The 1H,13C HSQC and HBMC spectra of the polysaccharide showed minor CH3/CH3 and CH3/CO cross-peaks at δ 2.08/21.9 and δ 2.08/174.2, respectively, which indicated the presence of a minor O-acetyl group. However, its position could not be determined owing to its too low content and, as a result, the lack of NMR signals potentially useful for determination of the site of O-acetylation. The data obtained indicated that the O-antigen of P. alcalifaciens
O40 has the structure 1 shown in Fig. 4, where d-Qui3NFo stands for 3,6-dideoxy-3-formamido-d-glucose. This monosaccharide derivative occurs rarely in bacterial polysaccharides; to the best of our knowledge, Crenolanib cost earlier it has been reported only once as a component of the O-antigen of Hafnia alvei 1204 (Katzenellenbogen et al., 1995). The O-antigen structure and the presence of an O-acetyl group Gefitinib solubility dmso were confirmed independently by negative ion high-resolution ESI MS of oligosaccharide
fractions A and B. The mass spectrum of fraction B showed a major ion peak for a Hex4HexA1Hep3Kdo1Ara4N1P1PEtN3 oligosaccharide (where Ara4N indicates 4-amino-4-deoxyarabinose, Hep – heptose, Hex – hexose, HexA – hexuronic acid, Kdo – 2-keto-3-deoxyoctonic acid, PEtN – phosphoethanolamine) (experimental and calculated molecular masses 2200.55 and 2200.54 Da, respectively). The major causes of structural heterogeneity were the presence of compounds having one less or one more PEtN group (∆m 123.01) and the occurrence of incomplete core glycoforms lacking one or two hexose residues
(∆m 162.05 u each). Therefore, fraction B represents a core oligosaccharide with composition typical of Providencia species (Kondakova et al., 2006; Ovchinnikova et al., 2011), which was derived from the R-form LPS devoid of any O-antigen. The mass spectrum of Sinomenine fraction A demonstrated ion peaks for compounds with the molecular masses 3037.78 and 3079.80 Da accompanied by related species with one less and one more PEtN group. The mass differences of 714.23 and 756.24 Da between these compounds and the core oligosaccharide corresponded to the Qui3NFo1Gal1GlcA1GalNAc1 and Qui3NFo1Gal1GlcA1GalNAc1Ac1 tetrasaccharide O-units, respectively. Therefore, the fraction A oligosaccharides were derived from the SR-form LPS and consist of an O-unit, either O-acetylated or not, attached to the core. In addition, fraction A was found to contain the cyclic Fuc4N4ManNA4GlcN4Ac11 (where Fuc4N indicates 4-amino-4-deoxyfucose, ManNA – mannosaminuronic acid) dodecasaccharide enterobacterial common antigen (experimental and calculated molecular mass 2386.88 Da) and two minor dodecasaccharides having one less and one more acetyl group (∆m 42.01 Da). Enzyme-immunosorbent assay with rabbit polyclonal O-antiserum against P. alcalifaciens O40 was used to characterize the O-antigen specificity of this bacterium and to reveal possible relationships of the O40-antigen with those of other Providencia O-serogroups.