Structure of the O-polysaccharide of Photorhabdus temperata subsp. temperata XlNach T containing a novel branched monosaccharide, 3,6-dideoxy-4-C-[(S)-1,2-dihydroxyethyl]-D-xylo-hexose Nikolay P. Arbatsky a , Alexander S. Shashkov a , Nadezhda A. Kirsheva b , Anna N. Kondakova a , Rima Z. Shaikhutdinova b , Sergey A. Ivanov b , Andrey P. Anisimov b , Yuriy A. Knirel a,⇑ a N.D. Zelinsky Institute of Organic Chemistry, Russian Academy of Sciences, Moscow, Russia b State Research Center for Applied Microbiology and Biotechnology, Obolensk, Moscow Region, Russia article info Article history: Received 8 January 2014 Received in revised form 11 February 2014 Accepted 13 February 2014 Available online 28 February 2014 Keywords: Photorhabdus temperata Lipopolysaccharide O-Antigen Bacterial polysaccharide structure Branched monosaccharide abstract O-Polysaccharide was isolated from the lipopolysaccharide of an entomopathogenic bacterium Photorhabdus temperata subsp. temperata XlNach T . Sugar analysis after full acid hydrolysis of the polysaccharide revealed D-glucose, D-mannose, D-galactose, D-GalNAc, and a branched monosaccharide, 3,6-dideoxy-4-C-[(S)-1 0 ,2 0 -dihydroxyethyl]-D-xylo-hexose (Sug), which was isolated as a 1,2 0 -anhydro furanose derivative. The following structure of the polysaccharide was established by 1D and 2D 1 H and 13 C NMR spectroscopy: Ó 2014 Elsevier Ltd. All rights reserved. 1. Introduction The genus Photorhabdus from the family Enterobacteriaceae in- cludes three species of entomopathogenic bioluminescent bacteria Photorhabdus temperata, Photorhabdus luminescens, and Photorhab- dus asymbiotica. They all have a mutualistic relationship with entomophagous nematodes from the family Heterorhabditis. 1 These bacteria synthesize an S-form lipopolysaccharide, which consists of lipid A, core oligosaccharide, and O-specific polysaccha- ride (O-antigen) and is important for both pathogenicity and sym- biosis. 2 Recently, aiming at creation of the chemical basis for classification of Photorhabdus species, structures of the O-polysac- charides of representatives of P. asymbiotica subsp. asymbiotica and subsp. australis 3 and P. luminescens subsp. laumondii 4 have been elucidated. In this work, we established the O-polysaccharide structure of P. temperata subsp. temperata XlNach T . 2. Results and discussion The O-polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide (1 % HOAc, 100 °C) isolated from bacterial cells by phenol–water extraction. Full acid hydrolysis of the O-polysaccharide with 3M CF 3 CO 2 H (120 °C) released almost equal amounts of glucose, mannose, and galactose as well as 2-amino-2-deoxygalactose (GalN), which were identified by GLC of the acetylated alditols. Their D configuration was established by GLC of the acetylated (S)-2-octyl glycosides. After hydrolysis under milder conditions (1 M CF 3 CO 2 H, 100 °C), a branched deoxy sugar derivative 1 also was isolated by reversed-phase HPLC and studied by NMR spectroscopy. The 1 H and 13 C NMR spectra and the 2D heteronuclear 1 H, 13 C HSQC spectrum of 1 revealed 9 proton and 8 carbon signals, includ- ing those for one quaternary carbon at d 90.3, one anomeric center at d H /d C 5.17/105.0, and one group each of CH 3 –C, C–CH 2 –C, and OCH 2 –C (Table 1). The signals were assigned using 2D COSY, TOCSY, 1 H, 13 C HSQC, and HMBC experiments, and three separate http://dx.doi.org/10.1016/j.carres.2014.02.020 0008-6215/Ó 2014 Elsevier Ltd. All rights reserved. ⇑ Corresponding author. Tel.: +7 499 1376148; fax: +7 499 1355328. E-mail address: yknirel@gmail.com (Y.A. Knirel). Carbohydrate Research 403 (2015) 202–205 Contents lists available at ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres