A unique approach to the synthesis of a dengue vaccine and the novel tetrasaccharide that results

Article abstract of DOI:10.1016/j.tetasy.2009.02.024

An approach to the development of a dengue vaccine by synthesizing the hexasaccharide epitope on the viral surface is examined. The stereochemical and structural challenges include the synthesis of a β-mannoside bond. Synthesis of this bond is approached

Full text of DOI:10.1016/j.tetasy.2009.02.024

Tetrahedron: Asymmetry 20 (2009) 867–874
Contents lists available at ScienceDirect
Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
A unique approach to the synthesis of a dengue vaccine and the novel
tetrasaccharide that results
*
Nigel Kevin Jalsa, Gurdial Singh
The Department of Chemistry, The University of the West Indies, St. Augustine, Trinidad and Tobago
a r t i c l e i n f o
a b s t r a c t
Article history:
An approach to the development of a dengue vaccine by synthesizing the hexasaccharide epitope on the
viral surface is examined. The stereochemical and structural challenges include the synthesis of a b-man-
noside bond. Synthesis of this bond is approached via a trisaccharide analogue portion of the epitope. A
novel tetrasaccharide with a mannose–mannose anomeric linkage results in the course of the synthetic
attempts.
Received 4 February 2009
Accepted 19 February 2009
Available online 18 March 2009
This paper is dedicated to Professor George
Fleet, who is an inspirational chemist, on the
occasion of his 65th birthday
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
the probability by at least 15-fold of the individual developing
DHF. Consequently, for any vaccine to be considered adequate, it
In the last 20 years, dengue fever, and its more severe counter-
part, dengue haemorrhagic fever (DHF), have emerged as the most
important arthropod-borne viral diseases of humans. It is esti-
mated that there are of up to 100 million cases worldwide annu-
ally, greater than the combined cases of malaria and West Nile
infections.1 The dengue virus belongs to the Flaviviridae family2
and there are at present four known antigenically distinct sero-
types. Infection by one serotype guarantees lifelong immunity to-
wards that serotype, but not towards the other three serotypes.
In fact, infection by any one of three remaining serotypes increases
must of necessity be tetravalent in formulation.3 There have been
two general approaches to address these problems. Foremost of
these have been the attempts aimed at controlling the Aedes aegyp-
ti mosquito vector.4 The second approach has been focused on the
preparation of synthetic vaccines.5 Unfortunately, both approaches
have had limited success; the former being plagued by a combina-
tion of social and political obstacles,6 while the latter suffers from
the various formulations not being tetravalent.7
It is known that the four serotypes share the same 3D structure
of their envelope glycoprotein8 (Fig. 1).
Figure 1. Semitransparent surface representation of the Dengue Virus E Protein. The b-N-octyl-glucoside molecule is added during crystallization of the structure to
significantly improve the abundance and diffraction limit of the crystals. Copyright (2003) National Academy of Sciences, USA.
* Corresponding author. Tel.: +1 868 662 2002x3538; fax: +1 868 645 3771.
E-mail address: Gurdial.Singh@sta.uwi.edu (G. Singh).
0957-4166/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2009.02.024

868
N. K. Jalsa, G. Singh / Tetrahedron: Asymmetry 20 (2009) 867–874
ent in an oligosaccharide significantly increase its overall
immunogenicity.18
We therefore proposed to synthesize the structural analogue 1
of the native molecule. This molecule maintains all the critical
and interaction determining linkages as the original, as well as
the immunogenic fucosyl residue. We postulate that any sterically
constraining and electron-withdrawing groups at the 4- and 6-
positions of the donor mannose molecule should have a similar
effect on b-mannoside selectivity as the benzylidene ring. This ana-
logue should therefore also be synthetically simpler, as it allows
the introduction of a b-mannoside bond as part of a trisaccharide.
OH
OH
O
HO
HO
OH
OH
O
OH
O
OH
H
C
3
OH
Figure 2. Carbohydrate distribution on the viral surface. Copyright (2003) National
Academy of Sciences, USA.
O
HO
HO
OH
O
OH
O
O
O
O
HO
O
O
HO
HO
Asn
Furthermore, there are several glycosylation sites on the surface
of the dengue virion8 (Fig. 2). The conserved oligosaccharide at the
Asn-153 glycosylation site has been elucidated and modelled as a
hexasaccharide consisting of three mannose, two N-acetyl glucosa-
mine and one fucose residues9 (Fig. 3).
We suggest that an alternative approach to the development of
a dengue vaccine would be to synthesize an analogue of the con-
served hexasaccharide residue.10 This concept is not without prec-
edent, as the literature is replete with examples of the success of
this approach.11 The third mannoside residue of the hexasaccha-
ride has a b-mannoside bond, widely acknowledged as the most
challenging glycoside bond in carbohydrate chemistry.12
Numerous approaches to the stereoselective synthesis of a
b-mannoside bond have been reported over the years. Of particular
interest to us is the use of a constraining 4,6-O-benzylidene ring
proposed and developed by Crich.13 The benzylidene ring has been
shown to impart both torsional and electronic effects on stereose-
lectivity.14 Another methodology is that employing the use of gly-
cosyl phosphates as the anomeric leaving group, namely the
propane-1,3-diyl phosphate coupling protocol developed by
Singh.15 The appeal of this approach is that the use of phosphates
is biomimetic as per the Leloir biosynthetic pathway for the con-
struction of glycoside bonds.16
NHAc
NHAc
1
To test this hypothesis, we set out to construct trisaccharide 2
(Scheme 1), a glycosyl phosphate donor.
Herein we report our approach to the synthesis of donor 2, and
the results of our endeavours.
2. Results and discussion
2.1. Attempts at synthesis involving a glycosyl acceptor having
a free anomeric hydroxyl
We envisioned the synthesis of trisaccharide 2 through the cou-
pling of monosaccharide derivatives 3 and 5 in a one-pot process,
followed by subsequent phosphorylation of the anomeric centre.
The partially protected synthon 5 was obtained in higher overall
yield than that obtained through the reported literature synthe-
sis19 by adopting the modified simpler process (Scheme 1). Our
synthesis utilized the readily available methyl-
side 6. Protection of the C-4 and C-6 hydroxyls was accomplished
with -dimethoxytoluene and afforded the mono-benzylidene-
a-D-mannopyrano-
a,a
It is well known that a major part of oligosaccharides involved in
biological interactions are made up of hexopyranoses and their
overall conformation (and hence interactions) can be described by
bonds to the exocyclic groups, specifically the 6-OH and N-acetyl
groups.17 In addition, it has been shown that fucosyl residues pres-
protected monosacccharide 7. The remaining C-2 and C-3 hydroxyl
groups were protected as benzyl ethers to afford 8. Subsequent
treatment with TFA in acetonitrile and water gave the desired
partially protected mannoside 5 in an improved overall yield
(Scheme 2).
OH
OH
O
HO
HO
OH
OH
OH
O
OH
H C
3
O
OH
O
HO
HO
OH
O
OH
O
HO
O
O
O
O
O
HO
HO
Asn
NHAc
NHAc
Figure 3. Hexasaccharide attached to Asn-153 of the Dengue E Glycoprotein

N. K. Jalsa, G. Singh / Tetrahedron: Asymmetry 20 (2009) 867–874
869
OAc
OAc
O
AcO
AcO
OAc
OAc
O
O
AcO
AcO
OBn
O
O
BnO
O
O
O
P
O
2
OAc
OAc
OH
O
OBn
AcO
AcO
O
HO
BnO
+
O
O
O
O
O
O
4
P
O
P
3
O
OH
OBn
O
HO
BnO
OH
5
Scheme 1. Retrosynthetic analysis of trisaccharide mannosyl donor.
In Scheme 3 we detail the synthesis of the mannosyl phosphate
donor 3. We reasoned that its use in glycoside bond formation
ing to the typically greater nucleophilicity of the anomeric hydroxyl
over the remaining secondary hydroxyls of a monosaccharide.
would yield the
a-mannoside exclusively and in high yield due
to the anchimeric participation of the C-2 acetate group.
2.2. Synthesis of a trisaccharide donor via selective anomeric
benzyl group deprotection
The peracetylated mannose derivative 10 was obtained through
iodine-promoted Lewis acid-catalyzed acetylation of D-mannose,
9.20 The monosaccharide 11 was obtained by the selective removal
We were therefore still left with the issue of how to efficiently
synthesize our target trisaccharide 2. The use of ammonium for-
mate in catalytic transfer hydrogenation (CTH) for the deprotection
of benzyl groups is well documented in the literature.23 However,
there appears to be only one example of this method being applied
to the regioselective removal of an anomeric benzyl group from a
perbenzylated carbohydrate.24 This methodology had been applied
successfully to prepare monosaccharides and disaccharides.
Intrigued by this, we decided to investigate the utility of this ap-
proach for the preparation of the desired trisaccharide derivative
bearing an anomeric benzyl group which on selective removal
would provide a free anomeric hydroxyl. To this end, an acceptor
bearing free hydroxyls at the 4- and 6-positions only was required
(Scheme 5).
of the anomeric acetate group that resulted in the sole formation of
the
a
-anomer.21 This was then reacted with propane-1,3-diyldi-
oxyphosphoryl chloride to yield the novel monosaccharide donor
3 in excellent overall yield.
With synthons 3 and 5 available, we coupled 1.0 equiv of 5 with
3.0 equiv of 3 (Scheme 4). We anticipated that such a reaction
would result in the formation of a trisaccharide analogue of 2.
The outcome of this reaction was somewhat surprising in that
we obtained tetrasaccharide 12 containing an
a,a-anomeric–ano-
meric linkage. Support for the formation of this structure was evi-
denced by its 1H and 13C NMR data. In its 13C spectrum, resonances
at 91.01, 93.84, 99.03 and 100.75 were observed for the anomeric
carbons. In addition, the MS gave a (M+Na)+ of 1373.3901. Such a
linkage is known in trehalose (a well-known glucose disaccharide),
and has been reported for mannose disaccharides.22
Coupling of 5 with 2.0 equiv of 3 produced an inconsistent mix-
ture of disaccharides, varying with each repetition when carried
out on a gram scale. In hindsight, these results are not surprising ow-
Mannose was mono-benzylideneated to afford the hitherto un-
known mannosides 13, the low yield being a reflection of the ready
formation of the thermodynamic product: the 2,3:4,6-O-dibenzy-
lidene derivative; in addition to the unreacted mannoses which
were isolated. During the course of the protection reaction of the

870
N. K. Jalsa, G. Singh / Tetrahedron: Asymmetry 20 (2009) 867–874
OH
OH
OH
O
Ph
O
O
O
HO
HO
(i)
HO
OMe
Ph
OMe
6
7
OBn
O
O
(ii)
O
(iii)
5
BnO
OMe
8
Scheme 2. Reagents and conditions: (i)
48 h, 98 °C (74%).
a,
a-dimethoxytoluene, pTSA, DMF, 4 h, 50–70 °C (78%); (ii) BnBr, NaH, TBAI, DMF, 24 h, 0 °C to rt (78%); (i) H2O: CH3CN: TFA (4:3:3),
OH
OAc
OAc
OH
O
O
(i)
HO
HO
AcO
AcO
OH
OAc
9
10
(α:β;19:1)
OAc
OAc
O
(iii)
(ii)
AcO
AcO
3
11 (α only)
OH
Scheme 3. Reagents and conditions: (i) I2, Ac2O, 1 h, rt (100%); (ii) NH3, CH3CN, 6 h, rt (98%); (iii) N-MeIm, propane-1,3-diyldioxyphosphoryl chloride, DCM, 0 °C to rt, 24 h
(93%).
OAc
OAc
O
AcO
AcO
OAc
OAc
O
O
AcO
AcO
OBn
O
O
BnO
5
(i)
+
3.0 equivalents 3
O
AcO
AcO
O
OAc
OAc
12
Scheme 4. Reagents and conditions: (i) 1.5 equiv TMSOTf, DCM, 3 h, ꢀ78 °C to rt (93%).
free hydroxyl functions as their benzyl ethers, it was interesting to
note that the b-anomer was obtained exclusively affording 14 in
81% yield. Given that the anomeric mixture 13 was predominately
the a-anomer, the fact that derivatization of the anomeric centre
gives only the b-product lends further support to the directing
effect of the 4,6-O-benzylidene ring. Subsequent benzylation fur-

N. K. Jalsa, G. Singh / Tetrahedron: Asymmetry 20 (2009) 867–874
871
OH
O
Ph
O
O
(i)
(ii)
OH
9
HO
13
OBn
OBn
HO
O
Ph
O
O
HO
O
(iii)
OBn
OBn
BnO
BnO
15
14
Scheme 5. Reagents and conditions: (i) a,a-dimethoxytoluene, pTSA, DMF, 4 h, 50–70 °C (53%); (ii) BnBr, NaH, TBAI, DMF, 24 h, 0 °C to rt (81%); (iii) 80% AcOH, 2 h, 80 °C
(90%).
nished the saccharide 14. Treatment of the latter with 80% acetic
acid resulted in the removal of the benzylidene ring and afforded
the target glycosyl acceptor 15.
1,3-diyldioxyphosphoryl chloride provided our target donor mole-
cule 2.
The partially protected monosaccharide 15 was then coupled
with 2.0 equiv of phosphate 3 to give the protected trisaccharide
16 (Scheme 6). Extending the CTH principle, we were pleased
when the anomeric benzyl group was selectively removed in good
yield to afford 17. In this process we did not observe any evidence
of deprotection of the acetates or secondary benzyl groups. It is
important to note that the methanol used was of HPLC grade and
not dried methanol stored under molecular sieves. The reason
being that the latter system deprotected the acetates, as has been
previously reported.25 Reaction of this compound with propane-
3. Conclusion
Whilst not our originally intended target, the synthesis of tetra-
saccharide 12 provides further insight into the synthetic utility of
glycosyl phosphates as anomeric leaving groups. Normally re-
garded as being mild coupling agents, these phosphates can be
very active and efficient. Furthermore, the nature of this tetrasac-
charide presents a cage-like structure which we believe opens up
the possibility for encapsulation properties not unlike lipid vesi-
cles; thereby conferring the ability to sequester and deliver small
OAc
OAc
O
AcO
AcO
OAc
OAc
O
O
2.0 equiv. 3
(i)
AcO
AcO
+
OBn
15
O
O
BnO
OBn
OAc
16
OAc
OAc
OAc
OAc
O
O
AcO
AcO
AcO
AcO
(ii)
(iii)
OAc
OAc
OAc
O
O
O
O
AcO
AcO
AcO
AcO
OBn
OBn
O
O
O
BnO
O
BnO
OH
O
O
O
P
17
2
O
Scheme 6. Reagents and conditions: (i) 1.5 equiv TMSOTf, DCM, 3 h, ꢀ78 °C to rt (77%); (ii) Pd/Al2O3, NH4HCO2, MeOH, 12 h, rt, (71%); (iii) N-MeIm, propane-1,3-
diyldioxyphosphoryl chloride, DCM, 0 °C to rt, 24 h (71%).

872
N. K. Jalsa, G. Singh / Tetrahedron: Asymmetry 20 (2009) 867–874
molecules. Further work is currently being undertaken to examine
its novel biological properties.
and saturated NaHCO3 was added until effervescence ceased. The
mixture was extracted into ethyl acetate (100 ml) and washed
with water (4 ꢁ 50 ml). It was then dried over Na2SO4, filtered
and concentrated. The residue was subjected to column chroma-
tography; pet ether/ethyl acetate (1:4) to give 102 (2.9 g, 74%), a
Additionally, our findings highlight that a benzyl group, not
usually considered for protection of the anomeric centre, is indeed
useful and is applicable for higher oligosaccharides without ero-
sion of the regioselectivity of the catalytic transfer hydrogenation
process. It allows one ready access to a free anomeric hydroxyl,
and is compatible with commonly used protecting groups. Work
is already underway to examine the b-mannoside selectivity of tri-
saccharide 2 and will be reported in due course.
colourless oil, as an
a:b mixture in the ratio 3.7:1. Rf = 0.22. [a]
D
(anomeric mixture, at equilibrium) = ꢀ27.2 (c, 1.03, CHCl3). [lit.19
[a]
D = ꢀ20.1,
a
-isomer only, (c 1.02, CHCl3)]; 1H NMR (600 MHz,
CDCl3): d
a
-(major anomer): 5.20 (1H, s, H-1), 4.59–4.71 (3H, m),
4.56 (1H, d, J = 11.4 Hz, PhCH2O–), 4.48 (1H, d, J = 11.4, Hz
PhCH2O–), 4.26–4.34 (1H, m), 3.89 (4H, dd, J = 9.0 Hz, J = 9.6 Hz),
3.72–3.78 (2H, m), 3.29 (1H, bs), 7.26–7.32 (10H, m, Ar); 13C
4. Experimental
NMR (150 MHz, CDCl3): d
a-: [92.83 (1C, JC1–H1 = 169.8 Hz, C-1),
4.1. General experimental
62.79, 67.42, 71.85, 72.52, 72.86, 74.33, 79.27 (7C)]; b-: [94.06
(1C, JC1–H1 = 161.9 Hz, C-1), 62.27, 66.78, 72.43, 75.01, 75.59,
All chemicals used were of reagent or HPLC grade and were
used as supplied without prior purification unless otherwise sta-
ted. The solvents: DCM, diethyl ether and DMF were dried over cal-
cium hydride for 24 h, distilled and stored over 4 Å molecular
sieves. Flash column chromatography was carried out using forced
flow of the indicated solvent on Merck (230–400 mesh) silica gel.
TLC was performed using pre-coated Silica Gel 60 F254 plates; com-
pounds were visualized using acidic ammonium molybdate solu-
tion (ammonium molybdate (VI) tetrahydrate (25 g) in 1 M
H2SO4 (500 ml)). Anhydrous reactions were performed under ar-
gon in oven-dried apparatus; anhydrous transfers were done with
standard syringe techniques.
75.88, 82.49 (7C)];
a- and b-mixture: 127.82, 127.85, 127.89,
127.99, 128.13, 128.15, 128.22, 128.31, 128.42, 128.53, 128.60,
128.69 (20C, Ar), 137.59, 137.81, 137.99 (4C, Ar quat). HRMS
(NH3) calcd for C20H28NO6: 378.1917. Found: 378.1904 (M+NH4)+.
4.1.3. 20,30,40,60-Tetra-O-acetyl-
(200,300,400,600-tetra-O- -acetyl-mannopyranosyl-(100?4))-
(2000,3000,4000,6000-tetra-O-acetyl- -mannopyranosyl-(1000?1))-2,3-
di-O-benzyl- -mannopyranoside 12
a-D
-mannopyranosyl-(10?6)-
a-D
a-D
a
-D
The phosphate 3 (0.7 g, 1.5 mmol) was dissolved in DCM
(10 ml) at ꢀ78 °C under an argon atmosphere. TMSOTf (0.14 ml,
0.75 mmol) was then added and after 5 min, a solution of the gly-
cosyl acceptor 7 (0.18 g, 0.5 mmol) in DCM (10 ml) was added at
ꢀ78 °C. The reaction mixture was stirred for 30 min at ꢀ78 °C, at
0 °C for a further 30 min and then for 2 h at rt. The solvent was re-
moved in vacuo. The residue was redissolved in DCM (30 ml) and
washed with saturated NaHCO3 (3 ꢁ 25 ml) and then with water
(3 ꢁ 25 ml). The DCM layer was dried over Na2SO4, filtered and
the solvent was removed in vacuo to furnish crude 12. The residue
was purified by column chromatography (elution with 6:4 petro-
leum ether/ethyl acetate) to yield 12 (0.63 g, 93%) as a dark orange
1H, 13C and 31P NMR spectra were recorded on Bruker 600, 400
or 300 MHz spectrometers in the deuterated solvent stated. IR
spectra were recorded on a Perkin Elmer Spectrum RXI FT-IR spec-
trometer. High resolution mass data were obtained using a Bruker
Daltonics micrOToF-Q instrument in the electron spray ionization
mode. Optical rotations were measured on a Bellingham & Stanley
ADP 220 polarimeter at 24 °C. Specific rotations are reported in
10ꢀ1 deg cm2/g and concentrations in g/100 mL and for anomeric
mixtures the equilibrated (eq), values are reported.
viscous oil, Rf = 0.20.
[a]
D = +42.2 (c 10.6, CHCl3). 1H NMR
4.1.1. 2,3,4,6-Tetra-O-acetyl-
propane-1,3-diyl phosphate 3
a-D-mannopyranosyl-1-O-
(400 MHz, CDCl3): d 2.015 (3H, s), 2.020 (3H, s), 2.052 (3H, s),
2.055 (3H, s), 2.060 (3H, s), 2.074 (3H, s), 2.089 (3H, s), 2.095
(3H, s), 2.102 (3H, s), 2.174 (3H, s), 2.177 (3H, s), 2.185 (3H, s),
3.58 (1H, add, J = 1.7 Hz, J = 5.0 Hz), 3.72 (1H, aq, J = 6.7 Hz), 3.79
(1H, add, J = 1.3 Hz, J = 5.4 Hz), 4.03 (3H, m), 4.10 (2H, dd,
J = 2.2 Hz, J = 5.3 Hz), 4.13 (2H, dd, J = 2.3 Hz, J = 5.1 Hz), 4.18 (2H,
m), 4.28 (4H, m), 4.53 (1H, add, J = 7.5 Hz, J = 12.2 Hz), 4.60 (1H,
dd, J = 6.8 Hz), 4.65 (1H, m), 4.75 (1H, m), 5.16 (2H, m), 5.27 (4H,
m), 5.34 (5H, td, J = 3.1 Hz, J = 6.37 Hz), 6.09 (1H, d, J = 1.8 Hz),
7.27–7.40 (10H, m). 13C NMR (100 MHz, CDCl3): d 21.140 (12C),
62.54, 62.99, 65.36, 65.93, 66.00, 66.03, 66.07, 68.74, 68.76,
69.11, 69.15, 69.24, 69.51, 69.65, 70.12, 70.15, 71.02, 71.04,
72.12, 74.49, 74.97, 79.30 (22C), 91.02, 93.84, 99.04, 100.75,
128.19, 128.23 (2C), 128.38, 128.71, 128.74, 128.84 (2C), 128.92,
128.94, 138.03, 138.24, 168.49, 169.95, 170.02, 170.12, 170.16,
170.23, 170.37, 170.41, 170.91, 171.01, 171.07, 171.13 (12C,
C@O). m/z HRMS (EI) calcd for C62H78O33Na: 1373.4323 Found:
1373.3870 (M+Na)+.
The tetra-acetylmannoside 11 (1.74 g, 5 mmol) was dissolved in
DCM (30 ml) under an argon atmosphere. Propane-1,3-diyldioxy-
phosphoryl chloride (2.0 g, 12.5 mmol) was added at 0 °C with stir-
ring and after 5 min, N-methylimidazole (1.0 ml, 12.5 mmol) was
added at 0 °C. Stirring was continued at rt for 24 h under an argon
atmosphere. The solvent was removed at 35 °C in vacuo. The resi-
due was redissolved in DCM (30 ml) and washed with saturated
NaHCO3 (3 ꢁ 25 ml) and water (3 ꢁ 25 ml). The DCM layer was
dried over Na2SO4, filtered and the solvent removed was at 35 °C
in vacuo. The resultant residue was purified by flash chromatogra-
phy (elution with 4:1 ethyl acetate/petroleum ether), Yield = 2.2 g,
93%, colourless thick oil, Rf = 0.27. [a]
D = +33.5 (c 1.0, CHCl3). 1H
NMR (400 MHz), (CDCl3): d 1.86–1.90 (1H, m, JP–H = 15.1 Hz,
Hax-5), 2.02 (3H, s, CH3CO), 2.07 (3H, s, CH3CO), 2.10 (3H, s, CH3CO),
2.18 (3H, s, CH3CO), 2.29–2.33 (1H, m, JP–H = 15.1 Hz, Heq-5), 4.09–
4.24 (1H, m), 4.25–4.32 (1H, m), 4.30–4.44 (4H, m), 4.45–4.55 (2H,
m), 5.22–5.40 (2H, m), 5.71 (1H, d, J1,2 = 6.7 Hz, H-1). 13C NMR
(100 MHz), (CDCl3): d 20.43, 20.55, 20.58, 20.64 (4C, CH3CO),
25.78 (1C, JP–C = 7.6 Hz), 61.99, 65.36, 68.29, 68.70, 68.80, 69.25,
70.38, 94.80 (1C, C-1, JP–C = 5.0 Hz), 169.49, 169.60, 169.94,
170.60 (4C, C@O). 31P NMR (CDCl3): d ꢀ11.27. HRMS calcd for
C17H25O13PNa: 491.0930. Found: 491.0756 (M+Na)+.
4.1.4. 4,6-O-Benzylidene-1,2,3-tri-O-benzyl-b-D-manno-
pyranoside 14
The partially protected mannoside 13 (5.0 g, 18.6 mmol) was
added to DMF (50 ml) and the mixture was stirred at 0 °C for
30 min. NaH in mineral oil (3.0 g, 74.6 mmol) was then added un-
der argon in small portions over 30 min, with the temperature
being maintained at 0 °C. Tetrabutyl ammonium iodide (1.72 g,
4.66 mmol) was then added and the mixture was stirred for a fur-
ther 2 h at 0 °C. Benzyl bromide (9.0 ml, 74.6 mmol) was then
added slowly. The reaction mixture was stirred at 0 °C for a further
4.1.2. 2,3-Di-O-benzyl-a,b-D-mannopyranoside 5
The mannoside 8 (5.0 g, 10.8 mmol) was added to a solvent
mixture of H2O (60 ml): CH3CN (45 ml): TFA (45 ml). The system
was heated at 98 °C for 48 h. The solvents were removed in vacuo

N. K. Jalsa, G. Singh / Tetrahedron: Asymmetry 20 (2009) 867–874
873
30 min, and then was allowed to warm to room temperature and
97.13 (1C, JC1–H1 = 173.5 Hz, a), 92.04 (1C, JC1–H1 = 172.9 Hz, a),
was stirred for 24 h. Methanol was added slowly to destroy the ex-
cess NaH and the solvents were then removed in vacuo. The resi-
due was subjected to column chromatography; petroleum ether/
ethyl acetate (3:2) to furnish 14 as a pale yellow oil (8.1 g, 81%).
127.48, 127.56, 127.64, 127.80, 127.87, 127.93, 127.99, 128.02,
128.15, 128.21, 128.35, 128.37, 128.49, 128.51, 128.54 (15C, Ar),
137.17, 137.24, 138.49 (3C, Ar quat), 169.52, 169.64, 169.81,
169.83, 169.98, 170.01, 170.06, 170.19 (8C, C@O). HRMS calcd for
C55H66O24Na: 1133.3842. Found: 1133.3492 (M+Na)+.
Rf = 0.64. [
a
]
D = ꢀ14.3 (c 0.5, CHCl3) 1H NMR (600 MHz, CDCl3): d
3.33 (1H, appt.t, J = 4.8 Hz), 3.57 (1H, d, J = 9.4 Hz), 3.95 (2H, m),
4.24 (1H, t, J = 9.4 Hz), 4.35 (1H, pseudot, J = 4.8 Hz), 4.51 (1H, s,
H-1), 4.58 (2H, d, J = 12.3 Hz, PhCH2O–), 4.68 (1H, d, J = 12.4 Hz,
PhCHHO–), 4.89 (1H, d, J = 12.4 Hz, PhCHHO–), 5.00 (2H, d,
J = 12.6 Hz, PhCH2O–), 5.63 (1H, s, PhCHO2–), 7.23–7.50 (20H, m,
Ar); 13C NMR (150 MHz, CDCl3): d 67.63, 68.62, 71.09, 72.39,
74.77, 75.87, 76.82, 78.67 (8C), 100.93 (1C, JC1–H1 = 155.0 Hz, C-
1), 101.43 (PhCHO2–), 127.45, 127.51, 127.61, 127.72, 127.76,
127.80, 127.84, 127.95, 127.99, 128.02, 128.06, 128.10, 128.17,
128.22, 128.29, 128.32, 128.36, 128.42, 128.48, 128.53 (20C, Ar),
137.20, 137.59, 138.32, 138.39 (4C, Ar quat). HRMS calcd for
C34H35O6: 539.2434. Found: 539.2801 (M+H)+.
4.1.7. 20,30,40,60-Tetra-O-acetyl-
(200,300,400,600-tetra-O- -acetyl-mannopyranosyl-(100?4))-2,3-tri-
O-benzyl- ,b- -mannopyranose 17
a-D
-mannopyranosyl-(10?6)-
a-D
a
D
At first, Pd/Al2O3 (0.08 g) and ammonium formate (0.079 g,
1.25 mmol) were added to methanol (5 ml) and were stirred under
an argon atmosphere for 5 min. Compound 16 (0.09 g, 0.08 mmol)
was then added and the mixture was stirred at rt for 12 h. The sys-
tem was then filtered through a pad of Celite, washed with meth-
anol (20 ml) and the solvent was removed in vacuo. The residue
was purified by column chromatography (elution with3:2 petro-
leum ether/ethyl acetate) to yield 17 (0.058 g, 71%) as a colourless
oil, Rf = 0.05. [a]
(at equilibrium) = +95.2 (c 0.01, CHCl3). 1H NMR
D
4.1.5. 1,2,3-Tri-O-benzyl-b-
D
-mannopyranoside 15
(300 MHz, CDCl3) for b-anomer: d 1.97 (6H, s, 2CH3CO), 2.03 (6H,
s, 2CH3CO), 2.07 (6H, s, 2CH3CO), 2.14 (6H, s, 2CH3CO), 3.78 (1H,
add, J = 2.9 Hz, J = 9.2 Hz), 3.83–3.89 (3H, m), 3.92 (1H, ad,
J = 2.9 Hz), 3.95–4.21 (6H, m), 4.23–4.34 (1H, m), 4.42–4.50 (1H,
m), 4.53–4.63 (2H, m), 4.67 (1H, d, J = 2.7 Hz), 4.69–4.76 (1H, m),
4.89–4.97 (2H, m), 5.24 (1H, dd, J = 2.7 Hz, J = 9.5 Hz), 5.28–5.31
(3H, m), 5.33–5.38 (1H, m), 5.40 (1H, appt.dd, J = 1.6 Hz,
J = 3.5 Hz), 7.27–7.40 (10H, Ar); 13C NMR (75 MHz, CDCl3): d
20.85, 20.70 (8C, CH3CO), 62.38, 62.51, 65.96, 66.31, 66.83, 67.49,
68.02, 68.44, 68.76, 69.07, 69.20, 69.39, 69.65, 69.85, 71.37,
72.04, 79.34, 92.38, 94.19, 98.09 (20C), 127.71, 127.83, 127.88,
128.02, 128.10, 128.18, 128.38, 128.46, 128.54, 128.69 (10C, Ar),
137.89, 138.09 (2C, Ar quat), 169.80, 169.88, 170.01, 170.62,
170.85 (8C, C@O). HRMS calcd for C48H60O24Na: 1043.3372. Found:
1043.2567 (M+Na)+.
The protected mannoside 14 (7.0 g, 13.0 mmol) was added to
80% AcOH (100 ml) and heated to 85 °C for 3 h. After the system
was cooled, the solvent was removed in vacuo. The crude residue
was subjected to column chromatography; petroleum ether/ethyl
acetate (1:4) to furnish 15 as white crystals (5.3 g, 90%).
[
a]
D = ꢀ29.9 (c 0.34, CHCl3) Rf = 0.41. mp = 132.5–133.5 °C. 1H
NMR (600 MHz, CDCl3): d 2.26 (1H, br s, OH), 2.42 (1H, s, OH),
3.31 (2H, m), 3.85 (1H, m), 3.98 (3H, m), 4.26 (1H, d, J = 12.0 Hz,
PhCHHO–), 4.47 (1H, d, J = 11.4 Hz, PhCHHO–), 4.53 (1H, s, H-1),
4.63 (1H, d, J = 12.0 Hz, PhCHHO–), 4.80 (1H, d, J = 12.0 Hz,
PhCHHO–), 4.98 (2H, d, J = 12.6 Hz, PhCH2O–), 7.24–7.41 (15H, m,
Ar); 13C NMR (150 MHz, CDCl3): d 63.10, 67.50, 71.03, 71.22,
73.14, 74.18, 75.85, 81.58 (8C), 100.64 (1C, JC1–H1 = 157.0 Hz, C-
1), 127.62, 127.71, 127.83, 127.92, 127.99, 128.23, 128.38,
128.49, 128.56 (15C, Ar), 137.26, 137.49, 138.36 (3C, Ar quat).
HRMS calcd for C27H30O6: 450.2042. Found: 450.2043 (M+).
Acknowledgment
4.1.6. 20,30,40,60-Tetra-O-acetyl-
(200,300,400,600-tetra-O- -acetyl-mannopyranosyl-(100?4))-1,2,3-
tri-O-benzyl-b- -mannopyranoside 16
a-D
-mannopyranosyl-(10?6)-
We gratefully acknowledge financial support by the University
of the West Indies, St. Augustine Campus, Trinidad and Tobago.
a-D
D
Phosphate 3 (0.20 g, 0.44 mmol) was dissolved in DCM (10 ml)
at ꢀ78 °C under an argon atmosphere. TMSOTf (0.06 ml,
0.33 mmol) was then added and after 5 min, a solution of the gly-
cosyl acceptor 7 (0.1 g, 0.22 mmol) in DCM (5 ml) was added at
ꢀ78 °C. The reaction mixture was stirred for 30 min at ꢀ78 °C, at
0 °C for a further 30 min and then for 2 h at rt. The solvent was re-
moved in vacuo. The residue was redissolved in DCM (30 ml) and
washed with saturated NaHCO3 (3 ꢁ 25 ml) and then with water
(3 ꢁ 25 ml). The DCM layer was dried over Na2SO4, filtered and
the solvent was removed in vacuo to furnish crude 16. The residue
was purified by column chromatography (elution with 3:2 petro-
leum ether/ethyl acetate) to yield 16 (0.19 g, 77%) as a dark orange
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