Total synthesis of a fully lipidated form of phosphatidyl-myo-inositol dimannoside (PIM-2) of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.tetlet.2009.07.109

Using an orthogonal protecting group approach to resolve racemic myo-inositol directly as its d-mannoside, we report a [2+n]-glycan/phosphatidylation strategy to assemble a bis-palmityl, tuberculostearyl form of phosphatidyl-myo-inositol dimannoside (PIM-

Full text of DOI:10.1016/j.tetlet.2009.07.109

Tetrahedron Letters 50 (2009) 5664–5666
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Tetrahedron Letters
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Total synthesis of a fully lipidated form of phosphatidyl-myo-inositol
dimannoside (PIM-2) of Mycobacterium tuberculosis
Asif Ali a,b, Markus R. Wenk b, Martin J. Lear a,
*
a Department of Chemistry, Faculty of Science, and Medicinal Chemistry Program of Life Sciences Institute, National University of Singapore, Singapore 117543, Singapore
b Department of Biochemistry and Department of Biological Sciences, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 117543, Singapore
a r t i c l e i n f o
a b s t r a c t
Article history:
Using an orthogonal protecting group approach to resolve racemic myo-inositol directly as its D-manno-
Received 26 June 2009
Revised 13 July 2009
Accepted 21 July 2009
Available online 23 July 2009
side, we report a [2+n]-glycan/phosphatidylation strategy to assemble a bis-palmityl, tuberculostearyl
form of phosphatidyl-myo-inositol dimannoside (PIM-2).
Ó 2009 Elsevier Ltd. All rights reserved.
A third of the world’s population are infected with the causative
organism of tuberculosis (TB), the bacillus Mycobacterium tubercu-
losis.1 In its active state, TB is responsible for over 2 million deaths
every year. Yet the molecular details of TB infection are still not
fully understood. One factor involved in the early pathogenicity
of infection is the interaction of complex glycolipids on the outer
cell wall envelope of the mycobacterium with the mammalian host
cell.2 Ubiquitous amongst the glycolipid core of this lipid envelope
are the phosphatidyl-myo-inositol mannosides (PIMs) and lipoara-
binomannans (LAMs).3
To advance our understanding of the biological roles of such
glycophospholipids,4,5 we have embarked on a program to investi-
gate the immuno-pathogenesis of PIMs. To this end, it was impor-
tant to devise a flexible strategy to synthetic PIMs that would
enable the incorporation of a diverse set of lipid and glycan do-
mains. It was further important to achieve domain homogeneity,
which typically cannot be achieved (or known with certainty)
within biologically derived material. We selected PIM-2 as a piv-
otal unit for making higher order PIMs and herein describe a con-
vergent total synthesis of PIM-2 (1).
successful in utilizing D-mannoside as a permanent resolving unit.
Our strategy to 1 was thus designed to allow 2 to be resolved and
lipidated en route to glycan and phospholipid attachment.
Having determined appropriate protecting groups,14 the
developed resolution method entailed the direct coupling of the
6-O-TBDPS-protected
1-O-PMB/6-O-allyl myo-inositol ( )-516 under
conditions (Scheme 2).12 This afforded a 1:1 mixture of
D
-mannosyl Schmidt donor 415 with the
-selective TMSOTf
-pseu-
a
a
do-disaccharides 6/7 in 72% yield. While these diastereomers
remained inseparable, Pd-mediated removal of the C6-allyl group
allowed for silica gel separation by flash chromatography. In this
way, the pure
a-mannosides 8 and 9 could each be isolated in
30% overall yield from ( )-5 (i.e., ca. 18% yield from commercial
1,2:4,5-dicyclohexylidene-myo-inositol).17
Encouraged by this direct resolution study, we turned our
attention to developing a viable strategy to construct a fully lipi-
dated D-inositide form of PIM-2 from the pseudo-disaccharide 8.
O
PG3O
6'
direct
OBn
O
lipidation
Several partial and total syntheses of PIMs in various lipidated
forms have been reported.6–9 Notably, Patil and Hung constructed
the bis-stearate form of PIM-2 through a new myo-inositol desym-
metrization strategy.10 In order to prepare fully lipidated forms 1
of PIM-2 (Scheme 1),11 we envisaged a resolvable pseudo-disac-
charide 2 that could be coupled to the H-phosphonate of a tuber-
culostearyl/palmityl glyceride 3. From prior experience,6,12 we
resolution
BnO
BnO
C15H31
OH
O
O
HO
6 OBnO
HO
PG2O
OBn
OBn
PG1O
OH
O
HO
[2+1]-glycan
strategy
2
1
HO
2 (pseudo-disaccarhide)
HO
O
OH
OH
6
O
OH
OH
speculated that a D
-mannosyl donor bearing a bulky C60-protecting
O
P
H
O
O
O
2
10R
1
group (PG3) could allow for the direct resolution of a C2-coupled
myo-inositide (2). Although chiral auxillaries have been attached
temporarily to achieve such resolutions,13 the orthogonal combi-
nation of PMB (PG1), allyl (PG2) and TBPDS (PG3) eventually proved
O
P
O
O
O
8
7
O
O
C15H31
O
8
7
O
C15H31
O
1 (PIM-2)
3 (H-phosphonate)
O
* Corresponding author. Tel.: +65 6516 3998; fax: +65 6779 1691.
E-mail address: chmlmj@nus.edu.sg (M.J. Lear).
Scheme 1. Retrosynthetic disassembly of PIM-2 (1).
0040-4039/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2009.07.109

A. Ali et al. / Tetrahedron Letters 50 (2009) 5664–5666
5665
OBn
O
Subsequent attachment of the palmitate ester by reacting pal-
mitoyl chloride with 12 in pyridine afforded 13 in good yields.
Again, somewhat surprisingly, standard oxidative conditions (pH-
buffered DDQ or CAN) to remove the 1-O-PMB-protecting group
in 13 proved problematic. Care was also needed to prevent cleav-
age of the newly installed fatty ester side-arm of the 2-O-manno-
side. Eventually we succeeded in the selective removal of the
1-O-PMB group with TFA/CH2Cl2 (9:1) to yield the known glycan
14.8
TBDPSO
BnO OH
BnO
BnO
AllylO
PMBO
OBn
OBn
X
4
( )-5
TMSOTf, CH2Cl2, 4 Å MS
5, -10ºC, 45 min (72%)
(X=OC(=NH)CCl3)
OBn
O
OBn
TBDPSO
BnO
BnO
TBDPSO
O
BnO
BnO
Important to future biological work was the differential selec-
tion of lipids associated with TB. We thus prepared 10R-methyloc-
tadecanoic acid, commonly known as tuberculostearic acid
(TBSA),19 and subsequently constructed the desired diacyl H-phos-
phonate 38 using established methods.12 Initial attempts to couple
the phospholipid moiety 3 with the palmitylated glycan 14 were
unsuccessful; for example, mixing 3 and 14 in the presence of piva-
loyl chloride and pyridine over 12 h at room temperature, typically
gave no reaction.20 We reasoned that the TBSA and palmitate es-
ters might hinder nucleophilic attack of the C1-alcohol of 14 onto
the mixed anhydride intermediate derived from 3. We thus added
a stronger base (Et3N) to promote the reaction. Under these condi-
tions, the H-phosphonate diester formed reliably over 18 h, which
was subsequently oxidized in situ with iodine in wet pyridine to
afford the phosphate diester 15 in 78% yield (Scheme 3).21 Com-
pound 15 was isolated as its triethylammonium salt (HR-MS found
2314.906 and 2314.902; calcd 2314.370 for C142H194O24P) and cor-
related well with that reported by the Seeberger group.8 Subse-
quent controlled global hydrogenolysis of the benzyl-protecting
+
BnO
6
O
6
OOBn
AllylO
OAllyl
OPMB
OBn
OBn
BnO
BnO
PMBO
1
1
α-6
inseparable
α-7
PdCl2, NaOAc
AcOH, rt, 18 h
D-series
resolution
L-series
OBn
O
OBn
O
TBDPSO
BnO
BnO
TBDPSO
BnO
BnO
BnO
O 6
6
OBnO
HO
OBn
OBn
BnO
BnO
OH
OPMB
PMBO
1
1
8 (42%)
9 (44%)
Scheme 2. Direct
a-mannoside resolution of myo-inositol.
(Scheme 3). First, we determined suitable conditions to form a
higher order [2+1] glycan domain. Under the established
i
TMSOTf-promoted conditions,12
a
-mannosidation of the
-manno-pyranosyl trichloroace-
timidate 10 occurred smoothly at the 6-position to give the
dimannoside 11 of -myo-inositol in excellent yield. Removal of
D-isomer
groups over Pd/C (H2, EtOAc/THF, PrOH, H2O, rt, 3 days) gave 1
in 91% yield, in fully lipidated D-myo-inositide form. For the first
8 with 2,3,4,6-tetra-O-benzyl-a-D
a, -
a0
time, the exact mass of m/z 1413.9016 (calcd m/z of 1413.9003
for C72H134O24P-) was confirmed by FT-MS-ESI analysis of 1 from
a 1:1 solution of CH3CN/H2O (Thermo Scientific LTQ-Orbitrap XL,
negative mode).
In summary, under a program to provide homogenous materials
for glycolipid-based immunomodulators and carbohydrate-based
vaccines against TB, we have reported the synthesis of a fully lipi-
dated, tuberculostearate form 1 of the phosphatidyl-myo-inositol
dimannoside (PIM-2) ofM. tuberculosis. Our approach implemented
an orthogonal set of protecting groups to identify the optimal
D
the TBDPS-ether group in 11 proved to be problematic. A number
of acidic and fluoride-based desilylation conditions were screened,
but the yield of 12 could not be improved beyond 42% in a clean
fashion. Conceivably, the 6-O-
a
-linked mannose hindered the
-linked mannoside, an
cleavage of the TBDPS ether of the 2-O-
a
observation which has some precedence.18 Importantly, this glyco-
sylation strategy has potential to be expanded to higher order
[2+n] glycan domains by using oligosaccharide donors akin to 10.
6' OBn
O
6' OBn
O
TBDPSO
HO
OBn
O
BnO
BnO
BnO
BnO
BnO
6'
BnO
OBn
O
BnO
OBn
O
OBn
O
TBDPSO
1N TBAF, THF
rt, 36 h (42%)
BnO
BnO
BnO
BnO
BnO
BnO
BnO
BnO
OC(=NH)CCl3
10
OBnO
6
O
O
HO
OBn
OBn
OBn
OBn
6
TMSOTf, CH2Cl2, 4 Å MS
-10ºC, 40 min (96%)
PMBO
O
OBn
OBn
O
OBn
OBn
PMBO
PMBO
2
8
11
12
O
C15H31COCl, py
rt, 18 h (93%)
O
O
O
C15H31
C15H31
C15H31
OR
O
O
6'
OBn
OBn
O
RO
O
O
RO
OR
BnO
BnO
BnO
BnO
TFA, CH2Cl2 (9:1)
rt, 30 min (81%)
3, PivCl, py
Et3N, 18 h;
RO
OBn
O
OBn
O
O
BnO
BnO
BnO
BnO
BnO
BnO
RO
RO
O
I2, py, H2O
4 h, rt (78%)
OR
O
O
OBn
OBn
O
OR
O
OR
O
OBn
OBn
O
OBn
OBn
1
O
HO
PMBO
1
O
P
O
14
13
O
O
8
7
15 R = Bn
H2, Pd/C
(91%)
O
C15H31
1
R = H
O
Scheme 3. Assembly of PIM-2 (1) via [2+1]-glycan construction, lipidation and phosphatidylation.

5666
A. Ali et al. / Tetrahedron Letters 50 (2009) 5664–5666
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(8), we established a convergent assembly of the C60-O-palmitylat-
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12. Ali, A.; Gowda, C.; Vishwakarma, R. A. Chem. Commun. 2005, 519–521.
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[2+n] glycan strategy is capable of providing a versatile range of
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17. Direct
a
-mannoside resolution of myo-inositol: the
D-mannosyl imidate 4
(0.645 g, 0.77 mmol) and racemic inositol
5
(0.400 g, 0.65 mmol) were
both D- and L-inositide forms will be reported elsewhere.
combined and co-evaporated (3 times) with toluene. After this pre-drying
process, freshly activated MS (4 Å) were added under argon. The reaction
mixture was then dissolved in anhydrous CH2Cl2 (10 mL) and cooled to ꢀ10 °C
Acknowledgements
followed by the dropwise addition of TMSOTf (12.0 lL, 0.065 mmol). After
stirring for 45 min, the reaction mixture was quenched by the addition of Et3N
(100 L) and filtered through Celite. The solvents were removed in vacuo and
the resulting crude material was purified by flash silica gel column
chromatography to afford the isomers -6/7 (710 mg, 72%). The isomers -6/
(0.525 g, 0.41 mmol), anhydrous NaOAc (550 mg, 6.7 mmol) and PdCl2
(515 mg, 2.85 mmol) were dissolved in a mixture of AcOH–H2O (19:1, 20 mL)
under argon with the aid of sonication. After stirring at room temperature for
18 h, the reaction mixture was quenched carefully by the addition of cold
saturated NaHCO3 solution. The aqueous layer was extracted with EtOAc
(3 ꢁ 50 mL) and the combined organic layers were dried over Na2SO4 and
evaporated in vacuo. The residue was purified by flash silica chromatography
This research was supported in part by the Novartis Institute for
Tropical Diseases (NITD), and in part by the Competitive Research
Program (CRP) of the National Research Foundation (NRF) of Singa-
pore (NRF-G-CRP 2007-04). We thank Karthik Sekar and Ravi K.
Sriramula for preparing several starting materials and intermedi-
ates for this project.
l
a
a
7
Supplementary data
(hexane/EtOAc, 85:15) to provide the desired
D-myo-inositide 8 (210 mg, 42%;
Rf = 0.40) and -inositide 9 (220 mg, 44%; Rf = 0.42). TLC (hexane/EtOAc, 3:1).
See Supplementary data for characterization data.
L
Experimental procedures, characterization data and scanned
spectra for key compounds and intermediates are given. Supple-
mentary data associated with this article can be found, in the on-
line version, at doi:10.1016/j.tetlet.2009.07.109.
18. Ainge, G. D.; Parlane, N. A.; Denis, M.; Hayman, C. M.; Larsen, D. S.; Painter, G. F.
Bioorg. Med. Chem. Lett. 2006, 14, 7615–7624.
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20. Martin, S. F.; Josey, J. A.; Wong, Y. L.; Dean, D. W. J. Org. Chem. 1994, 59, 4805–
4820.
21. Phosphatidylation of lipidated glycan: the pseudo-trisaccharide 14 (15 mg,
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mixture was dissolved in anhydrous pyridine (2 mL) followed by the addition
0
of freshly activated molecular sieves (4 AÅ). Pivaloyl chloride (27
and dry Et3N (150 L) were added to the resulting mixture with stirring. After
18 h at room temperature, a solution of iodine (40 mg, 155 mol) in pyridine/
lL, 225 lmol)
l
l
water (19:1, 0.2 mL) was added (to oxidize P(III) to the P(V) species) and the
reaction mixture was stirred for another 4 h at the same temperature. The
reaction mixture was then diluted with CHCl3 and washed with 5% aqueous
Na2S2O3 solution, and the aqueous layer was re-extracted with CH2Cl2
(3 ꢁ 20 mL). The combined organic layers were washed with TEAB buffer
(10 mL) and dried over Na2SO4. Evaporation under reduced pressure gave a
crude residue, which was purified by flash column chromatography with Et3N-
deactivated silica gel to give the known glycan 158 (17 mg, 78%) as a pale-
yellow syrup. See Supplementary data for characterization data.
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11. For earlier total syntheses of PIM-2 forms and related synthetic studies, see:
Elie, C. J. J.; Verduyn, R.; Dreef, C. E.; Brounts, D. M.; Vandermarel, G. A.; van