Synthesis and mycobacterial growth inhibition activities of bivalent and monovalent arabinofuranoside containing alkyl glycosides

Article abstract of DOI:10.1039/b803409e

Arabinofuranosides constitute one of the important components of cell wall structures of mycobacteria. With this importance of arabinofuranosides in mind, alkyl glycosides bearing arabinofuranoside trisaccharides were prepared, wherein the sugars were presented either in the monovalent or bivalent forms. Following the synthesis, the monovalent and bivalent alkyl glycosides were tested for their activities in a mycobacterial growth assay. The growth of the mycobacterial strain M. smegmatis was assessed in the presence of the alkyl glycosides and it was realized that the alkyl glycosides acted as inhibitors of the mycobacterial growth. The inhibition of the growth, caused by the above alkyl glycosides, was not observed for the arabinofuranose trisaccharide alone, without the alkyl groups, and for an alkyl glycoside bearing maltose as the sugar component.

Full text of DOI:10.1039/b803409e

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthesis and mycobacterial growth inhibition activities of bivalent and
monovalent arabinofuranoside containing alkyl glycosides
Kottari Naresh,a Binod Kumar Bharati,b Narayanaswamy Jayaraman*a and Dipankar Chatterjib
Received 29th February 2008, Accepted 20th March 2008
First published as an Advance Article on the web 2nd May 2008
DOI: 10.1039/b803409e
Arabinofuranosides constitute one of the important components of cell wall structures of
mycobacteria. With this importance of arabinofuranosides in mind, alkyl glycosides bearing
arabinofuranoside trisaccharides were prepared, wherein the sugars were presented either in the
monovalent or bivalent forms. Following the synthesis, the monovalent and bivalent alkyl glycosides
were tested for their activities in a mycobacterial growth assay. The growth of the mycobacterial strain
M. smegmatis was assessed in the presence of the alkyl glycosides and it was realized that the alkyl
glycosides acted as inhibitors of the mycobacterial growth. The inhibition of the growth, caused by the
above alkyl glycosides, was not observed for the arabinofuranose trisaccharide alone, without the alkyl
groups, and for an alkyl glycoside bearing maltose as the sugar component.
biological functions.7 In this respect, the presence of multivalent
Introduction
effects, seen commonly in carbohydrate–protein interactions,8 are
Alkyl glycosides comprising arabinofuranosyl residues have im-
apparent in arabinomannan bearing alkyl glycoside–protein inter-
mense significance as they are major components of mycobacterial
actions. Multivalent effects provide significantly enhanced binding
affinities, often through cross-linking processes.9 Studies of the
multivalent effects in alkyl glycosides containing arabinofuranosyl
residues were thus considered appropriate. Accordingly, the syn-
thesis of bivalent and monovalent arabinofuranosyl trisaccharides
containing alkyl glycosides was accomplished. In an earlier study,
the ability of the synthetic alkyl glycosides was assessed in
a mycobacterial growth assay. This report provides details of
the synthesis of arabinofuranosyl trisaccharide containing alkyl
glycosides and the influence of these ligands on the growth profile
of the M. smegmatis.
cell walls. Seminal works of Brennan, Chatterjee and coworkers
have established that arabinofuranose residues are present promi-
nently in the arabinogalactan and lipoarabinomannan compo-
nents of the mycobacterial cell wall structures.1 For example, it
was shown that the lipoarabinomannans from Mycobacterium
smegmatis are comprised of 40–70 arabinofuranosyl residues, in
addition to 11 mannopyranosyl residues, phosphatidyl inositols
and lipidic chains. The non-reducing end of the arabinofuranosyl
oligomer portion requires the presence of a few mannopyranosyl
oligosaccharides that act as caps and the caps are important
in order to activate virulence to the mycobacteria. There are
no structural homologues of arabinofuranoside containing alkyl
glycosides in mammalian systems and, in this context, the
mycobacterial originated alkyl glycosides are inherently foreign to
the mammalian immune systems.2 The importance associated with
the arabinan structures in mycobacterial cell walls led to the inves-
tigations, aimed at, for example, (i) to delineate the functions of
arabinofuranosyl transferase enzymes that are responsible for the
construction of arabinans;3 (ii) to construct structural analogues
that act as arabinofuranosyl transferase inhibitors that could be
useful to prevent the growth of the mycobacterium4 and (iii) to
evolve highly immunogenic lipoarabinomannan oligosaccharides
protein conjugates as potential TB vaccines.5 Studies previously
had shown that individual mannose, arabinose and lipid residues
are essential as components in order to engage a preferential
binding to, for example, pulmonary surfactant proteins present
in the alveolar macrophages.6 The requirement of the lipid
component in the alkyl glycosides denotes the essential role
of forming alkyl glycoside–protein aggregates necessary for the
Results and discussion
Syntheses of monovalent and bivalent alkyl glycosides incor-
porated with the arabinofuranose trisaccharide were targeted.
Incorporation of linkers between the branch points and the sugar
moieties was desired in the case of the bivalent ligand. It was
˚
shown previously that through bond distances of 15 A between the
sugars were required for their effective lectin binding, from studies
of the binding a-D-mannopyranoside containing bivalent ligands
binding to lectin concanavalin A.10 Further, in order to achieve
the hydrophobic–hydrophilic balance in the alkyl glycosides,
the presence of double alkyl chains were deemed necessary.
Arabinofuranosyl trisaccharide containing glycosides 1 and 2
(Fig. 1), were thus planned. In addition to the alkyl glycosides
1 and 2, a maltose based alkyl glycoside was also considered to be
useful for a comparison purpose in the biochemical studies.
Synthesis
aDepartment of Organic Chemistry, Indian Institute of Science, Bangalore,
560 012, India. E-mail: jayaraman@orgchem.iisc.ernet.in; Fax: +91-80-
2360-0529
The target alkyl glycoside structures have three distinct structural
components, namely, (i) the sugar component; (ii) the ethylene
glycol functionalized glycerol moiety and (iii) the long alkyl
lyophilic component.
bMolecular Biophysics Unit, Indian Institute of Science, Bangalore, 560 012,
India
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Fig. 1 Molecular structures of monovalent (1) and bivalent alkyl
glycosides (2).
Scheme 3 Reagents and conditions: (i). TMSOTf, rt, 2 h, CH2Cl2, MS
˚
Diethylene glycol and long alkyl group functionalized glycerol.
The synthesis of this derivative was planned through O-alkylations
of long alkyl group functionalized glycerol with an appropriately
derivatized diethylene glycol. The long alkyl group functionalized
glycerol was planned, in turn, through bis-O-alkylation of diol
311 with the long alkyl group moiety. Diol 3 was subjected
to O-alkylation with 1-bromohexadecane, followed by de-O-
benzylation (H2 and Pd–C (10%)), to afford O-alkylated derivative
4 (Scheme 1). Derivative 4 was subjected to bis-O-alkylation
with a protected diethylene glycol tosylate12 and a further de-
O-benzylation afforded the diethylene glycol and long chain alkyl
group functionalized diol 5 (Scheme 2).
(4 A); (ii). H2, Pd–C, EtOAc–MeOH (1 : 1), rt, 24 h; (iii). CCl3CN, DBU,
CH2Cl2, −10 ◦C, 20 min; (iv). NaOMe–MeOH : THF, rt, 24 h.
reaction of one and two molar equivalents of trichloroacetimidate
9 with the acceptor diol 5, in the presence of TMSOTf, followed
by the deprotection led to the formation of monovalent alkyl
arabinofuranoside 1 and bivalent alkyl arabinofuranoside 2,
respectively, in good yields (Scheme 4).
˚
Scheme 4 Reagents and conditions: (i) TMSOTf, CH2Cl2, MS (4 A),
−10 ◦C, 2 h; (ii). NaOMe–MeOH : THF, rt, 24 h.
Synthesis of the monovalent alkyl glycoside bearing the dis-
accharide maltose 12 was also accomplished. Accordingly, the
glycosylation of diol 5 with the acetobromo maltose 11,14 using
Ag2CO3 as the activator, followed by a deprotection, afforded the
monovalent alkyl disaccharide 12 (Scheme 5).
Scheme 1 Reagents and conditions: (i) C16H33Br, NaH, THF, 36 h, reflux;
(ii) H2, Pd–C, EtOAc–MeOH (1 : 1), rt, 24 h.
Scheme 2 Reagents and conditions: (i) NaH, THF, reflux, 36 h; (ii). H2,
Pd–C, EtOAc–MeOH (1 : 1), rt, 18 h.
Synthesis of arabinofuranoside trisaccharide. The required
glycosyl acceptor benzyl-6-O-acetyl-a-D-arabinofuranoside 6 was
prepared by the reaction of benzyl-a-D-arabinofuranoside13 with
Ac2O in the presence of Bu2SnO. The arabinofuranoside 6 served
as the acceptor for a glycosylation with 2,3,5-tri-O-benzoyl-a-D-
arabinofuranosyl trichloroacetimidate (7)13 and the glycosylation
was conducted in the presence of TMSOTf, to afford the protected
trisaccharide (Scheme 3). De-O-benzylation at the reducing end
of the trisaccharide afforded the lactol 8, which was converted
subsequently to an activated trichloroacetimidate glycosyl donor
9. The complete deprotection of the protecting groups in 8 afforded
free hydroxyl group containing trisaccharide 10.
Scheme 5 Reagents and conditions: (i). Ag2CO3, AgClO4, CH2Cl2, rt, 36 h,
78%; (ii). NaoMe–MeOH, rt, 18 h.
The identities and structural homogeneities of the alkyl glyco-
sides 1, 2, 10 and 12 were established by NMR spectroscopy and
mass spectrometry. In the ES-MS spectrum of 1, the molecular
ion peak was identified as the base peak. For 2, mass spectral
peaks corresponding to the consecutive loss of arabinofuranosyl
oxonium ions were observed, in addition to the presence of
the molecular ion peak. The molecular structures were further
confirmed by 1H and 13C NMR spectroscopy.
The preparation of monovalent (1) and bivalent (2) alkyl
arabinofuranosides was performed with the acceptor 5 and con-
trolled molar equivalents of the trisaccharide donor 9. Thus, the
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The critical micellar concentrations (CMCs) at which the
alkyl arabinofuranosides 1 and 2 undergo transformation into
micelles were assessed, using a fluorescence probe 8-anilino-1-
naphthalene sulfonic acid (ANSA).15 The measured CMC of each
alkyl glycoside was 1: 3.7 lM and 2: 5.2 lM. The increase in the
hydrophilic component in 2 was found to increase the CMC value,
in comparison to 1.
Effect of monovalent (1) and bivalent (2) alkyl arabinofuranosides
on the growth profile of M. smegmatis. The newly synthesized
alkyl arabinofuranosides 1 and 2 were studied in a mycobacterial
growth assay, in order to identify their effects on the mycobacterial
growth pattern. In addition to 1 and 2, the arabinofuranose trisac-
charide 10 and the alkyl glycoside bearing the maltosyl sugar unit
12 were also taken up for the studies. The growth of the wild type
M. smegmatis, which is a rapidly growing mycobacterium, was
examined in Middlebrook broth. The synthetic alkyl glycosides
1, 2 and 12 and the arabinofuranose trisaccharide 10 were
incorporated into the growth medium as a solution in aq. methanol
(50%). It was observed that alkyl arabinofuranosides 1 and 2
reduced the growth rate of the mycobacterium significantly. Fig. 2
shows the growth profiles for solutions having differing amounts
of the alkyl glycosides 1 and 2. Arabinofuranose trisaccharide
10 and the alkyl glycoside 12 did not reduce the growth rate of
mycobacterium and the growth was comparable to that of the
wild type. A maximum retardation of 62% was observed for the
monovalent alkyl glycoside 1 in the solution containing 200 lg
mL−1 of the ligand 1 and this maximum retardation of growth
required ∼ 60 h. On the other hand, it was observed that the
bivalent sugar ligand 2 exhibited a maximum inhibition of 35% in
the solution containing 100 lg mL−1 of the ligand and the time
required for this maximum inhibition was ∼ 30 h. In comparison
to the inactivity of the arabinofuranosyl trisaccharide 10 and the
alkyl glycoside 12 in the mycobacterial growth pattern, the above
results showed that 1 and 2 inhibited the growth. Interestingly,
the maximum inhibition did not persist throughout the duration
of the analysis. Specifically, it was observed that the bivalent
ligand 2 showed a reduction in inhibition after ∼ 40 h and
the mycobacterium continued to grow further after the initial
retardation in the exponential phase. The recovery of growth after
a retardation in the presence of the alkyl glycoside is unlikely
to result from a substrate-like activity of the alkyl glycoside
beyond a period in the exponential phase of the growth. The fact
that the arabinofuranose trisaccharide 10, constituting the sugar
component of the ligands 1 and 2, did not lead an effect in the
growth rate augment the above inference on the activity profile
of the ligands 1 and 2. A possible reason for the retardation in
the mycobacterial growth in the presence of the ligands 1 and
2 could be due to a slow diffusion of the nutrients mediated by
porins penetrating the cell wall. An important controlling factor
influencing the growth is the thick hydrophobic cell wall. Diffusion
of nutrients through the cell wall is promoted by porins, which are
channels formed from stable oligomeric protein, localized in the
cell wall.16 Studies previously have shown that the mycobacterial
porins are the major determinants of permeability to hydrophilic
molecules and nutrients.17 The incorporation of ligands 1 and 2
in the cell wall, in some form, might thus affect the diffusion
of the nutrients through the cell wall. The ligands 1 and 2 may
incorporate at different rates, which could depend on the extent of
Fig. 2 Effect of (a) monovalent alkyl glycoside 1 and (b) bivalent alkyl
glycoside 2 on the growth profile of M. smegmatis. Inset in each figure
shows the percentage inhibition of the growth of M. smegmatis in the
presence of alkyl glycosides 1 and 2 relative to the profile for the growth in
methanol alone.
the hydrophilicities and hydrophobicities. The more hydrophilic 2
might thus incorporate into the cell wall in a process analogous
to faster association and dissociation, thereby leading to the
inhibition at an early period of the growth. On the other hand, the
relatively more hydrophobic 1 requires higher concentrations and
also inhibition of growth occurs at the later period of the growth.
Alternatively, the inhibition of growth by ligands 1 and 2 could
be due to some other factors. Clearly, it is difficult to ascertain
the origin of the inhibitory effect by these ligands at present.
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The fact that the arabinofuranose trisaccharide 10 alone and the
alkyl glycoside 12 did not have an effect on the growth imply that
both the nature of the sugar ligand and the long alkyl groups are
essential for the functional nature of the alkyl arabinofuranosides
1 and 2.
31.8, 60.3, 62.1, 62.3, 69.8, 70.1, 71.8, 77.9, 81.4; ES-MS Calcd.
for C38H78O5Na [M + Na]: 637.5747, found 637.5745.
10-O-Hexadecyl-8-(7ꢀ-hydroxy-2ꢀ,5ꢀ-dioxaheptyl)-3,6,9,13-
tetraoxanonacosane-1,10-diol (5)
Derivative 4 (1.29 g, 2.1 mmol) in THF (5 mL) was added to
a suspension of NaH (0.11 g, 60% in mineral oil, 2.75 mmol)
in THF (20 mL). After refluxing for 30 min, 1-O-benzyl-5-O-
tosyl-3-oxa-pentane-1,5-diol12 (1.7 g, 5 mmol) in THF (5 mL)
was added slowly, stirred at reflux for 36 h, cooled, filtered and
filtrate was concentrated in vacuo. The crude residue was dissolved
in CH2Cl2 (50 mL), washed with water (3×30 mL). The organic
phase was dried, filtered and the filtrate was concentrated in vacuo.
A solution of the crude residue in EtOAc–MeOH (1 : 1) (30 mL)
was treated with Pd–C (10%) (0.15 g), stirred under H2 (1 bar) for
18 h. Filtration of the reaction mixture, followed by purification
(EtOAc–MeOH, 19 : 1), afforded 5, as a white solid. Yield: 1.02 g
(62%); Rf (EtOAc) 0.3; mp 36–38 ◦C; 1H NMR (CDCl3, 300 MHz)
d 0.88 (t, J = 6.6 Hz, 6 H), 1.91–1.22 (br s, 52 H), 1.51–1.56 (m,
4 H), 3.38–3.71 (m, 32 H); 13C NMR (CDCl3, 75 MHz) d 13.8,
21.2, 22.9, 25.5, 26.0, 29.4, 29.6, 31.6, 60.9, 61.1, 66.2, 68.2, 69.0,
69.9, 70.4, 71.2, 71.6, 72.3, 76.6, 77.6, 78.0; ES-MS Calcd. for
C46H94O9Na [M + Na]: 813.6796, found 813.6835; Anal. Calcd.
for C46H94O9: C 69.83, H 11.97; found: C 69.52, H 11.79.
Conclusion
Arabinofuranosyl trisaccharides containing alkyl glycosides, rel-
evant to the class of mycobacterial cell wall components, were
designed. A monovalent alkyl glycoside (1), with one trisaccharide
moiety, and a bivalent alkyl glycoside (2), bearing two trisaccha-
ride moieties, were synthesized and their CMCs assessed. The
alkyl glycosides were tested in a mycobacterial growth assay
and it was found that the alkyl glycosides were able to inhibit
the mycobacterial growth to varying degrees. The observations
of this report open up a possibility that arabinofuranoside
containing alkyl glycosides may form a new class of inhibitors
of mycobacterial growth.
Experimental section
General information
Solvents were dried and distilled according to literature proce-
dures. All chemicals were purchased from commercial sources and
were used without further purification. Silica gel (100–200 mesh)
was used for column chromatography and TLC analysis was
Benzyl 5-O-acetyl-a-D-arabinofuranoside (6)
performed on commercial plates coated with silica gel 60 F254
.
To a solution of benzyl-a-D-arabinofuranoside13 (0.12 g, 0.5 mmol)
in PhMe–C6H6 (9 : 1) (20 mL), Bu2SnO (0.16 g, 0.64 mmol) was
added. The solution was refluxed for 18 h, after which half the
amount of the solvents was removed. Ac2O (0.06 mL, 0.64 mmol)
was then added to the reaction mixture at room temperature and
the solution was stirred for 2 h, concentrated in vacuo and purified
(pet. ether–EtOAc 2 : 3) to afford 6, as a gum. Yield: 0.09 g (71%);
Rf (pet. ether–EtOAc 1 : 3) 0.33; [a]D = −33 (c 1, CHCl3); 1H NMR
(CDCl3, 300 MHz) d 2.0 (s, 3 H), 3.7 (br s, 2 H), 4.0–4.23 (m, 5 H),
4.42 (d, J = 11.7 Hz, 1 H), 4.67 (d, J = 11.7 Hz, 1 H), 4.95 (br s,
1 H), 7.0–7.4 (m, 5 H); 13C NMR (CDCl3, 75 MHz) d 20.8, 64.1,
69.2, 77.8, 80.8, 82.3, 106.6, 128.0, 128.4, 136.9, 171.3; ES-MS
Calcd. for C14H18O6Na [M + Na]: 305.1002, found 305.1001.
Visualization of the spots on TLC plates was achieved by
UV radiation or spraying 5% sulfuric acid in ethanol. High-
resolution mass spectra were obtained from Q-TOF instrument
by electrospray ionization (ESI). 1H and 13C NMR spectral
analyses were performed on a spectrometer operating at 300 MHz,
400 MHz, and 75 MHz, 100 MHz, respectively in CDCl3 solutions
unless otherwise stated. Chemical shifts are reported with respect
to tetramethylsilane (TMS) for 1H NMR and the central line
(77.0 ppm) of CDCl3 for 13C NMR. Coupling constants (J) are
reported in Hz. Standard abbreviations s, d, t, dd, br s, m refer
to singlet, doublet, triplet, doublet of doublet, broad singlet,
multiplet.
5-O-Hexadecyl-2-hydroxymethyl-3,7-dioxatricosane-1,5-diol (4)
5-O-Acetyl-[2,3,5-tri-O-benzoyl-a-D-(1→2)-arabinofuranosyl]-
[2,3,5-tri-O-benzoyl-a-D-(1→3)-arabinofuranosyl]-D-
arabinofuranose (8)
Diol 311 (1.32 g, 3.82 mmol) in THF (5 mL) was added to a
suspension of NaH (0.3 g, 7.5 mmol, 60% in mineral oil) in THF
(25 mL). After stirring for 30 min. at 60 ◦C, 1-bromohexadecane
(3 mL, 10 mmol) in THF (5 mL) was added slowly, stirred at
the same temperature for 36 h, cooled, filtered and filtrate was
concentrated in vacuo. The crude residue was dissolved in CH2Cl2
(20 mL) and washed with water (3×20 mL). The organic phase was
dried (Na2SO4), filtered and the filtrate was concentrated in vacuo.
A solution of the crude reaction mixture in EtOAc–MeOH (1 : 1)
(15 mL) was treated with Pd–C (10%) (0.25 g) and stirred under
H2 (1 bar) for 18 h. Filtration of the reaction mixture, followed
by purification (pet. ether–EtOAc 1 : 4) afforded 4, as a white
powder. Yield: 1.29 g (54%); Rf (pet. ether–EtOAc 1 : 4) 0.4; mp
48–50 ◦C; 1H NMR (300 MHz, CDCl3) d 0.82 (t, J = 6.6 Hz, 6 H),
A solution of 6 (0.24 g, 0.85 mmol), 713 (1.24 g, 2.04 mmol) and MS
˚
4 A, (0.3 g) in CH2Cl2 (30 mL) was stirred for 15 min. TMSOTf
(0.032 mL in 2 mL CH2Cl2, 0.17 mmol) was then added dropwise
at −10 ◦C under N2 atmosphere, the reaction mixture was stirred
for 2 h at room temperature, neutralized with Et3N, filtered and
filtrate concentrated in vacuo and purified (pet. ether–EtOAc 2 :
1) to afford the protected derivative of 8, as a foamy solid. Yield:
0.52 g (54%); Rf (pet. ether–EtOAc 6.5 : 3.5) 0.5; [a]D = +17.4 (c
1
1, CHCl3); H NMR (CDCl3, 300 MHz) d 2.01 (s, 3 H), 4.36 (s,
2 H), 4.46–4.57 (m, 2 H), 4.64–4.82 (m, 8 H), 5.24 (s, 1 H), 5.51
(s, 1 H), 5.55 (br s, 1 H), 5.6–5.64 (m, 5 H), 7.1–8.0 (m, 35 H);
13C NMR (CDCl3, 75 MHz) d 20.5, 60.3, 62.1, 63.5, 69.2, 78.8,
81.3, 81.6, 82.1, 82.4, 86.9, 105.5, 105.8, 127.6–129.7, 132.0, 133.3,
1.17–1.22 (br s, 52 H), 1.51–1.56 (m, 4 H), 3.36–3.74 (m, 16 H); 13
NMR (75 MHz, CDCl3) d 14.0, 20.9, 22.6, 25.1, 26.0, 29.3, 29.5,
C
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137.2, 165.3–166.1, 170.6; ES-MS Calcd. for C66H58O20Na [M +
Na]: 1193.3, found 1193.4.
with Amberlite ion-exchange (H+) resin, filtered and filtrate
concentrated to afford 1, as a foamy solid. Yield: 0.02 g, (95%);
1
A solution of above foamy solid (0.54 g, 0.45 mmol) in EtOAc–
MeOH (1 : 1) (50 mL) was treated with Pd–C (10%) (0.05 g),
stirred under H2 (1 bar) for 18 h. Filtration of the reaction mixture,
followed by purification (pet. ether–EtOAc 2 : 1) afforded 8, as a
white glassy solid. Yield: 0.49 g (94%); Rf (pet. ether–EtOAc 1 : 1):
0.50; [a]D = +8.6 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz) d 2.03
(s, 3 H), 3.1 (br s, 1 H), 4.1–4.3 (m, 1 H), 4.37–4.53 (m, 4 H), 4.62–
4.79 (m, 6 H), 5.44–5.53 (m, 4 H), 5.60–5.66 (m, 3 H), 7.26–8.06
(m, 30 H); 13C NMR (CDCl3, 100 MHz) d 20.6, 60.3, 63.5, 63.7,
64.9, 77.5, 78.2, 79.3, 80.4, 80.8, 81.4, 81.6, 81.9, 82.1, 85.6, 96.5,
101.6, 105.3, 105.7, 128.3–129.9, 133.0, 133.4, 133.6, 165.3–166.1,
170.6; ES-MS Calcd. for C59H52O20Na [M + Na]: 1103.3, found
1103.3.
[a]D = +47.3 (c 1, MeOH); H NMR (D2O, 300 MHz) d 0.86 (t,
J = 6.4 Hz, 6 H), 1.2–1.39 (m, 52 H), 1.51–1.56 (m, 4 H), 3.3–4.2
(m, 45 H), 5.04–5.2 (m, 3 H); 13C NMR (CD3OD, 75 MHz) d14.4,
23.7, 27.2, 30.4, 30.7, 33.1, 62.2, 62.6, 62.8, 63.0, 71.4, 71.9, 72.0,
73.7, 78.6, 79.5, 79.8, 82.0, 83.6, 85.6, 85.8, 87.8, 107.9, 108.7,
109.3; ES-MS Calcd. for C61H118O21Na [M + Na]: 1209.8, found
1209.9.
[11-O-Hexadecyl-8-({7ꢀ-[a-D-(1→2)-arabinofuranosyl]-[a-D-
(1→3)-arabinofuranosyl]-a-D-arabinofuranosyl}-2ꢀ,5ꢀ-
dioxaheptyl)-3,6,9,13-tetraoxanonacosyl]-[a-D-(1→2)-
arabinofuranosyl]-[a-D-(1→3)-arabinofuranosyl]-a-D-
arabinofuranoside (2)
[a-D-(1→2)-Arabinofuranosyl]-[a-D-(1→3)-arabinofuranosyl]-D-
A solution of 9 (0.06 g, 0.04 mmol), 5 (0.016 g, 0.02 mmol) and
MS 4 A, (0.1 g) in CH2Cl2 was stirred for 15 min. TMSOTf
˚
arabinofuranose (10)
(5.7 lL, 5 lmol) (0.057 mL from a stock solution of 0.1 mmol
in CH2Cl2) was added drop-wise at −10 ◦C, under N2 atmosphere.
The reaction mixture was stirred at room temperature for 2 h,
neutralized with Et3N, filtered and filtrate concentrated in vacuo
and purified (pet. ether–EtOAc, 2 : 1) to afford the protected
derivative of 2. Yield: 0.07 g (74%); Rf (pet. ether–EtOAc 1 : 1):
0.7; [a]D = +8.7 (c 1, CHCl3); 1H NMR (CDCl3, 300 MHz) d 0.82
(t, J = 6.6 Hz, 6 H), 1.17–1.22 (br s, 52 H), 1.51–1.55 (m, 4 H), 2.02
(s, 6 H), 3.2–3.8 (m, 30 H), 4.3–4.8 (m, 20 H), 5.17 (s, 2 H), 5.52–
5.82 (m, 14 H), 7.2–8.3 (m, 60 H); 13C NMR (CDCl3, 75 MHz), d
14.0, 20.2, 22.5, 25.6, 25.9, 29.2, 29.5, 29.9, 31.7, 61.5, 63.5, 69.1,
69.3, 70.1, 70.5, 70.7, 70.9, 71.5, 72.5, 77.8, 80.9, 81.3, 82.2, 86.9,
105.3, 105.6, 106.2, 128.2–129.8, 132.4, 132.6, 132.9, 133.4, 165.1–
166.1, 170.6; MALDI-TOF-MS Calcd. for C164H194O47Na2 [M +
2Na]: 2961.3, found 2961.7.
To a solution of 8 (0.10 g, 0.091 mmol) in THF–MeOH (1 :
1) (5 mL), NaOMe in MeOH (0.01 mL) was added and stirred
at room temperature for 18 h, neutralized with Amberlite ion-
exchange (H+) resin, filtered and concentrated in vacuo to afford
10, as a foamy solid. Yield: 0.04 g (95%); 1H NMR (D2O, 300 MHz)
d 3.1–4.04 (m, 15 H), 4.7–5.1 (m, 3 H); 13C NMR (D2O, 75 MHz)
d 62.1, 62.3, 63.4, 66.1, 67.4, 68.4, 69.5, 69.7, 70.5, 72.1, 72.8,
73.4, 77.3, 81.0, 82.1, 84.9, 93.6, 97.7, 110.2; ES-MS Calcd. for
C15H26O13Na [M + Na]: 437.1, found 437.1.
[11-O-Hexadecyl-8-(7ꢀ-hydroxy-2ꢀ,5ꢀ-dioxaheptyl)-3,6,9,13-
tetraoxanonacosyl]-[a-D-(1→2)-arabinofuranosyl]-[a-D-(1→3)-
arabinofuranosyl]-a-D-arabinofuranoside (1)
To a stirred solution of trisaccharide 8 (0.05 g, 0.05 mmol) and
CCl3CN (0.17 mL, 0.17 mmol) in CH2Cl2 (10 mL), DBU (7.6 lL,
5 lmol) (0.075 mL from a stock solution of 0.1 mmol in CH2Cl2)
as added at −10 ◦C and stirred for 20 min. The crude reaction
mixture was separated using pet. ether–EtOAc (7 : 3) to afford
the trichloroacetimidate 9, as a foamy solid. Yield: 0.06 g (92%).
A solution of 9 (0.06 g, 0.047 mmol), 5 (0.03 g, 0.04 mmol)
To a solution of the protected derivative of 2 (0.03 g, 0.01 mmol)
in THF–MeOH (1 : 1) (10 mL), NaOMe in MeOH (0.01 mL)
was added at room temperature and stirred for 18 h, neutralized
with Amberlite ion-exchange (H+) resin, filtered and concentrated
in vacuo to afford 2, as a foamy solid. Yield: 0.015 g (94%); [a]D =
1
+45.4 (c 1, MeOH); H NMR (D2O, 300 MHz) d 0.86 (t, J =
6.4 Hz, 6 H), 1.2–1.49 (m, 52 H), 1.51–1.56 (m, 4 H), 3.3–4.2 (m,
60 H), 5.05–5.19 (m, 6 H); 13C NMR (CD3OD, 75 MHz), d 14.4,
23.7, 27.2, 30.5, 30.8, 31.1, 33.0, 62.2, 62.7, 63.9, 71.4, 71.9, 72.1,
72.5, 73.4, 73.7, 78.6, 79.5, 79.8, 82.0, 83.6, 85.4, 85.7, 87.8, 108.0,
108.7, 109.1. MALDI-TOF-MS Calcd. for C76H142O33Na [M +
Na]: 1605.9, found 1605.9, 1341.0 [M − 2 (C5H9O3) + Na + 2 H+],
1208 [M − 3 (C5H9O3) + Na + 2 H+].
˚
and MS 4 A (0.1 g) in CH2Cl2 (10 mL) was stirred for 15 min.
TMSOTf (5.7 lL, 5 lmol) (0.057 mL from a stock solution of
0.1 mmol in CH2Cl2) was added dropwise at −10 ◦C, under N2
atmosphere. The reaction mixture was stirred at room temperature
for 2 h, neutralized with Et3N, filtered and filtrate concentrated
in vacuo and purified (pet. ether–EtOAc 1 : 1) to afford the
protected derivative of 1. Yield: 0.05 g (88%); Rf (pet. ether–EtOAc
1 : 1) 0.3; [a]D = +14.2 (c 1, CHCl3); 1H NMR (CDCl3, 400 MHz)
d 0.82 (t, J = 6.6 Hz, 6 H), 1.17–1.21 (br s, 52 H), 1.51–1.56
(m, 4 H), 2.02 (s, 3 H), 3.24–3.8 (m, 31 H), 4.3–4.8 (m, 10 H),
5.21 (s, 1 H), 5.50–5.82 (m, 7 H), 7.2–8.3 (m, 30 H); 13C NMR
(CDCl3, 100 MHz) d 14.0, 20.1, 22.5, 26.0, 28.9, 29.1, 29.6, 30.0,
31.8, 61.6, 63.0, 63.5, 69.3, 70.2, 70.4, 70.8, 71.6, 74.6, 77.4, 78.3,
78.8, 80.9, 81.2, 81.4, 82.5, 86.9, 105.6, 105.9, 106.2, 127.2–129.8,
133.0, 133.6, 165.4–165.8, 170.7; MALDI-TOF-MS Calcd. for
C105H144O28Na [M + Na]: 1876.0, found 1875.9.
[11-O-Hexadecyl-8-(7ꢀ-hydroxy-2ꢀ,5ꢀ-dioxaheptyl)-3,6,9,13-
tetraoxanonacosyl]-[a-D-(1→4)-glucopyranosyl]-b-D-
glucopyranoside (12)
Hepta-O-acetyl maltosyl bromide 1113 (0.1 g, 0.143 mmol) and 5
(0.12 g, 0.143 mmol) were dissolved in CH2Cl2 (20 mL), Ag2CO3
(0.04 g, 0.143 mmol) and AgClO4 (4 mg, 0.0143 mmol) were
added, stirred for 18 h at room temperature, filtered and the filtrate
concentrated in vacuo and purified (pet. ether–EtOAc, 1 : 3). The
resulting product (0.16 g) in THF–MeOH (1 : 1) (8 mL), NaOMe
in MeOH (0.01 mL) was added and stirred at room temperature for
To a solution of the protected derivative of 1 (0.04 g, 0.02 mmol)
in THF–MeOH (1 : 1) (10 mL), NaOMe in MeOH (0.010 mL)
was added at room temperature and stirred for 24 h, neutralized
2392 | Org. Biomol. Chem., 2008, 6, 2388–2393
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18 h, neutralized with Amberlite ion-exchange (H+) resin, filtered
and concentrated in vacuo to afford 12, as a foamy solid. Yield:
(0.13 g, 74%); [a]D = +9 (c 1, MeOH); 1H NMR (400 MHz,
CD3OD) d 0.90 (t, J = 6.9 Hz, 6 H), 1.29 (br s, 52 H), 1.53–1.58
(m, 4 H), 3.21–3.38 (m, 4 H), 3.39–3.64 (m, 35 H), 3.82–3.88 (m,
3 H), 4.19 (d, J = 7.6 Hz, 1 H), 5.15 (d, J = 4 Hz, 1 H). 13C NMR
(CD3OD, 100 MHz) d 14.4, 21.3, 23.7, 27.3, 30.5, 30.7, 33.0, 62.1,
62.7, 71.1, 71.5, 71.9, 72.5, 73.7, 74.1, 74.7, 75.0, 76.6, 77.8, 79.5,
79.8, 81.3, 102.9, 105.3. ES-MS Calcd. for C58H114O19Na [M +
Na]: 1137.8, found 1137.8.
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Acknowledgements
We thank Department of Science and Technology, New Delhi, for
a financial support. KN and BK thank the Indian Institute of
Science, Bangalore, for a research fellowship. We acknowledge the
help of Mr Prakash with the mass spectra.
This journal is
The Royal Society of Chemistry 2008
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