Concise synthesis of an arabinofuranose hexasaccharide present in the cell wall of Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.tetlet.2012.03.016

Mycobacterium cell wall consists of three major polysaccharide portions and arabinofuranose (Araf) is present in two of the major portions, arabinogalactan (AG) and lipoarabinomannan (LAM). A peculiar Araf hexasaccharide possessing two β-linked Araf units

Full text of DOI:10.1016/j.tetlet.2012.03.016

Tetrahedron Letters 53 (2012) 2461–2464
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Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Concise synthesis of an arabinofuranose hexasaccharide present in the cell
wall of Mycobacterium tuberculosis
Karnakar C. Reddy a, Narra Padmaja a, Vibha Pathak a, Ashish K. Pathak a,b,
⇑
a Department of Chemistry, Western Illinois University, Macomb, IL 61455, USA
b Organic Chemistry Department, Southern Research Institute, 2000 9th Avenue South, Birmingham, AL 35205, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Mycobacterium cell wall consists of three major polysaccharide portions and arabinofuranose (Araf) is
present in two of the major portions, arabinogalactan (AG) and lipoarabinomannan (LAM). A peculiar Araf
hexasaccharide possessing two b-linked Araf units are present in both AG and LAM polysaccharides.
Herein, we report an efficient and concise synthesis of this Araf hexasaccharide using single starting
3,5-TIPDS protected Araf thioglycoside precursor. Double b-glycosylation was achieved using strained
cyclic 2-p-methoxybenzyl-3,5-TIPDS Araf thioglycoside donor.
Received 1 February 2012
Revised 2 March 2012
Accepted 5 March 2012
Available online 10 March 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Mycobacterium tuberculosis
Oligosaccharide
Arabinofuranose
Thioglycoside
Glycosylation
Mycobacterium tuberculosis has attracted renewed attention
over the last three decades owing to increasing infections world-
wide.1 Additionally, AIDS patients and others with compromised
immune systems are susceptible to opportunistic infections caused
by Mycobacterium avium and Mycobacterium kansaii, resulting in a
mortality rate of millions per year.2 Drugs with novel targets are
urgently needed especially with the emergence of multiple drug
resistant (MDR-TB)3 and extreme drug resistant (XDR-TB)4 strains.
Biosynthesis of the mycobacterial cell wall is one such vital drug
target which protects the bacillus within the macrophage and also
functions as an effective barrier to antibiotics.5 The mycobacterial
cell envelope is typically composed of complex polysaccharides
bound to mycolic acids and the three major macromolecular com-
ponents of the cell wall are lipoarabinomannan (LAM), arabinoga-
lactan (AG), and peptidoglycan.6 Arabinan portion in AG and LAM
bacteria to construct cell wall AG and LAM are critical survival
pathway and the inhibitors of the involved glycosyltransferase en-
zymes are good targets for drug discovery.8
A complete synthesis of the arabinan portion and its fragments
has already been reported.9 The key installation of b-Araf units was
achieved by several methods and initially it was achieved by the
Intermolecular Aglycon Delivery (IAD) method.10 Other methods
employed for the synthesis of major fragments of the arabinan
with b-Araf units have successfully used p-tolyl 1-thio-2,3,5-tri-
O-benzyl arabinofuranoside or conformationally anchored 2-ben-
zyl thioarabinoglycoside.11 A reliable and general direct method
for the stereoselective b-arabinofuranosylation employing a 20-
carboxybenzyl arabinofuranoside as the glycosyl donor has also
been reported.12
Here, we report an efficient synthesis route to synthesize neo-
glycolipid hexasaccharide 1 (Fig. 1) containing two b-Araf units,
which can be utilized to study biosynthesis of arabinan portion
present in cell wall of TB. The ether protective group at C-2 position
on arabinosyl donors is essential for the b-stereoselectivity.10–13
The protective groups at 3- and 5-positions were also found to af-
fect the stereoselectivity of arabinofuranosylation. Conformational
restraint introduced by the eight-membered ring by tetraisopro-
pyldisilylene at 3,5-O-protection can aid for the enhanced b-selec-
tivity. The b-selectivity was drastically enhanced by using donors
protected with 3,5-TIPDS, possibly due to conformational con-
straints on the furanose ring.13 To simplify synthesis of 1, we chose
an approach in which it was assembled via protected hexasaccha-
ride 2 utilizing donors 3–5 containing the suitable functional
is a linear oligomer (
inan pentasaccharide at the terminus that consists of (
1?3), and (b1?2) linked Araf units.7 The assembly of the arab-
a1?5) linked Araf units and a branched arab-
a1?5),
(a
inan portions of cell wall polysaccharides in mycobacteria involves
a family of arabinosyltransferases (AraTs) that promote the poly-
merization of decaprenolphosphoarabinose (DPA) as the substrate,
and each enzyme constructing different glycosidic linkages. These
known AraTs include the Emb (EmbA, EmbB, and EmbC) proteins,
AftA, AftB, and AftC enzymes, which are responsible for biosynthe-
sis of arabinan with unique glycosyl linkages.7 The ability of myco-
⇑
Corresponding author. Tel.: +1 205 581 2542; fax: +1 205 581 2447.
E-mail addresses: pathaka@southernresearch.org, pathaka@sri.org (A.K. Pathak).
0040-4039/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tetlet.2012.03.016

2462
K. C. Reddy et al. / Tetrahedron Letters 53 (2012) 2461–2464
OBz
OH
O
OH
O
O
i-Pr
i-Pr Si
O
i-Pr
i-Pr Si
O
OH
HO
O
c
O
b
O
a
STol
STol
HO
HO
STol
O
O
Si
i-Pr
Si
i-Pr
HO
OH
O
i-Pr
i-Pr
3
7
8
O
d
OH
O
O
OH
OPMB
OLev
i-Pr
i-Pr Si
O
O
OH
i-Pr
i-Pr Si
O
O
O
O
OH
O
O
O
CO2Me
STol
O
4
STol
O
Si
O
O
OH
Si
i-Pr
HO
i-Pr
4
i-Pr
i-Pr
O
5
OH
HO
1
O
Scheme 1. Reagents and conditions: (a) Ref.12; (b) BzCl, dry Py, rt, 0 °C–rt, 3 h, 88%;
(c) Levulinic acid, DCC, DMAP, CH2Cl2, 0 °C, 1 h, 90%; (d) entry 9 in Table 1.
OH
thioglycoside 5 was obtained in 77% yield upon reaction of 7 with
p-methoxybenzyl bromide and NaH in DMF at ꢀ70 °C after column
chromatographic purification.
Figure 1. Structure of neo-glycolipid Araf hexasaccharide similar to Araf branched
polysaccharide present in cell wall of Mycobacterium tuberculosis.
The next task was to synthesize the diol acceptor, methyl 6-
hexanoate anchored glycoside 9, which was achieved in a two step
reaction sequence from thioglycoside 3 (Scheme 2). The glycosyla-
tion of 3 with methyl 6-hydroxyhexanoate at 0 °C with activator
groups at C-2 position for 1,2-trans and -cis regio- and chemoselec-
tive glycosylations (Fig. 2). The p-methoxybenzyl (PMB) protecting
group at C-2 in the donor 5 was selected over benzyl or silyl ether
because if the direct coupling reaction does not produce cleaner b-
arabinosylation, then an indirect activation and coupling IAD ap-
proach10 can be exploited to install desired b-Araf units. The syn-
thesis of all three donor precursors 3–5 can be achieved from
one single 3,5-TIPDS protected Araf thioglycoside 7.
NIS and Lewis acid promoter AgOTf gave exclusive
a-isomer of
arabinofuranoside 6. The cyclic siloxane protecting group in 6
was removed by Et4N+Fꢀ to obtain acceptor 3,5-diol glycoside 9
in quantitative yield.
With gram quantities of all the building arbinofuranosyl units in
hand, we carried out assembly of hexaarabinofuranoside
1
The monosaccharide building blocks 3–5 were synthesized
(Scheme 3). In the first step, selective glycosylation of n-octyl Araf
3,5-diol 9 at C-5 hydroxyl, to preclude several protection and
deprotection steps, was undertaken with 3 using several Lewis acid
activators at various temperatures. Coupling condition was found
to be optimal at ꢀ20 °C in CH2Cl2 promoted by a stoichiometric
amount of Sn(OTf)2 in the presence of NIS. Under these conditions
the coupling reaction gave desired disaccharide 10 with complete
starting from easily accessible p-tolyl 1-thio-a-D-arabinofurano-
side 814 as shown in Scheme 1. Thioglycoside 8 was treated with
1,3-dichloro-1,1;3,3-tetraisopropyldisiloxane (TIPDSCl2) in pyri-
dine for 2 h at 0 °C and after usual workup and purification by col-
umn chromatography provided the 3,5-disiloxane product 713 in
66% yield. The donor thioglycosides 3 and 4 were conveniently
synthesized in large scale from thioglycoside 7. The 2-benzoylation
of 7 with benzoyl chloride at room temperature gave thioglycoside
donor 3 in 98% yield and the reaction of 7 with levulinic acid in the
presence of DCC and DMAP produced 2-levulinoyl ester donor
thioarabinoside 4 in 90% yield. The synthesis of donor thioglyco-
side 5, for b-arabinosylation, seemed very straightforward but
usual conditions did not produce the desired product. Several opti-
mization reactions were performed to achieve of p-methoxybenzy-
lation on thioglycoside 7 as shown in Table 1. Finally, donor
a-selectivity, aided by benzoyl participating group at C2, in 73%
yield. To sustain further glycosylation reactions, the 3-OH group
in 10 was firstly protected with benzoyl group and further treat-
ment of crude product with Et4N+Fꢀ in THF at ꢀ20 °C unmasked
the C3 and C4 hydroxyl groups on the non-reducing end terminus
sugar of disaccharide. Purification of crude product from de-silyla-
tion reaction on Silica gel G column provided disaccharide 11 in
80% yield over two steps.
i-Pr
i-Pr
Si
O
O
i-Pr Si
i-Pr
O
O
OBz
OLev
i-Pr
i-Pr Si
O
O
O
PMBO
O
i-Pr
i-Pr Si
O
O
O
O
O
i-Pr
i-Pr Si
O
O
STol
3
STol
O
O
O
Si
Si
OH
i-Pr
i-Pr Si
O
O
OBz
O
O
i-Pr
i-Pr
4
O
O
O
i-Pr
i-Pr
Si
O
i-Pr
1
i-Pr
STol
O
OPMB
OBz
Si
i-Pr
O
OBz
i-Pr
i-Pr Si
O
O
i-Pr
O
i-Pr
i-Pr Si
O
O
O
O
i-Pr
i-Pr
Si
O
OR
OR
7
STol
5
O
Si
i-Pr
OBz
R =
Si
Si
i-Pr
i-Pr
O
i-Pr
6
i-Pr
i-Pr
O
PMBO
O
CO2Me
O
i-Pr
i-Pr Si
O
4
2
O
Si
i-Pr
i-Pr
Figure 2. Synthetic plan for target hexasaccharide 1.

K. C. Reddy et al. / Tetrahedron Letters 53 (2012) 2461–2464
2463
Table 1
Reaction optimization of p-methoxybenzylation of thioglycoside 7
Entry
Reagent (1.1 equiv)
Base (1.0 equiv)
Solvent
Temp (°C)
Time (h)
Yield (%)b
1
2
3
4
5
6
7
8
9
PMBCl
PMBCl
PMBCl
PMBBr
PMBBr
PMBBr
PMBBr
PMBBr
PMBBr
NaH
NaH
CsCO3
NaH
NaH
NaH
NaH
NaH
NaH
DMF
THF
DMF
DMF
THF
DMF
DMF
DMF
DMF
0–rt
0
0–50
0–rt
0–rt
0
ꢀ20
ꢀ50
ꢀ70
0.5
0.5
12
1
1
1
1
12
20
0
10
12
10
15
20
25
60
77
a
Isolated yield; purification was performed on neutral Alumina media.
OBz
OBz
i-Pr
i-Pr Si
O
O
HO
O
O
b
OBz
O
a
i-Pr
i-Pr Si
O
O
3
O
O
CO2Me
O
CO2Me
O
3
Si
a
OH
3
9 + 3
OBz
O
9
i-Pr
6
i-Pr
O
Si
i-Pr
i-Pr
Scheme 2. Reagents and conditions: (a) NIS, AgOTf, dry CH2Cl2, ꢀ20 °C, 15 min,
82%; (b) Et4N+Fꢀ, dry THF, 0 °C, quant, 3 h.
O
CO2Me
10
OH
3
b
To achieve the synthesis of tetrasaccharide 12, double glycosyl-
ation with 2-levulinoyl containing Araf donor 4 was carried out at
3- and 5-positions on the disaccharide 11. Tetrasaccharide 12 was
obtained in 81% yield after purification. Removal of levulinoyl
groups on 12 with hydrazine acetate gave acceptor 13 which was
then further used in double b-arabinofuranosylation with donor
thioglycoside 5. In a small scale trial reaction, tetrasaccharide 13
was reacted for 1.5 h with 2.5 equiv of donor thioglycoside 5 in
the presence of activator NIS and TMSOTf at ꢀ78 °C. The reaction
was monitored on TLC which showed complete consumption of
donor glycoside 5 and formation of two products, one major hexa-
saccharide 2 and other a pentasaccharide (supported by MS analy-
sis of reaction mixture). Thereafter, one more equivalent of donor
thioglycoside 5 was added at ꢀ78 °C to the above reaction mixture
and was further stirred for 30 min. The TLC of the reaction mixture
showed the complete conversion of mixture to hexasaccharide 2.
Next, the above reaction was repeated again with 150 mg of tetra-
saccharide 13 which was treated for 2 h with 3.5 equiv of donor
thioglycoside 6 in the presence of activator NIS and TMSOTf at
ꢀ78 °C and TLC showed the formation of one product. After usual
workup and column chromatographic purification on silica gel,
double b-arabinosylated hexasaccharide 2 was obtained in 79%
yield as an oil with excellent stereoselectivity. In the 13C NMR
OBz
O
HO
O
OBz
O
OH
11
O
CO2Me
OBz
3
c
OR
O
i-Pr
O
i-Pr Si
OBz
O
O
i-Pr
O
O
O
Si
O
i-Pr
i-Pr
Si
OBz
O
i-Pr
O
O
O
O
CO2Me
Si
i-Pr
OBz
3
i-Pr
O
OR
12. R = Lev
d
13.
R = H
i-Pr
Si
i-Pr
O
i-Pr Si
i-Pr
O
e
O
O
PMBO
O
O
spectrum of 2, the four anomeric carbon signals of
a-linked Araf
i-Pr
i-Pr Si
O
O
units were seen at d 106.08, 105.66, 105.63, and 105.29 ppm
whereas, the signals from two anomeric carbons of b-linked Araf
units were observed at d 98.95 and 98.77 ppm.9 Cyclic siloxane
TIPDS protecting groups in hexasaccharide 2 were removed by
reacting overnight at ꢀ20 °C with Et4N+Fꢀ in THF. After removal
of the reaction solvent, the crude product was purified by a quick
column chromatography on Silica gel. The desilylated hexasaccha-
ride product thus obtained was treated with ceric ammonium ni-
trate in CH3CN–H2O mixture to remove p-methoxybenzyl groups
and after usual workup the product was used without purification
in the final de-benzoylation step. The crude product obtained from
the above reaction was treated overnight with 7 N NH3 in MeOH
solution and desired hexaarabinofuranoside 1 was obtained in
82% yield after column chromatographic purification. The 1H and
13C NMR spectra of 1 showed the signals corresponding to the de-
sired product. In the 13C NMR spectra of 1 in CD3OD, the four ano-
OBz
O
O
O
Si
O
i-Pr
i-Pr
i-Pr
OBz
f
O
i-Pr
O
O
1
Si
O
O
O
CO2Me
Si
3
i-Pr
OBz
i-Pr
O
O
PMBO
O
2
i-Pr
O
i-Pr Si
O
O
i-Pr
Si
i-Pr
Scheme 3. Reagents and conditions: (a) NIS, Sn(OTf)2, dry CH2Cl2, 10 min, ꢀ20 °C,
73%; (b) (i) BzCl, dry Py, rt, 4 h; (ii) Et4N+Fꢀ, THF, ꢀ20 °C–rt, 3 h, overall 80% in two
steps; (c) 4 (2.5 equiv), NIS, AgOTf, dry CH2Cl2, 0 °C, 2 h, 81%; (d) hydrazinium
acetate in MeOH, CH2Cl2, rt, 4 h, 82%; (e) 5 (3.5 equiv), NIS, TMSOTf, dry CH2Cl2,
ꢀ78 °C, 2 h, 79%; (f) (ii) Et4N+Fꢀ, dry THF, rt, overnight; (ii) CAN, THF–H2O, 0 °C, 6 h;
(iii) 7 N NH3/MeOH, rt, overnight, overall 82% in three steps.
meric carbon signals from a-Araf units were observed at d 108.19,
108.07, 106.12, and 105.83 ppm, and two anomeric carbon signals
at d 101.27 and 101.19 ppm were assigned to b-Araf units.9

2464
K. C. Reddy et al. / Tetrahedron Letters 53 (2012) 2461–2464
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manipulations and several reaction steps were combined together
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Supplementary data
Supplementary data (experimental and analytical details of all
new compounds) associated with this article can be found, in the
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