Structures of the O‐specific Polysaccharide Chains of Pectinatus cerevisiiphilus and Pectinatus frisingensis Lipopolysaccharides

Abstract

Mild acid hydrolysis of the smooth-type lipopolysaccharide (LPS) of Pectinatus ,frisingensis afforded no polysaccharide but monomeric 6-deoxy-~-altrose (L-6dAlt) which was identified by anion-exchange chromatography in borate buffer, GLC/MS, 'H-NMR spectroscopy, and optical rotation. LPS was degraded with alkali under reductive conditions to give a completely 0-deacylated polysaccharide, which was studied by methylation analysis, 'H-NMR and IT-NMR spectroscopy, including sequential, selective spin-decoupling, two-dimensional correlation spectroscopy (COSY), COSY with relayed coherence transfer, two-dimensional heteronuclear "C,'H-COSY, one-dimensional NOE and two-dimensional rotating-frame NOE spectroscopy. It was found that the 0-specific polysaccharide chain of l? frisingensis LPS is a homopolymer of 6-deoxy-~-altrofuranose built up of tetrasaccharide-repeating units having the following structure:Similarly, mild acid degradation of smooth-type LPS of Pectinatus cerevisiiphilus resulted in depolymerisation of the polysaccharide chain to give a disaccharide consisting of D-glUCoSe and D-fucose. Study of the disaccharide by methylation analysis and alkali-degraded LPS by one-dimensional and twodimensional 'H-NMR and "C-NMR spectroscopy showed that the 0-specific polysaccharide of I? cerevisiiphilus has the following structure : --.2)-~-~-Fucf-(l-+2)-a-~-Glcp-(l--,Keywords: Pectinatus ; lipopolysaccharide; 0-specific polysaccharide; structure; methylation.Recently, outer-membrane lipopolysaccharides (LPS) of two species of the strictly anaerobic beer spoilage bacteria of the genus Pectinatus have been isolated and chemically characterised [l]. Both species (l? cerevisiiphilus and 19 frisingensis) elaborated mainly smooth-type LPS, but small amounts of chemically distinct rough-type LPS was also extracted from the cells. Sugar analysis showed that the 0-specific polysaccharide chains of the smooth-type Pectinatus LPSs are rich in 6-deoxyhexoses [I]. We now report the structures of the 0-specific polysaccharides of P. cerevisiiphilus and 19 frisingensis. The former contains, amongst others, D-fucofuranose, whereas the latter is a homopolymer of 6-deoxy-~-altrofuranose. EXPERIMENTAL PROCEDURESChromatography. Gel-permeation chromatography was carried out on a column (40 cmX2.S cm) of Sephadex G-50 or on a column (40 cmX1.5 cm) of TSK HW-40 using 0.5 M pyridine acetate, pH 4.5, or water, respectively as eluent; monitoring was performed using a Knauer differential refractometer. Anionexchange chromatography was performed with a column (15 cmX0.6 cm) of Durrum DAX4 resin using 0.S M sodium borate, pH 9, at 55 "C and monitored using a Biotronik LC-2000 sugar analyser. GLC was performed using a Hewlett-Packard 5890 instrument equipped with a glass capillary column (25 mX0.2 mm) coated with Ultra 1 stationary phase.NMR spectroscopy. NMR spectra of D,O solutions were run with a Bruker WM-250 instrument at 60°C. Acetone was used as internal standard (~3~2 . 2 3 ppm, 6,31.45 ppm). Selective spin decoupling [2], one-dimensiona...

Rare 6-deoxy-D-altrose from the folk medicinal mushroom Lactarius akahatsu

“…The values for the chemically synthesized authentic sample, as well as the material isolated from L. lividatus, were +18.4° (0.13%, at 25°C) and +18.2° (0.11%), respectively. The specific rotation of the 6-deoxy-l-altrose has been reported as a negative value [17][18][19]. Therefore, this result indicates that the 6-deoxy-sugar isolated from L. akahatsus was identified as 6-deoxy-d-altrose.…”

Pectinatus, Megasphaera and Zymophilus

“…For example, on the basis of the occurrence D-fucose in the O-specific chains of Pectinatus lipopolysaccharides, it was speculated (Helander et al, 1992) that Pectinatus originated from plant material, inasmuch as 6-deoxy Dhexoses are uncommon in nature except in plant pathogenic Gram-negative bacteria. The Ospecific sugar in P. frisingensis, however, later proved to be 6-deoxyaltrose in the L configuration (Senchenkova et al, 1995), showing that the presence of D-6-deoxyhexoses is not an obligatory component within the genus.…”

“…Complete structures of the core oligosaccharides in Pectinatus LPSs are not known, but each strain presumably elaborates two structurally distinct cores, only one of which carries polymeric O-specific chains; these two LPS populations can be separated from each other during processing of initial phenol/chloroform/petroleum ether extracts (Helander et al, 1992). The O-specific chain of P. cerevisiiphilus is composed of repeating disaccharides of 1 → 2 linked β-D-fucofuranose and α-D-glucopyranose, whereas the repeating unit of P. frisingensis O-specific chain is a branched tetrasaccharide consisting of a single sugar type, the rare 6-deoxy-L-altrofuranose (Senchenkova et al, 1995). It is worthy of note that the Ospecific chains of Pectinatus are highly labile towards acid, whereas the normally acid-labile lipid A-core linkage is exceptionally stable.…”

“…Several exceptional properties are assigned to Pectinatus LPSs, including the production of at least two distinct types of LPS by one strain (Helander et al, 1992), the presence of the phosphorylated disaccharide α-D-GlcpN-(1'-4)-Kdo (3-deoxy-D-manno-oct-2-ulopyranosonic acid) in the LPS core (Helander et al, 1993), the resistance of the lipid A-polysaccharide linkage to acid (Helander et al, 1994), and the predominance of furanosidic 6-deoxyhexoses in the O-specific chains (Senchenkova et al, 1995). The lipid A backbone of P. cerevisiiphilus and P. frisingensis is composed of the common bisphosphorylated β1'-6-linked glucosamine (GlcN) disaccharide, with almost quantitative substitution of the ester-linked phosphate by 4-amino-4-deoxyarabinose (Helander et al, 1994).…”