Synthesis of a pentasaccharide repeating unit of the extracellular polysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus 291

Article abstract of DOI:10.1080/07328300701410676

A pentasaccharide methyl glycoside has been synthesized efficiently using a modified glycosylation strategy. This pentasaccharide is a repeating unit of the exopolysaccharides produced by Lactobacillus delbrueckii subsp. bulgaricus 291.

Full text of DOI:10.1080/07328300701410676

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Synthesis of a Pentasaccharide Repeating
Unit of the Extracellular Polysaccharide
Produced by Lactobacillus Delbrueckii Subsp.
Bulgaricus 291
Pallavi Tiwari a & Anup Kumar Misra a
a Medicinal and Process Chemistry Division , Central Drug Research
Institute , Lucknow, India
Published online: 21 Jun 2007.
To cite this article: Pallavi Tiwari & Anup Kumar Misra (2007) Synthesis of a Pentasaccharide Repeating Unit
of the Extracellular Polysaccharide Produced by Lactobacillus Delbrueckii Subsp. Bulgaricus 291 , Journal of
Carbohydrate Chemistry, 26:4, 239-248, DOI: 10.1080/07328300701410676
To link to this article: http://dx.doi.org/10.1080/07328300701410676
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Journal of Carbohydrate Chemistry, 26:239–248, 2007
Copyright # Taylor & Francis Group, LLC
ISSN: 0732-8303 print 1532-2327 online
DOI: 10.1080/07328300701410676
Synthesis of a
Pentasaccharide Repeating
Unit of the Extracellular
Polysaccharide Produced
by Lactobacillus
Delbrueckii Subsp.
Bulgaricus 291
Pallavi Tiwari and Anup Kumar Misra
Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow,
India
A pentasaccharide methyl glycoside has been synthesized efficiently using a modified
glycosylation strategy. This pentasaccharide is a repeating unit of the exopolysacchar-
ides produced by Lactobacillus delbrueckii subsp. bulgaricus 291.
Keywords Pentasaccharide, Glycosylation, Exopolysaccharide, Lactobacillus del-
brueckii subsp. bulgaricus 291
INTRODUCTION
Lactic acid bacteria (LAB) are the organisms that beneficially affect the host
animal by improving the intestinal microbial balance[1] by producing an
abundant variety of exopolysaccharides (EPSs), which provide an important con-
tribution to human health by acting as prebiotic substrates. EPSs produced by
LAB have several medicinal values, possessing antitumor,[2] antimutagenic,[3]
antiulcer,[4] and antibacterial activities.[5] In addition, LAB have been shown
to be effective as immunostimulators[6] and blood cholesterol-lowering
Received July 31, 2006; accepted October 2, 2006.
C.D.R.I. communication no. 7019.
Address correspondence to Anup Kumar Misra, Medicinal and Process Chemistry
Division, Central Drug Research Institute, Chattar Manzil Palace, Lucknow, 226001
UP, India. E-mail: akmisra69@rediffmail.com
239

P. Tiwari and A. K. Misra
240
agents.[7] It has been found that the above-mentioned medicinal events arise
from the whole microorganisms or cell wall components or extracellular polysac-
charides.[8] Exopolysaccharides produced by lactic acid bacteria are also used to
improve the body and texture of dairy products.[9] The positive role of dairy lac-
tobacilli in human health was suggested nearly a century ego. Various brands of
yogurt are consumed as prophylactics or for treatment of common intestinal
infections, such as diarrhea.[10] Among several LAB, a combination of Streptococ-
cus thermophilus and Lactobacillus delbrueckii subsp. bulgaricus, which grow
synergistically, are widely used as starter cultures for the industrial production
of yogurt.[11] In another aspect, exopolysaccharides secreted by LAB play a sig-
nificant role in the protection of microbial cells against phagocytosis, phage
attacks, antibiotics, toxic compounds (e.g., toxic metal ions, sulfur dioxide,
andethanol), osmotic stress, and bacteriocins such as nisin.[12]
Recently, the structure of the extracellular polysaccharide produced by Lac-
tobacillus delbrueckii subsp. bulgaricus 291 has been reported by Faber
et al.,[13] which is a pentasaccharide composed of D-galactose and D-glucose.
In view of the importance of the exopolysaccharides for their biological use
and limited natural availability, it is essential to synthesize them chemically
in a concise manner. As a part of our ongoing program toward the synthesis
of bacterial oligosaccharides, we report herein a chemical synthesis of the extra-
cellular pentasaccharide as its methyl glycoside produced by Lactobacillus del-
brueckii subsp. bulgaricus 291 using a robust glycosylation protocol (Figure 1).
RESULTS AND DISCUSSIONS
The synthesis of pentasaccharide 7 is shown in Scheme 1 and began with
methyl a-D-glucopyranoside (1). Methyl 2,3-di-O-benzyl-4,6-O-benzylidene-a-
D-glucopyranoside (2) was prepared from methyl a-D-glucopyranoside (1) fol-
lowing a two-step reaction sequence involving the treatment with benzal-
dehyde dimethylacetal in the presence of p-toluene sulfonic acid followed by
benzylation using benzyl bromide and sodium hydroxide in 85% overall yield.
[14]
Treatment of compound 2 with HClO4-SiO2
in acetonitrile at rt furnished
methyl 2,3-di-O-benzyl-a-D-glucopyranoside (3) in 95% yield. N-Iodosuccini-
mide (NIS)-HClO4-SiO2 promoted[15] condensation of compound 3 with ethyl
2,3,6,20,30,40,60-hepta-O-acetyl-1-thio-b-D-lactopyranoside (5)[16] prepared
Figure 1: Structure of the extracellular pentasaccharide produced by Lactobacillus
delbrueckii subsp. bulgaricus 291.

Synthesis of Lactobacillus Repeating Unit
241
Scheme 1: Reagents: (a) PhCH(OCH3)2, p-TsOH, CH3CN, rt, 5 h; (b) BnBr, 50% aq. NaOH,
n-Bu4NBr, CH2Cl2, rt, 5 h, 85% in two steps; (c) HClO4-SiO2, CH3CN, rt, 20 min, 95%; (d) C2H5SH,
.
BF3 OEt2, CH2Cl2, 08C, 12 h, 80%; (e) NIS, HClO4-SiO2, CH2Cl2, 08C, 2 h, 78%; (f) CH3ONa, CH3OH,
rt, 5 h; (g) H2, 20% Pd(OH)2-C, CH3OH, rt, 12 h, 72% in two steps.
from D-lactoseoctaacetate and gave methyl [2,3,4,6-tetra-O-acetyl-b-D-galacto-
pyranosyl-(1 ! 4)-2,3,6-tri-O-acetyl-b-D-glucopyranosyl)-(1 ! 6)]-2,3,4,6-
tetra-O-acetyl-b-D-galactopyranosyl-(1 ! 4)-2,3,6-tri-O-acetyl-b-D-glucopyr-
ano-syl-(1 ! 4)-2,3-di-O-benzyl-a-D-glucopyranoside (6) in 78% yield. This gly-
cosylation methodology has been applied in a 10 millimolar scale to achieve
large quantities of pentasaccharide (6). Furthermore, this protocol is equally
effective, as NIS-trifluoromethanesulfonic acid (TfOH) promoted glycosylation,
which has been extensively used for the activation of thioglycosides. The
advantages of using HClO4-SiO2 in place of TfOH include no requirement of
controlled low temperature, in some cases the catalyst system can be recovered
and reused, the low cost of the catalyst, and stability toward the moisture.
Zemple0n transesterification of pentasaccharide derivative 6 with sodium
methoxide followed by hydrogenolysis with H2/Pd(OH)2-C furnished target
pentasaccharide methyl [b-D-galactopyranosyl-(1 ! 4)-b-D-glucopyranosyl)-
(1 ! 6)]-b-D-galactopyranosyl-(1 ! 4)-b-D-glucopyranosyl-(1 ! 4)-a-D-gluco-
pyranoside (7) in 72% yield (Sch. 1). The structure of the pentasaccharide (7) was
confirmed from its NMR and mass spectra. The presence of five anomeric signals
in the 1H NMR spectrum [d 4.75 (d, J ¼ 3.6 Hz, 1H, H-1), 6.52 (d, J ¼ 7.8 Hz, 1H,
H-10), 4.47 (d, J ¼ 8.1 Hz, 1H, H-1000), 4.39 (d, J ¼ 7.8 Hz, 1H, H-100), 4.34
(d, J ¼ 7.8 Hz, 1H, H-10000)] and 13C NMR spectrum [d 103.0 (C-100), 102.9 (C-10000),
102.1 (C-1000), 101.4 (C-10), 99.2 (C-1)] confirmed the formation of the required
pentasaccharide (7) (Figs. 2 and 3).
In summary, synthesis of a pentasaccharide as its methyl glycoside
produced by Lactobacillus delbrueckii subsp. bulgaricus 291 has been success-
fully achieved in a concise manner. Although methyl glycoside is not always

P. Tiwari and A. K. Misra
242
Figure 2: (A) 1H NMR spectrum of compound 7. (B) Magnified form of anomeric region of
compound 7. (C) 2D-COSY spectrum of compound 7.
suitable for biological studies, in the synthetic scheme it can be replaced
by other functional groups, such as 4-methoxyphenyl or 2-trimethylsilylethyl
group, which can be removed after formation of the pentasaccharide to attach
it with a spacer. The glycosylation protocol is considerably robust to be used for
a scale-up reaction.
EXPERIMENTAL
General Procedure
All the reactions were monitored by thin-layer chromatography over silica
gel-coated TLC plates. The spots on TLC were visualized by warming ceric
sulphate (2% Ce(SO4)2 in 2 N H2SO4)-sprayed plates in hot plate. Silica gel

Synthesis of Lactobacillus Repeating Unit
243
Figure 3: (A) 13C NMR spectrum of compound 7. (B) DEPT 135 spectra of compound 7.
1
230–400 mesh was used for column chromatography. H and 13C NMR was
recorded on a Brucker Advance DPX 300 MHz using CDCl3 and D2O as
solvents and TMS as internal reference and 1,4-dioxane as external reference.
Chemical shift value is expressed in (ppm. ESI-MS were recorded on a MICRO-
MASS QUTTRO II triple quadrupole mass spectrometer. Elementary analysis
was carried out on Carlo ERBA-1108 analyzer. Optical rotations were
measured at 258C on a Rudolf Autopol III polarimeter. Commercially available
grades of organic solvents of adequate purity are used in many reactions.
Preparation of HClO4-SiO2
HClO4 (1.8 g, 12.5 mmol, as a 70% aq solution) was added to a suspension of
SiO2 (230–400 mesh, 23.7 g) in Et2O (70.0 mL). The mixture was concentrated

P. Tiwari and A. K. Misra
244
and the residue was heated at 1008C for 72 h under vacuum to furnish HClO4-
SiO2 (0.5 mmol/g) as a free flowing powder.
Methyl 2,3-di-O-benzyl-4,6-O-benzylidene-a-D-
glucopyranoside (2)
To a solution of methyl a-D-glucopyranoside (1; 5 g, 25.7 mmol) in anhy-
drous CH3CN (20 mL) were added benzaldehyde dimethylacetal (5.85 mL,
39.0 mmol) and p-toluene sulfonic acid (100 mg) and the reaction mixture
was stirred at rt for 5 h. The reaction was quenched with triethyl amine and
evaporated to dryness. To a solution of the crude mass in CH2Cl2 (100 mL)
were added 50% aq. NaOH solution (50 mL) followed by benzyl bromide
(9.0 mL, 75.8 mmol) and tetrabutylammonium bromide (100 mg) and the
reaction mixture was stirred vigorously at rt for 5 h. The reaction mixture
was diluted with water (100 mL) and extracted with CH2Cl2 (150 mL). The
organic layer was washed with water, dried (Na2SO4), and concentrated
under reduced pressure. The crude product was purified over SiO2 using
hexane-EtOAc (7:1) as eluant to furnish pure compound 2 (10 g, 85%) as a
white solid, [a]D þ21.9 (c 1.0, CHCl3); IR (KBr): 2926, 2368, 1595, 1368,
1
1087, 1052, 739, 693 cm21; H NMR (CDCl3, 300 MHz): d 7.44–7.23 (m, 15H,
aromatic proton), 5.49 (s, 1H, PhCH), 4.86 (d, J ¼ 11.5 Hz, 1H), 4.81–4.76
(m, 2H), 4.65 (d, J ¼ 12.1 Hz, 1H), 4.53 (d, J ¼ 3.3 Hz, 1H, H-1), 4.23 (dd,
J ¼ 9.3 and 3.8 Hz, 1H), 3.99 (t, J ¼ 9.1 and 9.1 Hz, 1H), 3.84–3.69 (m, 2H),
3.64–3.46 (m, 2H), 3.37 (s, 3H, OCH3); 13C NMR (CDCl3, 75 MHz): d 139.2,
138.7, 137.9, 129.2–126.5 (aromatic carbon), 101.7, 99.6, 82.7, 79.7, 78.9,
75.6, 74.0, 69.4, 62.8, 55.7; ESI-MS (462): m/z 485 [M þ Na]; Anal. Calcd. for
C28H30O6: C, 72.71; H, 6.54; found: C, 72.55; H, 6.75.
Methyl 2,3-di-O-benzyl-a-D-glucopyranoside (3)
To a solution of compound 2 (1.5 g, 3.25 mmol) in CH3CN (15 mL) was
added HClO4-SiO2 (250 mg) and the reaction mixture was stirred at rt for
20 min. The reaction mixture was filtered through a celite bed and
evaporated to dryness to give the crude product. Column chromatography of
the crude product over a short pad of silica gel using hexane-EtOAc (1:1) furn-
ished pure compound 3 (1.15 g, 95%); [a]D þ17.4 (c 1.0, CHCl3); IR (neat): 2924,
1
1719, 1454, 1363, 1198, 1054, 743, 700 cm21; H NMR (CDCl3, 300 MHz): d
7.30–7.25 (m, 10H, aromatic proton), 4.95 (d, J ¼ 11.5 Hz, 1H), 4.75
(d, J ¼ 11.5 Hz, 1H), 4.69 (d, J ¼ 11.5 Hz, 1H), 4.58 (d, J ¼ 11.5 Hz, 1H), 4.54
(d, J ¼ 3.5 Hz, 1H, H-1), 3.77–3.60 (m, 3H), 3.56–3.51 (m, 1H), 3.47–3.38
(m, 2H), 3.34 (s, 3H); 13C NMR (CDCl3, 75 MHz): d 139.3, 138.6, 128.7–127.9
(aromatic carbon), 98.5, 81.8, 80.2, 76.3, 73.3, 71.6, 70.3, 62.0, 55.5; ESI-MS

Synthesis of Lactobacillus Repeating Unit
245
(374): m/z 397 [M þ Na]; Anal. Calcd. for C21H26O6: C, 67.36; H, 7.0; found: C,
67.10; H, 7.28.
Ethyl 2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-(1 ! 4)-
2,3,6-tri-O-acetyl-1-thio-b-D-glucopyranoside (5)
To a solution of b-D-lactose octaacetate (5.0 g, 7.37 mmol) in dry CH2Cl2
(20 mL), ethanethiol (1.6 mL, 21.5 mmol) was added under inert atmosphere.
The reaction mixture was cooled to 08C and borontrifluoride diethyletherate
(1.85 mL, 14.74 mmol) was added to it and the reaction mixture was stirred
at 08C for 5 h. The progress of the reaction was monitored by thin-layer
chromatography over silica gel-coated plates. After completion of the
reaction, the reaction mixture was diluted with CH2Cl2 and washed with
aq. sodium bicarbonate solution and water in succession. The organic layer
was dried over anhydrous Na2SO4 and concentrated to dryness under
reduced pressure. The crude reaction mixture was purified over SiO2 using
hexane-EtOAc as eluant to furnish desired ethyl thioglycosides 5 (4.0 g,
80%); m.p.: 728C; [a]D 26 (c 1.0; CHCl3); IR (neat): 2930, 1750, 1372, 1225,
1
1051, 769 cm21; H NMR (CDCl3, 300 MHz): d 5.23 (d, J ¼ 1.2 Hz, 1H), 5.10
(t, J ¼ 9.0 and 9.3 Hz, 1H), 5.09–4.94 (m, 1H), 4.89 (dd, J ¼ 10.5 and
3.3 Hz, 1H), 4.82 (t, J ¼ 9.6 and 9.6 Hz, 1H), 4.48 (d, J ¼ 7.5 Hz, 1H), 4.43
(d, J ¼ 9.9 Hz, 1H), 4.39 (dd, J ¼ 10.4 and 2.0 Hz, 1H), 4.07–3.99 (m, 3H),
3.85 (t, J ¼ 6.6 and 6.9 Hz, 1H), 3.71 (t, J ¼ 9.0 and 9.6 Hz, 1H), 3.58–3.54
(m, 1H), 2.66–2.51 (m, 2H), 2.09, 2.04, 2.0 (3 s, 9H, 3 COCH3), 1.99 (s, 3H,
COCH3), 1.97 (s, 6H, 2 COCH3), 1.96, 1.89 (2 s, 6H, 2 COCH3), 1.23–1.17
(m, 3H); 13C NMR (CDCl3, 75 MHz): d 170.1 (3 C), 169.8, 169.6, 169.5,
168.9, 101.3, 83.4, 77.0, 76.7, 74.2, 71.2, 70.8, 70.5, 69.4, 66.9, 62.7, 61.0,
24.3, 21.0 (2 C), 20.9 (2 C), 20.8 (2 C), 20.7, 15.2; ESI-MS (680): m/z 703.4
[M þ Na]; Anal. Calcd. for C28H40O17S: C, 49.41; H, 5.92; found: C, 49.12;
H, 6.20.
Methyl [2,3,4,6-tetra-O-acetyl-b-D-galactopyranosyl-
(1 ! 4)-2,3,6-tri-O-acetyl-b-D-glucopyranosyl)-(1 ! 6)]-
2,3,4,6-tetra-O-acetyl-b-D-galacto-pyranosyl-(1 ! 4)-
2,3,6-tri-O-acetyl-b-D-glucopyranosyl-(1 ! 4)-2,3-di-O-
benzyl-a-D-glucopyranoside (6)
To a solution of compound 3 (600 mg, 1.60 mmol) and thioglycoside donor 5
˚
(2.8 g, 4.12 mmol) in anhydrous CH2Cl2 (20 mL) was added powdered MS-4A
(4 g) and the reaction mixture was stirred at rt under argon for 1 h. After
cooling the reaction mixture to 08C, N-iodosuccinimide (1.2 g, 5.33 mmol) was
added to it followed by HClO4-SiO2 (150 mg) and it was allowed to stir at 08C

P. Tiwari and A. K. Misra
246
for 2 h. The reaction mixture was quenched by adding 5% aq. Na2S2O3, diluted
with CH2Cl2 and filtered through a celite bed. The organic layer was washed
successively with aq. NaHCO3 and water, dried (Na2SO4), and concentrated
under reduced pressure. The crude product was purified over SiO2 using
hexane-EtOAc (2:1) to afford pure pentasaccharide 6 (2.0 g, 78%); [a]D þ 5.9
1
(c 1.0, CHCl3); IR (neat): 2942, 1752, 1595, 1377, 1232, 1055, 601 cm21; H
NMR (CDCl3, 300 MHz): d 7.35–7.34 (m, 4H, aromatic protons), 7.28–7.26
(m, 6H, aromatic protons), 5.37–5.35 (m, 2H), 5.21 (t, J ¼ 7.8 Hz, 1H), 5.12–
5.07 (m, 3H), 5.00–4.99 (d, J ¼ 3.3 Hz, 1H), 4.94–4.92 (m, 4H), 4.89–4.87
(m, 1H), 4.70–4.67 (m, 2H), 4.60 (bs, 1H), 4.57–4.50 (m, 4H), 4.41
(d, J ¼ 7.8 Hz, 1H), 4.19 (d, J ¼ 12 Hz, 1H), 4.15–4.09 (m, 7H), 3.95–3.92
(m, 1H), 3.90–3.86 (m, 3H), 3.83–3.78 (m, 4H), 3.75–3.65 (m, 2H), 3.41–3.39
(m, 1H), 3.36 (s, 3H, OCH3), 2.18, 2.17, 2.14, 2.08 (4 s, 12H, 4 COCH3), 2.07
(s, 6H, 2 COCH3), 2.06, 2.05, 2.04, 2.03, 2.01, 2.00, 1.99, 1.98 (8 s, 24H, 8
COCH3); 13C NMR (CDCl3, 75 MHz): d 170.3 (3 C), 170.2, 170.1 (2 C), 170.0
(2 C), 169.7 (2 C), 169.5 (2 C), 169.0, 168.9, 139.2, 137.8, 128.3 (2 C), 128.2 (2
C), 128.0 (2 C), 127.9, 127.2, 126.6 (2 C), 101.0 (2C), 100.8, 99.8, 97.6, 79.4,
78.9, 78.1, 76.0, 75.7, 74.3, 73.1, 72.8, 72.7, 72.6, 72.4, 72.2, 71.6, 70.9 (2C),
70.5 (2C), 69.5, 69.0, 68.9, 68.3, 66.5 (2 C), 61.9, 61.7, 60.7 (2 C), 55.1, 20.8 (3
C), 20.6 (6 C), 20.5 (5 C); ESI-MS (1610): m/z 1633.4 [M þ Na]; Anal. Calcd.
for C73H94O40: C, 54.41; H, 5.88; found: C, 54.10; H, 6.14.
Methyl [b-D-galactopyranosyl-(1 ! 4)-b-D-glucopyranosyl)-
(1 ! 6)]-b-D-galactopyranosyl-(1 ! 4)-b-D-
glucopyranosyl-(1 ! 4)-a-D-glucopyranoside (7)
To a solution of pentasaccharide derivative 6 (500 mg, 0.31 mmol) in
CH3OH (10 mL), solid CH3ONa was added until the pH was ꢀ10. The
reaction mixture was allowed to stir at rt for 5 h followed by neutralization
with Amberlite IR-120 (Hþ) cation exchange resin. The reaction mixture was
filtered and evaporated to dryness. To a solution of the crude product in
CH3OH (5 mL) was added 20% Pd(OH)2-C (100 mg) and the reaction mixture
was stirred at rt under a positive pressure of hydrogen for 12 h. The reaction
mixture was filtered through a celite bed and concentrated to a white
powder, which was further purified through a Sephadex LH-20 using
CH3OH-H2O (4:1) as eluent to furnish pure pentasaccharide 7 as an amor-
phous powder (190 mg, 72%); [a]D þ10.5 (c 1.0, H2O); 1H NMR (CDCl3,
300 MHz): d 4.75 (d, J ¼ 3.6 Hz, 1H, H-1), 4.52 (d, J ¼ 7.8 Hz, 1H, H-10), 4.47
(d, J ¼ 8.1 Hz, 1H, H-1000), 4.39 (d, J ¼ 7.8 Hz, 1H, H-100), 4.34 (d, J ¼ 7.8 Hz,
1H, H-10000), 4.17 (d, J ¼ 11.1 Hz, 1H, H-6a), 3.96–3.95 (m, 1H), 3.93–3.90
(m, 1H), 3.87–3.84 (m, 3H), 3.81–3.66 (m, 11H), 3.64–3.55 (m, 6H),
3.51–3.45 (m, 3H), 3.37 (s, 3H, OCH3), 3.32–3.27 (m, 2H); 13C NMR (CDCl3,
75 MHz, 1,4-dioxan as external standard): d 103.0 (C-100), 102.9 (C-100000000),

Synthesis of Lactobacillus Repeating Unit
247
102.1 (C-1000), 101.4 (C-10), 99.2 (C-1), 79.9, 78.8, 78.4, 75.4, 75.3, 74.9 (2C), 74.6,
74.3, 74.2, 73.3, 72.6, 72.5 (2C), 70.9 (3C), 69.5, 68.5 (2C), 66.8, 61.0 (2C), 60.5,
60.0, 55.2; ESI-MS (842): m/z 865.3 [M þ Na]; Anal. Calcd. for C31H54O26: C,
44.18; H, 6.46; found: C, 43.90; H, 6.70.
ACKNOWLEDGEMENTS
Instrumentation facilities from SAIF, CDRI is gratefully acknowledged. P.T.
thanks CSIR, New Delhi, for providing a Senior Research fellowship. This
project was funded by the Department of Science and Technology (DST),
New Delhi (SR/FTP/CSA-10/2002), India.
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