Synthesis and biotinylation of oligosaccharide fragments of mannosylated and 5-deoxy-5-methylthio-xylofuranosylated lipoarabinomannan from Mycobacterium tuberculosis

Article abstract of DOI:10.1016/j.carres.2015.01.003

The attachment of biotin to a molecule provides a powerful tool in biology. Here, we report an efficient synthesis and biotinylation of mannosylated and 5-deoxy-5-methylthio-xylofuranosylated Lipoarabinomannan from Mycobacterium tuberculosis. Preparation

Full text of DOI:10.1016/j.carres.2015.01.003

Carbohydrate Research 407 (2015) 104e110
Contents lists available at ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Note
Synthesis and biotinylation of oligosaccharide fragments of
mannosylated and 5-deoxy-5-methylthio-xylofuranosylated
lipoarabinomannan from Mycobacterium tuberculosis
, b,
Pintu Kumar Mandal a *, Pratik Rajesh Chheda a
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, PO Box 173,
Lucknow 226 031, India
b Academy of Scientific and Innovative Research, New Delhi 110001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The attachment of biotin to a molecule provides a powerful tool in biology. Here, we report an efficient
synthesis and biotinylation of mannosylated and 5-deoxy-5-methylthio-xylofuranosylated Lip-
oarabinomannan from Mycobacterium tuberculosis. Preparation of the oligosaccharides involved the
sequential addition of thioglycoside donors with arabinofuranosyl-containing acceptors. Methylthio
group was introduced near the end of the synthesis.
Received 5 November 2014
Received in revised form
6 January 2015
Accepted 11 January 2015
Available online 11 February 2015
© 2015 Elsevier Ltd. All rights reserved.
Keywords:
Tuberculosis (TB)
5-Deoxy-5-methylthio-xylofuranose (MTX)
Glycosylation
Biotin
Lipoarabinomannan (LAM)
1. Introduction
oxygen free radicals.7 In some species the peripheral portion of LAM
consisting of arabinofuranosyl residue is capped with short man-
Tuberculosis (TB) continues to be world's deadliest communi-
cable disease, with an estimated 9 million new cases and 1.5 million
deaths in 2013.1 The hallmark of anti-mycobacterial treatment is
the use of multiple antibiotics over several months,2 which has led
to development of Multi-Drug-Resistant Tuberculosis (MDR-TB)
and Extremely-Drug-Resistant Tuberculosis (XDR-TB).1 The reason
behind difficulty in treatment of mycobacterial infections is the
unusual structure of its cell wall, which provides a daunting barrier
for the passage of antibiotics.3,4
The cell wall of mycobacteria is composed of a complex mixture of
polysaccharides, proteins, and lipids. Arabinogalactan (AG) and lip-
oarabinomannan (LAM) are the major structural polysaccharide
components of the cell wall.5 The polysaccharide component of LAM
is composed of mannopyranosyl and arabinofuranosyl residues,
linked via inositol moiety to the lipid portion of mycobacterial cell
wall. LAM has shown the ability to inhibit protein kinases6 and
macrophage activation along with the ability to neutralize cytotoxic
nopyranosyl oligosaccharides, together being termed Mannosylated
Lipoarabinomannan (ManLAM). It has been noted that adherence of
ManLAM to human cells through its interactions with mannose
binding proteins plays an important role in initial stages of infection.8
Drugs inhibiting the enzymes that assemble these oligosaccharides
can be useful in the prevention and treatment of mycobacterial dis-
ease, design of which would be greatly aided by the isolation and
characterization of respective mannosyltransferases. In addition to
this, there are reports involving binding of mycobacterium specific
antibodies to oligosaccharide fragments of LAM, making them po-
tential haptens for anti-tuberculosis vaccines.9
The detailed structural features of LAM demonstrated in
1990's10,11 were later modified by the discovery of a novel natural
thiosugar i.e. 5-deoxy-5-methylthio-pentose, being attached to
LAM (Fig. 1).12 The 5-deoxy-5-methylthio-xylofuranose (MTX)
sugar has been identified in both, laboratory strains (H37Rv and
H37Ra)12 as well as clinical isolates (CSU20 and MT103)12,13 of
Mycobacterium tuberculosis. Studies on MTX have indicated its
ability to inhibit cytokine response similar to ManLAM,14 and their
immuno-modulatory potential has been demonstrated by their
ability to inhibit cytokine response induced by Staphylococcus
* Corresponding author.
E-mail address: pintuchem06@gmail.com (P.K. Mandal).
http://dx.doi.org/10.1016/j.carres.2015.01.003
0008-6215/© 2015 Elsevier Ltd. All rights reserved.

P.K. Mandal, P.R. Chheda / Carbohydrate Research 407 (2015) 104e110
105
Fig. 1. Representative fragment structure for lipoarabinomannan (LAM) based on the typical carbohydrate composition of Mtb.
aureus Cowan strain (SAC) and Interferon-
g
(IFN-
g
) in human
containing acceptor 5 was synthesized employing the process re-
ported by Fletcher et al.21, which involves chlorination of 1022 at the
anomeric position, by bubbling hydrochloride gas through its so-
lution in dry CH2Cl2 followed by simple concentration to give the
labile chloride 11.22 This was reacted without purification with
benzyl N-(3-hydroxypropyl)carbamate in dry CH2Cl2 in the absence
mono-cytic cell line (THP-1).15
MTX, like other furanose sugars, would be inherently less stable
than the pyranose form. Mycobacteria host a number of furanosidic
oligosaccharides, which are biosynthesized from UDP-
galactopyranose with the help of specific mutase enzymes.16,17 But
in case of MTX, due to the presence of a thioether substituent at C-5,
the pyranose ring form is not possible and thus mycobacterium
would be investing a lot of effort in the biosynthesis of MTX. Thus
the study of the biosynthetic pathway for MTX would be helpful in
developing newer antibiotics and certain MTX specific antibodies,
which would ease the process of quantitation of MTX substituents.
The progress in this direction has been rather slow mainly due to the
lack of synthetic substrates useful for fundamental biochemical
studies leading to clarification of these biosynthetic pathways.
Venisse et al. have reported the utilization of biotinylated
ManLAM for studying its antigenic properties and its ability to
interact selectively with murine phagocytes.18 Biotinylation
involving a disulphide bond is useful for labelling and purifying
proteins and carbohydrates on the cell surface, as it attaches the
desired molecule on the surface, without permeating the cell
membrane. To add to this, binding of biotinylated nucleotides to
avidin-agarose affinity column, followed by subsequent elution by
reduction of the disulphide bond using reducing agents such as
Dithiothreitol (DTT),19 provided us with further rationale for bio-
tinylation of synthesized oligosaccharides.
of
a
promoter to give an intermediate
b
-glycoside product,
isomer. This crude
contaminated with minor quantity (~5%) of the
a
23
ꢀ
product was deacetylated under Zemplen conditions and then
purified by column chromatography to give desired compound 5 in
42% overall yield (Scheme 1).
2.2. Synthesis of trisaccharide 1
With all the required building blocks in hand, arabinofuranosyl
derivative 5 was glycosylated with thioglycoside derivative 624 in
the presence of N-iodosuccinimide and silver triflate25 to provide
the protected disaccharide derivative 12 in 82% yield with trace
amounts of
b isomer. The disaccharide 12 was then deacetylated
ꢀ
under Zemplen conditions23 to furnish the disaccharide alcohol 13
in 93% yield. The trisaccharide 15 was synthesized using a similar
procedure involving initial glycosylation of disaccharide 13 with
the thioglycoside donor 726 under the same reaction conditions, to
furnish the trisaccharide 14 in 86% yield.
Trisaccharide 14 was deacetylated using sodium methoxide to
furnish the trisaccharide 15 in 90% yield. A portion of this product
was completely deprotected by hydrogenation27 providing 1 in 82%
yield (Scheme 2).
With the aim of recognising antibodies generated against LAM,
and to determine their specificity towards ManLAM, Gadikota et al.20
had reported the synthesis of some oligosaccharide fragments of
ManLAM, covalently conjugated to 8-aminooctyl aglycone, having
the ability to form neoglycoconjugates. Adding to this; with the
additional motive of providing synthetic substrates for biochemical
studies, herein we describe an accessible synthesis of compounds 1
& 2 along with their biotinylated analogs 3 & 4 containing a cleav-
able disulfide bond in the 12-atom linker connecting them (Fig. 2).
2.3. Synthesis of tetrasaccharide 2
To synthesize the tetrasaccharide 2 containing the D-enantiomer
of MTX, an approach in which the methylthio substituent would be
introduced at the final step was developed. The process required
the synthesis of tetrasaccharide 2 having a leaving group at 5-
position of the xylofuranosyl moiety. The penultimate tetra-
saccharide 16 was synthesized by glycosylation between remaining
trisaccharide 15 and monosaccharide 815 using N-iodosuccinimide
and silver triflate. A minor quantity of 1,2-trans isomer product was
also formed under these reaction conditions. The crude product
was purified by column chromatography to give 16 in 77% yield and
the stereochemistry of compound 16 was confirmed from its
2. Result and discussion
2.1. Synthesis of monosaccharide 5
The target molecules 1 & 2 were straightforwardly synthesized
using
a
combination of sequential glycosylation with
arabinofuranosyl-containing acceptors. The arabinofuranosyl-
spectroscopic analysis [signals at
d
5.24 (d, J¼4.2 Hz, H-1D), 5.18 (br

106
P.K. Mandal, P.R. Chheda / Carbohydrate Research 407 (2015) 104e110
Fig. 2. Synthetic targets and their precursors.
s, H-1C), 4.91 (br s, H-1B), 4.79 (d, J¼3.8 Hz, H-1A) in the 1H NMR and
3. Conclusion
d
100.8 (C-1D), 100.7 (C-1A), 99.4 (C-1B), 99.2 (C-1C) in the 13C NMR
spectra] (Scheme 2). Finally the methylthio group was introduced
on the xylofuranose residue by refluxing tetrasaccharide 16
together with sodium thiomethoxide and 18-crown-6 in acetoni-
trile.15 The desired benzylated tetrasaccharide was purified by
passing through a short pad of SiO2. With the required methylthio
group in place, the benzyl ethers and benzyloxycarbonyl (CBz)
groups were cleaved by Birch reduction employing sodium and
ammonia at ꢀ78 ꢁC in THF.28 The final tetrasaccharide 2 was iso-
lated after purification over Sephadex LH-20 column using
CH3OHeH2O (4:1 v/v) as eluent in 72% overall yield (Scheme 2).
In summary, we carried out the synthesis of mannosyl-
arabinoside and MTX containing mannosyl-arabinoside fragments
of LAM from M. tuberculosis and their biotinylated analogs 3 & 4,
containing a disulfide bond in a 12-atom linker joining biotin. These
biotinylated oligosaccharides can be used as probes to investigate the
role of MTX and ManLAM in mycobacterial pathogenicity and will
find application in studies involving development of vaccines against
M. tuberculosis. A readily prepared p-tolyl 2,3-di-O-benzyl-5-O-tol-
uenesulfonyl-1-thio-b-D-xylofuranoside (8) was used as final glycosyl
donor having a leaving group at the 5-position. The methylthio group
was introduced on xylofuranose at 5-position in the last step.
2.4. Biotinylation of compound 1 and 2
4. Experimental
Finally, compounds 1 & 2 were coupled with sulfosuccinimidyl
2-(biotinamido) ethyl-l,3-dithiopropionate (9) in methanol and the
resulting solution was allowed to stir at room temperature for 8 h
(Scheme 3). The resulting biotin labelled glycoconjugates 3 & 4
were isolated in 82% and 72% respectively.
4.1. General methods
All reactions were monitored by thin layer chromatography over
silica gel-coated TLC plates. The spots on TLC were visualized by
Scheme 1. Reagents and conditions: (a) HCl, CH2Cl2, room temperature; (b) benzyl N-(3-hydroxypropyl)carbamate, CH2Cl2, room temperature; (c) 0.1 M CH3ONa, CH3OH, room
temperature, 42% overall yield.

P.K. Mandal, P.R. Chheda / Carbohydrate Research 407 (2015) 104e110
107
Scheme 2. Reagents and conditions: (a) N-iodosuccinimide (NIS), AgOTf, MS-4Å, CH2Cl2, ꢀ40 ꢁC/0 ꢁC, 82% for compound 12 and 86% for compound 14; (b) 0.1 M CH3ONa, CH3OH,
room temperature, 93% for compound 13 and 90% for compound 15; (c) H2, 20% Pd(OH)2eC, CH3OH, room temperature, 82%; (d) NIS, AgOTf, MS-4Å, CH2Cl2, 0 ꢁC/room tem-
perature, 77%; (e) NaSCH3, 18-crown-6, CH3CN, reflux; (f) Na, NH3, THF, ꢀ78 ꢁC, 72% overall yield.
warming ceric sulfate [2% Ce(SO4)2 in 5% H2SO4 in EtOH]-sprayed
plates on a hot plate. Silica gel 230e400 mesh was used for col-
umn chromatography. 1H and 13C NMR, spectra were recorded on
Brucker Avance 500 MHz and Bruker Avance 300 MHz instrument
spectrometers using CDCl3 and D2O as solvents and TMS as internal
reference unless stated otherwise. Chemical shift values are
additional 10 min. After completion of reaction, the reaction
mixture was concentrated to yield 5-O-acetyl-2,3-di-O-benzyl-a-D-
arabinofuranosyl chloride 11 as a light yellow syrup. The concen-
trated crude product 11 was dissolved in dry CH2CI2 (50 mL)
without any purification and benzyl N-(3-hydroxypropyl)carba-
mate (6.1 g, 28.98 mmol) was added to it. After 6 h of stirring, the
reaction mixture was diluted with CH2C12 (80 mL) and washed
with water (30 mL) and then saturated NaHCO3 solution (2ꢂ50 mL)
and water (50 mL). The organic layers were collected and dried over
anhydrous Na2SO4, then filtered and concentrated as light yellow
syrup. De-acetylation was done by adding few drops of 0.1 M
NaOMe to a solution of crude product in methanol (90 mL). The
reaction was stirred for 1 h. The reaction mixture was concentrated
and the light yellow oil obtained was purified by chromatography
expressed in
d parts per million. ESI-MS were recorded on a JEOL
spectrometer. Elementary analysis was carried out on Carlo ERBA
analyzer. IR spectra were recorded on Shimadzu Spectrophotome-
ters. Optical rotations were determined on Autopol III polarimeter.
Commercially available grades of organic solvents of adequate pu-
rity are used in all reactions.
4.2. 3-(Benzyloxycarbonylamino)propyl 2,3-di-O-benzyl-b-D-
arabinofuranoside (5)
(2:1 hexane: EtOAc) to give 5 (4.5 g, 42%) as a colorless oil.
25
[a
]
ꢀ47.5 (c 1.5, CHCl3); IR (neat): 2837, 1738, 1303, 1218, 1224,
D
1088, 1078, 986, 760, 699 cmꢀ1
;
1H NMR (500 MHz, CDCl3):
Compound 10 (8 g, 19.32 mmol) was dissolved in dry
dichloromethane (30 ml) and HCI gas was bubbled through the
solution for 30 min. The reaction mixture was further stirred for an
d
7.33e7.23 (m, 15H, AreH), 5.55e5.54 (m, 1H, NHCbz), 5.05 (ABq,
2H, COOCH2Ph), 4.80 (d, J¼4.4 Hz, 1H, H-1A), 4.64 (d, J¼11.6 Hz, 1H,
PhCH2), 4.59e4.57 (m, 2H, PhCH2), 4.54 (d, J¼11.6 Hz, PhCH2), 4.19
(dd, J¼6.2, 6.7 Hz, 1H, H-3A), 4.05 (dd, J¼4.5, 7.0 Hz, 1H, H-4A),
4.03e3.99 (m, 1H, H-2A), 3.83e3.78 (m, 1H, OCH2R), 3.67e3.65 (m,
1H, H-5aA), 3.57e3.54 (m, 1H, H-5bA), 3.40e3.38 (m, 1H, OCH2R),
3.37e3.32 (m, 1H, NCH2), 3.27e3.23 (m, 1H, NCH2), 2.64 (dd, J¼4.8,
7.1 Hz, 1H, H-5OH), 1.77e1.75 (m, 2H, eCH2e); 13C NMR (75 MHz,
CDCl3): d 156.6 (COOCH2Ph), 138.3e126.1 (AreC), 100.8 (C-1A), 84.0
(C-4A), 82.0 (C-2A), 81.2 (C-3A), 72.5 (PhCH2), 67.1 (OCH2R), 66.5
(COOCH2Ph), 63.9 (C-5A), 38.9 (NCH2), 29.5 (eCH2e); ESI-MS: m/z
544.2 [MþNa]þ; Anal. Calcd for C30H35NO7 (521.24): C, 69.08; H,
6.76%; found: C, 68.95; H, 6.87%.
4.3. 3-(Benzyloxycarbonylamino)propyl 5-O-(2-O-acetyl-3,4,6-tri-
O-benzyl-
a-D-mannopyranosyl)-2,3-di-O-benzyl-b-D-
arabinofuranoside (12)
Thioglycoside 6 (4.9 g, 9.21 mmol) and alcohol 5 (4.0 g,
7.68 mmol) were placed in a round-bottom flask to which
powdered MS-4Å (4.0 g) were added. The contents were dried in
vacuo overnight. To this, dry CH2C12 (80 mL) was added and the
Scheme 3. Biotinylation of compounds 1 & 2.

108
P.K. Mandal, P.R. Chheda / Carbohydrate Research 407 (2015) 104e110
mixture was stirred for 10 min at ꢀ40 ꢁC. N-iodosuccinimide
(2.49 g, 11.10 mmol) was added at ꢀ40 ꢁC and the reaction was
stirred for 20 min followed by addition of silver triflate (594 mg,
2.30 mmol). The reaction mixture was gradually warmed to 0 ꢁC
and then quenched by addition of triethylamine (0.5 mL). The
yellow solution formed was filtered, diluted with CH2Cl2 (100 mL)
and washed with 5% aq Na2S2O3 and water. The organic layers were
collected, dried (Na2SO4) and evaporated to light yellow syrup. The
crude product was purified by column chromatography using
hexane-ethylacetate (5:1) as eluent to furnish disaccharide 12
atmosphere at room temperature. The reaction mixture was then
cooled to ꢀ40 ꢁC and stirred for 10 min. To this, N-iodosuccinimide
(1.53 g, 6.79 mmol) was added and the reaction was allowed to stir
for 20 min before adding silver triflate (365 mg, 1.42 mmol). The
reaction mixture was then allowed to warm gradually to 0 ꢁC
followed by quenching using triethylamine (0.5 mL). The yellow
solution was filtered and diluted with CH2C12 (90 mL). This diluted
solution was washed with saturated Na2S2O3 solution (2ꢂ30 mL),
followed by brine (50 mL) and water (40 mL). The organic layers
were collected, dried (Na2SO4), filtered and concentrated over
vacuum to light yellow syrup. The crude product was purified by
(6.2 g, 82%) as a colourless liquid; [
a
]
25 þ3.7 (c 1.5, CHCl3); IR (neat):
D
2907, 1848, 1473, 1248, 1124, 1107, 1088, 986, 750, 699 cmꢀ1
;
1H
column chromatography (4:1 hexane:EtOAc) to give 14 (5.8 g, 86%)
25
NMR (500 MHz, CDCl3):
d
7.36e7.27 (m, 28H, AreH), 7.17e7.16 (m,
as a colourless syrup; [
a
]
þ4.5 (c 1.5, CHCl3); IR (neat): 2911,
D
2H, AreH), 5.59e5.57 (m, 1H, NHCbz), 5.40 (dd, J¼1.8, 3.0 Hz, 1H, H-
2B), 5.08 (br s, 2H, COOCH2Ph), 4.87 (br s, 1H, H-1B), 4.86 (d,
J¼10.8 Hz, 1H, PhCH2), 4.84 (d, J¼4.4 Hz, 1H, H-1A), 4.68 (dd,
J¼11.6 Hz, 2H, PhCH2), 4.62 (dd, J¼11.6 Hz, 2H, PhCH2), 4.57 (dd,
J¼11.6 Hz, 2H, PhCH2), 4.49 (m, 3H, PhCH2), 4.09 (dd, J¼6.2, 6.7 Hz,
1H, H-3A), 4.06e4.02 (m, 2H, H-2A, H-4A), 3.99 (dd, J¼3.3, 9.4 Hz,
1H, H-3B), 3.91 (t, J¼9.5 Hz, 1H, H-4B), 3.85e3.73 (m, 4H, H-5abA, H-
5B, OCH2), 3.69e3.67 (m, 1H, H-6aB), 3.51 (dd, J¼5.6, 10.1 Hz, 1H, H-
1818, 1303, 1278, 1204, 1137, 1038, 1048, 976, 750, 679 cmꢀ1
;
1H
NMR (500 MHz, CDCl3): 7.24e7.05 (m, 45H, AreH), 5.44e5.42
d
(m, 1H, NHCbz), 5.25 (dd, J¼9.7, 9.3 Hz, 1H, H-4C), 5.05 (br s, 1H, H-
1C), 4.96 (br s, 2H, COOCH2Ph), 4.91 (br s, 1H, H-1B), 4.75 (d,
J¼10.7 Hz, 1H, PhCH2), 4.69 (d, J¼4.2 Hz, 1H, H-1A), 4.55 (d,
J¼12.2 Hz, 1H, PhCH2), 4.53e4.37 (m, 12H, PhCH2), 4.35 (d,
J¼12.2 Hz, 1H, PhCH2), 4.27 (d, J¼12.2 Hz, PhCH2), 3.99e3.97 (m,
1H, H-2A), 3.95e3.88 (m, 4H, H-2B, H-2C, H-3A, H-4A), 3.85e3.83
(m, 1H, H-4B), 3.75e3.54 (m, 8H, H-3B, H-3C, H-5aA, H-5B, H-6aB, H-
6
bB), 3.44e3.33 (m, 2H, OCH2, NCH2), 3.24e3.17 (m, 1H, NCH2), 2.16
(s, 3H, COCH3), 1.77e1.75 (m, 2H, eCH2e); 13C NMR (75 MHz,
CDCl3): 170.6 (COCH3), 156.6 (COOCH2Ph), 138.5e127.8 (AreC),
6
abC, OCH2), 3.49 (dd, J¼6.3, 6.70 Hz, 1H, H-5C), 3.41 (dd, J¼3.3,
d
11.2 Hz, 1H, H-5bA), 3.35 (dd, J¼5.6, 10.0 Hz, 1H, H-6bB), 3.29e3.19
(m, 2H, NCH2, OCH2), 3.08e3.03 (m, 1H, NCH2), 1.78 (s, 3H, COCH3),
101.2 (C-1A), 98.5 (C-1B), 84.1 (C-4A), 83.2 (C-3A), 80.0 (C-2A), 78.4
(C-3B), 75.5 (PhCH2), 74.4 (C-4B), 73.7 (PhCH2), 72.7 (2C, PhCH2),
72.1 (PhCH2), 72.0 (C-5B) 70.2 (C-6B), 69.0 (OCH2R), 68.9 (C-2B), 67.2
(C-5A), 66.7 (COOCH2Ph), 39.6 (NCH2), 29.6 (eCH2e), 21.4 (COCH3);
ESI-MS: m/z 1018.4 [MþNa]þ; Anal. Calcd for C59H65NO13 (995.44):
C, 71.14; H, 6.58%; found: C, 71.03; H, 6.67%.
1.62e1.59 (m, 2H, eCH2e); 13C NMR (75 MHz, CDCl3):
d 170.3
(COCH3), 156.8 (COOCH2Ph), 138.7e127.8 (AreC), 101.1 (C-1A),
100.2 (C-1C), 99.4 (C-1B), 84.1 (C-4A), 83.4 (C-2A), 80.3 (C-3A), 80.1
(C-3B), 77.4 (C-2B), 75.6 (PhCH2), 75.1 (C-4B), 75.0 (C-3C), 74.8 (C-
2C), 73.7 (PhCH2), 73.6 (2C, PhCH2), 72.8 (PhCH2), 72.7 (2C, PhCH2),
72.3 (PhCH2), 72.0 (C-5C), 71.2 (C-5B), 70.4 (C-6C), 70.1 (OCH2R),
69.4 (C-6B), 69.2 (C-4C), 67.3 (C-5A), 66.8 (COOCH2Ph), 39.8 (NCH2),
29.7 (eCH2e), 21.3 (COCH3); ESI-MS: m/z 1450.5 [MþNa]þ; Anal.
Calcd for C86H93NO18 (1427.63): C, 72.30; H, 6.56%; found: C, 72.18;
H, 6.67%.
4.4. 3-(Benzyloxycarbonylamino)propyl 5-O-(3,4,6-tri-O-benzyl-
-mannopyranosyl)-2,3-di-O-benzyl- -arabinofuranoside (13)
a-
D
b-D
To a solution of compound 12 (5.5 g, 5.52 mmol) in MeOH
(80 mL), few drops of 0.1 M NaOMe were added and the reaction
was stirred at room temperature for 3 h. The reaction was then
neutralised with Amberlite-IR 120 (Hþ) resin, filtered and evapo-
rated to give the crude product, which was purified by column
4.6. 3-(Benzyloxycarbonylamino)propyl 2,3-di-O-benzyl-5-O-[2-O-
(2,3,6-tri-O-benzyl- -mannopyranosyl)-3,4,6-tri-O-benzy-
a-
D
a-D-
chromatography using hexane-EtOAc (2:1) as eluent, to give 13
mannopyranosyl]-b-D-arabinofuranoside (15)
25
(4.9 g, 93%); [
a]
þ3.5 (c 1.5, CHCl3); IR (neat): 2987, 1848, 1273,
D
1218, 1140, 1107, 1048, 986, 750, cmꢀ1; 1H NMR (500 MHz, CDCl3):
7.23e7.17 (m, 28H, AreH), 7.09e7.06 (m, 2H, AreH), 5.49e5.46
To a solution of 14 (4.2 g, 2.94 mmol) in MeOH (80 mL), few
drops of 0.1 M MeONa solution were added. The reaction mixture
was stirred for 5 h at room temperature and then concentrated to
yield a syrup. The crude product was purified by chromatography
d
(m, 1H, NHCbz), 4.97 (br s, 2H, COOCH2Ph), 4.84 (d, J¼1.4 Hz, 1H, H-
1B), 4.72 (d, J¼10.7 Hz, 1H, PhCH2), 4.71 (br s, 1H, H-1A), 4.56e4.51
(m, 5H, PhCH2), 4.47 (d, J¼12.5 Hz, 1H, PhCH2), 4.42 (dd, J¼11.9 Hz,
3H, PhCH2), 4.01 (dd, J¼6.2, 6.8 Hz, 1H, H-3A), 3.98e3.90 (m, 3H, H-
(4:1 hexane:EtOAc) to give 15 (3.7 g, 90%) as a syrup; [
a
]
25 þ4.9 (c
D
1.5, CHCl3); IR (neat): 2997, 1858, 1373, 1238, 1204, 1137, 1088, 1048,
2A, H-2B, H-4A), 3.77e3.56 (m, 7H, H-3B, H-4B, H-5abA, H-5B, H-6aB
,
976, 760 cmꢀ1 1H NMR (500 MHz, CDCl3):
; d 7.25e7.09 (m, 45H,
OCH2), 3.43 (dd, J¼5.6, 10.1 Hz, 1H, H-6bB), 3.34e3.22 (m, 2H, NCH2,
PhCH2), 5.46e5.44 (m, 1H, NHCbz), 5.10 (br s, 1H, H-1C), 4.96 (br s,
2H, COOCH2Ph), 4.86 (d, J¼1.4 Hz, 1H, H-1B), 4.77 (d, J¼10.8 Hz, 1H,
PhCH2), 4.70 (d, J¼3.6 Hz, 1H, H-1A), 4.57 (d, J¼12.2 Hz, 1H, PhCH2),
4.55e4.42 (m, 11H, PhCH2), 4.37 (d, J¼12.2 Hz, 1H, PhCH2), 4.34 (d,
J¼12.2 Hz, 1H, PhCH2), 4.27 (d, J¼12.2 Hz, PhCH2), 4.00e3.92 (m,
5H, H-2A, H-2B, H-2C, H-3A, H-4A), 3.86e3.78 (m, 2H, H-4B, H-4C),
OCH2), 3.13e3.07 (m, 1H, NCH2), 2.48 (br s, 1H, OH), 1.68e1.65 (m,
2H, eCH2e); 13C NMR (75 MHz, CDCl3):
d 156.7 (COOCH2Ph),
138.4e127.7 (AreC), 101.0 (C-1A), 99.8 (C-1B), 84.1 (C-3A), 83.2 (C-
4A), 80.5 (C-2A), 80.1 (C-3B), 75.5 (PhCH2), 74.5 (C-4B), 73.8 (PhCH2),
72.9 (PhCH2), 72.8 (PhCH2), 72.3 (PhCH2), 71.6 (C-5B), 69.7 (C-6B),
69.1 (OCH2R), 68.5 (C-2B), 67.3 (C-5A), 66.8 (COOCH2Ph), 39.6
(NCH2), 29.5 (eCH2e); ESI-MS: m/z 976.4 [MþNa]þ; Anal. Calcd for
3.75e3.57 (m, 10H, H-3B, H-3C, H-5abA, H-5B, H-5C, H-6aB, H-6abC
,
OCH2), 3.46 (dd, J¼5.5, 10.0 Hz, 1H, H-6bB), 3.31e3.21 (m, 2H, NCH2,
C
57H63NO12 (953.43): C, 71.75; H, 6.66%; found: C, 71.64; H, 6.78%.
OCH2), 3.10e3.04 (m, 1H, NCH2), 1.63e1.59 (m, 2H, eCH2e); 13C
NMR (75 MHz, CDCl3):
d 156.8 (COOCH2Ph), 138.8e127.9 (AreC),
4.5. 3-(Benzyloxycarbonylamino)propyl 2,3-di-O-benzyl-5-O-[2-O-
(4-O-acetyl-2,3,6-tri-O-benzyl- -mannopyranosyl)-3,4,6-tri-O-
benzy- -mannopyranosyl]- -arabinofuranoside (14)
101.1 (C-1A), 99.9 (C-1C), 99.5 (C-1B), 84.1 (C-4A), 83.5 (C-2A), 80.4
(C-3A), 80.1 (C-3B), 79.6 (C-2B),75.5 (PhCH2), 75.1 (C-4B), 74.8 (C-2C),
74.2 (C-3C), 73.8 (2C, PhCH2), 72.9 (3C, PhCH2), 72.8 (PhCH2), 72.4
(C-5B), 72.1 (PhCH2), 72.0 (C-5C), 70.6 (C-6C),70.4 (OCH2R), 69.5 (C-
6B), 67.9 (C-4C), 67.2 (C-5A), 66.8 (COOCH2Ph), 39.8 (NCH2), 29.7
(eCH2e); ESI-MS: m/z 1408.5 [MþNa]þ; Anal. Calcd for C84H91NO17
(1385.62): C, 72.76; H, 6.61%; found: C, 72.62; H, 6.74%.
a-D
b-D
a-D
MS-4Å (3.00 g) were added to a solution of compound 13 (4.5 g,
4.72 mmol) and thioglycoside 7 (3.0 g, 5.66 mmol) in anhydrous
CH2Cl2 (60 mL). The reaction mixture was stirred for 1 h under N2

P.K. Mandal, P.R. Chheda / Carbohydrate Research 407 (2015) 104e110
109
4.7. 3-(Benzyloxycarbonylamino)propyl 5-O-[3,4,6-tri-O-benzy-2-
O-[2,3,6-tri-O-benzyl-4-O-(2,3-di-O-benzyl-5-O-toluenesulfonyl-
-xylofuranosyl)- -mannopyranosyl]- -mannopyranosyl]-2,3-
di-O-benzyl- -arabinofuranoside (16)
4.9. 3-Aminopropyl 5-O-{2-O-[4-O-(5-deoxy-5-methylthio-
xylofuranosyl)- -mannopyranosyl]- -mannopyranosyl}-
arabinofuranoside (2)
a-D-
b-D-
a
-
a-D
a-
D
D
a-D
a-D
b-D
To a solution of 16 (367 mg, 0.20 mmol) in acetonitrile, 18-
crown-6 (40 mg) was added followed by sodium thiomethoxide
(48 mg, 0.69 mmol). This mixture was then refluxed for 12 h and
after completion of reaction, cooled to room temperature. It was
then diluted with acetonitrile and filtered through Celite® bed. The
filtrate after concentrating to a syrup was purified by passing
through a short pad SiO2. The resulting intermediate was dissolved
in THF and cooled to ꢀ78 ꢁC. Then using a dry ice trap, ammonia
was condensed into the flask followed by addition of sodium metal
in three portions till a deep blue colour persisted. After stirring the
solution for 1.5 h at ꢀ78 ꢁC, methanol was added and the flask was
left open to atmosphere overnight to allow NH3 to evaporate. Then
the remaining solution was concentrated, dissolved in methanol
and neutralized with glacial acetic acid. The mixture was recon-
centrated and purified over Sephadex® LH-20 column using
After drying thioglycoside 8 (1.75 g, 3.03 mmol) and alcohol 15
(3.5 g, 2.52 mmol) for 6 h over vacuum, both were dissolved in
CH2Cl2 (40 mL), and the resulting solution was cooled to 0 ꢁC. To
this solution, powdered 4 Å molecular sieves (2.5 g) were added,
and the suspension was stirred for 20 min at 0 ꢁC. N-iodosuccini-
mide (819 mg, 3.63 mmol) was added, followed by silver triflate
(196 mg, 0.76 mmol). The reaction mixture was stirred for 15 min
and then neutralized with Et3N. The reaction mixture was further
diluted with CH2Cl2 (90 mL) and filtered through Celite® bed. The
filtrate was washed successively with saturated aqueous Na2S2O3
solution (50 mL) followed by water (60 mL). The organic layers
were collected, dried over Na2SO4 and concentrated to give a syrup
that was purified by column chromatography (4:1 hexane:EtOAc)
25
to afford 16 (2.8 g, 77%), as a syrup; [
a]
þ6.2 (c 1.5, CHCl3); IR
D
(neat): 2637, 1708, 1333, 1278, 1214, 1137, 1048, 986, 770, 699 cmꢀ1
;
CH3OHeH2O (4:1 v/v) as eluant to give pure compound 2 (99 mg,
25
1H NMR (500 MHz, CDCl3):
d
7.59e7.57 (m, 2H, AreH), 7.24e7.05
72%); [
a
]
þ6.5 (c 1.5, CHCl3); IR (neat): 3307, 1948, 1873, 1248,
D
(m, 55H, AreH), 6.90e6.88 (m, 2H, AreH), 5.24 (d, J¼4.2 Hz, 1H, H-
1D), 5.18 (br s,1H, H-1C), 5.10 (br s, 2H, COOCH2Ph), 4.91 (br s, 1H, H-
1B), 4.79 (d, J¼3.8 Hz, 1H, H-1A), 4.76 (d, J¼11.5 Hz, 1H, PhCH2),
4.59e4.47 (m, 8H, PhCH2), 4.45e4.41 (m, 5H, PhCH2), 4.40e4.31 (m,
6H, H-2A, H-2C, H-4A, PhCH2), 4.24e4.09 (m, 6H, H-2B, H-3A, H-4D
PhCH2), 4.04e3.87 (m, 6H, H-2D, H-3D, H-4B, H-4C, H-6abC),
3.85e3.56 (m, 9H, H-3B, H-3C, H-5abA, H-5B, H-5C, H-5abD, H-6aB),
3.51e3.47 (m, 2H, H-6bB, OCH2-), 3.44e3.41 (m, 2H, OCH2-, NCH2),
3.27e3.24 (m, 1H, NCH2), 2.27 (s, 3H, CH3), 1.86e1.78 (m, 2H,
1224, 1137, 1088, 1048 cmꢀ1 1H NMR (500 MHz, D2O):
; d 5.39 (d,
J¼4.4 Hz,1H, H-1D), 5.12 (d, J¼1.8 Hz,1H, H-1C), 5.02 (br s,1H, H-1B),
5.01 (d, J¼4.5 Hz, 1H, H-1A), 4.37 (ddd, J¼5.0, 4.8, 8.4 Hz, 1H, H-4D),
4.24 (dd, J¼4.2, 5.0 Hz, 1H, H-3D), 4.22e4.16 (m, 2H, H-2D, H-3A),
4.14e4.05 (m, 4H, H-2A, H-2B, H-2C, H-4A), 4.00e3.92 (m, 5H, H-3B,
H-3C, H-6aB, H-6abC), 3.89e3.81 (m, 5H, H-4B, H-4C, H-5aA, H-5C, H-
6bB), 3.77e3.57 (m, 4H, H-5bA, H-5B, OCH2), 3.04e2.96 (m, 2H,
NCH2), 2.78 (dd, J¼4.8, 13.8 Hz, 1H, H-5aD), 2.67 (dd, J¼8.4, 13.8 Hz,
1H, H-5bD), 2.16 (s, 3H, SCH3), 1.90e1.88 (m, 2H, eCH2e); 13C NMR
eCH2e); 13C NMR (75 MHz, CDCl3):
d
157.1 (NHCOOCH2Ph),
(125 MHz, D2O): d 104.3 (C-1D), 104.1 (C-1B), 102.9 (C-1A), 100.0 (C-
144.8e125.0 (AreC), 100.8 (C-1D), 100.7 (C-1A), 99.4 (C-1B), 99.2 (C-
1C), 84.5 (C-4D), 83.7 (C-2C), 82.3 (C-4A), 80.9 (C-4C), 80.6 (C-5C),
79.9 (C-2D), 79.7 (C-3D), 75.4 (PhCH2), 75.2 (C-3C), 74.4 (C-2A), 74.3
(C-3B), 74.0 (C-3A), 73.8 (PhCH2), 73.4 (PhCH2), 73.1 (PhCH2), 72.8
(PhCH2), 72.6 (PhCH2), 72.4 (2C, C-2A, C-3B), 72.3 (PhCH2), 72.1
(PhCH2), 71.7 (C-3A), 70.8 (PhCH2), 70.5 (PhCH2), 69.9 (OCH2-),
69.6(C-6B), 69.1 (2C, C-6C, COOCH2Ph), 68.5 (C-5D), 65.2 (C-5A), 51.6
(NCH2), 29.1 (eCH2e), 22.1 (CH3); ESI-MS: m/z 1874.7 [MþNa]þ;
Anal. Calcd for C110H117NO23S (1851.77): C, 71.29; H, 6.36%; found: C,
71.18; H, 6.45%.
1C), 81.5 (C-4A), 80.5 (C-2B), 79.6 (C-4D), 78.4 (C-2D), 77.8 (C-3D),
77.4 (C-3A), 76.1 (C-2A), 76.0 (C-5C), 74.9 (C-5B), 73.7 (C-3C), 72.3 (C-
3B), 72.1 (C-2C), 72.0 (OCH2), 70.2 (C-4B), 68.8 (C-4C), 67.9 (C-5A),
63.1 (C-6B), 62.8 (C-6C), 38.8 (NCH2), 34.8 (C-5D), 29.8 (eCH2e), 16.8
(SCH3); ESI-MS: m/z 694.2 [MþH]þ; Anal. Calcd for C26H47NO18
S
(693.25): C, 45.02; H, 6.83%; found: C, 44.93; H, 6.91%.
4.10. Biotinylation of compound 1
To a solution of 1 (20 mg, 0.04 mmol) in methanol, sulfosucci-
nimidyl 2-(biotinamido) ethyl-l,3-dithiopropionate (9) (27.4 mg,
0.05 mmol) was added and the resulting solution was allowed to
stir at room temperature for 8 h. After completion, the reaction was
concentrated to a syrup, which was purified by Sephadex® LH-20
4.8. 3-Aminopropyl 5-O-[2-O-(a-D-mannopyranosyl)-a-D-
mannopyranosyl]- -arabinofuranoside (1)
b-D
column using CH3OHeH2O (10:1 v/v) as eluant, to afford 3
25
To a solution of compound 15 (500 mg, 0.36 mmol) in CH3OH,
20% Pd(OH)2eC (500 mg) was added. The reaction mixture was
then stirred at room temperature for 7 h under positive pressure of
hydrogen. The reaction mixture was filtered through Celite® bed
and then evaporated to dryness. The crude product was purified
over Sephadex_LH-20 column using CH3OH-H2O (8:1 v/v) as eluant,
(28.4 mg, 82%); [
a
]
D
þ4.9 (c 1.5, H2O); IR (KBr): 3317, 1948, 1873,
1558, 1224, 1137, 1088, 1008 cmꢀ1; 1H NMR (500 MHz, D2O):
d 5.20
(d, J¼1.6 Hz, 1H, H-1C), 5.08 (br s. 2H, H-1B, H-1A),4.65e4.61 (m, 1H,
H-3A), 4.48e4.45 (m, 1H, NHCH-), 4.39e4.36 (m, 1H, NHCH-),
4.16e4.10 (m, 3H,, H-2A, H-2C, H-4A), 4.08e3.98 (m, 2H, H-2B, H-3B),
3.96e3.85 (m, 4H, H-3C, H-6abB, H-6aC), 3.83e3.74 (m, 6H, H-4B,H-
to give pure compound 1 (100 mg, 82%); [
a
]
D
25 þ5.5 (c 1.5, H2O); IR
5aA, H-5B, H-5C, H-6bC, OCH2R), 3.73e3.53 (m, 5H, H-4C, H-5bA,
(neat): 3200, 1948, 1393, 1348, 1224, 1088, 1048, 986 cmꢀ1; 1H NMR
SCH2-, OCH2R), 3.29e3.23 (m, 2H, NCH2-), 3.19e3.02 (m, 3H,
eCH2e, SCH2-), 2.95e2.91 (m, 2H, eCH2e), 2.84e2.78 (m, 2H,
eCH2e), 2.70e2.68 (m, 2H, eCH2e), 2.34e2.30 (m, 2H, eCH2e),
2.04e1.99 (m 2H, eCH2e),1.77e1.62 (m, 2H, eCH2e), 1.50e1.44 (m,
2H, eCH2e), 1.23e1.20 (m, 2H, eCH2e); 13C NMR (125 MHz, D2O):
(500 MHz, D2O):
d
5.15 (d, J¼1.6 Hz, 1H, H-1C), 5.02 (br s. 2H, H-1A,
H-1B), 4.10e4.06 (m, 3H, H-2A, H-2C, H-3A), 4.03e3.98 (m, 2H, H-2B,
H-4A), 3.94e3.82 (m, 5H, H-3B, H-3C, H-6abB, H-6aC), 3.79e3.65 (m,
6H, H-4B, H-5aA, H-5B, H-5C, H-6bC, OCH2R), 3.62e3.58 (m, 3H, H-4C,
H-5bA, OCH2R), 3.14e3.11 (m, 2H, NCH2), 1.99e1.95 (m, 2H, eCH2e);
d
174.2 (NHCO-), 172.7 (NHCO-), 164.1 (NHCONH), 104.9(C-1A),
13C NMR (125 MHz, D2O):
d
104.9 (C-1A), 101.6 (C-1B), 98.8 (C-1C),
101.9(C-1B), 99.2(C-1C), 82.4(C-4A), 81.3(C-2B), 79.4(C-3A), 76.4(C-
2A), 73.9(C-5C), 73.5(C-5B), 70.9(C-3C), 70.8(C-3B), 70.6(C-2C), 67.5
(2C, H-4B, OCH2R), 66.4 (C-4C), 62.7(C-5A), 61.8(C-6B), 61.5(C-6C),
60.9 (NHCHCH-), 57.0 (NHCHCH-), 56.0 (SCH2CH-), 40.3 (eCH2e),
38.4(NCH2), 37.3 (NCH2-), 36.5 (2C, eCH2e), 33.4 (eCH2e), 30.2 (2C,
eCH2e), 28.4 (eCH2e), 28.3 (eCH2e), 25.7 (eCH2e); ESI-MS: m/z
82.9 (C-4A), 81.2 (C-2B), 79.1 (C-3A), 76.8 (C-2A), 73.8 (C-5C), 73.5 (C-
5B), 70.8 (C-3C), 70.7 (C-3B), 70.5 (C-2C), 67.4 (OCH2R), 67.3 (C-5A),
66.9 (C-4B), 66.3 (C-4C), 61.7 (C-6B), 61.4 (C-6C), 38.4 (NCH2), 27.1
(eCH2e); ESI-MS: m/z 532.2 [MþH]þ. Anal. Calcd for C20H37NO15
(531.22): C, 45.20; H, 7.02%; found: C, 45.07; H, 7.15%.

110
P.K. Mandal, P.R. Chheda / Carbohydrate Research 407 (2015) 104e110
943.3 [MþNa]þ; Anal. Calcd for C35H60N4O18S3 (920.31): C, 45.64;
Division of CSIR-CDRI for providing the spectroscopic and analytical
data. CDRI communication no. 8889.
H, 6.57%; found: C, 45.51; H, 6.68%.
Supplementary data
4.11. Biotinylation of compound 2
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.carres.2015.01.003.
To a solution of 2 (10 mg, 0.01 mmol) in methanol, sulfosucci-
nimidyl 2-(biotinamido) ethyl-l,3-dithiopropionate (9) (10.5 mg,
0.02 mmol) was added and the resulting solution was allowed to
stir at room temperature for 8 h. It was then concentrated to a syrup
that was purified by Sephadex® LH-20 column using CH3OHeH2O
References
(6:1 v/v) as eluant, to afford 4 (11.2 mg, 72%); [
a
]
25 þ3.5 (c 1.5, H2O);
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d
175.4 (NHCO-), 174.7 (NHCO-), 164.5 (NHCONH), 104.1 (C-1D),
104.0 (C-1B), 102.6 (C-1A), 99.7 (C-1C), 80.2 (C-4A), 79.3 (C-2B), 78.3
(C-4D), 77.1 (C-2D), 76.7 (C-3D), 76.1 (C-2A), 75.0 (C-2A), 74.8 (C-5C),
73.5 (C-5B), 72.4 (C-3C), 70.9 (C-3B), 70.8 (2C, C-2C, C-4C), 68.9
(OCH2-), 67.4 (C-4B), 66.5 (C-5A), 62.7 (NHCHCH-), 61.8 (C-6B), 61.4
(C-6C), 60.8 (NHCHCH2-), 55.9 (SCHCH2-), 40.3 (SCH2-), 38.4 (NCH2),
37.4 (NCH2), 37.2 (eCH2e), 36.0 (SCH2-), 35.6 (C-5D) 33.9 (eCH2e),
33.5 (eCH2e), 28.9 (eCH2e), 28.4 (eCH2e), 28.2 (eCH2e), 25.7
(eCH2e), 15.6 (SCH3); ESI-MS: m/z 1105.3 [MþNa]þ; Anal. Calcd for
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Author gratefully acknowledges financial support by Depart-
ment of Science and Technology (DST), India under Fast Track
Proposal Scheme for Young Scientists (CS-127/2012) and SAIF