Total synthesis of the fully lipidated glycosylphosphatidylinositol (GPI) anchor of malarial parasite Plasmodium falciparum

Article abstract of DOI:10.1016/j.tet.2010.04.014

We report a new and convergent strategy for the total synthesis of fully lipidated glycosylphosphatidylinositol (GPI) anchor, the major pro-inflammatory factor of malarial parasite (Plasmodium falciparum). The key features of our approach include, the acc

Full text of DOI:10.1016/j.tet.2010.04.014

Tetrahedron 66 (2010) 4357–4369
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Total synthesis of the fully lipidated glycosylphosphatidylinositol (GPI) anchor of
malarial parasite Plasmodium falciparum
,b,
Asif Ali a, Ram A. Vishwakarma a
*
a Bio-organic Chemistry Lab, National Institute of Immunology, Aruna Asaf Ali Marg, JNU Complex, New Delhi 110067, India
b Medicinal Chemistry Division, Indian Institute of Integrative Medicine (Council of Scientific & Industrial Research), Jammu 180001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We report a new and convergent strategy for the total synthesis of fully lipidated glycosylphos-
phatidylinositol (GPI) anchor, the major pro-inflammatory factor of malarial parasite (Plasmodium
falciparum). The key features of our approach include, the access to the key glucosamine–inositol
intermediate by a novel route without a priori resolution of myo-inositol, convergent assembly of the
tetramannose glycan domain, flexibility for the placement of the three fatty acids in the desired order in
the final steps, and the opportunity to construct GPI analogues/mimics to probe the biosynthesis,
immunology and cell biology of the GPI anchor pathway in the malaria parasite.
Received 19 March 2010
Received in revised form 1 April 2010
Accepted 2 April 2010
Available online 8 April 2010
Keywords:
Glycosylphosphatidylinositol
GPI anchor
Ó 2010 Elsevier Ltd. All rights reserved.
Malaria
Plasmodium falciparum
1. Introduction
Malaria affects 600 million people worldwide and kills 2 million
annually,1 mostly children in Africa and Asia, and most of the
mortalities are due to the cerebral malaria caused2 by Plasmodium
falciparum species. Furthermore, the parasite (P. falciparum) has
developed resistance against most of the currently used antima-
larial drugs and major global vaccine efforts have not succeeded.
Severe disease pathology of malarial infection (periodic fever and
cerebral malaria) and fatalities have been linked to the unique cell-
surface glycosylphosphatidylinositol (GPI) molecules (Fig. 1), the
so-called malaria toxins, that are expressed/released by P. falcipa-
rum during its blood stage life-cycle.3
A number of landmark studies have demonstrated that the P.
falciparum glycosylphosphatidylinositols (Pf-GPIs) trigger a major
pro-inflammatory cytokine cascade4–6 in the host and an anti-GPI
antibody was shown to provide protection7 to the host from the
disease. The unique chemical structure of GPI anchor of P. falcipa-
rum with three fatty acids in the lipid domain (Fig. 1), coupled with
its remarkable biosynthetic pathway and immunological mecha-
nism has generated worldwide interest as a potential therapeutic
target for anti-disease vaccine and drug design.
Figure 1. Structure of GPI anchor of P. falciparum.
intermediates from the malaria parasite culture, deciphering their
biosynthetic assembly and their role in disease pathology and im-
munology critically depends on the efficient methods for chemical
synthesis of Pf-GPIs and the biosynthetic intermediates. Two key
structural features that distinguish malarial-GPIs from that of other
parasitic species include; the presence of an extra fatty acid at 2-
position of the myo-inositol residue rendering Pf-GPIs resistant to
the host phosphatidylinositol-specific phospholipase C (PI-PLC)
mediated hydrolysis, and an additional fourth mannose at the
Since it is extremely difficult to isolate relevant amounts of
homogeneous and pure GPI molecules and their biosynthetic
* Corresponding author. Tel.: þ91 11 26703614; fax: þ91 11 26162125; e-mail
address: ravboc@gmail.com (R.A. Vishwakarma).
0040-4020/$ – see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2010.04.014

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A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
non-reducing end of the phosphoglycan domain. These features of
Pf-GPIs imparting remarkable (nM) pro-inflammatory activity4–6
(comparable to that of the bacterial lipopolysaccharide) present
substantial difficulty for synthesis.
product of this reaction on deacylation and 4,6-benzylidenation
provided clean separation of two diastereomeric pseudodi-
saccharides by a simple silica chromatography. This gave us the
confidence to extend this approach for the synthesis of 2-azido-2-
Although a number of approaches have been reported8–12 by
various leading groups for the synthesis of GPI anchors of various
parasitic species, including our own approach,13 the GPI molecules
of the malarial parasite present increasing challenges due to the
deoxyglycosyl-(1/6)-myo-D-inositol with an additional protecting
group at the 2-position of the myo-inositol residue for the place-
ment of the third fatty acid in the target Pf-GPI anchor. For this, the
first intermediate, racemic 1-O-PMB-2-O-allyl-3,4,5-tri-O-benzyl-
presence of a third fatty acid group at the 2-position of the myo-
D
-
(
DL)-myo-inositol (3), was prepared (Scheme 1) from the known25
inositol residue. For this reason, so far only one total synthesis of
fully lipidated Pf-GPI has been reported (Seeberger et al.).14 In
addition to this, a synthesis of a model GPI (lacking fourth mannose
and with short-chain fatty acid lipids) has been reported (Fraser-
Reid et al.).15 We have recently reviewed16 the previous work on
synthesis of GPI molecules of various species. Keeping in view the
remarkable biological role4–7 of Pf-GPIs in the malarial biology and
immunology, efficient methods for synthesis of Pf-GPIs and their
structural and functional mimics are critical for the further
advances in this field of glycobiology. We have had continuing
interest in the chemical biology of GPI molecules and related
phospholycans, including new synthesis,13,16–18 biosynthesis,19–22
and cell biology.23
1,2:4,5-bis-cyclohexylidene-(DL)-myo-inositol (2a). The 3-OH of
the starting material 2a was selectively protected by BaO/Ba(OH)2
mediated benzylation followed by the allylation of the remaining 6-
OH. Next three steps, acidic hydrolysis of the 4,5-cyclohexylidene
trans-ketal (p-TSA, CH2Cl2/CH3OH, 3:1, rt), benzylation, and hy-
drolysis of the 1,2 cis-ketal (p-TSA, 50 ꢀC) were carried out as de-
scribed by Brimacombe et al.25 to afford compound 2b.
2. Results and discussion
We now report a new and convergent total synthesis of the fully
lipidated GPI anchor of the malarial parasite P. falciparum. The key
features of our approach include: (a) access to the glucosamine–
inositol intermediate by a novel route without a priori resolution of
myo-inositol; (b) convergent assembly of the tetramannose glycan;
(c) flexibility to place three fatty acids in the desired order in the
final steps; (d) possibility to construct [4-deoxy-Man-III]-GPI
analogues. Such deoxy-GPI analogues may serve as valuable
conformational probes to address a fundamental biological ques-
tion relevant to the GPI biology: why the proteins are transferred24
to the terminal primary alcohol (6-OH) of the third mannose resi-
due irrespective to whether the nascent GPI precursor contains
either three mannoses as in Leishmania and human species or four
mannoses as in Trypanosoma and Plasmodium species. It needs
mention here that the GPI molecules isolated from the culture of
P. falciparum predominantly contain palmitate and stearate (or
their combinations) in their lipid domain. We have chosen in the
present study to synthesize the target Pf-GPI anchor with the
palmitate residues. However, the approach described in this paper
is suitable for the placement of other saturated and mixed fatty
acids as well.
Scheme 1. (a) Bu2SnO, CsF, KI, MeOH, PMBCl, DMF, rt, 24 h, 94%; (b) t-BuOK, DMSO,
80 ꢀC, 3 h, 85%; (c) Allyl Br, NaH, DMF, 3 h, 90%; (d) acetone/1 N HCl (9:1), 50 ꢀC, 0.5 h,
83%.
The 1-OH of 2b was selectively alkylated with PMB group using
dibutyltin chemistry to get 2c in excellent yield. Next step involved
the ‘half’ deprotection of an allyl group to a vinyl group under basic
condition (2c/2d) enabling the placement of an additional allyl
group at the 2-OH (2d/2e); this allyl group would eventually be
unmasked in the final steps of the synthesis to install the critical
third fatty acid characteristic of the Pf-GPI anchor. The removal of
the vinyl from the 6-OH of 2e under mild acidic condition provided
the desired myo-(DL)-inositol building block 3 in racemic form.
The racemic myo-inositol intermediate 3 was now glycosylated
(TMSOTf, CH2Cl2, ꢁ5 ꢀC) with known26 glycosyl donor (2-azidogly-
cosyl trichloroacetimidate, 4) providing the pseudodisaccharide 5 in
85% yield (Scheme 2) as a diastereoisomeric mixture containing
D
and
compound 3 proceeded with excellent
of 95:5) as established by detailed 1H and 13C NMR analysis. The
desired -isomeric product was purified by silica column chroma-
tography to remove 5% impurity of the -isomer. Moreover, the
L
myo-inositol residues. The glycosylation at the 6-OH of
To address the key issue of the placement of the third fatty acid
in the myo-inositol residue of the Pf-GPI, we have devised a new
way to assemble the optically pure glucosamine–inositol in-
termediate. Arguably, the most demanding aspect of the GPI syn-
thesis has been to access suitably protected glucosamine–inositol
a
stereoselectivity ( ratio
a/b
a
b
presence of an allyl group at the 2-OH position did not impact on the
anomeric selectivity at 6-OH. The next steps involved removal of
three acetyl groups (5/6) followed by reaction with benzaldehyde
dimethylacetal under acid catalysis, which provided diastereomeric
mixture of 4,6-cyclic acetals 7a and 7b. These diastereoisomers
showed excellent chromatographic separation, as observed13 pre-
viously by us in the case of their 2-O-benzylated counterparts. The
pseudodisaccharides 7a and 7b were isolated by silica column and
rigorously characterized by detailed 1H and 13C NMR analysis.
The benzylation of the free hydroxyl at the 3-position of both
isomers (7a/8a and 7b/8b) followed by the regioselective
cleavage of 4,6-benzylidene acetal of the corresponding 8a and 8b
by NaCNBH3 under acid catalysis provided desired 2-azido-2-
block requiring optically pure protected D-myo-inositol acceptor
and 2-azido-2-deoxyglucosyl donor. This has mainly been accom-
plished by previous workers8–12 either by the a priori resolution
of bis-cyclohexylidene-myo-inositols by chiral auxiliaries and
enzymes or through a multi-step synthesis from
Ferrier reaction.
D-glucose by the
We addressed this issue differently and instead of resorting to
a priori resolution of myo-inositol, we reasoned based on structural
modeling that if sufficient strain is built through a cyclic benzyli-
dene protecting group, the 2-deoxy-2-azidoglycosyl residue itself
could function as an efficient chiral auxiliary, making a number of
early resolution steps redundant. This methodology was success-
fully tested, as reported in our preliminary communication13 with
the glycosylation of racemic 1-O-PMB-2,3,4,5-tetra-O-benzyl-myo-
inositol with 2-azido-2-deoxyglycosyl trichloroacetimidate. The
deoxyglucopyranosyl-(1/6)-myo-
natural counterpart 2-azido-2-deoxyglucopyranosyl-(1/6)-myo-
-inositol 9b in excellent yields.
D-inositol 9a and its non-
L

A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
4359
Scheme 2. (a) TMSOTf, CH2Cl2, 4 Å MS, ꢁ5 ꢀC, 0.5 h, 85%; (b) NaOMe, CH2Cl2/CH3OH
(1:3), 2 h, rt, 91%; (c) PhCH(OMe)2, CSA, CH3CN, rt, 24 h, 79%; (d) BnBr, NaH, DMF, rt,
4 h, 98%; (e) NaBH3CN, HCl/Et2O, 0 ꢀC, 0.5 h, 94%.
Scheme 4. (a) BnBr, NaH, DMF, 24 h, 83%; (b) t-BuOK, DMSO, 80 ꢀC, 2 h, acetone/1 M
HCl (9:1), 40 ꢀC, 0.5 h, 95%; (c) CCl3CN, CH2Cl2, DBU, rt, 1.5 h, 98%; (d) PhCH(OMe)2,
CSA, CH3CN, rt, 18 h, 74%; (e) BnBr, NaH, DMF, 0 ꢀC, 65%; (f) TMSOTf, Et2O, rt, 2 h, 81%;
(g) t-BuOK, DMSO, 80 ꢀC, 3 h, acetone/1 M HCl (9:1), 55 ꢀC, 2 h, 82%; (h) Ac2O, pyridine,
rt, 24 h, 93%; (i) Me2NH, CH3CN, ꢁ20 ꢀC, 1 h, 94%; (j) CCl3CN, CH2Cl2, DBU, rt, 1 h, 96%.
To confirm the absolute stereochemistry of 7a/8a/9a series,
compound 8a was transformed (Scheme 3) to a previously
reported27 compound, (2-azido-3-O-benzyl-4,6-di-O-benzylidene-
2-deoxy-
a
-D
-glucopyranosyl)-(1-6)-2,3,4,5-tetra-O-benzyl-1-O-(4-
-myo-inositol (9c), by replacing 2-O-allyl of 8a
methoxybenzyl)-
D
Scheme 3. (a) t-BuOK, DMSO, 80 ꢀC, 3 h, HgCl2, HgO, acetone/H2O (3:1), 10 min, rt, 74%; (b) BnBr, NaH, DMF, rt, 3 h, 92%; (c) NaBH3CN, HCl/Et2O, 0 ꢀC, 30 min, 74%.
with a benzyl group. Compound 9c showed all the spectral data
and [
D value identical to that reported27 for this compound by an
alternative route. Furthermore, compound 9c on regiospecific
opening of the benzylidene ring afforded (2-azido-3,6-di-O-benzyl-
The simultaneous removal of anomeric allyl and 4,6-benzylidine
groups (t-BuOK, DMSO, 80 ꢀC; 1 M HCl/acetone, 1:9 v/v) from 16
provided the triol 17, and no unwanted deprotection of benzyloxy
groups was observed under the acidic conditions (1 M HCl/acetone;
1:9 v/v). The per-acetylation (17/18), selective removal of
anomeric acetyl group by freshly prepared dimethylamine solution
in anhyd acetonitrile at ꢁ20 ꢀC provided compound 19 with free
anomeric hydroxyl group. The glycosyl activation of compound 19
by Schmidt method (CCl3CN, DBU) provided desired mannobiose
donor 20. Notably, two acetyl groups were placed strategically at 4-
and 6-OH to open the avenue for the synthesis of [4-deoxy-Man-
III]-GPI probes.
a]
2-deoxy-
(4-methoxybenzyl)-
the reported27 compound.
After the efficient access to the optically pure key 2-azido-2-
deoxyglucopyranosyl-(1/6)-myo- -inositol 9a in practical yields,
a
-
D
-glucopyranosyl)-(1/6)-2,3,4,5-tetra-O-benzyl-1-O-
D
-myo-inositol (9d) with [ matching with
a]
D
D
a convergent [2þ2] approach for synthesis of tetramannose glycan
29 was designed from two suitably protected mannobiosides, the
activated donor 20 and the acceptor 26 (Schemes 4 and 5,
respectively). Compound 20 was prepared by the coupling of
The mannobiose intermediate 26 was synthesized (Scheme 5) by
tetra-O-benzyl-
a
-
D
-mannosyl trichloroacetimidate28 (13, prepared
glycosylation of allyl-2,3,4-tri-O-benzyl-
a-D-mannopyranoside (24)
from known29 allyl mannopyranoside 10 by perbenzylation, allyl
(prepared from
D
-mannose by anomeric allylation29 (10), selective
deprotection, and trichloroacetimidation) with allyl-3-O-benzyl-
tritylation at the 6-OH, and perbenzylation followed by the removal
of trityl group) with known31 pentenyl mannose orthoester (22).
The glycosylation of 24 with 22 went cleanly using triethylsi-
lyltriflate (TESTOf) and N-iodosuccinimide (NIS)-mediated condi-
tion, which provided the desired disaccharide 25 in excellent yield
and isomeric purity. The stereochemistry of new glycoside bond in
25 was confirmed by NMR (dH 5.19, J¼1.9 Hz; dC 96.83, COSY and
4,6-O-benzylidene-a-D-mannoside (15, prepared from 10 by
4,6-benzylidenation,30 and benzylation27 at 3-OH). The above gly-
cosylation (TMSOTf, Et2O, rt, 2 h) went smoothly to afford 16 and
major
a anomer was isolated by silica column chromatography, the
stereochemistry of new glycoside bond ascertained by NMR (dH
5.41, br s; dC 98.23, COSY and HSQC).

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A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
NMR analysis. This was followed by the deprotection of the anomeric
allyl group (PdCl2, AcOH) providing compound 28. The activation of
the free anomeric hydroxyl by the Schmidt method afforded the
tetramannose trichloroacetimidate donor 29.
Now, the above tetramannose donor 29 was reacted with 2-
azido-2-deoxy glycosyl-(1/6)-myo-D-inositol acceptor (9a). To our
satisfaction, this important glycosylation went smoothly (TMSOTf,
CH2Cl2, 0 ꢀC) with acceptable stereocontrol (80:20 anomeric ratio of
a
and
b
isomers), the desired
a
isomer pseudohexasaccharide 30 was
-isomer dis-
isolated by silica chromatography and the undesired
b
carded. The stereochemistry of four mannosyl residues (dH 5.26, 5.28,
5.37, 5.38) and azidoglycosyl residue (dH 5.80, J¼3.8 Hz) was
determined by NMR analysis. The two acetyl groups from the third
mannose residue of 30 were now removed to expose two hydroxyls
in compound 31, which could be easily purified. Selective protection
of primary 6-OH with TBDPS group went smoothly (TBDPSCl,
imidazole, THF, 5 h, 89%) and the remaining 4-OH was blocked with
careful benzylation (BnBr, NaH, DMF, 0 ꢀC, 1 h) to afford pseudo-
hexasaccharide 32 in 97% yield. Now the removal of PMB group from
the 1-position of inositol residue by ceric ammonium nitrate pro-
vided fully functional pseudohexasaccharide 33, ready for phos-
phorylation with the required glycerolipid domain.
Initially we made several attempts at phosphorylation of
hexasaccharide-glycan 33 with freshly prepared 1,2-dipalmitoyl-
sn-glycero-H-phosphonate and 1,2-disteroyl-sn-glycero-H-phos-
phonate and their phophoroamidite counterparts (as reported in all
previous syntheses of GPI anchors) but failed to obtain desired
phosphorylation at 1-position of the myo-inositol residue of ac-
ceptor 33; the reaction being too sluggish and gave very poor
yields. Therefore, we changed our approach and decided to attempt
a novel method of using simpler and known32 intermediate, 1,2-
isopropylidene-sn-glycero-H-phosphonate (34), for placing first the
phosphoglycerol moiety at the 1-position with the approach that
the required fatty acids could be brought at the later stage after
simultaneous hydrolysis of 1,2-isopropylidene acetal along with the
allyl group from 2-position of the myo-inositol residue. Luckily for
us, this approach (Scheme 7) worked very nicely and provided us
greater flexibility in linking fatty acids.
Scheme 5. (a) (i) BzCl, pyridine, rt, 24 h; 30% HBr/AcOH, CH2Cl2, 0 ꢀC, 12 h; (ii) 4-
pentenol, 2,6-lutidine, CH2Cl2, 60 h, 82%; (b) (i) NaOMe, CH2Cl2/CH3OH, 2 h; (ii) BnBr,
NaH, DMF, rt, 24 h, 93%; (c) (i) allyl alcohol, BF3$Et2O, 80 ꢀC, 4 h; (ii) TrCl, pyridine,
80 ꢀC, 16 h, 70%; (iii) BnBr, NaH, DMF, 4 h, 89%; (d) p-TSA, CH2Cl2/CH3OH, rt, 12 h, 93%;
(e) TESOTf, NIS, CH2Cl2, 4 Å MS, rt, 0.5 h, 72%; (f) NaOMe, CH2Cl2/CH3OH, rt, 3 h, 92%.
HSQC). Finally, the lone benzoyl group from the 2-position of the
upper mannose residue was easily removed (NaOMe/MeOH) to
expose free 2-OH in compound 26.
The glycosylation of the mannobiose donor 20 with the man-
nobioseacceptor26(Scheme6)requiredconsiderableefforts. Various
glycosylation conditions (temperature variation, solvents, and cata-
lysts such as BF3$Et2O, triflic acid, TBDMOSTf, etc.) failed to provide
the desired product. Finally, the optimum yield (69%) of the desired
Accordingly, the glycan 33 was phosphorylated with the freshly
prepared 1,2-isopropylidene-sn-glycero-H-phosphonate (34)32 to af-
ford compound 35 in excellent yield. The glycero-1,2-acetonide and
allyl groups were now simultaneously deprotected using palladium
a
-anomer of the tetrasaccharide (27) was obtained using TMSOTf at
0 ꢀC inanhyd Et2O for 0.5 hand freshlyactivated powderedmolecular
sieves (4 Å), the stereochemistry of new glycoside bond confirmed by
Scheme 6. (a) TMSOTf, Et2O, 4 Å MS, 0 ꢀC, 0.5 h, 69%; (b) PdCl2, NaOAc, AcOH/H2O, 19:1, 16 h, 91%; (c) CCl3CN, CH2Cl2, DBU, rt, 98%; (d) TMSOTf, Et2O, 4 Å MS, 0 ꢀC, 0.5 h, 53%; (e)
NaOMe, CH3OH/CH2Cl2 (3:1), rt, 1 h, 96%; (f) (i) TBDPSCl, imidazole, THF, 5 h, 89%; (ii) BnBr, NaH, DMF, 0 ꢀC, 1 h, 97%; (g) CAN, CH3CN/Tol/H2O (9:5:4), 1.5 h, 77%.

A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
4361
Scheme 7. (a) 1,2-Isopropylidene-sn-glycerol-H-phosphonate, pivaloyl chloride, pyridine, I2, pyridine/H2O (19:1), 94%; (b) PdCl2, NaOAc, AcOH/H2O, 19:1, 16 h, rt, 88%; (c) CdCl2,
palmitic anhydride, DMAP, rt, 18 h, 90%; (d) palmitic acid, DCC, DMAP, rt, 48 h, 80%.
chloride in acetic acid exposing three OH groups in intermediate 36,
and this turned out to be rather clean and robust reaction. Initial at-
tempts to place all the three fatty acids together by standard DCC/
DMAP condition failed even after prolonged (5 days) reaction. After
attempting several acylation conditions on 36, we discovered that the
activation of charged phosphoryl group as cadmium chloride com-
plex followed by reaction with palmitic anhydride cleanly provided
a di-acylated product 37 in very high yield; the idea inspired by the
seminal work33 of Khorana on synthesis of glycerolipid analogues.
Having two fatty acids in place at required 1,2-glycero positions, the
third fatty acid at 2-OH of the inositol residue could now be easily
placed by standard DCC/DMAP conditionproviding fullylipidated GPI
(38). This selectivity provides an interesting and novel method to
synthesize Pf-GPIs with mixed fatty acids, critical for decoding their
role in remarkable pro-inflammatory activity.
In the final steps, the TBDPS group was removed from the third
mannose of compound 38 (Scheme 8) and the exposed primary OH
was phosphorylated with freshly prepared34 Cbz-NH-ethanol-
amine-H-phosphonate (40) in presence of pivaloyl chloride fol-
lowed by in situ oxidation with iodine to get fully protected Pf-GPI
anchor (41). Final global deprotection (17 benzyls, 1 Cbz group, and
azide reduction) by hydrogenolysis turned out to be most de-
manding. Eventually it was accomplished by the method reported14
by Seeberger using hydrogenolysis (Pd(OH)2, DCM/MeOH/H2O, H2)
with slightly acidic pH, which provided the fully deprotected target
malarial GPI anchor (1) of P. falciparum. The target Pf-GPI anchor
was fully characterized by 1H and 31P NMR and ESMS analysis. The
synthetic approach enabled us to initiate chemical and biological
studies to decipher the role of the extra fatty acid in pathophysio-
logy of the malaria infection.
3. Conclusion
We have designed a new total synthesis of the full-length and fully
lipidated GPI anchor of malarial parasite (P. falciparum). This conver-
gent synthesis provides access to key building blocks and flexibility to
bring in three fatty acids in later steps in an efficient manner.
Currently, we are exploiting this strategy to synthesize GPI analogues
and fluorescent labeled probes to address key issues related to the
biosynthetic pathway, cell biology, and membrane biology of this
important class of cell-surface glycoconjugates. The immunological
studies using this synthetic Pf-GPI will be reported in due course.
4. Experimental
4.1. General procedure
Solvents were purified according to the standard procedures,
and reagents used were of highest purity available. All reactions
were performed in flame-dried glass apparatus under argon

4362
A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
Scheme 8. (a) 1 M TBAF, THF, rt, 18 h, 85%; (b) Cbz-ethanolamine-H-phosphonate 40, Piv Cl, pyridine, iodine, pyridine/H2O (95:5), rt, 6 h, 83%; (c) H2, 20% Pd(OH)2, CHCl3, CH3OH,
H2O, rt, 18 h, 73%.
atmosphere unless mentioned otherwise. Anhydrous solvents like
CH2Cl2, Et2O, THF, CH3OH, CH3CN, DMF, pyridine, Et3N were freshly
dried using standard methods. NMR measurements (1H, 13C, 31P, 2D
1H–1H and 1H–13C COSY, HMQC) were recorded on a 300 MHz
spectrometer (Bruker) fitted with pulse-field gradient probe, and
trimethylsilane (TMS) or residual resonance of deuterated solvent
were used as internal reference. For 31P NMR spectra, phosphoric
acid was used as external reference. 13C NMR spectra were broad-
band 1H decoupled or inverse HMQC experiments. Chemical shifts
are expressed in parts per million and coupling constants J in hertz.
Mass spectra (ESI) and high-resolution mass spectra (HRMS) were
obtained on quadrupole and LCT-TOF (time of flight) spectrometers,
respectively, using acetonitrile/water (1:1) mobile phase. Optical
rotations were measured on a digital Perkin–Elmer 241 polarime-
ter. Analytical TLC was performed on Merck Kieselgel 60 F254 plates,
and compounds visualized by ammonium molybdate/ceric sulfate
developing reagent. Preparative TLC was conducted on Analtech
Uniplate silica gel plates (20ꢂ20 cm). Silica column chromatogra-
phy was carried out with silica gel 60 (60–120 mesh) or flash silica
gel (230–400 mesh). Analytical and semi-preparative HPLC purifi-
cation were carried out on a Shimadzu system using RP-18 columns
and a photodiode array detector.
83.15, 113.85, 113.97, 116.63, 127.59–130.12 (multiple peaks), 135.33,
137.97,138.70,138.78,159.35; MS (positive ion ESMS, MþNaþ) calcd
for C38H42O7Na: 633.2828, found 633.2751.
4.3. 6-O-Vinyl-3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-(DL)-
myo-inositol (2d)
Compound 2c (4.42 g, 7.3 mmol) was dissolved in anhyd DMSO
(74 ml) and treated with potassium tert-butoxide (11.76 g). The
reaction mixture was heated at 80 ꢀC after 3 h, poured into ice-cold
water, and extracted with EtOAc. The organic layer was washed
with brine and water, dried (Na2SO4), and concentrated. The resi-
due after silica column purification provided desired vinyl com-
pound 2d (3.75 g, 85%) as white solid. TLC (hexane/EtOAc, 7:3):
Rf¼0.34. 1H NMR (300 MHz, CDCl3): 2.40 (d, 3H), 3.35 (dd, J¼2.4 and
9.6 Hz, 1H), 3.38 (t, J¼9.5 Hz, 1H), 3.81 (s, 3H), 3.82 (dd, 1H), 4.05
(dd, 1H), 4.15 (m, 1H), 4.56–4.90 (m, 9H), 6.25 (d, 1H), 6.87 (m, 2H),
7.25–7.36 (m, 17H); 13C NMR (75 MHz, CDCl3): 55.30, 68.03, 72.57,
72.70, 75.82, 75.93, 77.22, 79.44, 80.76, 82.31, 84.08, 98.38, 113.81,
127.56–129.96 (multiple peaks), 137.91, 138.40, 138.80, 147.66,
159.37; MS (positive ion ESMS, MþNaþ) calcd for C38H42O7Na:
633.2828, found 633.2833.
4.2. 6-O-Allyl-3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-(DL)-
myo-inositol (2c)
4.4. 2-O-Allyl-6-O-vinyl-3,4,5-tri-O-benzyl-1-O-(4-
methoxybenzyl)-(DL)-myo-inositol (2e)
A solution of compound 2b25 (5.11 g, 10.4 mmol) and dibutyltin
oxide (3.70 g, 15 mmol) in anhyd CH3OH (280 mL) was heated to
reflux for 4 h. Excess of solvent was removed and the residue was
dried by repeated evaporation through toluene. The residue was
dissolved in anhyd DMF (300 mL) followed by the addition of pre-
dried CsF (2.23 g), KI (2.34 g), and PMBCl (1.99 mL, 15.6 mmol). The
suspension was vigorously stirred for 24 h at rt. The solvent was
removed under reduced pressure and the residue was purified by
silica column chromatography (85:15 to 80:20 hexane/EtOAc) to
provide 2c (5.9 g, 94%) as white solid. TLC (hexane/EtOAc, 7:3):
Rf¼0.32. 1H NMR (300 MHz, CDCl3): 2.40 (s, 1H), 3.30 (dd, J¼2.4 and
9.5 Hz, 1H), 3.35 (dd, J¼2.4 and 9.5 Hz, 1H), 3.38 (t, J¼9.5 Hz, 1H),
3.81 (s, 3H), 3.82 (dd, 1H), 3.94 (dd, 1H), 4.15 (m, 1H), 4.35 (m, 2H),
4.56–4.90 (m, 8H), 5.14–5.28 (m, 2H), 6.00 (m, 1H), 6.86–6.89 (m,
2H), 7.27–7.40 (m, 17H); 13C NMR (75 MHz, CDCl3): 55.30, 67.70,
67.71, 72.47, 72.70, 74.58, 75.93, 75.97, 79.26, 79.74, 80.89, 81.10,
Compound 2d (4.43 g, 7.3 mmol) was dissolved in anhyd DMF
(62 mL) and solution was brought to 0 ꢀC and then NaH (0.77 g,
33 mmol) and allyl bromide (0.77 ml) were added. The reaction
mixture was stirred for 3 h at rt and brine (500 ml) was added,
and extracted with EtOAc (150 mlꢂ3). The organic extract was
dried over anhyd Na2SO4, concentrated followed by column puri-
fication to provide 2e (4.2 g, 90%) as white solid. 1H NMR
(300 MHz, CDCl3): 2.07 (d, 3H), 3.25 (dd, J¼2.4 and 9.6 Hz, 1H),
3.33 (t, J¼9.5 Hz, 1H), 3.83 (s, 3H), 3.90 (br s, 1H), 4.05 (dd, 1H),
4.15 (m, 1H), 4.35 (d, 1H), 4.48 (dd, 1H), 4.56–4.90 (m, 9H), 5.14–
5.28 (m, 2H), 5.99 (m, 1H), 6.25 (d, 1H), 6.87 (m, 2H), 7.25–7.36 (m,
17H); 13C NMR (75 MHz, CDCl3): 55.28, 70.88, 72.45, 72.73, 73.39,
74.11, 75.75, 75.80, 80.41, 81.30, 82.88, 84.43, 98.06, 113.73, 116.80,
127.46–130.29, 135.72, 138.28, 138.56, 138.97, 147.77, 159.20
(multiple peaks); MS (positive ion ESMS, MþNaþ) calcd for
C41H46O7Na: 673.3141, found 673.3240.

A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
4363
4.5. 2-O-Allyl-3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-(DL)-
myo-inositol (3)
(m, 2H), 4.40–5.20 (m, 10H), 4.92 (m, 1H), 5.40–5.43 (m, 1H), 5.64
(d, J¼3.6 Hz, 0.5H), 5.79 (d, J¼3.6 Hz, 0.5H), 6.00 (m, 1H), 6.82–6.86
(m, 2H, Ph), 7.23–7.45 (m, 17H); 13C NMR (75 MHz, CDCl3): 55.29,
60.89, 61.61, 66.63, 71.43, 71.89, 72.76, 72.88, 73.51, 73.54, 75.06,
75.46, 75.74, 79.69, 80.69, 81.47, 81.58, 81.91, 97.46, 97.16, 113.83,
116.59, 127.47–129.78 (multiple peaks), 138.09–138.81 (multiple
peaks), 159.39; MS (ESMS, MþNaþ) calcd for C44H51O11N3Na
820.3421, found 820.3395.
Compound 2e (4.2 g, 6.46 mmol) was dissolved in a solution of
1 M HCl/acetone (1:9, 50 ml) and kept at 50 ꢀC for 30 min, neu-
tralized with triethylamine, and concentrated. The residue was
purified by silica column (hexane/EtOAc, 95:05 to 80:20) afforded
3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-2-O-allyl-(DL)-myo-ino-
sitol 3 (2.4 g, 83%) as white solid. TLC (hexane/EtOAc, 7:3):
Rf¼0.34. 1H NMR (300 MHz, CDCl3): 2.59 (s, 1H, OH), 3.15 (dd,
J¼1.8 and 9.8 Hz, 1H, 3-H), 3.35 (dd, J¼9.8 Hz, 1H, 5-H), 3.37 (dd,
J¼2.0 and 9.6 Hz, 1H, 1-H), 3.79 (s, 3H, OMe), 4.10 (br s, 1H), 4.08
(dd, J¼9.8 Hz, 1H), 4.14 (m, 1H, 6-H), 4.42–4.92 (m, 10H, CH2Ph),
5.14–5.28 (m, 2H), 5.99 (m, 1H), 6.84–6.88 (m, 2H), 7.20–7.40 (m,
18H); 13C NMR (75 MHz, CDCl3): 55.31, 71.96, 72.77, 72.86, 73.25,
73.35, 75.34, 75.82, 76.63, 79.66, 80.90, 81.42, 83.42, 113.96, 116.76,
127.32–129.96 (multiple peaks), 135.64, 138.30, 138.85, 138.89,
159.40; MS (positive ion ESMS, MþNaþ) calcd for C38H42O7Na
633.2828, found 633.2990.
4.8. (2-Azido-4,6-di-O-benzylidene-2-deoxy-
glucopyranosyl)-(1/6)-3,4,5-tri-O-benzyl-1-O-(4-
methoxybenzyl)-2-O-allyl- -myo-inositol (7a) and other
a-D-
D
diastereoisomer (2-azido-4,6-di-O-benzylidene-2-deoxy-
a-D-
glucopyranosyl)-(1/6)-3,4,5-tri-O-benzyl-1-O-(4-
methoxybenzyl)-2-O-allyl-L-myo-inositol (7b)
This intermediate 6 (1.0 g, 1.25 mmol) was dissolved in anhyd
CH3CN (30 mL) followed by addition of benzaldehyde dimethyl-
acetal (1.5 mL, 9.86 mmol) and camphorsulphonic acid (220 mg,
0.94 mmol). The reaction mixture was stirred for 24 h at rt, neu-
tralized with Et3N, and concentrated. The residue was diluted with
EtOAc, washed with saturated NaHCO3 solution, brine, and H2O.
Organic layer was dried (Na2SO4), concentrated, and purified on
a flash silica column (hexane/EtOAc, 85:15) provided desired
4.6. (3,4,6-Tri-O-acetyl-2-azido-2-deoxy-a-D-glucopyranosyl)-
(1/6)-3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-2-O-allyl-
(DL)-myo-inositol (5)
A mixture of 3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-2-O-
D-inositol isomer 7a (400 mg, 36%) as white solid. TLC (hexane/
allyl-(DL)-myo-inositol acceptor 3 (1.0 g, 1.63 mmol) and 3,4,6-tri-O-
acetyl-2-azido-2-deoxy- -glucopyranosyl trichloroacetimidate
(4, 1.1 g, 2.31 mmol, 1.41 equiv, prepared from tri-O-acetyl- -glucal
EtOAc, 7:3): Rf¼0.32; [
a
]
D
þ52 (c 1.0, CHCl3). 1H NMR (300 MHz,
b-
D
CDCl3): 3.10–3.14 (dd, J¼3.9 and 10 Hz, 1H), 3.38 (m, 1H), 3.40–3.46
(m, 4H), 3.82 (s, 3H), 3.96 (br s, 1H), 4.17–4.27 (m, 4H), 4.28 (dd,
J¼9.3 Hz, 1H), 4.45 (br s, 2H), 4.54–4.95 (m, 8H), 5.14–5.28 (m, 2H),
5.41 (s, 1H), 5.75 (d, J¼3.9 Hz, 1H), 6.00 (m, 1H), 6.88–6.91 (m, 2H),
7.19–7.34 (m, 22H); 13C NMR (75 MHz, CDCl3): 55.21, 61.93, 68.51,
72.72, 72.73, 73.44, 74.98, 76.53, 76.96, 80.63, 81.23, 81.68, 81.84,
81.91, 97.75, 101.85, 113.83, 116.73, 126.38, 126.39–129.68 (multiple
peaks), 135.66, 138.16, 138.50, 138.80, 159.34; HRMS (ESMS,
MþNaþ) calcd for C51H55O11N3Na 908.3734, found 908.3790; and
D
by a reported11 azidonitration method), and freshly activated
powdered 4 Å molecular sieves was dried by azeotropic removal of
residual moisture through toluene. The mixture was dissolved in
anhyd CH2Cl2 (20 mL), stirred under argon at rt for 0.5 h, and then
cooled to ꢁ5 ꢀC. To the above suspension was added a solution of
TMSOTf (1.1 mL, 0.2 M solution in CH2Cl2) drop wise and the mix-
ture was stirred further for 0.5 h at ꢁ5 ꢀC. After completion, the
reaction mixture was neutralized with triethylamine, filtered
through Celite, and concentrated. The silica column chromatogra-
phy (hexane/EtOAc, 3:1) provided the product 5 (1.8 g, 85%,
diastereoisomeric mixture) as colorless solid. TLC (hexane/EtOAc,
7:3): Rf¼0.3. 1H NMR (300 MHz, CDCl3): 1.83–2.07 (3ꢂs, 9H, OAc),
3.11–3.15 (m, 1H), 3.39–3.42 (m, 2H), 3.44–3.46 (dd, J¼9.6 Hz, 1H),
3.47–3.50 (dd, J¼1.8 and 9.5 Hz, 1H), 3.60–3.63 (m, 2H), 3.82 (s, 3H,
OCH3), 4.06 (s, 1H), 4.13–4.14 (dd, J¼9.5 Hz, 1H), 4.20–4.33 (m, 2H),
4.40–5.18 (m, 10H), 4.92 (m, 1H), 5.40–5.43 (m, 1H), 5.64 (d,
J¼3.6 Hz, 0.5H), 5.79 (d, J¼3.6 Hz, 0.5H), 6.00 (m, 1H), 6.84–6.87 (m,
2H, Ph), 7.22–7.43 (m, 18H); 13C NMR (75 MHz, CDCl3): 20.54, 20.58,
20.63, 55.19, 60.89, 61.17, 66.73, 67.99, 68.99, 68.99, 70.57, 71.62,
71.66, 71.84, 72.77, 72.85, 73.61, 73.97, 75.04, 75.23, 75.69, 79.69,
80.74, 81.04, 81.33, 81.96, 83.41, 83.97, 97.35, 97.16, 113.83, 127.72–
129.29 (multiple peaks), 138.09–138.81 (multiple peaks), 159.37,
169.48, 169.98, 170.64; MS (ESMS, MþNaþ) calcd for
C50H57O14N3Na 946.3738, found 946.3687.
the
Rf¼0.39; [
L-inositol isomer 7b (480 mg, 43%). TLC (hexane/EtOAc, 7:3):
a]
þ38 (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): 3.15
D
(dd, J¼3.9 and 9.8 Hz, 1H), 3.34 (dd, J¼1.8 and 9.8 Hz, 1H), 3.39
(dd, J¼2.1 and 9.6 Hz, 1H), 3.45 (t, J¼9.3 Hz, 1H), 3.71 (dd, J¼10 Hz,
2H), 3.83 (s, 3H), 3.86 (t, J¼5 Hz, 1H), 3.94 (br s, 1H), 4.10
(dd, J¼9.6 Hz, 2H), 4.17–4.28 (m, 2H), 4.33 (t, J¼9.6 Hz, 1H), 4.53–
5.48 (m, 10H), 5.11–5.30 (m, 2H), 5.48 (s, 1H), 5.59 (d, J¼3.9 Hz, 1H),
6.00 (m, 1H), 6.79–6.82 (d, 2H), 7.25–7.40 (m, 22H); 13C NMR
(75 MHz, CDCl3): 55.11, 61.61, 63.00, 68.84, 72.59, 73.36, 74.71,
74.65, 76.53, 76.96, 77.38, 80.47, 81.75, 82.02, 84.26, 97.77, 101.85,
113.90, 116.39, 126.26–129.70 (multiple peaks),135.60, 138.46,
138.59, 159.25; HRMS (ESMS, MþNaþ) calcd for C51H55O11N3Na
908.3734, found 908.3883.
4.9. (2-Azido-3-O-benzyl-4,6-di-O-benzylidene-2-deoxy-
a-
D-glucopyranosyl)-(1/6)-3,4,5-tri-O-benzyl-1-
O-(4-methoxybenzyl)-2-O-allyl- -myo-inositol (8a)
D
4.7. (2-Azido-2-deoxy-
benzyl-1-O-(4-methoxybenzyl)-2-O-allyl-(DL)-myo-inositol (6)
a
-
D
-glucopyranosyl)-(1/6)-3,4,5-tri-O-
Compound 7a (350 mg, 0.39 mmol) was dissolved in anhyd DMF
(6 mL) followed by the addition of benzyl bromide (0.16 mL,
1.3 mmol) and NaH (0.12 g, 5 mmol) at 0 ꢀC. After 30 min, the
reaction mixture was brought to rt and stirred for 4 h. After com-
pletion, the excess of NaH was destroyed by addition of a few drops
of methanol. The mixture was diluted with excess of EtOAc, which
was washed with saturated solution of NaHCO3, water, dried over
Na2SO4, and concentrated. Silica column chromatography (hexane/
EtOAc, 9:1) afforded 8a (380 mg, 98%), as colorless syrup. TLC
The above pseudodisaccharide (5, 1.2 g, 1.3 mmol) dissolved in
a solvent mixture of anhyd CH2Cl2 (10 mL) and CH3OH (30 mL) and
added sodium methoxide (0.8 g, 14.8 mmol) and the reaction
mixture was stirred for 2 h at rt. After completion of reaction, it was
neutralized with cation exchange resin (Amberlite IR 120Hþ),
filtered, and concentrated to give (2-azido-2-deoxy-a-D-glucopyr-
anosyl)-(1/6)-3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-2-O-al-
lyl-(DL)-myo-inositol (6) in 91% yield as colorless solid. 1H NMR
(300 MHz, CDCl3): 3.00 (m, 1H), 3.33–3.42 (m, 2H), 3.44–3.63 (m,
4H), 3.80 (s, 3H, OCH3), 3.94 (br s, 1H), 4.01–4.14 (m, 1H), 4.20–4.33
(hexane/EtOAc, 8:2): Rf¼0.46; [
a]
þ48 (c 1.0, CHCl3). 1H NMR
D
(300 MHz, CDCl3): 3.27 (dd, J¼3.8 and 10.0 Hz, 1H), 3.39 (dd, J¼2.2
and 9.8 Hz, 1H), 3.45 (dd, J¼2.2 and 9.8 Hz, 1H), 3.46 (dd, J¼9.3 Hz,
1H), 3.63 (m, 2H), 3.82 (s, 3H, OCH3), 3.96–4.04 (m, 2H), 4.06–4.29

4364
A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
(m, 4H), 4.57–4.96 (m, 10H), 5.15–5.25 (m, 2H), 5.49 (s, 1H), 5.72 (d,
J¼3.8 Hz, 1H), 6.00 (m, 1H), 6.91–6.89 (m, 2H), 7.14 (m, 2H), 7.25–
7.37 (m, 25H, Ph); 13C NMR (75 MHz, CDCl3): 55.21, 62.25, 62.83,
68.67, 72.70, 73.41, 74.71, 74.92, 75.46, 75.52, 75.56, 76.54, 76.96,
77.38, 80.81, 81.13, 81.84, 82.92, 97.75,101.15,113.83, 116.70, 126.16–
129.64 (multiple peaks), 135.67, 137.48, 137.85, 137.97, 138.15,
138.59, 159.31; HRMS (ESMS, MþNaþ) calcd for C58H61O11N3Na
998.4204, found 998.4366.
97.71, 101.14, 113.86, 126.03, 126.15, 127.60, 127.63, 127.80, 127.92,
128.13, 128.17, 128.20, 128.24, 128.31, 128.34, 129.60, 130.00, 137.48,
137.85, 137.97, 138.15, 138.59, 138.81, 159.30; HRMS (ESMS, MþNaþ)
calcd for C62H63O11N3Na 1048.4360, found 1048.4366. The spectral
and optical data were identical to that reported for this compound
by Schmidt et al.27 by an alternative route.
4.13. (2-Azido-3,6-di-O-benzyl-2-deoxy-a-D-glucopyranosyl)-
(1/6)-2,3,4,5-tetra-O-benzyl-1-O-(4-methoxybenzyl)-
D-myo-
4.10. (2-Azido-3,6-di-O-benzyl-2-deoxy-
a
-D
-glucopyranosyl)-
inositol (9d)
(1/6)-3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-2-O-allyl-
D
-myo-inositol (9a)
Colorless solid. TLC (hexane/EtOAc, 5:2): Rf¼0.46; [
a
]
þ33.2 (c
D
1.4, CHCl3); reported27
[
a]
þ34 (c 1.4, CHCl3). 1H NMR (300 MHz,
D
To a solution of above compound 8a (350 mg, 3.88 mmol), so-
CDCl3): 1.92 (d, J¼3.7 Hz,1H, OH), 3.17 (dd, J¼3.8 and 9.9 Hz,1H, 2b-
H), 3.22 (m, 1H, 6b-H), 3.28 (dd, J¼3.8 and 10.5 Hz, 1H, 60b-H), 3.38
(dd, J¼2.2 and 9.8 Hz, 1H, 3a-H), 3.44 (m, 1H, 5a-H), 3.47 (dd, J¼2.2
and 9.8 Hz,1H,1a-H), 3.68 (m, 1H, 4b-H), 3.76 (m, 1H, 3b-H), 3.80 (s,
3H, OCH3), 3.95 (m, 1H, 5b-H), 4.01 (dd, J¼2.2 Hz, 1H, 2a-H), 4.11
(dd, J¼9.4 Hz, 1H, 4a-H), 4.25 (m, 1H, 6a-H), 4.23–5.04 (m, 14H,
CH2Ph), 5.72 (d, J¼3.7 Hz, 1H, 1b-H), 6.82–6.88 (m, 2H, Ph), 7.17–
7.45 (m, 32H, Ph); 13C NMR (75 MHz, CDCl3): 55.20, 62.66, 69.00,
69.30, 71.78, 72.18, 72.64, 73.32, 73.45, 74.16, 74.21, 74.68, 74.76,
75.49, 75.60, 79.27, 80.80, 81.54, 81.84, 81.90, 84.35, 97.30, 113,78,
127.49–129.96 (multiple peaks), 137.92, 138.17, 138.33, 138.43,
138.57, 138.83, 159.28; HRMS (ESMS, MþNaþ) calcd for
C62H65O11N3Na 1050.4517, found 1050.4521.
dium cyanoborohydride (270 mg, 4 mmol), freshly activated mo-
lecular sieves (4 Å) in anhyd THF (14 mL), at 0 ꢀC under argon, was
added drop wise a saturated solution of HCl in diethyl ether until
pH 1 was reached. After stirring at 0 ꢀC for 30 min, the reaction
mixture was neutralized with Et3N, diluted with EtOAc, and con-
centrated. The residue was purified on a flash silica column
(hexane/EtOAc, 85:15) to provide desired compound 9a (330 mg,
94%) as a colorless solid. TLC (hexane/EtOAc, 7:3): Rf¼0.29; [
a]
D
þ33 (c 1.4, CHCl3). 1H NMR (300 MHz, CDCl3): 3.17 (dd, J¼3.8 and
9.9 Hz, 1H), 3.29 (m, 2H), 3.42 (dd, J¼2.2 and 9.8 Hz, 1H), 3.45 (m,
1H), 3.50 (m, 2H), 3.76 (m, 1H), 3.80 (s, 3H, OCH3), 3.96 (m, 1H),
4.01 (t, 1H), 4.11 (dd, 1H), 4.28 (m, 1H), 4.29–5.00 (m, 14H), 5.15–
5.25 (m, 2H), 5.69 (d, J¼3.7 Hz, 1H), 6.00 (m, 1H), 6.82–6.88 (m,
2H), 7.23–7.41 (m, 27H, Ph); 13C NMR (75 MHz, CDCl3): 55.19,
62.57, 69.00, 69.30, 71.73, 72.63, 73.32, 73.40, 73.64, 74.66, 74.21,
76.51, 76.93, 81.79, 81.83, 81.84, 81.90, 97.24, 113,79, 116.65,
127.52–129.48 (multiple peaks), 135.67, 138.17, 138.33, 138.43,
138.57, 138.83, 159.26; HRMS (ESMS, MþNaþ) calcd for
C58H63O11N3Na 1000.4360, found 1000.4321.
4.14. Allyl (2,3,4,6-tetra-O-benzyl)- -mannopyranosyl-(1/
a
-D
2)-3-O-benzyl-4,6-O-benzylidene-a-D-mannopyranoside (16)
The known28 glycosyl donor 13 (2,3,4,6-tetra-O-benzyl-
mannopyranosyl trichloroacetimidate, 4.1 g, 6.0 mmol) and the
acceptor 15 (allyl-3-O-benzyl-4,6-O-benzylidene- -mannopyr-
a-D-
a-D
anoside, 1.75 g, 4.39 mmol) were dried through anhyd toluene and
then dissolved in anhyd diethyl ether (100 mL) under argon. After
addition of TMSOTf solution (2.95 mL, 0.1 N in anhyd diethyl ether),
the reaction mixture was stirred for 2 h at rt. This was followed by
neutralization with Et3N, concentration, and silica column chro-
matography (hexane/EtOAc, 7:1) to obtain desired compound 16
(4.4 g, 81%) as a colorless crystalline solid. TLC (hexane/EtOAc, 3:1):
4.11. (2-Azido-3,6-di-O-benzyl-2-deoxy-
(1/6)-3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-2-O-allyl-
a-D-glucopyranosyl)-
L-myo-inositol (9b)
An identical method described above for compound 9a was used
for the preparation of diastereoisomeric compound 9b as white
solid; [
a]
þ24 (c 1.5, CHCl3). 1H NMR (300 MHz, CDCl3): 3.15 (dd,
Rf¼0.36; [
a]
D þ40 (c 1.5, CHCl3). 1H NMR (300 MHz, CDCl3): 3.70 (m,
D
J¼3.8 and 9.9 Hz, 1H), 3.34 (m, 2H), 3.45 (dd, J¼2.2 and 9.8 Hz, 1H),
3.51 (m, 2H), 3.71 (m, 1H), 3.83 (s, 3H, OCH3), 3.86 (m, 1H), 4.00 (t,
1H), 4.25 (m, 2H), 4.29–5.00 (m, 14H), 5.15–5.25 (m, 2H), 5.59 (d,
J¼3.9 Hz, 1H), 6.00 (m, 1H), 6.79–6.82 (m, 2H), 7.27–7.40 (m, 27H,
Ph); 13C NMR (75 MHz, CDCl3): 55.11, 61.61, 68.84, 71.73, 72.59,
73.36, 73.41, 73.64, 74.66, 74.71, 76.53, 76.96, 81.75, 82.02, 84.26,
98.00, 113,90, 116.39, 127.45–128.36 (multiple peaks), 135.60,
138.45–138.83 (multiple peaks), 159.25; HRMS (ESMS, MþNaþ)
calcd for C58H63O11N3Na 1000.4360, found 1000.4350.
2H), 3.75–4.00 (m, 6H), 4.10–4.25 (m, 3H), 4.30 (m, 1H), 4.40 (t, 1H),
4.45–5.00 (m, 11H), 5.15–5.30 (m, 2H), 5.41 (s, 1H), 5.58 (s, 1H), 5.80
(m,1H), 7.09–7.50 (m, 30H); 13C NMR (75 MHz, CDCl3): 64.05, 67.94,
68.03, 68.69, 68.79, 69.32, 71.60, 72.41, 73.23, 73.34, 73.42, 73.69,
74.07, 74.77, 74.81, 77.55, 78.51, 79.23, 79.50, 98.23, 98.69, 102.03,
117.47, 126.13, 127.20, 127.27, 127.33, 127.48, 127.52, 127.64, 127.68,
127.74, 127.87, 128.00, 128.12, 128.16, 128.19, 128.22, 128.27, 128.32,
128.39, 128.49, 129.12, 133.37, 137.54, 137.75, 138.11, 138.38, 138.41,
138.72; HRMS (ESMS, MþNaþ) calcd for C57H60O11Na 943.4033,
found 943.4040.
4.12. (2-Azido-3-O-benzyl-4,6-O-benzylidene-2-deoxy-
a
-D-glucopyranosyl)-(1/6)-2,3,4,5-tetra-O-benzyl-1-O-
4.15. 2,3,4,6-Tetra-O-benzyl-
a-D-mannopyranosyl-(1/2)-
(4-methoxybenzyl)- -myo-inositol (9c) (for direct
D
3-O-benzyl- -mannopyranoside (17)
a-D
correlation of stereochemistry)
The intermediate 16 (2.0 g, 2.19 mmol) dissolved in DMSO
(90 mL) was treated with potassium tert-butoxide (7 g, 62.5 mmol)
and the reaction mixture was stirred at 80 ꢀC for 3 h. After com-
pletion of the reaction, ice was added and the mixture extracted
with EtOAc. The organic layer was dried with Na2SO4 and con-
centrated to provide thick syrup. This was dissolved in a mixture of
acetone/1 M HCl (9:1, 79 mL) and heated at 55 ꢀC for 2 h, the
progress of reaction checked by TLC. The reaction mixture was
cooled, neutralized with NEt3, and concentrated. Flash chroma-
tography (hexane/EtOAc,1:1) provided desired compound 17 (1.4 g,
Colorless solid. TLC (hexane/EtOAc, 8:2): Rf¼0.50; [
a
]
þ48 (c
D
1.0, CHCl3); reported27
[
a
]
D
þ50 (c 1.0, CHCl3). 1H NMR (300 MHz,
CDCl3): 3.24 (dd, J¼3.8 and 10.0 Hz, 1H, 2b-H), 3.38 (dd, J¼2.2 and
9.8 Hz, 1H, 1a-H or 3a-H), 3.45 (dd, J¼2.2 and 9.8 Hz, 1H, 1a-H or 3a-
H), 3.46 (dd, J¼9.3 Hz, 1H), 3.52 (m, 2H), 3.80 (s, 3H, OCH3), 3.96–
4.04 (m, 2H, 2a-H, 3a-H), 4.06–4.29 (m, 4H), 4.48 (s, 2H, CH2Ph),
4.57–4.96 (m, 10H, CH2Ph), 5.47 (s, 1H), 5.72 (d, J¼3.8 Hz, 1H, 1b-H),
6.83–6.89 (m, 2H, Ph), 7.08–7.13 (m, 2H, Ph), 7.17–7.45 (m, 30H, Ph);
13C NMR (75 MHz, CDCl3): 55.20, 62.83, 68.67, 71.77, 72.70, 73.50,
74.16, 74.72, 74.99, 75.49, 75.57, 77.36, 80.81, 81.13, 81.84, 82.92,
82%) as colorless foam. TLC (hexane/EtOAc, 3:7): Rf¼0.34; [ D þ35
a
]

A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
4365
(c 1.5, CHCl3). 1H NMR (300 MHz, CDCl3): 3.58 (m, 2H), 3.75 (m, 5H),
4.19. Allyl-2,3,4-tri-O-benzyl-a-D-mannopyranoside (24)
3.85 (m, 3H), 4.00 (br s, 2H), 4.43–4.54 (m, 10H), 5.06 (br s, 1H), 5.34
(d, J¼1.9 Hz, 1H), 7.12–7.24 (m, 25H); 13C NMR (75 MHz, CDCl3):
62.38, 62.84, 67.34, 67.70, 69.57, 69.73, 71.56, 72.12, 72.32, 72.43,
72.58, 72.67, 73.31, 74.53, 74.87, 75.12, 77.32, 77.65, 79.27, 92.37,
98.70, 127.44–128.31 (multiple peaks), 137.93–138.08 (5 peaks);
HRMS (positive ion ESMS, MþNaþ) calcd for C47H52O11Na 815.3407,
found 815.3412.
1-O-Allyl-
a-D-mannopyranose 10 (24 g, 109 mmol, prepared
from
D
-mannose by reported29 method) dissolved in anhyd pyri-
dine (100 mL) was treated with trityl chloride (26 g, 93 mmol) and
the reaction mixture was stirred overnight at 80 ꢀC. The excess of
pyridine was removed by distillation at reduced pressure and
residue diluted with EtOAc, washed with saturated solution of
NaHCO3, brine and water, dried over Na2SO4, and concentrated.
Silica column chromatography (CH2Cl2/MeOH, 95:5) afforded allyl-
4.16. Acetyl-(2,3,4,6-tetra-O-benzyl- -mannopyranosyl)-
a-
D
6-O-trityl-a-D-mannopyranoside (35 g, 70%) as pale yellow syrup,
(1/2)-3-O-benzyl-4,5-di-O-acetyl-a-D-mannopyranoside (18)
which was dissolved in anhyd DMF (400 mL) followed by the
addition of benzyl bromide (58 mL) and NaH (21 g) at 0 ꢀC. After
30 min, the reaction mixture was brought to rt and stirred for 4 h.
After completion, the excess of NaH was destroyed by addition of
methanol and the solution concentrated. The mixture was diluted
with EtOAc, which was washed with saturated solution of NaHCO3,
water, dried over Na2SO4, and concentrated. Silica column chro-
matography (hexane/EtOAc, 3:1) afforded allyl-2,3,4-tri-O-benzyl-
A solution of preceding deprotected 17 (1.4 g, 1.78 mmol) in
a mixture of pyridine and acetic anhydride (1:1, 6 equiv) was stirred
at 0 ꢀC for 15 min and then kept at rt overnight. This was followed
by addition of ice and stirring for 30 min, extraction with EtOAc.
The organic layer was washed with saturated solution of NaHCO3
and water, dried with Na2SO4, and concentrated. Flash chroma-
tography (hexane/EtOAc, 3:1) afforded desired triacetylated com-
pound 18 (1.45 g, 93%), as colorless syrup. TLC (hexane/EtOac, 7:3):
6-O-trityl-a-D-mannopyranoside (23) (49 g, 89%). This compound
Rf¼0.58; [
a
]
D þ43 (c 1.5, CHCl3). 1H NMR (300 MHz, CDCl3): 1.84 (s,
(49 g, 67 mmol) dissolved in a solvent mixture of CH2Cl2/CH3OH
(720 mL, 1:3) was treated with p-toulenesulponic acid (3.6 g) and
stirred for 12 h at rt. After completion of the reaction, it was neu-
tralized with Et3N and concentrated. Silica column chromatography
3H), 1.96 (s, 3H), 2,03 (s, 3H), 3.75 (m, 1H), 4.10 (dd, J¼3 and 12 Hz,
1H), 4.15 (dd, J¼5 Hz, 1H), 4.35 (m, 3H), 4.50–4.60 (m, 6H), 4.70 (t,
J¼11 Hz, 1H), 4.96 (d, J¼2 Hz, 1H), 5.23 (s, 1H), 5.30 (t, J¼10 Hz, 1H),
6.13 (d J¼2 Hz, 1H), 7.25–7.53 (m, 25H); 13C NMR (75 MHz, CDCl3):
20.54, 20.64, 20.71, 61.50, 62.47, 67.71, 69.63, 71.08, 71.93, 72.31,
72.40, 72.46, 72.69, 73.24, 74.01, 74.34, 75.04, 75.49, 75.80, 77.10,
79.49, 90.89,100.60,127.40–128.32 (multiple peaks),138.12–138.38
(5 peaks), 167.99, 168.54, 170.70; HRMS (positive ion ESMS,
MþNaþ) calcd for C53H58O14Na 941.3724, found 941.3730.
(hexane/EtOAc, 7:3) afforded allyl-2,3,4-tri-O-benzyl-a-D-man-
nopyranoside 24 (30 g, 93%) as colorless foam. TLC (hexane/EtOAc,
7:3): Rf¼0.25; [
a
]
þ23 (c 2.0, CHCl3). 1H NMR (300 MHz, CDCl3):
D
3.61 (m, 1H), 3.76 (m, 1H), 3.89 (m, 2H), 3.99 (m, 1H), 4.19 (dd, 1H),
4.05 (m,1H), 4.58–4.80 (m, 6H), 4.80 (d, J¼1.3 Hz,1H), 5.19–5.33 (m,
2H), 5.78 (m, 1H), 7.24–7.31 (m, 15H); 13C NMR (75 MHz, CDCl3):
61.86, 67.65, 72.01, 72.61, 72.78, 74.76, 74.95, 80.10, 97.24, 117.00,
127.43, 127.49, 127.55, 127.73, 127.86, 128.14, 128.23, 133.74, 138.26,
138.48, 138.53; MS (ESMS, MþNaþ) calcd for C30H34O6Na 513.2253,
found 513.2260.
4.17. 2,3,4,6-Tetra-O-benzyl-
a-D-mannopyranosyl-(1/2)-3-O-
benzyl-4,5-di-O-acetyl- -mannopyranoside (19)
a-D
A solution of compound 18 (1.0 g, 1.14 mmol) in a saturated
solution of dimethylamine in CH3CN (55 mL) was stirred at ꢁ20 ꢀC
for 1 h. The excess of solvent and reagent was carefully removed
by evaporation under reduced pressure without heating. Flash
chromatography (hexane/EtOAc, 3:1) provided compound 19
(0.9 g, 94%) as colorless foam. TLC (hexane/EtOAc, 7:3): Rf¼0.34;
4.20. Allyl (2-O-benzoyl-3,4,6-tri-O-benzyl-a-D-
mannopyranosyl)-(1/6)-2,3,4-tri-O-benzy-L-a-D-
mannopyranoside (25)
The known31 orthoester donor 22 (1.85 g, 3.11 mmol) and
mannobiose acceptor 24 (1.55 g, 3.16 mmol) were dissolved in
anhyd CH2Cl2 (15 mL) and freshly activated 4 Å molecular sieves
were added. The solution was stirred at rt for 30 min followed by
[
a]
þ47 (c 1.5, CHCl3). 1H NMR (300 MHz, CDCl3): 3.75 (m, 2H),
D
3.90 (m, 1H), 3.90–4.25 (m, 7H), 4.25–4.56 (m, 10H), 4.75 (t, 1H),
4.90 (d, J¼1.9 Hz, 1H), 5.08 (d, J¼1.9 Hz, 1H), 5.27 (t, 1H), 7.10–7.26
(m, 25H); 13C NMR (75 MHz, CDCl3): 20.67, 20.71, 62.67, 62.90,
68.38, 68.71, 69.65, 72.18, 72.27, 72.55, 72.60, 72.67, 72.92, 72.97,
73.27, 74.35, 74.44, 74.03, 75.22, 77.21, 79.66, 92.45, 100.33,
127.37–128.29 (multiple peaks), 138.02–138.30 (5 peaks), 169.47,
170.77; HRMS (positive ion ESMS, MþNaþ) calcd for C51H56O13Na
899.3619, found 899.3625.
addition of 0.1 N solution of TESOTf in CH2Cl2 (150 mL,15 mmol) and
N-iodosuccinimide (525 mg, 2.34 mmol). After 30 min stirring, the
solution was neutralized with Et3N, the solvent was evaporated,
and the residue purified by silica column (hexane/EtOAc, 85:15),
which provided 25 (2.31 g, 72%) as colorless syrup. TLC (hexane/
EtOAc, 7:3): Rf¼0.5; [
a
]
þ27 (c 1.5, CHCl3). 1H NMR (300 MHz,
D
CDCl3): 3.50–4.00 (m, 7H), 4.12–4.25 (m, 4H), 4.50–5.10 (m, 11H),
5.19 (d, J¼1.9 Hz, 1H), 5.20 (t, 1H), 5.90 (m, 1H), 7.24–8.10 (m, 35H);
13C NMR (75 MHz, CDCl3): 66.68, 67.78, 68.74, 69.00, 71.09, 71.65,
72.06, 72.70, 73.35, 74.23, 74.65, 74.76, 75.03, 75.11, 77.67, 80.30,
96.83, 98.07, 117.45, 127.51–128.35 (multiple peaks), 133.68, 138.49
(multiple peaks), 165.49; HRMS (positive ion ESMS, MþNaþ) calcd
for C64H66O12Na 1049.4452, found 1049.4463.
4.18. 2,3,4,6-Tetra-O-benzyl-
benzyl-4,5-di-O-acetyl- -mannopyranosyl
trichloroacetimidate (20)
a-D-mannopyranosyl-(1/2)-3-O-
a-D
To a solution of compound 19 (800 mg, 0.69 mmol) in anhyd
CH2Cl2 (10 mL) were added Cl3CCN (1.7 ml, 17 mmol) and DBU
(90
mL) and the reaction mixture was stirred at rt for 1 h. The sol-
4.21. Allyl (3,4,6-tri-O-benzyl-
a-D-mannopyranosyl)-(1/6)-
vent was removed and the residue was purified by a silica column
chromatography (hexane/EtOAc, 3:1) to afford compound 20
(890 mg, 96%) as colorless foam. TLC (hexane/EtOAc, 7:3): Rf¼0.50;
2,3,4-tri-O-benzyl- -mannopyranoside (26)
a-D
To a solution of compound 25 (2.3 g, 2.24 mmol) in a solvent
mixture of CH3OH/CH2Cl2 (20 mL, 7:1) was added sodium meth-
oxide (350 mg, 6.48 mmol). The solution was stirred at rt for 3 h.
After completion of the reaction, the solvent was evaporated and
the residue was purified by silica column (hexane/EtOAc, 3:1) to get
compound 26 (1.9 g, 92%) as colorless foam. TLC (hexane/EtOAc,
[
a]
D þ21 (c 1.7, CHCl3). 1H NMR (300 MHz, CDCl3): 3.76 (m, 2H), 3.93
(m, 1H), 3.95–4.25 (m, 7H), 4.26–4.56 (m, 10H), 4.75 (t, 1H), 5.08 (d,
J¼1.9 Hz, 1H), 5.27 (t, 1H), 6.30 (br s, 1H), 7.10–7.26 (m, 25H), 8.60 (s,
1H, NH); HRMS (ESMS, MþNaþ) calcd for C53H56O13NCl3Na
1042.2715, found 1042.2720.

4366
A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
7:3): Rf¼0.25; [
a
]
þ30 (c 1.5, CHCl3). 1H NMR (300 MHz, CDCl3):
4.24. (2,3,4,6-Tetra-O-benzyl-
(4,6-di-O-acetyl-3-O-benzyl-
(3,4,6-tri-O-benzyl- -mannopyranosyl)-(1/6)-(2,3,4-tri-O-
benzyl- -mannopyranosyl)trichloroacetimidate (29)
a
-
D
-mannopyranosyl)-(1/2)-
D
3.50–3.60 (m, 2H), 3.62–3.70 (m, 2H), 3.75–3.80 (m, 4H), 4.0 (m,
4H), 4.35–4.75 (m, 12H), 5.0 (br s, 1H), 5.10–5.15 (m, 2H), 5.80
(m, 1H), 7.18–7.29 (m, 30H); 13C NMR (75 MHz, CDCl3): 66.24,
67.76, 68.04, 68.87, 71.06, 71.44, 71.62, 72.08, 72.73, 73.32,
74.23, 74.63, 74.89, 75.01, 79.56, 80.20, 96.84, 99.59, 117.39, 127.62–
128.29 (multiple peaks), 133.65, 138.46–138.50 (multiple peaks);
HRMS (positive ion ESMS, MþNaþ) calcd for C57H62O11Na 945.4190,
found 945.4197.
a-D-mannopyranosyl)-(1/2)-
a-D
a-D
Compound 28 (300 mg, 0.172 mmol) was dissolved in anhyd
CH2Cl2 (5 mL) followed by addition of CCl3CN (0.51 mL, 5.1 mmol)
and DBU (150 mL) and the reaction mixture was stirred at rt for
1.5 h. After completion of the reaction, the solvent was removed
under reduced pressure and the residue was purified on a silica
column (hexane/EtOAc, 3:1) to give desired trichloroacetimidate
donor 29 (320 mg, 98%) as colorless foam. TLC (hexane/EtOAc, 3:1):
4.22. Allyl (2,3,4,6-tetra-O-benzyl-
2)-(4,6-di-O-acetyl-3-O-benzyl- -mannopyranosyl)-(1/2)-
(3,4,6-tri-O-benzyl- -mannopyranosyl)-(1/6)-2,3, 4-tri-O-
benzyl- -mannopyranoside (27)
a-D-mannopyranosyl)-(1/
a-D
a
-D
Rf¼0.53; [
a
]
D þ14 (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): 1.79 (s,
a
-D
3H), 2.00 (s, 3H), 3.30–4.25 (m, 22H), 4.30–4.90 (m, 24H), 5.06 (d,
2H), 5.16 (d,1H), 5.28 (t,1H), 6.32 (br s,1H), 7.08–7.29 (m, 55H), 8.61
(s, 1H, NH); MS (ESMS, MꢁC2NCl3þHþ) calcd for C105H112O23
1740.7594, found 1740.7599.
The mannobiosyl donor 20 (2,3,4,6-tetra-O-benzyl-
nopyranosyl-(1/2)-3-O-benzyl-4,5-di-O-acetyl- -mannopyra-
nosyl trichloroacetimidate, 1.0 g, 1.08 mmol) and the acceptor 26
(allyl (3,4,6-tri-O-benzyl- -mannopyranosyl)-(1/6)-2,3,4-tri-
O-benzyl- -mannopyranose, 650 mg, 0.63 mmol) were first
a-D-man-
a-D
a
-
D
4.25. (2,3,4,6-Tetra-O-benzyl-
(4,6-di-O-acetyl-3-O-benzyl-
(3,4,6-tri-O-benzyl- -mannopyranosyl)-(1/6)-(2,3,4-tri-O-
benzyl- -mannopyranosyl)-(1/4)-(2-azido-3,6-di-O-
benzyl-2-deoxy- -glucopyranosyl)-(1/6)-3,4,5-tri-O-
benzyl-1-O-(4-methoxybenzyl)-2-O-allyl- -myo-inositol (30)
a
-D-mannopyranosyl)-(1/2)-
a
-
D
a-D-mannopyranosyl)-(1/2)-
dried through co-evaporation with anhyd toluene, further dried
for 2 h under high vacuum, and then dissolved in anhyd CH2Cl2
(12 mL) and freshly activated molecular sieves (4 Å) were added
and the suspension stirred under argon at rt for 30 min. The
reaction mixture was cooled to 0 ꢀC and treated with 0.1 N
a-D
a-D
a-D
D
TMSOTf solution (760
m
L, 0.76 mmol) and stirred for 30 min. The
The above tetramannose trichloroacetimidate donor 29
reaction was neutralized with Et3N and solvent was evaporated.
The residue was purified by silica column (hexane/EtOAc, 85:15)
to provide target tetrasaccharide 27 (1.05 g, 69%) as white solid.
TLC (hexane/EtOAc, 7:3): Rf¼0.68. This glycosylation reaction
(421 mg, 0.22 mmol, 1.4 equiv) and the glucosamine–inositol ac-
ceptor 9a (2-azido-3,6-di-O-benzyl-2-deoxy-a-D-glucopyranosyl)-
(1/6)-(3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-2-O-allyl-
D
-
myo-inositol) (162 mg, 0.16 mmol) were dried by evaporation
through anhyd toluene and then dissolved in anhyd diethyl ether
(15 mL) and freshly activated molecular sieves (4 Å) under argon and
cooled to 0 ꢀC. To this was added a solution of 0.1 N TMSOTf (320
mL,
0.36 mmol) and reaction mixture was stirred for 30 min. After
completion of reaction, it was neutralized with Et3N and solvent
evaporated, and residue purified by a flash silica column (hexane/
EtOAc, 85:15) to provide desired pseudohexasaccharide 30 (320 mg,
required strictly anhyd conditions; [
a
]
þ22 (c 1.0, CHCl3). 1H
D
NMR (300 MHz, CDCl3); 1.80 (s, 3H), 2.01 (s, 3H), 3.52–4.32 (m,
22H), 4.35–500 (m, 26H), 5.05–5.40 (m, 5H), 5.29 (t, 1H), 5.77–
5.87 (m, 1H), 7.16–7.29 (m, 55H); 13C NMR (75 MHz, CDCl3):
20.57, 20.64, 62.87, 66.53, 67.69, 68.47, 68.86, 69.01, 69.13, 71.34,
71.41, 71.74, 72.01, 72.20, 72.24, 72.44, 72.59, 72.99, 73.18, 74.48,
74.61, 74.70, 74.88, 75.37, 75.76, 77.13, 79.03, 79.89, 80.27, 96.87,
99.05, 99.51, 99.81, 117.34, 127.30–128.24 (multiple peaks),
133.57, 137.98–138.76 (multiple peaks), 169.44, 170.69; HRMS
(positive ion ESMS, MþNaþ) calcd for C108H116O23Na 1803.7805,
found 1803.7811.
53%) as viscous substance. TLC (hexane/EtOAc, 7:3): Rf¼0.27; [
a]
D
þ27 (c 1.4, CHCl3). 1H NMR (300 MHz, CDCl3): 1.73 (s, 3H), 1.86 (s,
3H), 3.08–3.15 (m, 2H), 3.18–3.25 (m, 2H), 3.24–3.41 (m, 6H), 3.41–
3.56 (m, 4H), 3.71–4.01 (m, 24H), 3.82 (s, 3H), 4.33–5.08 (m, 34H),
5.14 (m, 2H), 5.26 (br s, 1H), 5.28 (br s, 1H), 5.37 (br s, 1H), 5.38 (br s,
1H), 5.80 (d, J¼3.8 Hz, 1H), 5.90 (m, 1H), 6.89 (d, 2H), 7.07–7.39 (m,
82H); 13C NMR (75 MHz, CDCl3): 20.57, 22.52, 55.19, 62.03, 62.76,
62.93, 65.73, 66.53, 68.48, 68.51, 68.84, 68.98, 69.15, 69.18, 69.58,
71.47, 71.64, 71.95, 72.09, 72.19, 72.45, 72.63, 73.00, 73.09, 73.18,
73.41, 74.16, 74.48, 74.59, 74.76, 74.93, 75.70, 76.48, 77.14, 79.02,
79.92, 80.01, 80.85, 81.45, 81.73, 81.81, 97.19, 99.21, 99.32, 99.78,
100.27, 113.84, 116.66, 127.18–128.34 (multiple peaks), 129.44,
135.63, 137.75, 137.95, 138.02–138.84 (multiple peaks), 159.30,
169.41, 170.68; MS (positive ion ESMS, MþNaþ) calcd for
C163H173O33N3Na 2723.1849, found 2723.1930.
4.23. 2,3,4,6-Tetra-O-benzyl-
di-O-acetyl-3-O-benzyl- -mannopyranosyl-(1/2)-(3,4,6-
tri-O-benzyl- -mannopyranosyl)-(1/6)-2,3,4-tri-O-benzyl-
-mannopyranose (28)
a-D-mannopyranosyl-(1/2)-4,6-
a-D
a-D
a-D
To a solution of tetrasaccharide 27 (425 mg, 0.238 mmol) in
AcOH/H2O (11 ml,19:1) were added PdCl2 (400 mg, 2.25 mmol) and
anhyd NaOAc (400 mg, 4.87 mmol). The reaction mixture was
stirred at rt for 16 h under argon atmosphere. After completion of
the reaction, it was diluted with EtOAc, organic layer washed with
saturated NaHCO3 solution and water, dried (Na2SO4), and evapo-
rated. The residue was purified by silica chromatography (hexane/
EtOAc, 7:3) to provide anomeric deprotected tetrasaccharide 28
(380 mg, 91%) as colorless foam. TLC (hexane/EtOAc, 6:4): Rf¼0.3;
4.26. (2,3,4,6-Tetra-O-benzyl-
(3-O-benzyl- -mannopyranosyl)-(1/2)-(3,4,6-tri-O-benzyl-
-mannopyranosyl)-(1/6)-(2,3,4-tri-O-benzyl-
mannopyranosyl)-(1/4)-(2-azido-3,6-di-O-benzyl-2-deoxy-
-glucopyranosyl)-(1/6)-3,4,5-tri-O-benzyl-1-O-
(4-methoxybenzyl)-2-O-allyl- -myo-inositol (31)
a-D-mannopyranosyl)-(1/2)-
a-D
a-
D
a-D-
[
a]
D þ21 (c 1.0, CHCl3). 1H NMR (300 MHz, CDCl3): 1.79 (s, 3H), 2.00
(s, 3H), 3.30–4.25 (m, 22H), 4.30–4.90 (m, 24H), 4.96 (d, 1H), 5.05
(d,1H), 5.09 (d,1H), 5.16 (d,1H), 5.28 (t,1H), 7.08–7.29 (m, 55H); 13C
NMR (75 MHz, CDCl3): 20.62, 20.76, 62.99, 68.12, 68.65, 68.87,
69.04, 69.17, 69.50, 71.34, 71.50, 71.55, 71.71, 72.01, 72.24, 72.37,
72.46, 72.62, 73.11, 73.20, 73.37, 74.02, 74.26, 74.52, 74.64, 74.78,
74.82, 74.90, 75.00, 75.09, 75.86, 75.99, 77.13, 78.59, 78.98, 79.86,
92.46, 99.09, 99.28, 99.89, 127.53–128.27 (multiple peaks), 138.24–
138.41 (multiple peaks), 169.51, 170.96; HRMS (positive ion ESMS,
MþNaþ) calcd for C105H112O23Na 1763.7492, found 1763.7503.
a-D
D
The above pseudohexasaccharide 30 (220 mg, 0.08 mmol) was
dissolved in a solvent mixture of CH3OH/CH2Cl2 (3:1,12 mL) followed
by addition of sodium methoxide (250 mg, 4.62 mmol). The reaction
mixture was stirred at rt for 1 h, solvent was evaporated under re-
duced pressure, and the residue purified by a silica column (hexane/
EtOAc, 65:35) to obtain the deacetylated pseudohexasaccharide 31

A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
4367
(205 mg, 96%) as colorless solid; [
a
]
þ28 (c 1.0, CHCl3). 1H NMR
CH2Cl2 and the combined organic layer was dried over Na2SO4 and
evaporated in vacuo. The residue was subjected to flash silica col-
umn (hexane/EtOAc, 85:15) to afford 33 (52 mg, 77%) as white
D
(300 MHz, CDCl3): 3.08–3.16 (m, 2H), 3.18–3.26 (m, 2H), 3.27–3.41
(m, 6H), 3.41–3.56 (m, 4H), 3.71–4.01 (m, 24H), 3.86 (s, 3H), 4.25–
5.10 (m, 34H), 5.14 (m, 2H), 5.26 (br s, 1H), 5.28 (br s, 1H), 5.37 (br s,
1H), 5.38 (br s, 1H), 5.80 (d, J¼3.8 Hz, 1H), 5.90 (m, 1H), 6.90 (d, 2H),
7.10–7.30 (m, 82H); 13C NMR (75 MHz, CDCl3): 55.20, 62.03, 62.74,
62.87, 66.04, 66.61, 68.48, 68.51, 68.84, 68.98, 69.08, 69.37, 69.66,
71.64, 71.72, 71.81, 71.90, 72.01, 72.08, 72.65, 72.78, 73.17, 73.26,
73.42, 73.41, 74.16, 74.52, 74.66, 74.79, 74.94, 75.47, 75.66, 76.55,
76.97, 78.07, 78.57, 79.29, 79.61, 79.91, 80.64, 80.48, 81.77, 81.78,
81.81, 97.22, 97.63, 98.81, 99.26, 99.78, 100.38, 113.84,116.71, 127.29–
128.24 (multiple peaks),129.43,135.66,137.78,138.00,138.03,138.24
(multiple peaks), 138.37, 138.42, 128.47, 138.55, 138.68, 138.84,
159.30; MS (positive ion ESMS, MþNaþ) calcd for C159H169O31N3Na
2639.1638, found 2639.1710.
foam. TLC (hexane/EtOAc, 70:30): Rf¼0.31; [
a
]
D
þ35 (c 1.5, CHCl3).
1H NMR (300 MHz, CDCl3): 1.04 (s, 9H, TBDPS), 3.15–3.85 (m, 36H),
3.96–4.88 (m, 36H), 4.94 (s, 1H), 5.14 (m, 2H), 5.17 (s, 2H), 5.29 (s,
1H), 5.71 (d, J¼3.6 Hz, 1H), 6.00 (m, 1H), 7.05–7.26 (m, 91H), 7.65–
7.72 (m, 4H); 13C NMR (75 MHz, CDCl3): 19.26, 26.80, 62.97, 63.90,
66.36, 68.44, 68.33, 69.01, 69.25, 69.64, 70.26, 71.67, 71.95, 72.10,
72.13, 72.55, 72.92, 72.97, 73.03, 73.24, 73.81, 74.06, 74.35, 74.66,
74.82, 74.97, 75.63, 75.50, 76.92, 77.07, 79.33, 79.75, 79.95, 80.45,
80.79, 81.26, 81.66, 81.86, 97.33, 98.71, 99.04, 99.31, 99.64, 99.92,
117.07, 127.13–128.33 (multiple peaks), 129.44, 138.54–138.83
(multiple peaks); MS (positive ion ESMS, MþNaþ) calcd for
C174H185O30N3SiNa 2847.2710, found 2847.2880.
4.27. (2,3,4,6-Tetra-O-benzyl-
(3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-
pyranosyl)-(1/2)-(3,4,6-tri-O-benzyl- -mannopyranosyl)-
(1/6)-(2,3,4-tri-O-benzyl- -mannopyranosyl)-(1/4)-(2-
azido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl)-(1/6)-
a
-
D
-mannopyranosyl)-(1/2)-
4.29. (2,3,4,6-Tetra-O-benzyl-
(3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-
pyranosyl)-(1/2)-(3,4,6-tri-O-benzyl- -mannopyranosyl)-
(1/6)-(2,3,4-tri-O-benzyl- -mannopyranosyl)-(1/4)-(2-
azido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl)-(1/6)-
(3,4,5-tri-O-benzyl-1-O-diisopropylidene-sn-glyceryl-
phosphonato)-2-O-allyl- -myo-inositol (35)
a
-
D
-mannopyranosyl)-(1/2)-
a
-D
-manno-
a-D-manno-
a
-D
a-D
a
-
D
a-D
a
-D
a-D
3,4,5-tri-O-benzyl-1-O-(4-methoxybenzyl)-2-O-allyl-D-myo-
inositol (32)
D
The above compound (75 mg, 28
THF (1 mL) followed by the addition of imidazole (25 mg,
0.36 mmol) and tert-butyldiphenylsilyl chloride (TBDPSCl, 60 L,
m
mol) was dissolved in anhyd
The above compound 33 (32 mg, 11
pared32 1,2-isopropylidene-sn-glyceryl-H-phosphonate (34, 12 mg,
60 mol) were dried by evaporation with anhyd pyridine three
mmol) and freshly pre-
m
m
0.23 mmol). The reaction mixture was stirred for 5 h, diluted with
EtOAc, organic layer washed with saturated aqueous solution of
NaHCO3 and H2O, dried over Na2SO4, and concentrated. The residue
was purified through a small silica column to provide selectively
mono-silylated compound (72 mg, 89%) as a colorless solid, which
was dissolved in anhyd DMF (2 mL) followed by addition of benzyl
times and dried under high vacuum for 2 h. The mixture was dis-
solved in anhyd pyridine (1.0 mL). This was followed by addition of
pivaloyl chloride (11
mL, 91
mmol). After stirring at rt for 30 min, the
reaction mixture was treated with iodine (6 mg, 23
mmol, 2 equiv)
in a mixture of pyridine/water (19:1, 0.1 ml), was added to oxidize
P(III) to P(V) and further stirred for 30 min. The reaction mixture
was diluted with CHCl3 and organic layer washed with 5% sodium
bisulfite solution to remove the excess of iodine. The organic layer
was dried over Na2SO4, concentrated, and purified on a flash col-
umn using 2% MeOH/CH2Cl2 (with 0.1% triethylamine) solvent
system, providing the desired compound 35 (32 mg, 94%) as
bromide (70 mL, 0.5 mmol) and sodium hydride (5 mg, 0.12 mmol).
The reaction mixture was stirred at 0 ꢀC for 1 h, quenched with
methanol, diluted with EtOAc, organic layer washed with brine and
H2O, dried over Na2SO4, and concentrated. Flash silica column
chromatography led to isolation of compound 32 (73 mg, 97%) as
a colorless solid. TLC (hexane/EtOAc, 7:3): Rf¼0.43; [
a
]
D þ27 (c 1.0,
a syrup. TLC (DCM/MeOH, 9.5:0.5): Rf¼0.35; [ D þ24 (c 1.5, CHCl3).
a
]
CHCl3). 1H NMR (300 MHz, CDCl3): 1.04 (s, 9H, TBDPS), 3.08–3.16
(m, 2H), 3.18–3.26 (m, 2H), 3.27–3.41 (m, 6H), 3.41–3.56 (m, 4H),
3.71–4.01 (m, 22H), 3.74 (s, 3H), 4.10–4.95 (m, 36H), 4.97 (br s, 1H),
5.14 (m, 2H), 5.17 (br s, 1H), 5.29 (br s, 1H), 5.42 (br s, 1H), 5.72 (d,
J¼3.6 Hz, 1H), 6.00 (m, 1H), 6.80–6.82 (d, 2H), 7.04–7.26 (m, 87H),
7.65–7.72 (m, 4H); 13C NMR (75 MHz, CDCl3): 19.26, 29.24, 55.17,
62.01, 62.89, 64.95, 68.61, 68.83, 69.11, 69.53, 71.55, 71.66, 71.78,
71.90, 72.05, 72.20, 72.65, 72.98, 73.05, 73.20, 74.13, 74.60, 74.75,
74.86, 75.01, 75.62, 76.18, 77.11, 77.81, 79.81, 79.53, 79.68, 79.91,
80.83, 81.52, 81.79, 81.89, 97.20, 98.29, 99.00, 99.17, 100.25, 113.89,
166.66, 127.01–128.15 (multiple peaks), 133.02, 135.55, 135.47,
137.75–138.89 (multiple peaks), 159.28; MS (positive ion ESMS,
MþNaþ) calcd for C182H193O31N3SiNa 2967.3285, found 2967.3221.
1H NMR (300 MHz, CDCl3): 1.02 (s, 9H, TBDPS), 137 (s, 3H), 1.41 (s,
3H), 3.05–3.16 (m, 2H), 3.18–3.26 (m, 2H), 3.27–3.41 (m, 6H), 3.41–
3.56 (m, 4H), 3.71–4.02 (m, 26H), 3.96–4.88 (m, 37H), 4.95 (s, 1H),
5.18 (s, 2H), 5.29 (s, 1H), 5.71 (d, J¼3.6 Hz, 1H), 7.05–7.26 (m, 91H),
7.65–7.72 (m, 4H); 13C NMR (75 MHz, CDCl3): 19.24, 26.63, 62.92,
63.91, 66.36, 68.45, 68.33, 69.05, 69.26, 69.64, 70.27, 71.70, 71.95,
72.11, 72.13, 72.56, 72.92, 72.98, 73.03, 73.24, 73.82, 74.06, 74.35,
74.66, 74.82, 74.97, 75.64, 75.51, 76.94, 77.09, 79.34, 79.75, 79.95,
80.51, 80.79, 81.27, 81.67, 81.86, 97.34, 98.71, 99.05, 99.31, 99.65,
99.92, 117.09, 127.12–128.33 (multiple peaks), 129.45, 138.54–
138.83 (multiple peaks); MS (negative ion ESMS, MꢁH)ꢁ calcd for
C180H195O35N3PSi 3017.3078, found 3017.3144.
4.30. (2,3,4,6-Tetra-O-benzyl-
(3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-
pyranosyl)-(1/2)-(3,4,6-tri-O-benzyl- -mannopyranosyl)-
(1/6)-(2,3,4-tri-O-benzyl- -mannopyranosyl)-(1/4)-(2-
azido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl)-(1/6)-
a
-D
-mannopyranosyl)-(1/2)-
4.28. (2,3,4,6-Tetra-O-benzyl-
(3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl
pyranosyl)-(1/2)-(3,4,6-tri-O-benzyl- -mannopyranosyl)-
(1/6)-(2,3,4-tri-O-benzyl- -mannopyranosyl)-(1/4)-(2-
azido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl)-(1/6)-
3,4,5-tri-O-benzyl-2-O-allyl- -myo-inositol (33)
a
-D
-mannopyranosyl)-(1/2)-
a-D-manno-
a
-D
-manno-
a-D
a
-D
a-D
a
-D
a-D
a
-D
(3,4,5-tri-O-benzyl-1-O-sn-glyceryl-phosphonato)-D-myo-
inositol (36)
D
The above pseudohexasaccharide 32 (70 mg, 23
dissolved in CH3CN/toluene/water (2 mL, 9:5:4) and cooled to 0 ꢀC.
Ceric ammonium nitrate (CAN) (65 mg, 110 mol) was added and
stirred for 30 min at same temperature and at rt for 1 h. The re-
action mixture was diluted with CH2Cl2, washed with saturated
NaHCO3 solution. The aqueous phase was further washed with
m
mol) was
Pseudohexasaccharide 35 (27 mg, 8.9 mmol), anhyd NaOAc
(75 mg, 0.91 mmol), and PdCl2 (65 mg, 0.36 mmol) were dissolved in
a mixture of AcOH/H2O (19:1, 2 mL) with the aid of sonication. The
reaction mixture was stirred at rt for 16 h under argon atmosphere.
After completion of the reaction, it was quenched carefully by ad-
dition of saturated NaHCO3 solution and solid Na2CO3. The aqueous
m

4368
A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
layer was extracted with EtOAc (3ꢂ15 mL) and the combined organic
layer was dried over Na2SO4 and evaporated in vacuo. The residue
was purified by flash silica chromatography (DCM/MeOH, 95:5)
afforded compound 36 (23 mg, 88%) as a syrup. TLC (DCM/MeOH,
TLC (CH2Cl2/CH3OH, 95:5): Rf¼0.46; [
a
]
D þ29 (c 1.0, CHCl3). 1H NMR
(300 MHz, CDCl3): 0.88 (t, 9H, 3CH3 of fatty acids), 1.10–1.27 (m,
72H, 36CH2 of fatty acids), 1.55–1.63 (m, 6H), 2.22–2.46 (m, 6H),
3.08–3.45 (m, 12H), 3.60–4.30 (m, 30H), 4.34–4.56 (m, 22H), 4.64 (s,
1H), 4.72–4.86 (m, 10H), 5.08 (d, 1H), 5.20 (m, 2H), 5.24 (d, 1H), 5.55
(m, 1H), 5.85 (br s, 1H), 6.00 (br s, 1H), 7.00–7.38 (m, 85H); 31P NMR
(125 MHz): ꢁ0.74; MS (negative ion ESMS, MꢁH)ꢁ calcd for
C206H259O38N3P 3413.8164, found 3413.9300.
9:1): Rf¼0.3; [
a]
D þ33 (c 1.4, CHCl3). 1H NMR (300 MHz, CDCl3): 1.14
(s, 9H, TBDPS), 3.05–3.16 (m, 2H), 3.18–3.26 (m, 2H), 3.27–3.41 (m,
6H), 3.41–3.56 (m, 4H), 3.71–4.02 (m, 26H), 3.96–4.88 (m, 37H), 4.95
(s, 1H), 5.18 (s, 2H), 5.29 (s, 1H), 5.71 (d, J¼3.6 Hz, 1H), 7.05–7.26 (m,
91H), 7.65–7.73 (m, 4H); 13C NMR (75 MHz, CDCl3): 19.25, 26.63,
62.92, 63.91, 66.37, 68.45, 68.33, 69.05, 69.26, 69.64, 70.27, 71.70,
71.95, 72.11, 72.13, 72.56, 72.92, 72.98, 73.04, 73.24, 73.82, 74.06,
74.35, 74.66, 74.82, 74.97, 75.64, 75.51, 76.94, 77.09, 79.35, 79.75,
79.95, 80.51, 80.79, 81.27, 81.67, 81.86, 97.34, 98.71, 99.05, 99.31,
99.66, 99.92, 117.09, 127.12–128.33 (multiple peaks), 129.45, 138.54–
138.83 (multiple peaks); MS (negative ion ESMS, MꢁH)ꢁ calcd for
C174H188O35N3PSi 2937.2452, found 2937.2354.
4.33. (2,3,4,6-Tetra-O-benzyl-
(3,4-di-O-benzyl-6-O-(2-(N-benzyloxycarbonyl)-amino-
ethylphosphonato)- -mannopyranosyl)-(1/2)-(3,4,6-tri-O-
benzyl- -mannopyranosyl)-(1/6)-(2,3,4-tri-O-benzyl-
mannopyranosyl)-(1/4)-(2-azido-3,6-di-O-benzyl-2-deoxy-
-glucopyranosyl)-(1/6)-3,4,5-tri-O-benzyl-2-O-
hexadecanoyl-1-O-(1,2-di-O-hexadecanoyl-sn-glyceryl-
phosphonato)- -myo-inositol (41, fully protected GPI anchor)
a-D-mannopyranosyl)-(1/2)-
a-D
a-
D
a-D-
a-D
D
4.31. (2,3,4,6-Tetra-O-benzyl-
(3,4-di-O-benzyl-6-O-tert-butyldiphenylsilyl-
pyranosyl)-(1/2)-(3,4,6-tri-O-benzyl- -mannopyranosyl)-
(1/6)-(2,3,4-tri-O-benzyl- -mannopyranosyl)-(1/4)-(2-
azido-3,6-di-O-benzyl-2-deoxy- -glucopyranosyl)-(1/6)-
3,4,5-tri-O-benzyl-2-O-hexadecanoyl-1-O-(1,2-di-O-
a
-
D
-mannopyranosyl)-(1/2)-
a-
D-manno-
Compound 39 (20 mg, 5.86
ethanolamine-H-phosphonate (40, 24 mg, 92 m
m
mol) and freshly prepared34 Cbz-
mol) were co-
a
-D
a
-
D
evaporated with anhyd pyridine (3ꢂ1 mL) and dried under high
a
-D
vacuum for 2 h. The mixture was dissolved in anhyd pyridine
(0.5 mL). This was followed by addition of pivaloyl chloride (16
120 mol). The reaction mixture was stirred for 6 h. After stirring at
rt for 6 h, the reaction mixture was treated with iodine (14 mg,
54 mol, 2 equiv) in a mixture of pyridine/water (19:1, 0.1 ml), was
mL,
hexadecanoyl-sn-glyceryl-phosphonato)-
D
-myo-inositol (38)
m
The previous compound 36 (20 mg, 6.8
mmol) was dissolved in
m
anhyd CH2Cl2 (2 ml) and CdCl2$H2O was added33 to it and mixture
stirred for 30 min at rt. The solvent was removed and the residue
was dried repeatedly through anhyd toluene (10 mg). To this was
added anhyd CH2Cl2 (1.5 ml) followed by freshly prepared palmitic
anhydride (40 mg) and dimethylaminopyridine (5 mg). The re-
action mixture was stirred at rt for 18 h under dark condition. After
completion of the reaction, solvent was removed and the residue
was purified by silica column chromatography (3–5% CH3OH in
CH2Cl2 with 0.1% Et3N) to provide dipalmitoylated intermediate 37
added to oxidize P(III) to P(V) and further stirred for 6 h at rt to
ensure the complete oxidation. The reaction mixture was diluted
with CHCl3 and organic layer washed with 5% sodium bisulfite
solution to remove the excess of iodine. The aqueous layer was
further washed with CHCl3 and the combined organic layer was
washed with TEAB buffer and dried over Na2SO4, concentrated, and
purified on a flash column using 15% MeOH/CH2Cl2 (with 0.1%
triethylamine) solvent system, afforded the desired compound 41
(18 mg, 83%) as white foam. TLC (CH2Cl2/CH3OH, 9:1): Rf¼0.2; [
a]
D
(21 mg). This intermediate (20 mg, 6.8
(1.5 mL) was treated with palmitic acid (20 mg, 78
dicyclohexylcarbodiimide (DCC) (20 mg, 76 mol) and catalytic
mmol) dissolved in CH2Cl2
þ27 (c¼1.0, CHCl3). 1H NMR (300 MHz, CDCl3-CD3OD): 0. 87 (t, 9H,
3CH3 of fatty acids), 1.10–1.40 (m, 78H, 36CH2 of fatty acids and
TEA), 1.50–1.59 (m, 6H), 2.10–2.31 (m, 6H), 3.17 (m, 2H), 3.27–4.00
(m, 34H), 4.05–5.00 (m, 47H), 5.12 (d, 1H), 5.21 (s, 1H), 5.23 (s, 1H),
5.28 (m,1H), 5.80 (d,1H), 5.92 (m,1H), 7.00–7.40 (m, 90H); 31P NMR
(125 MHz): ꢁ1.70, 0.80; MS (negative ion ESMS, Mꢁ2H)ꢁ calcd for
C216H270O43N4P2 3669.86, found 3669.8550.
m
mol) and
m
amount of DMAP. The reaction mixture was stirred for 48 h at rt.
After completion of the reaction, CH2Cl2 was removed and the
residue was subjected to flash column chromatography (3–5%
CH3OH in CH2Cl2 with 0.1% Et3N) to provide 38 (23 mg, 80%) as
white foam. TLC (DCM/MeOH, 95:5): Rf¼0.65; [
a
]
þ31 (c 1.4,
D
CHCl3). 1H NMR (300 MHz, CDCl3): 0.83 (t, 9H, 3CH3 of fatty acids),
1.14 (s, 9H, TBDPS), 1.13–1.27 (m, 72H, 36CH2 of fatty acids), 1.45–
1.53 (m, 6H), 2.20–2.26 (m, 6H), 3.08–3.59 (m, 12H), 3.60–4.30 (m,
30H), 4.34–4.56 (m, 22H), 4.64 (s, 1H), 4.72–4.86 (m, 10H), 5.12 (m,
2H), 5.15 (d, 1H), 5.24 (d, 1H), 5.25 (m, 1H), 5.85 (br s, 1H), 6.00 (br s,
1H), 7.00–7.38 (m, 85H), 7.65–7.73 (m, 4H); 31P NMR (125 MHz):
ꢁ0.75; MS (negative ion ESMS), [MꢁH]ꢁ calcd for C222H277O38N3PSi
3651.9342, found 3651.9399.
4.34. (
phosphonato)-
pyranosyl)-(1/6)-(
deoxy- -glucopyranosyl)-(1/6)-1-O-(2-hexadecanoyl-1-O-
octadecyl-sn-glyceryl-phosphonato)- -myo-inositol (1)
a
-
D
-Mannopyranosyl)-(1/2)-(6-O-(aminoethyl-
-mannopyranosyl)-(1/2)-( -manno-
-mannopyranosyl)-(1/4)-(2-amino-2-
a
-D
a-D
a-D
a-D
D
The protected and lipidated GPI anchor 41 (12 mg, 4.8 mmol) and
20% Pd(OH)2 (25 mg) were dissolved in a solvent mixture of CH3OH
(1 mL), CH2Cl2 (1 mL), and H2O (0.05 mL) and a trace amount of
4.32. (2,3,4,6-Tetra-O-benzyl-
(3,4-di-O-benzyl- -mannopyranosyl)-(12)-(3,4,6-tri-O-
benzyl- -mannopyranosyl)-(1/6)-(2,3,4-tri-O-benzyl-
mannopyranosyl)-(1/4)-(2-azido-3,6-di-O-benzyl-2-deoxy-
-glucopyranosyl)-(1/6)-3,4,5-tri-O-benzyl-2-O-
hexadecanoyl-1-O-(1,2-di-O-hexadecanoyl-sn-glyceryl-
phosphonato)- -myo-inositol (39)
a
-
D
-mannopyranosyl)-(1/2)-
formic acid (10 ml). The residual and dissolved air from the flask was
a-
D
removed by repeated evacuations by suction (three freeze–thaw–
pump cycles) and flushed with hydrogen gas. The reaction mixture
was then stirred under positive hydrogen atmosphere for 24 h at rt.
At this stage, another lot of CH3OH/CH2Cl2/H2O (1:1:0.3) (1 mL) and
Pd(OH)2 (15 mg) were added and stirring continued under hydro-
gen pressure for additional 24 h. After the completion of reaction,
the mixture was filtered through a small Celite pad, which was
washed several times with the solvent mixture, and concentrated
under reduced pressure. The residue was washed with anhyd
CH2Cl2 to remove non-polar impurities. This provided the desired
fully lipidated malarial GPI anchor 1 (8.2 mg, 73%) as colorless solid.
TLC (CH2Cl2/CH3OH/0.25% KCl; 5:4:1): Rf¼0.2. 1H NMR (300 MHz,
CD3OD/CDCl3/D2O): 0.81–1.00 (m, 9H), 1.16–1.35 (m, 72H),
a
-D
a-D-
a-D
D
Compound 38 (15 mg, 4.1 mmol) was dissolved in 0.4 ml of a 1 M
solution of tetrabutylammonium fluoride (TBAF) in anhyd THF and
stirred at rt for 18 h. After completion of reaction, solvent was
quickly removed under vacuum and the viscous yellow mass was
purified through a flash silica column (CH2Cl2/CH3OH, 98:2)
affording the desired compound 39 (12 mg, 85%) as colorless solid.

A. Ali, R.A. Vishwakarma / Tetrahedron 66 (2010) 4357–4369
4369
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Acknowledgements
Authors wish to acknowledge generous financial support from
the Department of Science & Technology, Govt. of India (Grant No.
SR/S0/BB-06/2005). The Department of Biotechnology (Govt. of
India) provided the core grant to the Institute for infrastructure and
instrumental facilities.
Supplementary data
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Supplementary data associated with this article can be found in
online version at doi:10.1016/j.tet.2010.04.014.
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