Synthetic arabinomannan glycolipids and their effects on growth and motility of the Mycobacterium smegmatis

Article abstract of DOI:10.1039/b917070g

Arabinomannan-containing glycolipids, relevant to the mycobacterial cell-wall component lipoarabinomannan, were synthesized by chemical methods. The glycolipids were presented with tri- and tetrasaccharide arabinomannans as the sugar portion and a double alkyl chain as the lyophilic portion. Following synthesis, systematic biological and biophysical studies were undertaken in order to identify the effects of the glycolipids during mycobacterium growth. The studies included mycobacterial growth, biofilm formation and motility assays. From the studies, it was observed that the synthetic glycolipid with higher arabinan residues inhibited the mycobacterial growth, lessened the biofilm formation and impaired the motility of mycobacteria. A surface plasmon resonance study involving the immobilized glycan surface and the mycobacterial crude lysates as analytes showed specificities of the interactions. Further, it was found that cell lysates from motile bacteria bound oligosaccharide with higher affinity than non-motile bacteria.

Full text of DOI:10.1039/b917070g

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PAPER
www.rsc.org/obc | Organic & Biomolecular Chemistry
Synthetic arabinomannan glycolipids and their effects on growth and motility
of the Mycobacterium smegmatis†
Kottari Naresh,a Binod Kumar Bharati,b Prakash Gouda Avaji,a Narayanaswamy Jayaraman*a and
Dipankar Chatterji*b
Received 18th August 2009, Accepted 22nd October 2009
First published as an Advance Article on the web 2nd December 2009
DOI: 10.1039/b917070g
Arabinomannan-containing glycolipids, relevant to the mycobacterial cell-wall component
lipoarabinomannan, were synthesized by chemical methods. The glycolipids were presented with tri-
and tetrasaccharide arabinomannans as the sugar portion and a double alkyl chain as the lyophilic
portion. Following synthesis, systematic biological and biophysical studies were undertaken in order to
identify the effects of the glycolipids during mycobacterium growth. The studies included mycobacterial
growth, biofilm formation and motility assays. From the studies, it was observed that the synthetic
glycolipid with higher arabinan residues inhibited the mycobacterial growth, lessened the biofilm
formation and impaired the motility of mycobacteria. A surface plasmon resonance study involving the
immobilized glycan surface and the mycobacterial crude lysates as analytes showed specificities of the
interactions. Further, it was found that cell lysates from motile bacteria bound oligosaccharide with
higher affinity than non-motile bacteria.
Introduction
Results and discussion
There are as many as 70 different mycobacterium strains known
currently and several of them are pathogenic. A morphologi-
cal feature common to all mycobacterial strains is the highly
orchestrated cell-wall, which provides protection against envi-
ronmental stresses.1 The cell-walls of mycobacterial strains are
composed largely of two glycolipids, namely, lipoarabinomannan
and lipoarabinogalactan.2–6 These glycolipids play critical roles
in the survival of organisms within the host.7,8 In this context,
compounds that inhibit the synthesis of the cell-wall components
have potential as drugs.9 Antibiotics such as isoniazid and
ethambutol inhibit the biosynthesis of mycobacterial cell-wall by
preventing the assembly of mycolyl-arabinogalactan (mAG).10,11
Studies of the effects of enzymes responsible for the biosynthesis
of cell-wall have been reported, using synthetic arabinogalactan
and arabinomannans.12–15
The molecular structures of tri- and tetra-saccharide glycolipids
of the present study are shown in Fig. 1. Both tri- and tetra-
saccharide glycolipids are built up with an arabinofuranoside core
moiety, which connects both the lipidic portion and the sugar
moieties of glycolipids. The mannopyranosyl-arabinofuranoside
glycolipids were chosen, as this combination of sugar moieties
are implicated to act as the capping region in native lipoarabi-
nomannan glycolipids, and the presence of the capping region is
important for the virulence properties of the species.
In our studies of synthetic glycolipid derivatives, we reported re-
cently glycolipids containing arabinofuranose trisaccharides and
their inhibitory effects on the growth of M. smegmatis strain.16 The
inhibitory effect was found to be specific with arabinofuranoside-
containing glycolipids. Continuing these efforts, we herein report
synthesis of arabinomannan glycolipids, followed by biological
and biophysical studies of these new glycolipids with a mycobac-
terial strain.
Fig. 1 Molecular structures of synthetic glycolipids.
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
Glycolipids 1 and 2 were synthesized by assembling individual con-
stituents, namely, the alkyl chain portion and the sugar portion.
The bis-O-alkylated glycerol 3 (Scheme 1) was synthesized from
solketal through: (i) O-benzylation of solketal; (ii) deprotection
of ketal protecting group; (iii) bis-O-alkylation of the resulting
diol with a long alkyl chain bromide and (iv) deprotection of the
bMolecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012,
India
† Electronic supplementary information (ESI) available: ES-MS and
NMR spectra of compounds 1, 2, 7, 10, 14, 16 and 19. See DOI:
10.1039/b917070g
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figuration of the newly formed glycosidic bond. De-O-benzylation
at the reducing end of tetrasaccharide 10 afforded lactol 11, which
was converted subsequently to an activated trichloroacetimidate
glycosyl donor 12. The preparation of tetrasaccharide glycolipid
2 was accomplished by glycosylation of 3 with 12, followed by
deprotection to afford 2, in an overall 83% yield. The 13C NMR
spectra of the protected derivative of 2 exhibited peaks at 97.9,
102.4, 105.3, 105.5 and 106.7 ppm. Whereas peaks at 97.9, 105.3
and 105.5 ppm corresponded to the a-anomeric carbon of the
glycosyl moieties at the non-reducing end, that of 102.4 and
106.7 ppm corresponded to b- and a-arabinofuranosyl moiety,19
respectively, coupled with the aglycone lipid moiety. Further, mass
spectrometric characterization confirmed the structure of 2.
In order to facilitate further biophysical studies on tri- and
tetrasaccharides with cell extracts of a mycobacterial strain,
the sugar derivatives functionalized with an amine were also
synthesized. Syntheses of amine tethered compounds 14, 16
and 19 were accomplished accordingly through glycosylation of
6-azido-hexan-1-ol with tri- and tetrasaccharide glycoside donors.
Thus, glycosylation of trichloroacetimidates 8, 12 and 1716 with
donor 6-azido-hexan-1-ol, in the presence of BF3·OEt2, followed
by deprotection of the protecting groups and a reduction of azide
to amine group (H2, Pd–C), afforded amine tethered derivatives
14, 16 and 19, in good yields (Schemes 3 and 4). The glycosylation
led to the formation of an anomeric mixture of products, which
was evident from the resonances for anomeric carbons in the
13C NMR spectra. Thus, in the case of 14 in addition to peaks
at 98.8, 99.0, 100.2 and 105.9 ppm for the a-anomeric carbons
of the glycosyl moieties, a resonance at 101.8 ppm indicated the
presence of the b-anomer of the arabinofuranoside moiety linked
to the aglycan portion of the molecule. The anomeric protons in
Scheme 1 Reagents and conditions: (i) BF3·OEt2, rt, 2 h, CH2Cl2, MS
˚
4 A; (ii) H2, Pd–C, EtOAc–MeOH (1 : 1), rt, 18 h; (iii) CCl3CN, DBU,
◦
˚
CH2Cl2, 0 C, 20 min; (iv) TMSOTf, rt, 30 min, CH2Cl2, MS 4 A;
(v) NaOMe–MeOH, rt, 2 h.
benzyl group.17 Derivative 3 was racemic and was used as such in
the preparation of glycolipids.
The tri- and tetrasaccharides were synthesized, using suitably
protected arabinofuranosides and mannopyranosides. The or-
thogonally protected sugar derivative 4 and the activated glycosyl
donor 5 were prepared by known methods.16,18 A glycosylation of
acceptor diol 4 with trichloroacetimidate donor 5, in the presence
of BF3·OEt2, led to the formation of trisaccharide 6, which
upon hydrogenolysis (H2, Pd–C) afforded lactol 7. Activation
of lactol 7 to trichloroacetimidate 8, followed by glycosylation
of acceptor 3 with 8, in the presence of TMSOTf, afforded the
protected derivative of glycolipid 1, in 88% yield. The anomeric
configuration of the protected derivative of 1 was ascertained
from the 13C NMR spectrum of the protected derivative of 1,
wherein the carbon nucleus of the newly formed glycosidic center
was observed at 105.6 ppm and 100.5 ppm, values typical of a-
and b-anomeric configurations of the arabinofuranosides (a : b ª
1 : 1).19 Subsequent deprotection, under Zemple´n conditions, led
to the isolation of glycolipid 1, in 94% yield. NMR spectroscopic
and mass spectrometric analyses confirmed the structure of
glycolipid 1.
1
the corresponding H NMR spectra could not be identified due
to several overlapping resonances. The structures of 14, 16 and 19
were confirmed further through mass spectrometry.
Preparation of tetrasaccharide 11 (Scheme 2) was initiated using
arabinofuranosyl trisaccharide 9.16 The glycosylation of 9 with
trichloroacetimidate5, mediated by BF3·OEt2, led to the formation
of tetrasaccharide 10, in 79% yield. The appearance of an apparent
1
singlet at 4.9 ppm in the H NMR spectrum and a resonance at
97.7 ppm in 13C NMR spectrum confirmed the a-anomeric con-
Scheme 3 Reagents and conditions: (i) BF3·OEt2, rt, 30 min, CH2Cl2, MS
˚
4 A; (ii) NaOMe–MeOH, rt, 2 h; (iii) H2, Pd–C, EtOAc–MeOH (1 : 1), rt,
6 h.
Effect of the arabinomannan glycolipids on growth, biofilm
formation and sliding motility in Mycobacterium smegmatis
Growth inhibition studies. Glycolipids 1 and 2, having moieties
that constitute natural lipoarabinomannan cell-wall polysaccha-
rides, were evaluated for their biological efficacies. The exper-
iments performed were mostly on the biological properties of
Scheme 2 Reagents and conditions: (i) BF3·OEt2, rt, 30 min, CH2Cl2,
˚
MS 4 A; (ii) H2, Pd–C, EtOAc–MeOH (1 : 1), rt, 18 h; (iii) CCl3CN,
DBU, CH2Cl2, 0 ◦C, 20 min; (iv) TMSOTf, rt, 30 min, CH2Cl2, MS 4 A;
˚
(v) NaOMe–MeOH, rt, 4 h.
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performed, using biofilm formation assays and sliding motility
tests.
Biofilm formation and quantitation studies. Bacterial station-
ary phase is an interesting biological system to study, as the
organism undergoes several metabolic changes during this period
and new molecules are generated to support its survival. For
pathogenic microorganisms such as mycobacteria, the stationary
phase adds a further complication, as many antibacterial com-
pounds become less effective.20,21 Work on planktonic cultures
under laboratory conditions showed that bacteria formed as
adherent multicellular aggregates called biofilms,22 which can form
on any moist surface exposed to biofilm-forming bacteria. Biofilms
are formed to protect the bacteria from host defenses, antibiotics
and harsh environmental conditions. The studies on biofilms
thus explore the possibilities for the development of antibiofilm
therapies. The formation of biofilm proceeds through different
steps. The first and most crucial step for biofilm formation is
the initial attachment of bacteria to an abiotic surface, followed
by multiplication and aggregate formation. The next step is the
formation of structured and matured biofilm. The final step is
the dispersion and reinitiation of the cycle.23 As biofilm forms a
complex of cell surface networks, we envisaged that it would be
worth studying the effect of 2 on them. Fig. 3 and Fig. 4 show
the effect of 2 (200 mg ml-1) on the formation and the quantitation
of biofilm, respectively. It can be seen from Fig. 3 that the biofilm
formation is affected significantly in the presence of 2, as compared
to the untreated bacteria (Wt). The biofilm formation is regulated
by several genes and their up and down regulation results in over-
expression and diminished expression of proteins, respectively. The
wild-type mycobacterium cultures started forming pellicles at the
air–liquid interface which can be seen as microcolonies floating
on the surface after 24 h of growth. These microcolonies spread
Scheme 4 Reagents and conditions: (i) BF3·OEt2, rt, 30 min., CH2Cl2, MS
˚
4 A; (ii) NaOMe–MeOH, rt, 4 h; (iii) H2, Pd–C, EtOAc–MeOH (1 : 1), rt,
6 h.
Mycobacterium smegmatis. Our earlier study on the monovalent
and bivalent arabinofuranoside glycolipids showed that these
glycolipids inhibited the growth of mycobacteria.16 Thus, we
undertook to evaluate the effects of glycolipids 1 and 2 on the
growth of the bacterial strain. Microbial growth is a global
indicator for metabolic regulations occurring inside the cell. The
growth of the bacteria is quantitated as a function of increase in the
cell density by spectrophotometric measurement. In the present
case, the cells of M. smegmatis were grown in a primary culture
and a secondary inoculation was carried out from a well grown
stock of primary culture. The growth profile of M. smegmatis was
subsequently followed in the presence of varying concentrations
of a solution 1 and 2 in MeOH. The growth rate in triplicate
was followed by measuring the optical density at 600 nm at
different time intervals. Growth of the bacteria without inhibitor
or in MeOH was used as a control. Fig. 2 shows the effects
observed on the growth profile of the strain in the presence of
1 and 2. It was observed that whereas 1 did not affect the growth,
glycolipid 2 inhibited the growth by about 65%, at a concentration
200 mg mL-1. Comparing this observation with our earlier report
on the arabinan glycolipid,16 it appears that the presence of a larger
proportion of arabinofuranose moiety in the glycolipid is required
for inhibiting the bacterial growth. It is unclear at present how
the inhibition of the bacterial growth occurs in the presence of the
glycolipids. In order to identify the effects of 2, further studies were
Fig. 3 Effect of 2 on biofilm formation of M. smegmatis.
Fig. 2 Effect of 1 and 2 on the growth of M. smegmatis.
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Fig. 4 Quantitation of biofilm formation.
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over the liquid surface to form a film covering the entire interface
surface in 5 to 7 days after inoculation (Fig. 3). It is also noticed
from Fig. 4 that the cultures treated with the inhibitor show a slow
growth of the biofilm, as compared to the untreated cultures or
those treated with MeOH. Following this observation, we allowed
the biofilm formation up to 240 h. It can be seen from Fig. 4 that
the formation of biofilm was reduced substantially in the presence
of 2 (200 mg ml-1), when compared to the untreated culture or
that in MeOH. The degree of inhibition of biofilm formation
by M. smegmatis with compound 2 is significant, in comparison
to our previous studies, where a gene was deleted and biofilm
formation was impaired.30 As such no other organic compound is
known to inhibit mycobacterial biofilms.
playing a role with the normal distribution of glycopeptidolipids
(GPLs) or the level of proteins which regulate the synthesis of
GPLs and results in the impaired sliding motility and biofilm
formation.
Surface plasmon resonance studies. Further to the observa-
tions that the glycolipid 2 inhibited mycobacterial growth, lessened
biofilm formation and impaired the motility of mycobacteria, we
were interested to study glycolipids 1 and 2 with cell lysates from
the motile and non-motile strain of mycobacteria, through surface
plasmon resonance (SPR) biophysical methods. SPR provides the
ability to evaluate the interactions between a receptor and a ligand,
under non-labeling and non-invasive conditions.28 The technique
allows the studies to be performed under flowing conditions, over
an immobilized surface, thereby mimicking the cell-surface envi-
ronments. Initially, glycolipids 1 and 2 were immobilized on to the
sensor chip, wherein the glycolipids interdigitated with long alkyl
chain functionalized HPA sensor chip. Although interdigitation of
the glycolipids occurred, interactions of mycobacterial cell lysates
were unsuccessful, possibly due to removal of the glycolipids
along with the flowing cell lysates. This difficulty led us to adopt
an alternative method of immobilization, wherein a covalent
functionalization of the surface was undertaken. Accordingly,
amine tethered oligosaccharides 14, 16 and 19 were used for
immobilization over the C1 sensor chip, presenting carboxylic
acid functionalities. The amine coupling methodology allowed
immobilization of 14, 16 and 19. Immobilization of the arabino-
mannans was performed uniformly to ~200 RU, corresponding to
a loading of ~200 pg mm-2 of the oligosaccharides immobilized
on the surface. The remaining activated succinimide esters, un-
bound by oligosaccharides, were blocked using ethanolamine. In
order to eliminate the bulk refractive change and non-specific
interactions, ethanolamine was immobilized in one of the flow
cells. After immobilization of 14, 16 and 19, physically adsorbed
materials were removed through purging with the buffer. Upon
immobilization, cell lysates from different types of bacteria were
used as analytes. Cell lysates from motile (mc2155) and non-motile
(mcdrz) bacteria, with 2 and 0.02% glucose, respectively, were
chosen.30 Cell lysates were extracted at two stages of the growth:
one was at the exponential phase of the growth, i.e., after 36 h
of the cell growth, and other at the late stationary phase of the
growth, i.e., after 88 h of the growth. The collected cell lysates
were dissolved in the running buffer (HEPES), and passed over
the flow cells. The cell lysates were passed for 60 s during the
association phase. The dissociation phase was monitored during
subsequent washing of the surface with buffer alone. Proteins
remaining after the dissociation phase were removed from the
chip, through an injection of an aqueous solution of sodium
dodecyl sulfate (SDS) (0.03%). Fig. 6 shows binding of the cell
lysate of the motile strain (wild type) in the presence of 19.
Different concentrations of the cell lysates were passed over the
flow cells containing trisaccharide 19 and ethanolamine alone.
Response units from the flow cell containing 19 were subtracted
from the flow cell containing ethanol amine, in order to eliminate
the refractive index change arising from the change in the buffer
and the buffer containing cell lysates, as well as the non-specific
interactions.
Sliding motility inhibition studies. The mycobacterial cell
membrane is surrounded by a thick and waxy envelope that
contains diverse lipids.24 The envelope is made up of three types of
insoluble macromolecular components: (i) heteropolysaccharide
arabinogalactan; (ii) peptidoglycan and (iii) mycolic acid.25 It
was demonstrated earlier that the glycolipids containing arabi-
nofuranose trisaccharide inhibited the growth of mycobacteria.16
Further, data presented herein show that an additional mannan
portion in the arabinomannan glycolipids does not alter the
growth pattern of the mycobacteria. The biofilm formation was
also affected in the presence of arabinomannan glycolipid 2. We
were interested to identify other phenotypic behaviors, such as
sliding motility. The sliding motility test was carried out to check
the spreading ability of mycobacteria on the surface. The motile
M. smegmatis was tested on the soft and moist surface of the
Petri-plates. The sliding motility was assayed in the presence of 2%
glucose as a carbon source. Glycolipid 2 was added in the MB7H9
base medium, supplemented with 0.3% high grade agarose. A
relationship between the defect in biofilm formation and sliding
motility was shown in M. smegmatis mutants defective in GPL
synthesis by Kolter and Recht.26 As the biofilm formation was
defective in our case, we were interested to look at the sliding
motility of mycobacteria. It was observed that the motility of the
bacteria was highly diminished in the presence of 2. Fig. 5 shows
the sliding motility of mycobacteria in the presence of different
concentrations of 2 up to 7 days. Whereas MeOH appeared to
show little inhibition of the motility, glycolipid 2, at concentrations
of 100 mg mL-1 and 200 mg mL-1, affected the sliding motility
remarkably. Thus, the glycolipid 2 not only exhibited the effect
of reducing the biofilm formation, but also induced an impaired
mobility of mycobacteria on the moist surface, as compared to the
untreated bacteria.
Fig. 5 Effect of 2 on the motility of M. smegmatis (7 days old).
The mechanism of sliding in mycobacteria has not been char-
acterized in detail. It is known that the acetylation of glycopep-
tidolipids affects the biofilm formation and sliding motility in
mycobacteria.27 It appears that these synthetic compounds may be
With the increase in the cell lysate concentration, i.e., from 0.16
to 0.4, an increase in the response units was observed, indicating
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Fig. 6 Sensorgrams of the binding of varying concentrations of the cell
lysates motile strain, after 36 h, and 19. (a) Cell lysates with the protein
absorbance 0.4; (b) cell lysates with absorbance 0.26 and (c) cell lysates
with absorbance 0.16.
Fig. 8 Sensorgrams of the binding of the cell extracts of WT, with the
glucose (2%) after 36 h with (a) 14; (b) 16 and (c) 19.
than the arabinofuranosides alone (19), or the mannopyranosyl
arabinofuranoside, having one mannopyranosyl unit (16).
that the interactions between the compound 19 and cell lysates
were specific.
Conclusion
Binding of the cell lysates extracted from the motile (wild type)
and non-motile (mcdrz) bacteria is shown in Fig. 7. All the cell
lysates were passed in a uniform concentration, in order to find
the relative affinity between different types of strains. In general,
motile bacteria showed stronger binding than the non-motile
strain of the mycobacteria. In the case of non-motile bacteria,
the cell extracts of the stationary phase exhibited a higher affinity
than the cell extracts of the exponential phase. On the other hand,
the motile bacterial cell extracts showed an almost similar binding
behavior for both the phases. Binding of cell lysates from motile
and non-motile strains, grown with a glucose concentration of
0.02%, with 19 was found to be low.
In conclusion, we have synthesized arabinomannan containing
glycolipids and studied their effects on the growth, biofilm
formation and motility of M. smegmatis. It was observed that the
synthetic glycolipid with higher arabinofuranoside composition
inhibited the growth, lessened the biofilm formation and impaired
the motility of the mycobacteria significantly, when compared
to the wild type. It appears that the synthetic glycolipids may
interrupt the normal profile of the glycopeptidolipids. An effort
to evaluate the interactions of the immobilized arabinomannan
oligosaccharide with mycobacterial cell lysates was undertaken.
It was found that the cell lysates from motile bacteria bound
oligosaccharide with higher affinity than the non-motile bacteria.
Future work will involve higher oligosaccharides of arabinofura-
nosides and mannopyranosyl arabinofuranosides, and studies of
their biological profiles.
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 performed on commercial plates coated with silica gel 60
F254. Visualization of the spots on TLC plates was achieved by
UV radiation or spraying with 5% sulfuric acid in ethanol. High-
resolution mass spectra were obtained from a 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 are
reported in Hz. Standard abbreviations s, app s, d, t, dd, br s,
m refer to singlet, apparent singlet, doublet, triplet, doublet of
doublets, broad singlet, multiplet.
Fig. 7 Sensorgrams of the binding of the protein extracts from the
wild-type and the mcdrz bacteria, with glucose (2%) after 36 h and 88 h of
the growth, with 19. (a) Wild-type strain with glucose (2%), after 36 h and
88 h of the growth; (b) mcdrz strain with glucose (2%), after 88 h of the
growth and (c) mcdrz strain with glucose (2%), after 36 h of the growth.
Fig. 8 shows the relative binding of the cell lysates of the motile
strain, with 2% glucose after 36 h with different arabinomannan
compounds. From the sensorgrams, it is clear that the binding is
relatively higher for the mannopyranosyl arabinofuranoside (14)
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5-O-Acetyl-[2,3,4,6-tetra-O-acetyl-a-D-(1→2)-mannopyra-
nosyl]-[2,3,4,6-tetra-O-acetyl-a-D-(1→3)-mannopyranosyl]-D-ara-
binofuranose (7). A solution of 416 (0.21 g, 0.75 mmol), 518
Benzyl-[2,3,4,6-tetra-O-acetyl-a-D-(1→5)-mannopyranosyl]-
[2,3,5-tri-O-benzoyl-a-D-(1→2)-arabinofuranosyl]-[2,3,5-tri-O-
benzoyl-a-D-(1→3)-arabinofuranosyl]-a-D-arabinofuranoside (10).
A solution of 916 (0.45 g, 0.40 mmol), 5 (0.23 g, 0.48 mmol) and MS
˚
(0.81 g, 1.66 mmol) and MS 4 A (0.5 g) in CH2Cl2 (60 mL) was
˚
stirred for 15 min, BF3·OEt2 (0.23 mL, 1.66 mmol) was added,
under N2 atmosphere and the reaction mixture was stirred at
room temperature for 2 h, filtered and the filtrate washed with
the saturated aq. NaHCO3 solution and with water. The organic
phase was separated, dried (Na2SO4), filtered and the filtrate
concentrated to afford 6. A solution of the crude reaction mixture
of 6 in EtOAc–MeOH (1 : 1) (50 mL) was treated with Pd–C (10%)
(0.45 g) and stirred under H2 (1 bar) for 18 h. The reaction mixture
was filtered, solvent removed in vacuo, the crude reaction mixture
purified (pet. ether–EtOAc 3 : 2), to afford 7, as a foamy solid.
Yield: 0.4 g (62%); Rf (pet. ether–EtOAc 3 : 7) 0.34; [a]D +41.2
4 A (0.5 g) in CH2Cl2 (30 mL) was stirred for 15 min., BF3·OEt2
(0.07 mL, 0.476 mmol) was added, under N2 atmosphere. The
reaction mixture was stirred at room temperature for 30 min,
filtered and filtrate washed with the saturated aq. NaHCO3
solution and with water. The organic phase was separated, dried
(Na2SO4), filtered and the filtrate concentrated and purified (pet.
ether–EtOAc 3 : 2), to afford 10, as a foamy solid. Yield: 0.45 g
(79%); Rf (pet. ether–EtOAc 3 : 2) 0.42; [a]D +25.7 (c 1, CHCl3);
1H NMR (400 MHz, CDCl3) d 1.86–2.11 (m, 12 H), 3.82 (d, J =
10.4 Hz, 1 H), 3.94 (dd, J = 4.8 Hz, J = 10.4 Hz, 1 H), 4.09–4.16
(m, 3 H), 4.28 (dd, J = 4.8 Hz, J = 7.6 Hz, 1 H), 4.35 (s, 2 H), 4.48–
4.77 (m, 9 H), 4.90 (app s, 1 H), 5.19 (br s, 1 H), 5.28 (t, J = 10 Hz,
1 H), 5.35 (s, 1 H), 5.38 (dd, J = 6.4 Hz, J = 10 Hz, 1 H), 5.51–5.64
(m, 5 H), 7.18–7.37 (m, 14 H), 7.4–7.6 (m, 10 H), 7.88 (dd, J =
7.2 Hz, J = 4 Hz, 3 H), 7.95–8.06 (m, 8 H); 13C NMR (100 MHz,
CDCl3) d 20.4, 20.6, 20.7, 62.4, 63.5, 63.7, 66.0, 68.5, 69.0, 69.2,
69.3, 77.2, 77.4, 80.1, 81.0, 81.4, 82.3, 82.7, 86.5, 97.7, 105.5, 125.2-
129.8, 132.9-133.5, 137.3, 165.3-169.7, 170.5, 170.6; ES-MS Calcd.
for C78H74O28Na: 1481.4264 [M + Na]; found: 1481.4232.
1
(c 0.5, CHCl3); H NMR (400 MHz, CDCl3) d 1.90–2.19 (m,
27 H), 3.88–4.09 (m, 5 H), 4.13–4.38 (m, 7 H), 4.92–4.96 (m,
1 H), 5.19 (br s, 1 H), 5.22–5.40 (m, 5 H), 5.45 (app s, 1 H); 13C
NMR (100 MHz, CDCl3) d 20.5, 20.6, 20.7, 62.3, 62.4, 62.8, 63.1,
63.5, 65.9, 66.1, 68.4, 68.7, 69.1, 69.4, 75.8, 79.0, 79.5, 80.8, 84.7,
85.9, 95.3, 96.9, 97.2, 99.2, 100.4, 169.5-171.7. ES-MS Calcd. for
C35H48O24Na: 875.2433 [M + Na]; found: 875.2427.
2-O-Hexadecyl-4-oxa-1,2-eicosanedioyl-[a-D-(1→2)-mannopy-
2,3,4,6-Tetra-O-acetyl-a-D-(1→5)-mannopyranosyl-[2,3,5-tri-
O-benzoyl-a-D-(1→2)-arabinofuranosyl]-[2,3,5-tri-O-benzoyl-a-D-
(1→3)-arabinofuranosyl]-D-arabinofuranose (11). A solution of
10 (0.32 g, 0.217 mmol) in EtOAc–MeOH (1 : 1) (20 mL) was
treated with Pd–C (10%) (0.1 g) and stirred under H2 (1 bar)
for 18 h. The reaction mixture was filtered, solvent removed in
vacuo, the crude reaction mixture purified (pet. ether–EtOAc
1 : 1), to afford 11, as a foamy solid. Yield: 0.28 g (90%); Rf
ranosyl]-[a-D-(1→2)-mannopyranosyl]-D-arabinofuranoside
(1).
To a stirred solution of trisaccharide 7 (0.06 g, 0.068 mmol)
and CCl3CN (0. 068 mL, 0.68 mmol) in CH2Cl2 (10 mL), DBU
(10 mL, 0.068 mmol) was added at 0 ◦C and stirred for 20 min.
The crude reaction mixture was purified using pet. ether–EtOAc
(1 : 1), to afford the trichloroacetimidate 8, as a foamy solid. Yield
(0.061 g, 90%). A solution of 8 (0.04 g, 0.039 mmol), 3 (0.02 g,
˚
0.039 mmol) and MS 4 A (0.1 g) in CH2Cl2 (10 mL) was stirred
1
(pet. ether–EtOAc 1 : 1) 0.45; H NMR (300 MHz, CDCl3) d
for 15 min, TMSOTf (7.1 mL, 10% solution in CH2Cl2, 4 mM)
was added at -10 ◦C, under N2 atmosphere. The reaction mixture
was stirred at room temperature for 30 min, neutralized with
Et3N, filtered and filtrate concentrated in vacuo and purified (pet.
ether–EtOAc 2 : 3), to afford the protected derivative of 1 (a :
b ª 1 : 1); Yield: 0.047 g (88%); Rf (pet. ether–EtOAc 1 : 1) 0.5;
1H NMR (400 MHz, CDCl3) d 0.88 (t, J = 6.8 Hz, 6 H), 1.25
(br s, 52 H), 1.53–1.60 (m, 4 H), 1.93–2.2 (m, 27 H), 3.36–3.60
(m, 7 H), 3.77–3.83 (m, 1 H), 3.90 (dd, J = 3.2 Hz, J = 12 Hz,
1 H), 3.96–3.99 (m, 1 H), 4.09–4.16 (m, 4 H), 4.18–4.4 (m, 6 H),
4.93–4.99 (m, 2 H), 5.13–5.19 (m, 2 H), 5.22–5.31 (m, 5 H); 13C
NMR (100 MHz, CDCl3) d 14.1, 20.6, 20.8, 21.0, 22.6, 26.1,
29.3-29.6, 30.1, 31.8, 60.3, 62.4, 65.7, 65.9, 66.0, 68.4, 68.8, 69.1,
69.2, 69.4, 70.4, 70.8, 71.7, 77.5, 80.4, 84.7, 85.0, 97.3, 99.5,
99.8, 100.5, 105.6, 165.5–171.1; ES-MS Calcd. for C70H118O26Na:
1397.7809 [M + Na]; found: 1397.7802.
1.90–2.11 (m, 12 H), 3.75–3.94 (m, 2 H), 4.03–4.18 (m, 4 H),
4.23–4.44 (m, 2 H), 4.51–4.98 (m, 8 H), 5.27–5.38 (m, 2 H), 5.39
(dd, J = 3.6 Hz, J = 9.9 Hz, 1 H), 5.46–5.68 (m, 7 H), 7.24–734
(m, 7 H), 7.42–7.61 (m, 12 H), 7.84–7.94 (m, 3 H), 8.0–8.09 (m,
8 H). 13C NMR (100 MHz, CDCl3) d 20.4, 20.6, 20.8, 21.0, 60.4,
62.8, 63.6, 66.0, 66.6, 67.8, 68.1, 68.8, 69.1, 69.7, 77.6, 79.1, 81.2,
81.4, 82.4, 82.7, 96.2, 97.7, 101.6, 105.2, 105.6, 128.2-129.9, 133.0,
133.4, 133.5, 133.6, 165.3–166.1, 169.6–171.1; ES-MS Calcd. for
C71H68O28Na: 1391.3795 [M + Na]; found: 1391.3731.
2-O-Hexadecyl-4-oxa-1,2-eicosanedioyl-[2,3,4,6-tetra-O-acetyl-
a-D-(1→5)-mannopyranosyl]-[2,3,5-tri-O-benzoyl-a-D-(1→2)-ara-
binofuranosyl]-[2,3,5-tri-O-benzoyl-a-D-(1→3)-arabinofuranosyl]-
D-arabinofuranoside (2). To a stirred solution of tetrasaccharide
11 (0.06 g, 0.043 mmol) and CCl3CN (0. 043 mL, 0.43 mmol)
◦
in CH2Cl2 (8 mL), DBU (7 mL, 0.043 mmol) was added at 0 C
To a solution of protected derivative of 1 (0.05 g, 0.037 mmol)
in THF–MeOH (1 : 1) (5 mL), NaOMe in MeOH (0.010 mL)
was added at room temperature and stirred for 2 h, neutralized
with Amberlite ion exchange (H+) resin, filtered and filtrate
concentrated to afford 1, as a foamy solid. Yield: 0.035 g (94%);
1H NMR (400 MHz, CD3OD) d 0.90 (t, J = 6.4 Hz, 6 H), 1.29
(br s, 52 H), 1.55–1.58 (m, 4 H), 3.47–3.87 (m, 24 H), 4.05–4.28 (m,
2 H), 4.94–5.01 (m, 3 H); 13C NMR (100 MHz, CD3OD) d 14.4,
23.7, 27.3, 30.4-31.0, 33.1, 62.8, 63.2, 64.9, 68.5, 71.5, 72.1, 72.3,
72.5, 72.6, 75.3, 79.1, 80.7, 82.0, 86.0, 101.9, 102.1, 103.8. ES-MS
Calcd. for C52H100O17Na 1019.6858 [M + Na]; found: 1019.6881.
and stirred for 20 min. The crude reaction mixture was purified
using pet. ether–EtOAc (1 : 1), to afford the trichloroacetimidate
12, as a foamy solid. Yield (0.06, 93%). A solution of 12 (0.057 g,
˚
0.038 mmol), 3 (0.02 g, 0.04 mmol) and MS 4 A (0.1 g) in CH2Cl2
(10 mL) was stirred for 15 min. TMSOTf (6.7 mL, 0.004 mmol)
was added at -10 ◦C, under N2 atmosphere. The reaction mixture
was stirred at room temperature for 30 min, neutralized with
Et3N, filtered and filtrate concentrated in vacuo and purified (pet.
ether–EtOAc 1 : 1), to afford the protected derivative of 2 (a :
b ª 9 : 1); Yield: 0.057 g (87%); Rf (pet. ether–EtOAc 6.5 : 3.5)
0.57; 1H NMR (400 MHz, CDCl3) 0.87 (t, J = 6.8 Hz, 6 H), 1.25
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(br s, 52 H), 1.4–1.53 (m, 4 H), 1.89–2.12 (m, 12 H), 3.22–3.57
(m, 9 H), 3.74–3.86 (m, 2 H), 3.96 (dd, J = 11.2 Hz, J = 4.8 Hz,
1 H) 4.04–4.14 (m, 3 H), 4.29 (dt, J = 12 Hz, J = 4.4 Hz, 1 H),
4.39–4.40 (m, 1 H), 4.48 (s, 1 H), 4.52–4.87 (m, 5 H), 4.91 (s, 1 H),
5.15 (s, 1 H), 5.22–5.37 (m, 3 H), 5.44–5.72 (m, 6 H), 7.25–7.56
(m, 18 H), 7.89–8.07 (m, 12 H). 13C NMR (100 MHz, CDCl3)
d 14.1, 20.4, 20.5, 20.6, 20.8, 21.0, 22.6, 26.1, 29.3-29.6, 30.1,
31.9, 50.8, 60.3, 62.2, 63.6, 66.1, 66.9, 68.6, 69.1, 69.4, 69.8, 70.1,
70.6, 70.9, 71.7, 71.8, 77.8, 80.1, 80.4, 81.2, 82.4, 82.9, 86.1, 97.9,
102.4, 105.3, 105.5, 106.7, 128.3–130.0, 133.3, 133.7, 165.3–171.1;
ES-MS Calcd. for C106H138O30Na: 1913.9171 [M + Na]; found
1913.9277.
To a solution of protected derivative of 2 (0.04 g, 0.02 mmol)
in THF–MeOH (1 : 1) (5 mL), NaOMe in MeOH (0.010 mL)
was added at room temperature and stirred for 4 h, neutralized
with Amberlite ion exchange (H+) resin, filtered and filtrate
concentrated to afford 2, as a foamy solid. Yield: 0.017 g (95%);
1H NMR (400 MHz, CD3OD) d 0.89 (t, J = 6.8 Hz, 6 H), 1.28
(br s, 52 H), 1.53–1.60 (m, 4 H), 3.43–3.50 (m, 4 H), 3.52–3.65 (m,
8 H), 3.67–3.79 (m, 7 H), 3.81–3.86 (m, 4 H), 3.90–3.95 (m, 2 H),
3.97–4.04 (m, 3 H), 4.09–4.22 (m, 2 H), 4.80–4.83 (m, 1 H), 4.96-
–5.12 (m, 3 H); 13C NMR (100 MHz, CD3OD) d 14.5, 23.7, 27.3,
30.1-31.2, 33.1, 62.9, 67.2, 68.6, 71.6, 72.0, 72.5, 72.6, 74.7, 78.7,
79.0, 82.4, 82.6, 82.9, 83.8, 85.6, 101.8, 108.4, 108.8, 109.7, 110.5;
ES-MS Calcd. for C56H106O20Na: 1121.7175 [M + Na]; found:
1121.7164.
of crude protected derivative of 15 in THF–MeOH (1 : 1) (5 mL),
NaOMe in MeOH (0.010 mL) was added at room temperature
and stirred for 4 h, neutralized with Amberlite ion exchange (H+)
resin, filtered and filtrate concentrated to afford 15, as a foamy
solid. Yield: 0.08 g (80%). A solution of 15 in EtOAc–MeOH
(1 : 1) (20 mL) was treated with Pd–C (10%) (0.1 g) and stirred
under H2 (1 bar) for 6 h. The reaction mixture was filtered, solvent
removed in vacuo, to afford 16 (a : b ª 1 : 1), as a foamy solid.
Yield: 0.066 g (85%); 1H NMR (D2O, 300 MHz) d 1.25 (br s, 4 H),
1.49 (br s, 4 H), 2.82 (t, J = 7.8 Hz, 2 H), 3.32–4.06 (m, 23 H), 4.76
(s, 1 H), 4.9–5.08 (m, 3 H); 13C NMR (D2O, 75 MHz) d 25.7, 26.4,
26.7, 29.2, 29.4, 62.0, 67.4, 69.2, 70.8, 71.3, 73.6, 77.4, 78.9, 82.3,
82.9, 84.9, 85.0, 100.7, 101.3, 107.8, 108.5, 109.3. ES-MS Calcd.
for C27H50NO18 [M + H]: 676.3, found 676.3.
6-Aminohexyl-[a-D-(1→2)-arabinofuranosyl]-[a-D-(1→3)-ara-
binofuranosyl]-D-arabinofuranoside (19).
A
solution of 1716
(0.133 g, 0.12 mmol), 6-azido1-hexanol (0.017 g, 0.12 mmol)
˚
and MS 4 A (0.1 g) in CH2Cl2 (20 mL) was stirred for 15 min,
BF3·OEt2 (15 mL, 0.12 mmol) was added dropwise at -10 ◦C,
under N2 atmosphere. The reaction mixture was stirred at room
temperature for 30 min, neutralized with Et3N, filtered and filtrate
concentrated in vacuo and purified (pet. ether–EtOAc 1 : 1), to
afford the protected derivative of 18. To a solution of protected
derivative of 18 in THF–MeOH (1 : 1) (5 mL), NaOMe in MeOH
(0.010 mL) was added at room temperature and stirred for 4 h,
neutralized with Amberlite ion exchange (H+) resin, filtered and
filtrate concentrated to afford 18 (a : b ª 1 : 1), as a foamy solid.
Yield: 0.045 g (74%). A solution of the in EtOAc–MeOH (1 : 1)
(20 mL) was treated with Pd–C (10%) (0.1 g) and stirred under H2
(1 bar) for 6 h. The reaction mixture was filtered, solvent removed
6-Aminohexyl-[a-D-(1→2)-mannopyranosyl]-[a-D-(1→2)-man-
nopyranosyl]-D-arabinofuranoside (14). A solution of 8 (0.2 g,
˚
0.2 mmol), 6-azido-1-hexanol (0.028 g, 0.2 mmol) and MS 4 A
(0.1 g) in CH2Cl2 (20 mL) was stirred for 15 min, BF3·OEt2
(26 mL, 0.2 mmol) was added dropwise at -10 ◦C, under N2
atmosphere. The reaction mixture was stirred at room temperature
for 30 min, neutralized with Et3N, filtered and filtrate concentrated
in vacuo and purified (pet. ether–EtOAc 2 : 3), to afford the
protected derivative of 13. To a solution of the crude protected
derivative of 13 in THF–MeOH (1 : 1) (5 mL), NaOMe in MeOH
(0.010 mL) was added at room temperature and stirred for 2 h,
neutralized with Amberlite ion exchange (H+) resin, filtered and
filtrate concentrated to afford 13, as a foamy solid. Yield: 0.12 g
(76%). A solution of 13 in EtOAc–MeOH (1 : 1) (20 mL) was
treated with Pd–C (10%) (0.1 g) and stirred under H2 (1 bar) for
6 h. The reaction mixture was filtered, solvent removed in vacuo,
to afford 14 (a : b ª 1 : 1), as a foamy solid. Yield (0.080 g, 90%);
1H NMR (D2O, 400 MHz) d 1.24 (br s, 4 H), 1.45–1.51 (m, 4 H),
2.83 (t, J = 7.2 Hz, 2 H), 3.33–4.17 (m, 19 H), 4.71–5.1 (m, 3 H);
13C NMR (D2O, 100 MHz) d 24.7, 24.9, 25.2, 26.5, 28.3, 39.3,
60.7, 61.3, 63.6, 66.5, 67.4, 69.9, 70.1, 70.3, 72.6, 73.4, 76.3, 79.4,
79.6, 79.9, 83.2, 98.8, 99.0, 100.2, 101.8, 105.9; ES-MS Calcd. for
C23H43NO15Na [M + Na]: 596.3, found 596.2.
1
in vacuo, to afford 19, as a foamy solid. Yield: 0.04 g (87%); H
NMR (D2O, 400 MHz) d 1.21 (br s, 4 H), 1.41–1.48 (m, 4 H),
2.81 (t, J = 7.6 Hz, 2 H), 3.32–4.06 (m, 17 H), 4.8–5.08 (m, 3 H);
13C NMR (D2O, 100 MHz) d 24.6, 24.8, 25.2, 26.5, 28.4, 39.4,
60.9, 61.1, 63.4, 68.0, 76.5, 79.6, 80.2, 81.1, 81.3, 82.4, 83.8, 84.0,
84.3, 85.3, 100.7, 106.1, 106.9, 107.6, 108.5; ES-MS Calcd. for
C21H40NO13 [M + H]: 514.2499, found 514.2492.
Bacterial strains, media, and growth conditions. Wild type
M. smegmatis mc2155 and mcdrz (rpoZ mutant) strains were
grown in Middlebrook 7H9 broth (MB7H9, Difco) supplemented
with 2% glucose, 0.05% Tween 80 and MB7H9 agar medium
supplemented with 2% glucose at 37 ◦C with agitation as and when
required. Kanamycin (25mg ml-1) was used as and when required.
The cells of M. smegmatis were grown in a primary culture and
secondary inoculation was carried out from a well grown stock
of primary culture. The growth profile of M. smegmatis was
followed at different concentrations of 1 and 2 and the growth
rate in triplicate was followed by measuring the optical density at
600 nm, at different durations. Growth of the bacteria without the
glycolipids or in MeOH was used as the control. The inhibitors
were dissolved in MeOH before addition to the M. smegmatis
culture.
6-Aminohexyl-[a-D-(1→5)-mannopyranosyl]-[a-D-(1→2)-arabi-
nofuranosyl]-[a-D-(1→3)-arabinofuranosyl]-D-arabinofuranoside
(16). A solution of 12 (0.24 g, 0.15 mmol), 6-azido-1-hexanol
˚
(0.02 g, 0.15 mmol) and MS 4 A (0.1 g) in CH2Cl2 (20 mL)
was stirred for 15 min, BF3·OEt2 (20 mL, 0.15 mmol) was added
Biofilm formation assay and quantitation. Inoculum of My-
cobacterium smegmatis mc2155 was added into primary cul-
tures containing 2% Glycerol and 0.05% Tween-80 in Sauton’s
medium.24,29 The well-grown culture was washed with Sauton’s
medium before secondary inoculation. For the development of
◦
dropwise at -10 C, under N2 atmosphere. The reaction mixture
was stirred at room temperature for 30 min, neutralized with Et3N,
filtered and filtrate concentrated in vacuo and purified (pet. ether–
EtOAc 3 : 2), to afford the protected derivative of 15. To a solution
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biofilm, Sauton’s medium (5 mL) was added to the 6-well cell
culture plate (Laxbro) and inoculated with 1 : 100 dilutions from
a well grown primary culture and inhibitors were added in
different concentrations. Petri-plates were incubated in a humid-
ified incubator at 37 ◦C and monitored at different points of
time.
Acknowledgements
We thank Department of Science and Technology, New Delhi
and Department of Biotechnology, New Delhi, for a financial
support. Council of Scientific and Industrial Research, New Delhi,
and Indian Institute of Science, Bangalore, are acknowledged for
research fellowships to KN and BKB, respectively.
The quantitation of biofilm formation was done as described
earlier.24,30 The well-grown culture was washed with Sauton’s
medium and diluted up to a final optical density (O.D.) of 0.0025
at 600 nm in Sauton’s medium. The inhibitors were added in
different concentration at zero time point and distributed into
96-well polystyrene plates. Eight wells were assayed for each
inhibitor at each time point and experiment was repeated thrice.
Inoculum (250 mL) was added in each well and biofilm formation
was monitored up to 240 h. The samples were emptied from the
wells, and the wells were washed twice thoroughly with water and
stained by the addition of 1% crystal violet solution (300 mL) and
incubated for 20 min. The wells were again washed thoroughly
with water and allowed to dry. The dye was quantitated after
incubating each well with aq. ethanol (80%) (300 mL) for an hour
and subsequently measuring the absorption at 590 nm using a
microplate reader (Spectra Max 340PC384, Molecular devices).
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The Royal Society of Chemistry 2010
Org. Biomol. Chem., 2010, 8, 592–599 | 599
©