearlman's hydrogenation.

Full text of DOI:10.1021/acs.joc.0c00402

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Article
Total synthesis of phospholipomannan of the Candida albicans
Veeranjaneyulu Gannedi, Asif Ali, Parvinder Pal Singh, and Ram A. Vishwakarma
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.0c00402 • Publication Date (Web): 19 May 2020
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Total synthesis of phospholipomannan of the Candida albicans
†
,‡
†,‡
†,‡
†,‡
Veeranjaneyulu Gannedi, Asif Ali, Parvinder Pal Singh, and Ram A. Vishwakarma *
†
Medicinal Chemistry Division, CSIR-Indian Institute of Integrative Medicine, Canal Road,
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Jammu-180001, India.
‡
Academy of Scientific and Innovative Research, Canal Road, Jammu-180001, India.
OBn
HO
O
BnO
BnO
OBn
O
BnO
O
AcO
BnO
BnO
OBn
OBn
O
BnO
O
O
P
OH
BnO
BnO
HO
O
BnO OBn
O
P
OBn
OBn
O
OBn
H
C14H29
HN(Bn)2
H
O
C14H29
O
H
C13H27
TBDPSO
HN
HO
NH OBn
O
C15H31
A3 coupling
C15H31
OH
O
HO
HO
HO
O
HO
OBn
O
BnO
BnO
BnO
OBn
O
HO
HO
O
OBn
O
O
Ph
O
Ph
O
O
O
O
BnO
HO
HO
HO
BnO
Ph
O
BnO
BnO
BnO
O
SPh
OH
O
O
O
O
O
O
One pot synthesis
O
HO
HO
Ph
O
BnO
Ph
BnO
BnO
BnO
O
O
O
BnO
O
O
O
HO
O
O
BnO
O
SPh
BnO
BnO
BnO
O
BnO
BnO
BnO
OH
O
O
BnO
BnO
BnO
O
O
P
OH
O
P
ONAP
O
ONAP
H
OH
OH
O
O
HO
HO
HO
O
HO
OH
OH
HO OH
O
HO
P
O
O
C14H29
O
HN
C15H31
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Abstract
First total synthesis of cell surface phospholipomannan anchor [β-Manp-(1→2)-β-Manp]n-
(1→2)-β-Manp-(1→2)-α-Manp-1→P-(O→6)-α-Manp-(1→2)-Inositol-1-P-(O→1)-phytocer-
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amide of Candida albicans is reported. The target PLM anchor poses synthetic challenges
such as unusual kinetically controlled (1→2)-β-oligomannan domain, anomeric
phosphodiester, and unique phytoceramide lipid tail linked to the glycan through a phosphate
group. The synthesis of PLM anchor was accomplished using a convergent block synthetic
approach using three main appropriately protected building blocks; (1→2)-β-tetramann
repeats, pseudodisaccharide, phytoceramide-1-H-phosphonate. The most challenging (1→2)-
β-tetramann domain was synthesized in one-pot by using pre-activation method. The
3
phytoceramide-1-H-phosphonate was synthesized through an enantioselective A three-
component coupling reaction. Finally, the phytoceramide-1-H-phosphonate moiety was
coupled with pseudodisaccharide followed by deacetylation to produce the acceptor, which
on subsequent coupling with tetramannosyl-H-phosphonate provided the fully protected PLM
anchor. Final deprotection was successfully achieved by Pearlman’s hydrogenation.
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Introduction:
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Candida species are harmless commensal colonisers of the gastrointestinal and genitourinary
tract and to a lesser extent of the human skin. Under normal circumstances, it lives in 80% of
the human population, but overgrowth of Candida species results in infectious diseases,
particularly in the immunocompromised individuals and there are around 60,000-70,000 such
cases per year in the US alone.1,2 Incidences of infection have risen with the increased
prevalence of immuno suppressive therapies and the use of broad-spectrum antibiotics. Since
C. albicans has rarely been isolated from the environment, it is considered to be obligatorily
associated with mammalian hosts. To fully understand its pathogenicity, a major key is to
explore and decipher the regulatory networks that support the transition from the commensal
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,4
to pathogenic state. The cell wall of C. albicans, as in other eukaryotic pathogens, plays an
vital role in pathogenesis by (a) protecting the pathogen from the host immune system (b)
initiating adherence to the host cells and (c) releasing immunosuppressive antigens and
virulence factors to facilitate the infection. One of the most important cell surface molecule
responsible for infectivity and pathology of C. albicans has been identified as a complex
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,6
glycosphingolipid called phospholipomannan (PLM 1, Figure 1). The PLM is comprised of
mannose-inositol-phosphoceramide lipid anchor (embedded in the cell wall) on which an
unusual oligomeric (1→2)-β-mannan is linked through an anomeric phosphodiester linkage
7
(
Figure 1). Interestingly the PLM anchor is quite distinct from the more widely occurring
GPI anchors in human biology in term of the following: (a) (1→2)-β-mannan in place of
(1→2)-α-mannan motif of GPI anchors; (b) (1→2)-α-mannose linked to myo-D-inositol in
place of (1→6)-α-glucosamine-inositol motif and (c) the presence of an unusual
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phytoceramide in place of the glycerolipid. More importantly, the PLM anchor is highly
immunogenic due to its (1→2)-β-mannan structural motif, which is absent in human host. It
has been shown that the deletion of GDP-mannose: inositol-phospho-ceramide mannose
transferase (MIT1) in C. albicans led to a decrease in virulence during both the acute and
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chronic phases of systemic disease in mice. Moreover short and truncated glycan analogues
of PLMs are also known to activate macrophages, indicating the critical role of PLM anchor
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in the pathogenic process. The enzyme involved in PLM anchor biosynthesis has not been
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0
purified yet, which require synthetic substrates and precursors. Also due to their absence in
human biology, PLM anchor presents a unique opportunity to design glycoconjugate vaccine
or utilizing its biosynthetic machinery as novel drug targets. In order to address these
questions (vaccine and drug design), one of the key hurdle is the access of synthetic PLM and
its intermediates as their isolation from C. albicans culture is difficult (limited quantities and
heterogeneity). The chemical synthesis can provide access to both native and novel PLM
structures, indispensable for biological studies.
OH
O
O
O
HO
HO
HO
O
P
Protein
O
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N
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OH
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H
HO
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NH2
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OH
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OH
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OH
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HO OH
HO
HO
2
H N
OH
OH
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HO
O
O
P OH
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OH
OH
HO
O
O
HO OH
O
O
C17H35
HO
P
O
C14H29
O
O
O
HN
C21H43
C15H31
1
Phospholipomannan (PLM) anchor of C. albicans
Glycosylphosphatidylinositol (GPI) anchor of
H.Sapiens
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Fig 1: Phospholipomannan of C. Albicans and GPI anchor of H. Sapiens
Early attempts on a synthesis of PLM were made by Bundle et al who reported synthesis of
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(1→2)-β-mannan domain of PLM and (1→2)-β-mannan glycoconjugates and studied them in
preclinical settings as vaccine candidates.11a, 11b Our group recently designed a new strategy to
construct (1 → 2)-β-Mannan domain using intra-molecular aglycon delivery (IAD).11c
However, the total synthesis of full-length PLM anchor has eluded success. We now report
the first synthesis of full-length PLM anchor of C. albicans, built on our previous experience,
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in making GPI anchors and Lipophosphoglycans (LPGs).
Results and Discussion:
The target PLM anchor presents key synthetic challenges such as unusual kinetically
controlled (1→2)-β-oligomannan, pseudodisaccharide, anomeric phosphodiester and
phytoceramide linked to saccharide through a phosphate. The successful synthesis of a
molecule of such complexity required new chemistry, a high degree of planning such as the
order of attachment of fragments, choice of glycosylation methods, protection groups, and
deprotection. The Scheme 1 depicts our retrosynthetic approach towards the synthesis of 1.
A convergent assembly of subunits was planned for the target PLM (1) from (1 → 2)-β-
Mannan domain 2, pseudodisaccharide 3, and phytoceramide-1-H-phosphonate 4. The most
challenging (1 → 2)-β-Mannan 2 was synthesized from teramannopyranoside 5, which in
turn was synthesized from intermediates 6, 7, and 8 via new diastereoselective glycosylation
reaction in one-pot. While pseudodisaccharide fragment 3 was synthesized from suitably
protected inositol acceptor 9 and the mannopyranoside donor 10 via the Schmidt
trichloroacetimidate method, both the fragments 9 and 10 could be synthesized from readily
available myo-inositol and mannose. On the other hand, the key phytoceramide-1-H-
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phosphonate fragment 4 could be synthesized from two main building blocks sphingoid base
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1 and palmitic acid 12, where the sphingoid base 11 can be synthesized from alkyne 13 via
dihydroxylation of the alkene, and Z-selective olefination.
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Scheme 1: Retrosynthetic analysis of Phospholipomannan (PLM) anchor 1.
OBn
BnO
BnO
OBn
O
Ph
O
O
OH
O
O
O
HO
HO
BnO
BnO
Ph
O
H
BnO
BnO
BnO
O
O
O
HO
O
O
O
H
C14H29
O
O
HO
HO
HO
BnO
Ph
H
15
TBDPSO 14
O
O
BnO
BnO
BnO
O
O
N(Bn)2
O
O
O
H
BnO
O
16
HO
HO
O
BnO
BnO
BnO
BnO
O
O
O
HO
BnO
Enantioselective
A3 coupling
HO
HO
HO
BnO
O
O
P
5
ONAP
C14H29
TBDPSO
O
Onepot synthesis by using
preactivation method
N(Bn)2
O
OHEt3N
1
3
2
O
P
OH
Sharpless asymmetric dihydroxylation,
Lndlar reduction
OBn
O
OH
O
BnO
BnO
BnO
OH
O
Ph
O
Ph
O
O
O
O
BnO
BnO
OH
O
OH
O
ONAP
HO
HO
SPh
SPh
7
6
8
TBDPSO
C13H27
HO
C15H31
12
NH2 OH
HO
O
OH
OH
HO OH
11
HO
AcO
BnO
OBn
O
O
O
O
BnO
BnO OBn
OBn
O
HO
P
O
O
C14H29
O
P
O
CCl3
NH
BnO
BnO
O
HN
10
H
O
O
C14H29
Glycosylation
C15H31
NEt3HO
HN
O
C15H31
BnO
HO
4
1
BnO
OBn
OBn
OH
BnO
BnO
PMBO
OBn
OBn
3
9
In turn, the alkyne fragment 13 could be synthesized from TBDPS protected aldehyde 14, C-
3
1
6 alkyne 15, and dibenzylamine 16 using an enantioselective A coupling.
The synthesis of the target molecule was started with suitably protected key intermediate
tetramannoside in an effective manner. Synthesis of tetramannoside was achieved by using
suitably protected mannose subunits 6-8 (Scheme 2). Thio mannopyranoside donor 6 was
1
3
obtained mannopyranoside derivative 17. Thio mannopyranosyl acceptor 7 was achieved
from the diol 18 via a stannylene acetal derived regioselective 3-O-benzylation in 80%
yield.14e The other mannopyranosyl acceptor 8 was derived from orthoester 19 (which in turn
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was obtained from 17 following known method),9b through the two-step sequence, and
cleavage of orthoester to hemiacetal, 2-naphthyl (NAP) protection of anomeric hydroxy and
deacetylation.
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Scheme 2: Synthesis of suitably protected mannopyranosyl subunits 6-8.
OBn
O
Ph
O
a
O
BnO
OH
O
SPh
Ph
O
6
O
HO
OH
O
Ph
O
SPh
18
b
O
BnO
SPh
Ref 11
7
O
OH
O
AcO
AcO
AcO
OAc
O
BnO
BnO
BnO
BnO
BnO
BnO
O
O
c
Ref 9b
O
17 OAc
19
ONAP
8
Reagents and Conditions: (a) BnBr, NaH, DMF, 95%; (b) i) Bu SnO, MeOH, 4 h, reflux ii)
2
o
BnBr, TBAB, toluene, DMF, 120 C, 80%; (c) i) pTSA, DCE, H O, ii) NAPBr, NaH, DMF, -
2
2
o
o
0 C, followed by MeOH, 60 C, 70%.
In literature, there are several reports for (1→2)-β-mannosylation by using different glycosyl
donors (anomeric thio, sulfoxide, pentenoate, carboxy-benzyl, dehydrative glycosylations and
inversion of gluco to manno).14 Moreover one-pot glycosylation method by using
preactivation were developed for the synthesis of oligosaccharides,15 however no such
attempts were made in the synthesis of (1→2)-β-mannosides. In this direction, we made
various attempts but resulted in poor overall yield. In the present study to establish the
effective conditions for diastereoselective (1→2)-β-mannosylation, thioglycosyl donor 6 was
selected as a glycosyl donor and fragment 8 was chosen as an acceptor (Table 1). The
glycosylation of glycosyl acceptor 8 with glycosyl donor 6 in presence of NIS/TfOH
proceeded moderately with 35% yield, providing 7.6/2.4 (52% de) mixture of the β and α
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disaccharides (Table 1, entry 1). The glycosylation of the above substrates in presence of
NIS/TMSOTf proceeded in 20% yield, with β and α selectivity in 7.7/2.3 (54% de) ratio
o
(Table 1, entry 2). In another attempts glycosylation was performed with Ph SO/Tf O/-78 C-
2
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o
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rt, PhSeBr/AgOTf/-78 C-rt, and PhSeBr/ Tf O/-78 C-rt proceeded in low yields (Table 1,
2
entry 3-5).
Table 1. Optimization conditions for (1→2)-β-mannosylation.
OH
O
BnO
BnO
BnO
OBn
O
Ph
O
O
O
BnO
OBn
O
Ph
O
ONAP
BnO
BnO
BnO
8
O
O
BnO
SPh
6
ONAP
2
0
Entries
Conditions
NIS, TfOH, -78 C
NIS, TMSOTf, -45 C
Yield (20)
35%
20%
-
*β/α
Diastereoselective%
o
1
2
3
4
5
6
7
7.6/2.4
7.7/2.3
-
7.8/2.2
8.05/1.95
8.9/1.1
9.4/0.6
52
54
-
56
61
78
88
o
o
Ph SO, Tf O, -78 C-rt
2
2
o
PhSeBr, AgOTf, -78 C-rt
15%
10%
90%
92%
o
PhSeBr, Tf O, -78 C-rt
2
o
BSP, TTBP, Tf O, -78 C-rt
2
o
PhSCl, AgOTf, -78 C-rt
*
Ratio was calculated by HPLC (SI, page no.8-13)
o
The glycosylation was performed with BSP/TTBP/Tf O/-78 C proceeded in the good yield
2
of 90%, providing 8.9/1.1 (78% de) mixture of the β and α disaccharides (Table 1, entry 6).
o
o
Then the glycosylation was performed with PhSOTf//-78 C - 0 C (generated in-situ from
1
6
PhSCl/AgOTf) proceeded in excellent yield of 92%, providing 9.4/0.6 (88% de) mixture of
the β and α disaccharides (Table 1, entry 7), this condition was found to be best for (1→2)-β-
mannosylation. The stereochemistry at anomeric centers of 20 was confirmed by 1J C-H
coupling constants 168.01Hz, 153.93 Hz for (1→2)-β and 170.03Hz, 172.17 Hz for (1→2)-α-
dimannoside (Supporting Information (SI), page no. 6). With the optimized conditions for
(1→2)-β-mannosylation in hand, the synthesis of tetramannoside 5 (Scheme 3) was achieved
in one-pot by using optimized preactivation conditions PhSOTf (generated in situ by using
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o
PhSCl and AgOTf) at -78 C by using donor 6 and acceptor 7. After 1h, the reaction was
o
brought to -20 C, and the continued till the consumption of 7 as confirmed by TLC, then the
o
reaction mixture was cooled to -78 C, and added AgOTf, PhSCl, and acceptor 7, with the
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
similar manner as followed in first addition and monitor for the consumption of acceptor 7 on
o
TLC. Subsequently, the reaction temperature was again lowered to -78 C, which was
followed by sequential addition of AgOTf, PhSCl, and acceptor 8. Glycosyl donors were
employed a slight excess for the first two glycosylations to ensure complete consumption of
the acceptor. On warming up the reaction causes the decomposition of the excess activated
glycosyl donor and doesn’t affect the following reactions. The desired tetramannoside 5 was
isolated from one pot reaction mixture by running the Sephadex LH-20 column followed by
flash column chromatography in 40% overall yield.
More importantly, the same acceptor 7 was used for the formation of first and second
glycosidic linkages without affecting the anomeric reactivity adjustment. The orientation of
glycosidic bonds in tetramannoside 5 was confirmed by converting it into 21 with pTSA in
methanol in 90% yield, and confirmed by 1JCH coupling constants 161.9, 155.0, 162.9 for
three (1→2)-β linkages and one 168.01 for (1→2)-α linkage (SI, page no. 16). Synthesis of
key tetramannosyl-H-phosphonate 2 was achieved from 21 via three steps, including
benzylation, and oxidative deprotection of anomeric 2-naphthyl group with DDQ in
methanol/DCM in 65%, and H-phosphonate preparation using PCl , imidazole in 75% yield.
3
Scheme 3: Synthesis of tetramannosyl fragment 2.
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2
3
4
5
6
7
8
9
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
OBn
O
Ph
O
O
BnO
Ph
O
O
O
O
O
BnO
Ph
O
OBn
O
O
Ph
O
O
7
a
7
a
8
b
O
O
BnO
BnO
a
BnO
BnO
BnO
Yield 40%
O
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
SPh
6
ONAP
5
OBn
O
BnO
BnO
BnO
OBn
BnO
BnO
BnO
OBn
O
HO
HO
BnO
O
O
O
O
BnO
BnO
BnO
O
BnO
BnO
BnO
HO
HO
BnO
O
O
O
O
O
BnO
BnO
BnO
O
BnO
BnO
BnO
HO
HO
BnO
c
O
d
O
O
O
O
BnO
BnO
BnO
BnO
BnO
O
BnO
BnO
BnO
O
BnO
O
O
P
OH
ONAP
HO
H
22
21
O
2
o
Reagents and Conditions: (a) PhSCl, AgOTf, TTBP, -78 C-rt; (b) pTSA, MeOH, 12 h,
5%; (c) i) BnBr, NaH, DMF, 10 h; ii) DDQ, DCM/MeOH, 4 h, 65%; (d) PCl , Imidazole,
9
3
o
Et N, DCM, -10 C, 75%.
3
En route to obtain optically pure inositol base key intermediates, we began from myo-inositol
(
Scheme 4), racemate (±) 23, which was synthesized from myo-inositol using a reported
procedure.17 The resolution of diol (±) 23 was commenced via the formation of a
camphanylidene ketal diastereomers, which were easily separated through column
chromatography. However (-)-24 was used for further synthesis and stereochemistry of (-)-24
was confirmed as a D-isomer by converting into known compound (-)-26.12d The intermediate
(
-)-24 was converted into inositol acceptor 9 in two-steps, by cleavage of camphanylidene
ketal to (-)-25 in 90% yield, and stannylene acetal derived regioselective 1-O-4-methoxy
benzylation to (-)-9 in 80% yield.
Scheme 4: Synthesis of pseudodisaccharide fragments 3, 28.
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7
8
9
O
HO
HO
O
BnO
BnO
HO
O
a
BnO
BnO
BnO
BnO
HO
c, d
OBn
OBn
b
OBn
OBn
OBn
OBn
O
23
(-) 24
(-) 25
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
H
OBn
BnO
BnO
HO
O P OHEt N
3
OBn
OBn
25
OBn
O
e, f
(-)26
[
[
]
]
= -9.2
= -9.0
O
D
BnO
BnO
2
5
D
HO
h, i
BnO
BnO
PMBO
OBn
OBn
OBn
O
BnO
BnO
O
AcO
BnO
BnO
OBn
OBn
(
-) 9
PMBO
28
1
0
g
OBn
BnO
O
AcO
BnO
PMBO
OBn
OBn
O
BnO
BnO
j
27
BnO
BnO
HO
O
OBn
OBn
3
Reagents and Conditions: (a) DCM, pTSA, reflux, 96% (48:48); (b) pTSA, MeOH, reflux,
0%; (c) Bu SnO, MeOH: (d) PMBCl, TBAB, Toluene, 80 C, 80%; (e) BnBr, NaH, DMF,
94%; (f) 15% TFA in DCM, 90%; (g) TMSOTF, DCM 4 MS, -20 C, to rt, 84%; (h)
NaOMe, MeOH, 0 C, 91%; (i) PCl , Imidazole, Et N, DCM, -10 C, 75%; (j) 10% TFA in
o
9
2
o
o
o
o
3
3
DCM, 88%.
The intermediate (-)-9 was coupled with mannopyranosyl imidate donor 10 (which was
18
synthesized from α-methylmannopyranoside by using reported method ) using TMSOTf as
o
activator under anhydrous conditions at -20 C to get 27 with α-stereochemistry in 84%
yield, which was confirmed through 2D-HSQC-NMR by observing anomeric peak as broad
1
13
singlet at 5.40 ppm in H-spectrum and 98.22 ppm in C-spectrum. The deacetylation of 27
was achieved by using sodium methoxide in 91% yield, followed by H-phosphonate
preparation with PCl , imidazole provided 28 in 75% yield. On the other hand
3
pseudodisaccharide acceptor 3 was synthesized from 27 by PMB deprotection with 10%
TFA/DCM in 88% yield (Scheme 3).
3
Table 2. Optimization conditions for A coupling.
C
15
H
29
1
4
O
C14H29
TBDPSO
TBDPSO
H
14
HN(Bn)2
N(Bn)2
16
13
Entry Conditions
yield %ee
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9
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
1
2
3
4
5
6
7
8
9
10
CuI, toluene, rt
CuI, toluene, 50 C
20%
45%
50%
30%
-
-
-
-
-
-
o
Cu(OTf) , toluene, rt
2
Zn(OTf) , toluene, rt
2
o
Zn(OTf) , toluene, 70 C 65%
2
o
CuBr, toluene, 40 C
L1, CuBr, toluene, rt
L2, CuBr, toluene, rt
L3, CuBr, toluene, rt
L4, CuBr, toluene, rt
L5, CuBr, toluene, rt
L6, CuBr, toluene, rt
L7, CuBr, toluene, rt
L8, CuBr, toluene, rt
92%
88%
88%
90%
89%
78%
85%
90%
90%
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
1
1
1
2
3
27.2*
39.2*
67.6*
92.8*
14
*
ee% was calculated by HPLC (SI, page no.32-36)
O
Ph
O
Ph
O
COOH
Fmoc
O
O
O
R-BINAP
L1
S-BINAP
L2
Ph2PO
N
HO
Ph2PO
L3
N
O
O
L4
O
L5
O
O
O
O
O
N
N
O
N
O
O
O
OR
RO
O
O
HO
L6
O
Ph
Ph
L7: R = H
L8: R = Ac
The synthesis was commenced with the preparation of the phytoceramide-1-H-phosphonate
. Phytoceramides are widely distributed in plants, yeasts, fungi, and even in mammalian
tissues and responsible for number of physiological processes including cell recognition,
4
1
9
adhesion, signal transduction, apoptosis, and control of immune response. Considering their
importance, several methods were reported regarding synthesis in the literature from the
carbohydrates or amino acid derived chiral starting materials, and few are based on
2
0
3
asymmetric synthesis. However, in the present study, A coupling strategy was employed
considering its ease of operation and high enantio-selectivity outcome, for the synthesis of
2
1
key intermediate 13 of phytoceramide. To achieve this, several conditions were tried (Table
3
2), to seek effective A coupling by using suitable stating partners such as glycoaldehyde 14,
1
-hexadecyne 15 and dibenzylamine 16. Different catalysts such as CuI, Cu(OTf) , Zn(OTf)
2 2
were employed (Table 2, entry1-5) where all the tried conditions are given low conversion
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(
20-65%). In the next attempt, CuBr when tried efficiently catalyzed the reaction with 92%
yield of the desired alkyne 13 (Table 2, entry 6). Next, to achieve the enantioselectivity
several asymmetric ligands was investigated in conjunction with a CuBr salt.in the initial
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
attempts, Phosphine ligands including (R)-BINAP,22a (S)-BINAP,22a FMOC-Proline,22b sugar
phosphines,22c sugar amino alcohol when tried,22d, 22e were not found promising results (Table
2
, entry 7-12). Interestingly, when Ac glucoBox was tried the desired alkyne 13, was
2
3
obtained in 90% yield with 92.8% ee (Table 2, entry 13). The stereochemistry of 13 was
confirmed by converting it into 32.
Scheme 5: Synthesis of phytoceramide-1-H-phosphonate 4.
OH
OH
a
C14H29
TBDPSO
Bn) N
C13H27
TBDPSO
(Bn) N
C13H27
TBDPSO
(
OH
9a, 82%
OH
2
2
N(Bn)2
2
29b, 18%
13
O
OH
OH
HO
C15H31
d
b
29a
C13H27
TBDPSO
C13H27
TBDPSO
c
NH OH
C15H31
NH2 OH
O
11
30
f
OAc
OBn
AcO
O
C13H27
OBn
O
NH OAc
e
C13H27
C13H27
H
P
O
HO
32
OH
NH OBn
NH OBn
C15H31
25
Experimental []
Literature []
= +26.0°
D
O
25
O
= +26.3°
C15H31
D
4
31
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8
9
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
Reagents and Conditions: (a) i) Lindlar Catalyst, EtOAc/ Pyridine, H ; ii) AD mix-β 1:1 t-
2
BuOH : H O, K OsO .2H O, MeSO NH , 80%; (b) Pd/C, H , 8 h, 95%; (c) i) EDC, HOBT,
2
2
4
2
2
2
2
DMAP, DCM, 12 h; (d) i) BnBr, NaH, DMF, 10 h, 65% for 3 steps; ii) TBAF, THF, 8 h,
70%; (e) PCl , Imidazole, Et N, DCM, 5 h, 74%; f) i) TBAF, THF; ii) Ac O, pyridine.
3
3
2
The alkyne 13 was treated with a lindlars catalyst to get (Z)-alkene (Scheme 5). The (Z)-
alkene was subjected to the sharpless assymetric dihydroxylation to afford a diastereomeric
mixture of the diol 29a and 29b, which were easily separated through column
chromatography. The stereochemistry of diol 29a was confirmed as D-ribo configuration by
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
24
converting into a known sphingoid base 32, through the intermediate 11. The intermediate
1
1 was converted into phytoceramide precursor 31 in a three steps sequence i.e., amidation,
benzylation of diol, TBDPS deprotection with TBAF in an overall yield of 65%. The
intermediate 31 was converted into the key fragment H-phosphonate 4 using PCl , and
3
imidazole in 74% yield.
Scheme 6: Synthesis of saccharide acceptor 34.
BnO
BnO
BnO
OBn
O
BnO
BnO
BnO
OBn
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
O
OHNEt3
O
O
O
OBn
O
H
P
O
BnO
BnO
BnO
BnO
BnO
BnO
OBn
O
BnO
BnO
BnO
O
O
O
O
O
O
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
O
O
O
O
O
BnO
BnO
PMBO
OBn
OBn
BnO
BnO
BnO
O
28
a
O
P
b
O
P
O
O
OH
O
OH
BnO
BnO
BnO
O
OBn
OBn
O
O
O
O
BnO
BnO
BnO
BnO
OH
22
BnO
O
BnO
BnO
HO
O
BnO
PMBO
OBn
OBn
OBn
OBn
33
34
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Reagents and Conditions: (a) Pivaloyl chloride, Py, I , Py/H O (19:1); (b) TFA, DCM,
2
2
6
0%.
To achieve the synthesis of PLM, tetramannoside acceptor 22 was coupled with H-
phosphonate donor of pseudo disaccharide 28 by using pivaloyl chloride as activator and
pyridine as a base, the unstable P(III) oxidation state was converted into P(V) 33 by using
Iodine/pyridine/water system in a crude yield of 70% yield. The crude reaction mixture was
subjected to the PMB group deprotection by using TFA/DCM 34 in 60% crude yield, but the
reaction mixture was found to be an inseparable mixture of products (Scheme 6). Later we
changed the strategy (Scheme 7), the pseudodisaccharide acceptor 3 was coupled to H-
phosphonate donor of phytosphingosine 4 by using pivaloyl chloride as activator and pyridine
as a base, the unstable P(III) oxidation state was converted into P(V) by using
Iodine/pyridine/water system. The crude material was deacetylated by using NaOMe/MeOH
to give 35 in 60% overall yield. The fully protected PLM 36 was achieved in 73% yield by
coupling acceptor 35 with H-phosphonate donor 2 using the same condition were used
earlier. The crude α, β mixture of fully protected PLM was subjected to flash column
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
chromatography with Et N-deactivated silica gel, which gave pure α-isomer of 36 as a white
3
waxy material, Rf 0.4(CHCl /MeOH/Et N = 7:3:0.1). To establish the synthesis of target
3
3
PLM 1, the benzylated compound 36 was debenzylated using Perlman’s catalyst in
THF/MeOH/H O and monitored on MS which confirm its formation.
2
Scheme 7: Synthesis of Phospholipomannan anchor 1.
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1
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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
BnO
BnO
BnO
OBn
O
O
BnO
BnO
BnO
O
O
BnO
BnO
BnO
O
O
BnO
BnO
BnO
O
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
OBn
O
HO
BnO
O
P
BnO
O
OH
O
BnO
BnO
O
OBn
O
O
OBn
OBn
OBn
O
O
O
BnO
BnO
BnO
BnO
2
4
O P OH
O
OBn
c
1
BnO
BnO
O
a, b
a, 73%
OBn
OBn
BnO
BnO
HO
O
O
OBn
OBn
C13H27
O P OH
O
3
O
NH OBn
OBn
C15H31
C13H27
35
O
NH OBn
C15H31
3
6
Reagents and Conditions: (a) Pivaloyl chloride, Py, I , Py/H O (19:1); (b) NaOMe/MeOH,
2
2
6
0%; (c) 30% Pd(OH) , THF, MeOH, H O, Formic acid (catalytic).
2 2
Conclusion:
In summary, the first total synthesis of PLM anchor of C. albicans was achieved via
3
convergent block approach. Developed an enantioselective A coupling approach and its
application for the synthesis of phytoceramide, and established the first iterative one pot
protocol for the synthesis of (1→2)-β-tetramannoside.
Experimental section:
General:
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Solvents were distilled in the standard way, and commercial reagents were used without any
purification. All reactions were performed in flame-dried glass apparatus under inert
atmosphere unless mentioned. Anhydrous solvents like CH Cl , Et O, THF, CH OH,
2
2
2
3
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
1
13
1
CH CN, DMF, pyridine, and Et N were dried in standard way. NMR spectra ( H, C, 2D H-
3
3
1
1
13
H-COSY and H- C HMBC, HMQC, and NOESY) were recorded on a 400 and 500 MHz
spectrometer, and Me Si used as an internal standard. NMR chemical shifts (δ) in ppm and
4
coupling constants J in Hz. High-resolution mass spectral data were obtained from Q-ToF
Mass Spectrometer coupled LC system. The following conditions were used: capillary
ο
voltage 3500 V, capillary temperature 350 C, auxiliary gas flow rate 7.0 L/min, spray
voltage 4.5 kV, mass range 100-1000 amu (maximum resolution 30000). Optical rotations
were measured on a Perkin Elmer polarimeter. Analytical TLC was performed on 60 F254
plates, and visualized in UV, staining solutions ceric-sulfate was used. Column
chromatography was carried out with silica (60-120, 230-400 mesh). Analytical and semi-
preparative HPLC purifications were carried out on normal and reversed-phase columns
connected to a binary pump and monitored using a photodiode array detector.
Experimental procedures:
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3
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4
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4
4
4
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5
5
6
Synthesis of suitably protected mannose-based building blocks:
Phenyl-4,6-O-Benzylidene-thio-α-D-mannopyranoside (18): D-(+)-mannose (10.0 g, 55.55
mmol) was suspended in acetic anhydride (40.0 mL, 423.52 mmol). At 0 °C (Ice bath)
several drops of perchloric acid (69%, aqueous) were added. The reaction was allowed to stir
for 1h at 0 °C (Immersion cooler). After completion of the reaction, diluted with 500 mL of
CH Cl , washed with water and saturated NaHCO solution. The combined organic phases
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
2
2
3
were dried over Na SO and concentrated in vacuum. The crude pentaacetate was used for
2
4
the subsequent glycosylation without further purification. Under an argon atmosphere the
crude penta acetate (21.65 g, 55.50 mmol) was dissolved in anhydrous CH Cl (250 mL) and
2
2
BF Et O (11.5 g, 81.02 mmol) was added. At 0 °C thiophenol (8.4 mL, 83.2 mmol) was
3
2
added dropwise, and the reaction mixture was allowed to stir at rt. After completion of the
reaction judged by TLC, the saturated NaHCO solution was added until BF Et O was
3
3
2
hydrolysed. The organic phase was washed with saturated NaHCO solution followed by
3
water several times. The combined organic phases were dried over Na SO , concentrated in
2
4
vacuum. To a solution of above crude thio-mannopyranoside in dry MeOH (250 mL) was
added 1M NaOMe (10 mL). The reaction mixture was stirred under an argon atmosphere for
+
1
0 h, then neutralized with DOWEX 50 H -form, filtered, and the filtrate was concentrated in
vacuum. The crude product was subjected to column chromatography (CH Cl /MeOH, 8:2)
2
2
to give thio mannopyranoside (13.42 g, 90%) as a solid (R 0.65CHCl /MeOH = 8:2).
f
3
To a solution of thio mannopyranoside (10.0 g, 36.76 mmol) in dry DMF (200 mL), p-TSA
0.63 g, 3.67 mmol) and benzaldehyde dimethylacetal (5.5 mL, 36.76 mmol) were added at
(
rt. The reaction mixture was stirred for 2 h on the rota at 60 °C under reduced pressure (200
mbar). Then the reaction was concentrated on vacuum, and the residue was diluted with
EtOAc (200 mL), the organic phase was washed with saturated NaHCO (200 mL), followed
3
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by water (2 x 100 mL), dried over Na SO , and concentrated in vacuum. The crude product
2
4
was subjected to column chromatography (CHCl /MeOH, 8:1) to give desired product 18 in
3
(7.26 g, 55%) as a white solid (R 0.55CHCl /MeOH = 9:1).
f
3
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
Phenyl-2,3-O-Benzyl-4,6-O-benzylidene-thio-α-D-mannopyranoside (6): To a solution of
1
8 (5.0 g, 13.88 mmol) in anhydrous DMF (100 mL), NaH (60% dispersion in mineral oil,
o
1
.99 g, 83.31 mmol) was added under N atmosphere at 0 C (Immersion cooler). After
2
stirring for 30 min, BnBr (3.96 mL, 33.33 mmol) was added dropwise. Then the reaction was
allowed to stir overnight at, reaction was quenched with ice, and diluted with EtOAc, and
subjected to aqueous workup, organic layer was dried over Na SO , and concentrated in
2
4
vacuum. The crude residue was subjected to column chromatography to afford compound 6
1
7
(7.12 g, 95%) as a foam (R 0.65 Hexanes/EtOAc = 9:1).
f
1
H NMR (400 MHz, CDCl ): δ 3.89 (t, J = 9.8 Hz, 1H), 3.93 - 3.99(m, 1H), 4.04 (dd, J =
3
3
.2, 1.4 Hz, 1H), 4.19 - 4.35 (m, 3H), 4.65 (d, J = 12.2 Hz, 1H), 4.72 (s, 2H), 4.82 (d, J =
1
2.2 Hz, 1H), 5.51 (d, J = 1.3 Hz, 1H), 5.65 (s, 1H), 7.20 - 7.43 (m, 18H), 7.52 (dd, J = 7.6,
1.8 Hz, 2H).
Phenyl-3-O-Benzyl-4,6-O-benzylidene-thio-α-D-mannopyranoside (7): A mixture of 18
(5.0 g, 13.88 mmol) and dibutyltin oxide (3.45 g, 13.88 mmol) in anhydrous methanol (100
mL) was refluxed for 4 h, then the reaction mixture was concentrated in vacuum, the residue
was dissolved in anhydrous toluene/DMF (1:1, 100 mL) and freshly activated 4Å MS were
added. The reaction mixture was cooled to 0 ºC (Immersion cooler), after which TBAB
(13.42 g, 41.65 mmol) and BnBr (1.98 mL, 2.84 mmol) were added to the solution. The
o
reaction was stirred at 120 C for 12h, then filtered through Celite and washed with EtOAc.
The organic layer was washed with water, dried over Na SO , and concentrated in vacuum,
2
4
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8
9
1
1
1
1
1
1
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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
the crude residue was subjected to column chromatography to give desired compound 7 (4.99
1
2e 1
g, 80%) as a foam (R 0.65Hexanes/EtOAc = 7:3).
H NMR (400 MHz, CDCl ): δ 2.84
3
f
(bs, 1H), 3.78 (t, J = 10.2 Hz, 1H), 3.89 (dd, J = 9.5, 3.3 Hz, 1H), 4.12 (dt, J = 12.3, 7.2 Hz,
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
2
5
H), 4.214.30 (m, 2H), 4.67 (d, J = 11.8 Hz, 1H), 4.82 (d, J = 11.8 Hz, 1H), 5.55 (s, 1H),
.52 (s, 1H), 7.18-7.45 (m, 15H).
2
-Naphthyl-3,4,6-tri-O-Benzyl-α-D-mannopyranoside (8): The compound 17 was
1
8
converted into 19 by using reported literature. To a solution of compound 19 (2.0 g, 3.95
mmol) in DCE/ H O, added catalytic amount p-TSA. The reaction was vigorously stirred at rt
2
over 3 h, after which the reaction mixture quenched with triethylamine and evaporated, the
crude residue was dissolved in EtOAc and washed with water, and concentrated in vacuum.
o
The crude residue was again dissolved in DMF 30 mL, and cooled to 0 C (Immersion
cooler), NaH (60%, 0.2 g, and 8.33 mmol) was slowly added under N atmosphere, after
2
stirring for 30 min, 2-bromomethylnapthalene (0.988 g, 4.47 mmol) was added. The reaction
o
was stirred overnight at rt, then quenched with methanol and heated to 60 C. The reaction
mixture was poured in water and extracted with 2 x 200 mL EtOAc, after which the
combined organic layer was washed with brine, dried over Na SO , and concentrated in
2
4
vaccum. The crude residue was subjected to column chromatography to give compound 8
1
(
1.63 g, 70%) as a colour less syrup (R 0.45Hexanes/EtOAc = 7:3). H NMR (400 MHz,
f
CDCl ): δ 2.52 (s, 1H), 3.72 (d, J = 10.5 Hz, 1H), 3.75 - 3.81 (m, 1H), 3.89 (d, J = 5.8 Hz,
3
2
H),3.96 (dd, J = 9.1, 3.2 Hz, 1H),4.09 (d, J = 1.6 Hz, 1H),4.54 (dd, J = 17.5, 11.5 Hz,
2H),4.62 - 4.71 (m, 4H),4.85 (dd, J = 17.3, 11.4 Hz, 2H),4.85 (dd, J = 17.3, 11.4 Hz, 2H),
5
5
.04 (d, J = 1.1 Hz, 1H), 7.15 - 7.19(m, 2H),7.26 - 7.37 (m, 13H), 7.46 (ddd, J = 18.7, 9.2,
.2 Hz, 3H), 7.75 (s, 1H), 7.82 (dd, J = 8.9, 4.4 Hz, 3H).
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2
-Naphthyl-(2,3-di-O-benzyl-4,6-O-benzylidene-β-D-mannopyranosyl)(1→2)-(3,4,6-tri-
O-benzyl-α-D-mannopyranoside (20):
PhSCl/AgOTf method: A solution of donor 6 (100 mg, 0.18 mmol) and freshly activated 4Å
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
molecular sieves (1.0 g) in CH Cl was stirred at room temperature for 30 min, and cooled to
2
2
o
-78 C (Immersion cooler), which was followed by addition of AgOTf (142.5 mg, 0.55
mmol) dissolved in CH CN (0.5 mL) without touching the wall of reaction vessel. After 5
3
min orange coloured solution of benzene sulfinyl chloride (PhSCl) (29.42 mg, 0.18 mmol)
was added to the solution through a micro syringe. This addition needs to be performed
quickly in order to PhSCl from freezing inside the syringe tip or on the flask wall. The yellow
colour of the solution quickly dissipated within a few seconds, which indicates the complete
consumption of PhSCl. After the donor was completely consumed, according to TLC (about
o
1
0-15 min at -78 C), a solution of acceptor 8 (98.3 mg, 0.16 mmol) and TTBP (41 mg, 0.18
mmol) In CH Cl was slowly added dropwise by using a syringe. The reaction mixture was
2
2
o
warmed to -20 C in 2h, the reaction was quenched with Et N, diluted with CH Cl , filtered,
3
2
2
and concentrated in vacuum. The residue was dissolved in CH CN and insoluble material was
3
filtered off, concentrated in vacuum. The crude residue was purified by silica gel column
chromatography with EtOAc and hexane (1:9) as the eluent to give 20 (174 mg, β/α: 9.4/0.6,
1
9
9% yield, ratio was determined by HPLC, SI page no. 13) as a foamy solid. H NMR
(CDCl , 400 MHz): δ 3.24-3.30 (m, 1H), 3.53 (dd, J = 4.0, 8.0 Hz, 1H), 3.67-3.75 (m, 2H),
3
3
.79-3.88 (m, 2H), 3.95 (t, J = 12.0 Hz, 1H), 4.0-4.03 (m, 2H), 4.18-4.23 (m, 2H), 4.30-4.33
(m, 2H), 4.47 (d, J = 12.0 Hz, 1H), 4.54 (d, J = 12.0 Hz, 1H), 4.59-4.69 (m, 5H), 4.78 (d, J =
2.0 Hz, 1H), 4.83-4.92 (m, 3H), 5.06-5.11 (m, 2H), 5.59 (s, 1H), 7.08-7.10 (m, 2H), 7.20-
1
1
3
7.54 (m, 31H), 7.76 (s, 1H), 7.82-7.86 (m, 3H)., C {1H} proton-decoupled NMR (CDCl
3
1
25 MHz): δ 138.7, 138.3, 138.1, 137.6, 134.4, 133.2, 133.0, 128.8, 128.7, 128.3, 128.3,
2
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9
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
1
1
28.1, 128.1, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.4, 127.0, 126.3, 126.1, 126.0,
25.9, 101.4, 100.4, 96.6, 78.3, 78.2, 77.2, 76.2, 75.1, 75.0, 74.3, 73.3, 73.0, 71.8, 71.7, 70.8,
69.4, 68.9, 68.5, 67.6., [α]D20 = -29.7 (c = 1.0, CHCl ); HRMS (ESI-TOF) m/z: [M + H]
+
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
calcd for C H O 1020.4449; found 1020.4445.
6
5
64 11
2
-Naphthyl-(2,3-di-O-benzyl-4,6-O-benzylidene-β-D-mannopyranosyl)(1→2)-(3-O-
benzyl-4,6-O-benzylidene-β-D-mannopyranosyl)(1→2)-(3-O-benzyl-4,6-O-benzylidene-
β-D-mannopyranosyl)(1→2)-(3,4,6-tri-O-benzyl-α-D-mannopyranoside (5): A solution of
donor 6 (200 mg, 0.37 mmol) and freshly activated 4Å molecular sieves (2.0 g) in CH Cl
2
2
o
was stirred at room temperature for 30 min, and cooled to -78 C (Immersion cooler), which
was followed by addition of AgOTf (285 mg, 1.11 mmol) dissolved in CH CN (1.0 mL)
3
without touching the wall of reaction vessel. After 5 min, orange coloured solution of PhSCl
(58.49 mg, 0.37 mmol) was added to the solution through a micro syringe. This addition
needs to be performed quickly in order to prevent freezing of PhSCl inside the syringe tip or
on the flask wall. The yellow colour of the solution quickly dissipated
within
a
few seconds, which indicates the complete consumption of
PhSCl. After the donor was completely consumed, according to TLC (about 10-15 min at -78
oC), a solution of acceptor 7 (151 mg, 0.336 mmol) and TTBP (82 mg, 0.336 mmol) in
CH Cl was slowly added dropwise by using a syringe. The reaction mixture was warmed to -
2
2
o
o
2
0 C in 2h, and then the mixture was cooled to -78 C, this was followed by another
glycosylation by the same protocol using sequential addition of AgOTf (256 mg, 1.0 mmol)
in CH CN (2.0 mL), PhSCl (52.6 mg, 0.33 mmol), acceptor 7 (137 mg, 0.306 mmol) and
3
TTBP (75.9 mg, 0.306 mmol) in anhydrous CH Cl (2.0 mL). Thereafter, another
2
2
glycosylation was achieved following the same protocol using AgOTf (267 mg, 0.925 mmol)
in CH CN (1.0 mL), PhSCl (48.3 mg, 0.306 mmol), acceptor 8 (164 mg, 0.278 mmol) and
3
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TTBP (69.18 mg, 0.278 mmol) in anhydrous CH Cl (5.0 mL). Finally, the reaction was
2
2
quenched with Et N, diluted with CH Cl , filtered, and concentrated in vacuum. The residue
3
2
2
was dissolved in CH CN and insoluble material was filtered off, concentrated in vacuum. The
3
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
crude residue was subjected to Sephadex LH-20 column, followed by flash column
chromatography with EtOAc and toluene (0.3:9.7) as the eluent to give 5 (251 mg, 40%
1
overall yield) as a foamy solid (R 0.7 Hexanes/EtOAc = 7:3). H NMR (CDCl , 400 MHz):
f
3
δ 3.31-3.37 (m, 1H), 3.39-3.44 (m, 1H), 3.53-3.65 (m, 6H), 3.73-3.87 (m, 6H), 3.91-4.01 (m,
H), 4.21-4.27 (m, 2H), 4.29-4.34 (m, 3H), 4.36-4.41 (m, 3H), 4.45-4.56 (m, 9H), 4.60 (d, J
4.0.0 Hz, 1H), 4.65-4.68 (m, 3H), 4.71-4.81 (m, 5H), 4.87 (d, J = 12.0 Hz, 1H), 5.02 (d, J =
5
=
12.0 Hz, 1H), 5.09 (d, J = 12.0 Hz, 1H), 5.38 (s, 1H), 5.43 (s, 1H), 5.53 (s, 1H), 5.62 (s,
13
1
H), 6.91 (d, J = 8.0 Hz, 2H), 7.07-7.52 (m, 60H), 7.75 (bs, 1H), 7.81-7.85 (m, 3H)., C
{1H} proton-decoupled NMR (CDCl 125 MHz): δ 139.6, 138.6, 138.5, 138.3, 137.9,
3
1
1
37.8, 137.7, 137.5, 137.3, 136.9, 133.9, 133.1, 133.0, 129.0, 128.7, 128.4, 128.4, 128.4,
28.3, 128.2, 128.2, 128.1, 128.0, 128.0, 128.04, 128.0, 127.9, 127.9, 127.69, 127.6, 127.5,
127.2, 127.2, 127.1, 127.04, 127.0, 126.9, 126.4, 12.3, 126.2, 126.1, 126.1, 125.9, 103.5,
1
7
02.0, 101.6, 101.3, 101.2, 99.2, 95.7, 79.3, 78.6, 78.4, 78.1, 77.2, 76.5, 76.1, 76.0, 75.5,
4.8, 74.3, 73.8, 72.4, 72.1, 71.5, 71.1, 70.8, 70.5, 69.6, 68.8, 68.8, 68.7, 67.8, 67.7, 67.7.
+
HRMS (ESI-TOF) m/z: [M + Na] calcd for C H NaO 1729.6968; found 1729.6976.
1
05 104
21
2-Naphthyl-(2,3-di-O-benzyl-β-D-mannopyranosyl)(1→2)-(3-O-benzyl-β-D-
mannopyranosyl)(1→2)-(3-O-benzyl-β-D-mannopyranosyl)(1→2)-(3,4,6-tri-O-benzyl-α-
D-mannopyranoside (21): To a solution of 5 (200 mg, 0.11 mmol) in CH Cl /MeOH (1:1,
2
2
20 mL), pTSA (200 mg, 1.16 mmol) was added, allowed to stir for 12 h, after completion of
reaction was confirmed by TLC analysis, then the reaction was stopped by adding Et N and
3
concentrated in vacuum. The crude residue was worked up with EtOAc/H O, organic layer
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was concentrated in vaccum. The crude residue was subjected to column chromatography
2
0
MeOH: CHCl (1:9) as a eluent to afford 21 (148.2 mg, 95% yield) as a foamy solid [α]
=
3
D
1
-29.7 (c = 1.0, MeOH). H NMR (400 MHz, C D N): 3.78-3.86 (m, 2H), 3.91 (t, J = 9.1 Hz,
5
5
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
2
8
8
6
3
H), 4.06 (dd, J = 10.6, 4.3 Hz, 1H), 4.09-4.15 (m, 2H), 4.20-4.51 (m, 12H), 4.55-4.67 (m,
H), 4.76 (dd, J = 12.1, 5.8 Hz, 3H), 4.80-5.18 (m, 75H, Including H O peak), 5.25 (d, J =
2
.5 Hz, 3H), 5.33 (d, J = 10.8 Hz, 2H),, 5.47 (s, 1H), 5.50 (s, 1H), 5.67 (s, 1H), 5.77 (s, 1H),
.02 (s, 1H), 7.02-7.07 (m, 3H), 7.09-7.15 (m, 4H), 7.17 (bs, 9H), 7.27 (dd, J = 11.0, 4.1 Hz,
H), 7.32 (d, J = 9.5 Hz, 5H), 7.37 (dd, J = 6.9, 4.1 Hz, 3H), 7.43-7.48 (m, 2H), 7.49-7.52
(m, 2H), 7.55 (d, J = 7.9 Hz, 6H), 7.60 (d, J = 7.4 Hz, 2H), 7.69 (t, J = 8.2 Hz, 3H), 7.88 (dd,
1
3
J = 16.7, 8.6 Hz, 4H). C {1H} proton-decoupled NMR (CDCl 125 MHz): δ 150.9, 150.7,
3
150.5, 142.0, 140.9, 140.6, 140.5, 140.34, 140.3, 139.9, 136.6, 136.4, 136.2, 136.0, 134.7,
1
1
8
34.4, 129.7, 129.6, 129.5, 129.4, 129.2, 129.17, 129.1, 129.0, 128.9, 128.8, 128.7, 128.4,
28.2, 128.1, 128.0, 127.7, 127.5, 127.3, 124.6, 124.4, 124.2, 104.8, 102.6, 100.7, 98.2, 84.4,
3.6, 82.4, 80.0, 79.9, 79.7, 78.9, 77.3, 76.6, 76.5, 75.9, 74.5, 73.4, 73.2, 73.1, 72.1, 71.5,
71.4, 71.1, 70.6, 70.4, 68.6, 68.3, 68.1, 64.6, 64.1, 63.5. HRMS (ESI-TOF) m/z: [M + Na]+
calcd for C H NaO 1459.6029; found 1459.6042.
8
4
92
21
2
,3,4,6-Tetra-O-benzyl-β-D-mannopyranosyl)(1→2)-(3,4,6-tri-O-benzyl-β-D-
mannopyranosyl)(1→2)-(3,4,6-tri-O-benzyl-β-D-mannopyranosyl)(1→2)-(3,4,6-tri-O-
benzyl-α-D-mannopyranoside (22): The solution of 21 (200 mg, 0.139 mmol) in anhydrous
o
DMF stirred under nitrogen at 0 C (Immersion cooler), and sodium hydride (60% dispersion
in mineral oil, 100 mg, 4.166 mmol) was slowly added. After stirring for 30 min, benzyl
bromide (0.120 mL, 0.99 mmol) was added dropwise. The reaction was stirred overnight at
room temperature, then quenched with ice, diluted with EtOAc, and poured into water. The
aqueous layer was extracted 2x with EtOAc, after which the combined organic layer was
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washed with brine, dried over Na SO , and concentrated in vaccum. R 0.55 (EtOAc/Hexane =
2
4
f
+
2
:8). HRMS (ESI-TOF) m/z: [M + Na] calcd for C126H NaO 1999.8846; found
128 21
1999.8858. The crude residue was dissolved in dichloromethane (10 ml) and methanol (3.0
ml). DDQ (163 mg, 0.718 mmol) was added in three equal portions at half-hour intervals.
Stirring continued for 2 h, the solvent was removed by evaporation, the residue taken up in
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
3
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3
4
4
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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
chloroform and washed with sat.NaHCO . After which the combined organic layer was
3
washed with brine, dried over Na SO , and concentrated in vaccum. Chromatography on
2
4
silica in dichloromethane– methanol 10:1 gave 22 (155 mg, 65%) as a foamy solid, (Rf
1
0
.2Hexanes/EtOAc = 7:3). [α]D20 = +16.57 (c = 0.33, CHCl ). H NMR (CDCl , 400 MHz):
3
3
δ 2.74 (bs, 1H), 2.96 (dd, J = 20.0, 5.0 Hz, 1H), 3.25 (dd, J = 20.0, 5.0 Hz, 1H), 3.44 (dd, J =
2
4
0.0, 5.0 Hz, 1H), 3.48-3.63 (m, 8H), 3.65-3.71 (m, 6H), 3.75-3.82 (m, 2H), 3.88-3.94 (m,
H), 4.02-4.05 (m, 3H), 4.19-4.22 (m, 2H), 4.26-4.31 (m, 4H), 4.34-4.45 (m, 10H), 4.48-4.56
(m, 4H), 4.60-4.64 (m, 3H), 4.73-4.83 (m, 5H), 4.93 (d, J = 20.0, 5.0 Hz, 1H), 5.0 (dd, J =
13
2
0.0, 5.0 Hz, 1H), 5.12-5.14 (m, 3H)., C {1H} proton-decoupled NMR (CDCl 125
3
MHz): δ 139.8, 139.7, 138.8, 138.8, 138.7, 138.7, 138.6, 138.5, 138.5, 138.3, 138.3, 138.27,
1
1
38.2, 138.0, 137.8, 137.83, 128.6, 128.67, 128.3, 128.35, 128.30, 128.2, 128.25, 128.22,
28.21, 128.1, 128.16, 128.09, 128.00, 127.9, 127.87, 127.8, 127.7, 127.73, 127.7, 127.5,
127.4, 127.3, 127.28, 127.2, 127.1, 127.0, 126.9, 126.93, 101.6, 98.8, 98.5, 98.4, 98.2, 93.8,
9
7
3.6, 83.2, 83.1, 79.8, 79.7, 77.99, 77.9, 77.2, 75.8, 75.6, 75.3, 75.2, 75.2, 75.0, 74.9, 74.7,
4.6, 74.6, 74.49, 74.4, 74.3, 74.2, 74.2, 73.56, 73.5, 73.4, 73.3, 73.2, 73.1, 72.6, 71.9, 71.4,
71.3, 71.2, 70.6, 70.5, 70.5, 70.4, 70.3, 70.3, 69.7, 69.7, 69.3, 69.1, 69.1, 68.8; HRMS (ESI-
+
TOF) m/z: [M + Na] calcd for C H NaO 1859.8220; found 1859.8238.
1
15 120
21
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Triethylammonium-O-Hydrogen-Phosphonato-2,3,4,6-Tetra-O-benzyl-β-D-
mannopyranosyl)(1→2)-(3,4,6-tri-O-benzyl-β-D-mannopyranosyl)(1→2)-(3,4,6-tri-O-
benzyl-β-D-mannopyranosyl)(1→2)-(3,4,6-tri-O-benzyl-α-D-mannopyranoside (2):
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
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7
8
9
0
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0
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7
8
9
0
o
To a stirred solution of imidazole (111 mg, 1.6 mmol) in anhydrous DCM (1.0 ml) at -10 C
were added PCl (30 ul, 0.326 mmol) and Et N (106 ul, 0.756 mmol). The mixture was
3
3
stirred for 20 min, after which alcohol 22 (200 mg, 0.108 mmol) in anhydrous DCM (2.0 ml)
o
was added dropwise over a period of 15 min. The mixture was stirred at -10 C for 3 h, and
quenched by addition of water/pyridine (1/4, 20.0 ml). The aqueous layer was extensively
washed with CHCl3 and the combined organic layers were further washed with
triethylammonium borate (TEAB) buffer and dried over Na SO . Evaporation in vacuum
2
4
gave the crude residue, which was subjected to flash column chromatography with Et N-
3
deactivated silica gel to afford H-phosphonate 28 as a white waxy material in 75% (163 mg)
2
0
1
(
R 0.55 CHCl /MeOH/Et N = 7:3:0.1). [α]
= +16.57 (c = 0.33, CHCl ). H NMR (400
3
f
3
3
D
MHz, CDCl ): δ 7.41 – 6.81 (m, 65H), 6.76 (d, J = 664.2 Hz, 1H), 5.61 (s, 1H), 5.33 (s, 1H),
3
4
.89 (dd, J = 28.3, 10.6 Hz, 3H), 4.74 – 4.53 (m, 8H), 4.52 – 4.18 (m, 13H), 4.10 (d, J = 11.5
Hz, 5H), 3.83 (s, 4H), 3.74 (s, 2H), 3.63 (s, 2H), 3.56 – 3.33 (m, 8H), 3.19 (d, J = 49.5 Hz,
1
3
9
1
H)., C {1H} proton-decoupled NMR (CDCl 125 MHz): δ 138.8, 138.7, 138.57, 138.5,
3
38.4, 138.3, 138.2, 128.4, 128.3, 128.3, 128.2, 128.2, 128.2, 128.1, 128.1, 128.0, 127.9,
127.8, 127.8, 127.7, 127.7, 127.6, 127.6, 127.5, 127.5, 127.4, 127.4, 127.3, 127.3, 100.9,
9
7
9.4, 94.2, 79.9, 79.8, 79.1, 78.9, 75.2, 75.0, 75.0, 75.0, 74.9, 74.8, 74.7, 74.5, 73.3, 73.2,
3
1
3.1, 73.0, 72.7, 72.5, 72.4, 72.3, 72.1, 72.0, 71.9, 71.8, 69.4, 69.2, 69.1, 68.8, 45.6, 8.5. P
+
NMR (CDCl , 161.98 MHz): δ 1.99. HRMS (ESI-TOF) m/z: [M + H] calcd for
3
C H NO P 2001.9241; found 2001.9265.
1
21 136
23
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,4,5,6-tetra-O-benzyl-myo-inositol (23): Synthesis is started from myoinositol, which was
converted into 1,2-cyclohexylidene-myoinositol by using known protocol. To a solution of
o
1,2-cyclohexylidene-myoinositol (10.0 g, 38.44 mmol) in anhydrous DMF (100 mL) at 0 C
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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3
3
4
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5
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5
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5
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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
(Immersion cooler) was slowly added sodium hydride (60%, 7.38 g, 307.54 mmol). After
stirring for 30 min, BnBr (21.91mL, 184.56 mmol) was added. The reaction was allowed to
stir overnight at rt, then quenched with ice, diluted with EtOAc, and poured into water. The
aqueous layer was extracted 2x with EtOAc, after which the combined organic layer was
washed with brine, dried over Na SO , and concentrated in vaccum. (HRMS (EI): m/z [M +
2
4
+
H] calculated for C H O 621.3216, observed: 621.3171. ) The residue was dissolved in
40 45
6
CH Cl /MeOH (2:1, 200mL), added acetyl chloride (2.0 mL) dropwise. After 3 h, the
2
2
reaction was stopped by adding Et N and concentrated in vacuum. The crude material was
3
purified by silica gel column chromatography to give ± 23 (18.69 g, 90%) as a white solid
1
(R 0.18Hexanes/EtOAc = 7:3). H NMR (CDCl , 500 MHz): δ 2.43 (d, J = 4.5 Hz, 1H), 2.51
f
3
(s, 1H), 3.45-3.50 (m, 3H), 3.84 (t, J = 10.0 Hz, 1H) 3.97 (t, J = 10.0 Hz, 1H), 4.20 (bs, 1H),
4
7
1
.69-4.75 (m, 3H), 4.83 (d, J = 5.0 Hz, 1H), 4.85 (d, J = 5.0 Hz, 1H ), 4.90-4.96 (m, 3H),
1
3
.28-7.37 (m, 20H)., C {1H} proton-decoupled NMR (CDCl 125 MHz): δ 138.6, 138.4,
3
37.7, 128.57, 128.5, 128.4, 128.3, 128.2, 127.9, 127.88, 127.8, 127.82, 127.6, 83.2, 81.6,
+
81.3, 79.9, 75.9, 75.7, 75.6, 72.7, 71.7, 69.1., HRMS (ESI-TOF) m/z: [M + H] calcd for
C H O 541.2590; found 541.2563.
3
4
37
6
(
-)-1,2-O--(R)-[camphanyldene]-3,4,5,6-tetra-O-benzyl-myo-inositol (24): To a Solution
of starting material 23 (5.0 g, 9.25 mmol) in dry DCM (100 ml) camphor dimethylacetal
(2.01 g, 10.18 mmol) was added followed by pTSA (0.318 g, 1.85 mmol). The reaction
mixture was refluxed for 2h and TLC was checked, substrate was completely consumed.
Reaction mixture was neutralized with Et N and evaporated on vaccum then purified by
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2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
3
4
4
4
4
4
4
4
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5
5
5
5
5
5
6
column chromatography (1, 2, 3, 4, 5, 6 % EtOAc / Hexane) to give (-)- 24 (2.90 g, 48%) as a
colourless syrup (R 0.5Hexanes/EtOAc = 9:1) and (+)- 24 (2.90 g, 48%) as a colourless
f
2
0
1
syrup (R 0.4Hexanes/EtOAc = 9:1) [α] = -7.7 (c = 0.66, CHCl ). H NMR (CDCl , 400
f
D
3
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
MHz) (-)- 24 : δ 0.83 (s, 3H), 0.87 (s, 3H), 1.08 (s, 3H), 1.26 (t, J = 8.0 Hz, 2H), 1.35-1.42
m, 1H), 1.48 (d, J = 12.0 Hz, 1H), 1.71-1.76 (m, 2H), 1.92-2.01 (m, 2H), 3.43 (t, J = 8.80
Hz, 1H), 3.70-3.78 (m, 2H), 3.83 (t, J = 8.40 Hz, 1H), 3.96 (t, J = 6.40 Hz, 1H), 4.30 (t, J =
(
1
3
4
.80 Hz, 1H), 4.68-4.82 (m, 7H), 4.90 (d, J = 11.20 Hz, 1H), 7.22-7.39 (m, 20H)., C {1H}
proton-decoupled NMR (CDCl 125 MHz): δ 138.8, 138.6, 138.4, 128.39, 128.36, 128.34,
3
1
7
28.3, 128.08, 128.0, 127.8, 127.7, 127.6, 127.59, 127.5, 117.7, 83.2, 82.1, 80.8, 77.4, 76.2,
5.2, 73.9, 73.2, 72.4, 51.5, 48.0, 45.2, 45.0, 29.8, 29.7, 27.0, 20.6, 20.4, 10.2., HRMS (ESI-
+
TOF) m/z: [M + H] calcd for C H O 675.3686; found 675.3661.
4
4
51
6
(-)-3,4,5,6-tetra-O-benzyl-myo-inositol (25): To a solution of (-)-24 (3.0 g, 4.45 mmol) in
CH Cl /MeOH (1:1, 100mL) at rt, was added pTSA (0.765 g, 4.45 mmol) and heated to 50
2
2
oC on oil bath, After 4 h, TLC indicates complete consumption of starting material, then the
reaction was quenched with TEA and concentrated in vacuum. The reaction mass was
dissolved in EtOAc and washed with water, dried over Na SO , and concentrated in vaccum.
2
4
The crude residue was subjected to column chromatography to afford (-)-25 (2.30 g, 96%) as
2
0
1
a white solid. (R 0.18Hexanes/EtOAc = 7:3). [α]
= -11.62 (c = 0.86, CHCl ). H NMR
3
f
D
(CDCl , 400 MHz): δ 2.43 (d, J = 3.3 Hz, 1H), 2.51 (s, 1H), 3.45-3.50 (m, 3H), 3.84 (t, J =
3
9
.5 Hz, 1H), 3.97 (t, J = 9.5 Hz, 1H), 4.20 (t, J = 2.7 Hz, 1H), 4.68-4.76 (m, 3H), 4.84 (dd, J
1
3
=
10.8, 3.9 Hz, 2H),4.93 (dd, J = 13.3, 11.0 Hz, 3H), 7.27-7.35 (m, 20H)., C {1H} proton-
decoupled NMR (CDCl 100 MHz): δ 138.6, 138.5, 137.8, 128.55, 128.5, 128.39, 128.3,
3
1
27.9, 127.88, 127.84, 127.8, 127.6, 83.2, 81.6, 81.3, 80.0, 75.9, 75.6, 75.5, 72.7, 71.7, 69.2.,
+
HRMS (ESI-TOF) m/z: [M + H] calcd for C H O 541.2590; found 541.2605.
3
4
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(
-)-3,4,5,6-tetra-O-benzyl-1-O-(4-methoxybenzyl)-myo-inositol (9): A mixture of (-)-25
(2.00 g, 3.70 mmol) and dibutyltin oxide (0.925 g, 3.70 mmol) in anhydrous methanol (60
mL) was refluxed on oil bath for 4 h, until solution becomes clear. After concentration in
vacuum, the residue was dissolved in dry Toluene/DMF (1:1, 40 mL) and freshly activated
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
4
Å molecular sieves were added. And cooled to 0 ºC on ice bath, after which tetrabutyl
ammonium bromide (2.37 g, 7.41 mmol) and p-methoxybenzyl chloride (0.60 mL, 4.4 mmol)
o
were added to the solution. After stirring overnight under a Nitrogen atmosphere at 80 C,
reaction mixture filtered through Celite and washed with EtOAc. The organic layer was
washed with water, dried over Na SO , and concentrated in vacuum. Which after silica gel
2
4
column chromatography gave compound (-)-9 (2.2 g, 90%)as a white solid (Rf
1
0
2
.55Hexanes/EtOAc = 7:3). H NMR (CDCl , 400 MHz):δ 1.91 (bs, 1H), 3.28-.3.32 (m,
3
H), 3.37 (t, J = 12.09 Hz, 1H),3.73 (s, 3H), 3.91 (dd, J = 8.0, 16.0, Hz, 2H), 4.12 (t, J = 4.0
Hz, 1H), 4.54-4.56 (m, 2H),4.64 (s, 2H), 4.74-4.84 (m, 6H), 6.78 (d, J = 8.0 Hz, 2H), 7.17-
13
7
.26 (m, 22H)., C {1H} proton-decoupled NMR (CDCl 125 MHz): δ 159.3, 138.8,
3
138.76, 138.7, 137.9, 130.0, 129.5, 128.4, 128.3, 128.04, 128.0, 127.9, 127.8, 127.6, 113.8,
8
3.1, 81.2, 79.8, 79.4, 75.9, 72.7, 72.4, 67.5, 55.3., [α]D20 = -1.3 (c = 0.75, CHCl ); HRMS
3
+
(ESI-TOF) m/z: [M + Na] calcd for C H NaO 683.2985; found 683.2963.
4
2
44
7
(
-)-2,3,4,5,6-penta-O-benzyl-myo-inositol (26): To a solution of (-)-9 (100 mg, 0.1515
o
mmol) in anhydrous DMF (100 mL) stirring under Nitrogen at 0 C (Immersion cooler) was
slowly added sodium hydride (60%, 15 mg 0.59 mmol). After stirring for 5 min, BnBr (22 ul,
0
.183 mmol) was added dropwise. The reaction was allowed to stir overnight at rt, then
quenched with ice, diluted with EtOAc, and poured into water. The aqueous layer was
extracted 2x with EtOAc, after which the combined organic layer was washed with brine,
dried over Na SO , and concentrated in vaccum. The residue was dissolved in CH Cl 5ml,
2
4
2
2
29
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