The centrifugate (250 mL) was stirred constantly and treated with EtOH (125 mL). The resulting precipitate was
separated, washed with EtOH, dehydrated by acetone, and dried in vacuo over P O to afford fraction 1 (0.43 g). Then, the
2
5
supernatant solution was treated with another portion of EtOH (125 mL). The resulting precipitate was worked up analogously.
Yield of fraction 2, 1.32 g. Fractions 3 (AG) and 4 were obtained by adding EtOH (250 mL) to the supernatant solutions. The
yields of fractions 3 and 4 were 2.0 and 0.73 g, respectively. The aqueous EtOH solution was evaporated to a syrup and
precipitated by EtOH. Yield of fraction 5, 3.68 g.
Gel-filtration of AG. AG (0.01 g) was dissolved in NaCl solution (1 mL, 0.3%), placed on a column of Sephadex
G-100 (2 ꢃ 55 cm), and eluted by the same solution. Fractions (3 mL) were collected and analyzed by the literature
method [5]. The column was calibrated by passing dextrans of molecular weights 110,000 (V = 21.8 mL), 80,000
1
(V = 24.6 mL), and 40,000 (V = 32.8 mL). The molecular weight (V = 28.6 mL) of 56,000 for C. repensAG was calculated
2
3
4
from a calibration curve.
Periodate Oxidation and Smith Degradation. AG (0.0322 g) was dissolved in H O (24.9 mL), treated with NaIO
2
4
(5.1 mL, 0.25 M), left for 24 h at 20°C, and then stored at 5°C. Aliquots (1 mL) were taken every day and titrated with sodium
thiosulfate solution (0.01 N). The consumption of NaIO after 26 d was 2.7 moles. Its value did not change further. The
4
formic acid that was released during the reaction was titrated with NaOH solution (0.01 N). Its amount was calculated as
0.33 moles for AG. Oxidant in the reaction mixture was destroyed by adding ethyleneglycol (0.1 mL). The solution was
+
dialyzed, treated with NaBH (0.1 g), and left overnight. The solution was treated with KU-2 cation-exchanger (H ) and
4
filtered. The filtrate was evaporated with MeOH. The dry solid was hydrolyzed in HCl (3 mL, 0.5 N) at 85°C for 4 h. PC of
the hydrolysis products (system 3, detectors 2 and 3) and GC (conditions a and b) detected mainly glycerin and arabinose.
Methylation of AG. AG (0.1 g) was methylated by the literature method [3]. The completeness of the methylation
–1
was checked by TLC (system 1, detector 1) and by IR spectra (OH absorption band at 3200–3600 cm missing). The yield of
permethylate was 0.08 g, [ꢀ] –24.4° (c 0.5%, acetone), OCH 40.8%.
D
3
AG permethylate (0.05 g) was refluxed in formic acid (1 mL, 85%) for 1.5 h, cooled, and evaporated. The solid was
dissolved in H SO solution (2 mL, 0.5 N) and hydrolyzed for 6 h at 100°C. The hydrolyzate was worked up as usual. Methyl
2
4
derivatives were separated over a column (L = 50 cm, d = 2 cm) of silica gel (L 100/250) with elution by CHCl :MeOH (9:1).
3
Aliquots (5 mL) were collected and checked by TLC using system 2. Pure 2,3,4,6-tetra-O-Me-D-Gal; 2,3,4-tri-O-Me-D-Gal;
2,3,5-tri-O-Me-L-Ara; and two compounds (di-O-Me-derivatives) with R values 1.0, 0.9, 0.55, 0.05, and 0.03, respectively,
f
were obtained.
Demethylation of Di-O-Me-derivatives of AG. The di-O-Me-derivatives (0.005 and 0.003 g) were dissolved
separately in HBr solution (2 mL, 45%, in a 2-mL ampul) and hydrolyzed for 1.5 h. The hydrolyzates were evaporated to
dryness and distilled with MeOH. PC (system 3, detector 2) identified arabinose and galactose.
Acetylation of AG. AG (0.1 g) was acetylated and oxidized by CrO as before [4]. PC (system 3, detector 3)
3
detected arabinose in the final products.
Partial Acid Hydrolysis of AG. AG (0.2 g) was dissolved in CF COOH solution (20 mL, 0.5 M) and hydrolyzed for
3
2.5 h at 100°C. The hydrolyzate was neutralized with anion-exchanger, condensed, and studied by PC (system 3, detector 2).
Arabinose, galactose, and three oligosaccharides with R
0.54, 0.32, and 0.1 were detected.
fGal
Total oligosaccharides were loaded onto a column of DEAE-cellulose-Sephadex A-25 (formate form) and eluted
sequentially by HCOOH solutions (0.1 M, 0.4, 0.6, and 0.8). The yields of oligosaccharides were monitored using phenol:H SO
2
4
[5]. The resulting oligosaccharides were rechromatographed to afford pure oligosaccharides in yields of 0.026 g (O-1),
0.053 g (O-2), and 0.0126 g (O-3). PC (system 3, detector 2) identified galactosylarabinose (O-1), galactotriose (O-2), and
galactotetraose (O-3) with [ꢀ] +17.8° (c 0.1%, H O) for O-1, lit. [6] [ꢀ] +7° (c 0.2%, H O); [ꢀ] +42.3° (c 0.25%, H O) for
D
2
D
2
D
2
O-2, lit. [7] [ꢀ] +48° (c 0.1%, H O); and [ꢀ] +34.2° (c 0.23%, H O) for O-3, lit. [6] [ꢀ] +28.2° (c 0.25%, H O).
D
2
D
2
D
2
Hydrolysis of C. repens Oligosaccharides. Oligosaccharides O-1, O-2, and O-3 (0.005 g each) were hydrolyzed by
HCl solution (0.5 N) at 100°C for 4 h and worked up appropriately. PC (system 3, detector 2) detected in the hydrolyzate of
O-1 galactose and arabinose; of O-2 and O-3, only galactose.
Periodate Oxidation of Oligosaccharides. Oligosaccharides O-1, O-2, and O-3 (0.03 g each) were oxidized by
sodium periodate. PC (system 3, detectors 2 and 3) of the hydrolyzate of the reduced product and GC (condition b) detected
glycerin and erythrite in ratios of 1:1, 1:2, and 1:3, respectively.
Methylation of Oligosaccharides. Oligosaccharides O-1, O-2, and O-3 (0.01 g each) were methylated according to
Hakomori [3].
730
Rakhmanberdyeva
