Structure of the repeat unit of the Alteromonas addita type strain KMM 3600T O-specific polysaccharide

Article abstract of DOI:10.1007/s10600-008-9149-1

The O-specific polysaccharide of Alteromonas addita type strain KMM 3600T is constructed of trisaccharide repeat units containing L-rhamnose, D-glucose, and D-galactose. It was established that the O-specific polysaccharide consists of trisaccharide repeat units with the structure →3)-α-D-Galp- (1→3)-α-L-Rhap-(1→3)-α-D-Glcp-(1→ based on monosaccharide analysis, Smith degradation, PMR and ¹³C NMR spectroscopy, and two-dimensional COSY, HSQC, and HMBC. Springer Science+Business Media, Inc. 2008.

Full text of DOI:10.1007/s10600-008-9149-1

Chemistry of Natural Compounds, Vol. 44, No. 5, 2008
STRUCTURE OF THE REPEAT UNIT OF THE Alteromonas addita
T
TYPE STRAIN KMM 3600 O-SPECIFIC POLYSACCHARIDE
R. P. Gorshkova, V. V. Isakov, V. A. Denisenko,
E. L. Nazarenko, E. P. Ivanova, and L. S. Shevchenko
UDC 577.114
*
T
O
Alteromonas addita
type strain KMM 3600 is constructed of trisaccharide
The -specific polysaccharide of
O
repeat units containing L-rhamnose, D-glucose, and D-galactose. It was established that the -specific
→
α
→
α
polysaccharide consists of trisaccharide repeat units with the structure 3)- -D-Galp-(1 3)- -L-Rhap-
13
→
α
→
(1 3)- -D-Glcp-(1
based on monosaccharide analysis, Smith degradation, PMR and C NMR
spectroscopy, and two-dimensional COSY, HSQC, and HMBC.
Key words: Alteromonas addita, O-specific polysaccharide, NMR spectroscopy.
The genus
has been described as an independent taxon with gram-negative marine aerobic gamma-
Alteromonas
proteobacteria with one polar flagellum [1]. This genus at present includes 14 validly described species. A strain assigned
according to phenotypic, chemotaxonomic, genetic, and phylogenetic features to the new species was isolated during
A. addita
taxonomic research of wild microbes from populations in Chasma Bay (Sea of Japan) [2, 3]. We have previously determined
T
the structure of a lipooligosaccharide from
polysaccharide from
strain KMM 3600 [4]. Herein a structural investigation of the -specific
A. addita
of this same strain is reported.
O
A. addita
A lipopolysaccharide (LPS) was isolated from wet bacterial cells of the marine proteobacterium
byextraction
A. addita
with hot aqueous phenol and was purified of nucleic acids by precipitation with TCA at pH 2. Mild acid degradation of the LPS
with subsequent anion-exchange and exclusion chromatography produced the -specific polysaccharide (PS). Carbohydrate
O
analysis of the PS using PC, GC, and GC—MS as the polyol acetates showed that the PS contained rhamnose, glucose, and
galactose residues in a 1:1:1 ratio. All components of the PS were isolated pure by preparative PC. Glucose and galactose were
assigned based on the specific optical rotations to the D-series; rhamnose, to the L-series.
13
The C NMR spectrum (Fig. 1, Table 1) exhibited three resonances for anomeric C atoms at 103.1, 96.5, and
96.4 ppm; resonances of two hydroxymethyls at 62.2 and 62.1 ppm; a resonance for CH of a 6-deoxysugar at 18.0 ppm; and
3
12 resonances of C ring atoms at 66-79 ppm. The spin—spin coupling constants (SSCC) for the anomeric C atoms that were
13
determined from the C NMR spectrum were recorded without suppression of C—H coupling (gated decoupling experiment)
were J
= 170-171 Hz, which was consistent with the α-configuration of all monosaccharide residues [5]. The lack of
C1,H1
13
resonances for non-anomeric C atoms at weak (>83 ppm) field in the C NMR spectrum indicated that all monosaccharide
residues had the pyranose form [6].
13
Smith degradation produced a polymer, the C NMR spectrum of which was identical to that of the starting PS, which
indicated that α-glycols were absent in the native PS. Therefore, all monosaccharide residues were α-1,3-bonded and the PS
had a linear structure.
The PMR spectrum of the native PS contained resonances for three anomeric protons at 5.19 ppm (2H,
J
= 3.1 Hz) and 5.10 ppm (J
= 1.2 Hz) in addition to a resonance for a CH –C group (H-6 of a 6-deoxysugar) at
H1-H2
H1-H2
3
13
1.31 ppm (J = 6.2 Hz). The PMR and C NMR spectra confirmed the PS structure as two hexose residues and one
H6
6-deoxyhexose residue in the pyranose form bonded α-1,3 through glycoside bonds. This correlated well with the PS
carbohydrate analysis.
Pacific Institute of Bioorganic Chemistry, Far-East Branch, Russian Academy of Sciences, 690022, Vladivostok, pr.
100-Letiya Vladivostoka, 159, fax (4232) 31 40 50, e-mail: elnaz@piboc.dvo.ru. Translated from Khimiya Prirodnykh
Soedinenii, No. 5, pp. 445-447, September-October, 2008. Original article submitted April 21, 2008.
0009-3130/08/4405-0549 ©2008 Springer Science+Business Media, Inc.
549

TABLE 1. NMR Spectra of O-Specific Polysaccharide from Alteromonas addita Strain 3600T (δ, ppm)
Monosaccharide residue
C1/H1
C2/H2
C3/H3
C4/H4
C5/H5
C6/H6
→ 3)-α-L-Rhap-(1 →
103.1
5.10
96.4
5.19
96.5
5.19
68.0
4.30
68.8
3.98
67.8
4.04
76.3
3.96
78.4
4.04
75.4
4.08
71.6
3.58
70.4
3.77
66.7
4.28
70.4
3.86
72.2
4.05
71.7
4.03
18.0
1.31
61.8
3.78
62.4
3.69
→3)-α-D-Glcp-(1 →
→ 3)-α-D-Galp-(1 →
1 00
9 0
8 0
70
6 0
5 0
4 0
3 0
2 0 ppm
13
Fig. 1. C NMR spectrum of O-specific polysaccharide
T
from Alteromonas addita strain 3600 (δ, ppm).
The two-dimensional (2D) COSY, HSQC, and HMBC experiments allowedresonancesofCandHatomstobe assigned
to the corresponding monosaccharide residues (Table 1). According to them, the C-3 resonances of all three monosaccharides
at 76.3, 78.4, and 75.4 ppm were shifted to relatively weak field at 71.3, 74.0, and 70.1 ppm compared with their positions in
the free monosaccharides (α-effect of glycosylation). This also was consistent with the presence of 1,3-glycosidic bonds.
Correlations were observed in the HMBC spectrum because of through-space inter-residue SSCC: H1 (δ 5.10) Rha/C3
(δ 78.4) Glc and H1 (δ 5.19) Glc/C3 (δ 75.4) Gal. This indicated that all monosaccharide residues were glycosylated in the
3-position with thefollowingbondingsequenceofmonosaccharideresiduesin thePS:→3)-α-D-Galp-(1→3)-α-L-Rhap-(1→3)-
α-D-Glcp-(1→.
The chemical shifts of the rhamnopyranose residue showed that the C-3 resonance at 76.3 ppm was shifted to weak
field by +5.3 ppm (α-effect of glycosylation) whereas the C-2 resonance; to strong field by -4 ppm (β-effect of glycosylation)
relative to their values in the unsubstituted rhamnose residue. Such values of the α- and β-effects of glycosylation are possible
only for α-D→L absolute configurations of monosaccharide residues [5]. Furthermore, the chemical shifts of the anomeric C
atoms agreed fully with the aforementioned sequence with respect to the glycosylation effects for 1,3-bonded monosaccharides
[5].
T
Thus, the repeat unit of the O-specific PS from A. addita strain 3600 had the structure →3)-α-D-Galp-(1→3)-α-L-
Rhap-(1→3)-α-D-Glcp-(1→ according to the obtained results.
EXPERIMENTAL
T
The type strain A. addita KMM 3600 from the Collection of Marine Microorganisms of PIBOC, FEB, RAS, was
isolated from marine waters of Chasma Bay (Sea of Japan). The isolation, cultivation, preliminary identification, and
13
preservation procedures have been published [3]. C NMR spectra in D O were recorded on a Bruker DPX-300 instrument
2
at 60°C with methanol internal standard (δ 50.15 ppm). PMRand 2D correlation spectra were recorded on a Bruker DRX-500
C
instrument with acetone internal standard (δ 2.225 ppm). Solutions were lyophilized or evaporated in vacuo. Specific optical
H
rotation was measured on a Perkin—Elmer 141 polarimeter in water. Descending paper chromatography (PC) was performed
550

on Whatman 1 and Whatman 3MM paper using n-butanol:pyridine:water (6:4:3). Monosaccharides were detected using basic
silver nitrate. Ion-exchange chromatography was performed on DEAE TSK 650M gel using tris-HCl buffer (0.5 M, pH 7.0).
Gel chromatography was carried out over a column (2.5 × 90 cm) with TSK HW 40 (F) gel in water. Elution curves were
constructed using an RIDK 101 (Czech Rep.) differential refractometer. LPS was isolated from wet microbial biomass by hot
aqueous phenol (45%) using the Westphal method [7]. Nucleic acids were removed by precipitation with trichloroacetic acid
(TCA) at pH 2. Yield of LPS was 50 mg from 1 L of medium.
Isolation of O-Specific Polysaccharide (PS). LPS (400 mg) was hydroyzed byCH COOH (1%, 40 mL, 100°C, 3 h).
3
The precipitate of lipid A was removed by centrifugation (yield of lipid A, 20 mg). The aqueous phase was lyophilized and
purified by anion-exchange chromatography over DEAE TSK 650M gel with subsequent gel chromatography over TSK HW
40 (F) gel. Yield of PS, 40 mg.
Smith Degradation. PS (20 mg) was dissolved in NaIO (3 mL, 0.1 N), stored in the dark for 90 h, reduced with
4
NaBH , neutralized byCH COOH, desalted bygel chromatographyover TSK HW 40 (F) gel, and lyophilized. The polyalcohol
4
3
was hydrolyzed byHOAc (1%, 1.5 h, 100°C) with subsequent gel chromatography over TSK HW 40 (F) gel and lyophilization.
Yield of modified PS, 15 mg.
Hydrolysis of PS (2 mg) was carried out in CF COOH (2 M, 0.5 mL, 100°C, 3 h). The acid was removed by
3
evaporation. The product was analyzed by PC, GC, and GC—MS as the polyol acetates. Preparative hydrolysis used PS
(100 mg) and CF COOH (1 M, 5 mL, 100°C, 3 h). The hydrolysate was purified bypreparative PC to afford D-glucose (14 mg),
3
[α] +65° (lit. [8], +52.7°); D-galactose (13 mg), [α] +72° (lit. [8], +81.1°), and L-rhamnose (10 mg), [α] +6.0° (lit. [8],
D
D
D
+9.1°). The last was converted to methyl-L-rhamnopyranoside (8 mg), [α] -65.5° (lit. [8], -62.5°), by methanolysis by an
D
acetylchloride:methanol (1:10) mixture (1 mL, 10 h).
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