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3.3. Optical rotation
was reduced with NaBH4 for 15 h and neutralized with acetic
acid. The resulting material was obtained by co-distillation with
methanol. The periodate-oxidized-reduced material was methyl-
ated by Ciucanu and Kerek method16 and the alditol acetates of
the methylated products were prepared. Alditol acetates were
analyzed by GLC and GLC–MS.
Optical rotation of the polysaccharide (PS-I) was measured on a
Jasco Polarimeter model P-1020 at 23.8 °C.
3.4. Colorimetric estimations23
Colorimetric estimations were carried out on a Shimadzu
UV–vis spectrophotometer, model-1601.
3.10. Absolute configuration of monosaccharides
The method used was based on Gerwig et al.15 The polysaccha-
ride (1.0 mg) was hydrolyzed with CF3COOH, and then the acid was
3.5. Paper chromatographic studies
removed. A solution of 250 lL of 0.625 (M) HCl in R-(+)-2-butanol
Paper chromatographic studies were performed on Whatmann
Nos. 1 and 3 mm sheets. Solvent systems used were (X) BuOH–
HOAc–H2O (4:1:5, upper phase) and (Y) EtOAc–pyridine–H2O
(8:2:1). The spray reagent used was alkaline silver nitrate solution.14
was added and heated at 80 °C for 16 h. The reactants were then
evaporated and TMS-derivatives were prepared with N,O-bis(tri-
methylsilyl)trifluroacetamide (BSTFA). The products were analyzed
by GLC using a capillary column SPB-1 (30 m ꢂ 0.26 mm), a tem-
perature program (3 °C/min) from 150 to 210 °C. The 2,3,4,6-tet-
ra-O-TMS-(+)-2-butylglycosides obtained were identified by
comparison with those prepared from the D and L enantiomers
of different monosaccharides.
3.6. Determination of molecular weight
The molecular weights of the polysaccharides were determined
by gel-chromatographic technique. Standard dextrans13 T-200,
T-70, and T-40 were passed through a sepharose 6B column, and
then the elution volumes were plotted against the logarithms of
their respective molecular weights. For the PS-I, the eluent was
water. But, in case of PS-II the eluent was 4% NaCl. At first, PS-II
was dissolved in 4% NaOH and then neutralized by HCl under cold
condition and freeze-dried. After freeze-drying, the freshly pre-
pared PS-II was passed through Sepharose S-6B column which
was previously saturated with 4% NaCl solution. The elution
volumes of the polysaccharides were then plotted in the same
graph and molecular weight of polysaccharides were determined.
3.11. GLC experiments
All gas liquid chromatography experiments were performed on
a Hewlett–Packard Model 5730 Å having a flame ionization detec-
tor and glass columns (1.8 m ꢂ 6 mm) packed with 3% ECNSS-M
(A) on Gas Chrom Q (100–120 mesh) and 1% OV-225 (B) on Gas
Chrom Q (100–120 mesh). All GLC analyses were performed at
170 °C.
3.12. GLC–MS experiments
3.7. Monosaccharide analysis
Gas–liquid chromatography–mass spectrometric (GLC–MS)
analysis was performed on Shimadzu GLC–MS Model QP2010 Plus
automatic system, using ZB-5MS capillary column (30 m ꢂ
0.25 mm). The program was isothermal at 150 °C; hold time
5 min, with a temperature gradient of 2 °C minꢃ1 up to a final tem-
perature of 200 °C.
The polysaccharide (3.0 mg) was hydrolyzed with 2 M CF3COOH
(2 mL) in a round-bottomed flask at 100 °C for 18 h in a boiling
water bath. The excess of acid was completely removed by co-
distillation with water. Then the hydrolyzed product was divided
into two parts. One part was examined by paper chromatography
in solvent systems X and Y. Another part was reduced with NaBH4
(9 mg), followed by acidification with dilute CH3COOH, and then
co-distilled with pure CH3OH to remove excess boric acid. The re-
duced sugars (alditols) were acetylated with 1:1 pyridine–acetic
anhydride in a boiling water bath for 1 h to give the alditol ace-
tates, which were analyzed by GLC and GLC–MS.
3.13. NMR studies
The pure polysaccharide was kept over P2O5 in vacuum for sev-
eral days, and then exchanged with deuterium25 by lyophilizing
with D2O (99.96% atom 2H, Aldrich) for four times. Using a Bruker
Avance DPX-500 spectrometer, 1H, TOCSY, DQF-COSY, NOESY,
ROESY, 13C (both 1H coupled and decoupled), DEPT-135, HMQC,
and HMBC NMR spectra of PS-I were recorded in D2O at 27 °C.
The 1H NMR spectrum of PS-I was recorded by suppressing the
HOD signal (fixed at d 4.74) using the WEFT pulse sequence.26
The 2D-DQF-COSY experiment was carried out using standard
BRUKER software at 27 °C. The TOCSY experiment was recorded
at mixing time of 150 ms, and complete assignment required sev-
eral TOCSY experiments having mixing times ranging from 60 to
300 ms. The NOESY and ROESY mixing delay was 300 ms. The 13C
NMR spectrum of the polysaccharide (PS-I) solution in D2O was re-
corded at 27 °C using acetone as internal standard, fixing the
methyl carbon signal at d 31.05. The delay time in the HMBC exper-
iment was 80 ms. The 13C and DEPT-135 NMR spectrum of the
polysaccharide (PS-II) was then carried out with Me2SO-d6 at
27 °C in a Bruker Avance DPX-500 spectrometer using acetone as
internal standard (d 31.05 ppm).
3.8. Methylation analysis
The polysaccharide (4.0 mg) was methylated using Ciucanu and
Kerek method16 using distilled DMSO and finely powdered dry
NaOH. The methylated products were isolated by partitioning be-
tween CHCl3 and H2O (5:2, v/v). The organic layer containing prod-
ucts was washed with 3 mL water for three times and dried. The
methylated products were then formylated with 90% formic acid
(1 mL) at 100 °C for 1 h, and excess formic acid was evaporated
by co-distillation with distilled water, and then reduced with so-
dium borohydride, acetylated with (1:1) acetic anhydride–pyri-
dine, and analyzed by GLC and GLC–MS using the same
temperature program indicated in GLC and GLC–MS experiments.
3.9. Periodate oxidation20,21
The polysaccharide (PS-II, 5 mg) was oxidized with 0.1 M so-
dium metaperiodate (2 mL) at 27 °C in the dark for 48 h. Excess
periodate was destroyed by adding 1,2-ethanediol, and the solu-
tion was dialyzed against distilled water. The dialyzed material
Acknowledgments
Dr. S. Roy, Director and Dr. A. K. Sen (Jr.), IICB, Kolkata are grate-
fully acknowledged for providing instrumental facilities. The