Synthesis, biological studies of linear and branched arabinofuranoside- containing glycolipids and their interaction with surfactant protein A

Glycobiology vol. 21 no. 9 pp. 1237–1254, 2011

doi:10.1093/glycob/cwr068

Advance Access publication on May 19, 2011

Synthesis, biological studies of linear and branched

arabinofuranoside-containing glycolipids and their

interaction with surfactant protein A

Kottari Naresh^2,*, Binod Kumar Bharati^3,*

,

Introduction

Prakash Gouda Avaji^2,*, Dipankar Chatterji³,

and Narayanaswamy Jayaraman^1,2

Oligosaccharides form an integral component of mycobacter-

ial cell wall and display rich structural and functional roles

(Jarlier and Nikaido 1994; Brennan and Nikaido 1995). The

cell walls of mycobacterial strains contain primarily lipoarabi-

nomannans (LAMs) and mycolyl arabinogalactan–peptidogly-

can (mAGP) complex (Nigou et al. 2004; Chatterjee and

Brennan 2009). The arabinan domain of both LAM and

mAGP consists of α-(1 → 3), α-(1 → 5) and β-(1 → 2) lin-

kages between ^D-arabinose moieties. Key intermediate in the

biosynthesis of the arabinan domain of LAM and mAGP is

the oligoarabinofuranoside, attached through α-(1 → 3) and

α-(1 → 5) linkages (Mikusová et al. 2000; Escuyer et al.

2001; Kremer and Besra 2005; Shi et al. 2006). Thus, under-

standing the polysaccharide components of LAM and mAGP

assumes signiﬁcance in studies directed toward mycobacterial

cell wall functions (Mereyala et al. 1998; Rose et al. 2002;

Jayaprakash et al. 2005; Lee et al. 2005; Hölemann et al.

2006; Ishiwata et al. 2006; Davis et al. 2007; Joe et al. 2007;

Zhang et al. 2007; Gandolﬁ-Donadío et al. 2008; Umesiri

et al. 2010).

We showed previously that glycolipids containing an arabi-

nofuranoside trisaccharide inhibited the growth of

Mycobacterium smegmatis. Both arabinofuranoside trisacchar-

ide and lipidic portion were essential for the biological

activity (Naresh et al. 2008). It was shown further that arabi-

nomannan glycolipids inhibited the sliding motility and

bioﬁlm formation of M. smegmatis (Naresh et al. 2010).

These studies showed that glycolipids with arabinan moeities

affected the growth more signiﬁcantly than glycolipids with

arabinomannan moeities. The inhibition activities were found

to be dependent on the presence of the lipid portion, as the

saccharide portion alone was not sufﬁcient. Continuing

the studies to identify the effects of synthetic glycolipids on

the growth and motilities of mycobacteria, we undertook

synthesis and studies of glycolipids constituted with higher

oligosaccharides. Glycolipids presenting linear tetra-, hexa-

and octasaccharide glycolipids, with α-(1 → 5) linkages, as

well as a branched heptasaccharide containing α-(1 → 2) and

α-(1 → 5) linkages (Chart 1) were synthesized, and their

effects on mycobacterial growth, motilities and bioﬁlm for-

mation were assessed. Further studies relating to the inter-

action of these synthetic glycolipids with a pulmonary

surfactant protein, namely surfactant protein A (SP-A), were

²Department of Organic Chemistry and ³Molecular Biophysics Unit, Indian

Institute of Science, Bangalore 560 012, India

Received on January 18, 2011; revised on May 2, 2011; accepted on May 3,

2011

Oligoarabinofuranoside-containing glycolipids relevant to

mycobacterial cell wall components were synthesized in

order to understand the functional roles of such glycolipids.

A series of linear tetra-, hexa-, octa- and a branched hepta-

saccharide oligoarabinofuranosides, with 1 → 2 and 1 → 5

α-linkages between the furanoside residues, were syn-

thesized by chemical methods from readily available

monomer building blocks. Upon the synthesis of glyco-

lipids, constituted with a double alkyl chain-substituted sn-

glycerol core and oligosaccharide fragments, biological

studies were performed to identify the effect of synthetic

glycolipids on the bioﬁlm formation and sliding motilities

of Mycobacterium smegmatis. Synthetic glycolipids and ara-

binofuranosides displayed an inhibitory effect on the

growth proﬁle, but mostly on the bioﬁlm formation and

maturation. Similarly, synthetic compounds also inﬂuenced

the sliding motility of the bacteria. Further, biophysical

studies were undertaken, so as to identify the interactions

of the glycolipids with a pulmonary surfactant protein,

namely surfactant protein A (SP-A), with the aid of the

surface plasmon resonance technique. Speciﬁcities of each

glycolipid interacting with SP-A were thus evaluated.

From this study, glycolipids were found to exhibit higher

apparent association constants than the corresponding

oligosaccharide portion alone, without the double alkyl

group-substituted glycerol core.

Keywords: bioﬁlm formation / glycolipids / sliding motility /

surface plasmon resonance / surfactant protein

¹To whom correspondence should be addressed: Fax: +91-80-2293-2578;

e-mail: jayaraman@orgchem.iisc.ernet.in

*Authors contributed equally to the work.

1237

K Naresh et al.

Chart 1. Molecular structures of synthetic oligoarabinan glycolipids.

conducted, with the aid surface plasmon resonance (SPR)

technique, so as to identify the interaction of glycolipids with

this receptor protein. Details of synthesis, biological and bio-

physical studies of arabinan glycolipids are presented herein.

Scheme 1. Synthesis of double alkyl chain containing lipid moiety 10.

Reagents and conditions: (i) NaH, THF, reﬂux, 36 h; (ii) Pd-C, H₂, EtOAc/

MeOH (1:1), room temperature, 24 h.

Results and discussion

The target arabinan glycolipids (Chart 1), both linear and

branched structures, are the components of repeat unit struc-

tures present in lipoarabinans and LAMs. Syntheses of target

oligoarabinofuranoside glycolipids 3, 4, 5 and 7 were accom-

plished through chemical methods. In the course of synthesis,

sugar and lipid portions were prepared individually ﬁrst and

were assembled subsequently to secure the target glycolipids.

Scheme 2. Synthesis of activated arabinofuranoside glycosyl donor 14.

Reagents and conditions: (i) (a) Bu₂SnO, MeOH, reﬂux, 5 h; (b) Ac₂O,

CH₂Cl₂, 35°C, 24 h; (ii) Py, BzCl, CH₂Cl₂, room temperature, 12 h; (iii) HBr/

AcOH, CH₂Cl₂, 0°C, 30 min; (iv) Ag₂CO₃, THF/H₂O (1:2), 18 h, room

temperature; (v) CCl₃CN, DBU, CH₂Cl₂, 0°C, 10 min.

Synthesis of glycolipids

Lipid moiety was synthesized using glycerol as the core.

Long alkyl chains functionalized sn-1,2-glycerol 8 and a

diethylene glycol spacer 9 were incorporated, so as to consti-

tute the lipid moiety 10 (Scheme 1). Double alkyl chain con-

taining glycerol 8 (Naresh et al. 2008), which was synthesized

from commercially available (+)-solketal, served as a precur-

sor for the synthesis of alcohol 10. Thus, O-alkylation of 8

with tosylate 9 (Huang and Tomalia 2006), followed by

O-debenzylation afforded alcohol 10.

Arabinofuranoside oligosaccharides were synthesized, initi-

ating from methyl-^D-arabinofuranoside 11. Protection of the

primary hydroxyl group in 11 afforded partially O-acetylated

derivative 12, which was subjected to O-benzoylation, bromi-

nation, followed by a hydrolysis to afford lactol 13. Lactol 13

was activated as a trichloroacetimidate glycosyl donor 14,

using CCl₃CN and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)

(Scheme 2). Synthesis of the glycosyl acceptor 17 was accom-

plished from triol 15 (D’Souza et al. 2000), which was con-

verted to 16. O-Benzoylation of secondary hydroxyl groups in

Scheme 3. Synthesis of glycosyl acceptor 17. Reagents and conditions: (i) (a)

Bu₂SnO, PhMe, reﬂux, 18 h; (b) Ac₂O, room temperature, 2 h; (ii) Py, BzCl,

CH₂Cl₂, room temperature, 12 h; (iii) AcCl; MeOH/CH₂Cl₂(1:1), room

temperature, 4 h.

16, followed by O-deacetylation, afforded the glycosyl accep-

tor 17, in a moderate yield (Scheme 3).

Glycosylation of 17 with the glycosyl donor 14, in the pres-

ence of BF₃·OEt₂(Schmidt and Michel 1980), afforded

1238

Synthetic glycolipids, biological and biophysical studies

disaccharide 19, in a good yield (Scheme 4). The anomeric

conﬁgurations in arabinofuranosides were adjudged from the

corresponding NMR spectra. Thus, observation of a doublet

Glycosylation of 20 with 22 afforded the linear tetrasac-

charide 26, presenting 1 → 5 α-linkage between furanoside

moieties (Scheme 5). The α-anomeric conﬁgurations of glyco-

sidic bonds at the non-reducing end in 26 were assigned on

the basis of signals at 105.8 and 106.0 ppm, with varied

intensities, in the ¹³C NMR spectrum. Glycosylation of 26

with dihexadecyl functionalized alcohol 10, followed by a

deprotection afforded the linear tetrasaccharide glycolipid 3,

in a good yield (Scheme 5, Chart 1). The α-anomeric con-

ﬁgurations of 3 was ascertained with the presence of signals

between 109.6 and 109.7 ppm in the corresponding ¹³C NMR

spectrum. The acetate and benzoate groups in 26 were hydro-

lyzed to afford the linear tetrasaccharide 1, in a good yield.

Linear hexasaccharide 2 (Chart 1) was obtained through (i)

hydrolysis of the thiocresyl group in 26 to lactol 27, (ii) con-

version of 27 to trichloroacetimidate 28, (iii) glycosylation of

the disaccharide acceptor 20 with 28 and (iv) deprotection of

the ester protecting groups (Scheme 5 and Chart 1).

1

at 5.71 ppm, as an apparent singlet, in the H NMR spectrum

and a resonance at 105.9 ppm in the ¹³C NMR spectrum cor-

responded to α-anomeric conﬁguration in 19. The α-anomeric

conﬁguration in furanosides is known to exhibit resonances at

such values previously (Mizutani et al. 1989; Joe et al. 2007).

O-Deacetylation of 19 afforded 20, which was used as a gly-

cosyl acceptor in the subsequent synthesis of linear tetrasac-

charides. Further, the thiocresyl group in 19 was hydrolyzed

to afford lactol 21, followed by an activation to trichloroaceti-

midate 22, toward the synthesis of 26. Similarly, disaccharide

lactol 24 was synthesized from a glycosyl donor 18 (Du et al.

2000), which, in turn, was prepared in situ prior to the con-

version of the same to an activated glycosyl donor 25, using

CCl₃CN/DBU.

Synthesis of linear hexa- and octasaccharides 4 and 5

(Chart 1) was achieved through the synthesis of disaccharide

30, containing dihexadecyl functionality at the reducing end

(Scheme 6). Glycosylation of alcohol 10 with trichloroaceti-

midate 22, in the presence of BF₃·OEt₂, afforded derivative

29. O-Deacetylation of 29 afforded alcohol 30 at the non-

reducing end, which was used further as a glycosyl acceptor

for the synthesis of linear hexasaccharide 31 (Scheme 7).

Glycosylation of 30 with thioglycoside 26 afforded 31, in a

good yield. The α-anomeric conﬁguration of glycosidic bonds

in 31 was ascertained by the presence of resonances at 105.8

and 105.9 ppm in the ¹³C NMR spectrum. Deprotection of

ester groups in 31 afforded the linear hexasaccharide glyco-

lipid 4 (Chart 1). O-Deacetylation of the primary acetate

group at the non-reducing end of hexasaccharide 31 afforded

acceptor 32 required for the synthesis of octasaccharide glyco-

lipid 5. Glycosylation of 32 was conducted with trichloroace-

timidate 25 (Scheme 4), followed by a deprotection of ester

groups, to afford the linear octasaccharide glycolipid 5

(Chart 1), in a moderate yield (Scheme 7). In the ¹³C NMR

spectrum of the protected derivative of 5, signals at 105.7 and

105.9 ppm indicated the α-anomeric conﬁguration.

Scheme 4. Synthesis of disaccharide 19 and their derivatives. Reagents and

conditions: (i) 17, BF₃·OEt₂, CH₂Cl₂, MS 4 Å, room temperature, 30 min; (ii)

AcCl; MeOH/CH₂Cl₂(1:1), room temperature, 4 h; (iii) NBS, THF/H₂O

(1:1), 1 h, room temperature; (iv) CCl₃CN, DBU, 0°C, 10 min.

Toward the synthesis of the branched heptasaccharide gly-

colipid 7 (Chart 1), thioglycoside 15 (Scheme 3) was con-

verted to 2,5-di-O-acetylated 33 (Scheme 8), which was

subjected to O-benzoylation, and O-deacetylation to afford

the diol acceptor 34. The presence of acetate groups at C-2

Scheme 5. Synthesis of tetrasaccharides 1 and 3 and their derivatives.

Reagents and conditions: (i) BF₃·OEt₂, MS 4 Å, CH₂Cl₂, room temperature,

30 min; (ii) NBS, THF/H₂O (1:1), room temperature, 1 h; (iii) CCl₃CN,

DBU, 0°C, 10 min; (iv) 10, AgOTf, NIS, MS 4 Å, CH₂Cl₂, −10°C, 30 min;

(v) NaOMe, MeOH/THF (1:1), 12 h.

Scheme 6. Synthesis of disaccharide 30, presenting a alcohol functionality at

the non-reducing end. Reagents and conditions: (i) BF₃·OEt₂, CH₂Cl₂, MS 4

Å, room temperature, 30 min; (ii) AcCl; MeOH/CH₂Cl₂(1:1), room

temperature, 4 h.

1239

K Naresh et al.

1

and C-5 in 33 was conﬁrmed through the comparison of H

NMR values with that in 15. Thus, a downﬁeld shift of H-2

(4.89 ppm) for 33, in comparison with 15 which showed H-2

at 4.0–4.1 ppm, indicated the presence of the O-acetyl group

at C-2 in 33. Similarly, downﬁeld shifts of H-5a and b of 33

were observed at 4.25 and 4.35 ppm, respectively, in compari-

son with 15, for which these protons resonated at 3.65–3.85

ppm. Further, a large downﬁeld shift of H-3 (5.23 ppm) was

Scheme 7. Synthesis of hexa- and octasaccharide glycolipids 4 and 5. Reagents and conditions: (i) AgOTf, NIS, MS 4 Å, CH₂Cl₂, −10°C, 30 min; (ii) AcCl;

MeOH/CH₂Cl₂(1:1), room temperature, 4 h; (iii) 25, BF₃·OEt₂, CH₂Cl₂, MS 4 Å, room temperature, 30 min; (iv) NaOMe, MeOH/THF (1:1), room temperature,

12 h.

Scheme 8. Synthesis of trisaccharide acceptor 35. Reagents and conditions: (i) (a) Bu₂SnO, PhMe, reﬂux, 18 h; (b) Ac₂O, PhMe, 40°C, 2 h; (ii) Py, BzCl,

CH₂Cl₂, room temperature, 12 h; (iii) AcCl, MeOH/CH₂Cl₂(1:1), room temperature, 18 h; (iv) BF₃·OEt₂, MS 4 Å, CH₂Cl₂, room temperature, 30 min; (v) AcCl,

MeOH/CH₂Cl₂(1:1), room temperature, 4 h.

Scheme 9. Synthesis of branched heptasaccahride glycolipid 7. Reagents and conditions: (i) BF₃·OEt₂, MS 4 Å, CH₂Cl₂, room temperature, 30 min; (ii) AgOTf,

NIS, MS 4 Å, CH₂Cl₂, −10°C, 30 min; (iii) NaOMe, MeOH/THF(1:1), room temperature, 12 h.

1240

Synthetic glycolipids, biological and biophysical studies

observed for 34, in comparison with H-3 of 33 (4.0 ppm).

H-2 nucleus of 34 upﬁeld shifted to 4.41 ppm, in comparison

with the same in 33 (4.89 ppm). Conﬁrmation was also

secured through two-dimensional NMR-correlated spec-

troscopy and heteronuclear multiple quantum coherence

experiments. Glycosylation of 34 with trichloroacetimidate 14

(Scheme 2), followed by O-deacetylation afforded trisacchar-

ide 35 (Scheme 8). Resonances at 104.5 and 105.7 ppm in the

¹³C NMR spectrum of 35 indicated the α-anomeric conﬁgur-

ation of glycosidic bonds.

multicellular aggregates of bacteria, embedded in a self-

produced extracellular polymeric matrix. Bioﬁlms act as the

protective coat for the growth and survival of a microorgan-

ism. They are formed usually during the stationary phase of

bacterial life cycle under limited nutrient and oxygen supply.

Bioﬁlms are formed to protect the bacteria from an external

agent, antibiotics and harsh environmental conditions. Bioﬁlm

formation is a multistep process and is an example of a coor-

dinated group behavior regulated by quorum sensing that

detect the density of other bacteria in a microenvironment

Trisaccharide 35 was further coupled with the disaccharide

donor 22 (Scheme 4) to afford the branched heptasaccharide

36, in a good yield (Scheme 9). Glycosylation of 36 with

alcohol 10, followed by deprotection of protecting groups

afforded glycolipid 7. Whereas the deprotection of ester groups

in 36 afforded heptasaccharide 6 (Scheme 9, Chart 1).

In summary, the synthesis of glycolipids was facile in

general, with moderate to good yields in each step. Each inter-

mediate of the multistep synthesis was characterized by

routine physical methods to conﬁrm their constitutions, struc-

tural and stereochemical homogeneities.

Effect of the glycolipids on the growth proﬁle of

Mycobacterium smegmatis

Subsequent to synthesis, biological studies were undertaken to

identify the effects of synthetic glycolipids on the growth

proﬁle, bioﬁlm formation and sliding motility of M. smegma-

tis. Two concentrations of glycolipids 1–7 in water were pre-

pared (50 and 100 µg/mL) and their effects on the growth

proﬁle were monitored over 72 h. As shown in Figure 1, syn-

thetic glycolipids (50 µg/mL) affected the growth proﬁle of

mycobacteria. The percentage inhibition of mycobacterial

growth was calculated in the stationary phase of bacterial

growth, and data are presented in Table I. The maximum inhi-

bition can be seen in the case of 4 and 5 (ꢀ43–45%) in the

log, as well as, stationary phases, followed by 6 and 7 (ꢀ19

and 22%, respectively).

Fig. 1. Effect of synthetic oligosaccharides and glycolipids 1–7 (50 µg/mL)

on mycobacterial growth.

Table I. Percentage inhibition of the mycobacterial growth and biofilm

Compound

Percentage inhibition of

mycobacterial growth

Percentage inhibition of the

mycobacterial biofilm formation

(100 µg/mL)

50 µg/mL

100 µg/mL

1

2

3

4

5

6

7

8

6

2

1

4

1

3

2

12

8

2

3

2

3

1

27

36

53

54

42

36

65

4

2

3

2

3

10

45

43

19

22

32

43

41

19

43

The culture did not grow well in the presence of 3 in the

initial log phage, although the growth was not affected much

in the late stationary phase. The cultures were found to be

least affected in the presence of oligosaccharides 1 and 2. We

have also tested synthetic glycolipids at a concentration of

100 µg/mL, a maximum inhibition was observed in the pres-

ence of the branched glycolipid 7 (ꢀ43), followed by 4 and 5

(ꢀ40%; Figure 2).

Glycolipid 3 (100 µg/mL) was also found to inhibit the

growth (ꢀ32%) and inhibition was observed in the late

stationary phase, whereas oligosaccharides 1 and 2 did not

much affect the growth of the bacterium. Further comparison

with our previous reports (Naresh et al. 2008, 2010) show

that glycolipids 37–40 (Figure 3) exhibit an inhibition of

ꢀ20–35%. Such comparisons among glycolipids also show

that anomeric linkage positions in arabinofuranoside contain-

ing glycolipids do not play a signiﬁcant role.

Effect of glycolipids on bioﬁlm formation

The effects of synthetic oligosaccharides were tested further

Fig. 2. Effect of synthetic oligosaccharides and glycolipids 1–7 (100 µg/mL)

on the mycobacterial bioﬁlm formation. Bioﬁlms are sessile

on mycobacterial growth.

1241

K Naresh et al.

Fig. 3. Molecular structures of synthetic arabinofuranoside and arabinofuranoside–mannopyranoside containing glycolipids (Naresh et al. 2010).

Fig. 4. Inhibitory effects of synthetic glycolipids and arabinofuranosides 1–7 on the bioﬁlm formation and maturation (7 days old, supplemented with 2%

glucose).

(Watnick and Kolter 1999; Nadell et al. 2008). Molecular

mechanisms that regulate the bioﬁlm formation vary from

species to species. All bioﬁlms contain an extracellular matrix

that holds cells together. This matrix is mostly composed of a

polysaccharide biopolymer, along with other components,

such as proteins, glycoproteins and DNA (Branda et al. 2005;

Monds and O’Toole 2009). The nature of matrix exopolysac-

charide greatly varies depending on growth conditions,

medium, substrates and species. Thus, it was of interest to

ﬁnd out how the synthetic glycolipids inﬂuence the formation

of bioﬁlms. Any compound that can reduce the bioﬁlm matu-

ration of a bacterial population will be, in principle, an effec-

tive tool to control the bacterial growth. The effect of

synthetic glycolipids on the bioﬁlm formation was assessed

ﬁrst. Figure 4 shows the effect of arabinan glycolipids 3, 4, 5

and 7 and oligoarabinofuranosides 1, 2 and 6 (each 100 μg/

mL) on the bioﬁlm formation and maturation. The bioﬁlm

maturation and stability were found to be severely affected in

the presence of glycolipids 3, 4, 5 and 7, when monitored

over a period of 7–10 days. Oligosaccharides without the

lipidic chain, namely 1, 2 and 6, did not show any signiﬁcant

change in the phenotype of the bioﬁlm, when compared with

the control bacteria.

Quantitative assay of bioﬁlm formation in the presence

of glycolipids

A quantitation of bioﬁlms was performed to measure the

degree of inhibition in the presence of glycolipids (100 µg/

mL; Figure 5). The percentage inhibition of the mycobacterial

bioﬁlm is presented in Table I. It can be seen from Figure 5

that the branched glycolipid 7 was affecting the bioﬁlm

1242

Synthetic glycolipids, biological and biophysical studies

(ꢀ65%), more than linear glycolipids 3, 4 and 5 (ꢀ53, 54

and 42%, respectively). Oligosaccharides without alkyl chains

1, 2, and 6 were not affecting the bioﬁlm formation in the

initial stage, although inhibition of ꢀ35% was observed in

the later stage. Also, it was found that heptasaccharide glyco-

lipid 7 exhibited higher inhibition at a lower concentration

(100 µg/mL), when compared with the branched tetrasacchar-

ide glycolipid 39 (Figure 3; 200 µg/mL), reported in our pre-

vious studies (Naresh et al. 2010). Further, it was found that

the bioﬁlm was unable to stick to the inner surface of Petri

plate when treated with 7, whereas the control bacteria,

without the glycolipids, attached to the inside wall of Petri

plate. These observations indicate that the branched

oligosaccharides prevent the bacterial attachment at the abiotic

surface, leading to a reduction in the bioﬁlm formation, in

comparison with wild type.

Sliding motility inhibition studies

Following the bioﬁlm studies, we studied another cell surface-

dependent phenomenon, sliding motility of the bacterial

species and the effect of synthetic glycolipids on them. The

sliding motility test was performed to verify the spreading

ability of mycobacteria on the moist surface, in the presence of

glycolipids 3, 4, 5 and 7 and oligoarabinofuranosides 1, 2 and

6. The motility of M. smegmatis was tested over the soft and

moist surface on the Petri plates. It was demonstrated earlier

that the glycolipids containing an arabinofuranose trisaccharide

inhibited the growth of mycobacteria (Naresh et al. 2008,

2010). Synthetic glycolipids not only affected the bioﬁlm for-

mation, but also affected the spreading ability of mycobacteria

on the moist surface. Arabinan glycolipids 3, 4, 5 and 7 and

oligoarabinofuranosides 1, 2 and 6 (each 100 µg/mL) were

added in the MB7H9 (Difco) base medium, solidiﬁed with

0.3% high-grade agarose (Sigma-Aldrich Inc., St. Louis, USA)

and 2% glucose was added as a carbon source. As shown in

Figure 6, glycolipids 3, 4, 5 and 7 and linear hexasaccharide 2

severely affected the motility of the bacteria (3 days old).

However, oligoarabinofuranosides 1 and 6 did not affect the

motility signiﬁcantly. The motility was observed over a period

of 10 days; however, the zone of spreading did not reach the

periphery of the plate, whereas the same in control bacteria

without glycolipids was found to spread in 3 days.

Fig. 5. Bioﬁlm quantitation assay showing the extent on inhibition by

synthetic glycolipids and arabinofuranosides. Wt, wild type.

The above biological studies show an inhibitory effect of

synthetic glycolipids on mycobacterial growth and sliding

Fig. 6. Sliding motility assay showing the spreading ability of mycobacteria (3 days old), in the presence of different synthetic oligoarabinofuranosides and

glycolipids 1–7.

1243

K Naresh et al.

motilities, as well as their bioﬁlm formation. It is difﬁcult to

ascertain a mechanism of mycobacterial growth inhibitions

mediated by synthetic arabinan glycolipids. We presume that

glycolipids may encounter blocking the mycobacterial porins.

Porins act as channels for the uptake of small hydrophilic

molecules through waxy mycobacterial cell wall. The outer

membrane of M. smegmatis has ꢀ1000 protein pores

(Engelhardt et al. 2004). The porin from M. smegmatis,

MspA, was cloned and crystallized, from which it was found

that porins formed a cone-like tetrameric complex structure,

with 10 nm in length, and a single central pore. The channel

diameter varies between 10 and 48 Å at the pore eyelet

(Faller et al. 2004). The blockage of porins by synthetic gly-

colipids might disturb the passage of nutrients through cell

wall, which, in turn, would catalyze inhibition of the bacterial

growth. Another possible reason for inhibition could be the

incorporation of synthetic glycolipids in the cell wall. The

integration of synthetic glycolipids might lead to a restriction

of chain elongation during the biosynthesis of mycobacterial

cell wall, which, in turn, would prevent the assembly of cell

wall. Monitoring these possibilities requires involved exper-

iments, for example, using radioactively enriched glycolipids

in mycobacterial growth inhibition studies. General mechan-

isms of sliding in mycobacteria are not known in detail so far.

It was shown previously that the acetylation of glycopeptidoli-

pids affected the bioﬁlm formation and sliding motility in

mycobacteria (Recht et al. 2000; Etienne et al. 2002). It

appeared that the synthetic glycolipids might be playing a role

with the normal proﬁle of glycopeptidolipids or proteins,

which regulates their synthesis and results in the impaired

sliding motility and bioﬁlm formation. It is noted that glyco-

lipids 3, 4, 5 and 7, having the alkyl chain, show the highest

degree of inhibition in the case of bioﬁlm formation and moti-

lity. It appears that synthetic oligoarabinofuranoside glyco-

lipids may be affecting the normal biosynthesis of the cell

wall involved in the maturation and re-initiation of the bioﬁlm

(Puskas et al. 1997).

immobilized on to the CM5 sensor chip, using the amine-

coupling method. Ethanolamine was immobilized in one of

the reference ﬂow cells. SP-A was immobilized to 3000

response units in NaOAc (pH 3.5) buffer. Varying concen-

trations of synthetic arabinan oligosaccharides 1–7 were dis-

solved in 4-(2-hydroxyethyl)-1-piperazineethane sulphonic

acid (HEPES)-P buffer and passed over ﬂow cells containing

SP-A and ethanolamine. Response units from SP-A-

immobilized surface were subtracted with that from ﬂow cell

containing ethanolamine. Corresponding sensorgrams are

shown in Figure 7. Absence of the mass transfer process was

ascertained through passing a ﬁxed concentration of the

analyte at varying ﬂow rates. Sensorgrams did not differ,

thereby excluding the possibility of mass transport phenom-

ena. With the aid of software suite provided with the instru-

ment, kinetic parameters were evaluated. Results of the

analysis are presented in Table II. The analysis was guided by

χ²values that refer to the best ﬁt of the theoretical model and

the experimental data. Among the kinetic models, the 1:1

Langmuir model was found to provide the best ﬁt.

Accordingly, further kinetic parameters were derived using

the 1:1 Langmuir model. The association rate constants (k_on)

were generally higher for the oligosaccharides without the

lipidic chain, and on the other hand, the dissociation rate con-

stants (k_off) were slower with oligosaccharides having the

lipidic chains. The branched oligosaccharide 6 and glycolipid

7 showed a higher apparent association constant (K_a) in the

series. The role of the lipidic chain could be adjudged from

the higher K_avalue for 7, when compared with 6. This obser-

vation is in line with the studies of Rivière and co-workers

(Sidobre et al. 2000, 2002), wherein the presence of lipid

moieties in native ManLAM glycoconjugates were shown to

exhibit higher binding afﬁnity to SP-A. Faster k_onis also

associated with a faster k_offfor oligosaccharides without the

lipidic chains. For the glycolipids, a relatively slower k_offwas

found to be the trend. A millimolar apparent association con-

stant was observed generally for the analytes. SPR studies so

far provide an indication of the binding constants pertaining

to the interaction of synthetic glycolipids with one of the

important pulmonary surfactant proteins, namely SP-A.

SPR studies of glycolipids

Following the biological studies of the arabinan glycolipids,

we conducted SPR studies to identify the interactions of these

glycolipids with an immunogenic protein, namely human pul-

monary SP-A. SP-A is a collectin of family of carbohydrate-

binding proteins and it mediates various immune cell

responses (Tino and Wright 1998; Crouch and Wright 2001;

Wright 2005). SP-A interacts with pathogens, such as myco-

bacteria (Sidobre et al. 2000, 2002) and HIV (Gaiha et al.

2008), through binding to lipopolysaccharides present on bac-

terial and viral cell surfaces (McCormack 1998). Additionally,

yet another surfactant protein, namely SP-D, is also known to

interact with mycobacteria (Carlson et al. 2009). Particularly,

binding studies of SP-A with LAM, mannosylated LAM

(ManLAM), two essential components of the mycobacterial

cell walls, showed that both these glycolipids bind strongly to

SP-A (Sidobre et al. 2002). The presence of lipid appeared

essential for high afﬁnity binding. SPR is a real-time tech-

nique (Rich and Myszka 2010) to evaluate the interaction of

an analyte with a ligand, wherein the analyte passed over an

immobilized ligand surface. For this purpose, SP-A was

Conclusion

A series of linear tetra-, hexa-, octa- and a branched heptasac-

charide oligoarabinofuranosides, with 1 → 3 and 1 → 5

α-linkages between the furanoside residues, were synthesized

by chemical methods from readily available monomer build-

ing blocks. The multistep synthesis afforded moderate to good

yields in each individual steps. Mycobacterial growth proﬁles,

bioﬁlm formation and sliding motility studies with M. smeg-

matis were evaluated in the presence of arabinofuranoside

containing oligosaccharides. In comparison with our previous

reports (Naresh et al. 2008; 2010), the hydrophobic alkyl

chains in glycolipids 3, 4, 5 and 7 were found to be essential

to affect the mycobacterial growth, bioﬁlm formation and

sliding motility. From our earlier and present studies, the pres-

ence of the lipidic portion on the oligosaccharide is found to

be essential for an efﬁcient inhibition of mycobacterial

growth, bioﬁlm formation and sliding motility. Mechanism of

1244

Synthetic glycolipids, biological and biophysical studies

Fig. 7. SPR sensorgrams of glycolipids 3–5 and 7 and oligosaccharides 1 and 6, upon interaction with SP-A. The superimposed dashed lines are ﬁtting curves.

inhibition mediated by synthetic glycolipids is unclear at

present. The binding afﬁnities of glycolipids with a pulmon-

ary surfactant protein, namely SP-A, were evaluated sub-

sequently, through the SPR technique. Evaluation of kinetic

parameters showed millimolar apparent association constants

and important differences in the association and dissociation

rate constants for the oligosaccharides with and without the

lipid chains. Studies presented herein, as well as, our previous

studies provide a possibility to evolve newer types of glyco-

lipids that can act as inhibitors of mycobacterial growth.

with silica gel 60 F₂₅₄. 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 quadrupole-time of ﬂight (Q-TQF) instrument by elec-

1

trospray ionization (ESI). H and ¹³C NMR spectral analyses

were performed on a spectrometer operating at 300 and 400

MHz, and 75 and 100 MHz, respectively, in CDCl₃solutions,

unless otherwise stated. Chemical shifts are reported with

1

respect to tetramethylsilane for H NMR and the central line

(77.0 ppm) of CDCl₃for ¹³C NMR. Coupling constants (J)

are reported in hertz. Standard abbreviations s, d, t, dd, br s

and m refer to singlet, doublet, triplet, doublet of doublet,

broad singlet and multiplet, respectively. (+)-Solketal was

from Sigma-Aldrich Inc.

Materials and methods

General information

Solvents were dried and distilled according to the literature

procedures (Armarego and Perrin 1996). All chemicals were

purchased from commercial sources and were used without

further puriﬁcation. Silica gel (100–200 mesh) was used for

column chromatography, and thin layer chromatography

(TLC) analysis was performed on commercial plates coated

8-O-Hexadecyl-3,6,10-trioxa-1,8-hexacosanediol (10)

8 (Naresh et al. 2008) (3 g, 5.5 mmol) in tetrahydrofuran

(THF) (10 mL) was added to a suspension of NaH (0.22 g,

60% in mineral oil, 5.5 mmol) in THF (50 mL). After stirring

for 30 min at room temperature, 9 (Huang and Tomalia 2006)

1245

K Naresh et al.

Table II. Kinetic data of the interactions between the synthetic glycolipids and SP-A

Compound

k_on(M⁻¹s⁻¹

)

k_d(s⁻¹) (10⁴)

K_a(M⁻¹) (10⁻³

)

χ²

3.9

7.91

4.9

8.3

1.5

3.98

0.22

3.77

17.5

2.9

6.7

0.384

27.3

5.79

47.2

4.5

11.3

23.3

6.14

11.6

18.4

20.1

2.3

2.4

53.6

17.9

29.9

5.4

Data fitted in the 1:1 Langmuir model using BIAevaluation software.

(1.92 g, 5.5 mmol) in THF (5 mL) was added slowly, stirred

at reﬂux for 36 h, cooled, ﬁltered and ﬁltrate concentrated in

vacuo. The crude residue was dissolved in CH₂Cl₂(100 mL)

and washed with water (3 × 50 mL). The organic portion was

dried, ﬁltered and ﬁltrate concentrated in vacuo. A solution of

the crude residue in EtOAc/MeOH (1:1) (30 mL) was treated

with Pd–C (10%; 0.4 g) and stirred under H₂(1 bar) for 24 h.

Filtration of the reaction mixture, followed by puriﬁcation

(pet. ether/EtOAc 9:1), afforded 10, as a white solid. Yield:

55.1, 55.2, 64.1, 65.4, 76.2, 77.2, 77.8, 79.6, 80.7, 82.3, 101.9,

108.7, 171.2, 171.3; ESI-MS calculated for C₈H₁₄O₆Na [M +

Na] 229.0688 and found 229.0688.

5-O-Acetyl-2,3-di-O-benzoyl-^D-arabinofuranose (13)

Pyridine (0.8 mL, 10 mmol) and BzCl (1.2 mL, 10 mmol)

were added to a solution of 12 (1 g, 4.8 mmol) in CH₂Cl₂at

0°C, and the reaction mixture was stirred for 12 h at room

temperature. The reaction mixture was diluted with CH₂Cl₂

(50 mL), washed with dil. HCl (2 × 20 mL), satd. aq.

NaHCO₃(2 × 20 mL) solution and water (20 mL). The

organic portion separated and dried (Na₂SO₄), ﬁltered and ﬁl-

trate concentrated in vacuo. HBr in AcOH (33%, 4.7 mL,

19.2 mmol) was added to the solution of crude residue in

CH₂Cl₂and the reaction mixture was stirred for 30 min at 0°

C. The reaction mixture was diluted with CH₂Cl₂(100 mL),

washed with satd. aq. NaHCO₃(2 × 20 mL) solution, brine

(20 mL) and water (20 mL). The organic portion was separ-

ated and dried (Na₂SO₄), ﬁltered and ﬁltrate concentrated in

vacuo. Ag₂CO₃(1.31 g, 4.8 mmol) was added to the solution

of the resulting bromide in THF and water (2:1) and stirred at

room temperature for 18 h. The reaction mixture was ﬁltered

and ﬁltrate concentrated in vacuo and puriﬁed to afford 13, as

a gum. Yield: 1.34 g (70%); α/β 2.5:1; R_f(pet. ether/EtOAc

1

2.1 g (61%); R_f(pet. ether/EtOAc 4:1) 0.7; H NMR (CDCl₃,

400 MHz) δ 0.87 (t, J = 7.2 Hz, 6 H), 1.24 (br s, 52 H), 1.50–

1.55 (m, 4 H), 2.59 (s, 1 H), 3.43–3.50 (m, 4 H), 3.54–3.62

(m, 7 H), 3.65 (s, 4 H), 3.72 (s, 2 H); ¹³C NMR (CDCl₃, 100

MHz) δ 14.1, 22.7, 26.1, 29.3, 29.5, 29.6, 29.7, 30.1, 30.8,

31.9, 61.7, 70.4, 70.6, 70.9, 71.4, 71.6, 72.5, 77.8; ESI-

molecular sieves (MS) calculated for C₃₉H₈₀O₅Na [M + Na]

651.5903 and found 651.5961.

Methyl 5-O-acetyl-^D-arabinofuranoside (12)

Bu₂SnO (1.95 g, 7.9 mmol) was added to a solution of

methyl-^D-arabinofuranoside 11 (Ness and Fletcher 1958) (1.3

g, 7.9 mmol) in MeOH (30 mL). The solution was reﬂuxed for

5 h and concentrated in vacuo. Ac₂O (0.8 mL, 7.9 mmol) was

added to the solution of the crude residue in CH₂Cl₂and the

reaction mixture was stirred at 35°C for 24 h, concentrated in

vacuo and puriﬁed (CHCl₃/MeOH 9:1) to afford 12, as a gum.

Yield: 0.93 g (57%); α/β 1:0.7; R_f(CHCl₃/MeOH 19:1) 0.38;

¹H NMR (CDCl₃, 400 MHz) δ 2.06 (s, 2.1 H), 2.08 (s, 3 H),

3.40 (s, 3 H), 3.42 (s, 2.1 H), 3.68 (br s, 2 H), 3.85 (br s, 1.4

H), 3.95–4.14 (m, 5.8 H), 4.2–4.3 (m, 2.7 H), 4.78 (d, J = 4

Hz, 0.7 H), 4.85 (s, 1 H); ¹³C NMR (CDCl₃, 100 MHz) δ 20.8,

1

3:2) 0.4; H NMR (CDCl₃, 400 MHz) δ 2.07 (s, 7.5 H), 2.10

(s, 3 H), 4.29–4.39 (m, 4 H), 4.55–4.65 (m, 6.5 H), 5.42 (d,

J = 3.6 Hz, 2.5 H), 5.48 (s, 3.5 H), 5.66 (s, 2.5 H), 5.75 (t, J

= 5.2 Hz, 1 H), 5.79 (d, J = 4 Hz, 1 H), 7.44–7.48 (m, 14 H),

7.58–7.62 (m, 7 H), 8.02–8.17 (m, 14 H); ¹³C NMR (CDCl₃,

100 MHz) δ 20.8, 20.9, 63.7, 65.3, 77.2, 77.5, 77.7, 79.0,

81.3, 82.0, 95.5, 101.0, 128.5, 128.9, 129.8, 129.9, 133.6,

1246

Synthetic glycolipids, biological and biophysical studies

133.7, 165.4, 165.7, 165.9, 170.7, 171.0; ESI-MS calculated

for C₂₀H₁₉O₈Na [M + Na] 423.1056 and found 423.1049.

¹³C NMR (CDCl₃, 75 MHz) δ 21.1, 62.0, 77.9, 82.2, 83.7,

91.7, 128.5, 129.0, 129.8, 130.0, 132.3, 132.9, 133.6, 138.1,

165.2, 165.9; ESI-MS calculated for C₂₆H₂₄O₆SNa [M + Na]

487.1191 and found 487.1186.

1-O-(5-O-Acetyl-2,3-di-O-benzoyl-α-^D-arabinofuranosyl)

trichloroacetimidate (14)

p-Tolyl-5-O-acetyl-2,3-di-O-benzoyl-α-D-arabinofuranosyl-

(1 → 5)-2,3-di-O-benzoyl-1-thio-α-^D-arabinofuranoside (19)

DBU (0.2 mL, 1.3 mmol) was added to a stirred solution of

13 (1.8 g, 4.5 mmol) and CCl₃CN (4.5 mL, 45 mmol) in

CH₂Cl₂(10 mL) at 0°C and stirred for 10 min. The crude

reaction mixture was puriﬁed using pet. ether/EtOAc (4:1) to

afford trichloroacetimidate 14. Yield: 2.2 g (90%) (Mei and

A solution of 14 (2.2 g, 4.05 mmol), 17 (1.69 g, 3.64 mmol)

and MS 4 Å (2 g) in CH₂Cl₂(50 mL) was stirred for 15 min

BF₃·OEt₂(0.52 mL, 4.05 mmol) was added dropwise at room

temperature, under N₂atmosphere. 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 7:3) to afford 19, as a foamy solid. Yield: 2.43 g

(79%); R_f(pet. ether/EtOAc 7:3) 0.53; [α]_D+39.9 (c 0.5,

1

Ning 2005); H NMR (CDCl₃, 400 MHz) δ 2.04 (s, 3 H),

4.44 (d, J = 5.6 Hz, J = 12 Hz, 1 H), 4.57 (d, J = 4.4 Hz, J =

12 Hz, 1 H), 4.67–4.68 (m, 1 H), 5.51 (d, J = 3.6 Hz, 1 H),

5.77 (d, J = 1.1 Hz, 1 H), 6.64 (s, 1 H), 7.44–7.64 (m, 6 H),

8.09–8.12 (m, 4 H), 8.72 (s, 1 H); ¹³C NMR (CDCl₃, 100

MHz) δ 20.4, 63.0, 76.9, 79.9, 84.0, 90.6, 102.6, 128.2,

128.3, 128.5, 129.6, 129.7, 133.2, 133.5, 133.6, 160.0, 164.6,

165.2, 170.3; ESI-MS calculated for C₂₃H₂₀Cl₃NO₈Na [M +

Na] 566.0 and found 566.1.

1

CHCl₃); H NMR (CDCl₃, 400 MHz) δ 2.02 (s, 3 H), 2.29 (s,

3 H), 3.98 (dd, J = 11.2 Hz, J = 3.2 Hz, 1 H), 4.25 (dd, J =

11.2 Hz, J = 4.2 Hz, 1 H), 4.37 (dd, J = 12 Hz, J = 5.2 Hz, 1

H), 4.55 (dd, J = 12 Hz, J = 3.2 Hz, 1 H), 4.60–4.61 (m, 1 H),

4.72 (q, J = 3.6 Hz, 1 H), 5.39 (d, J = 4.2 Hz, 1 H), 5.41 (s, 1

H), 5.58 (s, 1 H), 5.70 (s, 1 H), 5.71 (d, J = 3.6 Hz, 1 H), 5.73

(m, 1 H), 7.09 (d, J = 8 Hz, 2 H), 7.13–7.60 (m, 14 H), 7.92–

8.10 (m, 8 H); ¹³C NMR (CDCl₃, 100 MHz) δ 20.7, 21.1,

63.4, 65.9, 77.5, 81.1, 81.4, 81.9, 82.0, 91.5, 105.9, 128.3,

128.5, 128.8, 129.0, 129.7, 129.8, 129.9, 132.5, 133.3, 133.5,

137.9, 165.0, 165.3, 165.5, 165.6, 170.7; ESI-MS calculated

for C₄₇H₄₂O₁₃SNa [M + Na] 869.2244 and found 869.2245.

p-Tolyl-5-O-acetyl-1-thio-α-D-arabinofuranoside (16)

Bu₂SnO (4.3 g, 17.3 mmol) was added to a solution of

p-tolyl-1-thio-α-^D-arabinofuranoside 15 (D’Souza et al. 2000)

(4 g, 15.6 mmol) in PhMe (80 mL). The solution was reﬂuxed

for 18 h, after which half the amount of solvents was

removed. Ac₂O (1.61 mL, 17.3 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 1:2) to afford 16, as a gum. Yield: 3.34 g (72%); R_f

p-Tolyl–2,3-di-O-benzoyl-α-^D-arabinofuranosyl-(1 →

5)-2,3-di-O-benzoyl-1-thio-α-^D-arabinofuranoside (20)

1

(pet. ether/EtOAc 1:4) 0.5; [α]_D+190.5 (c 0.5, CHCl₃); H

NMR (CDCl₃, 400 MHz) δ 2.09 (s, 3 H), 2.32 (s, 3 H), 3.5–

3.8 (br s, 2 H), 3.97 (dd, J = 6.8 Hz, J = 5.2 Hz, 1 H), 4.13 (t,

J = 4.8 Hz, 1 H), 4.17 (dd, J = 4.4 Hz, J = 6.8 Hz, 1 H), 4.28

(s, 1 H), 4.29 (s, 1 H), 5.28 (d, J = 4.4 Hz, 1 H), 7.11 (d, J =

8 Hz, 2 H), 7.39 (d, J = 8 Hz, 2 H); ¹³C NMR (CDCl₃, 100

MHz) δ 20.9, 21.1, 63.5, 77.1, 80.1, 81.3, 91.3, 129.8, 132.4,

137.9, 171.6; ESI-MS calculated for C₁₄H₁₈O₈SNa [M + Na]

321.0773 and found 321.0777.

AcCl (0.25 mL) was added to a solution of 19 (1.4 g, 1.65

mmol) in MeOH/CH₂Cl₂(1:1) (20 mL) and stirred at room

temperature for 4 h. The reaction mixture was diluted with

CH₂Cl₂(100 mL), washed with the satd. aq. NaHCO₃(2 × 20

mL) solution, brine (20 mL) and water (20 mL). The organic

portion was dried (Na₂SO₄), ﬁltered and ﬁltrate concentrated in

vacuo and puriﬁed (pet. ether/EtOAc 7:3) to afford 20, as a

foamy solid. Yield: 1.1 g (85%); R_f(pet. ether/EtOAc 3:1) 0.3;

1

[α]_D+50.2 (c 0.5, CHCl₃); H NMR (CDCl₃, 400 MHz) δ

2.29 (s, 3 H), 3.92–4.02 (m, 4 H), 4.23 (dd, J = 4.4 Hz, J =

11.2 Hz, 1 H), 4.47 (q, J = 4.4 Hz, 1 H), 4.69 (q, J = 4.4 Hz, 1

H), 5.39 (s, 1 H), 5.44 (d, J = 5.2 Hz, 1 H), 5.63 (d, J = 1.2 Hz,

1 H), 5.71 (d, J = 1.2 Hz, 1 H), 5.72 (s, 1 H), 5.73 (s, 1 H),

7.25 (d, J = 8 Hz, 2 H), 7.37–7.60 (m, 14 H), 7.91–8.09 (m, 8

H); ¹³C NMR (CDCl₃, 100 MHz) δ 21.0, 62.2, 65.9, 77.6,

81.6, 81.9, 82.0, 83.7, 91.5, 105.8, 128.3, 128.4, 128.8, 128.9,

129.0, 129.8, 129.9, 130.0, 132.6, 133.3, 133.5, 137.9, 165.1,

165.3, 165.5, 166.0; ESI-MS calculated for C₄₅H₄₀O₁₂SNa

[M + Na] 827.2138 and found 827.2142.

p-Tolyl-2,3-di-O-benzoyl-1-thio-α-^D-arabinofuranoside (17)

Pyridine (1.27 mL, 16 mmol) and BzCl (1.85 mL, 16 mmol)

were added to a solution of 16 (2.2 g, 7.4 mmol) in CH₂Cl₂

(20 mL) at 0°C. The reaction mixture was stirred for 12 h,

diluted with CH₂Cl₂(50 mL), washed with dil. aq. HCl (2 ×

25 mL) solution, satd. aq. NaHCO₃solution (2 × 25 mL),

water (25 mL), dried (Na₂SO₄), ﬁltered and ﬁltrate concen-

trated in vacuo. AcCl (0.5 mL) was added to a solution of the

crude product in CH₂Cl₂/MeOH (1:1) (50 mL) and stirred at

room temperature for 4 h. The reaction mixture was diluted

with CH₂Cl₂(100 mL), washed with satd. aq. NaHCO₃(2 ×

20 mL) solution, brine (20 mL), water (20 mL), dried

(Na₂SO₄), ﬁltered and ﬁltrate concentrated in vacuo and puri-

ﬁed (pet. ether/EtOAc 3:1) to afford 17 (Cociorva and

Lowary 2004), as a foamy solid. Yield: 2.26 g (66%). R_f(pet.

5-O-Acetyl-2,3-di-O-benzoyl-α-^D-arabinofuranosyl-(1 →

5)-2,3-di-O-benzoyl-^D-arabino-furanose (21)

N-bromosuccinimide (NBS) (0.25 g, 1.4 mmol) was added to

a solution of 19 (1.2 g, 1.4 mmol) in THF and water (1:1) and

stirred at room temperature for 1 h. The reaction mixture was

ﬁltered and ﬁltrate was diluted with CH₂Cl₂(100 mL), washed

with satd. aq. sodium thiosulfate (2 × 20 mL) and water (1 ×

20 mL). The organic portion was dried (Na₂SO₄), ﬁltered and

1

ether/EtOAc 3:1) 0.6; H NMR (CDCl₃, 300 MHz) δ 2.32 (s,

3 H), 2.77 (br s, 1 H), 4.00–4.03 (m, 2 H), 4.56–4.60 (m, 1

H), 5.53–5.54 (m, 1 H), 5.71 (s, 2 H), 7.12 (d, J = 8 Hz, 2 H),

7.40–7.63 (m, 8 H), 8.02–8.04 (m, 2 H), 8.10–8.13 (m, 2 H);

1247

K Naresh et al.

ﬁltrate concentrated in vacuo followed by puriﬁcation (pet.

ether/EtOAc 3: 2) afforded 21, as a foamy solid. Yield: 0.83 g

(80%); α/β 1.8:1; R_f(pet. ether/EtOAc 7:3) 0.23; H NMR

(m, 8.2 H), 5.28 (s, 1 H), 5.44 (s, 1.3 H), 5.47 (s, 1 H), 5.54–

5.58 (m, 8.8 H), 5.62–5.68 (m, 1 H), 5.76 (d, J = 4.4 Hz, 1

H), 6.01 (t, J = 5.6 Hz, 1 H), 7.25–7.45 (m, 34.5 H), 7.97–

8.06 (m, 23 H); ¹³C NMR (CDCl₃, 100 MHz) δ 60.4, 63.7,

66.4, 67.4, 75.5, 77.2, 77.5, 77.8, 78.0, 79.8, 81.1, 81.9, 82.0,

82.3, 82.4, 95.3, 100.8, 105.9, 106.3, 128.3, 128.4, 128.5,

128.7, 128.8, 128.9, 129.0, 129.7, 129.8, 129.9, 130.0, 133.0,

133.3, 133.4, 133.5, 133.6, 165.3, 165.5, 165.6, 165.7, 165.8,

166.2, 166.3;. ESI-MS calculated for C₄₅H₃₈O₁₄Na [M + Na]

825.2159 and found 825.2163.

1

(CDCl₃, 400 MHz) δ 2.04 (s, 5.4 H), 2.07 (s, 3 H), 3.20 (br s,

1.8 H), 3.28 (br s, 1 H), 3.94–4.04 (m, 3.6 H), 4.08–4.11 (s, 1

H), 4.17–4.21 (m, 2 H), 4.29–4.30 (m, 1 H), 4.37–4.43 (m, 3.6

H), 4.48–4.69 (m, 7.6 H), 5.39–5.48 (m, 3.8 H), 5.49–5.53 (m,

1.8 H), 5.54 (s, 1.8 H), 5.55 (s, 1.8 H), 5.59–5.62 (m, 3.6 H),

5.65 (dd, J = 8.8 Hz, J = 6.8 Hz, 1 H), 5.98 (t, J = 6 Hz, 1 H),

7.30–7.6 (m, 33.6 H), 7.9–8.06 (m, 22.4 H); ¹³C NMR

(CDCl₃, 100 MHz) δ 20.7, 63.3, 63.5, 66.5, 67.5, 75.5, 77.2,

77.6, 78.0, 79.9, 81.0, 81.1, 81.4, 81.9, 82.1, 82.4, 95.3, 100.8,

105.9, 106.3, 128.3, 128.4, 128.5, 128.9, 129.0, 129.8, 129.9,

130.0, 133.4, 133.5, 133.6, 165.1, 165.5, 165.6, 165.8, 170.7;

ESI-MS calculated for C₄₀H₃₆O₁₄Na [M + Na] 763.2003 and

found 763.1998.

1-O-(5-O-(2,3,5-Tri-O-benzoyl-α-D-arabinofuranosyl)-

2,3-di-O-benzoyl-α- ^D-arabino-furanosyl)

trichloroacetimidate (25)

DBU (0.065 mL, 0.436 mmol) was added to a stirred sol-

ution of disaccharide 24 (0.35 g, 0.436 mmol) and CCl₃CN

(0.435 mL, 4.35 mmol) in CH₂Cl₂(10 mL) at 0°C and

stirred for 10 min. The crude reaction mixture was puriﬁed

using pet. ether/EtOAc (2:1) to afford trichloroacetimidate

25. Yield: 0.38 g (92%); R_f(pet. ether/EtOAc 7:3) 0.65;

[α]_D−14.2 (c 1, CHCl₃); ¹H NMR (400 MHz, CDCl₃) δ

4.00 (dd, J = 11.2 Hz, J = 2.8 Hz, 1 H), 4.29 (dd, J = 11.2

Hz, J = 4 Hz, 1 H), 4.62–4.72 (m, 3 H), 4.82 (dd, J = 11.2

Hz, J = 2.8 Hz, 1 H), 5.45 (s, 1 H), 5.56 (d, J = 4.8 Hz, 1

H), 5.60 (s, 1 H), 5.76 (d, J = 3.2 Hz, 1 H), 5.82 (s, 1 H),

6.60 (s, 1 H), 7.25–7.60 (m, 15 H), 7.89–8.09 (m, 10 H),

8.68 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 63.6, 65.9,

77.2, 77.7, 80.4, 81.3, 81.8, 85.2, 91.0, 103.0, 106.0, 128.3,

128.4, 128.5, 128.6, 128.9, 129.0, 129.7, 129.8, 129.9,

133.0, 133.0, 133.3, 133.5, 133.6, 133.7, 160.5, 165.0,

165.2, 165.5, 165.7, 166.2; ESI-MS calculated for

C₄₇H₃₈Cl₃NO₁₄Na [M + Na] 968.1 and found 968.2.

1-O-(5-O- (5-O-Acetyl-2,3-di-O-benzoyl-α-^D-

arabinofuranosyl)-2,3-di-O-benzoyl-α-^D-arabinofuranosyl)

trichloroacetimidate (22)

DBU (0.095 mL, 0.675 mmol) was added to a stirred sol-

ution of disaccharide 21 (0.5 g, 0.675 mmol) and CCl₃CN

(0.675 mL, 6.75 mmol) in CH₂Cl₂(10 mL) at 0°C and

stirred for 10 min. The crude reaction mixture was puriﬁed

using pet. ether/EtOAc (2:1) to afford trichloroacetimidate

22. Yield: 0.56 g (94%); R_f(pet. ether/EtOAc 7:3) 0.6; [α]_D

1

+3.7 (c 1, CHCl₃); H NMR (400 MHz, CDCl₃) δ 2.04 (s, 3

H), 3.98 (dd, J = 11.2 Hz, J = 3.2 Hz, 1 H), 4.27 (dd, J =

11.2 Hz, J = 4 Hz, 1 H), 4.36 (dd, J = 11.2 Hz, J = 4.8 Hz, 1

H), 4.53–4.59 (m, 2 H), 4.67 (d, J = 3.6 Hz, 1 H), 5.38 (d,

J = 4 Hz, 1 H), 5.42 (s, 1 H), 5.56 (s, 1 H), 5.73 (d, J = 3.6

Hz, 1 H), 5.80 (s, 1 H), 6.59 (s, 1 H), 7.26–7.32 (m, 2 H),

7.39–7.62 (m, 10 H), 7.89 (d, J = 8 Hz, 2 H), 8.01–8.09 (m,

6 H), 8.68 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 20.7,

63.5, 66.0, 77.5, 80.4, 81.2, 81.3, 85.2, 91.0, 103.0, 106.0,

128.3, 128.4, 128.5, 128.6, 128.9, 129.0, 129.7, 129.8,

129.9, 133.2, 133.3, 133.6, 133.7, 160.4, 165.0, 165.5,

165.6, 170.6; ESI-MS calculated for C₄₂H₃₆Cl₃NO₁₄Na [M

+ Na] 906.1099 and found 906.1078.

p-Tolyl-5-O-acetyl-2,3-di-O-benzoyl-α-^D-arabinofuranosyl-

(1 → 5)-2,3-di-O-benzoyl-α-^D-arabinofuranosyl-(1 → 5)-

2,3-di-O-benzoyl-α-^D-arabinofuranosyl-(1 → 5)-2,3-

di-O-benzoyl-1-thio-^D-arabinofuranoside (26)

A solution of 22 (0.50 g, 0.573 mmol), 20 (0.39 g, 0.5 mmol)

and MS 4 Å (1 g) in CH₂Cl₂(30 mL) was stirred for 15 min

BF₃·OEt₂(0.06 mL, 0.5 mmol) was added dropwise at room

temperature, under N₂atmosphere. 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 3:1) to afford 26, as a foamy solid. Yield: 0.63 g

(82%); R_f(pet. ether/EtOAc 7:3) 0.45; [α]_D+32.4 (c 0.25,

2,3,5-Tri-O-benzoyl-α-^D-arabinofuranosyl-(1 →

5)-2,3-di-O-benzoyl-^D-arabinofuranose (24)

A solution of 18 (2.8 g, 4.6 mmol), 17 (1.95 g, 4.2 mmol)

and MS 4 Å (2 g) in CH₂Cl₂(50 mL) was stirred for 15 min

BF₃·OEt₂(0.54 mL, 4.2 mmol) was added dropwise at room

temperature, under N₂atmosphere. The reaction mixture was

stirred at room temperature for 30 min, neutralized with Et₃N,

ﬁltered and ﬁltrate concentrated in vacuo to afford 23. NBS

(0.75 g, 4.2 mmol) was added to a solution of crude 23 in

THF and water (1:1) and stirred at room temperature for 1 h.

The reaction mixture was ﬁltered and ﬁltrate diluted with

CH₂Cl₂(200 mL), washed with satd. aq. sodium thiosulfate

(2 × 40 mL) and water (1 × 40 mL). The organic portion was

dried (Na₂SO₄), ﬁltered and ﬁltrate concentrated in vacuo and

puriﬁed (pet. ether/EtOAc 7:3) to afford 24, as a foamy solid.

Yield: 2.13 g (62%); α/β 1.3:1; R_f(pet. ether/EtOAc 7:3) 0.3;

¹H NMR (CDCl₃, 400 MHz) δ 3.04 (s, 1 H), 3.96–4.02 (m,

2.3 H), 4.19–4.23 (m, 2.3 H), 4.30–4.32 (m, 1 H), 4.64–4.88

1

CHCl₃); H NMR (CDCl₃, 400 MHz) δ 2.02 (s, 3 H), 2.29 (s,

3 H), 3.87–3.95 (m, 3 H), 4.13–4.23 (m, 3 H), 4.35 (dd, J =

5.2 Hz, J = 12 Hz, 1 H), 4.53 (d, J = 3.4 Hz, 1 H), 4.56 (s, 1

H), 4.58 (d, J = 3.4 Hz, 2 H), 4.68 (q, J = 3.4 Hz, 1 H), 5.37–

5.39 (m, 3 H), 5.43 (s, 1 H), 5.57 (s, 1 H), 5.62 (s, 4 H), 5.69

(s, 1 H), 5.71 (s, 2 H), 7.07 (d, J = 8 Hz, 2 H), 7.21–7.58 (m,

26 H), 7.86–8.13 (m, 16 H); ¹³C NMR (CDCl₃, 100 MHz) δ

20.7, 21.0, 63.5, 65.7, 77.2, 77.6, 81.1, 81.4, 81.6, 82.0, 91.5,

105.8, 106.0, 128.3, 128.5, 128.9, 129.0, 129.1, 129.7, 129.8,

129.9, 132.6, 133.2, 133.4, 133.5, 137.9, 165.1, 165.3, 165.5,

165.6, 170.7; ESI-MS calculated for C₈₅H₇₄O₂₅SNa [M + Na]

1549.4138 and found 1549.4210.

1248

Synthetic glycolipids, biological and biophysical studies

8-O-Hexadecyl-3,6,10-trioxa-1,8-

H), 5.54–5.75 (m, 19.8 H), 5.96 (t, J = 6 Hz, 1 H), 7.27–7.49

(m, 67.2 H), 7.9–8.06 (m, 44.8 H); ¹³C NMR (CDCl₃, 100

MHz) δ 20.7, 63.5, 66.0, 66.1, 66.3, 77.2, 77.6, 78.0, 81.2,

81.4, 81.7, 81.9, 82.1, 82.4, 95.3, 100.9, 105.8, 105.9, 106.2,

128.3. 128.4, 128.5, 128.9, 129.0, 129.2, 129.7, 129.8, 129.9,

133.3, 133.4, 133.5, 165.1, 165.2, 165.5, 165.6, 165.7, 165.8,

170.7; ESI-MS calculated for C₇₈H₆₈O₂₆Na [M + Na]

1443.3897 and found 1443.3895.

hexacosanedioyl–α-^D-arabinofuranosyl-(1 →

5)-α-^D-arabinofuranosyl-(1 → 5)-arabinofuranosyl-(1 →

5)-α-^D-arabinofuranoside (3)

A solution of 26 (0.15 g, 0.098 mmol), 10 (0.061g, 0.098

mmol) and MS 4 Å (0.1 g) in CH₂Cl₂(10 mL) was stirred for

15 min, and NIS (0.024 g, 0.11 mmol) and AgOTf (0.003 g,

0.01 mmol) were added at −10°C, under N₂atmosphere. The

reaction mixture was stirred at the same temperature for 30

min, neutralized with Et₃N, ﬁltered and ﬁltrate was diluted

with CH₂Cl₂(20 mL), washed with satd. aq. sodium thiosul-

fate (2 × 10 mL) and water (1 × 10 mL). The organic portion

was dried (Na₂SO₄), ﬁltered and ﬁltrate concentrated in vacuo

and puriﬁed (pet. ether/EtOAc 3:1) to afford the protected

derivative of 3, as a foamy solid. NaOMe in MeOH (1 M,

0.10 mL) was added to a solution of the protected derivative

in MeOH/THF (1:1) (10 mL) at room temperature and stirred

for 12 h, neutralized with Amberlite ion-exchange (H⁺) resin,

ﬁltered and ﬁltrate concentrated in vacuo and puriﬁed to

afford 3. Yield: 0.083 g (74%); [α]_D+24.4 (c 0.25, MeOH);

¹H NMR (CD₃OD, 400 MHz) δ 0.9 (t, J = 7.2 Hz, 6 H), 1.29

(br s, 52 H), 1.55 (br s, 4 H), 3.46–4.0 (m, 37 H), 4.94 (s, 4

H); ¹³C NMR (CD₃OD, 100 MHz) δ 14.4, 23.8, 27.3, 28.6,

29.8, 30.5, 30.6, 30.8, 31.1, 33.1, 62.2, 63.1, 67.9, 68.2, 71.3,

71.5, 71.9, 72.0, 72.2, 72.6, 73.7, 78.8, 79.2, 79.3, 83.1, 83.2,

83.4, 83.9, 84.2, 85.9, 109.6, 109.7; ESI-MS calculated for

C₅₉H₁₁₂O₂₁Na [M + Na] 1179.7954 and found 1179.8187.

1-O-(5-O-(5-O-(5- O-(5-O-Acetyl-2,3-di-O-benzoyl-α-

D-arabinofuranosyl)-2,3-di-O-benzoyl-α-D-

arabinofuranosyl)-2,3-di-O-benzoyl-α-^D-arabinofuranosyl)-

2,3-di-O-benzoyl-α-^D-arabinofuranosyl)

trichloroacetimidate (28)

DBU (0.028 mL, 0.190 mmol) was added to a stirred solution

of tetrasaccharide 27 (0.27 g, 0.190 mmol) and CCl₃CN

(0.19 mL, 1.90 mmol) in CH₂Cl₂(5 mL) at 0°C and stirred

for 10 min. The crude reaction mixture was puriﬁed using pet.

ether/EtOAc (2:1) to afford trichloroacetimidate 28. Yield:

0.27 g (90%); R_f(pet. ether/EtOAc 2:1) 0.5; [α]_D+4.5 (c 1,

1

CHCl₃); H NMR (400 MHz, CDCl₃) δ 2.02 (s, 3 H), 3.88–

3.95 (m, 3 H), 4.09–4.20 (m, 3 H), 4.24 (dd, J = 11.6 Hz, J =

4 Hz, 1 H), 4.36 (dd, J = 11.6 Hz, J = 4.8 Hz, 1 H), 4.53–4.57

(m, 4 H), 4.64 (d, J = 2.8 Hz, 1 H), 5.37–5.39 (m, 3 H), 5.43

(s, 1 H), 5.58–5.62 (m, 4 H), 5.74 (d, J = 3.2 Hz, 1 H), 5.79

(s, 1 H), 6.58 (s, 1 H), 7.26–7.59 (m, 24 H), 7.83–8.07 (m,

16 H), 8.67 (s, 1 H); ¹³C NMR (100 MHz, CDCl₃) δ 20.8,

60.4, 63.5, 65.7, 65.8, 66.0, 77.2, 77.6, 80.4, 81.2, 81.4, 81.6,

82.0, 82.1, 85.3, 91.0, 103.1, 105.8, 105.9, 106.0, 128.2,

128.4, 128.5, 128.6, 128.9, 129.0, 129.1, 129.7, 129.8, 129.9,

130.0, 133.1, 133.2, 133.3, 133.4, 133.5, 133.6, 133.7, 160.4,

165.0, 165.1, 165.5, 165.7, 170.7; ESI-MS calculated for

C₈₀H₆₈Cl₃NO₂₆Na [M + Na] 1586.3 and found 1586.8.

p-Tolyl–α-^D-arabinofuranosyl-(1 →

5)-α-^D-arabinofuranosyl-(1 → 5)-α-D-arabinofurano

syl-(1 → 5)-α-^D-1-thio-α-D-arabinofuranoside (1)

NaOMe in MeOH (1 M, 0.10 mL) was added to a solution of

the 26 (0.1 g, 0.063 mmol) in MeOH/THF (1:1) (10 mL) at

room temperature and stirred for 12 h, neutralized with

Amberlite ion-exchange (H⁺) resin, ﬁltered and ﬁltrate concen-

trated in vacuo to afford 1. Yield: (94%). [α]_D+67.3 (c 0.25,

p-Tolyl-α-^D-arabinofuranosyl-(1 → 5)-α-D-arabinofuranosyl-

(1 → 5)-α-^D-arabinofurano-syl-(1 → 5)-α-D-arabinofurano

syl-(1 → 5)-α-^D-arabinofuranosyl-(1 → 5)-1-thio-α-D-

arabinofuranoside (2)

1

MeOH); H NMR (D₂O, 400 MHz) δ 2.18 (s, 3 H), 3.48–4.0

(m, 20 H), 4.88–4.92 (m, 3 H), 5.10–5.12 (m, 1 H), 7.11 (d, J =

8 Hz, 2 H), 7.32 (d, J = 8 Hz, 2 H); ¹³C NMR (D₂O, 100 MHz)

δ 20.2, 61.2, 66.5, 66.8, 75.7, 76.6, 76.8, 79.9, 80.9, 82.4, 84.0,

90.3, 107.5, 130.1, 133.2, 139.3.; ESI-MS calculated for

C₂₇H₄₀O₁₆SNa [M + Na] 675.1935 and found 675.1939.

A solution of 28 (0.15 g, 0.096 mmol), 20 (0.07 g, 0.09

mmol) and MS 4 Å (0.2 g) in CH₂Cl₂(10 mL) was stirred for

15 min BF₃·OEt₂(0.013 mL, 0.1 mmol) was added dropwise

at room temperature, under N₂atmosphere. The reaction

mixture was stirred at room temperature for 30 min, neutral-

ized with Et₃N, ﬁltered and ﬁltrate concentrated in vacuo and

puriﬁed (pet. ether/EtOAc 2:1) to afford the protected deriva-

tive of 2, as a foamy solid. Yield: 0.16 g (81%); [α]_D+42.5 (c

0.5, CHCl₃);¹H NMR (CDCl₃, 400 MHz) δ 2.02 (s, 3 H),

2.28 (s, 3 H), 3.87–3.99 (m, 6 H), 4.12–4.23 (m, 5 H), 4.33–

4.46 (m, 1 H), 4.51–4.57 (m, 6 H), 4.67–4.68 (m, 1 H), 5.37–

5.43 (m, 6 H), 5.57–5.62 (m, 8 H), 5.67–5.74 (m, 3 H), 7.07

(d, J = 8 Hz, 2 H), 7.23–7.58 (m, 38 H), 7.86–8.09 (m, 24 H);

¹³C NMR (CDCl₃, 100 MHz) δ 20.7, 21.0, 63.4, 65.6, 65.9,

77.2, 81.1, 81.3, 81.5, 82.0, 91.4, 105.7, 105.8, 128.2, 128.3,

128.4, 128.9, 129.0, 129.7, 129.8, 132.5, 133.2, 133.3, 133.4,

133.5, 137.8, 164.9, 165.0, 165.2, 165.4, 165.5, 170.6;

matrix-assisted laser desorption/ionization time of ﬂight

(MALDI-TOF)-MS calculated for C₁₂₃H₁₀₆O₃₇SNa [M + Na]

2230.6 (100%) and found 2230.3. NaOMe in MeOH (1 M,

0.10 mL) was added to a solution of the protected derivative

5-O-Acetyl-2,3-di-O-benzoyl-α-^D-arabinofuranosyl-(1 →

5)-2,3-di-O-benzoyl-α-^D-arabinofuranosyl-(1 →

5)-2,3-di-O-benzoyl-^D-arabinofuranose (27)

NBS (0.12 g, 0.65 mmol) was added to a solution of 26 (1.0

g, 0.65 mmol) in THF/water (1:1) and stirred at room temp-

erature for 1 h. The reaction mixture was ﬁltered and ﬁltrate

diluted with CH₂Cl₂(100 mL), washed with satd. aq. sodium

thiosulfate (2 × 20 mL) and water (1 × 20 mL). The organic

portion was dried (Na₂SO₄), ﬁltered and ﬁltrate concentrated

in vacuo and puriﬁed (pet. ether/EtOAc 3:2) to afford 27, as a

foamy solid. Yield: 0.72 g (78%); α/β 1.8:1; R_f(pet. ether/

1

EtOAc 2:1) 0.28; H NMR (CDCl₃, 400 MHz) δ 2.04 (s, 5.4

H), 2.07 (s, 3 H), 3.30 (br s, 1 H), 3.91–3.97 (m, 8.2 H),

4.09–4.21 (s, 8.8 H), 4.28 (br s, 1.8 H), 4.35–4.38 (m, 2.8 H),

4.49–4.68 (m, 13.6 H), 5.37–5.45 (m, 11.2 H), 5.52 (s, 1.8

1249

K Naresh et al.

of 2 (0.08 g, 0.036 mmol) in MeOH/THF (1:1) (10 mL) at

room temperature and stirred for 12 h, neutralized with

Amberlite ion-exchange (H⁺) resin, ﬁltered and ﬁltrate con-

centrated in vacuo to afford 2. Yield: 0.03 g (95%). [α]_D

62.3. 66.1, 66.6, 70.2, 70.6, 70.8, 71.4, 71.6, 77.2, 77.8, 81.6,

81.8, 83.7, 105.7, 128.3, 128.4, 128.5, 129.0, 129.1, 129.2,

129.8, 129.9, 133.3, 133.4, 133.5, 165.0, 165.3, 165.7, 166.0;

ESI-MS calculated for C₇₇H₁₁₂O₁₇Na [M + Na] 1331.7797

and found 1331.7562.

1

−12.5 (c 0.5, MeOH); H NMR (D₂O, 400 MHz) δ 2.18 (s, 3

H), 3.48–4.0 (m, 30 H), 4.88–4.92 (m, 4 H), 5.07–5.14 (m, 2

H), 7.11 (d, J = 8 Hz, 2 H), 7.32 (d, J = 8 Hz, 2 H); ¹³C NMR

(D₂O, 100 MHz) δ 20.2, 61.2, 62.4, 66.4, 66.8, 68.5, 76.5,

76.7, 79.9, 80.8, 80.9, 82.3, 84.0, 90.3, 107.5, 130.1, 133.2,

139.2; ESI-MS calculated for C₃₇H₅₆O₂₄SNa [M + Na]

939.2780 and found 939.3249.

8-O-Hexadecyl-3,6,10-trioxa-1,8-hexacosanedioyl–5-O-

acetyl-2,3-di-O-benzoyl-α-^D-arabinofuranosyl-(1 → 5)-2,3-

di-O-benzoyl-α-^D-arabinofuranosyl-(1 → 5)-2,3-di-O-benz-

oyl-α-^D-arabinofuranosyl-(1 → 5)-2,3-di-O-benzoyl-α-D-

arabinofuranosyl-(1 → 5)-2,3-di-O-benzoyl-α-^D-arabinofur-

anosyl-(1 → 5)-2,3-di-O-benzoyl-α-^D-arabinofuranoside (31)

8-O-Hexadecyl-3,6,10-trioxa-1,8- hexacosanedioyl-5- O-

acetyl-2,3-di-O-benzoyl-α-^D-arabinofuranosyl-(1 → 5)- 2,3-

di-O-benzoyl-α-^D-arabinofuranoside (29)

A solution of 30 (0.1 g, 0.076 mmol), 26 (0.12 g, 0.08

mmol) and MS 4 Å (0.2 g) in CH₂Cl₂(20 mL) was stirred

for 15 min, and NIS (0.022 g, 0.1 mmol) and AgOTf (0.003

g, 0.01 mmol) were added at −10°C, under N₂atmosphere.

The reaction mixture was stirred at the same temperature for

30 min, neutralized with Et₃N, ﬁltered and ﬁltrate was

diluted with CH₂Cl₂(30 mL), washed with satd. aq. sodium

thiosulfate (2 × 15 mL) and water (1 × 20 mL). The organic

portion was dried (Na₂SO₄), ﬁltered and ﬁltrate concentrated

in vacuo and puriﬁed (pet. ether/EtOAc 3:1) to afford 31, as

a foamy solid. Yield: 0.15 g (75%); R_f(pet. ether/EtOAc

A solution of 22 (0.5 g, 0.573 mmol), 10 (0.31 g, 0.5 mmol)

and MS 4 Å (1 g) in CH₂Cl₂(30 mL) was stirred for 15 min

BF₃·OEt₂(0.06 mL, 0.5 mmol) was added dropwise at room

temperature, under N₂atmosphere. 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 3:1) to afford 29, as a foamy solid. Yield: 0.59 g

(88%); R_f(pet. ether/EtOAc 7:3) 0.59; [α]_D−2.6 (c 0.5,

1

CHCl₃); H NMR (CDCl₃, 400 MHz) δ 0.87 (t, J = 7.2 Hz, 6

H), 1.24 (br s, 52 H), 1.50–1.55 (m, 4 H), 2.04 (s, 3 H),

3.41–3.71 (m, 16 H), 3.89 (d, J = 6 Hz, 1 H), 3.94 (dd, J =

2.4 Hz, J = 11.2 Hz, 1 H), 4.22 (dd, J = 4.4 Hz, J = 11.2 Hz, 1

H), 4.38 (dd, J = 12 Hz, J = 5.2 Hz, 1 H), 4.46 (q, J =3.2 Hz,

1 H), 4.55 (dd, J = 3.2 Hz, J = 12 Hz, 1 H), 4.62 (q, J = 4.4

Hz, 1 H), 5.28 (s, 1 H), 5.40 (d, J = 4.4 Hz, 1 H), 5.43 (s, 1

H), 5.55 (s, 1 H), 5.59 (s, 1 H), 5.62 (d, J = 5.2 Hz, 1 H),

7.26–7.60 (m, 12 H), 7.92–8.08 (m, 8 H); ¹³C NMR (CDCl₃,

100 MHz) δ 14.1, 20.7, 22.6, 26.1, 29.3, 29.5, 29.6, 29.7,

30.0, 31.9, 63.5, 66.1, 66.6, 70.2, 70.4. 70.6, 70.8, 71.4, 71.6,

77.6, 77.8, 81.2, 81.4, 81.8, 105.7, 105.9, 128.2, 128.4,

128.5, 128.9, 129.0, 129.1, 129.3, 129.7, 129.8, 133.3, 133.4,

133.5, 165.0, 165.3, 165.6, 170.6; ESI-MS calculated for

C₇₉H₁₁₄O₁₈Na [M + Na] 1373.7903 and found 1373.7898.

1

7:3) 0.6; [α]_D+7.5 (c 0.5, CHCl₃); H NMR (CDCl₃, 400

MHz) δ 0.87 (t, J = 7.2 Hz, 6 H), 1.25 (br s, 52 H), 1.52–

1.55 (m, 4 H), 2.01 (s, 3 H), 3.40–3.55 (m, 11 H), 3.58–

3.70 (m, 6 H), 3.88–3.97 (m, 6 H), 4.14–4.20 (m, 5 H),

4.37 (dd, J = 11.2 Hz, J = 4.8 Hz, 1 H), 4.44–4.45 (m, 1 H),

4.52–4.58 (m, 6 H), 5.27 (s, 1 H), 5.38 (s, 4 H), 5.43 (t, J

= 5.2 Hz, 2 H), 5.55–5.64 (m, 10 H), 7.22–7.49 (m, 36 H),

7.84–8.04 (m, 24 H); ¹³C NMR (CDCl₃, 100 MHz) δ 14.1,

20.7, 22.6, 26.1, 29.3, 29.5, 29.6, 29.7, 30.0, 31.8, 63.5,

65.7, 66.0, 66.6, 70.2, 70.5, 70.6, 70.8, 71.3, 71.6, 77.2,

77.5, 77.7, 77.8, 81.1, 81.4, 81.6, 81.7, 81.8, 81.9, 82.0,

105.8, 105.9, 128.1, 128.2, 128.4, 128.8, 129.0, 129.3,

129.7, 129.8, 133.0, 133.2, 133.3, 133.5, 164.9, 165.0,

165.3, 165.5, 165.6, 170.6; MALDI-TOF-MS calculated for

C

₁₅₈H₁₇₈O₄₂Na [M + Na] 2735.1 (100%) and found 2735.0.

8-O-Hexadecyl-3,6,10-trioxa-1,8-hexacosanedioyl 2,3-di-O-

benzoyl-α-^D-arabinofurano syl-(1 → 5)-2,3-di-O-benzoyl-α-

D-arabinofuranoside (30)

8-O-Hexadecyl- 3,6,10-trioxa-1,8-hexacosanedioyl–α- ^D-

arabinofuranosyl-(1 → 5)-α-^D-arabinofuranosyl-(1 → 5)-α-

D-arabinofuranosyl-(1 → 5)-α-D-arabinofuranosyl-(1 → 5)-

α-^D-arabinofuranosyl-(1 → 5)-α-D-arabinofuranoside (4)

AcCl (0.4 mL) was added to a solution of 29 (0.5 g, 0.37

mmol) in MeOH/CH₂Cl₂(1:1) (40 mL) and stirred at room

temperature for 4 h. The reaction mixture was diluted with

CH₂Cl₂(100 mL), washed with satd. aq. NaHCO₃(2 × 20

mL) solution, brine (20 mL) and water (20 mL). The organic

portion was dried (Na₂SO₄), ﬁltered and ﬁltrate concentrated

in vacuo and puriﬁed (pet. ether/EtOAc 2:1) to afford 30, as a

foamy solid. Yield: 0.4 g (83%); R_f(pet. ether/EtOAc 3:2)

NaOMe in MeOH (1 M, 0.1 mL) was added to a solution of

31 (0.06 g, 0.022 mmol) in MeOH/THF (1:1) (10 mL), at

room temperature and stirred for 12 h, neutralized with

Amberlite ion-exchange (H⁺) resin, ﬁltered and ﬁltrate con-

centrated in vacuo to afford 4. Yield: 0.029 g (94%); [α]_D−8

1

0.6; [α]_D−2.0 (c 0.5, CHCl₃); H NMR (CDCl₃, 400 MHz) δ

1

0.87 (t, J = 7.2 Hz, 6 H), 1.24 (br s, 52 H), 1.50–1.55 (m, 4

H), 3.39–3.59 (m 12 H), 3.62–3.71 (m, 5 H), 3.86–4.04 (m, 4

H), 4.19 (dd, J = 4.4 Hz, J = 11.2 Hz, 1 H), 4.46 (dd, J = 4.4

Hz, J = 11.2 Hz, 1 H), 4.50 (q, J = 3.6 Hz, 1 H), 5.28 (s, 1 H),

5.41 (s, 1 H), 5.42 (d, J = 4.8 Hz, 1 H), 5.55 (s, 1 H), 5.61 (d,

J = 4.8 Hz, 1 H), 5.64 (s, 1 H),7.26–7.59 (m, 12 H), 7.93(d, J

= 8.4 Hz, 2 H), 8.01–8.06 (m, 6 H); ¹³C NMR (CDCl₃, 100

MHz) δ 14.1, 22.6, 26.1, 29.3, 29.5, 29.6, 29.7, 30.0, 31.9,

(c 0.5, MeOH); H NMR (DMSO-d₆, 400 MHz) δ 0.84 (t, J

= 7.2 Hz, 6 H), 1.21 (br s, 52 H), 1.42–1.44 (m, 4 H), 3.36–

3.84 (m, 47 H), 4.72 (br s, 6 H), 5.23 (s, 6 H), 5.41 (s, 7 H);

¹³C NMR (DMSO-d₆, 100 MHz) δ 13.8, 22.1, 25.6, 28.3,

28.7, 28.9, 29.0, 29.1, 29.2, 29.6, 29.8, 30.3, 31.3, 61.3, 66.3,

67.1, 67.4, 69.3, 69.7, 70.1, 70.5, 70.7, 77.0, 77.4, 81.4, 81.8,

81.9, 83.8, 108.0; MALDI-TOF-MS calculated for

C₆₉H₁₂₈O₂₉Na [M + Na] 1443.8 (100%) and found 1443.9.

1250

Synthetic glycolipids, biological and biophysical studies

8-O-Hexadecyl-3,6,10-trioxa-1,8-hexacosanedioyl 2,3-di-O-

benzoyl-α-^D-arabinofurano syl-(1 → 5)-2,3-di-O-benzoyl-α-

D-arabinofuranosyl-(1 → 5)-2,3-di-O-benzoyl-α-D-

arabinofuranosyl-(1 → 5)-2,3-di-O-benzoyl-α-^D-

arabinofuranoside (32)

temperature and stirred for 12 h, neutralized with Amberlite

ion-exchange (H⁺) resin, ﬁltered and ﬁltrate concentrated in

vacuo to afford 5. Yield: 0.035 g (92%); [α]_D−5.0 (c 0.25,

1

MeOH); H NMR (DMSO-d₆, 400 MHz) δ 0.83 (t, J = 7.2

Hz, 6 H), 1.21 (br s, 52 H), 1.39–1.46 (m, 4 H), 3.48–3.95

(m, 57 H), 4.73 (br s, 8 H), 5.30 (br s, 8 H), 5.52 (br s, 9 H);

¹³C NMR (DMSO-d₆, 100 MHz) δ 13.9, 22.1, 25.6, 28.6,

28.7, 28.8, 28.9, 29.0, 29.2, 29.5, 31.2, 61.2, 66.3, 67.1, 69.3,

69.6, 69.7, 69.8, 70.1, 70.5, 72.3, 77.0, 77.4, 81.5, 81.8, 82.0,

83.7, 108.0; MALDI-TOF-MS calculated for C₇₉H₁₄₄O₃₇Na

[M + Na] 1707.9 (100%) and found 1708.1.

AcCl (0.25 mL) was added to a solution of 31 (0.15 g, 0.05

mmol) in MeOH/CH₂Cl₂(1:1) (20 mL) and stirred at room

temperature for 4 h. The reaction mixture was diluted with

CH₂Cl₂(100 mL) and washed with the satd. aq. NaHCO₃(2 ×

20 mL) solution, brine (20 mL) solution and water (20 mL).

The organic portion was dried (Na₂SO₄), ﬁltered and ﬁltrate

concentrated in vacuo and puriﬁed (pet. ether/EtOAc 3: 2) to

afford 32, as a foamy solid. Yield: 0.11 g (84%); R_f(pet. ether/

p-Tolyl-2,5-di-O-acetyl-1-thio-α-^D-arabinofuranoside (33)

Bu₂SnO (4.3 g. 17.3 mmol) was added to a solution of 15

(4 g, 15.6 mmol) in PhMe (90 mL). The solution was reﬂuxed

for 18 h, after which half the amount of solvents was removed.

Ac₂O (3.22 mL, 34.2 mmol) was then added, solution stirred

at 40°C for 2 h, concentrated in vacuo and puriﬁed (pet. ether/

EtOAc 1:1) to afford 33, as a gum. Yield: 3.07 g (58%); R_f

1

EtOAc 3:2) 0.6; [α]_D+5.2 (c 0.5, CHCl₃); H NMR (CDCl₃,

400 MHz) δ 0.88 (t, J = 7.2 Hz, 6 H), 1.25 (br s, 52 H), 1.50–

1.55 (m, 4 H), 3.39–3.60 (m, 8 H), 3.64–3.70 (m, 3 H), 3.88–

3.95 (m, 7 H), 4.13–4.21 (m, 6 H), 4.44 (q, J = 2.6 Hz, 1 H),

4.56–4.67 (m, 6 H), 4.72 (q, J = 3.6 Hz, 1 H), 4.81 (dd, J = 2.6

Hz, J = 8.8 Hz, 1 H), 5.27 (s, 1 H), 5.30 (s, 1 H), 5.37–5.38

(m, 5 H), 5.44 (s, 1 H), 5.55 (s, 1 H), 5.63 (br s, 12 H), 7.19–

7.58 (m, 36 H), 7.83–8.08 (m, 24 H); ¹³C NMR (CDCl₃, 100

MHz) δ 14.1, 22.6, 26.1, 29.3, 29.5, 29.6, 29.7, 30.1, 30.8,

31.9, 62.3,65.8, 66.1, 66.7, 70.2, 70.6, 70.8, 70.9, 71.4, 71.6,

77.2, 77.7, 77.9, 81.6, 81.8, 82.0, 83.6, 105.8, 105.9, 128.2,

128.3, 128.4, 128.5, 129.0, 129.1, 129.7, 129.8, 133.1, 133.3,

133.4, 165.0, 165.6, 166.0; MALDI-TOF-MS calculated for

1

(pet. ether/EtOAc 1:1) 0.5; [α]_D+276.0 (c 0.5, CHCl₃); H

NMR (CDCl₃, 400 MHz) δ 2.09 (s, 3 H), 2.14 (s, 3 H), 2.33

(s, 3 H), 4.00 (dd, J = 3.6 Hz, J = 7.2 Hz, 1 H), 4.25 (dd, J =

5.6 Hz, J = 12 Hz, 1 H), 4.35 (t, J = 3.2 Hz, 1 H), 4.36–4.37

(m, 1 H), 4.89 (t, J = 3.6 Hz, 1 H), 5.48 (dd, J = 3.2 Hz, 1 H),

7.13 (d, J = 8 Hz, 2 H), 7.40 (d, J = 8 Hz, 2 H); ¹³C NMR

(CDCl₃, 100 MHz) δ 20.7, 21.1, 63.0, 77.1, 80.1, 86.2, 89.3,

129.8, 132.7, 138.1, 170.8, 171.8; ESI-MS calculated for

C₁₆H₂₀O₆SNa [M + Na] 363.0878 and found 363.0883.

C

₁₅₃H₁₇₆O₄₁Na [M + Na] 2693.1 (100%) and found 2693.1.

p-Tolyl-3-O-benzoyl-1-thio-α-^D-arabinofuranoside (34)

8-O-Hexadecyl-3,6,10-trioxa-1,8-hexacosanedioyl–α- ^D-

arabinofuranosyl-(1 → 5)-α-^D-arabinofuranosyl-(1 → 5)-α-

D-arabinofuranosyl-(1 → 5)-α-D-arabinofuranosyl-(1 → 5)-

α-D-arabinofuranosyl-(1 → 5)-α-D-arabinofuranosyl-(1 →

5)-α-^D-arabinofuranosyl-(1 → 5)-α-D-arabinofuranoside (5)

Pyridine (0.63 mL, 8.03 mmol) and BzCl (0.94 mL, 8.03

mmol) were added to a solution of 33 (2.5 g, 7.3 mmol) in

CH₂Cl₂(20 mL) at 0°C. The reaction mixture was stirred for 12

h, diluted with CH₂Cl₂(50 mL), washed with dil. aq. HCl (2 ×

25 mL) solution, satd. aq. NaHCO₃solution (2 × 25 mL) and

water (25 mL). The organic portion dried (Na₂SO₄), ﬁltered and

ﬁltrate concentrated in vacuo. AcCl (0.4 mL) was added to a sol-

ution of a crude residue in MeOH/CH₂Cl₂(1:1) (40 mL) and

stirred at room temperature for 18 h. The reaction mixture was

diluted with CH₂Cl₂(100 mL), washed with satd. aq. NaHCO₃

(2 × 20 mL) solution, brine (20 mL) and water (20 mL). The

organic portion separated and dried (Na₂SO₄), ﬁltered and ﬁl-

trate concentrated in vacuo and puriﬁed (pet. ether/EtOAc 2:1)

to afford 34, as a foamy solid. Yield: 1.62 g (62%); R_f(pet.

A solution of 32 (0.10 g, 0.037 mmol), 25 (0.04 g, 0.04

mmol) and MS 4 Å (0.1 g) in CH₂Cl₂(10 mL) was stirred for

15 min, and BF₃·OEt₂(0.005 mL, 0.04 mmol) was added

dropwise at room temperature, under N₂atmosphere. 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 3:1) to afford protected

derivative of 5, as a foamy solid. Yield: 0.097 g (76%); [α]_D

1

+9.2 (c 0.5, CHCl₃); R_f(pet. ether/EtOAc 7:3) 0.54; H NMR

1

(CDCl₃, 400 MHz) δ 0.87 (t, J = 7.2 Hz, 6 H), 1.24 (br s, 52

H), 1.50–1.55 (m, 4 H), 3.38–3.59 (m, 10 H), 3.63–3.69 (m,

4 H), 3.87–3.95 (m, 10 H), 4.11–4.19 (m, 7 H), 4.43–4.44

(m, 2 H), 4.56–4.58 (m, 6 H), 5.26 (s, 1 H), 5.29 (s, 2 H),

5.37–5.39 (m, 9 H), 5.54 (s, 1 H), 5.59–5.63 (m, 13 H),

7.19–7.59 (m, 51 H), 7.83–8.08 (m, 34 H); ¹³C NMR

(CDCl₃, 100 MHz) δ 14.1, 22.7, 26.1, 29.3, 29.5, 29.7, 30.1,

30.8, 31.9, 63.6, 65.8, 66.0, 66.7, 70.2, 70.6, 70.8, 71.4, 71.6,

77.2, 77.8, 77.9, 81.2, 81.5, 81.8, 81.9, 82.0, 105.7, 105.9,

128.2, 128.3, 128.4, 128.8, 129.0, 129.7, 129.8, 129.9, 133.0,

133.2, 133.3, 165.0, 165.2, 165.5, 165.6, 165.7, 166.1;

MALDI-TOF-MS calculated for C₁₉₈H₂₁₂O₅₄Na [M + Na]

3478.4 (100%) and found 3478.6. NaOMe in MeOH (1 M,

0.1 mL) was added to a solution of the protected derivative of

5 (0.08 g, 0.023 mmol) in MeOH/THF (1:1) (10 mL) at room

ether/EtOAc 3:1) 0.25; [α]_D+200.4 (c 0.5, CHCl₃); H NMR

(CDCl₃, 400 MHz) δ 2.29 (s, 3 H), 3.90–3.94 (m, 2 H), 4.41 (s,

1 H), 4.50 (d, J = 3.6 Hz, 1 H), 5.23 (d, J = 3.6 Hz, 1 H), 5.57

(s, 1 H), 7.09 (d, J = 8 Hz, 2 H), 7.42–7.47 (m, 4 H), 7.55–7.60

(m, 1 H), 8.07–8.09 (m, 2 H); ¹³C NMR (CDCl₃, 100 MHz) δ

21.0, 61.5, 80.1, 80.3, 83.5, 94.4, 128.5, 128.8, 129.7, 132.1,

133.5, 137.5, 166.5; ESI-MS calculated for C₁₉H₂₀O₅SNa [M +

Na] 383.0929 and found 383.0934.

p-Tolyl–2,3-di-O-benzoyl-α-^D-arabinofuranosyl-(1 → 5)-

[2,3-di-O-benzoyl-α-^D-arabino furanosyl-(1 → 2)]-3-O-

benzoyl-1-thio-^D-arabinofuranoside (35)

A solution of 34 (0.50 g, 1.3 mmol), 14 (1.52 g, 2.8 mmol)

and MS 4 Å (1 g) in CH₂Cl₂(30 mL) was stirred for 15 min

1251

K Naresh et al.

BF₃·OEt₂(0.36 mL, 2.8 mmol) was added dropwise at room

temperature, under N₂atmosphere. The reaction mixture was

stirred at room temperature for 30 min, neutralized with

Et₃N, ﬁltered and ﬁltrate concentrated in vacuo. AcCl (1.2

mL) was added to the solution of the crude residue in

MeOH:CH₂Cl₂(1:1) (60 mL), and stirred at room tempera-

ture for 4 h. The reaction mixture was diluted with CH₂Cl₂

(200 mL), washed with satd. aq. NaHCO₃(2 × 40 mL) sol-

ution, brine (40 ml) and water (40 mL). The organic portion

was dried (Na₂SO₄), ﬁltered and ﬁltrate concentrated in

vacuo and puriﬁed (pet. ether/EtOAc 3: 2) to afford 35, as a

foamy solid. Yield: 0.89 g (66%); R_f(pet. ether/EtOAc 7:3)

0.21; [α]_D+55.2 (c 0.5, CHCl₃); ¹H NMR (CDCl₃, 400

MHz) δ 2.25 (s, 3 H), 2.35 (t, J = 5.6 Hz, 1 H), 2.40 (t, J =

5.6 Hz, 1 H), 3.91–4.06 (m, 5 H), 4.07 (dd, J = 11.2 Hz, J =

5.6 Hz, 1 H), 4.30 (q, J = 4 Hz, 1 H), 4.41 (q, J = 4 Hz, 1

H), 4.61 (t, J = 2 Hz, 1 H),4.67 (q, J = 4.8 Hz, 1 H), 5.26 (s,

1 H), 5.42 (t, J = 6 Hz, 2 H), 5.51–5.52 (m, 1 H), 5.54 (d, J

= 1.2 Hz, 1 H), 5.59 (d, J = 1.2 Hz, 1 H), 5.62 (s, 1 H), 5.66

(d, J = 2.4 Hz, 1 H), 6.98 (d, J = 8 Hz, 2 H), 7.30–7.60 (m,

17 H), 7.94–8.1 (m, 10 H); ¹³C NMR (CDCl₃, 100 MHz) δ

21.0, 62.1, 62.3, 66.8, 77.2, 77.5, 77.6, 78.1, 81.7, 81.8,

81.9, 83.6, 84.2, 84.3, 91.9, 104.5, 105.7, 128.4, 128.5,

128.8, 128.9, 129.0, 129.7, 129.8, 129.9, 130.0, 132.2,

132.4, 133.5, 133.8, 137.9, 165.0, 165.2, 165.7, 166.1; ESI-

MS calculated for C₅₇H₅₂O₁₇Na [M + Na] 1063.2823 and

found 1063.2632.

p-Tolyl-α-^D-arabinofuranosyl-(1 → 5)-α-D-arabinofuranosyl-

(1 → 5)-α-^D-arabinofurano syl-(1 → 5)-[α-D-

arabinofuranosyl-(1 → 5)-α-^D-arabinofuranosyl-(1 → 5)-α-

D-arabinofuranosyl-(1 → 2)]-α-D-1-thio-arabinofuranoside

(6)

NaOMe in MeOH (1 M, 0.10 mL) was added to a solution of

36 (0.08 g, 0.032 mmol) in MeOH/THF (1:1) (10 mL) at

room temperature and stirred for 12 h, neutralized with

Amberlite ion-exchange (H⁺) resin, ﬁltered and ﬁltrate con-

centrated in vacuo to afford 6. Yield: 0.031 g (93%); [α]_D

1

−23.2 (c 0.25, MeOH); H NMR (D₂O, 400 MHz) δ 2.19 (s,

3 H), 3.53–4.05 (m, 35 H), 4.88–4.93 (m, 5 H), 5.05 (s, 1 H),

5.28 (d, J = 2.8 Hz, 1 H), 7.12 (d, J = 8 Hz, 2 H), 7.34 (d, J =

8 Hz, 2 H); ¹³C NMR (D₂O, 100 MHz) δ 20.2, 61.2, 66.5,

66.8, 75.2, 76.5, 76.7, 76.8, 79.9, 80.7, 80.8, 80.9, 81.2, 82.2,

82.3, 82.5, 84.0, 86.4, 89.7, 107.5, 130.1, 133.2, 139.2.; ESI-

MS calculated for C₄₂H₆₄O₂₈SNa [M + Na] 1071.3203 and

found 1071.3320.

8-O-Hexadecyl-3,6,10-trioxa-1,8-hexacosanedioyl–α- ^D-

arabinofuranosyl-(1 → 5)-α-^D-arabinofuranosyl-(1 → 5)-α-

D-arabinofuranosyl-(1 → 5)-[α-D-arabinofuranosyl-(1 → 5)-

α-^D-arabinofuranosyl-(1 → 5)-α-D-arabinofuranosyl-(1 →

2)]-α-^D-arabinofuranoside (7)

A solution of 36 (0.12 g, 0.048 mmol), 10 (0.03 g, 0.048

mmol) and MS 4 Å (0.3 g) in CH₂Cl₂(10 mL) was stirred for

15 min, and NIS (0.013 g, 0.06 mmol) and AgOTf (0.001 g,

0.06 mmol) were added at −10°C, under N₂atmosphere. The

reaction mixture was stirred at the same temperature for 30

min, neutralized with Et₃N, ﬁltered and ﬁltrate diluted with

CH₂Cl₂(20 mL) and washed with satd. aq. sodium thiosulfate

(2 × 10 mL) and water (1 × 10 mL). The organic portion was

dried (Na₂SO₄), ﬁltered and ﬁltrate concentrated in vacuo and

puriﬁed (pet. ether/EtOAc 3:1) to afford the protected deriva-

tive of 7, as a foamy solid. NaOMe in MeOH (1 M, 0.10 mL)

was added to a solution of the protected derivative of 7 (0.08

g, 0.036 mmol) in MeOH/THF (1:1) (10 mL) at room temp-

erature and stirred for 12 h, neutralized with Amberlite ion-

exchange (H⁺) resin, ﬁltered and ﬁltrate concentrated in vacuo

to afford 7. Yield: 0.046 g (62%); [α]_D−9 (c 0.25, MeOH);

¹H NMR (DMSO-d₆, 400 MHz) δ 0.84 (t, J = 7.2 Hz, 6 H),

1.21 (br s, 52 H), 1.42–1.44 (m, 4 H), 3.36–3.84 (m, 52 H),

4.73 (br s, 5 H), 4.85 (s, 1 H), 4.91 (s, 1 H), 5.47 (br s, 15

H); ¹³C NMR (DMSO-d₆, 100 MHz) δ 13.8, 22.1, 25.6, 28.7,

28.8, 29.0, 29.1, 29.2, 30.3, 31.3, 61.2, 67.0, 69.3, 69.6, 69.7,

70.1, 70.5, 70.7, 77.0, 77.3, 81.4, 81.8, 81.9, 83.7, 108.0;

MALDI-TOF-MS calculated for C₇₄H₁₃₆O₃₃Na [M + Na]

1575.9 (100%) and found 1576.2.

p-Tolyl-5-O-acetyl-2,3-di-O- benzoyl-α-^D-arabinofuranosyl-

(1 → 5)-2,3-di-O-benzoyl-α-^D-arabinofuranosyl-(1 → 5)-2,3-

di-O-benzoyl-α-^D-arabinofuranosyl-(1 → 5)-[5-O-acetyl-2,3-

di-O-benzoyl-α-^D-arabinofuranosyl-(1 → 5)-2,3-di-O-

benzoyl-α-^D-arabinofuranosyl-(1 → 5)-2,3-di-O-benzoyl-α-D-

arabinofuranosyl-(1 → 2)]-3-O-benzoyl-1-thio-^D-

arabinofuranoside (36)

A solution of 35 (0.14 g, 0.13 mmol), 22 (0.13 g, 0.14

mmol) and MS 4 Å (0.3 g) in CH₂Cl₂(20 mL) was stirred

for 15 min BF₃·OEt₂(0.018 mL, 0.14 mmol) was added

dropwise at room temperature, under N₂atmosphere. 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 36, as a

foamy solid. Yield: 0.284 g (88%); R_f(pet. ether/EtOAc 3:2)

0.3; [α]_D+38.0 (c 0.25, CHCl₃); ¹H NMR (CDCl₃, 400

MHz) δ 2.02 (s, 6 H), 2.21 (s, 3 H), 3.83–3.86 (m, 1 H),

3.91–3.95 (m, 4 H), 4.04–4.08 (m, 1 H), 4.17–4.21 (m, 3

H), 4.36 (dd, J = 5.2 Hz, J = 12 Hz, 2 H), 4.41–4.44 (m, 1

H), 4.49–4.52 (m, 1 H), 4.53 (d, J = 3.2 Hz, 1 H), 4.56–4.62

(m, 8 H), 5.35 (s, 1 H), 5.38 (t, J = 3.2 Hz, 4 H), 5.50 (s, 2

H), 5.51–5.53 (m, 2 H), 5.58–5.66 (m, 11 H), 6.96 (d, J = 8

Hz, 2 H), 7.22–7.58 (m, 41 H), 7.84–8.0 (m, 26 H); ¹³C

NMR (CDCl₃, 100 MHz) δ 20.7, 21.0, 60.3, 63.5, 65.7,

66.0, 66.6, 77.2, 77.6, 78.1, 81.2, 81.4, 81.6, 81.7, 81.9,

82.0, 82.1, 82.6, 84.0, 91.7, 104.7, 105.8, 105.9, 128.2,

128.3, 128.4, 128.5, 128.9, 129.0, 129.1, 129.2, 129.7,

129.8, 132.5, 133.1, 133.2, 133.3, 133.5, 137.7, 165.0,

165.1, 165.5, 165.6, 170.6; ESI-MS calculated for

Bacterial strains, media and growth conditions

Wild-type M. smegmatis mc²155 strain was grown in

Middlebrook 7H9 broth (MB7H9, Difco) supplemented with

2% glucose and 0.05% Tween-80 at 37°C with agitation 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 concen-

trations (50 and 100 µg/mL) of oligosaccharides and glyco-

lipids 1–7, and the growth rate in triplicate was followed by

C

₁₃₀H₁₂₀O₄₃Na [M + Na] 2507.6822 and found 2507.6824.

1252

Synthetic glycolipids, biological and biophysical studies

measuring the optical density (OD) at 600 nm, at different

durations. The oligosaccharides and glycolipids were added at

zero time point. Growth of the bacteria without the oligosac-

charide was used as the control.

sensor chip was activated with an injection of a solution con-

taining N-ethyl-N′-(3-diethylaminopropyl) carbodiimide (0.2

M) and N-hydroxysuccinimide (0.05 M). SP-A in NaOAc

buffer (100 μg/mL) was injected over the activated ﬂow cell.

The immobilization procedure was completed by an injection

of ethanolamine hydrochloride (1 M, 70 μL), followed by a

ﬂow of the buffer (100 μL/min), in order to eliminate the phys-

ically adsorbed compounds. Synthetic arabinan compounds

were dissolved in HEPES buffer and passed over ﬂow cells,

and binding studies were performed. Oligoarabinofuranosides

and glycolipids in a buffer were passed for 300 s for the associ-

ation phase and the buffer alone for 300 s for the dissociation

phase. The sensor chip was regenerated between each cycle

using ethylenediaminetetraacetic acid solution (5 mM) for 60

s, followed by an injection of buffer alone for 60 s. Primary

sensorgrams were analyzed by the 1:1 Langmuir model ﬁtting,

using the BIAevaluation software.

Bioﬁlm formation and quantitation assay

Inoculum of M. smegmatis mc²155 (wild type) was added

into primary cultures containing 2% glucose and 0.05%

Tween-80 in MB7H9 (Difco) media (Allen 1998; Piddington

et al. 2000). The well-grown culture was washed with

Sauton’s media before secondary inoculation. For the devel-

opment of a 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 sup-

plemented with 2% glucose. The ﬁnal concentrations of the

inhibitors were 100 µg/mL in all the studies and added at zero

time point. Culture plates were incubated in a humidiﬁed

incubator at 37°C and monitored at different points of time.

The quantitation of bioﬁlm formation was performed as

described earlier (Allen 1998; Mathew et al. 2006; Naresh

et al. 2010). The well-grown primary culture was washed with

Sauton’s media before secondary inoculation and diluted up

to a ﬁnal OD of 0.0025 at 600 nm in Sauton’s medium. The

oligosaccharides were added (100 µg/mL) at zero time point

and distributed into 96-well polystyrene plates. The quanti-

tation was performed in triplicates at each time point and

experiment was repeated thrice. Inoculum (250 μL) 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 300 μL of 1% crystal violet solution and incubated

for 20 min at room temperature. The wells were again washed

thoroughly with water and allowed to dry. The dye was quan-

titated after incubating each well with 300 μL of 80% ethyl

alcohol for an hour and subsequently measured the absorption

Acknowledgement

We are grateful to Professor Jo Rae Wright and Dr. Kathy

Evans, Department of Cell Biology, Pediatrics and Medicine,

Duke University, Durham, USA, for providing us the SP-A

protein. We thank Department of Biotechnology, New Delhi,

and Department of Science and Technology, New Delhi, for a

ﬁnancial support. Council of Scientiﬁc and Industrial

Research, New Delhi, is acknowledged for a research fellow-

ship to KN. Indian Institute of Science, Bangalore is acknowl-

edged for a research fellowship to BKB.

Conﬂict of interest statement

None declared.

Abbreviations

at 590 nm using a microplate reader (Spectra Max 340PC³⁸⁴

,

DBU, 1,8-diazabicyclo[5.4.0]undec-7-ene; ESI, electrospray

ionization; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane

sulphonic acid; LAM, lipoarabinomannan; mAGP, mycolyl

arabinogalactan–peptidoglycan; MALDI-TOF, matrix-assisted

laser desorption/ionization time of ﬂight; ManLAM,

mannosylated lipoarabinomannan; MS, molecular sieves;

NBS, N-bromosuccinimide; OD, optical density; Q-TOF,

quadrupole-time of ﬂight; SP-A, surfactant protein A; SPR,

surface plasmon resonance; THF, tetrahydrofuran; TLC, thin

layer chromatography.

Molecular Devices). The percentage inhibition that was calcu-

lated for the mycobacterial growth and bioﬁlm in the presence

of synthetic oligosaccharides using the stationary phase data

points as inhibition was observed in the stationary phase and

the average value is reported in Table I.

Sliding motility tests

Sliding motility test was carried out to check the spreading

ability of mycobacteria on the surface by using the method

described elsewhere (Etienne et al. 2002). Brieﬂy, MB7H9

base medium (Difco) was solidiﬁed with 0.3% high-grade

agarose (Sigma-Aldrich Inc.) supplemented with 2% glucose.

The oligosaccharides and glycolipids were added during

pouring the plate. Plates were inoculated into their centre with

the cultures (10 µL) after adjusting the OD₆₀₀to 0.5.

Spreading was evaluated after incubation for 3–10 days at 37°

C in a humidiﬁed incubator.

References

Allen BW. 1998. Mycobacteria: General culture methodology and safety con-

siderations. In: Parish T, Stoker N, editors. Mycobacteria Protocols.

Totowa, NJ: Humana Press. p. 15–30.

Armarego WLF, Perrin DD. 1996. Puriﬁcation of Laboratory Chemicals. 4th

ed. Oxford: Butterworth-Heinemann Press.

Branda SS, Vik S, Friedman L, Kolter R. 2005. Bioﬁlms: The matrix

revisited. Trends Microbiol. 13:20–26.

Brennan PJ, Nikaido H. 1995. The envelope of mycobacteria. Annu Rev

Biochem. 64:29–63.

Carlson TK, Torrelles JB, Smith K, Horlacher T, Castelli R, Seeberger PH,

Crouch EC, Schlesinger LS. 2009. Critical role of amino acid position 343

of surfactant protein-D in the selective binding of glycolipids from

Mycobacterium tuberculosis. Glycobiology. 19:1473–1484.

SPR studies

The studies were conducted using a Biacore 3000 SPR instru-

ment. A continuous ﬂow of HEPES-P buffer was maintained

over the sensor surface at a ﬂow rate of 10 μL/min. The CM5

1253

K Naresh et al.

Chatterjee D, Brennan PJ. 2009. Biosynthesis of the mycobacterial cell envel-

ope components. In: Moran A, Holst O, Brennan PJ, von Itzstein M,

editors. Microbial Glycobiology. Amsterdam: Elsevier. p. 375–392.

Cociorva OM, Lowary TL. 2004. Synthesis of oligosaccharides as potential

inhibitors of mycobacterial arabinosyltransferases. Di- and trisaccharides

containing C-5 modiﬁed arabinofuranosyl residues. Carbohydr Res.

339:853–865.

pleiotropic surface-related phenotypes in Mycobacterium smegmatis.

Microbiology. 152:1741–1750.

McCormack FX. 1998. Structure, processing and properties of surfactant

protein A. Biochim Biophys Acta. 1408:109–131.

Mei X, Ning J. 2005. An efﬁcient synthesis of a dimer of the tetrasaccharide

present in motif B of the mycobacterium tuberculosis cell wall. Synlett.

15:2267–2272.

Crouch E, Wright JR. 2001. Surfactant proteins A and D and pulmonary host

defense. Annu Rev Physiol. 63:521–554.

Davis CB, Hartnell RD, Madge PD, Owen DJ, Thomson RJ, Chong AKJ,

Coppel RL, von Itzstein M. 2007. Synthesis and biological evaluation of

galactofuranosyl alkyl thioglycosides as inhibitors of mycobacteria.

Carbohydr Res. 342:1773–1780.

D’Souza FW, Ayers JD, McCarren PR, Lowary TL. 2000. Arabinofuranosyl

oligosaccharides from mycobacteria: Synthesis and effect of glycosylation

on ring conformation and hydroxymethyl group rotamer populations. J Am

Chem Soc. 122:1251–1260.

Du Y, Pan Q, Kong F. 2000. Synthesis of a tetrasaccharide representing a

minimal epitope of an arabinogalactan. Carbohydr Res. 323:28–35.

Engelhardt H, Heinz C, Niederweis M. 2004. A tetrameric porin limits the

Mereyala HB, Hotha S, Gurjar MK. 1998. Synthesis of pentaarabinofuranosyl

structure motif A of Mycobacterium tuberculosis. Chem Comm. 685–686.

Mikusová K, Yagi T, Stern R, McNeil MR, Besra GS, Crick DC, Brennan PJ.

2000. Biosynthesis of the galactan component of the mycobacterial cell

wall. J Biol Chem. 275:33890–33897.

Mizutani K, Kasai R, Nakamura M, Tanaka O, Matsuura H. 1989. NMR spec-

tral study of α- and β-L-arabinofuranosides. Carbohydr Res. 185:27–38.

Monds RD, O’Toole GA. 2009. The developmental model of microbial bio-

ﬁlms: Ten years of a paradigm up for review. Trends Microbiol. 17:73–87.

Nadell CD, Xavier JB, Levin SA, Foster KR. 2008. The evolution of quorum

sensing in bacterial ﬁoﬁlms. PLoS Biol. 6:171–179.

Naresh K, Bharati BK, Avaji PG, Jayaraman N, Chatterji D. 2010. Synthetic

arabinomannan glycolipids and their effects on growth and motility of the

Mycobacterium smegmatis. Org Biomol Chem. 8:592–599.

Naresh K, Bharati BK, Jayaraman N, Chatterji D. 2008. Synthesis and myco-

bacterial growth inhibition activities of bivalent and monovalent arabino-

furanoside containing alkyl glycosides. Org Biomol Chem. 6:2388–2393.

Ness RK, Fletcher HG, Jr. 1958. The anomeric 2,3,5-tri-O-benzoyl-D-arabi-

nosyl bromides and other D-arabinofuranose derivatives. J Am Chem Soc.

80:2007–2010.

cell wall permeability of Mycobacterium smegmatis.

277:37567–37572.

J Biol Chem.

Escuyer VE, Lety MA, Torrelles JB, Khoo KH, Tang JB, Rithner CD, Frehel

C, McNeil MR, Brennan PJ, Chatterjee D. 2001. The role of the embA and

embB gene products in the biosynthesis of the terminal hexaarabinofurano-

syl motif of Mycobacterium smegmatis arabinogalactan. J Biol Chem.

276:48854–48862.

Etienne G, Villeneuve C, Billman-Jacobe H, Astarie-Dequeker C, Dupont

MA, Daffé M. 2002. The impact of the absence of glycopeptidolipids on

the ultrastructure, cell surface and cell wall properties, and phagocytosis of

Mycobacterium smegmatis. Microbiology. 148:3089–3100.

Faller M, Niederweis M, Schulz GE. 2004. The structure of a mycobacterial

outer-membrane channel. Science. 303:1189–1192.

Gaiha GD, Dong T, Palaniyar N, Mitchell DA, Reid KBM, Clark HW. 2008.

Surfactant protein A binds to HIV and Inhibits direct infection of CD4⁺

cells, but enhances dendritic cell-mediated viral transfer. J Immunol.

181:601–609.

Gandolﬁ-Donadío L, Gallo-Rodriguez C, de Lederkremer RM. 2008.

Synthesis of a tetrasaccharide fragment of mycobacterial arabinogalactan.

Carbohydr Res. 343:1870–1875.

Hölemann A, Stocker BL, Seeberger PH. 2006. Synthesis of a core arabino-

mannan oligosaccharide of Mycobacterium tuberculosis. J Org Chem.

71:8071–8078.

Huang B, Tomalia DA. 2006. Poly(ether) dendrons possessing phosphine

focal points for stabilization and reduced quenching of luminescent

quantum dots. Inorganica Chimica Acta. 359:1961–1966.

Nigou J, Gilleron M, Brando T, Puzo G. 2004. Structural analysis of myco-

bacterial lipoglycans. Appl Biochem Biotechnol. 118:253–267.

Piddington DL, Kashkouli A, Buchmeier NA. 2000. Growth of

Mycobacterium tuberculosis in a deﬁned medium is very restricted by acid

pH and Mg²⁺levels. Infect Immun. 68:4518–4522.

Puskas A, Greenberg EP, Kaplan S, Schaefer AL. 1997. A quorum-sensing

system in the free-living photosynthetic bacterium Rhodobacter sphaer-

oides. J Bacteriol. 179:7530–7537.

Recht J, Martínez A, Torello S, Kolter R. 2000. Genetic Analysis of Sliding

Motility in Mycobacterium smegmatis. J Bacteriol. 182:4348–4351.

Rich RL, Myszka DG. 2010. Grading the commercial optical biosensor

literature-class of 2008: ‘The mighty binders’. J Mol Recognit. 23:1–64.

Rose JD, Maddry JA, Comber RN, Suling WJ, Wilson LN, Reynolds RC. 2002.

Synthesis and biological evaluation of trehalose analogs as potential inhibi-

tors of mycobacterial cell wall biosynthesis. Carbohydr Res. 337:105–120.

Schmidt RR, Michel J. 1980. Facile synthesis of α- and β-O-glycosyl imi-

dates; preparation of glycosides and disaccharides. Angew Chem Int Ed

Engl. 19:731–732.

Shi L, Berg S, Lee A, Spencer JS, Zhang J, Vissa V, McNeil MR, Khoo KH,

Chatterjee D. 2006. The carboxy terminus of embC from Mycobacterium

smegmatis mediates chain length extension of the arabinan in lipoarabino-

mannan. J Biol Chem. 281:19512–19526.

Sidobre S, Nigou J, Puzo G, Rivière M. 2000. Lipoglycans are putative

ligands for the human pulmonary surfactant protein A attachment to myco-

bacteria. J Biol Chem. 275:2415–2422.

Sidobre S, Puzo G, Rivière M. 2002. Lipid-restricted recognition of mycobac-

terial lipoglycans by human pulmonary surfactant protein A: A surface-

plasmon-resonance study. Biochem J. 365:89–97.

Tino MJ, Wright JR. 1998. Interactions of surfactant protein A with epithelial

cells and phagocytes. Biochim Biophys Acta. 1408:241–263.

Umesiri FE, Sanki AK, Boucau J, Ronning DR, Sucheck S. 2010. Recent

advances toward the inhibition of mAG and LAM synthesis in

Mycobacterium tuberculosis. J Med Res Rev. 30:290–326.

Ishiwata A, Akao H, Ito Y. 2006. Stereoselective synthesis of a fragment of

mycobacterial arabinan. Org Lett. 8:5525–5528.

Jarlier V, Nikaido H. 1994. Mycobacterial cell wall: Structure and role in

natural resistance to antibiotics. FEMS Microbiol Lett. 123:11–18.

Jayaprakash KN, Lu J, Fraser-Reid B. 2005. Synthesis of a lipomannan com-

ponent of the cell-wall complex of mycobacterium tuberculosis is based on

Paulsen’s concept of donor/acceptor “match”. Angew Chem Int Ed.

44:5894–5898.

Joe M, Bai Y, Nacario RC, Lowary TL. 2007. Synthesis of the docosanasacchar-

ide arabinan domain of mycobacterial arabinogalactan and a proposed

octadecasaccharide biosynthetic precursor.

J Am Chem Soc. 129:

9885–9901.

Kremer L, Besra GS. 2005. A waxy tale by Mycobacterium tuberculosis. In:

Cole ST, editor. Tuberculosis and the Tubercle Bacillus. Washington, DC:

American Society for Microbiology. p. 287–305.

Lee YJ, Lee K, Jung EH, Jeon HB, Kim KS. 2005. Acceptor-dependent

stereoselective glycosylation: 2-CB glycoside-mediated direct β-D-arabino-

furanosylation and efﬁcient synthesis of the octaarabinofuranoside in

mycobacterial cell wall. Org Lett. 7:3263–3266.

Watnick PI, Kolter R. 1999. Steps in the development of a Vibrio cholerae

bioﬁlm. Mol Microbiol. 34:586–595.

Wright JR. 2005. Immunoregulatory functions of surfactant proteins. Nat Rev

Immunol. 5:58–68.

Zhang J, Khoo KH, Wu SW, Chatterjee D. 2007. Characterization of a distinct

arabinofuranosyltransferase in Mycobacterium smegmatis. J Am Chem Soc.

129:9650–9662.

Mathew R, Mukherjee R, Balachandar R, Chatterji D. 2006. Deletion of the

rpoZ gene, encoding the omega subunit of RNA polymerase, results in

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Article Doi

Synthesis, biological studies of linear and branched arabinofuranoside- containing glycolipids and their interaction with surfactant protein A

DOI: 10.1093/glycob/cwr068

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